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Archive for the ‘Cardiac Stem Cells’ Category

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Stem cell therapy for heart failure: first implant of …

16.01.2015 - Press release

Circulation, metabolism, nutrition

On the 21 October 2014, Professor Philippe Menasch and his team from the cardiovascular surgery service of the Georges Pompidou European Hospital, AP-HP, carried out a transplant of cardiac cells derived from human embryonic stem cells*, according to a method developed by the Department of Cell and Tissue Biotherapies of the Saint-Louis hospital, directed by Professor Jrme Larghero and through research led by this group within Inserm. The surgery, coupled with a coronary bypass*, was carried out on a woman of 68 years suffering from severe heart failure. Ten weeks after the intervention, the patient is feeling well, her condition has improved markedly, with no complications having been observed. This promising advance was presented this Friday, 16 January 2015 at the XXV European Days Conference of the French Society of Cardiology.

Human embryonic stem cells. Transplantation of undifferentiated human embryonic stem cells into rat heart organotypic cultures. Presence of human cells, in the cardiac parenchyma of the rat two months after injection. The human cells are positive for human nuclear antigen marking (red). Cardiac rat tissue is positive for cardiac troponin 1 marking (green). I-Stem (Institiute for Stem Cell Therapy), Evry Genopole. Inserm/Habeler, Walter

The transplant was carried out as part of a clinical trial developed by the Public Hospitals of Paris (AP-HP) and through the work of the teams from AP-HP, Inserm and the universities of Paris-Descartes and Paris-Diderot. The cardiac cells were prepared according to a technique developed by the Department of Cell and Tissue Biotherapies of the Saint-Louis hospital. The cytogenetics laboratory of the Antoine Bclre Hospital and the French General Agency for Health Products and Equipment also contributed to the preparation of this phase I trial which will enable the verification of the safety and feasibility of the procedure

For 20 years Professor Menasch, currently co-director of an Inserm team within PARCC (Paris Centre for Cardiovascular Research), and his colleagues have been involved in stem cell* therapy for heart failure.

The team first tested the implant of skeletal muscle stem cells in necrosed areas of the heart in the laboratory. These cells were implanted into the heart of a patient with heart failure for the first time in the world on 15 June 2000. Following an initial series of these implants, always coupled with a coronary bypass, the team coordinated a European multi-centre, randomised, placebo-controlled trial whose results have still not been able to establish any significant benefit of these cells on the contractile function of patients hearts. One of the conclusions drawn from this trial was that to be fully efficient, transplanted cells should resemble the cells of the tissue to be repaired as much as possible, in this instance cardiac tissue. It was then decided to venture along the path of embryonic stem cells. Derived from embryos conceived in in vitro fertilisation, these cells do in fact possess pluripotent properties, that is, they are capable of developing into any type of cell of the body, including of course cardiac cells, as soon as they receive the appropriate signals during the culture cycle in the laboratory.

In 2007, the team then composed of, among others, Michel Pucat, Director of Research at Inserm, and Philippe Menasch showed that human embryonic stem cells could be differentiated into cardiac cells after being transplanted into the failing hearts of rats. Since then, many experiments have been carried out on different animal species in order to validate the efficacity of these cells and to optimise conditions which can guarantee maximum safety. At the end of this stage, a bank of pluripotent embryonic stem cells was formed in the conditions which satisfied all regulatory constraints applying to biological products for human use. Then, the Department of Cell and Tissue Biotherapies of the Saint-Louis hospital, still in liaison with the Inserm teams, developed and tested specialisation procedures for cells in order to produce young cardiac cells from them. The focus was then on the purification of the cells directed like so in order to ensure that the final product was expunged of any remaining pluripotent cells which could be potentially tumorigenic.

Besides, as initial experience with muscular stem cells showed the limitations of administering cells by multiple injections, their transfer is now performed using a patch that the cells are incorporated into. This patch is then placed on the area of the infarction. To that end, after the purification stage, the cardiac cells are incorporated into a circular fibrin gel which is applied, during the surgical procedure, to the necrosed area with just a few sutures ensuring that it is anchored to the cardiac tissue.

This type of surgery is aimed at serious heart failure which doesnt respond to the usual medicinal treatments but is not at the stage of a complete heart transplant. This is a promising advance, which we hope will enrich the therapeutic arsenal available to treat heart failure today explains Prof. Menasch. We are continuing the trial, which authorises us to carry out four other transplants. It would seem already that the benefits of the cells are linked mainly to the substances that they secrete. The direct administration of the substances, without going through a transplant of productive cells, is a path to explore.

This project has been entirely financed by funds from public intstitutions and societies and was authorised by the French National Agency for the Safety of Medicines and Health Products (ANSM) after agreement with the Agency for Biomedicine for the importation and research on human embryonic cells.

Cell therapy: refers to cell transplants aiming to restore the function of tissue or an organ when it has been altered by an accident, illness or ageing. These therapies have benefited from recent scientific advances on stem cells and give millions of patients the hope of regenerative medicine.

Embryonic or pluripotent stem cells: they can renew indefinitely (self-renewal), multiply in a culture and be differentiated into more than 200 types of cell. In the course of development, they are destined to form all types of the bodys tissue.

Coronary bypass: a technique that enables the redirection of the bloodstream towards the cardiac muscle, by using a graft (coming from the saphenous vein or from a thoracic artery.) One end of the graft is connected to the aorta, the main artery supplying the coronary arteries; the other end is connected to the coronary artery, situated just behind the site of the obstruction. This creates a detour enabling the oxygenated blood to circulate towards the heart.

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Cardiac muscle cells as good as progenitors for heart …

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

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

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

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

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

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Cardiac muscle cells as good as progenitors for heart ...

Resident c-kit+ cells in the heart are not cardiac stem …

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A recent study reported that a subpopulation of endothelial cells yields progeny with CSC characteristics in the adult mouse heart34. This subpopulation purportedly arises from endothelialmesenchymal transition and gives rise to cardiomyocytes that contribute to heart renewal34. To determine whether c-kit+ endothelial cells produce CSCs that further differentiate into cardiomyocytes following cardiac injury, we performed LAD ligation on cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ mice (24 months old, n=4, Fig. 6b). cTnTMerCreMer/+ mediates specific and effective myocardial recombination after tamoxifen induction35. If c-kitnlacZ-H2B-GFP/+ cells become cardiomyocytes and if c-kit expression is maintained in these cells, then c-kitH2B-GFP-positive cells would be detected. However, after tamoxifen was injected at 37dps and 3135dps (three tamoxifen treatments for each period), we detected no c-kitH2B-GFP-positive cells in the infarcted region (Fig. 6c), although myocardial recombination was widely detected in and adjacent to the infarcted region (as revealed by ROSA26RtdTomato staining, Fig. 6c). Furthermore, examination of adult c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ mice after LAD ligation (35 months old, n=3, Fig. 6d) revealed

In the lineage tracing experiments used to determine the myocardial potential of c-kit+ cells during development and after cardiac injury in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animal models, very small number of cTnTH2B-GFP-positive cells was detected (Fig. 4g, ~66156 cells; and Fig. 6e, ~20 cells). In all cases, the number was extremely low when compared with the total number of c-kitH2B-tdTomato-positive cells (

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

Isolation and Expansion of Adult Cardiac Stem Cells From …

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Myocardial Repair (Echocardiography)

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

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

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

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

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

Human cardiac stem cells – PNAS

Communicated by Eugene Braunwald, Harvard Medical School, Boston, MA, July 19, 2007 (received for review May 2, 2007)

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.

