Archive for April, 2016

Request customized products to meet your specific research needs:

Please contact Technical Support for further details: techsupport@stemcell.com

Cryopreserved Leuko Pak and Whole Blood Products:

Donor Screening:Donors are screened for HIV (1 & 2), Hepatitis B, and Hepatitis C.

Cryopreserved products are shipped with negative test results from donor screening that is done within 90 days of collection.

Fresh Leuko Pak and Whole Blood Products:

Donor Screening:Donors are screened for HIV (1 & 2), Hepatitis B, and Hepatitis C.

If the donor was screened within 90 days of donation the product will be shipped with negative test results from donor screening.

If the donor was not screened within 90 days of collection, a test sample will be taken at the time of donation and the product will be shipped before the screening results are available. In the unlikely event that a test result is positive, the customer will be contacted as soon as possible (usually within 24-72 hours from the time of shipment).

Cryopreserved Cord Blood Products:

Donor Screening:Cord blood is only collected from mothers that have tested negative for HIV (1 & 2) and Hepatitis B during their pregnancy. Hepatitis C is tested for at the time of collection.

Cryopreserved products are shipped with negative test results from donor screening.

Fresh Cord Blood Products:

Donor Screening: Cord blood is only collected from mothers that have tested negative for HIV (1 & 2) and Hepatitis B during their pregnancy. Hepatitis C is tested for at the time of collection.

Fresh cord blood products are shipped with negative test results for HIV (1 & 2) and Hepatitis B donor screening. Hepatitis C test results are not available at the time of shipment. In the unlikely event that the Hepatitis C test result is positive, the customer will be contacted as soon as possible (usually within 24-72 hours from the time of shipment).

STEMCELL does not test for infectious diseases other than those listed above and the testing that is done cannot completely guarantee that the donor was virus-free. Therefore THESE PRODUCTS SHOULD BE TREATED AS POTENTIALLY INFECTIOUS and only used following appropriate handling precautions such as those described in biological safety level 2. When handling these products do not use sharps such as needles and syringes.

See the original post here:
Human Primary Cells - Stemcell Technologies

Bio-Identical Hormone Replacement Therapy, along with proper nutrition and exercise has the ability to return men and women to the optimal mental, physical, emotional and sexual health they had when they were younger adults. Bio-Identical hormone replacement BHRT therapy has renewed the lives of millions of men and women with hormonal imbalances by significantly improving their quality of life and their health.

Natural, Bio-Identical hormone replacement therapy (BHRT) has been used with great success by patients throughout Europe and the United States since the 1930s to improve the lives of millions of men and women with hormonal imbalances. Unlike conventional hormone therapy, which uses synthetic hormones, Bio-Identical hormone replacement therapy uses naturally derived hormones in the proper levels to eliminate symptoms of hormone imbalance. Bio-Identical hormones are identical to the hormones produced naturally by the human body.

Natural bio-identical hormone replacement therapy, under a doctor's supervision, is a safe and effective treatment that significantly improves the quality of life and health of people suffering from age-related hormone imbalance.

BENEFITS OF HORMONE REPLACEMENT THERAPY FOR WOMEN

Benefits of Bio-Identical hormone replacement therapy for women may include the elimination of night sweats and hot flashes, vaginal dryness and itching, improved energy levels, improved fat loss and muscle tone, improved mood and sex drive, improved memory and concentration and may reduce risk of heart disease.

BENEFITS OF HORMONE REPLACEMENT THERAPY FOR MEN

Benefits of Bio-Identical hormone replacement therapy for men may include improved muscle mass, tone and energy levels resulting in improved exercise endurance, improved fat loss, improved muscle tone, improved mood and sex drive, improved memory and concentration, improved sleep patterns, and improved cholesterol levels.

Bio-Identical hormones are most commonly prescribed to treat symptoms of hormonal imbalance in women and men, such as hot flashes, night sweats, decrease or loss of libido, weight gain, fatigue, mood swings and irritability.

Many patients choose Bio-Identical Hormone Replacement Therapy (BHRT) because it is prescribed on an individualized basis and does not fall into the category of a one-size-fits-all type of approach.

Dr. Jason Collins has extensive experience in both natural and conventional medicine and insures that each Bio-Identical hormone regimen is custom-compounded and is based on each patient's individual diagnostic results, making them not only safer, but also more effective than traditional synthetic hormones.

Studies show that natural Bio-Identical hormone replacement therapy for men and natural Bio-Identical hormone replacement therapy for women significantly improves ones quality of life and health while decreasing the risk of developing chronic illnesses in the future.

Read the rest here:
Bioidentical Hormone Replacement Therapy for Men and Women ...

April 28, 2016

A pair of molecular signals controls skin and hair color in mice and humansand could be targeted by new drugs to treat skin pigment disorders like vitiligo, according to a report by scientists at NYU Langone Medical Center.

Finding ways to activate these pathways, researchers say, could lead to therapies that repigment skin cells damaged in vitiligo, a disfiguring illness marked by the loss of skin pigmentation, leaving a blotchy, white appearance. The same pathways could serve as targets for drug therapies that repigment grayed hair cells for people seeking a younger look but who are allergic to cosmetic dyes. Such therapies might even one day reinforce pigment to correct discoloration around scars.

In experiments in mice and human cells, researchers found that control of these skin and early-stage hair cells, known as melanocyte stem cells, is regulated by cell-to-cell signaling reactions. These reactions are part of the endothelin receptor type B (EdnrB) and the Wnt signaling pathways.

Previous research had shown that endothelin proteins and the EdnrB pathway help control blood vessel development, as well as some aspects of cell growth and division, the scientists say. But they believe that their new findings, to be published in the journal Cell Reports online April 28, are the first evidence tying the signaling pathways to the routine growth of cells that produce pigment (melanocytes) and provide color to skin and hair.

They say the study is the first to outline the link between EdnrB and Wnt signaling, confirming that EdnrB coordinates the rapid reproduction of melanocyte stem cells.

"Our study results show that EdnrB signaling plays a critical role in growth and regeneration of certain pigmented skin and hair cells and that this pathway is dependent on a functioning Wnt pathway," says study senior investigator and cell biologist Mayumi Ito, PhD. Ito is an associate professor in the Ronald O. Perelman Department of Dermatology at NYU Langone and a member of NYU Langone's Helen L. and Martin S. Kimmel Center for Stem Cell Biology.

Among the study's key findings, Ito reports, was that mice bred to be deficient in the EdnrB pathway experienced premature graying of their fur.

Study co-lead investigator and postdoctoral fellow Wendy Lee, PhD, says the pathway's involvement in determination of hair color was "clearly evident" in the mice when she first examined them.

In further experiments in mice, stimulating the EdnrB pathway resulted in a 15-fold increase in melanocyte stem cell pigment production within two months, producing what Ito calls "hyperpigmentation." Wounded skin in normally white mice became dark upon healing.

In the latest study, Ito and her team found that blocking Wnt signaling stalled stem cell growth and the maturing of stem cells into normally functioning melanocytes, even when endothelin proteins were present. This led to mice with unpigmented grayish coats.

Ito says her team plans further investigations into how other cell repair and signaling pathways interact with EdnrB and melanocyte stem cells.

According to the National Institute of Arthritis and Musculoskeletal and Skin Diseases, vitiligo occurs in about 1 percent of people worldwide.

Explore further: New research provides clues on why hair turns gray

A new study by researchers at NYU Langone Medical Center has shown that, for the first time, Wnt signaling, already known to control many biological processes, between hair follicles and melanocyte stem cells can dictate ...

Mammals possess the remarkable ability to regenerate a lost fingertip, including the nail, nerves and even bone. In humans, an amputated fingertip can sprout back in as little as two months, a phenomenon that has remained ...

A pathway known for its role in regulating adult stem cells has been shown to be important for hair follicle proliferation, but contrary to previous studies, is not required within hair follicle stem cells for their survival, ...

Regenerative medicine may offer ways to banish baldness that don't involve toupees. The lab of USC scientist Krzysztof Kobielak, MD, PhD has published a trio of papers in the journals Stem Cells and the Proceedings of the ...

The human body maintains a healthy layer of skin thanks to a population of stem cells that reside in the epidermis. Previously, the signals responsible for regulating these so-called 'interfollicular epidermal stem cells' ...

Nicotinamide riboside (NR) is pretty amazing. It has already been shown in several studies to be effective in boosting metabolism. And now a team of researchers at EPFL's Laboratory of Integrated Systems Physiology (LISP), ...

Everything you eat or drink affects your intestinal bacteria, and is likely to have an impact on your health. That is the finding of a large-scale study led by RUG/UMCG geneticist Cisca Wijmenga into the effect of food and ...

In a major breakthrough, scientists at the Gladstone Institutes transformed skin cells into heart cells and brain cells using a combination of chemicals. All previous work on cellular reprogramming required adding external ...

For the first time, scientists studying stool transplants have been able to track which strains of bacteria from a donor take hold in a patient's gut after a transplant. The team, led by EMBL with collaborators at Wageningen ...

Researchers at the Icahn School of Medicine at Mount Sinai say that tiny doses of a cancer drug may stop the raging, uncontrollable immune response to infection that leads to sepsis and kills up to 500,000 people a year in ...

Age-related changes in the human pancreas govern how our bodies respond to rising and falling blood sugar levels throughout our lifetimes, and could affect whether we develop diabetes as adults. But it's been nearly impossible ...

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Read more from the original source:
Stem cell study finds mechanism that controls skin and ...

Frequently Asked Questions About Genetic Testing What is genetic testing?

Genetic testing uses laboratory methods to look at your genes, which are the DNA instructions you inherit from your mother and your father. Genetic tests may be used to identify increased risks of health problems, to choose treatments, or to assess responses to treatments.

There are many different types of genetic tests. Genetic tests can help to:

Genetic test results can be hard to understand, however specialists like geneticists and genetic counselors can help explain what results might mean to you and your family. Because genetic testing tells you information about your DNA, which is shared with other family members, sometimes a genetic test result may have implications for blood relatives of the person who had testing.

Top of page

Diagnostic testing is used to precisely identify the disease that is making a person ill. The results of a diagnostic test may help you make choices about how to treat or manage your health.

Predictive and pre-symptomatic genetic tests are used to find gene changes that increase a person's likelihood of developing diseases. The results of these tests provide you with information about your risk of developing a specific disease. Such information may be useful in decisions about your lifestyle and healthcare.

Carrier testing is used to find people who "carry" a change in a gene that is linked to disease. Carriers may show no signs of the disease; however, they have the ability to pass on the gene change to their children, who may develop the disease or become carriers themselves. Some diseases require a gene change to be inherited from both parents for the disease to occur. This type of testing usually is offered to people who have a family history of a specific inherited disease or who belong to certain ethnic groups that have a higher risk of specific inherited diseases.

Prenatal testing is offered during pregnancy to help identify fetuses that have certain diseases.

Newborn screening is used to test babies one or two days after birth to find out if they have certain diseases known to cause problems with health and development.

Pharmacogenomic testing gives information about how certain medicines are processed by an individual's body. This type of testing can help your healthcare provider choose the medicines that work best with your genetic makeup.

Research genetic testing is used to learn more about the contributions of genes to health and to disease. Sometimes the results may not be directly helpful to participants, but they may benefit others by helping researchers expand their understanding of the human body, health, and disease.

Top of page

Benefits: Genetic testing may be beneficial whether the test identifies a mutation or not. For some people, test results serve as a relief, eliminating some of the uncertainty surrounding their health. These results may also help doctors make recommendations for treatment or monitoring, and give people more information for making decisions about their and their family's health, allowing them to take steps to lower his/her chance of developing a disease. For example, as the result of such a finding, someone could be screened earlier and more frequently for the disease and/or could make changes to health habits like diet and exercise. Such a genetic test result can lower a person's feelings of uncertainty, and this information can also help people to make informed choices about their future, such as whether to have a baby.

Drawbacks: Genetic testing has a generally low risk of negatively impacting your physical health. However, it can be difficult financially or emotionally to find out your results.

Emotional: Learning that you or someone in your family has or is at risk for a disease can be scary. Some people can also feel guilty, angry, anxious, or depressed when they find out their results.

Financial: Genetic testing can cost anywhere from less than $100 to more than $2,000. Health insurance companies may cover part or all of the cost of testing.

Many people are worried about discrimination based on their genetic test results. In 2008, Congress enacted the Genetic Information Nondiscrimination Act (GINA) to protect people from discrimination by their health insurance provider or employer. GINA does not apply to long-term care, disability, or life insurance providers. (For more information about genetic discrimination and GINA, see http://www.genome.gov/10002328/genetic-discrimination-fact-sheet/).

Limitations of testing: Genetic testing cannot tell you everything about inherited diseases. For example, a positive result does not always mean you will develop a disease, and it is hard to predict how severe symptoms may be. Geneticists and genetic counselors can talk more specifically about what a particular test will or will not tell you, and can help you decide whether to undergo testing.

Top of page

There are many reasons that people might get genetic testing. Doctors might suggest a genetic test if patients or their families have certain patterns of disease. Genetic testing is voluntary and the decision about whether to have genetic testing is complex.

A geneticist or genetic counselor can help families think about the benefits and limitations of a particular genetic test. Genetic counselors help individuals and families understand the scientific, emotional, and ethical factors surrounding the decision to have genetic testing and how to deal with the results of those tests. (See: Frequently Asked Questions about Genetic Counseling)

Top of page

Talking Glossary of Genetic Terms

Genetic Testing From Genetics Home Reference: the benefits, costs, risks and limitations of genetic testing.

Genetic Testing Registry [ncbi.nlm.nih.gov] A publicly funded medical genetics information resource developed for physicians, other healthcare providers, and researchers.

Prenatal Screening [marchofdimes.com] Provides prenatal testing information, including ultrasound, amniocentesis and chorionic villus sampling (CVS).

National Newborn Screening & Genetics Resource Center [genes-r-us.uthscsa.edu] Provides information and resources in the area of newborn screening and genetics.

Genetic Alliance- Genes in Life [genesinlife.org] A guide from the Genetic Alliance with easy-to-read information about genetic testing.

Genetics and Cancer [cancer.gov] An information fact sheet from the National Cancer Institute about genetic testing for hereditary cancers.

Find a Genetic Counselor [nsgc.org] A search engine developed by the National Society of Genetic Counselors.

Top of page

Last Updated: August 27, 2015

Read the rest here:
genome.gov - FAQ About Genetic Testing

It is a critical moment for adoptive cell therapies. Clinical progress has been made with Chimeric Antigen Receptors (CAR), T Cell Receptors (TCR), and Tumor Infiltrating Lymphocytes (TIL), making these therapies the frontrunner for curing immune-based diseases. Still, many challenges remain. The Third Annual Adoptive T Cell Therapy event will bring together immunotherapy veterans and visionaries to not just address those challenges, but to provide solutions and showcase emerging opportunities. This years event will address topics such as developing adoptive cell therapies for solid tumors as well as new targets of interest. Emphasis will be placed on clinical case studies to further the understanding of T cell receptors and their biology. Overall, this event will uncover the critical components needed to make adoptive T cell therapies viable.

Day 1 | Day 2 | Download Brochure

WEDNESDAY, APRIL 27

7:00 am Registration and Morning Coffee

8:00 Chairpersons Remarks

Jeff Till, Ph.D., Director, External Innovation, EMD Serono R&D Institute

8:10 Jedi T Cells Provide a Universal Platform for Interrogating T Cell Interactions with Virtually Any Cell Population

Brian D. Brown, Ph.D., Associate Professor, Genetics and Genomic Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai

We recently generated the first GFP-specific T-cell mouse, called the Jedi (Agudo et al. Nat Biotech 2015). The Jedi technology is the first to facilitate direct visualization of a T-cell antigen, which enables unparalleled detection of antigen-expressing cells, and make it possible to utilize the 100s of cell type-specific GFP-expressing mice, tumors, and pathogens, to gain new insight into T-cell interactions with virtually any cell population in normal and diseased tissues.

8:40 The State-of-the-Art with T cell Receptor-Based Cancer Immunotherapies

Andrew K. Sewell, Ph.D., Distinguished Research Professor, Wellcome Trust Senior Investigator; Research Director, Institute of Infection and Immunity, Henry Wellcome Building, Cardiff University School of Medicine

The ab TCR enables cytotoxic T cells to scan the cellular proteome for anomalies from the cell surface. Tumor-specific TCRs can access a far greater range of targets than are available for antibodies. Engineered TCRs can be used in gene therapy and soluble molecule approaches. Next generation strategies allow circumvention of HLA-restriction. I will discuss future directions in the use of engineered T cells and TCRs in cancer immunotherapy.

9:10 Tumor Infiltrating Lymphocytes for Metastatic Cutaneous and Non-Cutaneous Melanoma: A UK Perspective

John S. Bridgeman, Ph.D., Director, Cell Therapy Research, Cellular Therapeutics Ltd.

We have established the UKs only GMP-compliant and MHRA (Medicines and Healthcare Products Regulatory Agency) licensed unit capable of producing multiple T cell product types (CAR or TCR-modified and natural T cells (TIL)) using clean room free technology. This unit has produced melanoma-derived TIL products which have been successfully returned to patients. This study supports the success of melanoma TIL therapy seen in other centers worldwide and suggests that this is a viable means of treating a disease which has few effective options.

9:40 Design of a Highly Efficacious, Mesothelin-Targeting CAR for Treatment of Solid Tumors

Boris Engels, Ph.D., Investigator, Exploratory Immuno-Oncology, Novartis Institutes for Biomedical Research

The treatment of solid tumors with CAR T cells has shown to be challenging. We describe the design of a fully human CAR targeting mesothelin, a tumor associated antigen overexpressed in mesothelioma, pancreatic and ovarian cancer. The screen of a scFv pool has identified two scFvs, which show enhanced efficacy as CARs, superior to what is currently being used by several groups. We have performed in-depth characterization of the scFvs and CARs to gain insight into structure-activity relationships, which may influence CAR design and efficacy.

10:10 Coffee Break in the Exhibit Hall with Poster Viewing

10:55 ACTR (Antibody Coupled T Cell Receptor): A Universal Approach to T cell Therapy

Seth Ettenberg, Ph.D., CSO, Unum Therapeutics

Fusing the ectodomain of CD16 to the co-stimulatory and signaling domains of 41BB and CD3z generates an Antibody Coupled T cell Receptor (ACTR). T cells expressing this receptor show powerful anti-tumor cytotoxicity when co-administered with an appropriate tumor-targeting antibody. Such cells have potential utility as a therapy to treat a wide range of cancer indications. We will describe efforts specifically targeting B-cell malignancies using a combination of ACTR T cells with rituximab.

11:25 Strategies to Optimize Tumor Infiltrating Lymphocytes (TIL) for Adoptive Cell Therapy

Shari Pilon-Thomas, Ph.D., Assistant Professor, Department of Immunology, Moffitt Cancer Center

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) has emerged as a powerful immunotherapy for cancer. TIL preparation involves surgical resection of tumors and in vitro expansion of TIL from tumor fragments. ACT depends upon the presence of TIL in tumors, successful expansion of TIL, and effective activation and persistence of T cells after infusion. In this presentation, I will discuss optimization of TIL infiltration into tumors and TIL expansion for ACT in melanoma and other cancers.

11:55 Engineered T Cell Receptors for Adoptive T Cell Therapy in Solid Tumors

Jo Brewer, Ph.D., Director, Cell Research, Adaptimmune Ltd.

NY-ESO-1 is a cancer antigen that is expressed by a wide array of solid and hematological tumors. An enhanced affinity TCR that recognizes this antigen is currently in Phase I/II trials for synovial sarcoma, multiple myeloma, melanoma, ovarian and esophageal cancers. Early clinical data demonstrate encouraging responses and a promising benefit/risk profile.

12:25 pm Cell Based Engineering of TCRs and CARs Using in vitro V(D)J Recombination

Michael Gallo, President, Research, Innovative Targeting Solutions

The ability to generate antibodies and TCRs specific to a MHC/peptide complex provides for new therapeutic opportunities. A novel approach using in vitro V(D)J recombination has been shown to be a robust strategy for targeting these ultra-rare epitopes by generating large de novo repertoires of fully human antibodies, CARs, or T-cell receptors on the surface of mammalian cells. The presentation highlights the advantages of cell based engineering for the generation of cell based adoptive therapies.

12:55 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:55 Session Break

2:10 Chairpersons Remarks

Jonathan Schneck, Ph.D., M.D., Professor, Pathology, Medicine and Oncology, Johns Hopkins

2:15 Artificial APCs: Enabling Adoptive T Cell Therapies

Marcela V. Maus, M.D., Ph.D., Director, Cellular Immunotherapy, Mass General Hospital Cancer Center

Adoptive T cell therapies require ex vivo T cell culture systems, which can include artificial antigen presenting cells. We will review several types of natural and artificial APCs and how they can be optimized to generate strong memory and effector T cells usable for adoptive transfer.

2:45 Immunoengineering of Artificial Antigen Presenting Cells, aAPC: From Basic Principles to Translation

Jonathan Schneck, Ph.D., M.D., Professor, Pathology, Medicine and Oncology, Johns Hopkins

Artificial antigen presenting cells (aAPCs) are immuno-engineered platforms which advance adoptive immunotherapy by reducing the cost and complexity of generating tumor-specific T cells. Our new approach, termed Enrichment and Expansion (E+E), utilizes paramagnetic nanoparticle-based aAPCs to rapidly expand both shared tumor antigen- and neoepitope-specific CTL. Streamlining the rapid generation of large numbers of T cells in a cost-effective fashion can be a powerful tool for immunotherapy.

3:15 Vector Free Engineering of Immune Cells for Enhanced Antigen Presentation

Armon Sharei, Ph.D., CEO, SQZ Biotech

In this work we describe the use of the vector-free technology to deliver antigen protein directly to the cytoplasm of antigen presenting cells to drive a powerful antigen specific T-cell response. Current efforts to use antigen presenting cells to drive T-cell responses rely on an inefficient process called cross-presentation that relies on material escaping the endosome and entering the cytoplasm. We believe that by delivering antigen directly to the cytoplasm of antigen presenting cells we can overcome this long standing barrier and drive powerful and specific T-cell responses. Our results show that by adoptively transferring antigen presenting cells that have antigen delivered into them we can drive a significant T-cell response. Specifically, we found that this results in a ~50x increase in antigen specific T-cells in vivo when compared to endocytosis. This advance has the potential to dramatically enhance the therapeutic potential of therapeutic vaccination with antigenic material for the treatment of a wide variety of cancers. Indeed, the ability to deliver structurally diverse materials to difficult-to-transfect primary cells indicate that this method could potentially enable many novel clinical applications.

3:45 Refreshment Break in the Exhibit Hall with Poster Viewing

4:45 Problem-Solving Breakout Discussions

Moving Adoptive T Cell Therapies Toward the End Game

Moderator: Richard S. Kornbluth, M.D., Ph.D., President & CSO, Multimeric Biotherapeutics, Inc.

Focusing CAR, TCR, and TIL for Effective Therapies

Moderator: John S. Bridgeman, Ph.D., Director, Cell Therapy Research, Cellular Therapeutics Ltd.

5:45 Networking Reception in the Exhibit Hall with Poster Viewing

7:00 End of Day

Day 1 | Day 2 | Download Brochure

THURSDAY, APRIL 28

8:00 am Morning Coffee

8:30 Chairpersons Remarks

Richard S. Kornbluth, M.D., Ph.D., President & CSO, Multimeric Biotherapeutics, Inc.

8:35 CD40 Ligand (CD40L) and 4-1BB Ligand (4-1BBL) as Keys to Anti-Tumor Immunity

Richard S. Kornbluth, M.D., Ph.D., President & CSO, Multimeric Biotherapeutics, Inc.

CD40 ligand (CD40L) and 4-1BB ligand (also called CD137L) activate immunity by binding to and clustering their receptors. We have solved the receptor clustering problem by creating fusion proteins that contain many TNFSF trimers. In this talk, we will discuss how soluble multi-trimer forms of TNFSFs such as CD40L and 4-1BBL have many important applications in cancer immunotherapy.

9:05 Portable Genetic Adjuvants Inspired by the EBV Latent Membrane Protein-1 (LMP1)

Richard S. Kornbluth, M.D., Ph.D., President & CSO, Receptome, LLC

The strongest CD8+ T cell response in humans occurs in Epstein-Barr Virus (EBV) infection and is due to LMP1, a CD40 receptor homologue. The LMP1 nucleic acid sequence activates dendritic cells and adjuvants RNA, DNA, and viral vaccines. Joining the LMP1 N-terminal domain with IPS-1 forms LMP1-IPS-1, a STING pathway activator and vaccine adjuvant. This technology provides a new approach for using CD40 and the STING pathway for cancer immunotherapy.

9:35 Overview of NK Cell and T Cell Therapies For Hematologic Malignancies After Hematopoietic Stem Cell Transplantation Conrad (Russell) Y. Cruz M.D., Ph.D., Assistant Professor of Pediatrics; Director, Translational Research Laboratory, Program for Cell Enhancement and Technologies for Immunotherapy (CETI), Children's National

10:05 Coffee Break in the Exhibit Hall with Poster Viewing

11:05 Genetic Modification of CAR-T cells for Solid Tumors: Challenges and Advancement

Pranay Khare, Ph.D., Independent Consultant

CAR-T cell engineering for adoptive T cell therapy have consistently shown exciting results by several groups in hematologic malignancies. But, limited success has been achieved in solid tumor field with CAR-T cell therapy. Efforts have been focused to improve CAR-T cells specificity, potency and persistence with variety of non-viral and viral vectors. This talk will focus on different strategies and lessons learned from hematologic malignancies and other novel ways to overcome the obstacles in solid tumor field.

11:35 Engineering Human T Cell Circuitry

Alex Marson, Ph.D., UCSF Sandler Fellow, University California, San Francisco

T cell genome engineering holds great promise for cancer immunotherapies and for cell-based treatments for immune deficiencies, autoimmune diseases and HIV. We have overcome the poor efficiency of CRISPR/Cas9 genome engineering in primary human T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs can promote targeted genome sequence replacement in primary T cells by homology-directed repair (HDR), which was previously unattainable with CRISPR/Cas9. This provides technology for diverse experimental and therapeutic applications.

12:05 pm Engineering the Genome of CAR T Cells: From Therapeutic Procedures to Products

Andr Choulika, Ph.D., CEO and Chairman, Cellectis

Cellectis therapeutics programs are focused on developing products using TALEN-based gene editing platform to develop genetically modified T cells that express a Chimeric Antigen Receptors (CAR) for cancer treatment. The first product, UCART19, T cells has been gene-edited to suppress GvHD and enable resistance to an Alemtuzumab treatment. The objective of this first product is to convert the CART cell therapy for an autologous approach to an off-the-shelve allogeneic CART product that can be produced in a cost effective fashion, stored, shipped anywhere in the world and immediately available to patient with an immediate unmet medical need.

