Archive for the ‘IPS Cell Therapy’ Category

Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3'-untranslated region (3' UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.Molecular Therapy (2016); doi:10.1038/mt.2016.97.

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Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for ...

Research is ongoing in Japan and overseas with the aim of realizing cell transplantation therapy using iPS cells. One safety issue of concern is the risk of tumor formation. CiRA in particular has focused its resources on this issue.

Broadly speaking, there are two main theories as to the mechanism whereby iPS cells may form tumors. One theory is that iPS cells form tumors in response either to reactivation of the reprogramming factors inserted into the cell or through damage caused to the original cell genome through the artificial insertion of the reprogramming factors. In response, a search was launched for optimal reprogramming factors which do not cause reactivation, and a method of generating iPS cells was developed in which reprogramming factors are not incorporated into the cell chromosomes and damage to the host genome is therefore avoided.

The other theory is that residues of undifferentiated cells - cells which have not successfully completed differentiation to the target cell type - or other factors lead to the formation of teratomas, a kind of benign tumor. This theory requires research on iPS cell proliferation and differentiation.

1. Search for optimal reprogramming factors When Professor Shinya Yamanaka and his research team announced the successful generation of mouse iPS cells, one of the reprogramming factors they used was c-Myc, which is known to be an oncogene, that is a cancer-causing gene. There have been suggestions that this gene may be activated within the cell and cause a tumor to form. However, in 2010, CiRA Lecturer Masato Nakagawa and his team reported that L-Myc was a promising replacement factor for c-Myc. iPS cells created using L-Myc not only display almost no tumor formation, they also have a high rate of successful generation and a high degree of pluripotency.

2. Search for optimal vectors When the reprogramming factors required to generate iPS cells were inserted into the cells of the skin or other body tissues, early methods employed a retrovirus or lentivirus as a "vector," or carrier. In these methods, the target genes are inserted into the viruses with the which the cells were then infected in order to deliver the target genes. When a retrovirus or lentivirus is used as a vector, however, the viruses are incorporated into the cells genomic DNA in a random fashion. This may cause some of the cells original genes to be lost, or in other cases activated, resulting in a risk of cancerous changes.

In 2008, to remedy this risk, CiRA Lecturer Keisuke Okita and his team explored the use of a circular DNA fragment known as a plasmid, which is not incorporated into the cell chromosome, as a substitute to the retrovirus or lentivirus methods. In this way, they developed a method of generating iPS cells in which the reprogramming factors are not incorporated into the cell chromosome. In 2011, Okita and his team further improved the efficiency generation by introducing into a self-replicating episomal plasmid six factors - OCT3/4, SOX2, KLF4, LIN28, L-MYC, and p53shRNA.

3. Establishing a method for generating and screening safe cells Once iPS cells have been induced to differentiate into the target somatic cells using the appropriate genes and gene insertion methods as explained above, the differentiated cells can be relied upon not to revert to the undifferentiated state. However, there may sometimes be a residue of undifferentiated cells which have not completed the process of differentiation into the target cells, and it is possible that these cells, however few, may form a tumor. Scientists had already established that different iPS cell lines, even if generated from the same individual using the same method, might nevertheless display differences in proliferation and differentiation potentials. This meant that, if iPS cells with low differentiation potential were used, there was a risk that a residue of cells in the cell group might fail to fully differentiate and result in the formation of a teratoma. In 2013, a team led by CiRA Lecturer Kazutoshi Takahashi and Dr. Michiyo Aoi, now an assistant professor at Kobe University, developed a simple method to screen for iPS cell lines that have high potential to differentiate into nerve cells. There is also a risk of tumorigenesis from genomic or other damage arising at the iPS cell generation stage or at the subsequent culture stage. CiRA Assistant Professor Akira Watanabe and his team have developed a sensitive method to detect genomic and other damage in iPS cells using the latest equipment.

4. Developing a reliable method of differentiation into the target cell type In cell transplantation therapy, iPS cells are not transplanted directly into the human body. Instead, cells are transplanted after first being differentiated into the target cell type. It is therefore important to develop a reliable method of inducing iPS cells to differentiate into the target cell type. CiRA is currently working to develop technology for differentiation into a range of different cell types from iPS cells. CiRA Professor Jun Takahashi and his team have developed a highly efficient method of inducing iPS cells to differentiate into dopamine-producing nerve cells. In 2014, CiRA Professor Koji Eto and his team reported a method of producing platelets from iPS cells that is both reliable and can yield high volumes. These findings represent a major step toward iPS cell-based regenerative medicine for nerve diseases such as Parkinsons disease and blood diseases such as aplastic anemia.

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Our Associate Medical Director, Professor Jeremy Pearson,discusses the parallels between todays news about treating paralysis and our hopes for mending broken hearts.

21 October 2014

This morning I woke to the news that a paralysed man could walk again. A medical miracle had been performed thanks to laboratory and clinical research. But when you break down the scientific journey thats got us to this point, you realise it isnt a miracle at all but decades of dedication and excellent science. This is the journey our funded researchers are on now as they work towards repairing and regenerating hearts damaged by heart attack.

A University College London (UCL) researcher, Professor Geoffrey Raisman, has been the driving force behind the paralysis breakthrough. Back in 1985 he discovered special cells in the nose that have a unique ability for allowing new nerve cells to grow. Almost three decades later, after developing a technique through studies in rats, we now have a potential treatment to regenerate a severed spinal cord.

This breakthrough is an excellent example of how persistence pays off in medical research. Laboratory science youre helping us to fund now could become a patient treatment in the future but the researchers need time and they need continued funding.

Professor Raisman was searching for a solution to a problem that seemed unsolvable something that our funded researchers can relate to. Right now, once a heart is damaged, like the spinal cord, it cannot be repaired. The heart doesnt spontaneously repair itself. A damaged heart cant pump blood around the body as well as it should, which can lead to heart failure. Heart failure can be severely disabling and prevent people carrying out basic tasks like going to the shops or washing without becoming totally exhausted.

Right now a BHF Professor Paul Riley(pictured) is moving us closer to a solving our unsolvable problem. In 2011, while at UCL, Professor Riley showed in mice how heart muscle can be regenerated in the adult heart after damage. Now at Oxford, he and his team are further investigating the outer layer of the heart, where these regenerative heart cells lie. We hope this work will eventually lead to a treatment that could be given to people after a heart attack to trigger the repair of any damage and prevent heart failure.

Due to difficulties in securing funding Professor Raismans progress was perhaps delayed by many years. With your support we hope to accelerate the progress that Professor Riley and his fellow researchers are making. We have already committed to funding 7.5 million across three Centres of Regenerative Medicine, one led by Professor Riley, that bring top researchers together with a common aim of repairing damaged heart muscle and blood vessels. And now we hope to raise a further 10 million towards a dedicated regenerative medicine facility for Professor Riley and his colleagues at Oxford.

This facility, called the Institute of Developmental and Regenerative Medicine, will bring experts from three separate disciplines under one roof where they can share facilities, ideas and resources making new treatments a reality much sooner.

Continue reading here: Regenerative medicine

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Regenerative medicine IPS Cell Therapy IPS Cell Therapy

DNA damage resulting in multiple broken chromosomes

DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day.[1] Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cells ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cells genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages (interstrand crosslinks or ICLs).[2][3]

The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:

The DNA repair ability of a cell is vital to the integrity of its genome and thus to the normal functionality of that organism. Many genes that were initially shown to influence life span have turned out to be involved in DNA damage repair and protection.[4]

The 2015 Nobel Prize in Chemistry was awarded to Tomas Lindahl, Paul Modrich, and Aziz Sancar for their work on the molecular mechanisms of DNA repair processes.[5][6]

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day.[1] While this constitutes only 0.000165% of the human genomes approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cells ability to carry out its function and appreciably increase the likelihood of tumor formation and contribute to tumour heterogeneity.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around packaging proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

DNA damage can be subdivided into two main types:

The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable (except in the rare case of a back mutation, for example, through gene conversion).

There are several types of damage to DNA due to endogenous cellular processes:

Damage caused by exogenous agents comes in many forms. Some examples are:

UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift.

In human cells, and eukaryotic cells in general, DNA is found in two cellular locations inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of eukaryotic cells.

Senescence, an irreversible process in which the cell no longer divides, is a protective response to the shortening of the chromosome ends. The telomeres are long regions of repetitive noncoding DNA that cap chromosomes and undergo partial degradation each time a cell undergoes division (see Hayflick limit).[10] In contrast, quiescence is a reversible state of cellular dormancy that is unrelated to genome damage (see cell cycle). Senescence in cells may serve as a functional alternative to apoptosis in cases where the physical presence of a cell for spatial reasons is required by the organism,[11] which serves as a last resort mechanism to prevent a cell with damaged DNA from replicating inappropriately in the absence of pro-growth cellular signaling. Unregulated cell division can lead to the formation of a tumor (see cancer), which is potentially lethal to an organism. Therefore, the induction of senescence and apoptosis is considered to be part of a strategy of protection against cancer.[12]

It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA damages and mutation are fundamentally different. Damages are physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damages can be recognized by enzymes, and, thus, they can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. If a cell retains DNA damage, transcription of a gene can be prevented, and, thus, translation into a protein will also be blocked. Replication may also be blocked or the cell may die.

In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and, thus, a mutation cannot be repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce. Although distinctly different from each other, DNA damages and mutations are related because DNA damages often cause errors of DNA synthesis during replication or repair; these errors are a major source of mutation.

Given these properties of DNA damage and mutation, it can be seen that DNA damages are a special problem in non-dividing or slowly dividing cells, where unrepaired damages will tend to accumulate over time. On the other hand, in rapidly dividing cells, unrepaired DNA damages that do not kill the cell by blocking replication will tend to cause replication errors and thus mutation. The great majority of mutations that are not neutral in their effect are deleterious to a cells survival. Thus, in a population of cells composing a tissue with replicating cells, mutant cells will tend to be lost. However, infrequent mutations that provide a survival advantage will tend to clonally expand at the expense of neighboring cells in the tissue. This advantage to the cell is disadvantageous to the whole organism, because such mutant cells can give rise to cancer. Thus, DNA damages in frequently dividing cells, because they give rise to mutations, are a prominent cause of cancer. In contrast, DNA damages in infrequently dividing cells are likely a prominent cause of aging.[13]

Single-strand and double-strand DNA damage

Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when non-essential genes are missing or damaged). Depending on the type of damage inflicted on the DNAs double helical structure, a variety of repair strategies have evolved to restore lost information. If possible, cells use the unmodified complementary strand of the DNA or the sister chromatid as a template to recover the original information. Without access to a template, cells use an error-prone recovery mechanism known as translesion synthesis as a last resort.

Damage to DNA alters the spatial configuration of the helix, and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules bind at or near the site of damage, inducing other molecules to bind and form a complex that enables the actual repair to take place.

Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can occur in only one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone. The formation of pyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue/UV light (300500nm wavelength) to promote catalysis.[14] Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans,[15] who instead use nucleotide excision repair to repair damage from UV irradiation. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called ogt. This is an expensive process because each MGMT molecule can be used only once; that is, the reaction is stoichiometric rather than catalytic.[16] A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes.[17] The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.[16]

Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination.[16] PVN Acharya noted that double-strand breaks and a cross-linkage joining both strands at the same point is irreparable because neither strand can then serve as a template for repair. The cell will die in the next mitosis or in some rare instances, mutate.[2][3]

In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the cofactor XRCC4, directly joins the two ends.[21] To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate.[22][23][24][25] NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms insertions or translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are backup NHEJ pathways in higher eukaryotes.[26] Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system.[27]

MMEJ starts with short-range end resection by MRE11 nuclease on either side of a double-strand break to reveal microhomology regions.[28] In further steps,[29] PARP1 is required and may be an early step in MMEJ. There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps. This is followed by recruitment of XRCC1LIG3 to the site for ligating the DNA ends, leading to an intact DNA.

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination (HR) and non-homologous end joining (NHEJ).[30] In an in vitro system, MMEJ occurred in mammalian cells at the levels of 1020% of HR when both HR and NHEJ mechanisms were also available.[28] MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair.[31]

Homologous recombination requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination.

Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNAs state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them.

A team of French researchers bombarded Deinococcus radiodurans to study the mechanism of double-strand break DNA repair in that bacterium. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination.[32]

Translesion synthesis (TLS) is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites.[33] It involves switching out regular DNA polymerases for specialized translesion polymerases (i.e. DNA polymerase IV or V, from the Y Polymerase family), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) on undamaged templates relative to regular polymerases. However, many are extremely efficient at inserting correct bases opposite specific types of damage. For example, Pol mediates error-free bypass of lesions induced by UV irradiation, whereas Pol introduces mutations at these sites. Pol is known to add the first adenine across the T^T photodimer using Watson-Crick base pairing and the second adenine will be added in its syn conformation using Hoogsteen base pairing. From a cellular perspective, risking the introduction of point mutations during translesion synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross chromosomal aberrations or cell death. In short, the process involves specialized polymerases either bypassing or repairing lesions at locations of stalled DNA replication. For example, Human DNA polymerase eta can bypass complex DNA lesions like guanine-thymine intra-strand crosslink, G[8,5-Me]T, although can cause targeted and semi-targeted mutations.[34] Paromita Raychaudhury and Ashis Basu[35] studied the toxicity and mutagenesis of the same lesion in Escherichia coli by replicating a G[8,5-Me]T-modified plasmid in E. coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these specialized DNA polymerases. A bypass platform is provided to these polymerases by Proliferating cell nuclear antigen (PCNA). Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site of lesion, PCNA is ubiquitinated, or modified, by the RAD6/RAD18 proteins to provide a platform for the specialized polymerases to bypass the lesion and resume DNA replication.[36][37] After translesion synthesis, extension is required. This extension can be carried out by a replicative polymerase if the TLS is error-free, as in the case of Pol , yet if TLS results in a mismatch, a specialized polymerase is needed to extend it; Pol . Pol is unique in that it can extend terminal mismatches, whereas more processive polymerases cannot. So when a lesion is encountered, the replication fork will stall, PCNA will switch from a processive polymerase to a TLS polymerase such as Pol to fix the lesion, then PCNA may switch to Pol to extend the mismatch, and last PCNA will switch to the processive polymerase to continue replication.

Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to acquire multiple sites of bulky DNA lesions and double-strand breaks. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage.[38] The global response to damage is an act directed toward the cells own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.

After DNA damage, cell cycle checkpoints are activated. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure,[39] whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified.[40] These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.

DNA damage checkpoint is a signal transduction pathway that blocks cell cycle progression in G1, G2 and metaphase and slows down the rate of S phase progression when DNA is damaged. It leads to a pause in cell cycle allowing the cell time to repair the damage before continuing to divide.