The recent identification of different classes of cardiac progenitor cells has suggested that the heart is not a terminally differentiated, postmitotic organ but an organ regulated by a stem cell compartment (1). The possibility has also been raised that stem cells are present in the normal and pathological human heart (2, 3). Together, these results point to a shift in paradigm concerning the biology of the heart and put forward potential therapeutic strategies for the failing heart. However, the actual existence of a human cardiac stem cell (hCSC) remains to be demonstrated. By definition, stem cells have to be self-renewing, clonogenic, and multipotent in vitro and in vivo (4, 5), and no studies to date have shown that the human heart contains primitive cells with these properties. Cells with limited growth and differentiation ability may acquire only the myocyte, endothelial cell (EC) or smooth muscle cell (SMC) lineage in vitro, and may not be capable of forming functionally competent myocardium in vivo. hCSCs have to be able to replace dead tissue with contracting myocardium composed of cardiomyocytes and vascular structures, independently from cell fusion. Heterokaryons divide poorly and have, at best, a transient positive impact on the age of the fused cells (6). Here, we report that these issues have been resolved, and hCSCs may represent a form of therapy for the diseased heart.

The documentation of hCSCs requires the identification of interstitial structures with the characteristics of stem cell niches and the recognition of the mechanisms of stem cell division that regulate niche homeostasis and the self-renewing properties of the human heart in vivo (7). We have found that the human heart contains clusters of hCSCs that are intimately connected by gap junctions and adherens junctions to myocytes and fibroblasts (Fig. 1 AC); myocytes and fibroblasts represent the supporting cells within the cardiac niches (7). Additionally, symmetric and asymmetric division of hCSCs was detected, respectively, by the uniform and nonuniform localization of the cell-fate determinants Numb and -adaptin (7) at one or both poles of hCSCs in mitosis (Fig. 1 D and E). The commitment to the myocyte lineage of hCSCs was also found within the niches. The coexpression of the stem cell antigen c-kit and myocyte transcription factors and sarcomeric proteins [see supporting information (SI) Fig. 6] is consistent with a lineage relationship between hCSCs and myocyte formation. C-kit POS cells expressing transcription factors for SMCs and ECs were also detected (data not shown). In the niches, hCSCs and committed cells were negative for hematopoietic markers and KDR (SI Table 1). These findings in the normal human heart, together with earlier observations in the diseased heart (3, 8), support the notion of a resident hCSC compartment that gives rise to the various cardiac cell progenies.

Cardiac niches and hCSC division. Sections of normal human myocardium. (AC) Cluster of c-kit POS cells (green). Arrows in A define the areas in B and C. Gap (connexin 43: Cx43, white; arrowheads) and adherens (N-cadherin: N-cadh, magenta; arrowheads) junctions are shown at higher magnification. Cx43 and N-cadh are present between c-kit POS cells and myocytes (-SA, red) and fibroblasts (procollagen, light blue); fibronectin, yellow. (D and E) Mitosis (phospho-H3, magenta; arrows) in c-kit POS cells; -adaptin (D, white) and Numb (E, yellow) show a uniform (D) and nonuniform (E) localization in the mitotic c-kit POS cells.

C-kit POS cells, i.e., hCSCs, were prepared with two methodologies. The first consisted of the enzymatic dissociation of myocardial samples from which c-kit POS cells were sorted with immunobeads and plated at low density (SI Fig. 7 AC ) to obtain multicellular clones from single founder cells. This procedure was dictated by the small size of the samples, 30 mg, which precluded FACS analysis at the outset. Successful isolation was obtained in 8 of 12 cases. The phenotype of the freshly isolated cells was characterized by FACS in 6 additional cases in which larger samples, 60 mg, were available. C-kit POS cells comprised 1.1 1.0% of the entire cell population. They were different from human bone marrow cells and were negative for markers of hematopoietic cells and KDR (Fig. 2 A and B; SI Table 2). Only small fractions of hCSCs expressed GATA4 and Nkx2.5, 0.5%.

Human CSCs. (A and B) Scatter plots of hCSCs (A) and human bone marrow cells (B). hCSCs do not express hematopoietic markers, KDR, GATA4, and Nkx2.5. (C) Nuclei (blue) of hCSCs were stained with a telomere probe (magenta). Lymphoma cells with short (7-kbp) and long (48-kbp) telomeres are shown for comparison. (D) Products of telomerase activity in hCSCs start at 50 bp and display a 6-bp periodicity. Samples treated with RNase and CHAPS buffer were used as negative controls, and HeLa cells were used as positive control. The band at 36 bp corresponds to an internal control for PCR efficiency. Optical density (arbitrary units, AU) is shown as mean SD.

With the second protocol, samples were cultured by the primary explant technique (SI Fig. 7 D and E ). Successful cell outgrowth was obtained in 46 of 70 cases. A monolayer of 6,000 cells was present at the periphery of each tissue aggregate, 3 weeks after seeding. C-kit POS cells accounted for 1.6 1.4%. Adherent cells at passage P0 were analyzed by immunocytochemistry and FACS (SI Fig. 8; SI Tables 1 and 2). In enzymatically dissociated cells, lineage negative (Lin) c-kit POS cells were 41 14%, and early committed cells (GATA4-positive) were 59 14%. Corresponding values with the primary explant technique were 52 12% and 48 12% (SI Fig. 9A ). In the presence of serum, hCSCs obtained with both protocols attached rapidly and continued to grow up to P8, undergoing 24 population doublings (PDs); the majority of experiments were concluded at P8. Cells maintained a stable phenotype and did not reach growth arrest. The percentage of c-kit POS cells did not vary from P1 to P8, averaging 71 8%. Undifferentiated cells constituted 63 6%. Ki67POS cycling-cells averaged 48 10%. p16INK4a, a cdk inhibitor, was present in 6 4% of the cells (SI Fig. 9 BD ). Thus, hCSCs are distinct from bone marrow cells and can be isolated and expanded in vitro.

To determine whether hCSCs reach senescence in culture, telomeric length was evaluated by Q-FISH (Fig. 2 C). From P3P4 (912 PDs) to P5P6 (1518 PDs) and P8P9 (2427 PDs), average telomere length in hCSCs decreased from 9.3 to 8.2 and 6.9 kbp, respectively (SI Fig. 10). From P3 to P9 there were 18 PDs with an average telomeric shortening of 130 bp per PD. This rate of telomere attrition is comparable with that of human bone marrow stem cells (9). Additionally, nearly 50% of the telomerase activity in hCSCs at P3P4 was still present at P8P9 (Fig. 2 D).

Critical telomere length associated with growth arrest and cellular senescence of hCSCs and human hematopoietic SCs varies from 2.0 to 1.5 kbp (3, 9). The fraction of hCSCs with critical telomeric shortening increased from 1% at P3P4 to 2% at P5P6 and 5% at P8P9. However, after 2427 PDs at P8P9, 69% hCSCs had telomere length 5.0 kbp (SI Fig. 10). It can be predicted that cells at P8P9 can undergo 23 additional PDs (52 = 3kbp/0.13kbp = 23 PDs) before irreversible growth arrest (10). In theory, 50 PDs can result in the formation of 1 1015 hCSCs before replicative senescence is reached. Thus, hCSCs can be extensively grown in vitro in the absence of a major loss in their expansion potential.

hCSCs obtained by enzymatic digestion and explant technique were plated at limiting dilution and in Terasaki plates, respectively. In the first case, 1,530 c-kit POS cells were seeded, and after 34 weeks, 11 clones were generated. In the second case, cells were placed in individual wells, and 53 clones were formed from 6,700 seeded cells. Thus, hCSCs had 0.70.8% cloning efficiency (Fig. 3 AC). Clones were expanded and characterized. Doubling time was 29 10 h, and 90 7% of cells after 5 days were BrdUPOS. Clonogenic hCSCs retained largely their primitive state and were negative for hematopoietic markers, KDR, and transcription factors and cytoplasmic proteins of cardiac cells (SI Fig. 11 A and B; SI Table 1).