12:35 End of Adoptive T Cell Therapy

5:15 Registration for Dinner Short Courses

Day 1 | Day 2 | Download Brochure

Visit link:
Adoptive T Cell Therapy Conference

Finding a gene can be like a treasure hunt.

At first it might seem weird that researchers found a bit of DNA involved in baldness but that they can't figure out why it is involved. The reason for this has to do with the way people find DNA involved in disease.

Human DNA is a long string of 3 billion letters (or bases). Each human is unique because these letters are arranged in a certain order*.

It is too expensive to figure out all of the bases of the DNA from the hundreds or thousands of people involved in a typical study. So what scientists have done is figured out millions of places in human DNA where these letters are often different between people. (This is called the HapMap.)

These differences or SNPs (single nucleotide polymorphisms) work like landmarks to help scientists find which part of the DNA to focus on. They are like clues on a treasure map.

The first part in using a treasure map is narrowing down what part of the world the treasure is in. Imagine the map shows that the treasure is in San Francisco. Then there might be clues that the treasure is near a certain hill or near an oddly shaped tree. Perhaps the treasure is buried near the tower on Mt. Sutro.

With this information, the treasure seekers can get digging. If they know a treasure is in San Francisco, they can't just dig up the whole city. But if they know it is near the tower on Mt. Sutro, then they can dig all over that area.

This is how DNA searches work too. Scientists use SNPs as landmarks to narrow down DNA regions to focus on.

Instead of a treasure map, scientists use the HapMap. They use this map to compare the DNA of people with and without the condition they are interested in. In these studies, scientists compared the DNA of balding and not balding men.

The first study looked at German men. One experiment in this study compared 296 balding men to 347 German men and women who were not seriously bald. The researchers looked at over 500,000 different spots on their DNA and found that bald people shared a number of landmarks in a 1.7 million base chunk of chromosome 20. They had narrowed it down to San Francisco.

More clues led them to a single letter difference that was shared by many of the balding men. A second experiment looked at 319 bald men and compared them to 234 men who weren't bald by the age of 60. This second experiment confirmed the results of the first one.

The second study was done similarly. They compared 578 Swiss men with male pattern baldness to 547 Swiss men who weren't balding. They found a different SNP near the one the first study found. They confirmed that this DNA difference as associated with baldness in over 3000 other individuals from a variety of Northern European countries.

So these two studies have narrowed down where the "treasure" is. They made it to Mt. Sutro. They know that something on a small section of chromosome 20 is partly responsible for balding in Northern European men.

The next steps will be to do some serious digging and to find the treasure. In other words, the researchers need to figure out what in this region is causing these men to bald early. And once they do that, they need to find out why these men go bald. With that information, they might be able to create medicines that can treat baldness.

Usually there is a gene nearby that researchers can investigate. In this case, there isn't. The SNPs are in the middle of nowhere with the nearest gene being at least 350,000 bases away. So researchers have their work cut out for them.

In doing these studies, the researchers also rediscovered the DNA difference that men can inherit from their mom's dad that can lead to early balding.

*The exception is identical twins who have essentially the same DNA but are still unique for environmental reasons.

See original here:
The Genetics of Balding | Understanding Genetics

Stem cell therapies for post-heart attack tissue repair have had modest success at best. Clinical trials have primarily used bone marrow cells, which can promote the growth of new blood vessels, but many studies have shown no benefit. A better alternative may be to use human heart muscle cells (cardiomyocytes), suggests a study published October 22 in Stem Cell Reports, the journal of the International Society for Stem Cell Research.

The authors compared how well human embryonic stem cell-derived cardiomyocytes, embryonic stem cell-derived cardiovascular progenitors, and bone marrow cells worked to repair tissue damage post-heart attack in a rat. The verdict is that both cardiomyocytes and progenitor cells surpassed the healing power of bone marrow cells. And despite the progenitors' abilities to differentiate into more cell types, they demonstrated no statistically significant improvement in heart tissue function, which means the more mature and stable heart muscle cells are a viable option for future therapies.

"There's no reason to go back to more primitive cells, because they don't seem to have a practical advantage over more definitive cell types in which the risk for tumor formation is lower," says senior study author Charles Murry of the University of Washington, Seattle. "The other important finding is that both of these populations are far superior to bone marrow cells. This work is a go signal that tells us to keep moving on to more promising and more powerful cell types in human trials."

The experiments, led by first authors Sarah Fernandes and James J.H. Chong, involved injecting the cells in the walls of the heart and measuring how well heart muscle tissue contracted in follow-up tests 4 weeks later. About ten animals receiving each of the three treatment variables and ten controls receiving a non-therapeutic cell population were included in the study. Injections were given 4 days after heart attacks occurred in the rats, as interventions that are given later don't have as much impact.

James Chong, now an interventional cardiologist at the University of Sydney, added: "We have recently had success in regenerating hearts of monkeys using a similar approach of transplanting stem cell-derived cardiomyocytes. The next goals will be to determine if these large animal experiments show similar improvements in cardiac function, and if so, to begin testing these cells in human patients."

Story Source:

The above post is reprinted from materials provided by Cell Press. Note: Materials may be edited for content and length.

Go here to see the original:
Cardiac muscle cells as good as progenitors for heart ...

Cancer is a condition triggered by mutations (changes) in the genes of a cell that result in uncontrolled, abnormal cell growth. Some families have gene mutations that are passed down from one generation to the next.

Genetic testing may help you determine if your cancer was due to an inherited gene mutation and if you are at an increased risk of developing a second cancer.

During your initial evaluation period at Cancer Treatment Centers of America (CTCA), you will fill out a family history questionnaire, which includes questions regarding your personal and family history of cancer. This information will help determine if you are a candidate for genetic testing.

The following are some red flags for a hereditary cancer predisposition:

Genetic testing consists of a mouthwash or blood test. Analysis of the sample can determine if you inherited a gene mutation that contributed to your diagnosis of cancer. Genetic testing might also help determine if you are at greater risk of developing the same cancer again or of developing another type of cancer.

Genetic testing can help you make informed decisions about how to manage future risks of cancer. For example, if it is determined that you are at greater risk than the average patient for breast cancer recurrence, we may recommend adding breast MRIs to your routine screenings.

Also, if you are a woman who has breast cancer and you find out that you have an inherited risk, you may be at an increased risk for developing ovarian cancer. We will present you with options to reduce that risk.

The test results can help your CTCA doctor develop a plan of care individualized just for you. Test results can also be of great value to family members. Before and after genetic testing, you may have a genetic counseling session.

Read about genomic tumor assessment at CTCA.

Read the rest here:
Genetic Testing - Cancer Treatment | CTCA

Advances in genetics have the potential to revolutionize how physicians diagnose and treat illness. But while the ability to repair defective genes remains far in the future, genetic testing can help patients determine the likelihood of passing on or inheriting certain disorders today. Genetic testing usually refers to the analysis of DNA to identify changes ingene sequence (deletions, additions, or misspellings) or expression levels.Genetic testing can also refer to biochemical tests for gene products (proteins) and for microscopic analysis of stained chromosomes. Genetic testing still is in its early stages, so both patients and experienced physicians may need guidance when it comes to navigating this new and complex territory.

How is genetic testing used clinically? Diagnostic medicine: identify whether an individual has a certain genetic disease. This type of test commonly detects a specific genealteration butis often not able to determine disease severity or age of onset. It is estimated that there are >4000 diseases caused by a mutation in a single gene. Examples of diseases that can be diagnosed by genetic testing includes cystic fibrosis andHuntington's disease.

Predictive medicine: determine whether an individualhas an increased risk for a particular disease. Results from this type of test are usually expressed in terms of probability and are therefore less definitive since disease susceptibility may also be influenced by other genetic and nongenetic (e.g. environmental, lifestyle) factors. Examples of diseases that use genetic testing to identify individuals with increased risk include certain forms of breast cancer (BRCA) andcolorectal cancer.

Pharmacogenomics:classifies subtle variations in an individual's genetic makeup todeterminewhether a drug is suitable for a particular patient, and if so, what would be the safest and most effective dose. Learnmore aboutpharmacogenomics.

Whole-genome and whole-exome sequencing: examines the entire genome or exome to discover genetic alterations that may be the cause of disease. Currently, this type of test is most often used in complex diagnostic cases, but it is being explored for use in asymptomatic individuals to predict future disease. Read more in this article.

How many different types of genetic tests are currently available? There are >2000genetic tests available to physicians to aid in the diagnosis and therapy for >1000 different diseases. Genetic testing is performed for the following reasons:

What are geneticcounselors? Genetic counselors are health professionals with specialized graduate degrees and experience in the areas of medical genetics and counseling. They are an integral part of the healthcare team providing information and support to individuals and families who have members with birth defects, genetic disorders, or may be at risk for a variety of inherited disorders. Genetic counselors also serve as educators and a resource for other healthcare professionals and for the general public.

Additional resources

NIH Genetic Testing Registry

National Cancer Institute - Understanding Cancer Series: Gene Testing

US Department of Health and Human Services - Understanding Gene Testing

Here is the original post:
Genetic Testing - American Medical Association

In recent years, much energy has been put into genetic research both through the individual efforts of interested scientists and through the collaboration of international teams in the Human Genome Project. Through this work, we have learned a great deal about how genes function and how they can cause certain problems. We now know how to look for mutations (changes in the gene) that can lead to specific disorders. Genetic testing is possible for some conditions because we can recognize the difference between a normal gene and a disease gene.

Genetic testing presents us with both opportunity and concern. There is opportunity for diagnoses and definitive information and, indeed, a hope that cures may ultimately be possible. On the other hand, we have seen that genetic information can have far-reaching effects on individuals being tested and on their familiesemotionally, socially, ethically.

Genes are specific pieces of information that tell our bodies how to grow, function, and develop. It is estimated that each person has between 50,000-100,000 genes. These genes, which are contained on our 23 pairs of chromosomes, make up our genetic blueprint. Each gene codes for a particular set of instructions, and a genes function is determined by its unique DNA code. DNA consists of four basic building blocks called bases that are linked in a specific order. When a change occurs in the ordering or number of bases, a gene may not function properly. A gene change which can cause a disease is called a mutation.

Genes come in pairs, with one copy inherited from each parent. A condition is called dominantly inherited when only one copy of a disease gene is needed to lead to symptoms of that disease. One example of dominant inheritance is Huntingtons Disease (HD). The HD gene can be passed from one generation to the next and a person who has the HD gene has a 50% chance of passing that gene on to each of his or her children. A person affected by a recessively inherited condition inherits a particular disease gene from each parent. One example is cystic fibrosis in which both parents, by chance, have passed on a CF gene.

Some diseases do not follow simple patterns of inheritance. Many factors influence how a gene works or who will get a disease and when. Mutations in several different genes can lead to the same disease, as we see in some forms of Alzheimers disease. Genes that increase ones risk of getting a certain disease are called susceptibility genes.

Genetic testing involves analyzing a persons DNA. Usually a blood sample is taken, and a molecular genetics lab performs special tests to look for mutations in a gene that lead to disease. Genetic testing is available for only a fraction of the many genetic conditions in existence. There is no test that analyzes a persons DNA and gives him or her a clean bill of health.

Genetic testing can be done to confirm or rule out a certain diagnosis. Testing might interest a person who knows or suspects that he/she is at risk for a genetic disease for which treatment options or preventative measures are available. Also, couples considering having children may wish to know the risk of passing on an inherited disorder (e.g., Huntingtons disease) to offspring.

Some of the more common genetic diseases for which genetic tests are available include sickle cell disease, myotonic dystrophy, cystic fibrosis, Duchennes muscular dystrophy, and Fragile X syndrome.

There are also tests available for some inherited adult-onset disorders, including those described below:

At this time, routine predictive testing of Alzheimers disease genes is not recommended. The APOE4 gene is only a risk factor and it cannot provide definitive information. Since there is no cure for Alzheimers disease, the benefit of learning about a possible predisposition to the disease is questionable.

ALS is inherited in aproximately 10% of cases in an autosomal dominant or autosomal recessive manner. Familial ALS (FALS) has been studied closely to determine that in some families, a mutation in a gene called SOD1 (on chromosome 21) is likely the cause. The vast majority of ALS cases are sporadic with no clear cause. The hope now is that the discovery of a gene causing a disease in certain families may give scientists the lead they have been searching for to reach a cure.

Ataxia Ataxia means a lack of coordination and can be associated with a degenerative disorder. Testing is currently available for spinocerebellar ataxia (SCA) Types1, 2 and 3. Type3 is also known as Machado-Joseph disease. Dementia is not typically seen in SCA Types1, 2 and 3. They are inherited in an autosomal dominant manner, meaning that either men or women can be affected and that an affected person has a 50% chance of passing the gene on to each of his/her children. The genes for SCA Types1, 2 and 3, like the HD gene, have repeated sections of DNA that are larger than those in the normally functioning gene.

Cerebrovascular Disease (Stroke) Scientists studying cerebrovascular disease have suggested that many risk factors for stroke are under genetic influence, for example, having a family history of stroke may be associated with an increased risk. Greater understanding of these factors may lead to early recognition of and intervention in stroke. Genetic effects are subject to environmental influences (e.g., diet, weight).

A person with symptoms of Huntingtons disease may have a genetic test to confirm that he/she has HD. People at risk for HD (meaning that one of their parents has HD) may consider presymptomatic testing to learn if they carry the HD gene and therefore will ultimately develop HD symptoms.

After many years of intense research, the HD gene was identified in 1993. It was discovered that a three base pair section of the DNA of the HD gene is repeated many times in individuals who have HD. The normal functional gene does not have this enlargement. Current testing analyzes the HD gene to look for the presence or absence of this enlargement (or expanded repeat). At this time, the function of the HD gene and how it causes HD is not known.

Multiple Sclerosis (MS) Multiple sclerosis is a disease that randomly attacks the central nervous system. Familial occurrence (not necessarily genetic) in MS is documented, but uncommon. It is thought that the major causes for MS will prove to be immunological and possibly infectious, but certain genes may be required for susceptibility.

Although there are no cures for these adult-onset disorders, genetic testing for actual gene mutations can provide an accurate diagnosis or rule out a specific condition. Having a clear diagnosis can allow a person and his/her family to anticipate disease progression and make informed decisions about the future. In some cases, treatment options may be available to slow the progression of symptoms.

Persons at risk (e.g., a person with a parent with Huntingtons disease) might feel uncertain about their own future and that of their children. A negative test (indicating that a person does not have the gene) can give a tremendous sense of relief. A positive test result can relieve uncertainty and let the person plan for the future.

There are not tests available for every adult-onset disorder. One important limitation for gene testing is that diagnostic information often is not matched by effective treatment strategies or therapies.

Since most genetic tests involve only a blood sample, there is no significant physical risk. Any potential risks have more to do with the way the results of the test might change a persons life.

There can be a major psychological impact on people considering and undergoing genetic testing. The knowledge that one does or does not carry a disease gene can provoke many emotions. Many people with a family history of certain diseases have already seen relatives become affected by the disorder. The news that they have the disease gene can lead to depression or anger. These emotions can impact the person and reverberate throughout the family. A person who finds he/she does not carry a disease gene may feel guilty.

There is also concern about confidentiality. People have expressed concern that testing information could someday be used against them.

As knowledge about the genetic basis of common disorders grows, so does the potential for discrimination in obtaining health or life insurance. People also have concerns about discrimination in employment.

At the state and federal levels, legislation is being pursued to help ensure that genetic information is not used against people. The Americans with Disabilities Act (ADA) provides employment anti-discrimination protection for people with disabilities and neurological disorders. In addition, as an example of state law, the State of California prohibits insurers, to varying degrees, from requiring or requesting genetic tests or their results, from denying coverage on the basis of genetic tests, and from using tests to determine rates and benefits. California law has provisions to protect the privacy of genetic information. However, in this time of flux and changing health care systems, it is not clear to what extent consumers are protected. People considering genetic testing need to consider potential risks for discrimination.

Your primary care physician may be able to make a referral to a specialist such as a neurologist and genetic counselor as appropriate. The National Society of Genetic Counselors may also be a helpful source of referrals. A trained professional can help evaluate family history, document diagnosis and discuss whether testing options are available. In addition, in California there is a Genetically Handicapped Persons Program (see Resources section of this fact sheet).

Genetic counselors are specially trained health professionals who help families learn about and cope with genetic conditions. If a person is considering testing, a genetic counselor would discuss risks, benefits, and limitations and provide balanced information for the individual to make an informed decision about whether to proceed with testing. There are many issues to consider including psychological impact, family issues, and privacy. Genetic counseling can be helpful in addressing these issues. Genetic counselors support families and individuals in making decisions about genetic testing and in adjusting to test results.

The decision about whether to have testing is a very personal one. It should also be voluntary; people should have the test only if they want the information and should not be pressured into testing by relatives or health care providers.

Because the issues are so complex and the consequences so profound, the decision to have a genetic test deserves careful preparation and thought.

As a final note, it is also important to understand that the available information is changing rapidly as genetic research continues. It is likely that more information and genetic tests will be available in the future. Please use the Resource listings below to help stay informed and up to date.

Family Caregiver Alliance 785 Market Street, Suite 750 San Francisco, CA 94103 (415) 434-3388 (800) 445-8106 Web Site: caregiver.org E-mail: [emailprotected]

Family Caregiver Alliance (FCA) seeks to improve the quality of life for caregivers through education, services, research and advocacy.

Through its National Center on Caregiving, FCA offers information on current social, public policy and caregiving issues and provides assistance in the development of public and private programs for caregivers.

For residents of the greater San Francisco Bay Area, FCA provides direct family support services for caregivers of those with Alzheimer's disease, stroke, head injury, Parkinson's and other debilitating disorders that strike adults.

Huntingtons Disease Society of America 140 West 22nd St., 6th Flr. New York, NY 10011-2420 (212) 242-1968 (800) 345-HDSA HDSA maintains a list of genetic testing centers across the U.S.

Genetically Handicapped Persons Program State of California Department of Health Services 714 P St., Rm. 300 Sacramento, CA 95814 (916) 654-0503 (800) 639-0957

Alliance of Genetic Support Groups 35 Wisconsin Circle, Suite 440 Chevy Chase, MD 20815 (800) 336-4363 (301) 652-5553

National Alliance for Rare Disorders P.O. Box 8923 New Fairfield, CT 06812 (800) 999-6673 (203) 746-6518

National Society of Genetic Counselors 233 Canterbury Dr. Wallingford, PA 19086-6617 (610) 872-7608

Human Genome Management Information System Oak Ridge National Lab 1060 Commerce Park MS 6480 Oak Ridge, TN 37830 (423) 576-6669

Publishes a Primer on Molecular Genetics.

Prepared by Ann Bourguignon, M.S., Genetic Counselor, Kaiser Permanente, Oakland, California, for Family Caregiver Alliance and California's Caregiver Resource Centers, a statewide system of resource centers serving families and caregivers of brain-impaired adults. Funded by the California Department of Mental Health. Printed October 1997. All rights reserved.

More:
Genetic Testing | Family Caregiver Alliance

There are hundreds of diseases that are related to changes in our genetic code, but most of them are extremely rare. or alterations in specific may prevent the genes from creating vital or cause alterations in the proteins that they produce. These changes can affect the way that the body functions and cause specific diseases. Some of the disease-related gene mutations are , while others are . Some are or sex-linked, associated with the X or Y that determines our sex, and are found only in males. Some mutations have arisen and been passed down in specific families, and some are more prevalent in individuals of certain ethnic descent.

Genetic testing is a personal choice. Blood tests for some of the more common genetic diseases may be performed on a woman and her partner before a pregnancy if they wish to know if they are . Many times, genetic testing is done first on the woman and only done on the partner if the woman is a carrier. Couples should talk to a genetic counselor about their ethnicity and family medical history to determine which tests are the most appropriate and to help them make an informed decision. For more information on genetics and genetic testing, see The Universe of Genetic Testing.

Individuals of Ashkinazi (East European) Jewish descent, for example, are at increased risk of carrying the genes for Tay-Sachs, Gaucher, Canavan disease, and familial dysautonomia. These genetic diseases can occur when both parents have an abnormal gene and their child inherits two copies of the abnormal gene, one from each parent. In both Tay-Sachs and Canavan diseases, there is a buildup of a substance in the childs brain that prevents normal development. There is no known cure for either disease. Children with Tay-Sachs rarely live past five years of age; children with Canavan disease may live to early adolescence. There are three types of Gaucher disease, each causing too much fatty substance to be stored in the bone marrow, spleen, and liver. Although one type of Gaucher disease is fatal, the most common type is not. Treatments are available for individuals with Gaucher disease. Familial dysautonomia is caused by incomplete development of nerve fibers in the autonomic and sensory nervous systems. There are a variety of symptoms (which range in severity), the most noticeable of which is the lack of tears during crying.

Links National Human Genome Research Institute: FAQ About Genetic Testing March of Dimes: Tay-Sachs and Sandhoff diseases Genetic and Rare Diseases Information Center

Excerpt from:
Pregnancy & Prenatal Testing: Genetic Testing for Inherited ...

What are hematopoietic cell transplantation (HCT) and peripheral blood stem cell transplantation (PBSCT)?

HCT and PBSCT are procedures that use stem cells to treat a patient's malignancy or to repair diseased or defective bone marrow. A patient receives intensive chemotherapy with or without total body irradiation therapy in an attempt to kill all cancerous cells, but which also destroy his/her own bone marrow function. This therapy also causes immunosuppression, which prevents rejection of the newly transplanted stem cells from a related or unrelated donor.

There is little risk of rejection of a patient's own stem cells following autologous transplant. After transplantation, the new stem cells replace the damaged bone marrow and cells of the immune system.

How do HCT and PBSCT help patients?

HCT and PBSCT allow a patient to receive very high doses of chemotherapy and radiation designed to kill cancer cells. The high doses of therapy lead to the destruction of a patient's own marrow and immune system, which is then replaced by marrow from a donor or from peripheral blood stem cells that have been harvested before therapy.

How many HCTs and PBSCTs are performed at City of Hope?

City of Hope has performed more than 12,000 transplants for patients from virtually every state as well as from numerous countries. HCT and PBSCT patients at City of Hope have ranged in age from less than 1 year old to 79 years old. City of Hope's HCT program is one of America's largest, dedicated solely to the traditional and newer uses of this procedure.

Which diseases are HCT and PBSCT most frequently used to treat?

What is the difference between autologous and allogeneic HCT?

How are donors for allogeneic transplantations found?

About 30 percent of patients needing a transplant get one from a family member whose HLA testing has identified compatibility between a patient and donor. This matching of donor and recipient reduces the chance of marrow rejection and greatly increases the likelihood of a successful transplant. The remaining 70 percent of patients must find an unrelated donor whose marrow is compatible.

Currently, there are nearly 5 million volunteer donors in the National Marrow Donor Program (NMDP) Be The Match Registry. Almost 50 percent of patients searching the registry have at least one identically matched, unrelated donor. The NMDP is conducting a major effort at the 97 donor centers around the United States (of which City of Hope is one) to increase minority registration.

Because HLA types vary greatly between people of different ethnic backgrounds, increasing minority and ethnic representation will increase minority patients' chances of finding matches.

What is a mini-HCT?

Mini-HCT is a procedure that allows successful transplant of bone marrow without the use of high-dose chemo and radiation therapy. It is less intensive but allows transplant to be utilized in the treatment of older patients who may not be able to endure the intensity of traditional HCT transplant regimens.

Because many diseases, such as leukemia, lymphoma, myeloma and myelodysplasia, are more common in older patients, mini-HCTs allow these patients to potentially benefit from transplant.

What is bone marrow?

Bone marrow is the soft, spongy material found inside bones. Bone marrow contains stem cells that give rise to white blood cells (to fight infections), red blood cells (for oxygenation) and platelets (to prevent hemorrhaging). The chief function of bone marrow is to produce blood cells.

What are platelets?

Platelets are critical in the clotting process and to help control bleeding. Platelets are commonly used to treat leukemia and cancer patients undergoing chemotherapy and bone marrow transplants. Platelets are also used for trauma patients.

What are stem cells?

All blood cells develop from very immature cells called stem cells. Most stem cells are found in the bone marrow, although some, called peripheral blood stem cells, circulate in blood vessels throughout the body. Stem cells can divide to form more stem cells, or they can go through a series of cell divisions by which they become fully mature blood cells.

Who can donate bone marrow or peripheral blood stem cells?

Donating bone marrow or stem cell to someone suffering from a life-threatening disease is one of the greatest gifts you can provide, the gift of life. The first step is to join the National Marrow Donor Program (NMDP) Be The Match Registry. The NMDP maintains the registry of potential donors and searches this when people need a match. To join the registry, you need to complete a brief health questionnaire, sign a consent form, and provide a small blood sample to determine your tissue type.

At City of Hope, we ask that you donate a unit of blood or platelets to help offset the cost of a tissue-type test. Your tissue type will be compared to the tissue types of thousands of patients awaiting a bone marrow transplant. If you are ever a potential match, the City of Hope Donor Center will notify you to see if you are still interested in continuing with the process. If you are, a City of Hope staff member will request an additional blood sample. This sample will determine if the donor matches well enough to continue with the process.

Will patients need blood and platelet donations?

Blood donations from friends and family are a great source of encouragement and support for a patient needing transfusions. If your blood type is compatible with the patient, your donated blood can be given directly to your loved one. If your blood is not the same type, it is still important that you donate to help other City of Hope patients who are a blood type match and seriously in need of your help.

In most circumstances, platelet donations do not need be the same blood type. Therefore, most friends and family members can direct their platelet donations to their loved one. Because platelets can only be stored for 3-5 days, consistent support for our patients is crucial. You can help rally friends and family members by sponsoring blood drives for patients as well as arranging for group donations in our Donor Center.

Encourage friends and family members to call the CityofHopeBloodDonorCenter at 626- 471-7171 and schedule an appointment to donate blood and/or platelets or make arrangements for a blood drive in your community. To find a blood drive in your community, please call 626-301-8385.

Why do patients need platelets?

Before a patient receives a donor's marrow, his or her own marrow must be destroyed by a rigorous treatment of chemotherapy and/or radiation. Once the patient receives the donated marrow, it takes about 4 to 8 weeks for the new marrow to produce platelets. During that time period, the patient needs transfusions of platelets to help his/her blood to clot. City of Hope patients sometimes receive platelet transfusion on a daily basis.

What are the risks to marrow donors?

Virtually none. Bone marrow is extracted under general anesthesia in a procedure that takes less than an hour. Donors have commented that their buttocks felt sore for several days after aspiration. Contrary to organ donations, marrow is completely replenished by the body within a couple of weeks. There are no increased risks to the donor during this period. Historically, at HCT centers around the world, marrow has been donated by individuals less than 1 year old to 60 or 70 years old.