Checkpoint Proteins can be separated into four groups: phosphatidylinositol 3-kinase (PI3K)-like protein kinase, proliferating cell nuclear antigen (PCNA)-like group, two serine/threonine(S/T) kinases and their adaptors. Central to all DNA damage induced checkpoints responses is a pair of large protein kinases belonging to the first group of PI3K-like protein kinases-the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia- and Rad-related) kinases, whose sequence and functions have been well conserved in evolution. All DNA damage response requires either ATM or ATR because they have the ability to bind to the chromosomes at the site of DNA damage, together with accessory proteins that are platforms on which DNA damage response components and DNA repair complexes can be assembled.

An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA damage.[41] The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and p53-independent mechanisms and can arrest the cell cycle at the G1/S and G2/M checkpoints by deactivating cyclin/cyclin-dependent kinase complexes.[42]

The SOS response is the changes in gene expression in Escherichia coli and other bacteria in response to extensive DNA damage. The prokaryotic SOS system is regulated by two key proteins: LexA and RecA. The LexA homodimer is a transcriptional repressor that binds to operator sequences commonly referred to as SOS boxes. In Escherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and recA genes.[43] The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like the Spirochetes.[44] The most common cellular signals activating the SOS response are regions of single-stranded DNA (ssDNA), arising from stalled replication forks or double-strand breaks, which are processed by DNA helicase to separate the two DNA strands.[38] In the initiation step, RecA protein binds to ssDNA in an ATP hydrolysis driven reaction creating RecAssDNA filaments. RecAssDNA filaments activate LexA autoprotease activity, which ultimately leads to cleavage of LexA dimer and subsequent LexA degradation. The loss of LexA repressor induces transcription of the SOS genes and allows for further signal induction, inhibition of cell division and an increase in levels of proteins responsible for damage processing.

In Escherichia coli, SOS boxes are 20-nucleotide long sequences near promoters with palindromic structure and a high degree of sequence conservation. In other classes and phyla, the sequence of SOS boxes varies considerably, with different length and composition, but it is always highly conserved and one of the strongest short signals in the genome.[44] The high information content of SOS boxes permits differential binding of LexA to different promoters and allows for timing of the SOS response. The lesion repair genes are induced at the beginning of SOS response. The error-prone translesion polymerases, for example, UmuCD2 (also called DNA polymerase V), are induced later on as a last resort.[45] Once the DNA damage is repaired or bypassed using polymerases or through recombination, the amount of single-stranded DNA in cells is decreased, lowering the amounts of RecA filaments decreases cleavage activity of LexA homodimer, which then binds to the SOS boxes near promoters and restores normal gene expression.

Eukaryotic cells exposed to DNA damaging agents also activate important defensive pathways by inducing multiple proteins involved in DNA repair, cell cycle checkpoint control, protein trafficking and degradation. Such genome wide transcriptional response is very complex and tightly regulated, thus allowing coordinated global response to damage. Exposure of yeast Saccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct transcriptional profiles. Similarities to environmental shock response indicates that a general global stress response pathway exist at the level of transcriptional activation. In contrast, different human cell types respond to damage differently indicating an absence of a common global response. The probable explanation for this difference between yeast and human cells may be in the heterogeneity of mammalian cells. In an animal different types of cells are distributed among different organs that have evolved different sensitivities to DNA damage.[46]

In general global response to DNA damage involves expression of multiple genes responsible for postreplication repair, homologous recombination, nucleotide excision repair, DNA damage checkpoint, global transcriptional activation, genes controlling mRNA decay, and many others. A large amount of damage to a cell leaves it with an important decision: undergo apoptosis and die, or survive at the cost of living with a modified genome. An increase in tolerance to damage can lead to an increased rate of survival that will allow a greater accumulation of mutations. Yeast Rev1 and human polymerase are members of [Y family translesion DNA polymerases present during global response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in eukaryotes.[38]

DNA repair rate is an important determinant of cell pathology

Experimental animals with genetic deficiencies in DNA repair often show decreased life span and increased cancer incidence.[13] For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms get lymphoma and infections more often, and, as a consequence, have shorter lifespans than wild-type mice.[47] In similar manner, mice deficient in a key repair and transcription protein that unwinds DNA helices have premature onset of aging-related diseases and consequent shortening of lifespan.[48] However, not every DNA repair deficiency creates exactly the predicted effects; mice deficient in the NER pathway exhibited shortened life span without correspondingly higher rates of mutation.[49]

If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging,[13] increased sensitivity to carcinogens, and correspondingly increased cancer risk (see below). On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ.[50]

Most life span influencing genes affect the rate of DNA damage

A number of individual genes have been identified as influencing variations in life span within a population of organisms. The effects of these genes is strongly dependent on the environment, in particular, on the organisms diet. Caloric restriction reproducibly results in extended lifespan in a variety of organisms, likely via nutrient sensing pathways and decreased metabolic rate. The molecular mechanisms by which such restriction results in lengthened lifespan are as yet unclear (see[51] for some discussion); however, the behavior of many genes known to be involved in DNA repair is altered under conditions of caloric restriction.

For example, increasing the gene dosage of the gene SIR-2, which regulates DNA packaging in the nematode worm Caenorhabditis elegans, can significantly extend lifespan.[52] The mammalian homolog of SIR-2 is known to induce downstream DNA repair factors involved in NHEJ, an activity that is especially promoted under conditions of caloric restriction.[53] Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents,[54] although similar effects have not been observed in mitochondrial DNA.[55]

It is interesting to note that the C. elegans gene AGE-1, an upstream effector of DNA repair pathways, confers dramatically extended life span under free-feeding conditions but leads to a decrease in reproductive fitness under conditions of caloric restriction.[56] This observation supports the pleiotropy theory of the biological origins of aging, which suggests that genes conferring a large survival advantage early in life will be selected for even if they carry a corresponding disadvantage late in life.

Defects in the NER mechanism are responsible for several genetic disorders, including:

Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:

All of the above diseases are often called segmental progerias (accelerated aging diseases) because their victims appear elderly and suffer from aging-related diseases at an abnormally young age, while not manifesting all the symptoms of old age.

Other diseases associated with reduced DNA repair function include Fanconi anemia, hereditary breast cancer and hereditary colon cancer.

Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer.[57][58] There are at least 34 Inherited human DNA repair gene mutations that increase cancer risk. Many of these mutations cause DNA repair to be less effective than normal. In particular, Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. BRCA1 and BRCA2, two famous genes whose mutations confer a hugely increased risk of breast cancer on carriers, are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.

Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side-effect is that other non-cancerous but rapidly dividing cells such as progenitor cells in the gut, skin, and hematopoietic system are also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body).

Classically, cancer has been viewed as a set of diseases that are driven by progressive genetic abnormalities that include mutations in tumour-suppressor genes and oncogenes, and chromosomal aberrations. However, it has become apparent that cancer is also driven by epigenetic alterations.[59]

Epigenetic alterations refer to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications are changes in DNA methylation (hypermethylation and hypomethylation) and histone modification,[60] changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1)[61] and changes caused by microRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlying DNA sequence. These changes usually remain through cell divisions, last for multiple cell generations, and can be considered to be epimutations (equivalent to mutations).

While large numbers of epigenetic alterations are found in cancers, the epigenetic alterations in DNA repair genes, causing reduced expression of DNA repair proteins, appear to be particularly important. Such alterations are thought to occur early in progression to cancer and to be a likely cause of the genetic instability characteristic of cancers.[62][63][64][65]

Reduced expression of DNA repair genes causes deficient DNA repair. When DNA repair is deficient DNA damages remain in cells at a higher than usual level and these excess damages cause increased frequencies of mutation or epimutation. Mutation rates increase substantially in cells defective in DNA mismatch repair[66][67] or in homologous recombinational repair (HRR).[68] Chromosomal rearrangements and aneuploidy also increase in HRR defective cells.[69]

Higher levels of DNA damage not only cause increased mutation, but also cause increased epimutation. During repair of DNA double strand breaks, or repair of other DNA damages, incompletely cleared sites of repair can cause epigenetic gene silencing.[70][71]

Deficient expression of DNA repair proteins due to an inherited mutation can cause increased risk of cancer. Individuals with an inherited impairment in any of 34 DNA repair genes (see article DNA repair-deficiency disorder) have an increased risk of cancer, with some defects causing up to a 100% lifetime chance of cancer (e.g. p53 mutations).[72] However, such germline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers.[73]

Deficiencies in DNA repair enzymes are occasionally caused by a newly arising somatic mutation in a DNA repair gene, but are much more frequently caused by epigenetic alterations that reduce or silence expression of DNA repair genes. For example, when 113 colorectal cancers were examined in sequence, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration).[74] Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region.[75][76][77][78][79]

Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, PMS2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1).[80] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.[81]

In further examples (tabulated in Cancer epigenetics), epigenetic defects were found at frequencies of between 13%-100% for the DNA repair genes BRCA1, WRN, FANCB, FANCF, MGMT, MLH1, MSH2, MSH4, ERCC1, XPF, NEIL1 and ATM. These epigenetic defects occurred in various cancers (e.g. breast, ovarian, colorectal and head and neck). Two or three deficiencies in the expression of ERCC1, XPF or PMS2 occur simultaneously in the majority of the 49 colon cancers evaluated by Facista et al.[82]

The chart in this section shows some frequent DNA damaging agents, examples of DNA lesions they cause, and the pathways that deal with these DNA damages. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes.[83] Of these, 83 are directly employed in the 5 types of DNA repair processes illustrated in the chart. The more well studied genes central to these repair processes are also shown in the chart. As indicated by the DNA repair genes shown in red, many of the genes in these repair pathways are regulated by epigenetic mechanisms, and these are frequently reduced or silent in various cancers (marked by an asterisk). Two review articles,[65][84] and two broad experimental survey articles[85][86] document most of these epigenetic DNA repair deficiencies.

It appears that epigenetic repression of DNA repair genes in accurate DNA repair pathways are central to carcinogenesis. However microhomology-mediated end joining (MMEJ) is an additional error-prone repair pathway for double-strand breaks. In MMEJ repair of a double-strand break, an homology of 5 25 complementary base pairs on both strands is identified and used as a basis to align the strands, but with mismatched ends. MMEJ removes extra nucleotides (flaps) where strands are joined, then ligates the strands to create an intact DNA double helix. MMEJ always involves at least a small deletion, so that it is a mutagenic pathway.[30]FEN1, the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast,[87] prostate,[88] stomach,[89][90] neuroblastomas,[91] pancreatic,[92] and lung.[93] Other genes in the MMEJ pathway are also over-expressed in a number of cancers (see MMEJ for summary), and are shown in cyan (blue) in the chart in this section.

The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophage (viruses that infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.[94] The ability of a large number of protein structural motifs to catalyze relevant chemical reactions has played a significant role in the elaboration of repair mechanisms during evolution. For an extremely detailed review of hypotheses relating to the evolution of DNA repair, see.[95]

The fossil record indicates that single-cell life began to proliferate on the planet at some point during the Precambrian period, although exactly when recognizably modern life first emerged is unclear. Nucleic acids became the sole and universal means of encoding genetic information, requiring DNA repair mechanisms that in their basic form have been inherited by all extant life forms from their common ancestor. The emergence of Earths oxygen-rich atmosphere (known as the oxygen catastrophe) due to photosynthetic organisms, as well as the presence of potentially damaging free radicals in the cell due to oxidative phosphorylation, necessitated the evolution of DNA repair mechanisms that act specifically to counter the types of damage induced by oxidative stress.

On some occasions, DNA damage is not repaired, or is repaired by an error-prone mechanism that results in a change from the original sequence. When this occurs, mutations may propagate into the genomes of the cells progeny. Should such an event occur in a germ line cell that will eventually produce a gamete, the mutation has the potential to be passed on to the organisms offspring. The rate of evolution in a particular species (or, in a particular gene) is a function of the rate of mutation. As a consequence, the rate and accuracy of DNA repair mechanisms have an influence over the process of evolutionary change.[96] Since the normal adaptation of populations of organisms to changing circumstances (for instance the adaptation of the beaks of a population of finches to the changing presence of hard seeds or insects) proceeds by gene regulation and the recombination and selection of gene variations alleles and not by passing on irreparable DNA damages to the offspring,[97] DNA damage protection and repair does not influence the rate of adaptation by gene regulation and by recombination and selection of alleles. On the other hand, DNA damage repair and protection does influence the rate of accumulation of irreparable, advantageous, code expanding, inheritable mutations, and slows down the evolutionary mechanism for expansion of the genome of organisms with new functionalities. The tension between evolvability and mutation repair and protection needs further investigation.

A technology named clustered regularly interspaced short palindromic repeat shortened to CRISPR-Cas9 was discovered in 2012. The new technology allows anyone with molecular biology training to alter the genes of any species with precision.[98]

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DNA repair Wikipedia, the free encyclopedia IPS Cell ...

In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinsons disease and Alzheimers disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.

The 2012 Nobel Prize in Physiology or Medicine was awarded for The discovery that mature cells can be reprogrammed to become pluripotent. First, we would like to consider the significance of this research. The lives of mammals, including humans, begin with the fertilization of an egg by a sperm cell. In humans, a blastocyst composed of 70-100 cells forms by approximately 5.5 days after fertilization. The blastocyst is composed of the inner cell mass, the cell population that has the ability to differentiate into the various cells that constitute the body (pluripotency), and the trophoblast, the cells that develop into the placenta and extra-embryonic tissues and do not contribute cells to the body. In the embryonic stage, the pluripotent cells of the inner cell mass differentiate into the three germ layers, endoderm, mesoderm, and ectoderm, which will form specific organs and tissues containing somatic stem cells with limited differentiation potencies. These somatic stem cells continue to divide and differentiate, and, by adulthood, an individual composed of 60 trillion cells is produced. Somatic stem cells born in the fetal period actively divide, and are involved in the formation and growth of various organs. However, even in the adult, somatic stem cells persist in niches in every organ and tissue, and play an important role in maintaining organ and tissue homeostasis. When cells in the inner cell mass are removed at the blastocyst stage and cultured in vitro, pluripotent embryonic stem (ES) cells are obtained. Thus, in the normal process of development, cell differentiation of the three germ layers proceeds from the simple stages of the fertilized egg and blastocyst, and ultimately produces an individual consisting of a complex cellular society.

In 1893, August Weismann argued that only germ cells (eggs and spermatozoa) maintain a determinant, which was described as heritable information essential to decide on the functions and features of all somatic cells in the body [1]. In his germ plasm theory, the determinants are lost or irreversibly inactivated in differentiated somatic cells.