In vitro properties of hCSCs. (AC) Clones formed by hCSCs (c-kit, green) isolated by enzymatic digestion (A and C) or primary explant (B). The number of cells increased with time (C). (D) hCSCs generate myocytes positive for cardiac myosin heavy-chain (MHC), -SA, and -cardiac-actinin (-actinin). Sarcomeres are apparent (Insets); phalloidin, green. (E) Myocyte shortening in cells derived from clonogenic hCSCs was recorded by two-photon microscopy and laser line-scan imaging (Left). The line scan is shown (Right), and arrowheads point to individual contractions. (F) Myocytes derived from EGFP-positive hCSCs, cocultured with neonatal myocytes. EGFP-positive human myocytes shorten (arrowheads) with electrical stimulation. (G) Calcium transients in EGFP-positive human myocytes and EGFP-negative rat myocytes (calcium indicator Rhod-2, red).

In differentiating medium, hCSCs gave rise to myocytes, SMCs, and ECs (Fig. 3 D; SI Fig. 11 C and D ). Developing myocytes had sarcomere striation (Fig. 3 D) and, after electrical stimulation at 1 Hz, showed contractile activity (Fig. 3 E). Moreover, hCSCs were infected with a lentivirus expressing EGFP and cocultured with neonatal rat myocytes. Two weeks later, cultures were stimulated, and 9% shortening of EGFP-positive human myocytes was detected (Fig. 3 F). In the presence of the calcium indicator Rhod-2, calcium transient was identified in EGFP-positive human myocytes and EGFP-negative rat myocytes (Fig. 3 G). Thus, hCSCs form multicellular clones and differentiate into contracting myocytes.

Nonclonogenic hCSCs, collected from eight patients, were injected in the infarcted mouse or rat heart to form chimeric organs containing human myocytes and coronary vessels. Cell treatment led to areas of myocardial regeneration that were located within the infarct and were positive for -sarcomeric actin (-SA) and human AluDNA sequences (Fig. 4 A). Human myocardium was found in 17 of 25 treated mice (68%), and 14 of 19 treated rats (74%). hCSCs were delivered with rhodamine-labeled microspheres for the recognition of the sites of injection and correct administration of cells (1). The absence of myocardial regeneration was due to technical failure to properly inject hCSCs in the rodent heart. Conversely, successful cell implantation was invariably associated with the presence of human myocardium.

hCSCs regenerate infarcted myocardium. (A) Mouse heart 21 days after infarction and injection of hCSCs. Human myocardium (arrowheads) is present within the infarct (MI). BZ, border zone. Areas in rectangles are shown at higher magnification below. Human myocytes are -SA- (red) and Alu- (green) positive. Asterisks indicate spared myocytes. (B) Expression of human (h) genes by real-time RT-PCR in treated infarcted rats at 511 and 1221 days. Clonogenic hCSCs were used for comparison of human transcripts. (C) Electrophoresis of real-time RT-PCR products (for sequences see SI Fig. 11J ).

The human myocardium comprised 1.3 0.9 mm3 in mice and 3.7 2.9 mm3 in rats. Accumulation of new cells was also determined by BrdU labeling because BrdU was given to the animals throughout the experiment (SI Fig. 12 A and B ). The human myocardium consisted of myocytes that occupied 84% of the tissue, whereas arterioles and capillaries accounted for 8%. The human origin of the myocardium was confirmed by the detection of human Alu and Mlc2v DNA by PCR in sections of regenerated infarcts (SI Fig. 12C ). PCR products had the expected molecular weight, and the nucleotide sequences confirmed the specificity of the assay (SI Fig. 12 DF ).

Three control groups were used: (i) unsuccessfully treated-animals (eight mice, five rats); (ii) immunodeficient infarcted mice (n = 12) and immunosuppressed infarcted rats (n = 9) injected with PBS; and (iii) immunosuppressed infarcted rats injected with c-kit-negative cells obtained from the unfractionated cell population at P1 (n = 16). Infarct size was similar in all groups: 48 9% in mice and 54 11% in rats. Myocardial regeneration was absent in control hearts with the exception of 3 of the 16 hearts treated with c-kit-negative cells. In one case, a few -SA and Alu-positive cells were found within the infarct, whereas, in the other two, a small band of human myocardium was identified near the border zone (SI Fig. 12 G and H ).

For completeness, clonogenic hCSCs were injected in infarcted rats shortly after coronary ligation to determine their multipotentiality in vivo (n = 6) and establish whether multipotentiality persisted when cell implantation was performed 5 days after coronary occlusion under the condition of a fully developed ischemic injury (n = 10). In both cases, clonogenic hCSCs regenerated the infarcted myocardium (SI Fig. 12I ) by forming human myocytes and coronary vessels (see below).

Real-time RT-PCR was used to demonstrate human transcripts for myocyte (MLC2v, connexin 43), SMC (smooth-muscle myosin heavy-chain 11 = Mhc 11) and EC (vWF) genes, and the housekeeping gene GAPDH in infarcted rat hearts treated with clonogenic hCSCs. Because there is no baseline in the rat myocardium for the analysis of human genes, clonogenic hCSCs were used for comparison (n = 4). With respect to clonogenic hCSCs, there was a substantial up-regulation of human myocardial transcripts for parenchymal and vascular cells in the infarcted heart (Fig. 4 B). The expression of human MLC2v, connexin 43, Mhc 11, and vWF increased from 511 days (n = 8) to 1221 days (n = 15) after infarction and cell implantation. RT-PCR products had the expected molecular weight (Fig. 4 C), and the nucleotide sequences confirmed the specificity of the assay (SI Fig. 12J ). Thus, hCSCs generate human myocardium.

After the identification of AluDNA, cardiac myosin heavy-chain (MHC), troponin I, and -SA were detected in human myocytes. Moreover, GATA4, MEF2C, connexin 43, and N-cadherin were identified (SI Figs. 13 and 14). Human myocytes varied in size from 100 to 2,900 m3 (SI Fig. 14). Human coronary arterioles and capillaries were also found (SI Figs. 13 and 14). The number of human arterioles and capillaries was comparable in rats and mice; there was one capillary/eight myocytes (SI Fig. 14), and the diffusion distance for oxygen averaged 18 m. These parameters are similar to those found in the late fetalneonatal human heart. Thus, hCSCs differentiate into human myocytes and coronary vessels, leading to the formation of a chimeric heart.

Two protocols were used to test whether the generation of human myocardium involved fusion events between hCSCs and rodent cells. hCSCs were infected with a lentivirus carrying Cre-recombinase (infection efficiency = 90%) and injected in the infarcted heart of mice expressing loxP-flanked EGFP (n = 6). If fusion were to occur, EGFP transcription would be activated in the recipient cells by Cre-mediated excision of the stop codon in the EGFP promoter (1). At 10 days after infarction and cell implantation, newly formed human myocardial cells showed a nuclear localization of Cre protein (Fig. 5 A; SI Fig. 15). However, human myocytes and vessels were negative for EGFP, indicating that the formation of heterokaryons was not involved in cardiac repair.