What are the possible complications associated with HCT and PBSCT?

Immediately following allogeneic transplantation, patients are immunosuppressed and unable to fight infection. Different drugs are administered during this critical period and isolation is sometimes necessary for the patient.

Another possible complication for patients receiving allogeneic transplantation is known as graft-versus-host disease (GvHD). Despite the close match between patient and donor, in GvHD, the donated marrow may recognize its new home as foreign and react against the host.

In addition, patients can acquire post-transplant cytomegalovirus (CMV) pneumonia. City of Hope has pioneered several outstanding advances for the prevention and treatment of this potentially fatal complication. Recurrent disease also is possible if the pre-transplant chemotherapy and irradiation therapy were not successful in killing all malignant cells.

In autologous transplantations there are few complications once the patient leaves the hospital, and the only risk is whether the disease will return, causing relapse.

Read more from the original source:
Bone Marrow and Blood Stem Cell Transplants - City of Hope

c-kit is expressed in the developing and adult mouse heart

We first generated a knock-in mouse model, c-kitH2B-tdTomato/+, by gene targeting (Fig. 1a and Supplementary Fig. 1). In this animal, the H2B-tdTomato cassette was inserted into the c-kit start codon without deleting any genomic sequences, thereby expressing tdTomato under the control of the full complement of endogenous c-kit regulatory elements. Since tdTomato is fused to histone H2B gene24, its expression is localized to the nucleus.

(a) Diagram of the c-kitH2B-tdTomato/+knock-in allele. (bi) Sections of c-kitH2B-tdTomato/+hearts at embryonic days (E) 8.5, 9.5, 12.5 and 14.5 (be) and at postnatal (P) days 1, 30, 60 and 120 (fi). c2, e2, g2 and i2 are high-magnification images (without DAPI) of the areas outlined in c1, e1, g1 and i1, respectively. c-kitH2B-tdTomato cells are denoted by arrows. LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle; VS, ventricular septum. n=3 for each stage. Scale bar, 100m.

To confirm the fidelity of the c-kitH2B-tdTomato signal to the endogenous c-kit expression pattern, we performed whole-mount RNA in situ hybridization on the wild-type mice from embryonic day (E) 9.5 to E13.5. By comparing c-kitH2B-tdTomato signals to c-kit mRNA expression, we found that the signals overlapped in all known regions of c-kit expression25, 26, such as the pharyngeal arches, liver, umbilical cord and melanocytes (Supplementary Fig. 2ac). Furthermore, H2B-tdTomato expression was detected in other organs, including the lung, stomach, intestine and spleen (Supplementary Fig. 2e), as well as the neural tube and yolk sac during embryogenesis. This finding is consistent with previous reports of c-kit expression in these organs25, 26. Immunostaining of sectioned c-kitH2B-tdTomato/+ mouse tissues revealed that the c-kitH2B-tdTomato-positive cells co-localized with c-kit antibody in the liver, lung and melanocytes (Supplementary Fig. 3). Further support for the sensitivity and fidelity of this reporter is the observation that cells with low c-kit expression detected by antibody exhibited bright H2B-tdTomato fluorescence (Supplementary Fig. 3b,c).

Next, we examined the location of c-kit+ cells in the hearts of c-kitH2B-tdTomato/+mice (Fig. 1). Endocardial cells with nuclear tdTomato expression were observed as early as E8.5 and 9.5 (Fig. 1b,c). Starting from E12.5, cells with strong c-kitH2B-tdTomato expression were dispersed throughout the heart, with the highest density in the inner layers of the atrial and ventricular chambers at all embryonic stages tested (Fig. 1d,e). At postnatal day (P) 1, P30, P60 and P120, c-kitH2B-tdTomatoexpressing cells were consistently detected in all chambers of the heart (Fig. 1fi). The broad distribution of c-kitH2B-tdTomato-positive cells in the heart from embryonic stages to adulthood is inconsistent with previous studies reporting that c-kit+ cells represent a small population of CSCs in the mammalian heart7, 12, 13, 14, 15, 27.

In the initial characterization of cardiac resident c-kit+ cells in the adult rat, c-kit+ cells were shown to contain a mixed population of cells exhibiting early stages of myogenic differentiation as demonstrated by the active expression of the early cardiac transcription factors Nkx2.5, Gata4 and Mef2c in the nucleus and of sarcomeric proteins in the cytoplasm of these cells7, 15. To determine whether c-kitH2B-tdTomato-positive cells express the cardiac progenitor marker Nkx2.5, we crossed Nkx2.5H2B-GFP/+knock-in mice28 with c-kitH2B-tdTomato/+mice to obtain compound heterozygotes (c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+). H2BGFP expression in Nkx2.5H2B-GFP/+mice faithfully recapitulates the endogenous Nkx2.5 pattern28. We examined cardiac tissues throughout the embryonic (E9.518.5) and postnatal (P1120) stages (Supplementary Fig. 4). All histological sections from E9.5 to 13.5 hearts and more than 30 sections from E14.5 to P120 hearts were inspected (n=3 for each stage). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found (Supplementary Fig. 4b,dg), except at E12.5, wherein only 11 double-positive cells were detected in the ventricular septum (Supplementary Fig. 4c, ~0.007% of total Nkx2.5H2B-GFP-positive cells).

To determine whether any c-kit+ cells produce sarcomeric or myocardial proteins7, 15, we applied a cTnTH2B-GFP/+ knock-in mouse model with insertion of an H2BGFP cassette into the start codon of cTnT (Tnnt2; Supplementary Fig. 5a). On examining heart sections from c-kitH2B-tdTomato/+;cTnTH2B-GFP/+ compound heterozygous animals at embryonic and postnatal stages (E8.5P120), we did not detect any cells in which both markers were co-localized (Supplementary Fig. 5), with the exception of E13.5, where an average of 15 double-positive cells were found within the ventricular septum (Supplementary Fig. 5d, ~0.009% of total cTnTH2B-GFP-positive cells). These observations reveal that c-kit+ cells in c-kitH2B-tdTomato/+mice very rarely co-express either Nkx2.5 or cTnT in the embryonic heart and do not co-express these markers in foetal or adult hearts.

To further determine the identity of c-kit+ cells, we performed immunostaining with antibodies against the endothelial marker PECAM (CD31) and the smooth muscle marker, -SMA. Surprisingly, at all the stages examined (E8.5P120), c-kitH2B-tdTomato-positive cells were PECAM+(Fig. 2a-f) but -SMA (Fig. 2g,h). This finding suggests that cardiac c-kitH2B-tdTomato-positive cells are endothelial cells. Quantitative flow cytometric analysis of 4-month-old hearts demonstrated that ~43% PECAM+ cells in the ventricles were also c-kit+ (Supplementary Fig. 6). Thus, our results indicate that c-kitH2B-tdTomato-positive cells represent a subset of cardiac endothelial cells.

(a,b) At E8.5 and E9.5, c-kitH2B-tdTomato cells are endocardial (PECAM+). (cf) c-kitH2B-tdTomato cells express PECAM at E16.5 (c) and at P1120 (df). Arrows indicate PECAM+ and tdTomato+ double-positive cells. Arrowheads indicate PECAM+ and tdTomato cells. (g,h) Cardiac smooth muscle cells (-SMA+) are tdTomato at P120 (arrowheads). a2h2 are high-magnification images of the areas outlined in a1h1 (without DAPI), respectively. n=3 for each stage. Scale bar, 100m.

tdTomato is a bright fluorescent protein29, 30. We were concerned that the long stability of tdTomato could complicate the detection of transient c-kit expression. To confirm the identity of c-kit+ cells identified by c-kitH2B-tdTomato/+, we generated another reporter line, c-kitnlacZ-H2B-GFP/+, by inserting a LoxP-nlacZ-4XPolyA-LoxP-H2BGFP cassette into the c-kit start codon (Fig. 3a and Supplementary Fig. 7). H2BGFP is not detected in this line unless the nlacZ-4XPolyA stop cassette is removed by Cre-mediated recombination. We performed whole-mount X-gal staining on c-kitnlacZ-H2B-GFP/+ embryos and found that the c-kitnlacZ signal was not only reliably recapitulated by c-kit mRNA expression, but also consistent with the H2BtdTomato expression patterns in c-kitH2B-tdTomato/+mice (Supplementary Fig. 2). Furthermore, X-gal staining of whole-mount and sectioned hearts at E15.5P90 readily detected a broad distribution of c-kitnlacZ-positive cells throughout the heart (Fig. 3b,d,f,h, and j), including the endocardium (Fig. 3b,h), similar to the pattern observed in c-kitH2B-tdTomato/+mice. X-gal staining of compound heterozygous littermate hearts bearing an endothelial-specific Tie2-Cre allele (c-kitnlacZ-H2B-GFP/+;Tie2Cre) could not detect c-kitnlacZ-positive cells (Fig. 3c,e,g,i and k; less than 10 randomly distributed c-kitnlacZ-positive cells were found in the adult heart, representing ~0.0002% of total c-kit+ cells). Consistent with the endothelial nature of c-kit+ cells in the heart, c-kitH2B-GFP-positive cells generated by Tie2Cre excision were all co-stained with anti-PECAM antibody (Supplementary Fig. 8). Thus, the c-kitnlacZ-H2B-GFP/+ reporter line confirms the endothelial identity of cardiac c-kit+ cells.

(a) Diagram of the c-kitnlacZ-H2B-GFP/+reporter allele (a1). The c-kitH2B-GFP/+ allele is generated when the nlacZ cassette is removed by Cre excision (a2). (bk) X-gal staining of c-kitnlacZ-H2B-GFP/+ and c-kitnlacZ-H2B-GFP/+;Tie2Cre littermate hearts at E15.5 (b,c, sections) and at P190 (dk). Arrows indicate comparable regions to X-gal+ or X-gal staining. Arrowheads indicate rare X-gal+ cells on c-kitnlacZ-H2B-GFP/+;Tie2Cre hearts, suggesting that most c-kit+ cells lose the nlacZ gene because they are in the Tie2Cre lineage. f2k2 are high-magnification images of the areas outlined in f1k1, respectively. n=35 for each stage. Scale bar, 400m (black) and 200m (white).

To further address the issue of stability of both H2BtdTomato and nlacZ proteins, we analysed cardiac c-kit cells with the third reporter allele c-kitMerCreMer/+, in which an inducible MerCreMer cassette was inserted into the c-kit start codon (Fig. 4a and Supplementary Fig. 9). c-kitMerCreMer/+;ROSA26RtdTomato/+mice were subsequently generated by crossing with ROSA26RtdTomato/+ mice. In the absence of tamoxifen treatment, no tdTomato-expressing cells were detected in the adult hearts. To confirm whether c-kit is actively expressed in the postnatal heart, we injected tamoxifen at P30, P60 or P90 for 3 consecutive days (days 1, 2 and 3), and immediately collected cardiac tissues for analysis at day 4 (P3034, P6064) or 14 (P90104). This treatment consistently resulted in tdTomato labelling of a large number of cells in the heart (Fig. 4b,d,e) that also expressed PECAM (Fig. 4c). This result further confirms that cardiac c-kit+ cells are endothelial (Figs 2 and 3), and supports the previous observation that cardiac c-kit+ cell progeny are endothelial19.

(a) Diagram of the c-kitMerCreMer/+ allele. c-kitMerCreMer/+ animals were crossed to the ROSA26RtdTomato reporter line to obtain c-kitMerCreMer/+;ROSA26RtdTomato/+. (be) Cre activity was transiently induced in c-kitMerCreMer/+;ROSA26RtdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 13. Hearts were harvested on days 4 and 14. Many tdTomato+ cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato+ cells were PECAM+ (c2, arrows, P3034). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. (f) Diagram of the cTnTnlacZ-H2B-GFP/+allele and lineage tracing using c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). (g) cTnTH2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100m (white).

c-kitH2B-tdTomato/+, c-kitnlacZ-H2B-GFP/+ and c-kitMerCreMer/+ animals are heterozygous null for c-kit (c-kit+/). Haploinsufficiency of c-kit could affect c-kit regulation in vivo20, 31, 32, 33, possibly leading to ectopic cardiac expression. To determine whether ectopic c-kit expression occurs in the reporter mouse hearts, we performed immunostaining at embryonic (E11.515.5) and postnatal stages (P160) using c-kit antibody on mice of four different genotypes: wild type, c-kitH2B-tdTomato/+ (c-kit+/), c-kitH2B-tdTomato/MerCreMer(c-kit/) and c-kitMerCreMer/MerCreMer(c-kit/). Using c-kit antibody, we frequently detected cells in wild-type hearts that were dually labelled with c-kit and PECAM (Supplementary Fig. 10a4,d4,g2 and Supplementary Fig. 11a,f,h,i). In c-kitH2B-tdTomato/+ animals, c-kit antibody immunoreactivity co-localized with c-kitH2B-tdTomato (Supplementary Fig. 10b2, e2,h2 and Supplementary Fig. 11b,c), although the immunofluorescence was decreased compared with that in wild-type animals. Reduced c-kit immunoreactivity in c-kitH2B-tdTomato/+ tissues is consistent with the c-kit+/ genetic background (theoretically 50% c-kit protein reduction in c-kit+/). Importantly, c-kit antibody staining was completely undetectable in c-kit/mutant hearts or lungs, even with Tyramide Signal Amplification (TSA) amplification (Supplementary Figs 10c,f and 11d,e), demonstrating the specificity of the antibody staining. Therefore, immunostaining with c-kit antibody also reveals that cardiac c-kit+ cells are endothelial and indicates that no ectopic cardiac c-kit expression occurs in the new knock-in mouse models employed.

To further determine the myogenic potential of c-kit+ cells during heart formation, we applied cTnTnlacZ-H2B-GFP/+ cardiomyocyte-specific reporter mice with the LoxP-nlacZ-4XPolyA-LoxP-H2B-GFP cassette targeted into cTnT start codon. cTnTH2B-GFP expression is detected in cardiomyocytes when Cre is expressed in the myocardium or myogenic precursor cells (Fig. 4f). We crossed c-kitMerCreMer/+ mice with cTnTnlacZ-H2B-GFP/+mice and injected tamoxifen in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animals. After two doses of tamoxifen administration (days 1 and 2) to pregnant mice (E11.5 embryos) or four doses (days 1, 2, 3 and 5) to P30, P60 and P90 mice, we collected hearts for analysis at E13.5 or at P37, P67 and P97, respectively. All cardiac sections were assessed for cTnTH2B-GFP-positive cells. On average, approximately 50, 324, 156 and 66 cells were found in hearts (n=3 for each group) at E13.5, P37, P67 and P97, respectively (Fig. 4g), representing <0.04% of cardiomyocytes at corresponding stages (<0.007% after P90). This finding demonstrates that the myogenic potential of c-kit+ cells, if any, is extremely low in both embryonic and postnatal hearts.

Previous studies have reported that within 4 weeks of myocardial infarction in adult mouse hearts, the number of c-kit/Nkx2.5 double-positive myogenic precursors significantly increased in the injured region, and some of these myogenic precursors transformed into proliferative cardiomyocytes7, 15. To directly investigate the differentiation potential of cardiac c-kit+ cells post myocardial infarction, we ligated the left anterior descending (LAD) coronary artery of c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+ mice (25 months old, n=12, Fig. 5a,b). Examination of cardiac sections at 1, 3, 7, 21, 30 and 60 days post-surgery (dps) revealed many c-kitH2B-tdTomato-positive cells in the infarcted region (Fig. 5cf). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found in the injured area at any stage tested (Fig. 5c1f1). To further determine the cell identity of these c-kit+ cells, we performed LAD ligation on Tie2Cre;c-kitnlacZ-H2B-GFP/+ mice (24 months old, n=3). c-kitH2B-GFP-positive cells were readily detected in the infarcted region, demonstrating that they retained their endothelial nature after injury (Fig. 6a).

(a) Diagram of LAD ligation. (b) Masson trichrome staining shows the infarcted region of a c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+heart at 60 days post-surgery (dps). b1 and b2 are high-magnification images of the numbered outlined areas in b. (cf) No c-kitH2B-tdTomato/Nkx2.5H2B-GFP double-positive cells were found in the infarcted regions at 3 (c), 21 (d), 30 (e) and 60dps (f). c1/c2, d1/d2, e1/e2, and f1/f2 are high-magnification images of the numbered outlined areas in c, d, e and f, respectively. Scale bar, 500m (black) and 50m (white).

(a) c-kitH2B-GFP-positive cells were present in the infarcted region of Tie2Cre;c-kitnlacZ-H2B-GFP/+ hearts at 30dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. (b) Masson trichrome staining of cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ hearts at 60dps shows the infarcted region. (c) Adjacent section of b. ROSA26RtdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kitH2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. (d) Masson trichrome staining of c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts at 60dps. (e) Adjacent section of d shows a few cTnTH2B-GFP cells (<20) that were found in the infarcted zone (e1, arrowhead). cTnTH2B-GFP cells were also present in a remote, uninjured region (e2, arrowhead). Scale bar, 100m.

A recent study reported that a subpopulation of endothelial cells yields progeny with CSC characteristics in the adult mouse heart34. This subpopulation purportedly arises from endothelialmesenchymal transition and gives rise to cardiomyocytes that contribute to heart renewal34. To determine whether c-kit+ endothelial cells produce CSCs that further differentiate into cardiomyocytes following cardiac injury, we performed LAD ligation on cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ mice (24 months old, n=4, Fig. 6b). cTnTMerCreMer/+ mediates specific and effective myocardial recombination after tamoxifen induction35. If c-kitnlacZ-H2B-GFP/+ cells become cardiomyocytes and if c-kit expression is maintained in these cells, then c-kitH2B-GFP-positive cells would be detected. However, after tamoxifen was injected at 37dps and 3135dps (three tamoxifen treatments for each period), we detected no c-kitH2B-GFP-positive cells in the infarcted region (Fig. 6c), although myocardial recombination was widely detected in and adjacent to the infarcted region (as revealed by ROSA26RtdTomato staining, Fig. 6c). Furthermore, examination of adult c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ mice after LAD ligation (35 months old, n=3, Fig. 6d) revealed <20 cTnTH2B-GFP-positive cells per heart (~0.002% of total myocardial cells) throughout the injured region (Fig. 6e). cTnTH2B-GFP-positive cells could also be detected in remote uninjured regions (~30 cells, ~0.003% of total myocardial cells, Fig. 6e), suggesting that the cTnTH2B-GFP-positive cells found in the injured region are likely not a response to cardiac injury. These cardiac injury mouse models revealed that the myocardial potential of c-kit+ endothelial cells, if any, is extremely low. However, these data do not preclude the possibility that c-kit cardiac endothelial cells may have the potential for endothelialmesenchymal transition and myocardial differentiation.

In the lineage tracing experiments used to determine the myocardial potential of c-kit+ cells during development and after cardiac injury in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animal models, very small number of cTnTH2B-GFP-positive cells was detected (Fig. 4g, ~66156 cells; and Fig. 6e, ~20 cells). In all cases, the number was extremely low when compared with the total number of c-kitH2B-tdTomato-positive cells (<0.005%) or myocardial cells (<0.015%) in whole hearts. The origin of these rare cells is unknown. These cells may be derived from uncommitted cells originally expressing c-kit, or they could be cardiomyocytes that express both c-kit and cTnT due to a rare stochastic event. To explore these possibilities, we examined cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+ adult mouse hearts (24 months old, uninjured) after tamoxifen injection for 2 consecutive days (days 1 and 2). At days 3, 7 and 30, we detected ~2030 c-kitH2B-GFP-positive cells per heart after examining all the heart sections (n=3, Supplementary Fig. 12). This result suggests that a very small number of resident c-kit cells are cardiomyocytes (~0.005% of total c-kit+ cells and ~0.002% of total myocardial cells in the heart). Notably, the number of c-kitH2B-GFP-positive cells detected in cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+ hearts (~2030, Supplementary Fig. 12) is less than the number of cTnTH2B-GFP-positive cells in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts (~66156, Fig. 4g3). This is probably due to much higher levels of cTnT expression than c-kit expression and/or to differential sensitivity of the reporters to Cre-mediated recombination.

View post:
Resident c-kit+ cells in the heart are not cardiac stem ...

Stem Cell Research & Therapy20112:13

DOI: 10.1186/scrt54

BioMed Central Ltd.2011

Published: 14March2011

The vast majority of hematopoietic stem cells (HSCs) reside in specialized niches within the bone marrow during steady state, maintaining lifelong blood cell production. A small number of HSCs normally traffic throughout the body; however, exogenous stimuli can enhance their release from the niche and entry into the peripheral circulation. This process, termed mobilization, has become the primary means to acquire a stem cell graft for hematopoietic transplant at most transplant centers. Currently, the preferred method of HSC mobilization for subsequent transplantation is treatment of the donor with granulocyte colony-stimulating factor. The mobilizing effect of granulocyte colony-stimulating factor is not completely understood, but recent studies suggest that its capacity to mobilize HSCs, at least in part, is a consequence of alterations to the hematopoietic niche. The present article reviews some of the key mechanisms mediating HSC mobilization, highlighting recent advances and controversies in the field.

The online version of this article (doi:10.1186/scrt54) contains supplementary material, which is available to authorized users.

Higher organisms have the remarkable capacity to produce and maintain adequate numbers of blood cells throughout their entire lifespan to meet the normal physiological requirements of blood cell turnover, as well as to respond to needs for increased blood cell demand as a consequence of injury or infection. At the center of lifelong blood cell production is the hematopoietic stem cell (HSC), with the capacity to give rise to all mature circulating blood cell types. Regulation of HSC function is a highly complex process involving not only intrinsic cues within the HSC themselves, but signaling from the surrounding microenvironment in which they reside. It was first postulated by Schofield that defined local microenvironments created specialized stem cell niches that regulated HSCs [1]. Bone marrow is the primary HSC niche in mammals and is composed of stromal cells and an extracellular matrix of collagens, fibronectin, proteoglycans [2], and endosteal lining osteoblasts [36]. HSCs are thought to be tethered to osteoblasts, other stromal cells, and the extracellular matrix in this stem cell niche through a variety of adhesion molecule inter-actions, many of which are probably redundant systems.

Disruption of one or more of these niche interactions can result in release of HSCs from the niche and their trafficking from the bone marrow to the peripheral circulation, a process termed peripheral blood stem cell mobilization. Mobilization can be achieved through administration of chemotherapy [79], hematopoietic growth factors, chemokines and small-molecule chemokine receptor inhibitors or antibodies against HSC niche interactions [1012].

The process of mobilization has been exploited for collection of hematopoietic stem and progenitor cells (HSPCs) and is widely used for hematopoietic trans-plantation in both the autologous and allogeneic settings. Mobilized peripheral blood hematopoietic stem cell grafts are associated with more rapid engraftment, reduction in infectious complications and, in patients with advanced malignancies, lower regimen-related mor-tality [1315] compared with bone marrow grafts. In many transplantation centers, mobilized HSC grafts are now the preferred hematopoietic stem cell source used for human leukocyte antigen-identical sibling transplants as well as for matched related and unrelated donor transplants [16, 17]. Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor and - more recently, for patients who fail to mobilize with a G-CSF or granulocyte-macrophage colony-stimulating factor - plerixafor (AMD3100) are the only US Food and Drug Administration-approved agents for mobilizing HSCs. Despite the clinical prevalence of peripheral blood stem and progenitor cell mobilization, the mechanisms orchestrating the release of these cells from the hematopoietic niche are still not completely understood. In the following sections, we highlight some of the key mechanistic findings concerning HSPC mobilization, with an emphasis on the effects of mobilizing agents on bone marrow niche interactions.

The most explored HSC niche interaction is between the CXC4 chemokine receptor (CXCR4) and its ligand, stromal cell-derived factor 1 (SDF-1). SDF-1 is produced by osteoblasts [18], a specialized set of reticular cells found in endosteal and vascular niches [19], endothelial cells and bone itself [20, 21], and high levels of SDF-1 were observed recently in nestin-positive mesenchymal stem cells [22]. HSPCs express CXCR4 and are chemoattracted to and retained within the bone marrow by SDF-1 [2325]. Genetic knockout of either CXCR4 [26] or SDF-1 [27] in mice is embryonically lethal, with a failure of HSPCs to tracffic to the bone marrow niche during development. In addition, conditional CXCR4 knockout in mice results in a substantial egress of hematopoietic cells from the bone marrow [28] and impaired ability of CXCR4 knockout HSPCs to be retained within the bone marrow after transplantation [29].

Many agents reported to mobilize HSCs have been shown to disrupt the CXCR4/SDF-1 axis. Most notably, the CXCR4 antagonist AMD3100 (Plerixafor; Mozobil, Genzyme Corporation, Cambridge, MA, USA) mobilizes HSPCs [3035]; and similarly, the CXCR4 antagonists T140 [36] and T134 [37] are both capable of mobilization. Partially agonizing CXCR4 with SDF-1 mimetics including (met)-SDF-1 [38], CTCE-0214 [39], and CTCE-0021 [35] also mobilizes HSCs through CXCR4 receptor desensitization and/or downregulation of surface CXCR4 expression. Intriguingly, these agents that directly disrupt the CXCR4/SDF-1 axis lead to rapid mobilization of HSPCs - that is, hours after treatment - in contrast to other mobilization agents like G-CSF, which take several days to maximally mobilize HSPCs.

Despite the abundance of evidence supporting a key role for the CXCR4/SDF-1 axis in HSPC retention/trafficking/mobilization, it is still not clear which population of cells within the bone marrow niche is the pre-dominate source of SDF-1. Some studies have demonstrated that SDF-1 production by osteoblasts is reduced after G-CSF treatment [21, 40, 41], and seminal work by Katayama and colleagues suggests that this reduction in osteoblast SDF-1 is at least partly mediated by the sympathetic nervous system [21]. Notwithstanding the fact that decreased levels of SDF-1 production by osteoblasts are routinely seen following G-CSF administration, however, other studies have questioned the relative importance of osteoblast-derived SDF-1 in HSC maintenance and mobilization [19, 22, 42]. A recent study by Christopher and colleagues indicated that reduction in osteoblast production of SDF-1 is a common mechanism of cytokine-induced HSC mobilization and showed a specific reduction in SDF-1 production in Col2.3-expressing osteoblasts with no reduction in Col2.3-negative stromal cells [43]. Mendez-Ferrer and colleagues, however, showed, using a similar approach, a substantial decrease in SDF-1 in a novel population of nestin-expressing mesenchymal stem cells [22], relative to a similar population of stromal cells described by Christopher and colleagues [43], although a direct comparison with defined osteoblasts was not made. Future studies are clearly required in order to define the specific niche cells responsible for SDF-1 production and HSC retention, and may identify specific targets for future HSC therapies.