It took more than 50 years for researchers to rewrite this dogma. In 1962, Sir John Gurdon demonstrated the acquisition of pluripotency by reprogramming cells to their initial stage using a novel research technique, i.e., producing cloned individuals by transferring somatic cell nuclei into eggs [2]. However, for many years, that result was regarded as a special case limited to frogs alone. The production of Dolly the sheep by transferring the nucleus of a somatic cell (mammary gland epithelial cell) by Sir Ian Wilmut in the late 1990s [3] showed that cloning could also be applied to mammals.

These brilliant previous works led to our studies that culminated in the induction of pluripotency in mouse somatic cells in 2006, using retroviral vectors to introduce four genes that encode transcription factors i.e., Oct4, Sox2, Klf4, and c-Myc. We designated these cells as iPS cells [4]. In 2007, we succeeded in generating human iPS cells using genes encoding the same four transcription factors [1]. The results of this research showed that although the developmental process was thought to be irreversible, by introducing key genes into differentiated adult cells the cells could be reset to a state in the extremely early stage of development in which they possessed pluripotency. That is, the results demonstrated that the differentiation process was reversible. This startling discovery made it necessary to rewrite the embryology textbooks.

Three major lines of research led us to the production of iPS cells [

] (Figure

). The first, as described above, was nuclear reprogramming initiated by Sir John Gurdon in his research of cloning frogs by nuclear transfer in 1962 [

] and by Sir Ian Wilmut, who cloned a mammal for the first time in 1997 [

]. In addition, Takashi Tada showed that mouse ES cells contain factors that induce reprogramming in 2001 [

]. The second line of research was factor-mediated cell fate conversion, initiated by Harold Weintraub, who showed that fibroblasts can be converted into the muscle lineage by transduction with the

gene, which encodes a muscle lineage-specific basic helix-loop-helix transcription factor in 1987 [

]. The third line of research was the development of mouse ES cells, initiated by Sir Martin Evans and Gail Martin in 1981 [

,

]. Austin Smith established culture conditions for mouse ES cells and identified many factors essential for pluripotency including leukemia inhibitory factor (LIF) in 1988 [

]. Later, he developed the method to induce the ground state of mouse ES cell self-renewal using inhibitors for mitogen-activated protein kinase and glycogen synthase kinase 3 [

], which supports the establishment of fully reprogrammed mouse iPS cells. Furthermore, James Thomson generated human ES cells [

] and established their optimal culture conditions using fibroblast growth factor-2 (FGF-2). Without these previous studies, we could never have generated iPS cells. Interest rapidly escalated, and, in tandem with the birth of iPS cell technology, pluripotency leapt into the mainstream of life sciences research in the form of reprogramming technology [

]. However, there remain many unanswered questions regarding reprogramming technology. What are the reprogramming factors in the egg cytoplasm that are active in cloning technology? What do they have in common with the factors required to establish iPS cells and what are the differences? What kind of epigenetic changes occur in association with the reprogramming?

The history of investigations of cellular reprogramming that led to the development of iPS cells. Our generation of iPS cells in 2006 [4] became possible due to three scientific lines of investigation: 1) nuclear reprogramming, 2) factor-mediated cell fate conversion, and 3) ES cells. See the text for details (modified from Reference [5] with permission).

Apart from basic research in embryology, broad interest has been drawn to the following possible applications of iPS cell research: (1) regenerative medicine, including elucidating disease pathologies and drug discovery research using iPS cell disease models, and (2) medical treatments (Figure

). In this review, we describe these potential applications in the context of the results of our own research.

The application of iPS cell technologies to medical science. iPS cell technologies can be used for medical science including 1) cell therapies and 2) disease modeling or drug development. See the text for details.

iPS cells can be prepared from patients themselves and therefore great expectations have been placed on iPS cell technology because regenerative medicine can be implemented in the form of autografts presumably without any graft rejection reactions. Although there have been some controversies [

], the immunogenicity of terminally differentiated cells derived from iPS cells can be negligible [

]. Moreover, there has been substantial interest in the possibility of regenerative medicine without using the patients own cells; that is, using iPS cell stocks that have been established from donor somatic cells that are homozygous at the three major human leukocyte antigen (HLA) gene loci and match the patients HLA type [

]. The development of regenerative medicine using iPS cells is being pursued in Japan and the USA for the treatment of patients with retinal diseases, including age-related macular degeneration [

], spinal cord injuries [

], Parkinsons disease (PD) [

,

], corneal diseases [

], myocardial infarction [

,

], diseases that cause thrombocytopenia, including aplastic anemia and leukemia [

,

], as well as diseases such as multiple sclerosis (MS) and recessive dystrophic epidermolysis bullosa [

] (Table

).

Planned clinical trials of iPS cell-based therapies

Masayo Takahashi, (RIKEN)

Retinal Pigment Epithelium (sheet)

Age-related macular degeneration (wet type)

Alfred Lane, Anthony Oro, Marius Wernig (Stanford University)

Keratinocytes

Recessive dystrophic epidermolysis bullosa (RDEB)

Mahendra Rao (NIH)

DA neurons

Parkinsons disease

Koji Eto (Kyoto University)

Megakaryocyte

Thrombocytopenia

Jun Takahashi (Kyoto University)

DA neurons

Parkinsons disease

Steve Goldman, (University of Rochester)

Oligodendrocyte precursor cell

Multiple Sclerosis

Hideyuki Okano, Masaya Nakamura (Keio University)

Neural stem/progenitor cells

Spinal Cord Injury

Shigeto Shimmura (Keio University)

Corneal endothelial cells

Corneal endothelial dysfunction

Koji Nishida (Osaka University)

Corneal epithelial cells (sheet)

Corneal epithelial dysfunction and trauma (e.g. StevensJohnson syndrome)

Yoshiki Sawa (Osaka University)

Cardiomyocytes (sheet)

Heart Failure

Keiichi Fukuda (Keio University)

Cardiomyocytes (sphere)

Heart Failure

Yoshiki Sasai and Masayo Takahashi (RIKEN)

Neuroretinal sheet including photoreceptor cells

Retinitis pigmentosa

Advanced Cell Technology

Megakaryocytes

Refractory thrombocytopenia

In 1998, Hideyuki Okano, in collaboration with Steven Goldman, demonstrated for the first time the presence of neural stem/progenitor cells (NS/PCs) in the adult human brain using a neural stem cell marker, the ribonucleic acid (RNA)-binding protein Musashi1 [30, 31]. Research on nerve regeneration then commenced in earnest. That same year, we began regenerative medicine research on neural stem cell transplantation in a rat model of SCI, and have since made progress in developing NS/PC transplantation therapies in experiments on animal models of SCI. First, motor function was restored by transplanting rat fetal central nervous system (CNS)-derived NS/PCs into a rat SCI model [32]. The same study also showed that the sub-acute phase is the optimal time for NS/PC transplantation after SCI. In this study, at least part of the putative mechanism by which NS/PC transplantation restored function was identified in animal models of SCI. Both the cell autonomous effect (such as synaptogenesis between graft-derived neurons and host-derived neurons) and non-cell autonomous (trophic) effects mediated cytokines released from the graft-derived cells are likely contributing to tissue repair and functional recovery. Subsequently, a non-human primate SCI model was developed using the common marmoset, and motor function in that model was restored by transplanting human fetal CNS-derived stem cells [33]. In the same study, a behavioral assay for motor function associated with SCI was developed. Based on these studies, a preclinical research system for cell transplantation therapy was established in a non-human primate SCI model.

Given these findings, we began preparations for clinical studies of human fetal CNS-derived NS/PC transplantation to treat SCI patients. However, with the guidelines for clinical research on human stem cells of the Japanese Ministry of Health, Labor and Welfare that came into effect in 2006, human fetus-derived cells and ES cells became ineligible for use in regenerative medicine. Thus, we had no choice but to change our strategy (human ES cells became eligible for use in the 2013 guidelines). In 2006, one of our research groups (Yamanakas group) established iPS cells from adult mouse skin cells. Hypothesizing that it might be possible to induce NS/PCs from iPS cells, we (Okanos group) turned our attention to iPS cells as a means of obtaining NS/PCs without using fetal or ES cells. Based on conditions that were developed for experiments on mouse ES cells [34, 35], NS/PCs were induced from mouse iPS cells [36]. The following year, we succeeded in restoring motor function by transplanting these mouse iPS cell-derived NS/PCs into a mouse model of SCI, and reported that when good iPS cells -derived NS/PCs, which had been pre-evaluated as non-tumorigenic by the transplantation into the brains of immunocompromised mice, were used for transplantation, motor function was restored for a long period of time without tumors developing [37]. In 2011, we succeeded in restoring motor function by transplanting human iPS cell-derived stem cells into a mouse SCI model [38]. Moreover, in 2012, motor function was restored by transplanting human iPS (line 201B7) cell-derived NS/PCs into the marmoset SCI model, and long-term motor function was recovered without observable tumor formation [39]. This finding was of great significance in terms of preclinical research, and provided a proof of concept that could potentially lead to a treatment method.

Collectively, when mouse or human iPS cells were induced to form NS/PCs and were transplanted into mouse or non-human primate SCI models, long-term restoration of motor function was induced, without tumorigenicity, by selecting a suitable iPS cell line [17, 40]. Considering the sub-acute phase (2-4 weeks after the injury) as the optimal time for iPS cells-derived NS/PCs transplantation for SCI patients, there are following major difficulties with autograft-based cell therapy. First, it takes about a few months to establish iPS cells. Second, it also takes three months to induce them into NS/PCs in vitro. Third, one more year would be required for the quality control including their tumorigenesis.

Considering these, our collaborative team (Okano and Yamanaka laboratories) are currently planning iPS-based cell therapy for SCI patients in the sub-acute phase using clinical-grade integration-free human iPS cell lines that will be generated by Kyoto Universitys Center for iPS Cell Research and Application (CiRA). We will establish a production method, as well as a storage and management system, for human iPS cell-derived NS/PCs for use in clinical research for spinal cord regeneration, build an iPS cell-derived NS/PC stock for regenerative medicine, establish safety screenings against post-transplantation neoplastic transformation, and commence clinical research (Phase IIIa) trials for the treatment of sub-acute phase SCI (Figure

). As these studies progress, the application of iPS cells to treat chronic phase SCI and stroke will be investigated. Significant therapeutic efficacy in the treatment of chronic phase SCI has not been achieved by cell transplantation alone [

]. However, clinical studies are planned using antagonists of axon growth inhibitors, such as Semaphorin3A inhibitors [

], followed by multidisciplinary rehabilitation combination therapies. We aim to perform a clinical trial based on the Pharmaceutical Affairs Act in collaboration with drug companies and to use iPS cell-derived NS/PC stocks for regenerative medicine to establish treatment methods for stroke, MS, and Huntingtons disease.

Strategies for the development of iPS cell-based cell therapy for SCI patients. Our collaborative team (Okanos group at Keio University and Yamanakas group at Kyoto University) have been developing an iPS cell-based cell therapy for SCI since 2006. Our previous preclinical studies have shown that long-term functional restoration can be obtained by transplantation of NS/PCs derived from appropriate iPS cells clones without observable tumor formation [10]. Currently, we aim to develop iPS cells-based cell therapy for SCI patients at sub-acute phase using the clinical grade iPS cell-derived NS/PCs (i.e., the role of Okanos group described in the blue box) which have been prepared from human iPS cell stock (i.e., the role of Yamanakas group described in the yellow box).

Lesion sites are difficult to access in patients with degenerative diseases of the nervous system. Therefore, in past studies, cell biological or biochemical analyses of their pathology centered on forced expression of the causative genes in non-nervous system cultured cell lines and on mice in which the causative gene was knocked out. However, in a few instances, the animal or cell models did not necessarily reflect the human pathology. Identifying cell biological or biochemical changes in the initial stages of the disease, before onset of symptoms, has been difficult given analyses conducted on postmortem brains. However, with the development of iPS cell technologies, it became possible to establish pluripotent stem cells from the somatic cells of anyone, irrespective of race, genetic background, or whether the person exhibits disease symptoms. Thus, it is no exaggeration to say that generation of disease-specific iPS cells using iPS cell technologies is the sole means of reproducing ex vivo phenomena that occur in patients in vivo, particularly for nervous system disorders. The result has been a tremendous desire by investigators who are conducting research on neurological diseases to become engaged in disease-specific iPS cell research [4345].

A variety of disease-specific iPS cells have been used to study nervous system diseases, including amyotrophic lateral sclerosis (ALS) [

], spinal muscular atrophy [

], spinobulbar muscular atrophy [

], Friedreichs ataxia [

], Alzheimers disease (AD) [

], PD [

], Huntingtons disease [

,

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iPS cell technologies: significance and applications to ...

The discovery of iPS cells

In 2006, Shinya Yamanaka made a groundbreaking discovery that would win him the Nobel Prize in Physiology or Medicine just six years later: he found a new way to reprogramme adult, specialized cells to turn them into stem cells. These laboratory-grown stem cells are pluripotent they can make any type of cell in the body - and are called induced pluripotent stem cells, or iPS cells. Only embryonic stem cells are naturally pluripotent. Yamanakas discovery means that theoretically any dividing cell of the body can now be turned into a pluripotent stem cell.

So how are these iPS cells made? Yamanaka added four genes to skin cells from a mouse. This started a process inside the cells called reprogramming and, within 2 3 weeks, the skin cells were converted into induced pluripotent stem cells. Scientists can now also do this with human cells, by adding even fewer than four genes.

This short clip introduces the science behind reprogramming. View the full 16-minute film to see the whole story of Shinya Yamanaka's discovery.

IPS cells and embryonic stem cells are very similar. They are self-renewing, meaning they can divide and produce copies of themselves indefinitely. Both types of stem cell can be used to derive nearly any kind of specialized cell under precisely controlled conditions in the laboratory. Both iPS cells and embryonic stem cells can help us understand how specialized cells develop from pluripotent cells. In the future, they might also provide an unlimited supply of replacement cells and tissues for many patients with currently untreatable diseases.

In contrast to embryonic stem cells, making iPS cells doesnt depend on the use of cells from an early embryo. Are there any other differences? Current research indicates that some genes in iPS cells behave in a different way to those in embryonic stem cells. This is caused by incomplete reprogramming of the cells and/or genetic changes acquired by the iPS cells as they grow and multiply. Scientists are studying this in more detail to find out how such differences may affect the use of iPS cells in basic research and clinical applications. More research is also needed to understand just how reprogramming works inside the cell. So at the moment, most scientists believe we cant replace ES cells with iPS cells in basic research.