Integration of human myocardium. (A) Human myocytes are Cre-recombinase-positive (white) but EGFP negative. (B) Human myocytes and vessels show, at most, two human X-chromosomes (X-Chr, white dots; arrowheads). Mouse X-Chr (magenta dots; arrows) are present in myocytes of the border zone (BZ). (C) Transmural infarct in a treated rat; human myocardium (arrowheads) is present within the infarct. The area in the rectangle is shown at higher magnification (Bottom); human myocytes are -SA- (red) and Alu- (green) positive. Echocardiogram shows contraction in the infarcted wall (arrowheads). (D) Ventricular function. Results are mean SD. * and , Difference, P

The second protocol consisted of the evaluation of the number of sex chromosomes by Q-FISH in human myocytes and coronary vessels. Because female human cells were injected in female mice and rats, human, rat and mouse X-chromosomes were measured. We never found a colocalization of a human X-chromosome with a mouse or rat X-chromosome in regenerated myocytes and vessels (Fig. 5 B). Human myocytes, SMCs, and ECs carried, at most, two human X-chromosomes. Thus, hCSCs form human myocardium independently from cell fusion.

Echocardiograms were examined retrospectively after the histological documentation of transmural infarcts and the presence of human myocardium (Fig. 5 C; SI Fig. 16 AC ). Tissue regeneration restored partly contractile function in the infarct, resulting in an increase of ejection fraction (SI Fig. 16D ), attenuation of chamber dilation (SI Fig. 16E ), and improvement of ventricular function (Fig. 5 D).

The interaction between human and rodent myocardium was determined by an ex vivo preparation and two-photon microscopy. EGFP-positive hCSCs were injected in infarcted mice, and the heart was studied 2 weeks later (n = 6). After the blockade of contraction and spontaneous activity, the heart was perfused with Rhod-2 and stimulated at 1 Hz. Calcium transient was recorded in EGFP-positive human myocytes and EGFP-negative mouse myocytes. The synchronicity in calcium tracings between these myocyte populations documented their functional integration (Fig. 5 E). hCSC-derived myocytes acquired the properties of the recipient rodent myocardium, indicating that primitive cells of human origin possess a high level of plasticity. Additionally, connexin 43 was found between human and rodent myocytes (Fig. 5 F) demonstrating their structural coupling. Thus, both myocardial components of the chimeric heart participate in the performance of the infarcted heart.

The current work demonstrates that the human heart possesses a pool of clonogenic hCSCs that can acquire the myocyte, SMC, and EC lineages in vitro and in vivo. The ability of hCSCs to create cardiomyocytes and coronary vessels in vivo provides strong evidence in favor of the role that hCSCs have in cardiac homeostasis and myocardial regeneration. Besides their therapeutic implications, these observations challenge the view of the heart as a postmitotic organ (11) and form the basis of a paradigm in which multipotent hCSCs modulate the physiological turnover of the heart. Understanding the mechanisms of cardiac homeostasis would offer the opportunity to potentiate this process and promote cardiac repair after injury.

Human cells with the ability to differentiate into cardiomyocytes have been obtained from myocardial biopsies and were claimed to possess the properties of stem cells (2). These cells express the typical markers of human circulating endothelial-progenitor cells (EPCs): CD34, CD31, and KDR, together with c-kit (12). The expression of CD34, CD31, and KDR does not compromise the ability of these circulating cells to acquire the myocyte lineage in vitro (13) and in vivo (14). The presence of these epitopes, however, suggests that these cells originate from the bone marrow and only subsequently accumulate within the heart. These early findings failed to provide evidence for the clonogenicity of these cells in vitro and their multilineage differentiation in vivo, which are critical for the recognition of a tissue-specific adult stem cell (1). The inability of these cells to generate a functional human myocardium in vivo is consistent with the role of EPCs in cardiac repair; they acquire, at low efficiency, the myocyte lineage and exert a paracrine effect on the infarcted heart (13).

Conversely, as demonstrated here, hCSCs are positive for the stem cell antigen c-kit but are negative for the hematopoietic and endothelial antigens CD45, CD34, CD31, and KDR; CD45 and KDR are typically expressed in a subset of bone marrow c-kit POS cells that have the ability to migrate to the heart after injury (12). Stem cell niches have been identified here in the normal human myocardium, and hCSCs divide symmetrically and asymmetrically and give rise to differentiating and lineage-negative cells. This provides evidence in favor of a linear relationship between hCSCs and myocyte formation. Additionally, these observations do not support the notion of dedifferentiation of mature myocytes with the acquisition of a stem cell phenotype. Importantly, clonogenic hCSCs have the inherent potential to form contracting myocardium integrated structurally and functionally with the recipient heart. Although CSCs with similar characteristics were shown in animal models (4, 5), the applicability of this information to humans was seriously questioned and considered a major limitation for the clinical implementation of CSCs (15).

In the current study, three possibilities were considered in the formation of human myocardium within the infarcted mouse and rat heart (1): (i ) Growth and differentiation of hCSCs; (ii ) Fusion of hCSCs with the surviving mouse or rat cardiac cells, followed by proliferation of the heterokaryons and generation of myocytes and coronary vessels; and (iii ) A combination of these two processes. The evaluation of human, mouse, and rat sex chromosomes together with the Cre-lox strategy has indicated that the generation of human myocardium involved only the commitment of hCSCs to cardiomyocytes, SMCs, and ECs. The unlikely involvement of cell fusion was supported by the size (1002,900 m3) of human myocytes. If fusion were to be implicated, the newly formed human myocytes should have had a volume of at least 25,000 m3 or larger, that is, the volume of adult mouse and rat cardiomyocytes. It is improbable that fusion of a primitive cell with a terminally differentiated myocyte can induce division of a highly specialized and rapidly contracting cell permanently withdrawn from the cell cycle (1, 6).

The identification of a resident hCSC pool in the human heart is apparently at variance with the small foci of myocardial regeneration present after acute and chronic infarcts or pressure overload in patients (3, 16). The limitation that resident hCSCs have in reconstituting myocardium after infarction has been interpreted as the unequivocal documentation of the inability of the adult heart to create cardiomyocytes (11). The inevitable evolution of ischemic injury is myocardial scarring with loss of mass and contractile function. A possible explanation of this apparent paradox has been obtained in animal models of the human disease (5). Stem cells are present throughout the infarcted myocardium but, despite the postulated resistance of these cells to death stimuli, they follow the same pathway of cardiomyocytes and die by apoptosis. The fate of hCSCs is comparable with that of the other cells, and myocyte formation is restricted to the viable portion of the human heart (3).

It might come as a surprise, but a similar phenomenon occurs in solid and nonsolid organs, including the skin, liver, intestine, and kidney. In all cases, occlusion of a supplying artery leads to scar formation mimicking cardiac pathology (1720). In the presence of polyarteritis nodosa and vasculitis, microinfarcts develop in the intestine and skin, and resident SCs do not repair the damaged organs (21). In nonsolid organs, infarcts of the bone marrow are seen with sickle cell anemia (21). Thus, the SC compartment appears to be properly equipped to modulate growth during postnatal development and regulate homeostasis in adulthood. However, SCs do not respond effectively to ischemic injury or, late in life, to aging and senescence of the organ and organism (22, 23).