Osteoblasts are important HSC regulators [36], and express numerous signaling molecules in addition to SDF-1 that regulate HSC function and retention in the bone marrow niche. Osteoblasts express vascular cell adhesion molecule 1 (VCAM-1), and targeting the inter-action between very late antigen 4 (VLA-4) and VCAM-1 with either antibodies against VLA-4 [44, 45], antibodies against VCAM-1 [46, 47], or a small molecule inhibitor of VLA-4 (BIO5192) [48] results in HPSC mobilization. In addition, the Eph-ephrin A3 signaling axis increases adhesion to fibronectin and VCAM-1, and disruption of this signaling axis in vivo with a soluble EphA3-Fc fusion protein mobilizes HSPCs [49].

Osteoblasts also express significant amounts of osteo-pontin, and HSPCs adhere to osteopontin via 1 integrins, such as VLA-4 [50]. Osteopontin is a negative regulator of HSC pool size within the bone marrow niche [50, 51], and knockout of osteopontin in mice results in endoge-nous HSPC mobilization and increases the mobilization response to G-CSF [52]. Future therapies that target osteopontin may not only increase the HSC pool size available for hematopoietic mobilization, but may also act to untether the expanded HSCs from the bone marrow niche, resulting in significantly enhanced HSC mobilization.

Mobilizing regimens of G-CSF are associated with suppression of niche osteoblasts [21, 41, 53], with increased osteoblast apoptosis [41] and osteoblast flattening [21], resulting in significant decreases in endosteal niche expression of many of the above-mentioned retention molecules. This suppression has been reported to be the result of altered sympathetic nervous system signaling to osteoblasts [21]. A recent report by Winkler and colleagues demonstrated that G-CSF treatment results in the reduction of endosteal-lining osteomacs, which results in suppression of osteoblasts [53]. This osteomac population of cells is F4/80+ Ly-6G+ CD11b+ and provides a yet to be determined positive supporting role for osteoblasts. When osteomacs are depleted using Mafia transgenic mice or by treatment of mice with clodronate-loaded liposomes, significant mobilization of HSPCs was observed. These findings support a mechanistic role for osteoblasts in mediating G-CSF-induced mobilization, independent of the sympathetic nervous system, and highlight that multiple mechanisms may be responsible for the mobilizing effects of G-CSF.

Osteoblasts and osteoclasts regulate/coordinate bone formation and bone resorption, respectively, within the bone marrow niche. A report from Kollet and colleagues suggested that osteoclasts can mediate HSPC mobilization [54], and proposed a model where the balance between osteoblasts and osteoclasts is required for homeostatic maintenance of the stem cell niche and HSPC pool size. In their model, increased osteoblasts - for example, after parathyroid hormone administration [3] - increase the stem cell pool size and adherence in the niche, whereas increased osteoclasts degrade the niche - facilitating release and egress of HSPCs.

A role for osteoclasts in mobilization was shown by treating mice with RANK ligand, which increased osteoclast activity that correlated with a moderate increase in hematopoietic progenitor cell (HPC) mobilization [54]. Similarly, bleeding mice or treating them with lipopoly-saccharide, two models of physiological stress, resulted in an increase in the number of bone marrow niche osteoclasts as well as HPC mobilization. Inhibition of osteoclasts, either by treatment with calcitonin or using a genetic knockout model of PTP in female mice, resulted in a reduced HPC mobilization response to G-CSF compared with controls, further suggesting that osteoclasts were involved in G-CSF-mediated mobilization. The authors proposed that osteoclast-derived proteolytic enzymes, such as cathepsin K, degraded important niche interaction components including SDF-1 and osteopontin, thereby facilitating mobilization [54]. A more recent study by the same laboratory demonstrated reduced osteoclast maturation and activity in CD45 knockout mice, which correlated with reduced mobilization to RANK ligand and G-CSF [55], providing an additional link between osteoclast activity and HSPC mobilization.

In contrast to studies showing that increased osteoclasts enhance HPC mobilization, an earlier report by Takamatsu and colleagues demonstrated that while G-CSF treatment increases osteoclast number and bone resorption in both BALB/c mice and humans, the increase in osteoclasts did not occur until 10 to 15 days or 6 to 8 days, respectively, after treatment with G-CSF [56] - a finding that has also been observed by other groups using similar systems [40, 57]. Since HSPC mobilization by G-CSF is typically evaluated after 4 to 5 days, the importance of osteoclasts to HSPC mobilization in response to G-CSF treatment remains unclear. Furthermore, treatment of mice with bisphosphonates, which inhibit osteoclast activity and/or number, prior to G-CSF administration does not result in an impaired HSPC mobilization response [53, 56]; in fact, in one case, bisphosphonate treatment increased mobilization by G-CSF [53]. These studies suggest that while osteoclasts elicit mechanisms that can induce hematopoietic stem and progenitor mobilization, their role in clinical HSC mobilization with G-CSF is not sufficiently defined and may not be a primary mechanism of mobilization.

The endosteal surface of bone, particularly at the site of resorbing osteoclasts, is a significant source of soluble extracellular calcium within the bone marrow niche. Studies by Adams and colleagues demonstrated that HSCs express calcium-sensing receptors and are chemo-attracted to soluble Ca2+ [58]. When the gene for the calcium-sensing receptor was knocked out, mice had reduced HSC content within the bone marrow niche and increased HSCs in peripheral blood. Moreover, calcium-sensing receptor-knockout HSCs failed to engraft in hematopoietic transplantation experiments. These results suggest that Ca2+ at the endosteal surface is an important retention signal within the hematopoietic niche and that pharmacologic antagonism of the HSC calcium-sensing receptor may represent a possible strategy for HSPC mobilization.

The bone marrow hematopoietic niche has been shown to be hypoxic [59, 60]. HSCs that reside in hypoxic niches have also been shown to have greater hematopoietic-repopulating ability than those that do not [61]. A known physiological response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 has been shown to upregulate erythropoietin production [62], numerous cell proliferation and survival genes [6365], the angiogenic vascular endothelial growth factor [66], and other genes. It has also been suggested that the hypoxic bone marrow niche maintains HIF-1 activity, thereby maintaining stem cells [67] - a hypothesis supported by the fact that hypoxic conditions expand human HSCs [68] and HPC populations [6971] in vitro. In response to G-CSF, both the hypoxic environment and HIF-1 expand within the bone marrow compartment [72] and increase production of vascular endothelial growth factor A; however, bone marrow vascular density and permeability are not increased [61]. HIF-1 also increases production of SDF-1 [73] and CXCR4 receptor expression [74], suggesting that hypoxia may be a physiological regulator of this important signaling axis within the hematopoietic niche.

HIF-1 has recently been reported to prevent hematopoietic cell damage caused by overproduction of reactive oxygen species [75], suggesting that the hypoxic niche helps maintain the long lifespan of HSCs. However, some small degree of reactive oxygen species signaling may be necessary for HSC mobilization. A recent report demonstrated that enhanced c-Met activity promotes HSPC mobilization by activating mTOR and increasing reactive oxygen species production in HSPCs [76], while inhibition of mTOR with rapamycin reduced HSC mobilization [76, 77]. Genetic knockout of the gene for thioredoxin-interacting protein also results in increased HSPC mobilization under stress conditions [78], suggesting a role for oxygen tension and reactive oxygen species in regulation of hematopoietic stem and progenitor mobilization. These findings clearly warrant additional exploration.

It has been known for some time that there is dynamic interaction between the bone marrow niche and the nervous system. Studies by Katayama and colleagues demonstrated that HSPC mobilization by G-CSF requires peripheral 2-adrenergic signals [21], showing that G-CSF mobilization was reduced in chemically sympathectomized mice treated with 6-hydroxydopamine, in mice treated with the -blocker propanolol, or in mice genetically deficient in the gene for dopamine -hydroxylase (Dbh), an enzyme that converts dopamine into norepinephrine. They also showed that treatment with the 2-adrenergic agonist clenbuterol reversed the phenotype of Dbh knockout mice [21]. Intriguingly, G-CSF attenuated osteoblast function via the sympathetic nervous system resulting in osteoblasts having a marked flattened appearance. The effects of nervous system signaling can also be mediated directly on HSCs, as human CD34+ hematopoietic cells express 2-adrenergic and dopamine receptors that are upregulated after G-CSF treatment [79]. Neurotransmitters serve as direct chemo-attractants to HSPCs, and treatment with norepinephrine results in HSC mobilization [79]. Norepinephrine treatment of mice has also been shown to increase CXCR4 receptor expression [80], perhaps suggesting that adrenergic signaling could directly affect CXCR4/SDF-1 signaling in HSPCs. Additional studies directly assessing effects of neurotransmitter signaling in HSPCs will help to further define the role of the nervous system in hematopoietic regulation.

Not only does the sympathetic nervous system affect HSC mobilization during stress situations, but it also regulates HSC trafficking via a circadian rhythm [81, 82]. 3-Adrenergic stimulations demonstrate regular oscillations controlling norepinephrine release, CXCR4 expression, and SDF-1 production, leading to rhythmic release of HSPCs from the bone marrow niche. Intriguingly, while optimal mobilization occurs in the morning in mice (Zeitgeber time 5), HSC mobilization circadian control is inverted in humans, with peak mobilization occurring later in the evening [81]. Mobilization by both G-CSF and AMD3100 is affected by circadian control of the CXCR4/SDF-1 axis. Recently, it was demonstrated that 2-adrenergic signaling upregulates the vitamin D receptor on osteoblasts; that expression of this receptor is necessary for the G-CSF-induced suppression of osteoblast function; and that vitamin D receptor knockout mice have reduced HSC mobilization [83]. Intriguingly, vitamin D receptor is an important regulator of extracellular calcium and HSPC localization [84] and the receptor is also regulated by circadian rhythms [85], possibly suggesting additional interconnected mobilization mechanisms. Further assessment of the role of nervous system signaling and vitamin D receptor signaling on other niche cells, particularly mesenchymal stem cells, should be performed.

There has been significant progress in understanding the mechanisms of action of G-CSF and other stimuli that increase HSPC trafficking/mobilization. As described in the present review, however, there is currently an abundance of proposed mechanisms that may be responsible for mobilization. This raises the question of whether the proposed mechanisms, be they HSPC intrinsic or manifested through the bone marrow niche, truly represent alternate and independent means to mobilize or enhance egress of HSPCs from bone marrow to the circulation, or whether we have not yet found the unifying mechanism.

Intriguingly, many of the proposed mechanisms of mobilization converge on the CXCR4/SDF-1 pathway (Figure

). Alterations of the osteoblast/osteoclast balance result in a reduction of SDF-1 production and/or degradation of SDF-1 by proteases. Signaling from the sympathetic nervous system, stimulated by G-CSF, can alter the osteoblast/osteoclast balance leading to reduced CXCR4/SDF-1 signaling and HSPC mobilization. Circadian rhythms act to reduce niche SDF-1 production and HSPC CXCR4 expression in an oscillating manner, suggesting that clinical mobilization should be performed at the trough of SDF-1 and CXCR4 expression (early night for humans) and perhaps suggesting that clinical transplantation should be performed at the peak of expression (early morning in humans). The hypoxic nature of the hematopoietic bone marrow niche may itself regulate the CXCR4/SDF-1 signaling axis, perhaps further identifying this axis as a unifying mobilization mechanism. The importance of CXCR4 signaling in HSPC retention and mobilization is certainly supported by the abundance of agents that directly antagonize, or compete with SDF-1 and partially agonize, the CXCR4 receptor and result in HSPC mobilization. Even a rapid mobilizing agent such as GRO (CXCR2 agonist) may function by increasing proteolytic cleavage of SDF-1 [

,

], or altering a homeostatic balance between the CXCR4 and CXCR2 signaling pathways [

].

Hematopoietic stem and progenitor mobilization converges on the CXCR4/SDF-1 signaling axis within the hematopoietic niche. Many of the proposed mechanisms for hematopoietic stem and progenitor mobilization function by altering the marrow microenvironmental CXC4 chemokine receptor (CXCR4)/stromal cell-derived factor 1 (SDF-1) signaling axis. Shown are representative mobilization mechanisms and their relationship to the CXCR4/SDF-1 axis. Question marks denote hypothetical linkage to the CXCR4/SDF-1 axis. G-CSF, granulocyte colony-stimulating factor; HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; ROS, reactive oxygen species.

While perhaps connecting many of the proposed mechanistic pathways for HSPC mobilization, however, the CXCR4/SDF-1 pathway does not appear to be an exclusive target for HSPC mobilization. Continued investigation of the molecular mechanism(s) for action of G-CSF and other HSPC mobilizers is warranted and may define new molecular targets that can be used to enhance the magnitude and/or ease of HSPC collection for hematopoietic transplant.

This article is part of a review series on Stem cell niche. Other articles in the series can be found online at http://stemcellres.com/series/ stemcellniche

CXC4 chemokine receptor

granulocyte colony-stimulating factor

hypoxia inducible factor 1

hematopoietic progenitor cell

hematopoietic stem cell

hematopoietic stem and progenitor cell

mammalian target of rapamycin

receptor activator NF-B

stromal cell-derived factor 1

vascular cell adhesion molecule 1

late antigen 4.

The present work was supported by NIH grants HL069669 and HL096305 (to LMP). JH is supported by training grant HL007910.

Below are the links to the authors original submitted files for images.

The authors declare that they have no competing interests.

Read the original:
Mobilization of hematopoietic stem cells from the bone ...

Comparison Between Bone Marrow or Peripheral Blood Stem Cells and Cord Blood Donated for Transplantation

Cord blood transplants, as all unrelated hematopoietic stem cell transplants, can be associated with serious complications, severe organ toxicity, and in some cases, death.

A transplant requires donation of a quart or more of bone marrow (mixed with blood).

After a formal search is started, it usually takes 2 or more months to transplant, if a donor is available.

When a match is found, it can take only a few days for confirmatory and special testing for shipment to the Transplant Center (less than 24 hours in an emergency).

Donor may be available to give a second transplant or to donate blood for T-cells if necessary.

Patient must begin conditioning before the bone marrow or peripheral bloods harvest. Coordination between donation and transplant is critical and complex.

Cord blood graft can be shipped to the transplant center before the patient enters the hospital and begins conditioning for transplantation. Coordination is simple. Cord blood units are shipped on demand.

No risk of transplanting a genetic disease.

There is a small probability that a rare, unrecognized genetic disease affecting the blood or immune system of the baby may be given with the cord blood transplant.

Generally requires a perfect match between donor and recipient for 8/8 HLA-A, -B, -C and -DRB1 antigens. Additional HLA factors (HLA-DQ and -DP) increasingly used to improve prognosis.

HLA-mismatched cord blood transplants are possible, making it easier to find a suitable match. Role of HLA-C, -DQ and -DP are not yet known.

Link:
Comparison Between Bone Marrow or Peripheral Blood Stem ...

vp`Dx #Bc+RG? V&yP-ir endstream endobj 5 0 obj 3609 endobj 2 0 obj > endobj 6 0 obj > /Font > >> endobj 12 0 obj > stream xwTS7" %z ;HQIP&vDF)VdTG"cEb PQDEk 5Yg}PtX4XXffGD=H.d,P&s"7C$ E63p H.Hi@A> A1vjpzN6pW pG@ K0iABZyCAP8C@&*CP=#t] 4}a ;GDxJ>,_@FXDBX$!k"EHqaYbVabJ0cVL6f3bX'?v 6-V``[a;p~2n5 &x*sb|! ' Zk! $l$T4QOt"yb)AI&NI$R$)TIj"]&=&!:dGrY@^O$ _%?P(&OJEBN9J@y@yCR nXZOD}J}/G3k{%Ow_.'_!JQ@SVF=IEbbbb5Q%O@%!ByM:e0G7 e%e[(R0`3R46i^)*n*|"fLUomO0j&jajj.w_4zj=U45n4hZZZ^0Tf%9->=cXgN].[7ASwBOK/X/_Q>QG[ `Aaac#*Z;8cq>[&IIMST`kh&45YYF9:>>>v}/avO8 FV>2 u/_$BCv{$vrK .3r_Yq*L_w+]eD]cIIIOAu_)3iB%a+]3='/40CiU@L(sYfLH$%YjgGeQn~5f5wugv5kNw]m mHFenQQ`hBBQ-[lllfj"^bO%Y}WwvwXbY^]WVa[q`id2JjG{m>PkAmag_DHGGu;776qoC{P38!9^rUg9];}}_~imp}]/}.{^=}^?z8hc' O*?f`gC/O+FFGGz)~wgbk?J9mdwi?cOO?w| x&mf endstream endobj 13 0 obj 2612 endobj 7 0 obj [ /ICCBased 12 0 R ] endobj 15 0 obj > stream x)p3swwey9/OCQ{g&@3Ggr_e_@f>&!?|?Kks|a]/7M Z{V@ r5W(npX&6n}yl>v&f]%Fsl ~eX"d77u]YD#1yCP60dy8u.X6Ojoxcyfj ;_sAVo #mi#I`1qmr=C$atw -w~SSL2[$/.n0Dn:1[I)sre4JM 4u{K W@qy#*21c.eJ M,wo:x(SOvQC2IK"h (2"'ANB.TjlX13-*Tg%7/7]>LE0uhc >BU0:5n:e#$~zfKt-%qAbr/&4=o|/1uq]l,mm"SH dBS])[*'k7W#,Wk8"bG C(4GKqgcPPtW.XDR;@pD0;TNc~nB!1uO6,"fAi,u+' #_w bC!m&&m0yRled~|hN>RXcl*4es1uUcyF]C$@VED]4^o!obxoqu}d Imw{@d?)'B&%iNo]V#ptcCHB7,U>:)nXumbPm?a#t4[O.wB.xo QoX}/hJI?>nAKB@Kb%1@yLFr$naXr {6N$H2*-qx|b -Sk_OV^.#Am,rz

!#h69T'BP(#FNx` MBd`70`8|yS a AAwFvUITX]NZXzzXvl!5"Tg?#0Oz>]F9|3 4xSL?8AGk@t9qb:cq2oPl53tgL*w=NkLEl6k_L^Hemvp~ a>U]4P/)BBGjwk%5/vg@7x?B7f]X@m!}O9*|G"?RC!PdoQUg ;$wik7;,9${sNhDB:8~~?Zy)tM{|.};B l )F2]CW #>E`LhOD ] m69zlV8hK3t#N.W} @EJZsVie_t4T*;^*|GGh!0w2/E5LUV{H.5:`"KP^K;bp)N]3_J>q>O!fmU&O^B+]#hj8w;gju~Rdh;

nX0]X TVyFxq~1^|F =yvD(0J+c*^;1CUUxM~9tfN9 S>"0Pc8 GhyZMxhA%;1ne !x-uQ6AOOk&F0SG H^U)E1`x?eGfI KaqYU+S|)*1E/9A6c%YpT0uj=T&x|"2&1&W|PO1{M7iX`,XV{D%zPSG?htfQFzmr$'%CnyQ~wL+d2XlNN/[~KE}**Qpts^dP(vYQz%8 $A#/!+2#v=F]_3Hen=ONSu&)[|vmo`/y"_.|!h>!N>2fs-re|Z>*(aPmn& c`W 2C{Biv9d3F@s |KNA 34"J,j*hD8HQ MSl[$B[; :DboPR^'{rU%YA}Xdb|CNp&+m1) Qv`3=/Y!2f`;i#@ePg'7[$-.VV=k+T gDv]mZb^rb){JF_x^D|*-|EJX0m(F a's%eZ{XtD';v!rf?lUA];Ou`nM(:Ush'E:R^z^vl*],n&[Tr)9z9^XTXIZ{8?0%7JA;}-dj#)JCO#&|:XM>0Ub$_8#~/y'&F^(B#TTkTz-fbVJrj[ ;J%*:YpHwvyD{x]QgnSY1tAt`I) SzON`iv7>"yC5q[_rp5:ZN#gQPEqV+gu*F='0,j]>#V"Itr|1j09#?#+8Q 8+)7>0 gT^UzYHm$}W|CjKV2Nbq3^3be`lAMp]?Y&>Kwe BAMyNm3xpX:R@D 1Q1Q K]he]uy5iWG'x_M-@tIU8hO]p=@5=Genpq's!Vi.!lyIQvdnLtx!7 j }pW-JRj rQ):H"St8/SdZ7&,PUW7r*#!dcO}dN=B D-a^6e ^v',"pd/!!Uc?C)sqzu TD& f?`;!A!DE[ tH$|dN L.:t,HILN~C._IY= W[@?fU=?$xgMuU^z.-Y? 5eMLS/;!oV=5Vf8t|*YP]cAS/oq"m_r`;A> S}v endstream endobj 20 0 obj 6929 endobj 18 0 obj > endobj 21 0 obj > /Font > >> endobj 23 0 obj > stream xm*!#svpI~FQG~wmtW__}rVwUe@Swvx~sCm/7o_}MV7_UvlW7]Wc?]XGc{f~(KN_W%l?VHy[wFuT@P}f~-|%WXiWmwK36r~cCV4N?Q`|RG!@lUL:K&O|~h}|ua@fj)XXx Fw*cg7 9{P>4 QM!f.OdA`R"Sw*uz7/xL _rI JI"M $$0>d}, vq1d7-?bTZaJgX ~y5aL_lpe8[tC'W`^b2 Z=&1}'nuZ3_0o`4sQNgh6B&lxG!Qi C)naBomm `#A_ZjCCaC4k{X6+qh` 8y-2sL}'_p>a}|.K_Bm,s]hW7H;gZhXd#h :aHp-DR}:u"gLCH8EB;Jfcv^8! Eo#cUj{"P"7Q;CM1b6u3X=w=tYs gn0nb$@/TqbkI!&3=1P7Rf {UN0(2}D"$d&HQ.z}ssLqq&2g=6Kqv_i2_~) hH K Gj]-c%a#vc5N-bR_kKuS3t=;~%`T_S!gwb9 j *2lG+Q1; kW]@dZfYX_2Gtjs?fP9s(8u; 91(N}O}1@vHNRGoe/m~a*nykN!!#_K=Z1#a8,8d9'qVDCqDzp6$buR6(&l?QMm_>P5i&,[mhz@^Tdlj:9L`Z7$L"}l.gj:Gnk #&VsKB0t%R'i1&5exj[xdPnbSL1,:0i_V&Zw+HQm0>l?r>!.8J8:l[JoO36CdO: xFI&Sy2RSa6V;h `QpNq2a#pq06'-0:$#x&du3_i"%>hl_~;BXH]x?)6|/)2K/Of3I-x[I|OF0f~!Cq `OqDKh q:=AbKT[vf&!.!izG.ba+eZee(.-Kml7(3H $7

sQF !-#'ggmMB~aP?E7(AjMI S}Mji@X1 cV =;eI_ c[u0Q?pTTIMq=*//}uvUU)ODq('Eog $Y6ym`C>~HNf8C=o{CS` Qr}}~"+]m7bl0{P2dk1)_7&=wjx>.c''lj"%p_b{I0C>6r]jX&JlW,Qw,B{P5#+xu20c1"b:3=}9Sbe$w CIotu|r KDZ/7ubQX6n)]h}KV/`W(xzmt|p^dD9=b'u_IS}Om+le.Ay]CWrGi%,__;JKu)~A[FG>@wjYk}vm5% MA5v.ZN`7[k(G~T.Ej 5 !Bvf3y:C ?_/,({a[H/f)r.!eP5GR6kfPKEW,^JK~!2u /m}VibU?&)u2hEK#ZB(*isL5P.4n[uc -cucRLh Gfh^xiK[rA| =s/9,{v[~hJZ@,p,cjf*OrR)$UNSM@I?` :ov9FTv2cm9;lg*]|xu;Zf]:N:kR gZuK/m]o].3)d,Tr PYI4x(+RE$ '{>u9^zjVH(Di>X^5ev "p/; ZR9KG2*Y0vE,>rH"o[dejE [i+f')FK]G4psu%:uLa]:>jsqqO@%6 endstream endobj 24 0 obj 7495 endobj 22 0 obj > endobj 25 0 obj > /Font > >> endobj 27 0 obj > stream xIsuX6Dsw/_ee~,&LJ>fsm4&2@Z/Xlve7T{+XnCOMyT[4}_j{_ l`nySn~*oQ2mvs0exmfDm-P'jn^}*rsUUy|rnR;Oy{1Z'{U%Ho P| ~ )eOWo"B=j~TVjWc2 pPL-e}A,fzQ(:lM[f {B>CAQ EhtXOZQ*r'xO@Mr/G{Ks"P9 X8@G_D$^1>gv}W c/?2 ,gy oaA5H1d.zDmUG^~b/@|QVql /c3q4}Gsw0tS :BMU > nIPd:6eS0b['QVn>)| 7]9)#M 42, xTPlp7YXbE}*{*+lex-RQRjf+Rw8%"w:5G):I%BVmjsAa x> &kkU J)l2!+MBR@e#/#%>qm `3y`N:F_|+B T0yW&,}y8l}uEGX QOI#BID2rh1`);`"kS>cQu|#DR-MLjqJj|tFdNc~H>C$?wI}5US%zseD-fP.IoTZ$i+3 9I*!) 4QFEwOXGRc1 ,`M@