An important step in developing a therapy for a given disease is understanding exactly how the disease works: what exactly goes wrong in the body? To do this, researchers need to study the cells or tissues affected by the disease, but this is not always as simple as it sounds. For example, its almost impossible to obtain genuine brain cells from patients with Parkinsons disease, especially in the early stages of the disease before the patient is aware of any symptoms. Reprogramming means scientists can now get access to large numbers of the particular type of neurons (brain cells) that are affected by Parkinsons disease. Researchers first make iPS cells from, for example, skin biopsies from Parkinsons patients. They then use these iPS cells to produce neurons in the laboratory. The neurons have the same genetic background (the same basic genetic make-up) as the patients own cells. Thus scientist can directly work with neurons affected by Parkinsons disease in a dish. They can use these cells to learn more about what goes wrong inside the cells and why. Cellular disease models like these can also be used to search for and test new drugs to treat or protect patients against the disease.

Reprogramming holds great potential for new medical applications, such as cell replacement therapies. Since iPS cells can be made from a patients own skin, they could be used to grow specialized cells that exactly match the patient and would not be rejected by the immune system.If the patient has a genetic disease, the genetic problem could be corrected in their iPS cells in the laboratory, and these repaired iPS cells used to produce a patient-specific batch of healthy specialized cells for transplantation. But this benefit remains theoretical for now.

Until recently, making iPS cells involved permanent genetic changes inside the cell, which can cause tumours to form. Scientists have now developed methods for making iPS cells without this genetic modification. These new techniques are an important step towards making iPS-derived specialized cells that would be safe for use in patients. Further research is now needed to understand fully how reprogramming works and how iPS cells can be controlled and produced consistently enough to meet the high quality and safety requirements for use in the clinic.

Stem cells the future: an introduction to iPS cells Research into reprogrammed stem cells: an interactive timeline Stem cell school - multimedia learning module on cellular reprogramming Alzheimer Research Forum 4-part article on iPS cells and disease (September 2010) Nature news feature on challenges in the iPS field (May 2011) News article on the first iPS cell clinical trial (which is currently halted) Update on halted iPS cell clinical trial

This factsheet was created by Manal Hadenfeld, updated in 2012 by Michael Peitz and Annette Pusch and reviewed by Oliver Bruestle. Shinya Yamanaka photograph by Rubenstein. Additional images byMichael Peitz, Johannes Jungverdorben and Michael Rossbach.

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iPS cells and reprogramming: turn any cell of the body ...

For an hour on Friday 12 September, Masayo Takahashi sat alone, calmly reflecting on the decade of research that had led up to this moment. (David Cyranoski, Nature doi:10.1038/516311a)

A year ago, the first historic transplantation of iPS cell-based product took place in Japan. Recently, the trial was halted due to RIKENs decision on changing strategy. Today, Id to summarize some new info about the first iPS cell trial, based on two recent articles.

Was treatment of the first patient safe and effective? Safe YES, as of 6 months post-transplant. One year safety report is coming soon. Some observations of potential efficacy were shared in todays issue of Cell & Gene Therapy Insights by Hardy Kagimoto (CEO of Healios KK):

She had a series of 18 anti-vascular endothelial growth factor (VEGF) ocular injections for both eyes to cope with the constant recurrence of the disease. The results presented by Dr Takahashi showed that, after the removal of the subretinal fibrotic tissue and implantation of the iPS-cell-derived retinal pigment epithelial (RPE) cell sheet, the patient experienced no recurrence of neovascularization at the 6 month point and was free from frequent anti-VEGF injections. Her visual acuity was stabilized and there have been no safety related concerns so far.

Mutations in the product from the 2nd patient Some mutations were detected in the iPS cell-derived RPE product, prepared for the second patient. Nobody knows if these mutations were prohibitive for product release, since nobody has a guidance. What we know about these mutations:

three single-nucleotide variations (SNVs) and three copy-number variants (CNVs) were present that were not detectable in the patients original fibroblasts. The CNVs were all single-gene deletions. With regards to the SNVs, one is listed in a curated database of cancer somatic mutations, but only linked to a single cancer, reports CiRA. The mutated genes were not driver genes for tumor formation, wrote Takahashi in an e-mail.

and more from Kagimotos piece:

The result of tumorigenicity testing has proven the final RPE cells to be safe. Furthermore, the presence of genetic mutation does not necessarily mean that these RPE cells can be tumorigenic.

Regulatory change As per trial PI, Masayo Takahashi, the main reason for trial halting was a regulatory change:

Although the mutations were identified before transplanting cells into the second patient, and the mutations may have contributed to RIKENs decision not to treat, the main reason not to go ahead with the trial was because of a regulatory change, says Takahashi.

New Japanese law on Regenerative Medicine became effective after iPS trial was started and after first product was transplanted. Perhaps, the trial design was not very suitable in this new regulatory framework. But still, there is no guidance in new regulation about allowable level of mutations and methods of their detection in iPS cell products:

there is no regulation with which medical professionals are obligated to check gene modification for organ transplantation, mesenchymal stem cell injections or autologous cell therapy. The fact remains that we do not have clear guidelines today on which the whole community can reach a consensus that the second RPE cells are safe enough for implantation .

RIKENs decision It seem to me RIKENs decision on halting a trial was wise and very strategic. The first and the most important thing here:

As a pioneer of iPS cell clinical application, Riken took the responsible decision not to rush ahead with the second patients RPE cells, which could potentially damage the whole field of regenerative medicine.

The second thing is if there is no guidance on mutations, why not develop it now?

Although therefore, the cells were widely thought to be safe to use, after careful consideration, they made the decision that they would not implant another autologous cell sheet until such guidelines could be officially authorized. They are now coordinating the discussions at the Ministry of Education, Culture, Sports, Science, and Technology, and also at the Ministry of Health, Labor and Welfare to carefully discuss these issues with key opinion leaders in the field including government officials, regulatory experts, scientists and toxicologists.

The third thing is realization that auto- model is not the way go in future:

As of now, autologous would not be a feasible way of providing wide level clinical therapy, says CiRA spokesperson Peter Karagiannis. At the experimental level its fine, but if its going to be mass produced or industrialized, it has to be allogeneic.

Moving forward So, RIKEN is moving forward with allo- iPS cell-derived RPE. Moving together with CiRA. Well characterized partially matched lines with safety clearance by rigorous QC testing. However, some concerns about potential immunogenicity were expressed by peers:

But the project is fraught with uncertainty, because HLA-matched cells might still suffer immune rejection. Were not going to know until those clinical trials, says Coffey. CiRA is not typing its cells for minor histocompatibility antigens, which can cause T cellmediated transplant rejection. The current effort is going for major [histocompatibility matching], says Kapil Bharti, a stem cell researcher at the National Eye Institute.

Expectations Im positive about future iPS cell-based trials. I like the approach, which RIKEN is taking. Here some of our expectation for the future (feel free to add to the list):

PS: Emphases throughout the text are mine.

Tagged as: clinical trial, genomic stability, iPS, iPS cell bank, safety, stem cell line, Takahashi M

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The first iPS cell clinical trial insights - Stem Cell Assays

What if doctors could grow a new working inner ear from a persons own skin cells? Or repair the damaged inner ear from within?

Solving this profound mystery is the driving force behind stem cell research and the promise of tissue engineering in otolaryngology. While hearing aids and cochlear implants can provide good recovery of hearing function, the development of a biological method to repair the damaged cochlea has the potential to restore normal hearing without any type of prosthesis.

One approach to restore hearing might be to surgically place stem cells within the cochlea in such a way that they would fuse with the remaining cochlear structures and develop and function as hair cells. Scientists believe this is a viable approach because, unlike most organs that are destroyed by disease, the inner ear remains structurally intactonly the hair cells are lost. By mimicking the steps involved in the formation of embryonic mouse ears, Stanford scientists have produced stem cells in the laboratory that look and act very much like hair cells, the sensory cells that normally reside in the inner ear. If they can generate hair cells in the millions, it could lead to significant scientific and clinical advances along the path to curing deafness in the future.

A promising source of creating hair cells comes from induced pluripotent stem cells (iPS)adult cells, taken for example from a patients own skin that have been genetically reprogrammed to revert back to stem cells. This breakthrough process represents a major opportunity to eventually treat a patient with his or her own cells.

Currently, our research team is working toward producing human hair cells for the first time in a culture dish. This work could lead, in the long run, to novel therapies based on cell transplantation.

Equally exciting is an ongoing approach to use embryonic stem cell-based approaches for discovery of novel drugs that could be used for treatment for deafness. More about this exciting new direction can be found under Molecular Therapy.

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Stem Cell Therapy | Stanford Initiative to Cure Hearing Loss

iPSC are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes. For example, iPSC can be prodded into becoming beta islet cells to treat diabetes, blood cells to create new blood free of cancer cells for a leukemia patient, or neurons to treat neurological disorders.

In late 2007, a BSCRC team of faculty, Drs. Kathrin Plath, William Lowry, Amander Clark, and April Pyle were among the first in the world to create human iPSC. At that time, science had long understood that tissue specific cells, such as skin cells or blood cells, could only create other like cells. With this groundbreaking discovery, iPSC research has quickly become the foundation for a new regenerative medicine.

Using iPSC technology our faculty have reprogrammed skin cells into active motor neurons, egg and sperm precursors, liver cells, bone precursors, and blood cells. In addition, patients with untreatable diseases such as, ALS, Rett Syndrome, Lesch-Nyhan Disease, and Duchenne's Muscular Dystrophy donate skin cells to BSCRC scientists for iPSC reprogramming research. The generous participation of patients and their families in this research enables BSCRC scientists to study these diseases in the laboratory in the hope of developing new treatment technologies.

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Induced Pluripotent Stem Cells (iPS) | UCLA Broad Stem ...

The most successful stem cell therapybone marrow transplanthas been around for more than 40 years. Johns Hopkins researchers played an integral role in establishing the methods for how bone marrow transplants are done, which you can read about in Human Stem Cells at Johns Hopkins: A Forty Year History. The latest developments in bone marrow transplants are Half-Matched Transplants, which may be helpful in treating more diseases than ever before. In The Promise of the Future, three Hopkins researchers who study blood diseases share their ideas about which technologies hold most promise for developing therapies.

Induced pluripotent stem cells, or iPS cells, are adult cells that are engineered to behave like stem cells and to regain the ability to differentiate into various cell types. Engineered Blood describes current research in generating blood cells that contain disease traits with Those Magic Scissors so we can learn more in the lab about diseases like sickle cell anemia.

Adult stem cells are being used in other applications as well. Stem Cells Enhance Healing tells of an undergraduate biomedical engineering team at Hopkins that has devised medical sutures containing stem cells which speed up healing when stitched in. And A New Path for Cardiac Stem Cells tells of how a patients own heart stem cells were used to repair his heart after a heart attack.

In the podcast What Anti-Depression Treatments Actually Target In The Brain, Hongjun Song reveals that current antidepressant therapies may have unknowingly been targeting stem cells all along.

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Cell Therapy - Hopkins Medicine

Scientists at the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research and Center for Duchenne Muscular Dystrophy at UCLA have developed a new approach that could eventually be used to treat Duchenne muscular dystrophy. The stem cell gene therapy could be applicable for 60 percent of people with Duchenne, which affects approximately 1 in 5,000 boys in the U.S. and is the most common fatal childhood genetic disease.

The approach uses a technology called CRISPR/Cas9 to correct genetic mutations that cause the disease. The study, which was led by co-senior authors April Pyle and Melissa Spencer and first author Courtney Young, was published in the journal Cell Stem Cell.

The researchers designed the approach to be useful in a clinical setting in the future.

This method is likely 10 years away from being tested in people, said Spencer, professor of neurology in the UCLA David Geffen School of Medicine, co-director of the Center for Duchenne Muscular Dystrophy at UCLA and member of the Broad Stem Cell Research Center It is important that we take all the necessary steps to maximize safety while quickly bringing a therapeutic treatment to patients in clinical trials.

Duchenne typically occurs through one mutation in a gene called dystrophin, which makes a protein with the same name. In people without the disease, the dystrophin protein helps strengthen and connect muscle fibers and cells. There are hundreds of mutations in the dystrophin gene that can lead to the disease, but in 60 percent of people with Duchenne, their mutation will occur within a specific hot spot of the gene.

Duchenne mutations cause abnormally low production of the dystrophin protein, which in turn causes muscles to degenerate and become progressively weaker. Symptoms usually begin in early childhood; patients gradually lose mobility and typically die from heart or respiratory failure around age 20. Some current medications can treat the diseases symptoms but none can stop the progression of the disease or significantly improve patients quality of life and there is currently no way to reverse or cure the disease.

The platform developed by the UCLA researchers focuses on the hot spot of the dystrophin gene.

To test the platform, they obtained skin cells from consenting patients at the Center for Duchenne Muscular Dystrophy, all of whom had mutations that fell within the dystrophin gene hot spot. The researchers reprogrammed the cells to create induced pluripotent stem cells in an FDA-compliant facility at the Broad Stem Cell Research Center; the use of this facility is an important step in the process as preclinical research moves toward human clinical trials. Induced pluripotent stem cells, or iPS cells, have the ability to become any type of human cell while also maintaining the genetic code from the person they originated from.

Next, the scientists removed the Duchenne mutations in the iPS cells using a gene editing platform they developed that uses the CRISPR/Cas9 technology. (CRISPR stands for clustered regularly interspaced short palindromic repeats.) The platform targets and removes specific regions of the hot spot of the dystrophin gene, which harbors 60 percent of Duchenne mutations, which restores the missing protein.

CRISPR/Cas9 is a naturally occurring reaction that bacteria use to fight viruses. In 2012, scientists discovered they could adapt the process to make cuts in specific human DNA sequences. One part of the CRISPR/Cas9 system acts like a navigation system and can be programmed to seek out a specific part of the genetic code a mutation, for example. The second part of the system can cut mutations out of the genetic code, and in some cases can replace the mutation with a normal genetic sequence.

UCLA Broad Stem Cell Research Center

April Pyle, Courtney Young and Melissa Spencer

Once the UCLA researchers had produced iPS cells that were free from Duchenne mutations, they differentiated the iPS cells into cardiac muscle and skeletal muscle cells and then transplanted the skeletal muscle cells into mice that had a genetic mutation in the dystrophin gene.

They found that the transplanted muscle cells successfully produced the human dystrophin protein.

The result was the largest deletion ever observed in the dystrophin gene using CRISPR/Cas9, and the study was the first to create corrected human iPS cells that could directly restore functional muscle tissue affected by Duchenne. (Previously, scientists had used CRISPR/Cas9 to repair mutations that affect smaller numbers of people with Duchenne, and in cell types that werent necessarily clinically relevant.)