Current knowledge supports the notion that primitive cells are present in the heart at the very beginning of embryonic life and regulate heart morphogenesis and postnatal development (24). By introducing the EGFP gene in the mouse embryo, at the stage of the morulablastocyst transition, the patterns of myocardial histogenesis have been defined and the presence of a common progenitor of cardiomyocytes in prenatal and postnatal life suggested (24). The documentation of myocardial specification of embryonic stem cells (25, 26), in particular c-kit POS Nkx2.5POS cells (26), supports the view that a pool of resident c-kit POS progenitors is implicated in cardiac morphogenesis. These findings are consistent with the existence of a pool of primitive cells in the adult human heart.

hCSCs have been isolated expanded and characterized in vitro and in vivo after implantation in the infarcted rodent heart. Protocols are described in SI Materials and Methods .

The lentivirus expressing Cre-recombinase was kindly provided by Drs. Chang and Terada (University of Florida, Gainesville, FL) and the lymphoma cells by Dr. Blasco (Spanish National Cancer Centre, Madrid, Spain). This work was supported by National Institutes of Health grants.

Author contributions: C.B., M.R., T.H., R.W.S., K.U., R.B., J.K., A.L., and P.A. designed research; C.B., M.R., T.H., J.T., A.N., A.D.A., S.Y.-A., I.T., R.W.S., N.L., S.C., A.P.B., D.A.D., E.Z., F.Q., K.U., R.E.M., J.K., and A.L. performed research; C.B., M.R., T.H., J.T., A.N., A.D.A., S.Y.-A., I.T., R.W.S., N.L., D.A.D., K.U., R.E.M., R.B., J.K., A.L., and P.A. analyzed data; and C.B., M.R., J.K., A.L., and P.A. wrote the paper.

Conflict of interest statement: P.A. has applied for a patent.

This article contains supporting information online at http://www.pnas.org/cgi/content/full/0706760104/DC1.

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Human cardiac stem cells - PNAS

Growing beating cardiac tissue from stem cells: New model …

Researchers at the University of California, Berkeley, in collaboration with scientists at the Gladstone Institutes, have developed a template for growing beating cardiac tissue from stem cells, creating a system that could serve as a model for early heart development and a drug-screening tool to make pregnancies safer.

In experiments to be published Tuesday, July 14, in the journal Nature Communications, the researchers used biochemical and biophysical cues to prompt stem cells to differentiate and self-organize into micron-scale cardiac tissue, including microchambers.

"We believe it is the first example illustrating the process of a developing human heart chamber in vitro," said Kevin Healy, a UC Berkeley professor of bioengineering, who is co-senior author of the study with Dr. Bruce Conklin, a senior investigator at the Gladstone Institute of Cardiovascular Disease and a professor of medical genetics and cellular and molecular pharmacology at UC San Francisco. "This technology could help us quickly screen for drugs likely to generate cardiac birth defects, and guide decisions about which drugs are dangerous during pregnancy."

Screening for drug toxicity

To test the potential of the system as a drug-screening tool, the researchers exposed the differentiating cells to thalidomide, a drug known to cause severe birth defects. They found that at normal therapeutic doses, the drug led to abnormal development of microchambers, including decreased size, problems with muscle contraction and lower beat rates compared with heart tissue that had not been exposed to thalidomide.

"We chose drug cardiac developmental toxicity screening to demonstrate a clinically relevant application of the cardiac microchambers," said Conklin. "Each year, as many as 280,000 pregnant women are exposed to drugs with evidence of potential fetal risk. The most commonly reported birth defects involve the heart, and the potential for generating cardiac defects is of utmost concern in determining drug safety during pregnancy."

The new milestone comes nearly four months after Healy and other UC Berkeley researchers publicly debuted a system of beating human heart cells on a chip that could be used to screen for drug toxicity. However, that heart-on-a-chip device used pre-differentiated cardiac cells to mimic adult-like tissue structure.

In this new study, the scientists mimicked human tissue formation by starting with stem cells genetically reprogrammed from adult skin tissue to form small chambers with beating human heart cells. Conklin's lab at Gladstone, an independent, nonprofit life science research organization affiliated with UC San Francisco, supplied these human induced pluripotent stem cells for this study.

The undifferentiated stem cells were then placed onto a circular-patterned surface that served to physically regulate cell differentiation and growth.

Location, location, location

By the end of two weeks, the cells that began on a two-dimensional surface environment started taking on a 3D structure as a pulsating microchamber. Moreover, the cells had self-organized based upon whether they were positioned along the perimeter or in the middle of the colony.

Compared with cells in the center, cells along the edge experienced greater mechanical stress and tension, and appeared more like fibroblasts, which form the collagen of connective tissue. The center cells, in contrast, developed into cardiac muscle cells. Such spatial organization was observed as soon as the differentiation started. Center cells lost the expression of octamer-binding transcription factor 4 (OCT4) and epithelial cadherin (E-cadherin) faster than perimeter cells, which are critical to the development of heart tissue.

"This spatial differentiation happens in biology naturally, but we demonstrated this process in vitro," said study lead author Zhen Ma, a UC Berkeley postdoctoral researcher in bioengineering. "The confined geometric pattern provided biochemical and biophysical cues that directed cardiac differentiation and the formation of a beating microchamber."

Could eventually replace animal models

Modeling early heart development is difficult to achieve in a petri dish and tissue culture plates, the study authors said. This area of study has typically involved the dissection of animals at different stages of development to study the formation of organs, and how that process can go wrong.

"The fact that we used patient-derived human pluripotent stem cells in our work represents a sea change in the field," said Healy. "Previous studies of cardiac microtissues primarily used harvested rat cardiomyocytes, which is an imperfect model for human disease."

The researchers pointed out that while this study focused on heart tissue, there is great potential for use of this technology to study other organ development.

"Our focus here has been on early heart development, but the basic principles of patterning of human pluripotent stem cells, and subsequently differentiating them, can be readily expanded into a broad range of tissues for understanding embryogenesis and tissue morphogenesis," said Healy.

The National Institutes of Health and a Siebel Postdoctoral Fellowship helped support this research.

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Growing beating cardiac tissue from stem cells: New model ...

Development of a scalable suspension culture for cardiac …

Highlights

We present a strategy to optimize cardiac differentiation in suspension for hiPSCs.

The matrix-free suspension platform integrates hPSC expansion and differentiation.

Cardiac production in suspension achieves >90% purity with 1L spinner flasks.

The production process in suspension is defined, scalable, and GMP compliant.

To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2109 CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

Myocardial infarction and heart failure are leading causes of death worldwide. As the myocardium has a very limited regenerative capacity, endogenous cell regeneration cannot adequately compensate for heart damage caused by myocardial infarction. The concept of cell replacement therapy is an appealing approach to the treatment of these cardiac diseases. HPSCs are an attractive cell source for cell replacement therapies because they can be expanded indefinitely in culture and efficiently differentiated into a variety of cell lineages, including cardiac cells. However, current hPSC expansion and differentiation methods rely on adherent cell culture systems that are challenging to scale up to the levels required to support pre-clinical and clinical studies.