TTnGRoYcl-U5Gpg#m.x| m k"?qH?ED6^#d ID#)R[BToWv>.BdZoQ5 Y5|`db@~07MK/94.ol drle'eE1 q4S|HXXVrb Js51AmB.CFxAJ-w.F{p3,o,jw.re/Yq-' s%brP=IE}4Ib[ i/lH[`}$&(ebD)j/ UJS*:0, '0i#hD8ZP 4(+l lJbFM/[>EqwI bmsn1QlfH( Xak|359yJ=B_{40=x A(A)gl^J>LX -&D5'@Xy,Kv~VK,[mPb,o&~A8KCrL"JlbvsVC9gYYk'sI7s%gA|edb+BZHH6&{Sluq=y_F9,3dRFX9-{9hAVu[0{sYAm;).X%s2mKKvv%29t=SM&ZyQ1O:7K?Sd&0RjCG,@acLPhB')*;d=gk,7_q-.(Pv& LD+VSP?dY@B.G=[>h[v0'kG/,l@qdh+Q`F@6 M cT`m>aZX/4Q$0l f) rn}3gK65G/E}*-+Y/n?g Cz8)$@"e[S2 ag}Ab1Oo't#fyk1PU:7'gnUb2_I!FYRcYkF;U2Fw"b[0(lY|70r.^c~1>,17rEc)"^kAASyn: "3Cj4>r=+ 0)tkzB:"jXk(XJzTaCB*+1]Zl,&L2wrl+wN $h6TY5q+Z"/ g?%cU;g _I[9u_#UHou#g,#psl4Oz"0kqs %e5 t1 gOAB"e@9n ,lSXDCVV>wz/*OB>1IC*] ;Q$B#i: ccE0"IvU@I8$&xm,'7`vO,oh9Q!E2@gA[9VnK;4u9I#9[GgW:5y}8F8X'q UuZ{^W{n$o`#dP%ASMw F8b z}~vH7@@/]mBeWj&:VjG~gN = OBB4DA/|a6 -L7 K-()6D4y^Fr8}qpLL {I01"Xf2T}aIr4^5b=8l/Yq ]^HL z4qV>A' "Dus#2O:?db:rC?~";Yy-7fN0t59=: l$x~*TU{9XiWIeI4qXY~U/m^,v;VSC_z;TDprivX11./'v.xfX$ME`G`Fh`-`~v"N2%)p%-)pnT[hQ[bQ;h|ejG+R_ZXjiK`ch=; ) `ogZLZW.7QnR)t;S)Lzy]2be[$]Ap-w{~O!`V2!GA1rK3jo KL#|{T !T9uOP pViJC!+g}s- 84{,ftLe?0[lYVhhX}5Iu4!A/r}V4WI%!/wa L8sO q:e I-6T+xmvQEey9bHHCt/Kb6e> endobj 29 0 obj > /Font > >> endobj 31 0 obj > stream x)pYAKuE$%)i49>JT5%IF"[.UWnOw:MkwO~T/^4}_]kvCu_wUS^mQ}|otL)U28;qsj/wet?{uyINLKZj3@x$zgx; kjgUf_3aH# _fC= ^$O]TMN*HeI]| ~]W!~#TzS4TxVO$x[B}h}}>U m@pmu])9Qm9wF]u*R Qn.C_^ m{2b6lnUiGGL)X}tWvD}7;L54l[aYSovjWa%rA4dYte1GlOJb3y[tI'/8haQ,.C=5,]`qL5*L,HXHLYR&&F)mm"Z###,|9]J?:K23mf6-6M(wE0A84/G -cTnR-m_4C $ys2%;("W,@$}4havb;6&9JBP^Qn LzfD0v]W sI7KVyHw3`[aAzf+{"08w2cxgFm:Y+3Nv5M"GDAIa+{ [Fp%YTa0V^8hz8.n9'eSB8f>,&l++!C}~_c_:sUbE X,rWPw# hK+SYg|m[PYSbGrXWCeqA(eiIMGs[*(Cz _tURWCPm@% EG))l`!S =YJN$D[5aJSwYU_NB @eC,l(pREt8 FY{Br)#Y-in SyJIS*_GpxO@sNI9JH2%Ilb.h m3qnyykKLxivBH1=Xev,#Zv^vir?YXM +AxkuOiVPk@ "EtGA=IwR=e{d8`iC2cc72#0cM@#VN1iD &xP'5)!Su@X P;+o]}q 1Az!33L `__6`OemIqD(}hw`u[U/jbE0DKx{cpqKXK|$'}98vk`5 Up}GbxTfM^b

ypYapx*(9~Hs6FcA'?fC8D^1jL&W26Sc)OMH>n$BxCu*-r(*qJpvxt(uA @#8`:|&E86Na_Ka2%vRT'}}JT^+/.P6]@P27'M5{s+/1=&8 kX!R3@3'};~3:D8bfg! ZQ'UZ*0]Xx!)xy G4*H_+~q-8WT9OZ99srG& FEO}06x$qn"t^Kw8=FTAE$'B2rBS{f!Z}Pc5~o0eTd,xq+j4ZH.U=y}>^#o/Z/qd@1! 3B"HDZ%Z, &bK~S5'I_9/(1jVl398cWe5w. -cCp6WFriCMmu0l_mIa1*3ElXYLy_q_-7bGNUV'u[f)*L}Fqrdm(49RY8hj@( n N=$!L lC 0f4GPT'K`2q-%f#k=%0f^c}9"!Dd#,nN >lD'DxD$OlJ! v6q;>67 n=.cz'uSzYs@c='| 5RVSY*ebH[FA-6-#XVu+!+ _CnEcl~p.j'M@_ -zT]qLnn ~!H|q(s8 aUz,Oggur/uCH'3&712.oW]6AKV2jc|b0U)z6,F2I*.#T`Y/cK{iHxf7"yR/d?de4y2jLmLd' 5z+K?Q%mQgTi*Y^17-UlG_()(M7.keR&?RAIIH'93@+pHsQytS%FD%["2YYxY]}^-C3.YbpQ]o DMz8:PMgsCSFgedVQ Q=@;y1"Uh0"0I` x@auB**zv2=XYR5T1regV2`.AK3j4)D!8$gZ_:(7 !Xb!j;EQeRswNu^U{p]!Y7cMe?E*A}EIOKjq2FFiz}i;lZLi-e|,Uc=q=oTB='DRgg,(^= ; e!J@1rzTGhNR0tjg(9CN[j; ,MuWyKCn;R"3F>"?6.y")FM#,3^O/Wn{7Oh p3cC?sqX =avOMX

AR:Z.Y,LYeW&=5L[>8FZ0Yp{iM}v=$5>`,!xo_tqMQN3= ~^9Ala8TWf2.11yHdt?;G>@1[?4Ejd=qfwsi}7P!sR5Aez8pZ]@F'f/j+kdX3Li& =UgToXf@yr#>wc/s> Ab' 7 "Rr1l] zjLRK L|TnOOjiy;jfga=,?=?*DtRr@L4wj7AvR1a=@fS 5 J~bwS_.ir3_5PU1g0lMohz;9,IqmbI tVj+I)US:4ldNi1# "$be2H)4HWx> T6n FJRB%PL}),4po(R1^tE@+.uU,-[u/(,U38P`v^'+/$rfCw.Cf;2N3kn+t/YniDjM]G*Kvk I`"r'HDAjs!77{@|H Z?=q~#+1'llPM 3_uH29X: SIuszxPPb>L(p&fBuLl@"Xud5ypPz_=Pt}D7%5#6LC]_jOWSJ8NM # ' rYP/?%25yC*P[-cPuutv4,ZI#S/8q8[d*!o:WH*Cj])>?72oFop*[f*J!v5l+HPjS_|+.Xei?G$RDS/JYS"25Bcu>"u(,8#F+M#}i8#|Q( BS%Vkpau>5R%l3;pOCgHI zw_pOMOh{:Gh9T(=uGlfV (qWS;K :^S%}=qxzO6 6 F8){i}:Ki"HCrI/J"0^|-T3c])br-pgZO*VRb7C>E:WbV^Lv~OSX5'XU2-s; ]mG} n@Gj#h:i jy%Jy ,vydU>Eqcl'ug'tj^] 5^dI>n5hdHI`+4N/[*VCm1b?/>V %4 u[w> ({aDKazAhJ}*^1cZxz _SSR,.7x#H?0 H2e| X%=9@BA$0E n.#qbqDBIFF=Q M}Tr'ER- E2$H O)HQl,uto8D8I[]GRTZ,dW1]'s.Uf$)8_r:u rX{Wl(zF@~mVprTuMTNgc_pu6kNM1^B25~x_:Xe66Yb lq+TEji VM (wF ]t c>lf8m)n L>p I(u]:qY/!Se;tBG} Cq05W4?b&5`f j+ytk q7:G0("ypxb=bdcqynKl.&};&@VADh8R/C1HMR9?|-'(sDz'ZozhN9a &T,tECtSV[Sl>L[3)Mu# Oawuj"_V|fy#5w'$NxwZg.+0[P87'#nBt IEb4K VVm&X"+NPa2.;UvX;"N LySO%j1X`.Gs>p_}5e7Fgh`7BVhZS]4M(8v%XY#GtV>~^;eK@ii=K fh/;n8^Jkt7Q)bfRKyn_WKqM6NIME2qb_%uywjhvbqDTvDytNNl.Cm,zhkQyiTiaE~]C-@}PpG1r]be?bmkb)/6MM_%FTOv= zf$bn^4HyxXPC'|;{"U#vnWTUOW.e"NNk,iJ%U|9AV$xwC4^Zr=l[T/:pNW"m%ZP1 (/$:xpq]:&Gm%sV5#0buXgKIGWX:_~Oo4)5>*~U]f:;)zwN =OyY*+FeK`RQ^CLA]*qR$PJcMS|ODxlHh$X_*!I,0^Xk2:gNue%]R&Qp )WxX>o{^6Fs2`Q cnb^~~oXuru+ }l/ 8B5%QtG]O~KdU sK#,? B0[r^Zp2ugKYVDQB.WDg[> endobj 37 0 obj > /Font > >> endobj 39 0 obj > stream x)x9asQN|6FBH^Yv$_3U3ikfu}WuuW_E}(l:uMEeVz3p9!l)@7X{)H6=pY32T[A ?OXh=|ei8+p/?+b}n>^?_|3=` xwb'O>ye?%j=5^4/G,,(5Q0(k%f 8NI{9RqY"o.r $o[r$AhZf#)k.*S,TQK0R+p|GnH hOxNh5XR#tD uOnb!z@`7yUw-ijl4_TJ}K:]lRjskl0e;@`IL*^>A30K(bTc_ZE*xrI 'C]R>s YTD5 aG!A|(5fNyDk"05Lkg;o-y`T/qD}40x(}fVjf^IZ1u@e@Smy4@m[)@8c1=I vA/(F1p0:|vtC`Y28z|9}V;bZ7q65ZFm_&^s~{&-hT$cf~::O[rgE2m >q6UWmC1ln{`xC}KZk(vZV>"H- ~CN-f^Go[U?6K#n1hbG.{R~.p]j) Uy-|yL~>jj]P?6g3P|Gol(]f2c!x4mpl-%-i:CCV;@prwYE |/Efmt5cGjDXP@utJME;;HaZ0iB4(NR]U[2GR{5@EN"X..bwBhJxu4,`sAqm7$V}G iJkakFbA*6t(t|8|&iL[zXOzwI@)#O9( dl3LP7xbqe8d@BAV8.]$#R(?t6rB Njk:-2Fdb]u30x G8 B]=IA;SI>k y9]aH[E|fi8+LX;Zn Mv{ivqnzBI n3L*FAE#5e)`[.O,6M7C)

`#SL,l||s3lZ J+45 ;E9pUh(m jGpc+5O" H1^ NGG#'sS}|'XN7Tz3)>(|Iqv 47ku_6p vw)X%g`/cj|gi3pbHR0ZR0@/tvSG-F#z5h:tin^a5dk5`osw[DxjV9T{:-*)-chfQP]MaL m >]Lz%O`-4Z+hB`V@(L,W'NWq"z&k23yz uK 3M"X*NS*pIFIm)[N3^]XMj>%?"4#%?R?+nK`|m(C'VPi;HmoXqdW|5IJ@K bXyOC"npRm_~3)I_5a9#q=C_4I:)XbLY|x8=#9g?z0s@CRhA5#!?LLD54GU7'$ypr 08|AtVk{Nrvdw.;o{KQ*(xbfejW[eALtn)[:SM8N5gv|,^R#k3Kb5Z vpl|0>#V4cA1Rs@_l n:O+4I_ v"lx/0omd-#{8xlhn DN1dQ,~)zu~ZK@[72];d%Is T3eo| %#jC_..|_QY]8zHdV9buhzc :Or(KHd[%>z_:#YiU]T>x&Fkt>2.!@396tLKUL^QEapJ"CIODg?WH!qQt]P6*o~B&z$PQzmxs{0NoXaES7]6_}fq86M:AtLF+$TO#g0;Suz-:BOnB,lOfO71/Us#Tx]v-U,68f^=m O IcAaF(2f4N%`F` kY 0 nMu='C}6r0w+jM]m NG*ile t%4 Ay?vlWE,UR:*u!Y.P tVQg[oo2dZ` 1lR4oWb86x8`syQaT*[,([9?x*Bi=#et vIG5ZT`4Mvh,'5#RbQdoQZy6Zk<3]G;Q[:j@ Jkq3v:p[66e2 G_v@U4c J$Qw0e (tRv97I`T}!0>G|~qAIgaAi0 Fnf[t)`j76@4ygbs7o"fQq?>_lWcv{JW&i5K."r.bkyBsG-+/+~2'*Y4;v'c[*Vs(i%y>Gj/7Id2}J9Oro-!9_Tk|Ke_e)UJ$P9&joGY9sJYp9VK&(|)-6zT~0zRsX*YJ-pi$0|;|=1d>Nfz)g^ |!(cqI9:)9|:LKNY)n2EfbS|uc*!?rR3zVdNbq7mQNh(#X7+a[.x3|- .H'suntmA/fs,?I endstream endobj 40 0 obj 7268 endobj 38 0 obj << /Type /Page /Parent 3 0 R /Resources 41 0 R /Contents 39 0 R /MediaBox [0 0 612 792] >> endobj 41 0 obj << /ProcSet [ /PDF /Text ] /ColorSpace << /Cs1 7 0 R >> /Font << /F2.0 9 0 R /F1.0 8 0 R /F4.0 11 0 R >> >> endobj 44 0 obj << /Length 45 0 R /Filter /FlateDecode >> stream xFPf {XE[rZIYU(H"DYE[][nwUiCP|}W[&UP6MmU5v-oq/vW// u,UUm~OVM_e.+_nF_0M7]nXH)1ppcc{qWxLz(Ev%!57]i]WELVDo: hx0kP{@"gH#0& y753"G*pcVvAA"m`MbeYU; A(pSHo4uR@+mX|KWi b+DPQ3va2#0w3wicOL@/.7c5;yQmW_aVQs,"dZ[`]pM4JZ`&3kekT4>=i+~S; !urKl)v3:f[QEozR[s>mDU.~ n#DN+&cOU[jLMbuPBxpy-Mx$NzCz)VMPxvi8&]O_nPY1^vkl&]P8zw)} ~GiHJX-z! [y?lQ`a_wJD"j=/7zI@ ^`o" bSh)}`Z|nIK2d=ZT6h_LW)/ G| lgaatMi<} tp/0*bu#z/{`n(4{a4T-+'R6k6hOd*7E>8N|oh.> $h0"&AV$~_qB Cnm'*oTM{04Da6wQ"t+b{NZPQok~eje+ `?DHS]0M(.chW*qgK$sNiL >d p$4Aoxx0={.;S8LulvvS>q{7x#{h^ca`~wNU LB(1&2{/|IejH#f?lI~m_PQm/3tI?M]I# 6zSYq6 < 4a5l]&R;[Wu8[+.!IVYIq-m()%EW HlCOI!7;J%XDXm)mv->mm+pF$uP6|ur|PhT1%-Z%}.)}?QTiCOw_*w'=EYi< X_`CzQ.iJ3!ih5_W+0hIgN%0B6yPP8I8.rtr"`_fC'jCRTRDXr]]sRn;pW`}'rDc0}/:$z gVmW2UAZEPuwL*;8P".EZ0%]5{009`z8SrD%%E0*BZ}B QR_NHJZ-eGCTw+@&YP=U[nq4`a+eR{>(:jVQ[hD_i"1(n)%"#*~HsS!VANYtZD,Yz~);rXq#w_I ]we=jE$Lw Cc~KK1RPkct2z0i1nJc :VW 0l nyW)SD+$lU 0WJFW^j4K7$xe4_:C%dm1,y5lhU,+C_$+?/p&i#5dC{epNG|SM YJgEI/YyYsvDj< D13 ME2K6ehpWlC|xk @7R]4g,>]-q4Q9_nH ^,yr" %&55;TX?5QUN3)HC&D) S)HMP_Q D9"0J_F`7VHk|Sk$3 7RXVJdJ)OGi#2 h$C 6%?s^U_W7ieASb9'$I0.wJ,&qI iAd3HAJRhcS`bJ (WzCe~zM{m-o%}W/Q2@A1y|$S9#vXDR2q._,B[qT7rh:U:n+>6}o[{vl@Q7}Ml4R3}U!6>1"PVWuz8 A6^B.1<2G&{&rQzme6c}~S]:G-IwE.hGMH]YoAR0F.UhR /W'PgwXb2a"Q=$bpli:v&DmVH("|d< +0A^b8gas.YJ(%^_>%Pu0Z4F@_,[e^c5d&,2ddBqL1ENB8P}u)5FL)k<1q UlPqC=z-2)8hTGd#@,#*:xsp X8dH]6!DZ 2].BI]|-9|S(s G}^*N]cA|2qUcdM31bltdZlUR>yxY1B], qiTImk@Z!}A,o8zkyf-R|_$_c3ia'1dzViLt~@O}O +cN2z$'n9GU{RLEYtm;&N;WQTr Oniwr1#^O;9egq/#Xkn3(Z;Fbmf QhHQykxP[wI]3C4meq"uk XyO}27(>Q#C)XCfOX*z3Xu!_*.PMf5eAahJ +K; L*/N8G5e?KF8xl+XWS !q!]ycqFPOO84G^1R9n[Vn2X>QO[8h>zZR*~{!(yDD$+=t(@LU7I!;k4*j-;u~gF?&Ldjo,[qhoyL(l]U,Q*_G$V "X<]dsGE{6S5K}|r&~`RBDy -W endstream endobj 45 0 obj 6990 endobj 42 0 obj << /Type /Page /Parent 43 0 R /Resources 46 0 R /Contents 44 0 R /MediaBox [0 0 612 792] >> endobj 46 0 obj << /ProcSet [ /PDF /Text ] /ColorSpace << /Cs1 7 0 R >> /Font << /F2.0 9 0 R /F1.0 8 0 R /F4.0 11 0 R >> >> endobj 48 0 obj << /Length 49 0 R /Filter /FlateDecode >> stream x[SM$eFgpX ,L;]A.DtO%+U/oTj}_oMEEwu]ai:4zzWq}U}f[w7};]X5uiunP=~/Q)>:ncCujf=.Ka|sh6}{8DVH{joSmP _IhOKE%P7XG)b Wro }nZf>Leuxy.t~9^t~S8^ipqZr f]z)H6^7R%x T3<{#k}qO_yt_x{S5zsXn^wf)/5H4awXx8eygqp+je7QQo~7h>Z4$QtR]Uj53!C-~3KU_ % 4xlXR1TZqQg^HToYoXvO4 D`Of|t-iNsl&70<,#BRR3w{8<,P3fxU"n 5#y/.3iplwsmU0hs9kP,g%*9*k/Lia*BTtcyn@0`5W1:4@chh1P7cOP:|EaSw#8 S|3"b{QcV8nn :7^]^mKY9M[T8J}%CdKX~i**M$ ryvuMOB"~^:t$QqvJd"?s%n3iC0S1CWNh(vMlR(2dS4.fv+VRW'$S#w <4M =6|t.g("$EZjJaaj:fXO('XlUpI0Y_=F"lGQ ZA#V@C] Mpw3A7kx?oe#@@Z`8r ^!Hrv6F]+<+ P|s${{6 *[u.mVQW=t-Z/j-5g+_oH0a#^xYaI!-B: gx@.A)TpbBm/I?#OPA ,a ^@;8bp3TGKa15E9){ac42m1A}#: B,/a|7Cp($1U6R(i=n8_9EaQj0,Oep.JtdRSF(L 'f'Q:,XR8"?zgK7SQ!D`cTk ;(5U>yOL=& $f&'s <{;YAPT!aACfVz,v <8$u (UnBCQOe+{zF WzrtcpuIN>R+--l+O$1whG Xl-jfwXuxObK9SDN4UPmrtMN 3 jhAosz:p96F kW3|cNOgI!}_&Jk3|L2l)>cX,Zht 5sD,x'@y*}]]uvUo^xKDgJ8 #N.Lx`5l!;) 1u`C2Q jC5"GN4: 8XpG[B wFCX!W*@8ytGNdNc &$NhcUk8 q- }F2K@zjCpH K^w"MbL1 S_K `i{pSMNe* ^iKdlHbDh*gGa=b4;{B;s[I%6n mo5s0i$'9X7{u fx]?NvP0|}''t[N&0W6"ZzqA^Cs~jTd]"''V1t%%o3.!S?XZm&P=+GNwd^$j6V %u:>AHey%mV]XDGo$%:]X?b2VT>fm(b80=GX0ER b)6uO,>1?@%/!1<=j_W)m9Q>ah$${[/xM6_%r?5;fA(e)|r/Gu4AOMj-:w6#C,5>FcNYh=)4Ka3)+8s81]cvz@3/.L|9fhkn3ErpF,fEdf;a U_iO_Kt"(a'fQ5 <)evcQF";+<5#Y,~}B?]EA&^j@`T+f[ o|#4^|+ KEhqjZSX)z9/ >9~ xm:^@4 f.h}pw&`)'^6{u;K~C#G5{S8M$tUohO65g#9R?qqe`w~qGp&$eEi=Mh2D6-9EKJV5Jz0Ptah-zG|4/ m97?hJ8w Ohe%+|^i>:x11 q>4f(bX^-I}!oNx=x Hksj.IxVKB{Zv`Az y >+Lh$ J$WT))u!zy>rT3i9xD#~wcg8%7e4E_/GO K)Vs8/gb4tfO:giC;- pVZ SyaNvn^&-xCo ,s$k~ 3]1U<]Zy2"3Q kx#`"#~}s3P[J+VB;d%Hxt".[6a#BFdG[l^])r"fc10)G)B^ &gsbgr55/|-_T:x=>cY>^1o: 9K0y|OE|nAP+WSB$*AdWbEIkNO9Dm(WYc`Fb'B*XRF~&yc_qH5f`9&&y2:tM.%H6tKZ_M6&Jngz ^zHLHC@x4j|3oS.i(;kq:Z49`A?}e8.9vQ+DvY=WpppN+'+ o%r*{C|w#L!1UchvI8?"5cA%,zs(IeAeR)AH$)IR xmm%DSZO)HPGkc-z&L~JpP|!@x}2Ur4vo&}6gtZ>^BTW:.[NDwAp>HC{|7ZqKW<#))5Bjn D5q.Z&|* y<;v803.>&iD:}$xPWtymKym$6fX&GKch6FKBt3 -C`yb,Qt='{lq7mS7Joh#BhD4nr)|4Y-n6{SrCau]rX!;0 uxe^"&0B = )_Z>D V-G)*ZCw6 -6^/(!u0mdl3OdYq!"Wf&VeUgmoMEX/f>Xo+'>Ip4|bi,2E? H;6yL5 npx3yk+hv,6i$'_j|Gx1yrD)_ x9lw[{f5k1,)*z)gfN+)1S Odgqz')c2lWgY/GyC7) endstream endobj 49 0 obj 8867 endobj 47 0 obj << /Type /Page /Parent 43 0 R /Resources 50 0 R /Contents 48 0 R /MediaBox [0 0 612 792] >> endobj 50 0 obj << /ProcSet [ /PDF /Text ] /ColorSpace << /Cs1 7 0 R >> /Font << /F2.0 9 0 R /F1.0 8 0 R /F4.0 11 0 R >> >> endobj 52 0 obj << /Length 53 0 R /Filter /FlateDecode >> stream xVrSSxZ5;Ak'&A#k#ididN"Cj[4bZuqwS|Q~*+J9U6^v_1uqh60.W^EU,uY5_]W^

Read the rest here:
STEM CELL THERAPY FOR ATHLETES - Mississippi Sports Law Review

Frequently Asked Questions About Genetic Testing What is genetic testing?

Genetic testing uses laboratory methods to look at your genes, which are the DNA instructions you inherit from your mother and your father. Genetic tests may be used to identify increased risks of health problems, to choose treatments, or to assess responses to treatments.

There are many different types of genetic tests. Genetic tests can help to:

Genetic test results can be hard to understand, however specialists like geneticists and genetic counselors can help explain what results might mean to you and your family. Because genetic testing tells you information about your DNA, which is shared with other family members, sometimes a genetic test result may have implications for blood relatives of the person who had testing.

Top of page

Diagnostic testing is used to precisely identify the disease that is making a person ill. The results of a diagnostic test may help you make choices about how to treat or manage your health.

Predictive and pre-symptomatic genetic tests are used to find gene changes that increase a person's likelihood of developing diseases. The results of these tests provide you with information about your risk of developing a specific disease. Such information may be useful in decisions about your lifestyle and healthcare.

Carrier testing is used to find people who "carry" a change in a gene that is linked to disease. Carriers may show no signs of the disease; however, they have the ability to pass on the gene change to their children, who may develop the disease or become carriers themselves. Some diseases require a gene change to be inherited from both parents for the disease to occur. This type of testing usually is offered to people who have a family history of a specific inherited disease or who belong to certain ethnic groups that have a higher risk of specific inherited diseases.

Prenatal testing is offered during pregnancy to help identify fetuses that have certain diseases.

Newborn screening is used to test babies one or two days after birth to find out if they have certain diseases known to cause problems with health and development.

Pharmacogenomic testing gives information about how certain medicines are processed by an individual's body. This type of testing can help your healthcare provider choose the medicines that work best with your genetic makeup.

Research genetic testing is used to learn more about the contributions of genes to health and to disease. Sometimes the results may not be directly helpful to participants, but they may benefit others by helping researchers expand their understanding of the human body, health, and disease.

Top of page

Benefits: Genetic testing may be beneficial whether the test identifies a mutation or not. For some people, test results serve as a relief, eliminating some of the uncertainty surrounding their health. These results may also help doctors make recommendations for treatment or monitoring, and give people more information for making decisions about their and their family's health, allowing them to take steps to lower his/her chance of developing a disease. For example, as the result of such a finding, someone could be screened earlier and more frequently for the disease and/or could make changes to health habits like diet and exercise. Such a genetic test result can lower a person's feelings of uncertainty, and this information can also help people to make informed choices about their future, such as whether to have a baby.

Drawbacks: Genetic testing has a generally low risk of negatively impacting your physical health. However, it can be difficult financially or emotionally to find out your results.

Emotional: Learning that you or someone in your family has or is at risk for a disease can be scary. Some people can also feel guilty, angry, anxious, or depressed when they find out their results.

Financial: Genetic testing can cost anywhere from less than $100 to more than $2,000. Health insurance companies may cover part or all of the cost of testing.

Many people are worried about discrimination based on their genetic test results. In 2008, Congress enacted the Genetic Information Nondiscrimination Act (GINA) to protect people from discrimination by their health insurance provider or employer. GINA does not apply to long-term care, disability, or life insurance providers. (For more information about genetic discrimination and GINA, see http://www.genome.gov/10002328/Genetic-Discrimination-Fact-Sheet).

Limitations of testing: Genetic testing cannot tell you everything about inherited diseases. For example, a positive result does not always mean you will develop a disease, and it is hard to predict how severe symptoms may be. Geneticists and genetic counselors can talk more specifically about what a particular test will or will not tell you, and can help you decide whether to undergo testing.

Top of page

There are many reasons that people might get genetic testing. Doctors might suggest a genetic test if patients or their families have certain patterns of disease. Genetic testing is voluntary and the decision about whether to have genetic testing is complex.