This work demonstrates the feasibility of using a single gene editing platform, plus the regenerative power of stem cells to correct genetic mutations and restore dystrophin production for 60 percent of Duchenne patients, said Pyle, associate professor of microbiology, immunology and molecular genetics and member of the Broad Stem Cell Research Center.

Young, a UCLA predoctoral fellow and president of a UCLA student group called Bruin Allies for Duchenne, is particularly passionate about Duchenne research because she has a cousin with the disease.

I already knew I was interested in science, so after my cousins diagnosis, I decided to dedicate my career to finding a cure for Duchenne, Young said. It makes everything a lot more meaningful, knowing that Im doing something to help all the boys who will come after my cousin. I feel like Im contributing and Im excited because the field of Duchenne research is advancing in a really positive direction.

Duchenne muscular dystrophy is the most common and severe of the 30 forms of muscular dystrophy.

The UCLA researchers plan to develop strategies to test the Duchenne-specific CRISPR/Cas9 platform to treat the disease in animals as the next step toward perfecting a method that can be used in humans.

The CRISPR/Cas9 platform for Duchenne developed by the UCLA scientists is not yet available in clinical trials and has not been approved by the FDA for use in humans.

The research was supported by the National Science Foundation Graduate Research Fellowship Program, the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health, the Rose Hills Foundation Research Award, the California Institute for Regenerative Medicines Bridges Program and the UCLA Broad Stem Cell Research Center. The FDA-compliant facility was supported by a grant from the California Institute for Regenerative Medicine.

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Stem cell gene therapy could be key to treating Duchenne ...

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.

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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

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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

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Adoptive T Cell Therapy Conference

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STEM CELL THERAPY FOR ATHLETES - Mississippi Sports Law Review

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 ...

Yesterday, Stem Cell Reports published must read paper, which describes manufacturing of GMP-grade iPS cell line for potential clinical use. We saw a few very similar paper titles in in the past, but this one is special. Here is why:

We didnt want Lonza to own the process, even though they helped develop it, Rao said, speaking on his own behalf. We wanted the government to be able to provide the process to people, so they could modify it or have access to the process at a reasonable cost. That was one reason why the government funded this All the basic processes will be free.

I have few general consideration about this protocol and few technical. Lets start from general: Since, Im as US taxpayer also funded this study, Id like to have an option for process tech transfer to my facility. If not Lonza, who is owner of this process? Can I get a license and do the same thing in-house? The process largerly developed on Lonzas reagents and platforms (such as nucleofection). Also, proprietary reagent and process were mentioned in the paper. Id be ok to buy Lonzas reagents for this wonderful process, but what if Id like alternative to reduce the cost? Also, in GMP must be back-up supplier for every reagent and material. Who is back-up for Lonza?

Now, some technical considerations:

Overall, I was enjoying reading this paper. Id highly recommend this protocol to every cell product developer, irrespective to type of your cells!

Tagged as: clinical protocols, donor eligibility, drug master file, GMP, iPS, iPS cell bank, manufacturing, master cell bank, protocol, QC

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A protocol for manufacturing of GMP-compliant iPS cell lines

Since their discovery/invention a little less than a decade ago, induced pluripotent stem (iPS) cells inspired hope to become a powerful tool for drug discovery and development applications. With advances in reprogramming and differentiation technologies, as well as with the recent availability of gene editing approaches, we are finally able to create more complex and phenotypically accurate cellular models based on iPS cell technology. This opens new and exciting opportunities for iPS cell utilization in early discovery, preclinical and translational research. Cambridge Healthtech Institutes inaugural iPS Cell Technology in Drug Discovery and Development conference is designed to bring together experts and bench scientists working with iPS cells and end users of their services, researchers working on finding cures for specific diseases and disorders.

Final Agenda

Day 1 | Day 2 | Speaker Biographies | Download Brochure

Wednesday, June 15

7:00 am Registration and Morning Coffee

8:25 Chairpersons Opening Remarks

8:35 KEYNOTE PRESENTATION: iPS CELL TECHNOLOGY, GENE EDITING AND DISEASE RESEARCH

Rudolf Jaenisch, M.D., Founding Member, Whitehead Institute for Biomedical Research; Professor, Department of Biology, Massachusetts Institute of Technology

The development of the iPS cell technology has revolutionized our ability to study human diseases in defined in vitro cell culture systems. A major problem of using iPS cells for this disease in the dish approach is the choice of control cells because of the unpredictable variability between different iPS / ES cells to differentiate into a given lineage. Recently developed efficient gene editing methods such as the CRISPR/Cas system allow the creation of genetically defined models of monogenic as well as polygenic human disorders.

9:05 iPSC Genome Editing: From Modeling Disease to Novel Therapeutics

Chad Cowan, Ph.D., Associate Professor, Harvard Department of Stem Cell & Regenerative Biology (HSCRB)

Our goal is to understand how naturally occurring human genetic variation protects (or predisposes) some people to cardiovascular and metabolic diseasethe leading cause of death in the worldand to use that information to develop therapies that can protect the entire population from disease.

9:35 Stem Cells and Genome Editing to Enable Drug Discovery

Jeffrey Stock, Ph.D., Principal Scientist, Global R&D Groton Labs, Pfizer

Significant advances have been made in recent years in the isolation/generation and differentiation of human pluripotent stem cells (hPSC). Similarly, powerful tools for in vitro genomic editing are now readily available. When combined, these technologies make it possible to generate physiologically relevant models of human disease to enable drug discovery. In this presentation, we provide some examples of how we have applied these technologies to produce models that are suitable for target validation as well as small molecule screening.

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

10:50 Phenotypic Diversity in a Large Cohort of iPSC-Derived Cardiomyocytes as a Platform for Response Modeling in Drug Development

Ulrich Broeckel, M.D., Professor of Pediatrics, Medicine and Physiology, Pediatrics, Medical College of Wisconsin

We will discuss the underlying concepts of phenotypic variation and the impact of genomic variation on common, complex phenotypes in iPSCs. To demonstrate this, we have established 250 iPSC cell lines from the NHLBI HyperGen study. We will discuss our approach to analyzing disease phenotypes on a molecular level using iPSC-derived cardiomyocytes. Furthermore we will present data, which provides a framework to use the obtained data for the selection of samples for compound screening and drug development.

11:20 Transcriptional and Proteomic Profiling of Human Pluripotent Stem Cell-Derived Motor Neurons: Implications for Familial Amyotrophic Lateral Sclerosis

Joseph Klim, Ph.D., Postdoctoral Scholar, Eggan Lab, Stem Cell and Regenerative Biology Department, Harvard University

We combined pluripotent stem cell technologies with both RNA sequencing and mass spectrometry-based proteomics to map alterations to mRNA and protein levels in motor neurons expressing mutant SOD1. This approach enabled us to study the effects of mutant SOD1 in purified populations of motor neurons using multiple molecular metrics over time. These investigations have afforded an unprecedented glimpse at the biochemical make-up of human stem cell-derived motor neurons and how they change in culture.

11:50 Presentation to be Announced

Yoko Ejiri, Researcher, Microdevice Team, New Business Development Division, Kuraray Co. Ltd.

12:05 Sponsored Presentation (Opportunity Available)

12:20 pm Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

12:50 Session Break

1:40 Chairpersons Remarks

Vikram Khurana, M.D., Co-Founder and Vice President, Discovery Technologies, Yumanity Therapeutics

1:50 High-Throughput Phenotyping of Human PSC Derived Neurons

Bilada Bilican, Ph.D., Investigator II, Neuroscience, Novartis Institutes for BioMedical Research (NIBR)

We established a fully automated human pluripotent stem cell (PSC) maintenance and excitatory cortical neuronal differentiation platform that enables parallel phenotyping of many different lines at once. This human disease-modeling platform is being integrated into Novartis lead discovery pipeline to identify new targets, molecules, and to elucidate cellular aspects of human neuronal biology.

2:20 Modeling ALS with Patient Specific iPSCs

Shila Mekhoubad, Ph.D., Scientist, Stem Cell Biology Lab, Biogen

Advances in stem cell biology and neuronal differentiations have provided a new platform to study ALS in vitro. Here we will describe our use of induced pluripotent stem cells (iPSCs) from patients with familial ALS to establish new models and tools that can contribute to the development and validation of novel ALS therapeutics.

2:50 Refreshment Break in the Exhibit Hall with Poster Viewing

3:35 Modeling Huntingtons Disease in IPS Cells: Development and Validation of Phenotypes Relevant for Disease

Kimberly B. Kegel-Gleason, Ph.D., Assistant Professor in Neurology, Massachusetts General Hospital & Harvard Medical School

Huntingtons disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HD gene. Using induced pluripotent stem (IPS) cells from controls and HD patients with low and medium CAG repeat expansions, we are developing assays for target validation and drug discovery based on phenotypic changes observed in PI 3-kinase dependent signaling, Rac activation and cell motility in microfluidic channels.

4:05 From Yeast to Patient iPS Cells: A Drug Discovery Pipeline for Neurodegeneration

Vikram Khurana, M.D., Co-Founder and Vice President, Discovery Technologies, Yumanity Therapeutics

Phenotypic screening in neurons and glia derived from patients is now conceivable through unprecedented developments in reprogramming, transdifferentiation, and genome editing. We outline progress in this nascent field, but also consider the formidable hurdles to identifying robust, disease-relevant and screenable cellular phenotypes in patient-derived cells. We illustrate how analysis in the simple bakers yeast cell Saccharaomyces cerevisiae is driving discovery in patient-derived neurons, and how approaches in this model organism can establish a paradigm to guide the development of stem cell-based phenotypic screens.

4:35 Sponsored Presentation (Opportunity Available)

5:05 PANEL DISCUSSION: iPSC-Based Neurodegenerative Disease Modeling

Moderator: Vikram Khurana, M.D., Co-Founder and Vice President, Discovery Technologies, Yumanity Therapeutics

Human neurodegenerative disorders are among the most difficult to study. This panel will discuss existing and future models for major neurodegenerative diseases.

5:35 Welcome Reception in the Exhibit Hall with Poster Viewing

6:45 Close of Day

Day 1 | Day 2 | Speaker Biographies | Download Brochure

Thursday, June 16

7:00 am Registration.

7:30 Interactive Breakout Discussion Groups with Continental Breakfast

This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.

Modeling neurodegenerative disorders for drug discovery and development

Moderator: Bilada Bilican, Ph.D., Investigator II, Neuroscience, Novartis Institutes for BioMedical Research (NIBR)

iPS Cell Technology Enabled Organ-on-Chip Models

Moderator: James Hickman, Ph.D., Professor, NanoScience Technology Center, University of Central Florida

Gene Editing in iPS Cells: Technology and Major Applications

Moderator:Joseph Klim Ph.D., Postdoctoral Scholar, Eggan Lab, Stem Cell and Regenerative Biology Department

8:35 Chairpersons Remarks

James J. Hickman, Ph.D., Founding Director, NanoScience Technology Center and Professor, Nanoscience Technology, Chemistry, Biomolecular Science, Material Science and Electrical Engineering, University of Central Florida

8:45 Utilization of iPSCs in Developing Human-on-a-Chip Systems for Phenotypic Screening Applications

James J. Hickman, Ph.D., Founding Director, NanoScience Technology Center and Professor, Nanoscience Technology, Chemistry, Biomolecular Science, Material Science and Electrical Engineering, University of Central Florida

Our lab is developing multi-organ human-on-a-chip systems for evaluating toxicity and efficacy compounds for drug discovery applications. Validation of the systems has already indicated good agreement with previous literature values, which gauges well for the predictive power of these platforms. Applications for neurodegenerative diseases, metabolic disorders as well as cardiac and muscle deficiencies will be highlighted in the talk.

9:15 Human-Induced Pluripotent Stem Cells Recapitulate Breast Cancer Patients Predilection to Doxorubicin-Induced Cardiotoxicity

Paul W. Burridge, Ph.D., Assistant Professor, Department of Pharmacology, Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine

The ability to predict which patients are likely to experience cardiotoxicity as a result of their chemotherapy represents a powerful clinical tool to attenuate this devastating side-effect. We report our progress towards this aim using the hiPSC cell model, a battery of in vitro assays, and machine learning.

9:45 Utilization of Induced Pluripotent Stem Cells to Understand Tyrosine Kinase Inhibitors (TKIs)-Induced Hepatotoxicity

Qiang Shi, Ph.D., Principal Investigator, Division of Systems Biology, National Center for Toxicological Research (NCTR), U.S. FDA

For cancer patients, the benefits of anti-cancer agents are often countered by hepatotoxicity. The purpose of current study is to predict tyrosine kinase inhibitors (TKIs)-induced toxicity using rat primary hepatocytes and human induced pluripotent stem cell (iPSC) -derived hepatocytes. Multi-parameter cellular endpoints have been used to examine the utilization of iPSC in safety screening. Data on cross-species comparison from rodent to human will be presented.

10:15 Coffee Break in the Exhibit Hall with Poster Viewing

10:55 Chairpersons Remarks

Joseph Klim, Ph.D., Postdoctoral Scholar, Eggan Lab, Stem Cell and Regenerative Biology Department, Harvard University

11:00 KEYNOTE PRESENTATION: STEM CELL PROGRAMMING AND REPROGRAMMING, AND APPLICATIONS OF iPSC TECHNOLOGIES TO MODELING OF THE NEUROMUSCULAR SYSTEM AND THE DISEASES THAT AFFECT IT

Kevin C. Eggan, Ph.D., Harvard Department of Stem Cell and Regenerative Biology, Howard Hughes Medical Institute

While iPSCs have created unprecedented opportunities for drug discovery, there remains uncertainty concerning the path to the clinic for candidate therapeutics discovered with their use. Here we share lessons that we learned, and believe are generalizable to similar efforts, while taking a discovery made using iPSCs into a clinical trial.

11:30 Trans-Amniotic Stem Cell Therapy (TRASCET) for the Treatment of Birth Defects

Dario O. Fauza, M.D., Ph.D., Associate in Surgery, Boston Children's Hospital; Associate Professor, Surgery, Harvard Medical School

Trans-Amniotic Stem Cell Therapy (TRASCET) is a novel therapeutic paradigm for the treatment of birth defects. It is based on the principle of harnessing/enhancing the normal biological role of amniotic fluid-derived mesenchymal stem cells (afMSCs) for therapeutic benefit. The intra-amniotic delivery of afMSCs in large numbers can either elicit the repair, or significantly mitigate the effects associated with major congenital anomalies such as neural tube and abdominal wall defects.