Activin/Nodal/TGF-, BMP, and Wnt signaling play pivotal roles in regulating mesoderm and cardiac specification during embryo development (Arnold and Robertson, 2009, Buckingham et al., 2005, Tam and Loebel, 2007, David et al., 2008, Naito et al., 2006, Ueno et al., 2007andBurridge et al., 2012). Significant progress has been made in the cardiac differentiation process by modulating Activin, BMP, and Wnt pathways, which can efficiently drive differentiation to over 80% purity of CM (Burridge et al., 2014, Kattman et al., 2011, Lian et al., 2012, Yang et al., 2008, Zhang et al., 2012andZhu et al., 2011). Using an adherent cell culture platform, one study revealed that using 2 small Wnt pathway modulators to sequentially activate and then inhibit Wnt signaling at different differentiation stages of the culture is sufficient to drive cardiac differentiation and generate CM with high purity (Lian et al., 2012). In spite of this, adherent culture systems have limited scalability and are not practical to support the anticipated CM requirements of clinical trials. Alternatively, using an embryoid body (EB) differentiation method, a complex cardiac induction procedure involving stage-specific treatments with growth factors and small molecules to modulate Activin/Nodal, BMP, and Wnt pathways has been reported to be effective in cardiac differentiation in a suspension culture system (Kattman et al., 2011andYang et al., 2008). However, the process of generating EBs is inefficient, rendering this method impractical for large scale CM production. An additional limitation of these approaches for scale-up application is that both methods are based on the expansion of the hPSCs in adherent culture and the subsequent CM differentiation process in either adherent culture or as EBs. The labor intensiveness and limited scalability of current processes have been the primary bottle necks to the large scale production of CM for clinical applications of hPSC-derived CM.

Pre-clinical studies suggest that doses of up to one billion CM will be required to achieve therapeutic benefit after transplantation (Chong et al., 2014andLaflamme and Murry, 2005). In order to meet the current CM demand for pre-clinical studies and the anticipated demand for foreseeable clinical studies, development of a robust, scalable and cGMP-compliant differentiation process for the production of both hPSCs and hPSC-derived CM is essential. Suspension cell culture is an attractive platform for large scale manufacture of cell products for its scale-up capacity. Application of a suspension culture platform to support hPSC growth in matrix-free cell aggregates has been well established (Amit et al., 2010, Krawetz et al., 2010, Olmer et al., 2010, Singh et al., 2010, Steiner et al., 2010andChen et al., 2012). We previously also reported the development of a defined, scalable and cGMP-compliant suspension system to culture hPSCs in the form of cell aggregates (Chen et al., 2012). With this suspension culture system, hPSC cultures can be serially passaged and consistently expanded. In the present study we adapted our suspension culture system to establish a robust, scalable and cGMP-compliant process for manufacturing CM. We were able to use hPSC aggregates generated in the suspension culture system directly to produce CM with high efficiency and yield in suspension with various scales of spinner flasks. We optimized various critical process parameters including: small molecule concentration, induction timing and agitation rates for differentiation cultures in spinner flasks with scales up to 1L. In this study, we integrated undifferentiated hPSC expansion and small molecule-induced cardiac differentiation into a scalable suspension culture system using spinner flasks, providing a streamlined and cGMP-compliant process for scale-up CM differentiation and production.

We routinely maintained the hPSCs lines H7 (WA07, WiCell), ESI-017 (BioTime), and a hiPSC line (a gift from Dr. Joseph Wu, Stanford) in the form of cell aggregates in suspension culture as previously described (Chen et al., 2012). Briefly, suspension-adapted hPSCs were seeded as single cells at a density of 2.53105 cells/mL in 125, 500, or 1000mL spinner flasks (Corning) containing culture medium (StemPro hESC SFM, Thermo Fisher Scientific, Life Technologies) with 40ng/mL bFGF (Life Technologies) and 10M Y27632 (EMD Millipore). Stirring rates were adjusted to between 5070rpm depending on the vessel size and hPSC line. Medium was changed every day by demi-depletion with fresh culture medium without Y27632. Cells were dissociated with Accutase (Millipore) into single cells and passaged every 34days when the aggregate size reached approximately 300m. Cell suspension cultures were maintained in 5% CO2 with 95% relative humidity at 37C.

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Development of a scalable suspension culture for cardiac ...

Adult Cardiac Stem Cells Are Multipotent and Support …

Abstract

The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin c-kitPOS cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.

Until recently, the accepted paradigm in cardiac biology considered the adult mammalian heart to be a postmitotic organ without regenerative capacity. It has been assumed that from shortly after birth to adulthood and senescence the heart has a relatively stable but slowly diminishing number of myocytes. This static view of the myocardium implied that both myocyte death and myocyte regeneration had little role in cardiac cellular homeostasis. Although stem cells have been isolated from many adult tissues including the blood, skin, central nervous system, liver, gastrointestinal tract, and skeletal muscle (see Rosenthal, 2003), the search for a cardiac stem cell has been considered futile given the accepted lack of regenerative potential of this tissue.

Evidence challenging the accepted wisdom has been slowly accumulating McDonnell and Oberpriller 1984andRumyantsev and Broisov 1987. In the past few years, we have documented the existence of cycling ventricular myocytes in the normal and pathologic adult mammalian heart of several species, including humans Kajstura et al. 1998, Beltrami et al. 2001andQuaini et al. 2002. Although these data provided an alternative view of cardiac homeostasis, they also raised questions because it required reconciliation of two apparent contradictory bodies of evidence: the well-documented irreversible withdrawal of cardiac myocytes from the cell cycle soon after birth on one hand MacLellan and Schneider 2000andChien and Olson 2002, and the presence of cycling myocytes undergoing mitosis and cytokinesis on the other. These results raised the question as to the origin of the cycling myocytes and their dramatic increase in response to an acute work overload.

In cases of sex-mismatched cardiac transplants in humans, the female hearts in the male hosts had a significant number of Y positive myocytes and coronary vessels (Quaini et al., 2002). Most likely due to technical differences (Anversa and Nadal-Ginard, 2002a), there are some discrepancies among groups about the degree of cardiac chimerism Muller et al. 2002, Glaser et al. 2002andLaflamme et al. 2002. It is likely that these male cells colonized the female heart after the transplant and subsequently differentiated, although alternative explanations have been raised. These male cells in the female heart presuppose the existence of mobile stem-like cells able to differentiate into the three main cardiac cell types: myocytes, smooth, and endothelial vascular cells.

Primitive cells of donor and recipient origin that express stem cell-related surface antigensc-kit, Sca-1, and MDR1were identified in the recipient hearts. More importantly, identical cells were found in human control hearts Quaini et al. 2002andAnversa and Nadal-Ginard 2002b. It is well known that in early fetal life, c-kitPOS cells colonize the yolk sack, liver, and probably other organs. The colonized organs express stem cell factor (SCF), the ligand of the c-kit receptor (Teyssier-Le Discorde et al., 1999); SCF mRNA is also present in fetal and neonatal myocardium (Kunisada et al., 1998), raising the possibility that stem-like cells could have been in the heart from fetal life. The rapid induction of SCF during myocardial ischemia (Frangogiannis et al., 1998) could be involved in the activation of these cells and explain the significant increase in new myocyte formation (Beltrami et al., 2001). However, the origin of these primitive cells, their presence in normal and pathological hearts, together with the identification of some of them having initiated the cardiomyocyte gene expression program, is suggestive that they might be true cardiac stem cells that give rise to the cycling myocytes detected in the adult heart. If this were the case, their manipulation might provide the opportunity to stimulate myocardial regeneration with endogenous cells. For this reason, we endeavored to establish a precursor-product relationship between these primitive cells and the fully differentiated cardiac cells and to determine, in vitro and in vivo, whether they behave like true adult cardiac stem cells.