A geneticist or genetic counselor can help families think about the benefits and limitations of a particular genetic test. Genetic counselors help individuals and families understand the scientific, emotional, and ethical factors surrounding the decision to have genetic testing and how to deal with the results of those tests. (See: Frequently Asked Questions about Genetic Counseling)

Top of page

Talking Glossary of Genetic Terms

Genetic Testing From Genetics Home Reference: the benefits, costs, risks and limitations of genetic testing.

Genetic Testing Registry [ncbi.nlm.nih.gov] A publicly funded medical genetics information resource developed for physicians, other healthcare providers, and researchers.

Prenatal Screening [marchofdimes.com] Provides prenatal testing information, including ultrasound, amniocentesis and chorionic villus sampling (CVS).

National Newborn Screening & Genetics Resource Center [genes-r-us.uthscsa.edu] Provides information and resources in the area of newborn screening and genetics.

Genetic Alliance- Genes in Life [genesinlife.org] A guide from the Genetic Alliance with easy-to-read information about genetic testing.

Genetics and Cancer [cancer.gov] An information fact sheet from the National Cancer Institute about genetic testing for hereditary cancers.

Find a Genetic Counselor [nsgc.org] A search engine developed by the National Society of Genetic Counselors.

Top of page

Last Updated: August 27, 2015

Follow this link:
Frequently Asked Questions About Genetic Testing - Genome.gov

At Childrens Hospital of Pittsburgh of UPMC, we believe parents and guardians can contribute to the success of this treatment and invite you to participate. Please read the following information to learn about the treatment and how you can help.

In order for a child to grow, a gland deep inside the brain, called the pituitary, must release enough growth hormone (GH). Natural growth hormone is released during deep sleep. Many factors influence the release of GH, including nutrition, sleep, exercise, stress, medications, blood sugar levels, and other hormones present in the body. When a childs body does not produce or release enough GH, he or she may have several symptoms, the most noticeable being slow or no growth or facial features that make the child look a lot younger than his or her peers. Although being small has no effect on a childs intelligence, it may cause self-esteem issues and interfere with the development of mature social skills. For that reason, GH treatment may be prescribed to help a child reach his or her fullest growth potentialboth in height and in personal development.

Once a child has been diagnosed with GH deficiency, Turner Syndrome, or other conditions treatable with GH therapy, the pediatric endocrinologist will discuss the pros and cons of, and usually recommend, GH therapy. The GH used in treatment is manufactured in the laboratory to be identical to that produced by the pituitary gland, so it is safe and effective. GH is given through a subcutaneous (sub-Q-TAIN-ee-us) injection, which means that it goes into the fatty tissue just beneath the surface of the skin. GH can be given by a special injection device that looks like a pen. Because it is such a shallow injection, the needle is very small and does not hurt much at all.

The main thing to expect is growth! Although it takes about 3 to 6 months to realize any height differences, the important thing is that your child will grow probably 1 to 2 inches within the first 6 months of starting treatment. There may be a few other things you notice:

It may take a number of years for your child to reach his or her adult height, so you should be aware that GH treatment is often a long-term commitment. Routine visits with the pediatric endocrinologist will be needed, as will periodic blood tests and x-rays to monitor your childs progress on the treatment. Although the length of treatment varies, your child probably will have to stay on GH treatment until he or she has:

GH injections are quick and almost pain-free, so children ages 10 and up may be able to and often prefer to give themselves their own injections. It is important that a parent supervises the injection to make sure the child gives the correct dosage each day. Parents should give the injections to younger children. Because natural growth hormone is released mainly during sleep in children, GH treatment is more effective when taken at bedtime.

Learning how to give GH injections may sound intimidating at first, but once you and your child get used to it, it becomes just another daily habit. There are, however, some tips that you should know when you start GH therapy:

Storage

Time of Day

Injection Sites

Finishing A Cartridge

Because GH is very expensive, you should use up all of the medication in every cartridge.

Since GH does not interfere with other medications, it can be taken even if your child is mildly ill (colds, flu), unless your PCP tells you to stop.

Although infrequent, there are some possible side effects that you should be aware of. They are:

If the headache is persistent or severe, however, call the Endocrinology Fellow on call immediately. If you have questions about a reaction, or your child is experiencing a reaction, call the Endocrinology Clinic or office.

GH is sold under a number of different prescription brand names, but all of them contain the same medication. Which brand name your child will use, and the shape and color of the pen that delivers the medication, will depend upon your medical insurance.

Because GH is very expensive, Childrens Hospital works with insurance reimbursement specialists to determine which brand will be covered under your medical insurance. Within 2 to 4 weeks after your child has been prescribed HG treatment, an insurance reimbursement specialist will call your home. It is very important that you speak with the specialist please pick up or return the call! Your childs prescription will not be filled until you have spoken with the reimbursement specialist. You should receive your childs GH with 2 to 4 weeks after approval; if you havent heard from the reimbursement specialist after 4 weeks, call the Endocrinology Clinic.

If your insurance changes during the course of GH treatment, please notify the Endocrinology Clinic as soon as possible or the continuity of your childs treatment could be interrupted.

As soon as your childs GH starter kit arrives, call the Endocrinology Clinic to schedule your familys GH injection training session. Your child and both parents or guardians should attend the training sessions before your child can begin GH treatment. At the training session, the nurse consultant will teach you and your child how to:

If you have any questions or if your child has any special needs you feel the Endocrinology Clinic needs to know about, please call the nurse consultant at Childrens Hospital before your childs clinic appointment.

Read more from the original source:
Growth Hormone Treatment

Hormone ImbalanceTest

Are you experiencing exhaustion, moodiness, low libido, weight gain or hot-flashes? Find out if your symptoms can be corrected with Bioidentical Hormone Therapy.

Learn More

BHRT utilizes hormones that are chemically identical to those already in your body. These natural hormones are not created in a lab, but come from plants and animals.

Learn More

Experiencing symptoms of low testosterone but not sure if you indeed have low levels? Check today with asimple Testosterone Test. You deserve to know!

Learn More

Stephen A. Goldstein, M.D., F.A.C.S. founded Denver Hormone Health on the central belief that no man or woman should have to suffer needlessly at the hands of hormone imbalances.

Dr. Goldstein has been studying Bioidentical Hormone Replacement Therapy (BHRT) and anti-aging medicine since 1999. He understands the detailed nuances of balancing hormones (tailored to meet your unique needs). He has also personally experienced the positive impact Bioidentical Hormone Therapy has had on his own health.

Born in Spartanburg, South Carolina and raised in Nashville, Tennessee, Dr. Goldstein was awarded an academic scholarship to the University of Tampa where he received a B.S. degree in Chemistry. H

e went on to receive his M.D. from the University of Tennessee College of Medicine, and completed his General Surgery and Plastic Surgery Residency at Brown University.

Dr. Goldstein firmly believes in continuing education and attends numerous professional meetings and conferences yearly to remain in touch with the latest technological advances. Read More

[testimonialswidget_widget refresh_interval=4]

See the rest here:
Denver Hormone Therapy | Denver Hormone Health

Introduction

The post-natal bone marrow has traditionally been seen as an organ composed of two main systems rooted in distinct lineagesthe hematopoietic tissue proper and the associated supporting stroma. The evidence pointing to a putative stem cell upstream of the diverse lineages and cell phenotypes comprising the bone marrow stromal system has made marrow the only known organ in which two separate and distinct stem cells and dependent tissue systems not only coexist, but functionally cooperate. Originally examined because of their critical role in the formation of the hematopoietic microenvironment (HME), marrow stromal cells later came to center stage with the recognition that they are the stem/progenitor cells of skeletal tissues. More recent data pointing to the unexpected differentiation potential of marrow stromal cells into neural tissue or muscle grant them membership in the diverse family of putative somatic stem cells. These cells exist in a number of post-natal tissues that display transgermal plasticity; that is, the ability to differentiate into cell types phenotypically unrelated to the cells in their tissue of origin.

The increasing recognition of the properties of marrow stromal cells has spawned a major switch in our perception of their nature, and ramifications of their potential therapeutic application have been envisioned and implemented. Yet, several aspects of marrow stromal cell biology remain in question and unsettled throughout this evolution both in general perspective and in detail, and have gained further appeal and interest along the way. These include the identity, nature, developmental origin and in vivo function of marrow stromal cells, and their amenability to ex vivo manipulation and in vivo use for therapy. Just as with other current members of the growing list of somatic stem cells, imagination is required to put a finger on the seemingly unlikely properties of marrow stromal cells, many of which directly confront established dogmas or premature inferences made from other more extensively studied stem cell systems.

Alexander Friedenstein, Maureen Owen, and their coworkers were the first to utilize in vitro culture and transplantation in laboratory animals, either in closed systems (diffusion chambers) or open systems (under the renal capsule, or subcutaneously) to characterize cells that compose the physical stroma of bone marrow [1-3]. Because there is very little extracellular matrix present in marrow, gentle mechanical disruption (usually by pipetting and passage through syringe needles of decreasing sizes) can readily dissociate stroma and hematopoietic cells into a single-cell suspension. When these cells are plated at low density, bone marrow stromal cells (BMSCs) rapidly adhere and can be easily separated from the nonadherent hematopoietic cells by repeated washing. With appropriate culture conditions, distinct colonies are formed, each of which is derived from a single precursor cell, the CFU-F.

The ratio of CFU-F in nucleated marrow cells, as determined by the colony-forming efficiency (CFE) assay [4], is highly dependent on the culture conditions, and there is a great deal of variability in the requirements from one animal species to another. In rodents, irradiated marrow feeder cells are absolutely required in addition to selected lots of serum in order to obtain the maximum number of assayable CFU-F (100% CFE), whereas CFE is feeder cell-independent in humans [5]. The mitogenic factors that are required to stimulate the proliferation of CFU-F are not completely known at this time, but do at least include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor, transforming growth factor-, and insulin-like growth factor-1 [6, 7]. Under optimal conditions, multi-colony-derived strains (where all colonies are combined by trypsinization) can undergo over 25 passages in vitro (more than 50 cell doublings), demonstrating a high capacity for self-replication. Therefore, billions of BMSCs can be generated from a limited amount of starting material, such as 1 ml of a bone marrow aspirate. Thus, the in vitro definition of BMSCs is that they are rapidly adherent and clonogenic, and capable of extended proliferation.

The heterogeneous nature of the BMSC population is immediately apparent upon examination of individual colonies. Typically this is exemplified by a broad range of colony sizes, representing varying growth rates, and different cell morphologies, ranging from fibroblast-like spindle-shaped cells to large flat cells. Furthermore, if such cultures are allowed to develop for up to 20 days, phenotypic heterogeneity is also noted. Some colonies are highly positive for alkaline phosphatase (ALP), while others are negative, and a third type is positive in the central region, and negative in the periphery [8]. Some colonies form nodules (the initiation of matrix mineralization) which can be identified by alizarin red or von Kossa staining for calcium. Yet others accumulate fat, identified by oil red O staining [9], and occasionally, some colonies form cartilage as identified by alcian blue staining [10].

Upon transplantation into a host animal, multi-colony-derived strains form an ectopic ossicle, complete with a reticular stroma supportive of myelopoiesis and adipocytes, and occasionally, cartilage [8, 11]. When single colony-derived BMSC strains (isolated using cloning cylinders) are transplanted, a proportion of them have the ability to completely regenerate a bone/marrow organ in which bone cells, myelosupportive stroma, and adipocytes are clonal and of donor origin, whereas hematopoiesis and the vasculature are of recipient origin [7] (Fig. 1). These results define the stem cell nature of the original CFU-F from which the clonal strain was derived. However, they also confirm that not all of the clonogenic cells (those cells able to proliferate to form a colony) are in fact multipotent stem cells. It must also be noted that it is the behavior of clonal strains upon transplantation, and not their in vitro phenotype, that provides the most reliable information on the actual differentiation potential of individual clones. Expression of osteogenic, chondrogenic, or adipogenic phenotypic markers in culture (detected either by mRNA expression or histochemical techniques), and even the production of mineralized matrix, does not reflect the degree of pluripotency of a selected clone in vivo [12]. Therefore, the identification of stem cells among stromal cells is only done a posteriori and only by using the appropriate assay. In this respect, chondrogenesis requires an additional comment. It is seldom observed in open transplantation assays, whereas it is commonly seen in closed systems such as diffusion chambers [11], or in micromass cultures of stromal cells in vitro [13], where locally low oxygen tensions, per se, permissive for chondrogenesis, are attained [14]. Thus, the conditions for transplantation or even in vitro assays are critical determinants of the range of differentiation characteristics that can be assessed.

FigureFigure 1.. Transplantation of ex vivo-expanded human BMSC into the subcutis of immunocompromised mice.A) Multi-colony and some single colony-derived strains attached to particles of hydroxyapatite/tricalcium phosphate ceramic (HA) form a complete bone/marrow organ composed of bone (B) encasing hematopoietic marrow (HP). B) The bone (B) and the stroma (S) are of human origin as determined by in situ hybridization using a human specific alu sequence as probe, while the hematopoietic cells are of recipient origin.

Download figure to PowerPoint

The ability to isolate the subset of marrow stromal cells with the most extensive replication and differentiation potential would naturally be of utmost importance for both theoretical and applicative reasons. This requires definitive linkage of the multipotency displayed in transplantation assays with a phenotypic trait that could be assessed prior to, and independently of, any subsequent assays. Several laboratories have developed monoclonal antibodies using BMSCs as immunogen in order to identify one or more markers suitable for identification and sorting of stromal cell preparations [15-18]. To date, however, the isolation of a pure population of multipotent marrow stromal stem cells remains elusive. The nearest approximation has been the production of a monoclonal antibody, Stro-1, which is highly expressed by stromal cells that are clonogenic (Stro-1+bright), although a certain percentage of hematopoietic cells express low levels of the antigen (Stro-1+dull) [19]. In principle, the use of the same reagent in tissue sections would be valuable in establishing in vivo-in vitro correlation, and in pursuing the potential microanatomical niches, if not anatomical identity, of the cells that are clonogenic. The Stro-1 reagent has limited application in fixed and paraffin-embedded tissue. However, preliminary data using frozen sections suggest that the walls of the microvasculature in a variety of tissues are the main site of immunoreactivity (Fig. 2), a finding of potentially high significance (see below).

FigureFigure 2.. Immunolocalization of the Stro-1 epitope in the microvasculature of human thymus.A) CD34 localizes to endothelial cells (E) forming the lumen (L) of the blood vessel. B) Stro-1 localizes not only to endothelial cells, but also the perivascular cells of the blood vessel wall (BVW).

Download figure to PowerPoint

Freshly isolated Stro-1+bright cells and multi-colony-derived BMSC strains, both of which contain but are not limited to multipotent stromal stem cells, have been extensively characterized for a long list of markers expressed by fibroblasts, myofibroblasts, endothelial cells, and hematopoietic cells in several different laboratories [20-24]. From these studies, it is apparent that the BMSC population at large shares many, but not all, properties of fibroblastic cells such as expression of matrix proteins, and interestingly, some markers of myofibroblastic cells, notably, the expression of -smooth muscle actin (-SMA) and some characteristics of endothelial cells such as endoglin and MUC-18. It has been claimed that the true mesenchymal stem cell can be isolated using rather standard procedures, and characterized using a long list of indeterminate markers [23]. However, in spite of this putative purification and extensive characterization, the resulting population was no more pure than multi-colony-derived strains isolated by simple, short-term adherence to plastic; the resulting clones displayed varying degrees of multipotentiality. Furthermore, the pattern of expressed markers in even clonal strains that are able to completely regenerate a bone/marrow organ in vivo is not identical, and changes as a function of time in culture. These results indicate that identifying the phenotypic fingerprint of a stromal stem cell may well be like shooting at a moving target, in that they seem to be constantly changing in response to their microenvironment, both in vitro and in vivo.

The primitive marrow stroma is established in development through a complex series of events that takes place following the differentiation of primitive osteogenic cells, the formation of the first bone, and the vascular invasion of bone rudiments [25]. This intimate relationship of the stromal cells with the marrow vascularity is also found in the adult marrow. In the post-natal skeleton, bone and bone marrow share a significant proportion of their respective vascular bed [26]. The medullary vascular network, much like the circulatory system of other organs, is lined by a continuous layer of endothelial cells and subendothelial pericytes [27]. In the arterial and capillary sections of this network, pericytes express both ALP (Fig. 3B, C, D, F, G) and -SMA (Fig. 3E), both of which are useful markers for their visualization in tissue sections. In the venous portion, cells residing on the abluminal side of the endothelium display a reticular morphology, with long processes emanating from the sinus wall into the adjacent hematopoietic cords where they establish close cell-cell contacts, that convey microenvironmental cues to maturing blood cells. These particular adventitial reticular cells express ALP (Fig. 3G) but not -SMA under normal steady-state conditions (Fig. 3H). In spite of this, but in view of their specific position along with the known diversity of pericytes in different sites, organs and tissues [28], reticular cells can be seen as bona fide specialized pericytes of venous sinusoids in the marrow. Hence, phenotypic properties of marrow pericytes vary along the different sections of the marrow microvascular network (arterial/capillary versus post-capillary venous sinusoids). In addition, adventitial reticular cells of venous sinusoids can accumulate lipid and convert to adipocytes, and they do so mainly under two circumstances: A) during growth of an individual skeletal segment when the expansion of the total marrow cavity makes available space in excess of what is required by hematopoietic cells, or B) independent of growth, when there is an abnormal or age-related numerical reduction of hematopoietic cells thereby making space redundant [29-31].

FigureFigure 3.. Anatomical and immunohistological relationship of marrow stromal cells to marrow pericytes.A) Marrow vascular structures as seen in a histological section of human adult bone marrow. hc = hematopoietic cells; ad = adipocytes; a = artery; VS = venous sinusoid; PCA = pre-capillary arteriole. Note the thin wall of the venous sinusoid. B) Semi-thin section from low-temperature processed glycol-methacrylate embedded human adult bone marrow reacted for ALP. Arrows point to three arterioles emerging from a parent artery (A). Note that while there is no ALP activity in the wall of the large size parent artery, a strong reaction is noted in the arteriolar walls. C, D) Details of the arterioles shown in A and B. Note that ALP activity is associated with pericytes (P). E) Section of human adult bone marrow immunolabeled for -SMA. Note the reactivity of an arteriolar wall, and the complete absence of reactivity in the hematopoietic cords (hc) interspersed between adipocytes (ad). F) Detail of the wall of a marrow venous sinusoid lined by thin processes of adventitial reticular cells (venous pericytes). Note the extension of cell processes apparently away from the wall of the venous sinusoid (vs) and into the adjacent hematopoietic cord ALP reaction. G, H) High power views of hematopoietic cords in sections reacted for ALP (G) and -SMA (H). Note the presence of ALP activity identifying reticular cells, and the absence of labeling for -SMA.

Download figure to PowerPoint

The ability of reticular cells to convert to adipocytes makes them a unique and specialized pericyte. Production of a basement membrane by adipocytes endows the sinus with a more substantial basement membrane, likely reducing the overall permeability of the vessel. Furthermore, the dramatic increase in cell volume through the accumulation of lipid during adipose conversion collapses the lumen of the sinus. This may exclude an individual sinus from the circulation without causing its irreversible loss. In general, the loss of pericyte coating on a microvessel is associated with vessel regression by apoptosis, while a normal pericyte coating is thought to stabilize them and prevent vessel pruning [32]. Adipose conversion is thus a mechanism whereby the size and permeability of the overall sinusoidal system is reversibly regulated in the bone marrow. Not surprisingly, regions of bone marrow that are hematopoietically inactive are filled with fat.

Given the similar location of pericytes and stromal cells, the significance of -SMA expression, a marker of smooth muscle cells, in marrow stromal cells takes on new meaning, although its expression is variable, both in vitro and in vivo. -SMA expression is commonly observed in nonclonal, and some clonal cultures of marrow stromal cells [33], where it appears to be related to phases of active cell growth [34], and may reflect a myoid differentiation event, at least in vitro [35]. However, the phenotype of -SMA-expressing stromal cells in culture resembles that of pericytes and subintimal myoid cells rather than that of true smooth muscle cells [35]. In the steady-state normal bone marrow, -SMA expressing stromal cells other than those forming the pericyte/smooth muscle coats of arteries and capillaries are not seen. In contrast, -SMA+ stromal cells not associated with the vasculature are commonly observed in the fetal bone marrow [36, 37], that physically grows together with the bone encasing it. -SMA+ marrow stromal cells are likewise seen in conjunction with a host of hematological diseases [37], and in some bone diseases, such as hyperparathyroidism [37] and fibrous dysplasia (FD) of bone (Riminucci and Bianco, unpublished results). In some of these conditions, these cells have been interpreted as myofibroblasts [34, 37]. More interestingly, at least some of these conditions also feature an increased vascularity, possibly related to angiogenesis [38], and an increased number of CFU-F, quantitated as discussed above (Bianco, Kuznetsov, Robey, unpublished results). Taken together, these observations seem to indicate that -SMA expression in extravascular marrow stromal cells (other than arterial/ capillary pericytes) is related to growth or regeneration events in the marrow environment, which is in turn associated with angiogenesis.

Angiogenesis in all tissues involves the coordinated growth of endothelial cells and pericytes. Nascent endothelial tubes produce EGF and PDGF-B, which stimulate the growth and migration of pericytes away from the subintimal myoid cell layer of the vascular section. A precise ligand-receptor expression loop of PDGF-B produced by endothelial cells and expression of the cognate receptor on pericytes regulates the formation of a pericyte coating and its occurrence in physical continuity with the nascent vascular network [39]. Interestingly, PDGF-receptor beta and EGF receptor are two of the most abundant tyrosine kinase growth factor receptors in BMSCs, and PDGF-B and EGF have been found to stimulate proliferation of BMSCs [6, 40], indicating a physiological similarity between pericytes and BMSCs.

In bone, as in any other organ, angiogenesis is normally restricted to phases of developmentally programmed tissue growth, but may reappear in tissue repair and regeneration or proliferative/neoplastic diseases. During normal bone growth, endothelial cell growth, pericyte coverage, and bone formation by newly generated bone-forming cells occur in a precise spatial and temporal sequence, best visualized in metaphyseal growth plates. Growing endothelial tubes devoid of pericytes occupy the foremost 200 microns of the developing metaphysis [41]. Actively dividing abluminal pericytes and bone-forming osteoblasts are next in line. Progression of endochondral bone formation is dependent on efficient angiogenesis, and is blocked if angiogenesis is blocked, as illustrated by both experimental and pathological conditions. Experimentally, inhibition of VEGF signaling initiated by chondrocytes with blocking antibodies to the cognate receptor on growing blood vessels in the metaphysis results in a blockade not only of bone growth, but also of the related activities in the adjacent cartilage growth plates [42]. A remarkably similar event occurs naturally in rickets, and can be mimicked by microsurgical ablation of the metaphyseal vasculature [41].

Taking into account the similarities in their physical relationship to the vasculature, the cellular response to growth factors, and expression of similar markers lead one to suspect that marrow pericytes and marrow stromal cells are the same entity. Pericytes are perhaps one of the most elusive cell types in the body, and their significance as potential progenitor cells has been repeatedly surmised or postulated [28, 43-46]. Elegant as much as unconventional, experimental proof of their ability to generate cartilage and bone in vivo, for example, has been given in the past [47, 48]. Likewise, it has been shown that retinal pericytes form cartilage and bone (and express Stro-1) in vitro [49]. But, there has been little definitive understanding of the origin of this elusive cell type. Current evidence suggests that there is most likely more than one source of pericytes throughout development and growth. First, during development, pericytes may be recruited during angiogenesis or vasculogenesis from neighboring resident mesenchymal cells [50]. Secondly, as recently shown, pericytes may arise directly from endothelial cells or their progenitors [51, 52]. Third, they can be generated during angiogenesis, either pre- or post-natally, through replication, migration and differentiation of other pericytes downstream of the growing vascular bud [32, 39, 53, 54]. With regards to bone marrow, this implies that marrow pericytes might also be heterogeneous in their mode of development and origin. Some may be recruited during blood vessel formation from resident, preexisting osteogenic cells; others may originate from endothelial cells; still others may grow from preexisting pericytes during vascular growth. Interestingly, it would be predicted from this model that a hierarchy of marrow stromal/progenitor cells exists. Some would be osteogenic in nature, while others would not. If so, one would expect to find multipotent cells with markers of osteogenic commitment, and multipotent cells with endothelial/pericytic markers. With respect to the phenotypic characterization of clonal stromal cells, evidence supporting a dual origin is indeed available.

As described above, stromal cells can take on many forms such as cartilage, bone, myelosupportive stroma, or fat. This behavior of marrow stromal cells, both in vitro and in vivo, has perhaps offered the first glimpse of the property now widely referred to as plasticity. It was shown, for example, that clonal strains of marrow adipocytes could be directed to an osteogenic differentiation and form genuine bone in an in vivo assay [55, 56]. Earlier, the ability of marrow reticular cells to convert to adipocytes in vivo had been noted [29, 57]. A number of different studies have claimed that fully differentiated chondrocytes can dedifferentiate in culture and then shift to an osteogenic phenotype [58, 59], and that similar or correlated events can be detected in vivo [60]. All of these data highlight the non-irreversible nature of the differentiation of several cell types otherwise seen as end points of various pathways/lineages (i.e., reticular cells, osteoblasts, chondrocytes, and adipocytes). The primary implication of these findings has remained largely unnoticed until recently. Commitment and differentiation are not usually thought of as reversible, but rather as multistep, unidirectional and terminal processes. This concept is reflected in the basic layout of virtually every scheme in every textbook depicting the organization of a multilineage system dependent on a stem cell. Here, a hierarchy of progenitors of progressively restricted differentiation potential is recognized or postulated. Lineages are segregated, leaving no room for switching phenotype at a late stage of differentiation, no way of turning red blood cells into white blood cells, for example. In contrast, it seems that one can turn an adipocyte or a chondrocyte into an osteoblast, and nature itself seems to do this under specific circumstances. If so, then some kind of reversible commitment is maintained until very late in the history of a single cell of the stromal systema notable and yet unnoticed singularity of the system, with broad biological significance.

There is a real physiological need for plasticity of connective tissue cells, namely the need to adapt different tissues that reside next to one another during organ growth, for example [30, 61], and it is likely that nature has evolved mechanisms for maintaining plasticity which remain to be fully elucidated. One example may be the key transcription factor controlling osteogenic commitment, cbfa1 [62, 63], which is commonly if not constitutively expressed in stromal cells derived in culture from the post-natal marrow [12], and maintained during differentiation towards other cell types such as adipocytes. This is perhaps the most stringent proof that a cell committed to osteogenesis (as demonstrated by expression of the key gene of commitment) may still enter other pathways of differentiation that were thought to be alternative ones [61]. Whether one can isolate a multipotent cbfa1-negative (non-osteogenically committed) stromal cell is at present unclear. However, freshly isolated stromal cells sorted as Stro-1bright have been shown to be cbfa1-negative by reverse transcriptase-polymerase chain reaction (Gronthos and Simmons, unpublished results). Interestingly, these cells also exhibit several endothelial markers, although never a true endothelial phenotype [21, 22].