12:00 pm Bridging Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

12:30 Session Break

1:00 Coffee and Dessert in the Exhibit Hall with Poster Viewing

1:45 PLENARY KEYNOTE SESSION

3:30 Refreshment Break in the Exhibit Hall with Poster Viewing

4:15 Close of Conference

Day 1 | Day 2 | Speaker Biographies | Download Brochure

Here is the original post:
Advances in iPS Cell Technology for Drug Development ...

Media in Japan are reporting that Waseda University will shortly revoke the Ph.D. of STAP cell scientist Haruko Obokata. A hat tip to blog reader Tom on this story. The thesis contained plagiarized material and problematic data as did the Nature papers on which she was first author. Those papers were retracted and now apparently her thesis will essentially find the same fate.

Obokata had been given 1 year to correct her thesis, a potentially impossible task, and Japan TImes quotes a source that she didnt meet the deadline:

The former Riken researcher was last October given a year in which to correct a thesis she wrote in 2011. She failed to submit the revisions in time, the sources said, and a request for an extension was refused.

This is a further step in the winding down of the STAP cell mess following on the recent publication of papers by Nature refuting STAP. There is a sense that the supervision of Obokata as a young scientist was not effective, which is perhaps the main STAP element that is as yet not entirely resolved.

The voters have spoken and below is the list of the top 12 vote getters from the larger pool of nominees for Stem Cell Person of the Year in 2015. These are some amazing people.

Look for more information, such as mini-bios, soonon some of the top finalists.

There were nearly 4,700 votes in total.

Now I have the tough task of picking from this dozen just one winner, who will receive the recognition as the top stem cell outside the box innovator of 2015 and of course the $2,000 prize.

A draft agenda is now publicly available for the upcoming National Academy of Sciences (NAS) meeting on human gene editing. We now know a lot more about what to expect from this international gathering, which is called the International Summit on Human Gene Editing: A Global Discussion.

The meeting will start on day 1 with context from David Baltimore as well as other scientists from around the world. There will be scientific background on the technology and information on applications. Social Implications will be discussed. Sprinkled throughout the first day will be opportunities for comments and questions from the floor totaling about 2 hours on this day.

Image from National Academy of Medicine. Oops they made the DNA left-handed.

Day 2 looks to build on the themes of the first day, but now bringing in the issues of governance and more emphasis on international perspectives.

Day 3 will be focused more squarely on societal implications and governance including the crucial issue of commercialization. These days also provide time for comments and questions from the floor.

These windows of time will include opportunities for members of the public to bring their voices into the discussion. I asked a NAS spokesperson about the role of the public in the meeting and received this reply:

The organizing committee and staff and leadership of the academies have been identifying experts/stakeholders/interested parties from a range of disciplines and perspectives to invite to attend and participate in the summit. In addition, a general public registration will open next week, it will be open to anyone although seating is limited and dozens of people have already expressed interest in attending. And yes, public may participate in breakout sessions, and will have an opportunity to speak in public comment sessions as appropriate.

There will also be other opportunities for involvement according to the spokesperson:

Also, the video webcast will allow many people to view the proceedings and we expect a lively conversation on social media including at #GeneEditSummit

Since I will be at the meeting and blogging it live here, I hope that this site will in addition provide a forum for discussion involving a diverse group and boost democratic deliberation on this important topic.

The myostatin gene has been getting quite a bit of attention lately.

The buzz surrounds the idea of inhibiting myostatin either through gene therapy or via germline human genetic modification.

In this way, some hope to create people with more muscle. Myostatin, which also goes by the acronym MSTN, has an inhibitory function on muscle. Inhibit and inhibitor of muscle and you should get more muscle, right?

Data backs it up.

Animals including humans with spontaneous mutations in myostatin have unusualmusculature including increases in muscle. This NEJM case report on a boy with a myostatin mutation describes a remarkable phenotype of drastically increased muscle and reduced fat. No clear pathology was associated with the condition, which is referred to as myostatin-related muscle hypertrophy. More on that condition more generally here.

Pop culture isfascinated with the idea of genetically modifying people to artificially create this kind of super-muscle condition. Would they be like superheroes? Just last week came the first report of a person, Liz Parrish, supposedly doing a DIY gene therapy to target myostatin.

Scientists have recently reported a string of super-muscled animals created through genetic modification includingGM dogs and pigs.

If this kind of trend continues and increasingly involves people, what might go wrong? One possibility is that GM people who have had the myostatin pathway targeted could have other phenotypes or even diseases that we cannot anticipate.

Ive written a new book on human genetic modification. This is my second book as the first one was Stem Cells: An Insiders Guide, which is currently the top stem cell book on Amazon.The new book iscalled GMO Sapiens: The Life-Changing Science of Designer Babies.

You can pre-order ithereat Amazon or overhere at my publishers site.The newly updated cover is shown at right.

The title was chosen as a portmanteau (mashup) of GMO andHomo sapiens.

Weve been aiming for the book to come outin mid-late December. Im optimistic based on what I hear from the publisher, but well see.

Why write a book on human genetic modification?

The science in this areahas changed dramatically and both the wider scientific community and the public need to know what is going on so that they can participate in the dialogue.

Unlike in past decades, today the possibility of heritable human genetic modification is very real. Based on all that we now know it is reasonable to predict that someone will attempt to create genetically modified people(aka designer babies) in coming years. The results could be great or disastrous, but in either case (or probably more likely some mixture of the two) the outcomes are going to be revolutionary.

Are we ready for what may come next? How will this affect individuals and society as a whole?

More thinking, discussion, and transparency are urgently needed.The goal of this upcoming book is to move in a constructive direction by educating and stimulating debate as well as dialogue.

At the same time in the book, I am not afraid to tackle the real, but tough issues that are integral to this topic. It seems that up until now in science it has been somewhat taboo to talk about the possibility of designer babies.However, we dont have time to close our eyes to the reasonable probability that someone will try to make designer babies in the near future nor to pretend that nothing could go wrong for individuals or society. Already this year we saw the creation of the first genetically-modified human embryos in the lab using the amazing gene editing toolbox that is CRISPR. That is just one step, but may have opened the door to much more.

It addition to beinga resource for learning, my newbook is written to be an enjoyable read that is approachable to both an educated lay audience and scientists alike.

Heres the draft back cover blurb (could still change):

Genetically modified organisms (GMOs) including plants and the foods made from them are a hot topic of debate today, but soon related technology could go much further and literally change what it means to be human. Scientists are on the verge of being able to create people who are GMOs.

Should they do it? Could we become a healthier and better species or might eugenics go viral leading to a real, new world of genetic dystopia? GMO Sapiens tackles such questions by taking a fresh look at the cutting-edge biotech discoveries that have made genetically modified people possible.

Bioengineering, genomics, synthetic biology, and stem cells are changing sci-fi into reality before our eyes. This book will capture your imagination with its clear, approachable writing style. It will draw you into the fascinating discussion of the life-changing science of human genetic modification.

See more here:
The Niche - Knoepfler lab stem cell blog

In the past year, Japan has accelerated its position as a hub for regenerative medicine research, largely driven by support from Prime Minister Shinzo Abe who has identified regenerative medicine and cellular therapy as key to the Japans strategy to drive economic growth. The Prime Minister has encouraged a growing range of collaborations between private industry and academic partners through an innovative legal framework approved last fall. He has also initiated campaigns to drive technological advances in drugs and devices by connecting private companies with public funding sources. The result has been to drive progress in both basic and applied research involving induced pluripotent stem cells (iPS cells) and related stem cell technologies.

Indeed, 2013 represented a landmark year in Japan, as it saw the first cellular therapy involving transplant of iPS cells into humans initiated at the RIKEN Center in Kobe, Japan.[1] The RIKEN Center is Japans largest, most comprehensive research institution, backed by both Japans Health Ministry and government. To speed things along, RIKEN did not seek permission for a clinical trial involving iPS cells, but instead applied for a type of pretrial clinical research allowed under Japanese regulations.

As such, this pretrial clinical research allowed the RIKEN research team to test the use of iPS cells for the treatment of wet-type age-related macular degeneration (AMD) on a very small scale, in only a handful of patients. Unfortunately, this trial was paused in 2015 due to safety concerns and is currently on hold pending further investigation. Regardless, the trial has set a new international standard for considering iPS cells as a viable cell type to investigate for clinical purposes.

If this iPS cell trial is ultimately reinstated, it will help to accelerate the acceptance of cellular therapies within other countries. Currently, the main concern surrounding iPS cell-based cellular therapy isthe fear of creating multiplying cell populations within patients. Even treatments using embryonic stem cells, which have been cultured and studied for decades, are still in very early clinical trials, so it is not surprising that clinical trials using iPS cells are being conducted on a small-scale, experimental level.[2]

Japan has a unique affection for iPS cells, as the cells were originally discovered by the Japanese scientist, Shinya Yamanaka of Kyoto University. Mr. Yamanaka was awarded the Nobel Prize in Physiology or Medicine for 2012, an honor shared jointly with John Gurdon, for the discovery that mature cells can be reprogrammed to become pluripotent. In addition, Japans Education Ministry said its planning to spend 110 billion yen ($1.13 billion) on induced pluripotent stem cell research during the next 10 years, and the Japanese parliament has been discussing bills that would speed the approval process and ensure the safety of such treatments.[3] In April, Japanese parliament even passed a law calling for Japan to make regenerative medical treatments like iPSC technology available for its citizens ahead of the rest of the world.[4] If those forces were not enough, Masayo Takahashi of the RIKEN Center for Developmental Biology in Kobe, Japan, who is heading the worlds first clinical research using iPSCs in humans, was also chosen by the journal Natureas one of five scientists to watch in 2014.[5]

In summary, Japan is the clear global leader with regard to investment in iPS cell technologies and therapies. While progress with stem cells has not been without setbacks within Japan, including a recent scandal at the RIKEN Institute that involved falsely manipulated research findings and the aforementioned hold on the first clinical trial involving transplant of an iPS cell product into humans, Japan has emerged from these troubles to become the most liberalized and progressive nation pursuing the development of iPS cell products and services.

To learn more about induced pluripotent stem cell (iPSC)industry trends and events, view the Compete 2015-16 Induced Pluripotent Stem Cell (iPSC) Industry Report.

To receive future posts about the stem cell industry, sign-up here. We will never share your information with anyone, and you can opt-out at any time. No spam ever, just great stuff.

BioInformant is the only research firm that has served the stem cell sector since it emerged. Our management team comes from a BioInformatics background the science of collecting and analyzing complex genetic codes and applies these techniques to the field of market research. BioInformant has been featured on news outlets including the Wall Street Journal, Nature Biotechnology, CBS News, Medical Ethics, and the Center for BioNetworking.

Serving Fortune 500 leaders that include GE Healthcare, Pfizer, Goldman Sachs, Beckton Dickinson, and Thermo Fisher Scientific, BioInformant is your global leader in stem cell industry data.

Footnotes [1] Dvorak, K. (2014).Japan Makes Advance on Stem-Cell Therapy [Online]. Available at: http://online.wsj.com/news/articles/SB10001424127887323689204578571363010820642. Web. 14 Apr. 2015. [2] Note: In the United States, some patients have been treated with retina cells derived from embryonic stem cells (ESCs) to treat macular degeneration. There was a successful patient safety test for this stem cell treatment last year that was conducted at the Jules Stein Eye Institute in Los Angeles. The ESC-derived cells used for this study were developed by Advanced Cell Technology, Inc, a company located in Marlborough, Massachusetts. [3] Dvorak, K. (2014).Japan Makes Advance on Stem-Cell Therapy [Online]. Available at: http://online.wsj.com/news/articles/SB10001424127887323689204578571363010820642. Web. 8 Apr. 2015. [4] Ibid. [5] Riken.jp. (2014).RIKEN researcher chosen as one of five scientists to watch in 2014 | RIKEN [Online]. Available at: http://www.riken.jp/en/pr/topics/2014/20140107_1/. Web. 14 Apr. 2015.

See the rest here:
Japan Most Liberalized Market for iPS Cell Therapy ...

In 2006, Shinya Yamanaka of Kyoto University in Japan was the first to disprove the previous notion that reversible cell differentiation of mammals was impossible. He reprogrammed a fully differentiated mouse cell into a pluripotent stem cell by introducing four genes, Oct-4, SOX2, KLF4, and Myc, into the mouse fibroblast through gene-carrying viruses. With this method, he and his coworkers created induced pluripotent stem cells (iPS cells), the key component in this experiment.[1] Scientists have been able to conduct experiments that show the ability of iPS cells to treat and even cure diseases. In this experiment, tests were run on mice with inherited sickle-cell anemia. Skin cells were turned into cells containing genes that transformed the cells into iPS cells. These replaced the diseased sickled cells, curing the test mice. The reprogramming of the pluripotent stem cells in mice was successfully duplicated with human pluripotent stem cells within about a year of the experiment on the mice.[citation needed]

Sickle-cell anemia is a disease in which the body produces abnormally shaped red blood cells. Red blood cells are flexible and round, moving easily through the blood vessels. Infected cells are shaped like a crescent or sickle (the namesake of the disease). As a result of this disorder the hemoglobin protein in red blood cells is faulty. Normal hemoglobin bonds to oxygen, then releases it into cells that need it. The blood cell retains its original form and is cycled back to the lungs and re-oxygenated.

Sickle cell hemoglobin, however, after giving up oxygen, cling together and make the red blood cell stiff. The sickle shape also makes it difficult for the red blood cell to navigate arteries and causes blockages.[2] This can cause intense pain and organ damage. The sickled red blood cells are fragile and prone to rupture. When the number of red blood cells decreases from rupture (hemolysis), anemia is the result. Sickle cells die in 1020 days as opposed to the traditional 120-day lifespan of a normal red blood cell.

Sickle cell anemia is inherited as an autosomal (meaning that the gene is not linked to a sex chromosome) recessive condition.[2] This means that the gene can be passed on from a carrier to his or her children. In order for sickle cell anemia to affect a person, the gene must be inherited from both the mother and the father, so that the child has two recessive sickle cell genes (a homozygous inheritance). People who inherit one sickle cell gene from one parent and one normal gene from the other parent, i.e. heterozygous patients, have a condition called sickle cell trait. Their bodies make both sickle hemoglobin and normal hemoglobin. They may pass the trait on to their children.

The effects of sickle-cell anemia vary from person to person. People who have the disease suffer from varying degrees of chronic pain and fatigue. With proper care and treatment, the quality of health of most patients will improve. Doctors have learned a great deal about sickle cell anemia since its discovery in 1979. They know its causes, its effects on the body, and possible treatments for complications. Sickle cell anemia has no widely available cure. A bone marrow transplant is the only treatment method currently recognized to be able to cure the disease, though it does not work for every patient. Finding a donor is difficult and the procedure could potentially do more harm than good. Treatments for sickle cell anemia are generally aimed at avoiding crises, relieving symptoms, and preventing complications. Such treatments may include medications, blood transfusions, and supplemental oxygen.