To determine whether the putative cardiac stem cells detected in human heart transplants and their controls are bona fide stem cells with cardiogenic potential, we isolated them to test their differentiation potential in vivo and in vitro. For experimental convenience, we chose the rat as the animal model system. We first analyzed whether cells with the cell surface markers commonly expressed by other stem cells could be identified in the adult rat myocardium. Based on the postulated higher number of proliferating stem and precursor cells with age (Morrison et al., 1996), we analyzed the myocardium from older animals. Histological sections of myocardium from Fisher rats 2023 months of age were examined by confocal microscopy for the presence of cells negative for the expression of blood lineage markers (Lin) but positive for the common stem cell markers c-kit (Kondo et al., 2003), Sca-1 (Morrison et al., 1997), and MDR-1 (Sellers et al., 2001). Small Lin cells with a very high nucleus/cytoplasm ratio and positive for each of the above markers were distributed throughout the ventricular and atrial myocardium with a higher density in the atria and the ventricular apex. Because of the role of bone marrow-derived Lin c-kitPOS cells in myocardial regeneration (Orlic et al., 2001), the mesodermal origin of both the heart and the bone marrow, and the use of c-kit as a hematopoietic stem cell marker Morrison et al. 1997, Weissman et al. 2001andKondo et al. 2003, we decided to concentrate on the cardiac cells expressing this marker, the receptor for SCF. Although the density of these cells varied among different regions of the heart, on average we identified one Lin c-kitPOS cell every 1 104 myocytes. It should be noted that most, if not all, of the detected c-kitPOS cells were negative for the pan leukocyte marker CD45 and the endothelial/hematopoietic progenitor marker CD34.

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Jan. 29, 2016 The mechanism used by specialized enzymes to remodel the extremely condensed genetic material in the nucleus of cells in order to control which genes can be used has been discovered. The research ... read more Ultrasound-Based Therapy for Cardiac Stem Cells Recovery Jan. 29, 2016 When cardiac stem cells undergo low-intensity pulsed ultrasound treatment, these cells can perform continuing modifications, tissue remodeling and regeneration of damaged cardiac tissue after a heart ... read more Assessing Stem Cells: New Biomarker Developed Jan. 29, 2016 A research team has found a way to assess the viability of 'manufactured' stem cells known as induced pluripotent stem cells (iPSCs). The team's discovery offers a new way to ... read more Jan. 29, 2016 Industry 4.0 requires comprehensive data collection in order to control highly automated process sequences in complex production environments. One example is the cultivation of living cells. But ... read more Protein Combination Improves Bone Regeneration, Study Shows Jan. 29, 2016 A combination of proteins that could improve clinical bone restoration, and could lead towards the development of therapeutic treatments for skeletal defects, bone loss and osteoporosis, report ... read more Cancer's Surprise Origins, Caught in Action Jan. 28, 2016 For the first time, researchers have visualized the origins of cancer from the first affected cell and watched its spread in a live animal. This work could change the way scientists understand ... read more Research Hints at a Nutritional Strategy for Reducing Autism Risk Jan. 28, 2016 Folic acid has long been touted as an important supplement for women of childbearing age for its ability to prevent defects in the baby's developing brain and spinal cord. In fact, folic acid is ... read more CRISPR Used to Repair Blindness-Causing Genetic Defect in Patient-Derived Stem Cells Jan. 28, 2016 Scientists have used a new gene-editing technology called CRISPR, to repair a genetic mutation responsible for retinitis pigmentosa (RP), an inherited condition that causes the retina to degrade and ... read more Jan. 27, 2016 The Achilles heel of hepatocellular carcinoma, a leading cause of cancer deaths worldwide, has been discovered by researchers. The key to disrupting chemo-resistant stem cells that become liver ... read more Scientists Make an Important Contribution to Decoding the Language of Cells Jan. 27, 2016 There are astonishing similarities between molecular mechanisms in neural stem cells and pancreatic islet cells, new research shows. This may lead to new forms of therapy, particularly for ... read more Jan. 25, 2016 A molecule that interrupts biochemical signals essential for the survival of a certain type of cancer stem cell has been discovered by ... read more How to Detect and Preserve Human Stem Cells in the Lab Jan. 22, 2016 Human stem cells that are capable of becoming any other kind of cell in the body have previously only been acquired and cultivated with difficulty. Scientists have now presented details of a method ... read more Jan. 21, 2016 A research team has now discovered how human macrophages can divide and self-renew almost indefinitely. As the researchers show in their new report, the macrophages achieve this by activating a gene ... read more Jan. 20, 2016 In 1917, Florence Sabin, the first female member of the US National Academy of Sciences, discovered hemangioblasts, the common precursor cells for blood cells and blood vessel endothelia. Her ... read more Breakthrough in Human Cell Transformation Could Revolutionize Regenerative Medicine Jan. 19, 2016 A breakthrough in the transformation of human cells by an international team of researchers could open the door to a new range of treatments for a variety of medical ... read more Jan. 19, 2016 Electrical stimulation of human heart muscle cells engineered from human stem cells aids their development and function, researchers have demonstrated for the first time. They used electrical ... read more Broken UV Light Leads to Key Heart Muscle Cell Discovery Jan. 18, 2016 For a team of investigators trying to generate heart muscle cells from stem cells, a piece of broken equipment turned out to be a good thing. The faulty equipment pushed the researchers to try a ... read more Jan. 14, 2016 Where and when do stem cells first appear during development? Researchers investigated this question by examining how cells organize as the hair follicle first appears in mouse embryos. They ... read more Donor's Genotype Controls Differentiation of Induced Pluripotent Stem Cells Jan. 14, 2016 Pluripotent stem cells derived from different cell types are equally susceptible to reprogramming, indicates a recent study. However, the genotype of the donor strongly influences the differentiation ... read more Mechanism That Controls Neuron Production from Stem Cells Revealed Jan. 13, 2016 The discovery of a mechanism enabling the production of cellular diversity in the developing nervous system has been announced by scientists. This discovery could improve the protocols to ... read more

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Cardiac muscle cell – Wikipedia, the free encyclopedia

Cardiac muscle cells or cardiomyocytes (also known as myocardiocytes[1] or cardiac myocytes[2]) are the muscle cells (myocytes) that make up the cardiac muscle. Each myocardial cell contains myofibrils, which are specialized organelles consisting of long chains of sarcomeres, the fundamental contractile units of muscle cells. Cardiomyocytes show striations similar to those on skeletal muscle cells. Unlike multinucleated skeletal cells, the majority of cardiomyocytes contain only one nucleus, although they may have as many as four.[3] Cardiomyocytes have a high mitochondrial density, which allows them to produce adenosine triphosphate (ATP) quickly, making them highly resistant to fatigue.