The fact that chondrocytes, osteoblasts, reticular cells, and adipocytes come from a single precursor cell carrying a marker of osteogenic commitment is consistent with the fact that all of these cell types are members of the same organ, even though of different tissues. A single skeletal segment contains all of these cell types either at different stages of its own organogenesis or simultaneously. Although heretical to some and novel to others, even the notion that each of these cell phenotypes can switch to another within the same family under specific circumstances is consistent with the development and maintenance of the organ from which they were derived. This kind of plasticity is thus orthodox, meaning that it remains within the context of the organ system.

Over the past 2 years, several studies have indicated or implied that progenitors can be found in a host of different post-natal tissues with the apparently unorthodox potential of differentiating into unrelated tissues. First, it was shown that the bone marrow contained systemically transplantable myogenic progenitors [64]. Second, it was shown that neural stem cells could reestablish hematopoiesis in irradiated mice [65]; third, that bone marrow cells could generate neural cells [66], and hepatocytes [67]; and fourth, that a neurogenic potential could be ascribed to marrow stromal cells [68, 69]. What is striking about these data is the developmentally distant nature of the source of these progenitors and their ultimate destination. Differentiation across germ layers violates a consolidated law of developmental biology. Although consolidated laws are not dogmas (which elicited the comment that germ layers are more important to embryologists than to embryos), it is still indisputable and remarkable that even in embryos, cells with transgermal potential only exist under strict temporal and spatial constraints, with the notable exception of neural crest cells, which in spite of their neuroectodermal nature generate a number of craniofacial mesodermal tissues including bone. Cells grown in culture from the inner cell mass self-renew and maintain totipotency in culture for extended periods of time. However, this is in a way an artifact, of which we know some whys and wherefores (feeder cell layers, leukemia inhibitory factor). Embryonic stem (ES) cells only remain multipotent and self-renewing in the embryo itself for a very short period of time, after which totipotent cells only exist in the germline.

Consequently, the first key question iswhere do the multipotent cells of post-natal organisms come from? All answers at this time are hypothetical at best. However, if marrow stromal cells are indeed members of a diffuse system of post-natal multipotent stem cells and they are at the same time vascular/pericytic in nature/origin, then a natural corollary would read that perhaps the microvasculature is a repository of multipotent cells in many, if not all, tissues [70]a hypothesis that is currently being tested.

A second question is that if multipotent cells are everywhere, or almost everywhere, then what are the mechanisms by which differentiated cells keep their multipotency from making every organ a teratoma? Phrased in another way, adult tissues must retain some kind of organizing ability previously thought of as specific to embryonic organizers. If indeed cells in the bone marrow are able to become muscle or liver or brain, then there must be mechanisms ensuring that there is no liver or brain or muscle in the marrow. Hence, signals for maintenance of a tissue's self must exist and be accomplished by differentiated cells. (That is, of course, if stem cells are not differentiated cells themselves).

A third question ishow much of the stemness (self-renewal and multipotency) observed in experimental systems is inherent to the cells that we manipulate, and how much is due to the manipulation? Are we discovering unknown and unexpected cells, or rather unknown and unexpected effects of manipulation of cells in culture? To what extent do cell culture conditions mimic the effects of an enucleated oocyte cytoplasm, which permits a somatic cell nucleus to generate an organism such as Dolly, the cloned sheep? For sure, a new definition of what a stem cell isa timely, and biotechnologically correct, oneshould incorporate the conditions under which phenomena are recorded, rather than guessing from ex vivo performance what the true in vivo properties are. This exercise also has important implications for understanding where and when stem cells come into action in physiology. Even for the mother of all stem cells, the ES cell, self-renewal and multipotency are limited to specific times and events in vivo, and are much less limited ex vivo. Are similar constraints operating for other stem cells? Marrow stromal stem cells for example, can be expanded extensively in culture, but the majority of them likely never divide in vivo once skeletal growth has ceased (except the few that participate in bone turnover, and perhaps in response to injury or disease). What physiological mechanism calls for resumption of a stem cell behavior in vivo in the skeleton and other systems?

All of these questions are important not only for philosophical or esoteric reasons, but also for applicative purposes. Knowing even a few of the answers will undoubtedly enable biotechnology to better harness the magical properties of stem cells for clinical applications.

In vivo transplantation under defined experimental conditions has been the gold standard for defining the differentiation potential of marrow stromal cells, and a cardinal element of their very discovery. Historically, studies on the transplantability of marrow stromal cells are inscribed into the general problem of bone marrow transplantation (BMT). The HME is created by transplantation of marrow stromal cell strains and allows for the ectopic development of a hematopoietic tissue at the site of transplantation. The donor origin of the microenvironment and the host origin of hematopoiesis make the ectopic ossicle a true reverse BMT.

Local transplantation of marrow stromal cells for therapeutic applications permits the efficient reconstruction of bone defects larger than those that would spontaneously heal (critical size). A number of preclinical studies in animal models have convincingly shown the feasibility of marrow stromal cell grafts for orthopedic purposes [71-77], even though extensive work lies ahead in order to optimize the procedures, even in their simplest applications. For example, the ideal ex vivo expansion conditions have yet to be determined, or the composition and structure of the ideal carrier, or the numbers of cells that are required for regeneration of a volume of bone.

In addition to utilizing ex vivo-expanded BMSCs for regeneration of bone and associated tissues, evidence of the unorthodox plasticity of marrow stromal cells has suggested their potential use for unorthodox transplantation; that is, for example, to regenerate neural cells or deliver required gene products at unorthodox sites such as the central nervous system (CNS) [78]. This could simplify an approach to cell therapy of the nervous system by eliminating the need for harvesting autologous human neural stem cells, an admittedly difficult procedure, although it is currently believed that heterologous cells may be used for the CNS, given the immune tolerance of the brain. Moreover, if indeed marrow stromal cells represent just a special case of post-natal multipotent stem cells, there is little doubt that they represent one of the most accessible sources of such cells for therapeutic use. The ease with which they are harvested (a simple marrow aspirate), and the simplicity of the procedures required for their culture and expansion in vitro may make them ideal candidates. For applicative purposes, understanding the actual differentiation spectrum of stromal stem cells requires further investigation. Besides neural cells, cardiomyocytes have been reported to represent another possible target of stromal cell manipulation and transplantation [79]. It also remains to be determined whether the myogenic progenitors found in the marrow [64] are indeed stromal (as some recent data would suggest, [80]) or non-stromal in nature [81], or both.

Given their residency in the marrow, and the prevailing view that marrow stromal cells fit into the hematopoietic paradigm, it was unavoidable that systemic transplantation of marrow stromal cells would be attempted [82] in order to cure more generalized skeletal diseases based on the successes of hematopoietic reconstitution by BMT. Yet major uncertainties remain in this area. Undoubtedly, the marrow stromal cell is the entity responsible for conveying genetic alterations into diseases of the skeleton. This is illustrated very well by the ability of these cells to recapitulate natural or targeted genetic abnormalities into abnormal bone formation in animal transplantation assays [83-85]. As such, they also represent a potential repository for therapy to alleviate bone disease. However, a significant rationale for the ability of stromal cells to colonize the skeleton once infused into the circulation is still missing.

The stroma is not transplanted along with hematopoiesis in standard BMT performed for hematological or oncological purposes [86-88]. Infusion of larger numbers of stromal cells than those present in cell preparations used for hematological BMT should be investigated further, as it might result, in principle, in limited engraftment. Stringent criteria must be adopted when assessing successful engraftment of systemically infused stromal cells [61]. The detection of reporter genes in tissue extracts or the isolation in culture of cells of donor origin does not prove cell engraftment; it proves cell survival. In this respect, it should be noted that even intra-arterial infusion of marrow stromal cells in a mouse limb may result in virtually no engraftment, even though abundant cells of donor origin are found impacted within the marrow microvascular network. Of note, these nonengrafted cells would routinely be described as engrafted by the use of any reporter gene or ex vivo culture procedure. Less than stringent definitions of stromal cells (for example, their identification by generic or nonspecific markers) must be avoided when attempting their detection in the recipient's marrow. Clear-cut evidence for the sustained integration in the target tissue of differentiated cells of donor origin must be provided. This is rarely the case in current studies claiming engraftment of marrow stromal cells to the skeleton. Some evidence for a limited engraftment of skeletal progenitors following systemic infusion has, however, been obtained in animal models [89, 90]. These data match similar evidence for the possible delivery of marrow-derived myogenic progenitors to muscle via the systemic circulation [64]. It should be kept in mind that both skeletal and muscle tissues are normally formed during development and growth by extravascular cells that exploit migratory processes not involving the circulation. Is there an independent circulatory route for delivery of progenitors to solid phase tissues, and if so, are there physiologically circulating mesodermal progenitors? From where would these cells originate, both in development and post-natal organisms, and how would they negotiate the vessel wall? Addressing these questions is mandatory and requires extensive preclinical work.

Even once these issues are addressed, kinetic considerations regarding skeletal growth and turnover represent another major hurdle that must be overcome in order to cure systemic skeletal diseases via systemic infusion of skeletal progenitors. Yet there is broad opportunity for the treatment of single clinical episodes within the context of skeletal disease. While curing osteogenesis imperfecta by replacing the entire population of mutated skeletal progenitors with normal ones may remain an unattainable goal, individual fractures or deformity in osteogenesis imperfecta or FD of bone could be successfully treated with ex vivo repaired stromal cells, for example. Towards this end, future work must focus on the feasibility of transducing or otherwise genetically correcting autologous mutated osteoprogenitors ex vivo, and studies are beginning to move in this direction.

Molecular engineering of cells, either transiently or permanently, has become a mainstay in cell and molecular biology, leading to many exciting insights into the role of a given protein in cell metabolism both in vitro and in vivo. Application of these techniques for correcting human deficiencies and disease is a challenge that is currently receiving much attention. BMSCs offer a unique opportunity to establish transplantation schemes to correct genetic diseases of the skeleton. They may be easily obtained from the future recipient, manipulated genetically and expanded in number before reintroduction. This eliminates not only the complications of xenografts, but also bypasses the limitations and risks connected with delivery of genetic repair material directly to the patient via pathogen-associated vectors. While a similar strategy may be applied to ES cells, the use of post-natal BMSCs is preferable considering that they can be used autologously, thereby avoiding possible immunological complications from a xenograft. Furthermore, there is far less concern of inappropriate differentiation as may occur with ES cell transplantation. Finally, ES cell transplantation is highly controversial, and it is likely that the ethical debate surrounding their usage will continue for quite some time.

Depending on the situation, there are several approaches that can be envisioned. If a short-lived effect is the goal, such as in speeding up bone regeneration, transient transduction would be the desired outcome, utilizing methods such as electroporation, chemical methods including calcium phosphate precipitation and lipofection, and plasmids and viral constructs such as adenovirus. Transducing BMSCs with adenoviral constructs containing BMP-2 has demonstrated at least partial efficacy of this approach in hastening bone regeneration in animal models [75, 91, 92]. Adenoviral techniques are attractive due to the lack of toxicity; however, the level at which BMSCs are transfected is variable, and problematic. It has been reported that normal, non-transformed BMSCs require 10 more infective agent than other cell types [93], which is often associated with cellular toxicity. Clearly, further optimization is needed for full implementation of this approach.

For treatment of recessive diseases in which a biological activity is either missing or diminished, long-lasting or permanent transduction is required, and has depended on the use of adeno-associated viruses, retroviruses, lentiviruses (a subclass of retrovirus), and more recently, adeno-retroviral chimeras [94]. These viruses are able to accommodate large constructs of DNA (up to 8 kb), and while retroviruses require active proliferation for efficient transfection, lentiviruses do not. Exogenous biological activity in BMSCs by transduction with retroviral constructs directing the synthesis of reporter molecules, interleukin 3, CD-2, Factor VIII, or the enzymes that synthesize L-DOPA has been reported [78, 95-102]. However, these studies also highlight some of the hurdles that must be overcome before this technology will become practical. The first hurdle is optimization of ex vivo transfection. It has been reported that lengthy ex vivo expansion (3-4 weeks) to increase cell numbers reduces transfectability of BMSCs, whereas short-term culture (10-12 days) does not [98]. Furthermore, high levels of transduction may require multiple rounds of transfection [95, 101]. The second hurdle relates to the durability of the desired gene expression. No reported study has extended beyond 4 months post-transplantation of transduced cells [99] (Gronthos, unpublished results), and in most instances, it has been reported that expression decreases with time [96], due to promoter inactivation [102] and/or loss of transduced cells (Mankani and Robey, unpublished results). While promising, these results point to the need for careful consideration of the ex vivo methods, choice of promoter to drive the desired biological activity, and assessment of the ability of the transduced BMSCs to retain their ability to self-maintain upon in vivo transplantation. It must also be pointed out that using retrovirally transduced BMSCs for this type of application, providing a missing or decreased biological activity, does not necessarily require that they truly engraft, as defined above. They may be able to perform this function by remaining resident without actually physically incorporating and functioning within a connective tissue. In this case, they can be envisioned as forming an in vivo biological mini-pump as a means of introducing a required factor, as opposed to standard means of oral or systemic administration.

Use of transduced BMSCs for the treatment of a dominant negative disease, in which there is actual expression of misfunctioning or inappropriate biological activity, is far more problematic, independent of whether we are able to deliver BMSCs systemically or orthotopically. In this case, an activity must be silenced such that it does not interfere with any normal activity that is present, or reintroduced by any other means. The most direct approach would be the application of homologous recombination, as applied to ES cells and generation of transgenic animals. The almost vanishing low rate of homologous recombination in current methodology, coupled with issues of the identification, separation, and expansion of such recombinants does not make this seem feasible in the near future. However, new techniques for increasing the rate of homologous recombinations are under development [103] which may make this approach more feasible. Another approach to gene therapy is based on the processes whereby mismatches in DNA heteroduplexes that arise sporadically during normal cell activity are automatically corrected. Genetic mutations could be targeted by introducing exogenous DNA with the desired sequence (either short DNA oligonucleotides or chimeric RNA/DNA oligonucleotides) which binds to homologous sequences in the genome forming a heteroduplex that is then rectified by a number of naturally occurring repair processes [104]. A third option exists using a specially constructed oligonucleotide that binds to the gene in question to form a triple helical structure, thereby disallowing gene transcription [105].

While it would be highly desirable to correct a genetic disease at the genomic level, mRNA represents another very significant target, and perhaps a more accessible one, to silence the activity of a dominant negative gene. Methods for inhibiting mRNA translation and/or increasing its degradation have been employed through the use of protein decoys to prevent association of a particular mRNA to the biosynthetic machinery and antisense sequences (either oligonucleotides or full-length sequences). Double-stranded RNA also induces rapid degradation of mRNA (termed RNA interference, RNAi) by a process that is not well understood [105]. However, eliminating mRNAs transcribed from a mutant allele with short or single-base mutations by these approaches would most likely not maintain mRNA from a normal allele. For this reason, hammerhead and hairpin ribozymes represent yet another alternative, based on their ability to bind to very specific sequences, and to cleave them and inactivate them from subsequent translation. Consequently, incorporating a mutant sequence, even one that transcribes a single base mutation, can direct a hammerhead or hairpin ribozyme to inactivate a very specific mRNA. This approach is currently being probed for its possible use in the treatment of osteogenesis imperfecta [106]. Taking this technology one step further, DNAzymes that mimic the enzymatic activity of ribozymes, which would be far more stable than ribozymes, are also being developed. Regardless of whether genomic or cytoplasmic sequences are the target of gene therapy, the efficacy of all of these new technologies will depend on: A) the efficiency at which the reagents are incorporated into BMSCs in the ex vivo environment; B) the selection of specific targets, and C) the maintenance of the ability of BMSCs to function appropriately in vitro.

In conclusion, the isolation of post-natal stem cells from a variety of tissues along with discovery of their unexpected capabilities has provided us with a new conceptual framework in which to both view them and use them. However, even with this new perspective, there is much to be done to better understand them: their origins, their relationships to one another, their ability to differentiate or re-differentiate, their physiological role during development, growth, and maturity, and in disease. These types of studies will most certainly require a great deal of interdisciplinary crosstalk between investigators in the areas of natal and post-natal development, and in different organ systems. Clearly, as these studies progress, open mindedness will be needed to better understand the nature of this exciting family of cells, as well as to better understand the full utilization of stem cells with or without genetic manipulation. Much to be learned. Much to be gained.

The rest is here:
Bone Marrow Stromal Stem Cells: Nature, Biology, and ...

Scientists at Oregon Health & Science University and the Oregon National Primate Research Center (ONPRC) have successfully reprogrammed human skin cells to become embryonic stem cells capable of transforming into any other cell type in the body. It is believed that stem cell therapies hold the promise of replacing cells damaged through injury or illness. Diseases or conditions that might be treated through stem cell therapy include Parkinson's disease, multiple sclerosis, cardiac disease and spinal cord injuries.

The research breakthrough, led by Shoukhrat Mitalipov, Ph.D., a senior scientist at ONPRC, follows previous success in transforming monkey skin cells into embryonic stem cells in 2007. This latest research will be published in the journal Cell online May 15 and in print June 6.

The technique used by Drs. Mitalipov, Paula Amato, M.D., and their colleagues in OHSU's Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, is a variation of a commonly used method called somatic cell nuclear transfer, or SCNT. It involves transplanting the nucleus of one cell, containing an individual's DNA, into an egg cell that has had its genetic material removed. The unfertilized egg cell then develops and eventually produces stem cells.

"A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells, into several different cell types, including nerve cells, liver cells and heart cells. Furthermore, because these reprogrammed cells can be generated with nuclear genetic material from a patient, there is no concern of transplant rejection," explained Dr. Mitalipov. "While there is much work to be done in developing safe and effective stem cell treatments, we believe this is a significant step forward in developing the cells that could be used in regenerative medicine."

Another noteworthy aspect of this research is that it does not involve the use of fertilized embryos, a topic that has been the source of a significant ethical debate.

The Mitalipov team's success in reprogramming human skin cells came through a series of studies in both human and monkey cells. Previous unsuccessful attempts by several labs showed that human egg cells appear to be more fragile than eggs from other species. Therefore, known reprogramming methods stalled before stem cells were produced.

To solve this problem, the OHSU group studied various alternative approaches first developed in monkey cells and then applied to human cells. Through moving findings between monkey cells and human cells, the researchers were able to develop a successful method.

The key to this success was finding a way to prompt egg cells to stay in a state called "metaphase" during the nuclear transfer process. Metaphase is a stage in the cell's natural division process (meiosis) when genetic material aligns in the middle of the cell before the cell divides. The research team found that chemically maintaining metaphase throughout the transfer process prevented the process from stalling and allowed the cells to develop and produce stem cells.

"This is a remarkable accomplishment by the Mitalipov lab that will fuel the development of stem cell therapies to combat several diseases and conditions for which there are currently no treatments or cures," said Dr. Dan Dorsa, Ph.D., OHSU Vice President for Research. "The achievement also highlights OHSU's deep reproductive expertise across our campuses. A key component to this success was the translation of basic science findings at the OHSU primate center paired with privately funded human cell studies."

One important distinction is that while the method might be considered a technique for cloning stem cells, commonly called therapeutic cloning, the same method would not likely be successful in producing human clones otherwise known as reproductive cloning. Several years of monkey studies that utilize somatic cell nuclear transfer have never successfully produced monkey clones. It is expected that this is also the case with humans. Furthermore, the comparative fragility of human cells as noted during this study, is a significant factor that would likely prevent the development of clones.

"Our research is directed toward generating stem cells for use in future treatments to combat disease," added Dr. Mitalipov. "While nuclear transfer breakthroughs often lead to a public discussion about the ethics of human cloning, this is not our focus, nor do we believe our findings might be used by others to advance the possibility of human reproductive cloning."

See original here:
Human skin cells converted into embryonic stem cells ...

Im attending annual IBC Cell Therapy Bioprocessing meeting. It is probably the best meeting for cell product manufacturers and developers. You can follow the real time updates via hashtag #IBC_CTB13. This year, iPS cell manufacturing session was added to the program for the first time. And it has been very interesting and informative. Scott Lipnick from the New York Stem Cell Foundation Research Institute (NYSCF) and Wen Bo Wang from Cellular Dynamics International, share their experience in iPS cell manufacturing approaches and automation of the process.

Are iPS cells ready for prime time? Yes, as research tools and disease models. Not quite yet for human therapies. NYSCF is non-profit organization with ~ 45 researchers, which focused on high-risk high-reward experiments in iPS cell field. NYSCF builds infrastructure to industrialize SC research. This is one of the first organizations, which applied automation in iPS cell manufacturing. Bringing automation in iPSC cell derivation and differentiation would allow to tackle standardization and scalability issues. NYSCF approaches this problem by high-throughput platform Global Stem Cell Array.

Lipnick told that they were able to create automated assembly line with only 2 manual steps left skin biopsy and seeding in the dish. The production rate is 200 lines / per month. The whole process is traceable and recorded as a batch record. Besides iPS cell lines generation, NYSCF is also working on automation of differentiation process. For example, beta-cells production and DOPA+ neurons. They are also looking into GMP manufacturing.

Wang of CDI gave an example of their current commercial production capacity per day: 2B (billions) of iPS cells, 1B icardiomyocytes, 1B ineurons, 0.5B iendothelial, 0.4B ihepatocytes. Two more products will be launched next year. She described how CDI changed research process to make it automated and clinical-grade.

Potential challenges in scaling out of autologous iPS line production that she has mentioned: choice of starting material, footprint-free (no transgene) lines, undefined components, spontaneous differentiation, abnormal karyotype, asynchronous growth, batch record/ information review. They decided to use blood as source material, because less risk of contamination and possibility of closed system. Optimization of source material allowed them to move from 0.5L of blood to few ml. of fresh blood. They expand mononuclear cells and freeze them down for scheduled manufacturing. CDI manufactures iPS cell lines by batches. Episomal vector used to generate footprint-free lines. In order to pick right colonies, they dilute 1 clone/ well in 96-well plate. Only 2 steps left non-automated in CDI process: transfection and colony picking.

Characterization of the line includes: morphology, markers, SNP (genotype), ID match, loss of reprogramming plasmid, karyotype, mycoplasma. They used robotic streamline of qPCR 39 genes for quality control. For creation of GMP lines, they changed a process: use of GMP-grade plasmid, reprogramming by small molecules, recombinant feeder-mimetic (ECM), antibiotic-free, xeno-free medium. Finally, CDI has started a HLA-matched iPS cell line banking project. Phase 2 of the project will utilize 200 donors and can cover 90% of US and EU population.

One question from the audience was very interesting: Dont you think, HLA iPS cell banking is racing ahead of science and realization of its usefulness? Wang said: Well, we dont know how useful they will be, we just want to show we can do it!

Tagged as: automation, cell line, conference, IBC, iPS, manufacturing

See the article here:
IBC Cell Therapy Bioprocessing 2013 moving to iPS cell ...

Many people decide to learn whether or not they have an abnormal gene that is linked to higher breast cancer risk. Three of the most well-known abnormal genes are BRCA1, BRCA2, and PALB2. Women who inherit a mutation, or abnormal change, in any of these genes from their mothers or their fathers have a much higher-than-average risk of developing breast cancer and ovarian cancer. Men with these mutations have an increased risk of breast cancer, especially if the BRCA2 gene is affected, and possibly of prostate cancer. Many inherited cases of breast cancer have been associated with these three genes.

The function of the BRCA and PALB2 genes is to keep breast cells growing normally and prevent any cancer cell growth. But when these genes contain the mutations that are passed from generation to generation, they do not function normally and breast cancer risk increases. Abnormal BRCA1, BRCA2, and PALB2 genes may account for up to 10% of all breast cancers, or 1 out of every 10 cases.

Remember that most people who develop breast cancer have no family history of the disease. However, when a strong family history of breast and/or ovarian cancer is present, there may be reason to believe that a person has inherited an abnormal gene linked to higher breast cancer. Some people choose to undergo genetic testing to find out. A genetic test involves giving a blood sample that can be analyzed to pick up any abnormalities in these genes.

In this section, you can read more about the following topics related to genetic testing:

If you want to learn more about family-related risk and genetics, you can visit the Lower Your Risk section of this site.

Researchers have discovered, and are continuing to discover, other abnormal genes that are less common than BRCA1, BRCA2, and PALB2 but also can raise breast cancer risk. Testing for these abnormalities is not done routinely, but it may be considered on the basis of your family history and personal situation. You can work with your doctor to decide whether testing for gene abnormalities besides BRCA1, BRCA2, and PALB2 is warranted.

To connect with others who have tested positive for a BRCA1 or BRCA2 gene abnormality, visit the Breastcancer.org Discussion Board forum BRCA1 or BRCA2 Positive.

The medical experts for Genetic Testing are:

These experts are members of the Breastcancer.org Professional Advisory Board, which includes more than 60 medical experts in breast cancer-related fields.

"Simply having a proven gene abnormality does not necessarily mean that a woman will develop breast cancer, or that her cancer will be any worse than cancer that does not stem from an inherited genetic flaw."

More here:
Genetic Testing - Breastcancer.org - Breast Cancer ...

Our vision is to provide our patients with personalized, physician-supervised care with an emphasis on preventive medicine, healthy aging and aesthetics. Our customized services and programs are created within a relaxed environment proven to give you superior results in your pursuit of optimal health and appearance.

Endorsements from celebrities like Suzanne Somers, Oprah Winfrey, Robin McGraw, Linda Evans and Rachel Ray have greatly increased public awareness of BHRT.

Like celebrities, many of our patients are busy juggling careers, family life and everything else. At the same time, they are trying to camouflage their symptoms trying to convince themselves that what they are experiencing is the normal aging process, when in fact, they are suffering from hormone imbalance associated with menopause or andropause.

Health conditions caused by hormone imbalances have started to generate a lot of attention. On her website, Oprah writes, After one day on bio-identical estrogen, I felt the veil lift. After 3 days the sky was bluer, my brain was no longer fuzzy, my memory was sharper. I was literally singing and had a skip in my step.

Although there is no magic recipe for relief from the symptoms of menopause and andropause, Doctor Trim feels that men and women should educate themselves about the options available and he applauds the celebrities that continue to keep the topic in the headlines.

Here are some of the benefits of BHRT : Improved libido (sex drive) Improved sleep Reduced risk of depression Better mood, concentration, and memory Help in the prevention of Osteoporosis and restoration of bone strength May protect against heart disease and stroke Reduced hot flashes and reduced vaginal dryness Muscle mass and strength are better maintained Improvement in cholesterol levels

View original post here:
Atlantic Age Management - New Jersey Hormone Doctor ...

Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy.1 In the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential.25 The origin and significance of the subpopulation of replicating myocytes are unknown; these issues could be relevant to understand the for mechanisms coaxing endogenous cardiomyocytes to reenter the cell cycle and to the search for strategies to transplant cardiac progenitor cells.6 In fact, although embryonic stem cells have an exceptional capacity for proliferation and differentiation, potential immunogenic, arrhythmogenic, and, particularly, ethical considerations limit their current use. Moreover, autologous transplantation of skeletal myoblasts has been considered because of their high proliferative potential, their commitment to a well-differentiated myogenic lineage, their resistance to ischemia, and their origin, which overcomes ethical, immunological, and availability problems. However, even if phase II clinical trials with autologous skeletal myoblasts are ongoing, several problems related to potentially life-threatening arrhythmia (perhaps reflecting cellular uncoupling with host cardiomyocytes7) must be taken into account when this approach is considered. Furthermore, although cardiomyocytes can be formed, at least ex vivo, from different adult stem cells, the ability of these cells to cross lineage boundaries is currently causing heated debate in the scientific community,8 with the majority of reports indicating neoangiogenesis as the predominant in vivo effect of bone marrow or endothelial progenitor cells.9,10

This report describes the identification and preliminary characterization of cells from the adult human and murine heart, which have the properties of cardiac stem cells. Because these cells also have been isolated and expanded from human heart biopsy specimens, they could have a significant impact on future clinical strategies to treat patients with heart disease.

Human tissue was derived from atrial or ventricular biopsy specimens belonging to patients (1 month to 80 years of age) undergoing heart surgery, in conformation with the guidelines of the Italian Department of Health. Murine tissue was derived from the hearts of previously characterized homozygous MLC1/3F-nlacZ11 and cTnI-nlacZ12 transgenic mice expressing a nuclear lacZ transgene under the transcriptional control of the striated muscle myosin light chain or cTnI promoters, respectively, homozygous B5-eGFP mice,13 homozygous GFP-cKit14 mice, MLC3F-nlacZ/B5-eGFP, MLC3F-nlac-Z/GFP-cKit, and cTnI-nlacZ/B5-eGFP cTnI-nlac-Z/GFP-cKit crossed mice, SCID mice, and SCID beige mice (Charles River Italia, Lecco, Italy).

Isolated myocardial tissue was cut into 1- to 2-mm3 pieces, washed with Ca2+-Mg2+free phosphate-buffered solution (PBS) (Invitrogen), and digested three times for 5 minutes at 37C with 0.2% trypsin (Invitrogen) and 0.1% collagenase IV (Sigma, Milan, Italy). The obtained cells were discarded, and the remaining tissue fragments washed with complete explant medium (CEM) (Iscoves Modified Dulbeccos Medium [IMDM] supplemented with 10% fetal calf serum, 100 U/mL penicillin G, 100 g/mL streptomycin, 2 mmol/L l-glutamine, and 0.1 mmol/L 2-mercaptoethanol) were cultured as explants in CEM at 37C and 5% CO2. After a period ranging from 1 (embryo) to 3 (adult) weeks, a layer of fibroblast-like cells was generated from adherent explants over which small, phase-bright cells migrated. These phase-bright cells were collected by pooling two washes with Ca2+-Mg2+free PBS, one wash with 0.53 mmol/L EDTA (Versene, Invitrogen) (1 to 2 minutes), and one wash with 0.5 g/L trypsin and 0.53 mmol/L EDTA (Invitrogen) (2 to 3 minutes) at room temperature under visual control. The cells obtained (from 104 to 4105 cells/explant) were seeded at 0.5 to 2105 cells/mL in poly-d-lysine-coated multiwell plates (BD Bioscences, Milan, Italy) in cardiosphere-growing medium (CGM) (35% complete IMDM/65% DMEMHam F-12 mix containing 2% B27, 0.1 mmol/L 2-mercaptoethanol, 10 ng/mL epidermal growth factor [EGF], 20 ng/mL basic fibroblast growth factor [bFGF], 40 nmol/L cardiotrophin-1, 40 nmol/L thrombin, antibiotics, and l-Glu, as in CEM). Isolation of the cardiosphere-forming cells could be performed at least 4 times at 6- to 10-day intervals from the same explant. Cardiospheres (CSs) were passaged every 2 to 3 days by partial changing of the medium and mechanical trituration of the larger clusters. Movies of cultured CSs, available in the online data supplement at http://circres.ahajournals.org, were recorded using a Nikon-COOLPIX-4500 digital camera connected to a Leitz inverted microscope. For cryopreservation, we used CEM/DMEMHam F12 at 50:50, 5% B27, and 10% DMSO as the freezing medium.

Extensive descriptions of BrdUrd labeling, clonal analysis, differentiation on substrate-coated surface, coculture experiment, immunocytochemistry, flow cytometric analysis, in vivo analysis, and heterotopic and orthotopic transplantation are provided in the online data supplement.

Sphere-generating cells were obtained by mild enzymatic digestion of explanted human atrial or ventricular biopsy specimens and embryo, fetal, and postnatal mouse hearts. Soon after the generation of a layer of fibroblast-like cells from well-adherent explants, some small, round, phase-bright cells began to migrate over this coat. These cells could be harvested periodically by treatment with EDTA and mild trypsinization and were allowed to grow on poly-d-lysinecoated culture surfaces in a low-serum (3.5% fetal calf serum) medium supplemented with a serum substitute (B27), growth factors (EGF and bFGF), cardiothrophin-1 (CT-1),15 and thrombin.16 During the first week of culture, the last factor led to a 7-fold increase in the number of spheres with respect to that obtained using the medium supplemented with the other factors, either alone or in combination. Time-course observations of cells derived from human and murine explants showed that early after their seeding (30 minutes), some of these cells began to divide while still in suspension. Most cells became loosely adherent, whereas others remained in suspension, and some contaminating fibroblast-like cells attached firmly to the poly-d-lysine coat. Cellular divisions also were evident from the loosely adherent cell population and produced clusters of small, round, phase-bright cells (that we termed CSs) after 10 to 12 hours (Figure 1a). Within 24 to 36 hours of their appearance, CSs increased in size and some of them detached from the culture surface; after 48 to 72 hours, most CSs were between 20 and 150 m in size, and, when not subjected to mechanical dissociation, the largest contained dark zones within the inner mass (Figure 1a).

Figure 1. CS proliferation. a, Phase micrograph of floating CSs (cultured from <24 hours to >48 hours) derived from primary culture of a human atrial biopsy sample. b, Proliferation curves of human and mouse CSs (derived from 8 different subjects [left] and from prenatal and postnatal hearts [middle and right], respectively) in the presence (middle) and absence (right) of 3.5% serum. Number of spheres refers to the mean number per well from which 90% of the spheres were withdrawn at each time point for further analysis. Note the different pattern of proliferation between the human and mouse CSs and the rapid rise of the curves, followed by an irreversible decline in the serum-free conditions.

Murine CSs started beating spontaneously soon after their generation (Supplementary Movie: mouse CSs movie 1a) and maintained this function during their life span (Supplementary Movie: mouse CSs movie 1b), whereas human CSs did so only when cocultured with rat cardiomyocytes (Supplementary Movie: human CSs movie 1a and 1b). To be sure that contraction was a new trait acquired by the CSs cells, GFP-labeled human CSs (partially or totally dissociated) were cocultured with cardiomyocytes prestained (Supplementary Human CSs Movie 2b through 2d) or not prestained (Supplementary Human CSs Movie 3a through 3d) with Dil. Contracting GFP-labeled cells were observed after 48 hours of coculture; furthermore, Cx-43 immunostaining performed on the cocultures of human GFP-transduced CSs with unlabeled neonatal rat cardiomyocytes showed the typical punctuate fluorescence pattern of the main gap junction protein of the heart along the cytoplasmatic membrane of the human cells (Figure 2d and Supplementary Figure VIII), suggesting that a functional connection is created between the two cellular populations.

Figure 2. Clonogenesis and coculture features. a, Fluorescence analysis of a single cell (upper right) (obtained from a dissociated GFP-expressing CS) when plated by limiting dilution on mitomycin-treated STO fibroblast-coated 96-well plates in CGM over the course of the generation of the GFP-labeled clone. This clone could be passaged and expanded on poly-d-lysine coat (lower left). b, X-Gal staining of a eGFP/MLC3F clone (obtained in the same way as were human clones) after 48 hours of exposure to growth factor-free medium. In these conditions, clone cells become more flattened, with many nuclei appearing blue, demonstrating that a differentiation process occurred (see also Supplementary Figure I and Supplementary clone movies). c, Fluorescence analysis of partially dissociated eGFP-labeled human CSs at 96 hours of coculture with rat cardiomyocytes. The same green cells that showed a synchronous contraction with cardiocytes (see supplementary human CSs movies) express cTnI. d, Fluorescent analysis of connexin-43 expression (red) in eGFP-labeled human CSs cocultured with rat cardiomyocytes, as in (c). A punctuate red fluorescence is present in the cell membrane of human cells (see Supplementary Figure VIII).

CSs were found to be composed of clonally derived cells and did not simply represent cellular aggregates. In fact, when human GFP-transduced CSs or murine CSs (derived from eGFP/MLC3F or eGFP/cTrI mice) were dissociated and plated as single cells on mitomycin-treated STO fibroblast-coated 96-well plates (or clonally diluted on 10-cm Petri dishes), fluorescent spheres were generated with a 1% to 10% efficiency (Figure 2a). These spheres could be subcloned on poly-d-lysine-coated surfaces, showing the same functional and phenotypic behavior in culture as the nonclone-derived CSs. In fact, 3 days after their appearance, some of the MLC3F-nlacZ/B5-eGFP or cTnI-nlacZ/B5-eGFP mice clonederived CSs started to beat (supplementary clone movie), and, after 48 hours of culture with CEM, the majority (6 of 7) of these showed expression of the lac-Z transgene within the nuclei after specific histochemical staining (Figure 2b1 and 2b2 and Supplementary Figure I). Moreover, human clones derived from a single GFP-labeled cell started a synchronous beating and expressed cTnI after 48 hours of coculture with rat cardiomyocytes (Supplementary Movie human CSs 2a and 2a1 and Supplementary Figure II).

Furthermore, when BrdUrd was added to the culture medium, virtually all cells in the small CSs and those of the inner part of the largest CSs were labeled (Figure 3a), indicating that these cells were newly generated (Supplementary Figures III through Va).

Figure 3. CSs BrdUrd incorporation and CSs characterization. a, Fluorescence confocal analysis of BrdUrd-labeled human CSs for cardiac differentiation markers: 6-m scans (from the periphery to the center of the sphere) and final pictures (small and large images, respectively) of BrdUrd (green) and cTnI (red) (see Supplementary Figures III through V). b, Confocal analysis of human CSs after 12 hours of culture: CD-34, CD-31, KDR, and c-Kit labeling of CS-generating cells at the beginning of sphere formation. c, fluorescence-activated cell sorting analysis of postnatal mouse CSs-derived cells. A time course at 0 and 6 days was used, and the phenotype profile for CD34, cKit, Cd31, and sca-1 expression was analyzed and shown as a percentage of positive events. Data are presented as meanSD (n=3). *Statistically significant difference from 0 days. See the graphics in the Table and in Figure 6.

Human CS-generating cells were capable of self-renewal. With periodical dissociation, together with partial substitution of CGM every 2 to 3 days, a log-phase expansion of spheres was obtained (Figure 1b). Mouse CS growth was slower (probably because of the more differentiated features assumed in culture, such as beating) and serum-dependent as for the human CSs (Figure 1b).

As shown in Figure 3a and Supplementary Figure V, confocal immunofluorescence analysis of BrdUrd-labeled human CSs with anti-BrdUrd (green) and cardiac-troponin I (cTnI) or atrial natriuretic peptide (ANP) (red) revealed BrdUrd-positive cells, particularly in the inner of the spheres, whereas cTnI-positive or ANP-positive cells were mainly localized in the external layers. Similar features are shown in Supplementary Figures III and IV. BrdUrd-labeled cells (red) mostly localized in the center of a CS and colocalize with the Hoechst-labeled nuclei, whereas cardiac myosin heavy chain (MHC)-expressing cells (green) were preferentially located in the boundary layers. Furthermore, several CS cells expressed cardiac differentiation markers (cTnI, ANP) while still dividing, as indicated by BrdUrd incorporation (Figure 3a and Supplementary Figure Va), suggesting that early cardiac differentiation already occurred during the proliferation phase of their growth. Usually within 10 days, some spheres became adherent, showing a more flattened morphology. Some small cells eventually migrated out from these sun-like spheres in the form of adherent (differentiated) or small, round cells that could generate new spheres. After thawing from cryopreservation, CSs proliferated again, maintaining their ability to beat (Supplementary Movie: human CSs movie).

Phenotypic analysis of newly developing human and mouse CSs revealed expression of endothelial (KDR (human)/flk-1 [mouse], CD-31) and stem cell (CD-34, c-kit, sca-1) markers. As shown in Figure 3b, CSs at the 2- to 10-cell stage strongly reacted with antibodies against these antigens. In larger spheres, the expression pattern of some of these markers (particularly cKit) was similar to that of the BrdUrd-labeling (positive staining in the center and in some peripheral zones, generating satellite spheres; data not shown).

A time course (0 and 6 days) of the quantitative characterization of CS cells with these stem and endothelial markers was performed by fluorescence-activated cell sorting analysis (Figure 3c and Supplementary Figure VI). As shown at the beginning of their formation (0 days), the phenotype of these cells seems to reflect the epifluorescent microscopy analysis with 10% of positive staining for all four phenotypes. However, at 6 days, cKit appears to be the only conserved marker, suggesting that the cKit+ cells could be the main ones contributing to the maintenance of proliferation. The initial cell-labeling may reflect an early activation state, as has been suggested for CD-34 in several systems.17 Fluorescence microscopy analysis performed on cryosectioned human CSs revealed expression of cardiac differentiation markers (cTnI, MHC) and endothelial markers (von Willebrand factor) (Supplementary Figure Vc1 through Vc3). When totally or partially dissociated into single cells and cultured on collagen-coated dishes in the same medium as the explants, mouse and human CS-derived cells assumed a typical cardiomyocyte morphology, phenotype (Supplementary Figures Vb1 through Vb2 and VIIc and VIId), and function documented (in the mouse only) by spontaneous contraction (Supplementary Movie: mouse CSs movie 2a and 2b).

Human CSs did not beat spontaneously; however, these began to beat within 24 hours when cocultured with postnatal rat cardiomyocytes, losing their spherical shape and assuming a sun-like appearance. Markers of cardiac differentiation were coexpressed within GFP in labeled human CSs cells (Figure 2c).

To follow the differentiation process of CSs during the prenatal and postnatal age, MLC3F-nlacZ and cTnI-nlacZ mice were used.1112 These mice express a form of lacZ transgene that localizes within the nucleus under the skeletal and cardiac muscle myosin light chain or cardiac troponin I promoter, respectively. CSs obtained from embryonic day 9 to 12, fetal day 17 to 18, and from neonatal and adult mice showed spontaneous expression of the reporter gene in variable percentages (10% to 60%) of spheres in the different culture conditions used (Figure 4a1 through 4a4 and Supplementary Figure VIIa1, VIIa2, VIIb1, and VIIb2). Moreover, regarding the human ones, CS-generating cells from mice expressed stem (CD-34, sca-1, cKit) and endothelial cell markers (flk-1, CD-31) (data not shown).

Figure 4. CSs features in transgenic mice. a, Phase micrograph of CSs from MLC3F-nlacZ and cTnI-nlacZ mice. Nuclear lacZ expression is mainly localized in the external layers of embryo and adult CSs soon after their formation (inserts) and after a few days of culture (right and central panels) (see Supplementary Figure VII). b, Fluorescence analysis of a spontaneously differentiated mouse CS. As suggested from the synchronous contraction showen in culture (supplementary mouse CSs movie), cTnI (red) is expressed in the sphere and the migrated cells; in these, last sarcomers are also evident. c, Fluorescence and phase analysis of CSs from GFP-cKit, GFP-cKit/MLC3F-nLacZ, and GFP-cKt/cTnI-nlacZ mice. GFP-labeled cells were present a few minutes after their seeding in culture with CGM, at the beginning of the generation of the CSs, later in their inner mass, and after their migration out from the oldest adherent spheres (arrows) (upper left, lower left, and central panels). GFP-labeled cells did not colocalize with the blue-stained ones (arrows) in CSs from GFP-cKit/MLC3F-nLacZ and GFP-cKit/cTnI-nlacZ mice. Fluorescent cells also were present in the growth area of the CSs (arrows) (right upper and right lower panels). Fluorescence, phase (small), and merged (large) images.

On this basis, we used transgenic mice expressing GFP under the control of the c-kit promoter14 to further clarify the cellular origin of these spheres and to follow the pattern of their growth process. As shown in Figure 4c1, GFP-positive cells were present from the beginning of the formation of the CSs and, albeit with reduced fluorescence intensity, also later within the mass of cells of the CSs and in cells migrating from old adherent sun-like CSs (Figure 4c2). Moreover, as suggested by the growth pattern of human CSs, when satellite secondary CSs appeared to detach from the primary ones, GFP-positive cells localized on the margins of the latter and in the inner part of the former.

We studied this process in double-heterozygous mice obtained from GFP-cKit/MLC3F-nlacZ or GFP-cKit/cTnI-nLacZ crossings. As shown in Figure 4c3 and 4c4, -Gal positivity did not colocalize with GFP in cells present within the growing areas.

To investigate the survival and morpho-functional potential of the CSs in vivo, two sets of experiments were performed. In the first, CS cells were injected in the dorsal subcutaneous region of SCID mice. In the second, they were injected into the hearts of SCID beige mice, acutely after myocardial infarction. The objective of ectopic transplantation experiments was to study the pattern and the behavior of growth of CSs in a neutral milieu (ie, without specific cardiac induction) to verify their unique potential of generation of the main cardiac cell types and to exclude the potential of neoplastic transformation. For these experiments, 60 pooled spheres/inoculum/mouse from prenatal and postnatal MLC3F-nlacZ/B5-eGFP mice, TnI-nlacZ/B5-eGFP mice, MLC3F-nlacZ/CD-1 mice, and cTnI-nlacZ/CD-1 mice were used. During the first 10 days, beating was appreciable through the skin over the injection site, distant from large blood vessels. On day 17, animals were euthanized and the inoculum recognized as a translucent formation, grain-like in size, wrapped in ramified vessel-like structures. Observation of unfixed cryosections by fluorescence microscopy (Figure 5a1 through 5a4) revealed the presence of open spheres from which cells appeared to have migrated. Clusters of black holes, particularly in the periphery of the structure, were evident. The tissue contained tubular formations, surrounded by nuclei (Hoechst-positive), identified as cardiac sarcomeres by cTnI and sarcomeric myosin immunostaining (Figure 5b3 through 5b6). -Smooth muscle actin (-SMA)-positive structures (known to be transiently expressed during cardiomyogenesis)2,18 were present in the remainder of the spheres and associated with the vasculature (the clusters of black holes) (Figure 5a3 through 5a5). This exhibited well-differentiated structures with a thin endothelium expressing vascular endothelialcadherin (Figure 5b1) and a relative large lumen containing erythrocytes (Figure 5a3), indicating the establishment of successful perfusion by the host. Light microscopic observation of the inoculum, after X-gal staining, showed strong nuclear expression of striated muscle-specific lacZ in the remainder of the spheres and in some cells close to them (Figure 5b2). No multidifferentiated structures suggesting the presence of tumor formation were observed.

Figure 5. In vivo analysis (ectopic CSs inoculum). a1 to a5, Ectopic transplantation of CSs from MLC3F-nlacZ/B5-eGFP mouse to SCID mouse (upper left panels). Fluorescence analysis of unfixed cryosections (a1, a2, and a4) from the subcutaneous dorsal inoculum (day 17). GFP cells seemed to have migrated from the spheres, whereas clusters of vessel-like structures (a2) could be observed mainly in the external area. Staining for SMA of one of these cryosections showed positive immunoreaction of the sphere and some cells within the inoculum (a5). b-1 to b6, Fluorescence (b3 to b4) and phase analysis (b5 to b6) of fixed and immunostained cryosections from dorsal inoculum of CSs from MLC3F-nlacZ/CD-1 and cTnI-lacZ/CD-1 mice. Tubular structures were stained for sarcomeric myosin (b3 to b5) and cTnI (b4 through b6). X-Gal staining labeled the cells within and those migrating from CS (b2). Endothelial markers (SMA and vascular endothelialcadherin) stained the vasculature (black holes) (a3 and b1).

To test the acquisition of functional competence and the cardiac regenerative potential of the CSs when challenged into an infarcted myocardium, orthotopic transplantation experiments with human CSs were performed. To perform these, thawed (cryopreserved) adult human CSs from three atrial (one male and two female) and one ventricular (one female) biopsy specimens were injected into the viable myocardium bordering a freshly produced infarct. Each mouse received CSs from a single passage of an explant (derived from a single subject). Four control infarcted animals were injected with an equal volume of PBS. Eighteen days after the intervention, the animals were euthanized and infarct size was determined. Infarct size was 34.97.1 (SEM, 3.6) and 31.96.9 (SEM, 3.5) in the CS-treated group and PBS-injected group, respectively (P=NS). However, echocardiography showed better preservation of the infarcted anterior wall thickness in the CS-treated group compared with the PBS-injected group (0.800.29 [SEM, 0.15] versus 0.600.20 [SEM, 0.08]) (P=NS), particularly of percent fractional shortening (36.8516.43 [SEM, 8.21] versus 17.875.95 [SEM, 2.43]) (P<0.05) (Figure 6 and the Table).

Figure 6. In vivo analysis (orthotopic transplantation of human CSs). Orthotopic transplantation performed in a SCID-beige mouse. Cryopreserved human CSs were transplanted into the viable myocardium bordering a freshly produced infarct. Confocal analysis of cryosectioned left ventricular heart 18 days after the coronary ligature shows that (a) cardiomyocytes expressing MHC (red) in the regenerating myocardium (particularly those indicated by the two central arrows) also stain positive for lamin A/C (green) (a specific human nuclear marker). In these cells, MHC expression is evident mainly in the perinuclear area (see Supplementary Figure X). Lamin A/C-labeled cells (red) are present in newly generated capillaries staining for -SMA (b1 through d), and platelet endothelial cell adhesion molecule (c). d, Confocal analysis of colocalization of lamin A/C-labeled cells (red) with the newly generated capillaries staining for -smooth muscle actin. e, Low-magnification image shows viable lamin A/C-expressing cells (green) in regenerating myocardium expressing MHC (red).

Myocardial Repair (Echocardiography)

At the time of evaluation, bands of regenerating myocardium were present (with different degrees of organization and thickness) throughout most of the infarcted areas, as evaluated with hematoxylineosin histochemistry (data not shown) and MHC immunofluorescence (Supplementary Figure IXa1 and IXa2). In the regenerating myocardium, cells expressing lamin A/C (a specific human nuclear marker) also colocalize with cardiomyocytes stained positive for MHC (Figure 6a and 6e and Supplementary Figures IXb1, IXb2, and X), newly generated capillaries stained for -SMA (Figure 6b1, 6b2, and 6d) and platelet endothelial cell adhesion molecule (Figure 6c), and with connexin-43expressing cells (data not shown).

CSs appear to be a mixture of cardiac stem cells, differentiating progenitors, and even spontaneously differentiated cardiomyocytes. Vascular cells were also present, depending on the size of the sphere and time in culture. It is possible that, as for neurospheres,19 differentiating/differentiated cells stop dividing and/or die, whereas stem cells continue to proliferate in an apparently asymmetric way, giving rise to many secondary spheres and to exponential growth in vitro. Mechanical dissociation favors this process. Death, differentiation, and responsiveness to growth factors of the different cells within the CSs could depend on the three-dimensional architecture and on localization within the CSs.20 The spontaneous formation of spheres is a known prerogative of neural stem cells, some tumor cell lines (LIM),21 endothelial cells,22 and fetal chicken cardiomyocytes.23 All these models (ours included) that mimic the true three-dimensional architecture of tissues consist of spheroids of aggregated cells that develop a two-compartment system composed of a surface layer of differentiated cells and a core of unorganized cells that first proliferate and then disappear over time (perhaps through apoptotic cell death). As well-documented in fetal chick cardiomyocytes and endothelial cell spheroid culture, three-dimensional structure affects the sensitivity of cells to survival and growth factors.21,22 In particular, central spheroid cells do not differentiate and are dependent on survival factors to prevent apoptosis, whereas the cells of the surface layer seem to differentiate beyond the degree that can be obtained in two-dimensional culture and become independent of the activity of survival factors.23 Furthermore, cellcell contact and membrane-associated factors, known to be important for the division of neural precursor cells,24 could be involved in our system. This is in accordance with the notion that stem cells (or cells with stem cell function) will only retain their pluripotency within an appropriate environment, as suggested by the niche hypothesis.25

Thus CSs can be considered clones of adult stem cells, maintaining their functional properties in vitro and in vivo after cryopreservation.

While the experiments performed for this article were ongoing, two articles were published concerning the isolation of cardiac stem cells or progenitor cells from adult mammalian hearts.26,27 Isolation of these cells was based exclusively on the expression of a stem cell-related surface antigen: c-kit in the first article and Sca-1 in the second one. In the first study,26 freshly isolated c-kit+ Lin cells from rat hearts were found to be self-renewing, clonogenic, and multi-potent, exhibiting biochemical differentiation into the myogenic cell, smooth muscle cell, or endothelial cell lineage but failing to contract spontaneously. When injected into an ischemic heart, these cells regenerated functional myocardium. In the second study,27 Sca-1+ cKit cells from mice hearts were induced in vitro to differentiate toward the cardiac myogenic lineage in response to 5-azacytidine. When given intravenously after ischemia/reperfusion, these cells targeted injured myocardium and differentiated into cardiomyocytes, with and without fusion with the host cells. Our data obtained on GFP-cKit transgenic mice also suggest that the adult cardiac stem cell is cKit+. It is possible that CSs enclose a mixed population of cells that, as in the niche, could promote the viability of cKit progenitors and contribute to their proliferation. The data obtained in the present article confirm the existence of adult cardiac stem cells/progenitor cells. More importantly, they demonstrate for the first time to our knowledge that it is possible to isolate cells from very small fragments of human myocardium and expand these cells in vitro many-fold (reaching numbers that would be appropriate for in vivo transplantation in patients) without losing their differentiation potential. Previously unforeseen opportunities for myocardial repair could now be identified.

Excerpt from:
Isolation and Expansion of Adult Cardiac Stem Cells From ...