During the first step of the experiment, skin cells (also known as fibroblasts) were collected from infected test mice and put in a culture. The fibroblasts were reprogrammed by infecting them with retroviruses that contained genes common to embryonic stem cells. These genes were the same four used by Yamanaka (Oct-4, SOX2, KLF4, and Myc) in his earlier study. The investigators were trying to produce cells with the potential to differentiate into any type of cell needed (i.e. pluripotent stem cells). As the experiment continued, the fibroblasts multiplied into identical copies of iPS cells. The cells were then treated to form the mutation needed to reverse the anemia in the mice. This was accomplished by restructuring the DNA containing the defective globin gene into DNA with the normal gene through the process of homologous recombination. The iPS cells then differentiated into blood stem cells, or hematopoietic stem cells. The hematopoietic cells were injected back into the infected mice, where they proliferate and differentiate into normal blood cells, curing the mice of the disease.[3][4][verification needed]

To determine whether the mice were cured from the disease, the scientists checked for the usual symptoms of sickle cell disease. They examined the blood for mean corpuscular volume (MCV) and red cell distribution width (RDW) and urine concentration defects. They also checked for sickled red blood cells. They examined the DNA through gel electrophoresis, checking for bands that display an allele that causes sickling. Compared to the untreated mice with the disease, which they used as a control, "the treated animals had marked increases in RBC counts, healthy hemoglobin, and packed cell volume levels".[5]

Researchers examined "the urine concentration defect, which results from RBC sickling in renal tubules and consequent reduction in renal medullary blood flow, and the general deteriorated systemic condition reflected by lower body weight and increased breathing."[5] They were able to see that these parts of the body of the mice had healed or improved. This indicated that "all hematological and systemic parameters of sickle cell anemia improved substantially and were comparable to those in control mice."[5] They cannot say if this will work in humans because a safe way to inject the genes for the induced pluripotent cells is still needed.[citation needed]

The reprogramming of the induced pluripotent stem cells in mice was successfully duplicated in humans within a year of the successful experiment on the mice. This reprogramming was done in several labs and it was shown that the iPS cells in humans were almost identical to original embryonic stem cells (ES cells) that are responsible for the creation of all structures in a fetus.[1] An important feature of iPS cells is that they can be generated with cells taken from an adult, which would circumvent many of the ethical problems associated with working with ES cells. These iPS cells also have potential in creating and examining new disease models and developing more efficient drug treatments.[6] Another feature of these cells is that they provide researchers with a human cell sample, as opposed to simply using an animal with similar DNA, for drug testing.

One major problem with iPS cells is the way in which the cells are reprogrammed. Using gene-carrying viruses has the potential to cause iPS cells to develop into cancerous cells.[1] Also, an implant made using undifferentiated iPS cells, could cause a teratoma to form. Any implant that is generated from using these iPS cells would only be viable for transplant into the original subject that the cells were taken from. In order for these iPS cells to become viable in therapeutic use, there are still many steps that must be taken.[5][7]

In the future, researchers hope that induced pluripotent cells may be used to treat other diseases. Pluripotency is a crucial part of disease treatment because iPS cells are capable of differentiation in a way that is very similar to embryonic stem cells which can grow into fully differentiated tissues. iPS cells also demonstrate high telomerase activity and express human telomerase reverse transcriptase, a necessary component in the telomerase protein complex. Also, iPS cells expressed cell surface antigenic markers expressed on ES cells. Also, doubling time and mitotic activity are cornerstones of ES cells, as stem cells must self-renew as part of their definition. As said, iPS cells are morphologically similar to embryonic stem cells. Each cell has a round shape, a large nucleolus and a small amount of cytoplasm. One day, the process may be used in practical settings to provide a fundamental way of regeneration.

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Induced pluripotent stem-cell therapy - Wikipedia, the ...

Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanakas lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells.[1] He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent." [2]

Pluripotent stem cells hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.

The most well-known type of pluripotent stem cell is the embryonic stem cell. However, since the generation of embryonic stem cells involves destruction (or at least manipulation) [3] of the pre-implantation stage embryo, there has been much controversy surrounding their use. Further, because embryonic stem cells can only be derived from embryos, it has so far not been feasible to create patient-matched embryonic stem cell lines.

Since iPSCs can be derived directly from adult tissues, they not only bypass the need for embryos, but can be made in a patient-matched manner, which means that each individual could have their own pluripotent stem cell line. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. While the iPSC technology has not yet advanced to a stage where therapeutic transplants have been deemed safe, iPSCs are readily being used in personalized drug discovery efforts and understanding the patient-specific basis of disease.[citation needed]

Depending on the methods used, reprogramming of adult cells to obtain iPSCs may pose significant risks that could limit their use in humans. For example, if viruses are used to genomically alter the cells, the expression of oncogenes (cancer-causing genes) may potentially be triggered. In February 2008, scientists announced the discovery of a technique that could remove oncogenes after the induction of pluripotency, thereby increasing the potential use of iPS cells in human diseases.[4] In April 2009, it was demonstrated that generation of iPS cells is possible without any genetic alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.[5] The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells).

iPSCs are typically derived by introducing a specific set of pluripotency-associated genes, or reprogramming factors, into a given cell type. The original set of reprogramming factors (also dubbed Yamanaka factors) are the genes Oct4 (Pou5f1), Sox2, cMyc, and Klf4. While this combination is most conventional in producing iPSCs, each of the factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even non-related genes such as lineage specifiers.

iPSC derivation is typically a slow and inefficient process, taking 12 weeks for mouse cells and 34 weeks for human cells, with efficiencies around 0.01%0.1%. However, considerable advances have been made in improving the efficiency and the time it takes to obtain iPSCs. Upon introduction of reprogramming factors, cells begin to form colonies that resemble pluripotent stem cells, which can be isolated based on their morphology, conditions that select for their growth, or through expression of surface markers or reporter genes.

Induced pluripotent stem cells were first generated by Shinya Yamanaka's team at Kyoto University, Japan, in 2006.[1] Their hypothesis was that genes important to embryonic stem cell function might be able to induce an embryonic state in adult cells. They began by choosing twenty-four genes that were previously identified as important in embryonic stem cells, and used retroviruses to deliver these genes to fibroblasts from mice. The mouse fibroblasts were engineered so that any cells that reactivated the ESC-specific gene, Fbx15, could be isolated using antibiotic selection.

Upon delivery of all twenty-four factors, colonies emerged that had reactivated the Fbx15 reporter, resembled ESCs, and could propagate indefinitely. They then narrowed their candidates by removing one factor at a time from the pool of twenty-four. By this process, they identified four factors, Oct4, Sox2, cMyc, and Klf4, which as a group were both necessary and sufficient to obtain ESC-like colonies under selection for reactivation of Fbx15.

Similar to ESCs, these first-generation iPSCs showed unlimited self-renewal and demonstrated pluripotency by contributing to lineages from all three germ layers in the context of embryoid bodies, teratomas, fetal chimeras. However, the molecular makeup of these cells, including gene expression and epigenetic marks, was somewhere between that of a fibroblast and an ESC, and the cells also failed to produce viable chimeras when injected into developing embryos.

In June 2007, the same group published a breakthrough study along with two other independent research groups from Harvard, MIT, and the University of California, Los Angeles, showing successful reprogramming of mouse fibroblasts into iPS cells. Unlike the first generation of iPS cells, these cells could produce viable chimeric mice and could contribute to the germline, the 'gold standard' for pluripotent stem cells. These cells were derived from mouse fibroblasts by retroviral-mediated expression of the same four transcription factors (Oct4, Sox2, cMyc, Klf4), but the researchers used a different marker to select for pluripotent cells. Instead of Fbx15, they used Nanog, a gene that is functionally important in ESCs. By using this different strategy, the researchers were able to create iPS cells that were more similar to ESCs than the first generation of iPS cells, and independently proved that it was possible to create iPS cells that are functionally identical to ESCs.[6][7][8][9]

Unfortunately, two of the four genes used (namely, c-Myc and KLF4) are oncogenic, and 20% of the chimeric mice developed cancer. In a later study, Yamanaka reported that one can create iPSCs even without c-Myc. The process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer.[10]

Induced pluripotent cells have been made from adult stomach, liver, skin cells, blood cells, prostate cells and urinary tract cells.[11]

In November 2007, a milestone was achieved[12][13] by creating iPSCs from adult human cells; two independent research teams' studies were released one in Science by James Thomson at University of WisconsinMadison[14] and another in Cell by Shinya Yamanaka and colleagues at Kyoto University, Japan.[15] With the same principle used earlier in mouse models, Yamanaka had successfully transformed human fibroblasts into pluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a retroviral system. Thomson and colleagues used OCT4, SOX2, NANOG, and a different gene LIN28 using a lentiviral system.

On 8 November 2012, researchers from Austria, Hong Kong and China presented a protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine on Nature Protocols.[16] This method of acquiring donor cells is comparatively less invasive and simple. The team reported the induction procedure to take less time, around 2 weeks for the urinary cell culture and 3 to 4 weeks for the reprogramming; and higher yield, up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) were found to show good differentiation potential, and thus represent an alternative choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.[16]

Although the methods pioneered by Yamanaka and others have demonstrated that adult cells can be reprogrammed to iPS cells, there are still challenges associated with this technology:

The table at right summarizes the key strategies and techniques used to develop iPS cells over the past half-decade. Rows of similar colors represents studies that used similar strategies for reprogramming.

One of the main strategies for avoiding problems (1) and (2) has been to use small compounds that can mimic the effects of transcription factors. These molecule compounds can compensate for a reprogramming factor that does not effectively target the genome or fails at reprogramming for another reason; thus they raise reprogramming efficiency. They also avoid the problem of genomic integration, which in some cases contributes to tumor genesis. Key studies using such strategy were conducted in 2008. Melton et al. studied the effects of histone deacetylase (HDAC) inhibitor valproic acid. They found that it increased reprogramming efficiency 100-fold (compared to Yamanakas traditional transcription factor method).[25] The researchers proposed that this compound was mimicking the signaling that is usually caused by the transcription factor c-Myc. A similar type of compensation mechanism was proposed to mimic the effects of Sox2. In 2008, Ding et al. used the inhibition of histone methyl transferase (HMT) with BIX-01294 in combination with the activation of calcium channels in the plasma membrane in order to increase reprogramming efficiency.[26] Deng et al. of Beijing University reported on July 2013 that induced pluripotent stem cells can be created without any genetic modification. They used a cocktail of seven small-molecule compounds including DZNep to induce the mouse somatic cells into stem cells which they called CiPS cells with the efficiency at 0.2% comparable to those using standard iPSC production techniques. The CiPS cells were introduced into developing mouse embryos and were found to contribute to all major cells types, proving its pluripotency.[27][28]

Ding et al. demonstrated an alternative to transcription factor reprogramming through the use of drug-like chemicals. By studying the MET (mesenchymal-epithelial transition) process in which fibroblasts are pushed to a stem-cell like state, Dings group identified two chemicals ALK5 inhibitor SB431412 and MEK (mitogen-activated protein kinase) inhibitor PD0325901 which was found to increase the efficiency of the classical genetic method by 100 fold. Adding a third compound known to be involved in the cell survival pathway, Thiazovivin further increases the efficiency by 200 fold. Using the combination of these three compounds also decreased the reprogramming process of the human fibroblasts from four weeks to two weeks. [29][30]

Another key strategy for avoiding problems such as tumor genesis and low throughput has been to use alternate forms of vectors: adenovirus, plasmids, and naked DNA and/or protein compounds.

In 2008, Hochedlinger et al. used an adenovirus to transport the requisite four transcription factors into the DNA of skin and liver cells of mice, resulting in cells identical to ESCs. The adenovirus is unique from other vectors like viruses and retroviruses because it does not incorporate any of its own genes into the targeted host and avoids the potential for insertional mutagenesis.[31] In 2009, Freed et al. demonstrated successful reprogramming of human fibroblasts to iPS cells.[32] Another advantage of using adenoviruses is that they only need to present for a brief amount of time in order for effective reprogramming to take place.

Also in 2008, Yamanaka et al. found that they could transfer the four necessary genes with a plasmid.[33] The Yamanaka group successfully reprogrammed mouse cells by transfection with two plasmid constructs carrying the reprogramming factors; the first plasmid expressed c-Myc, while the second expressed the other three factors (Oct4, Klf4, and Sox2). Although the plasmid methods avoid viruses, they still require cancer-promoting genes to accomplish reprogramming. The other main issue with these methods is that they tend to be much less efficient compared to retroviral methods. Furthermore, transfected plasmids have been shown to integrate into the host genome and therefore they still pose the risk of insertional mutagenesis. Because non-retroviral approaches have demonstrated such low efficiency levels, researchers have attempted to effectively rescue the technique with what is known as the piggyBac transposon system. The lifecycle of this system is shown below. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving any footprint mutations in the host cell genome. As demonstrated in the figure, the piggyBac transposon system involves the re-excision of exogenous genes, which eliminates issues like insertional mutagenesis

In January 2014, two articles were published claiming that a type of pluripotent stem cell can be generated by subjecting the cells to certain types of stress (bacterial toxin, a low pH of 5.7, or physical squeezing); the resulting cells were called STAP cells, for stimulus-triggered acquisition of pluripotency.[34]

In light of difficulties that other labs had replicating the results of the surprising study, in March 2014, one of the co-authors has called for the articles to be retracted.[35] On 4 June 2014, the lead author, Obokata agreed to retract both the papers [36] after she was found to have committed research misconduct as concluded in an investigation by RIKEN on 1 April 2014.[37]

Studies by Blelloch et al. in 2009 demonstrated that expression of ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhances the efficiency of induced pluripotency by acting downstream of c-Myc .[38] More recently (in April 2011), Morrisey et al. demonstrated another method using microRNA that improved the efficiency of reprogramming to a rate similar to that demonstrated by Ding. MicroRNAs are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Morriseys team worked on microRNAs in lung development, and hypothesized that their microRNAs perhaps blocked expression of repressors of Yamanakas four transcription factors. Possible mechanisms by which microRNAs can induce reprogramming even in the absence of added exogenous transcription factors, and how variations in microRNA expression of iPS cells can predict their differentiation potential discussed by Xichen Bao et al.[39]

[citation needed]

The generation of iPS cells is crucially dependent on the genes used for the induction.

Oct-3/4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.

Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed.[42]

Gene expression and genome-wide H3K4me3 and H3K27me3 were found to be extremely similar between ES and iPS cells.[43][citation needed] The generated iPSCs were remarkably similar to naturally isolated pluripotent stem cells (such as mouse and human embryonic stem cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity, authenticity, and pluripotency of iPSCs to naturally isolated pluripotent stem cells:

Recent achievements and future tasks for safe iPSC-based cell therapy are collected in the review of Okano et al.[55]

The task of producing iPS cells continues to be challenging due to the six problems mentioned above. A key tradeoff to overcome is that between efficiency and genomic integration. Most methods that do not rely on the integration of transgenes are inefficient, while those that do rely on the integration of transgenes face the problems of incomplete reprogramming and tumor genesis, although a vast number of techniques and methods have been attempted. Another large set of strategies is to perform a proteomic characterization of iPS cells. The Wu group at Stanford University has made significant progress with this strategy.[56] Further studies and new strategies should generate optimal solutions to the five main challenges. One approach might attempt to combine the positive attributes of these strategies into an ultimately effective technique for reprogramming cells to iPS cells.

Another approach is the use of iPS cells derived from patients to identify therapeutic drugs able to rescue a phenotype. For instance, iPS cell lines derived from patients affected by ectodermal dysplasia syndrome (EEC), in which the p63 gene is mutated, display abnormal epithelial commitment that could be partially rescued by a small compound[57]

An attractive feature of human iPS cells is the ability to derive them from adult patients to study the cellular basis of human disease. Since iPS cells are self-renewing and pluripotent, they represent a theoretically unlimited source of patient-derived cells which can be turned into any type of cell in the body. This is particularly important because many other types of human cells derived from patients tend to stop growing after a few passages in laboratory culture. iPS cells have been generated for a wide variety of human genetic diseases, including common disorders such as Down syndrome and polycystic kidney disease.[58][59] In many instances, the patient-derived iPS cells exhibit cellular defects not observed in iPS cells from healthy patients, providing insight into the pathophysiology of the disease.[60] An international collaborated project, StemBANCC, was formed in 2012 to build a collection of iPS cell lines for drug screening for a variety of disease. Managed by the University of Oxford, the effort pooled funds and resources from 10 pharmaceutical companies and 23 universities. The goal is to generate a library of 1,500 iPS cell lines which will be used in early drug testing by providing a simulated human disease environment.[61]

A proof-of-concept of using induced pluripotent stem cells (iPSCs) to generate human organ for transplantation was reported by researchers from Japan. Human liver buds (iPSC-LBs) were grown from a mixture of three different kinds of stem cells: hepatocytes (for liver function) coaxed from iPSCs; endothelial stem cells (to form lining of blood vessels) from umbilical cord blood; and mesenchymal stem cells (to form connective tissue). This new approach allows different cell types to self-organize into a complex organ, mimicking the process in fetal development. After growing in vitro for a few days, the liver buds were transplanted into mice where the liver quickly connected with the host blood vessels and continued to grow. Most importantly, it performed regular liver functions including metabolizing drugs and producing liver-specific proteins. Further studies will monitor the longevity of the transplanted organ in the host body (ability to integrate or avoid rejection) and whether it will transform into tumors.[62][63] Using this method, cells from one mouse could be used to test 1,000 drug compounds to treat liver disease, and reduce animal use by up to 50,000.[64]

Embryonic cord-blood cells were induced into pluripotent stem cells using plasmid DNA. Using cell surface endothelial/pericytic markers CD31 and CD146, researchers identified 'vascular progenitor', the high-quality, multipotent vascular stem cells. After the iPS cells were injected directly into the vitreous of the damaged retina of mice, the stem cells engrafted into the retina, grew and repaired the vascular vessels.[65][66]

In a study conducted in China in 2013, Superparamagnetic iron oxide (SPIO) particles were used to label iPSCs-derived NSCs in vitro. Labeled NSCs were implanted into TBI rats and SCI monkeys 1 week after injury, and then imaged using gradient reflection echo (GRE) sequence by 3.0T magnetic resonance imaging (MRI) scanner. MRI analysis was performed at 1, 7, 14, 21, and 30 days, respectively, following cell transplantation. Pronounced hypointense signals were initially detected at the cell injection sites in rats and monkeys and were later found to extend progressively to the lesion regions, demonstrating that iPSCs-derived NSCs could migrate to the lesion area from the primary sites. The therapeutic efficacy of iPSCs-derived NSCs was examined concomitantly through functional recovery tests of the animals. In this study, we tracked iPSCs-derived NSCs migration in the CNS of TBI rats and SCI monkeys in vivo for the first time. Functional recovery tests showed obvious motor function improvement in transplanted animals. These data provide the necessary foundation for future clinical application of iPSCs for CNS injury.[67]

In 2014, type O red blood cells were synthesized at the Scottish National Blood Transfusion Service from iPSC. The cells were induced to become a mesoderm and then blood cells and then red blood cells. The final step was to make them eject their nuclei and mature properly. Type O can be transfused into all patients. Each pint of blood contains about two trillion red blood cells, while some 107 million blood donations are collected globally every year. Human transfusions were not expected to begin until 2016.[68]

The first human clinical trial using autologous iPSCs is approved by the Japan Ministry Health and will be conducted in 2014 in Kobe. iPSCs derived from skin cells from six patients suffering from wet age-related macular degeneration will be reprogrammed to differentiate into retinal pigment epithelial (RPE) cells. The cell sheet will be transplanted into the affected retina where the degenerated RPE tissue has been excised. Safety and vision restoration monitoring is expected to last one to three years.[69][70] The benefits of using autologous iPSCs are that there is theoretically no risk of rejection and it eliminates the need to use embryonic stem cells.[70]

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As many of you know, the pioneering, first of its kind IPSC clinical study in Japan has been suspended as I first blogged about here.

In the comments section of that blog post there has been a helpful overall discussion that has involved Dr. Masayo Takahashi, the leader of the trial. It is great that Dr. Takahashi has been participating in this discussion and I commend her for that openness.

This comment stream has been particularly important because the media have only minimally reported on this important development. There have been only a few articles in Japanese (several months ago) and as far as I know only one in English, which was posted in the last day or so in The New Scientist. Unfortunately The New Scientist article, as many have noted here, used an inflammatory title invoking a supposed cancer scare and some over-the-top language. Although that article had some bits of important info, the negative bias in the article made it overall not very helpful. Some readers of that article were likely confused by how it was written and the title.

The clinical study in question is for macular degeneration and involves the use of sheets of retinal pigmented epithelial cells (RPE) made from IPSC (e.g. see image above from RIKEN).Several of us have been discussing the suspension of this trial over on Twitter too including Dr. Takahashi (@masayomasayo). Some tweets by the community have been constructive. Others not so much.

Two main possible issues have come up in the discussion of the reasons for the trial stopping: (1) six mutations were detected in the 2nd patients IPSC and (2) significant regulatory changes are on the way in Japan that apparently in some way will delimit IPSC research there. Dr. Takahashi has indicated that the latter reason was the dominant factor in their decision to suspend the trial. The fact that the 2nd patients IPSC reportedly had six mutations that were not present in the original somatic cells warrantsfurther discussion too. For example, when and how did these mutations arise? To be clear, however, I do not see (based on the information available) that there was a cancer scare by any stretch of the imagination as The New Scientist article had indicated.

At some point a restarted version of this study will likely focus on allogeneic use of IPSC perhaps via an IPSC bank being developed by Dr. Shinya Yamanaka. For many years the consensus, most exciting aspect of IPSCs in the field was considered to be their potential for use as the basis for powerful patient-specific autologous therapies. The apparent planned shift to non-autologous clinical use of IPSC in this case raises the question of how it would be superior or substantially different to the use of hESC, other than that making IPSC does not involve the use of a leftover IVF embryo.

This development also raises a 2nd question as to whether there will be a domino effect now of other clinical studies or trials that are in the works using IPSC switching to allogeneic paths as well. In other words, is this a historic, turning point moment for the IPSC field in Japan overall away from an autologous path?Or is the switch here to allogeneic just a one time, one study decision? More info on the regulatory changes is needed to help clarify the answer to this question and the path forward as well.

Hopefully the regulatory body in Japan (Ministry of Education?) that has made or is making the relevant regulatory changes will announce them publicly in detail soon. If that information is already out there (e.g. in Japanese on the web) perhaps someone can find it and well post it here.

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The ability to convert one cell type into another has caused great excitement in the stem cell field. Two main techniques exist: one reprograms somatic cells into pluripotent stem cells (iPS cells), the other converts somatic cells directly into other types of specialized cells (transdifferentiation). These techniques raise high hopes that patient-personalized cell therapies will become a reality in the not-so-distant future.

The first technique, developed by Yamanaka in 2006, makes it possible to convert essentially any cell type in the body back into pluripotent stem cells that are almost identical to embryonic stem cells. This is done by adding a quartet of proteins: the transcription factors Oct4, Sox2, Klf4 and Myc. The resulting iPS cells can be grown and multiplied almost indefinitely without losing their potential to differentiate (or change) into a broad range of cell types. If a clinician wanted to use this technology to treat a patient with say, Parkinson's disease, she/he would prepare a skin biopsy, grow skin-derived cells called fibroblasts in the lab, introduce the Yamanaka combination of four proteins and wait a couple of months for the formation of stable populations or iPS cell lines. Since iPS cells can proliferate indefinitely, they can be isolated relatively easily and a small initial population can be used to produce a large number of cells. In our hypothetical Parkinsons treatment, the multiplied iPS cells would then be made to differentiate into dopaminergic neurons, the cell type that is deficient in Parkinson's patients. As a final step the neurons would be purified and injected back into the patient.

Comparison of iPS reprogramming and transdifferentiation: These processes might eventually be applied in the clinic for cell replacement therapies.

An alternative to the iPS procedure is transdifferentiation. This approach uses transcription factors to convert a given cell type directly into another specialized cell type, without first forcing the cells to go back to a pluripotent state. Research in the 1980s and 90s showed that fibroblasts can be converted directly into muscle cells at very high efficiencies using the transcription factor MyoD. Similarly, scientists found they could use a transcription factor called C/EBP alpha to turn lymphocytes into macrophages (different types of white blood cell). However, this transdifferentiation approach has only more recently taken off in the stem cell community. One reason for the slow start is that it took the Yamanaka experiments on iPS cells to convince many skeptics that cell reprogramming is possible at all. Another is that for a long time it seemed that direct conversions could only be achieved between 'sister cells', such as between two types of blood cells. The relationship between cell types is sometimes pictured as a developmental or epigenetic landscape.

:Landscape of development: The four main germ layers in which cells develop are divided by tectonic plates. Transitions between cell types are hardest when they cross over tectonic plates. See also Graf & Enver, 2009 and Waddington, 1957.

In this view, pluripotent cells (iPS cells and embryonic stem cells) sit at the very top of the landscape and can produce cells that roll down the valleys to convert into the different types of specialized cells sitting on the bottom. Once the cells are settled in a particular area, travelling across a mountain ridge into a different region to become an unrelated cell type is a tough challenge, but as mentioned before, sister or neighbouring cell types can be moved relatively easily over a small hill from one neighbouring valley to the next, given the right encouragement. This restriction to 'small jumps' between related cell types kept transdifferentiation within the realm of basic research studies. Then, in 2010, another barrier was broken when a group of researchers at Stanford demonstrated that a combination of three neural transcription factors can induce 'large jumps' between distantly related cell types by converting fibroblasts into functional neurons, cell types that belong to different germ layers. This study showed that cells can be pushed to traverse tectonic plates, opening up the prospect that any desired specialized cell could be generated from essentially any other cell type, once the right transcription factor formula is known. Since then, several other transitions across germ layers have been reported.

So, which of the two approaches iPS or transdifferentiation will make it into the clinic, and which will be first? Because both technologies are patient-specific, there is virtually no risk of immune rejection. However, there are a number of hurdles that must be taken regardless of the technique used. For example, reprogrammed cells must be free of vectors inserted into the DNA that can potentially cause mutations and thus cancer. The cells produced must also be fully functional, engraft efficiently after transplantation and survive long-term. For these and other reasons, not all degenerative diseases might be amenable to treatment with cell replacement strategies.

The iPS approach has the advantage that it permits obtaining large numbers of cells. Also, genetic defects can be corrected at the iPS cell stage, meaning that specialized cells made for the patient would no longer have the defect. A disadvantage is its complexity, high costs and the length of time required to produce first iPS cells and then the specialized cells needed for transplantation. There is also a risk that residual iPS cells in the transplant could cause tumors. However, recently there have been major advances to bring pluripotent cell technology closer to the clinic. For example, in ongoing clinical trials, teams in the US and Japan have transplanted retinal pigmented epithelium derived from embryonic and iPS cell cultures into the eyes of patients with inherited or acquired macular degeneration and preliminary results about improving vision are encouraging. The eye is a privileged site for transplantation since it is largely protected from immune attack. In other ongoing efforts teams in Japan, USA and elsewhere are developing iPS cell derived platelets to prevent thrombocytopenia, such as often occurs after chemotherapy of cancer. A major advantage of using platelets is that they have no nuclei, essentially eliminating cancer risk after transplantation. Other labs are engaged in developing methods to generate dopaminergic neurons and insulin-producing cells from pluripotent cells with the goal to eventually ameliorate or even cure Parkinsons disease and diabetes.

The main advantages of converting a specialized cell directly into another are the relative simplicity and short times required, reducing the costs in a clinical setting. However, most current protocols use retro- or lentiviral vectors to introduce combinations of transcription factors, with a danger of introducing undesired mutations. Here a promising alternative approach is to induce cell fate conversions by transiently expressing microRNAs (Victor et al., Neuron 2014), although this method is so far is limited to the generation of specific types of neurons. More generally, it is presently unclear whether it will be possible to generate the large numbers of specialized cells required for cell therapy via the transdifferentiation route, and whether the new cell types are of similar quality as those generated from pluripotent cells.

In conclusion, it can safely be predicted that the iPS approach will make it into the clinic first. Time will tell
whether specialized cells generated by direct cell conversions will also be eventually used for cell therapy.

Related articles on EuroStemCell:

More from Thomas Graf:

Scientific papers The research papers mentioned in the above article:

Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts. Victor MB, Richter M, Hermanstyne TO, Ransdell JL, Sobiesky C, Deng PY, Klyachko VA, Nerbonne, JM and Yoo AS. Neuron, Neuron 84, 311-323 (2014)

Scientific review articles of interest (may require subscription):

First use of the landscape metaphor described above:

Landscape diagram provided by Debbie Maizels of Zoobotanica. Fibroblast image (header) by Tilo Kunath.

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Cell replacement therapies: iPS technology or ...