There are two types of cells within the heart: the cardiomyocytes and the cardiac pacemaker cells. Cardiomyocytes make up the atria (the chambers in which blood enters the heart) and the ventricles (the chambers where blood is collected and pumped out of the heart). These cells must be able to shorten and lengthen their fibers and the fibers must be flexible enough to stretch. These functions are critical to the proper form during the beating of the heart.[4]

Cardiac pacemaker cells carry the impulses that are responsible for the beating of the heart. They are distributed throughout the heart and are responsible for several functions. First, they are responsible for being able to spontaneously generate and send out electrical impulses. They also must be able to receive and respond to electrical impulses from the brain. Lastly, they must be able to transfer electrical impulses from cell to cell.[5]

All of these cells are connected by cellular bridges. Porous junctions called intercalated discs form junctions between the cells. They permit sodium, potassium and calcium to easily diffuse from cell to cell. This makes it easier for depolarization and repolarization in the myocardium. Because of these junctions and bridges the heart muscle is able to act as a single coordinated unit.[6][7]

Cardiac action potential consists of two cycles, a rest phase and an active phase. These two phases are commonly understood as systole and diastole. The rest phase is considered polarized. The resting potential during this phase of the beat separates the ions such as sodium, potassium and calcium. Myocardial cells possess the property of automaticity or spontaneous depolarization. This is the direct result of a membrane which allows sodium ions to slowly enter the cell until the threshold is reached for depolarization. Calcium ions follow and extend the depolarization even further. Once calcium stops moving inward, potassium ions move out slowly to produce repolarization. The very slow repolarization of the CMC membrane is responsible for the long refractory period.[8][9]

Myocardial infarction, commonly known as a heart attack, occurs when the heart's supplementary blood vessels are obstructed by an unstable build-up of white blood cells, cholesterol, and fat. With no blood flow, the cells die, causing whole portions of cardiac tissue to die. Once these tissues are lost, they cannot be replaced, thus causing permanent damage. Current research indicates, however, that it may be possible to repair damaged cardiac tissue with stem cells,[10] as human embryonic stem cells can differentiate into cardiomyocytes under appropriate conditions.[11]

Humans are born with a set number of heart muscle cells, or cardiomyocytes, which increase in size as our heart grows larger during childhood development. Recent evidence suggests that cardiomyocytes are actually slowly turned over as we age, but that less than 50% of the cardiomyocytes we are born with are replaced during a normal life span.[12] The growth of individual cardiomyocytes not only occurs during normal heart development, it also occurs in response to extensive exercise (athletic heart syndrome), heart disease, or heart muscle injury such as after a myocardial infarction. A healthy adult cardiomyocyte has a cylindrical shape that is approximately 100m long and 10-25m in diameter. Cardiomyocyte hypertrophy occurs through sarcomerogenesis, the creation of new sarcomere units in the cell. During heart volume overload, cardiomyocytes grow through eccentric hypertrophy.[13] The cardiomyocytes extend lengthwise but have the same diameter, resulting in ventricular dilation. During heart pressure overload, cardiomyocytes grow through concentric hypertrophy.[13] The cardiomyocytes grow larger in diameter but have the same length, resulting in heart wall thickening.

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Regenocyte, Stem Cells Used To Treat Cardiovascular Disease

About 128 million people suffer from diseases that might be cured or treated through stem cell therapy. About 58 million of these people suffer from cardiovascular disease.

Cardiovascular disease can manifest itself in many different ways because the blood vessels transport blood to every single part of the body. The heart is the organ that pumps the blood around the body, and it also receives nutrients from the blood vessels (via the coronary vessels). Any interruption of the supply of blood containing nutrients and oxygen to one of the bodys organs leads to functional impairment and, in the worst case scenario, the death of the tissue. One typical example is cardiac arrest, which occurs when the blood supply to the heart muscle is restricted.

Cardiovascular disease can have any number of causes. Some people are born with a susceptibility to vascular disease (e.g. varicose veins), which can be alleviated by taking medication. Other peoples heart and blood vessels can be damaged by external factors. The majority of vascular diseases these days, however, are caused by our modern-day lifestyles. The walls of the blood vessel are always in contact with the blood which flows through them, so they are most commonly affected by unhealthy lifestyles. If someone has an unfavorable haemogram, i.e. if their blood contains too much glucose, cholesterol, triglycerides (fats) or nicotine, this can put the blood vessels under an enormous amount of stress. Glucose adheres to the walls of the blood vessels and the blood constituents, and cholesterol and triglycerides also accumulate on the blood vessel walls. As a result, the blood clumps, the blood vessel walls calcify, turning porous and can no longer perform their biological function properly. Nicotine also constricts the blood vessels, so they narrow and the amount of blood circulating the body is reduced.

If the condition is aggravated by a lack of vessel-protecting substances, the damaged vessels lose their ability to regenerate. The consequences include arteriosclerosis, leg ulceration, dilation of the abdominal artery (aneurysm), cardiac insufficiency, cardiac arrest and stroke. Cardiovascular disease is still the number one cause of death in many other western industrial nations.

Adult Stem Cells derived from the patients own blood are potent and effective to treat heart disease.Patients who have severe cardiac disease with a history of coronary infarction, congestive heart failure, those with previous bypass surgery and stents, cardiomyopathy, and individuals with low ejection fraction (the rate at which the heart pumps) are candidates for this procedure. Patients that survive myocardial infarction have diminished cardiac reserve putting them at risk for subsequent heart failure. Doctors and scientists throughout the world now understand that myocardial repair and regeneration are possible and attainable.

Watch this informative video that highlights one of our patients and how their heart was treated with Stem Cell Therapy. Stem Cell Treatment for the heart saved this patients life.

Regenocyte generates healthy heart muscle cells in the laboratory and then transplants those cells into patients with chronic heart disease. Stem cells cultivated from the patients own blood and transplanted into a damaged heart, can generate new collateral vessels.

Treatment is non-invasive consisting of an intravenous infusion of precursor cardiomyocyte stem cells derived from a patients own blood through a specially designed catheter. This approach increases the engraftment, survival and proliferation of the stem cells to the heart muscle.

To find out more about how stem cells can treat the heart,click hereor call us at (866) 216-5710

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Regenocyte, Stem Cells Used To Treat Cardiovascular Disease

Challenges in identifying the best source of stem cells …

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Challenges in identifying the best source of stem cells ...

Endogenous cardiac stem cell – Wikipedia, the free …

Endogenous cardiac stem cells (eCSCs) are tissue-specific stem progenitor cells harboured within the adult mammalian heart.

They were first discovered in 2003 by Bernardo Nadal-Ginard, Piero Anversa and colleagues [1][2] in the adult rat heart and since then have been identified and isolated from mouse, dog, porcine and human hearts.[3][4]

The adult heart was previously thought to be a post mitotic organ without any regenerative capability. The identification of eCSCs has provided an explanation for the hitherto unexplained existence of a subpopulation of immature cycling myocytes in the adult myocardium. Indeed, recent evidence from a genetic fate-mapping study established that stem cells replenish adult mammalian cardiomyocytes lost by cardiac wear and tear and injury throughout the adult life.[5] Moreover, it is now accepted that myocyte death and myocyte renewal are the two sides of the proverbial coin of cardiac homeostasis in which the eCSCs play a central role.[6] These findings produced a paradigm shift in cardiac biology and opened new opportunities and approaches for future treatment of cardiac diseases by placing the heart squarely amongst other organs with regenerative potential such as the liver, skin, muscle, CNS. However, they have not changed the well-established fact that the working myocardium is mainly constituted of terminally differentiated contractile myocytes. This fact does not exclude, but is it fully compatible with the heart being endowed with a robust intrinsic regenerative capacity which resides in the presence of the eCSCs throughout the individual lifespan.

Briefly, eCSCs have been first identified through the expression of c-kit, the receptor of the stem cell factor and the absence of common hematopoietic markers, like CD45. Afterwards, different membrane markers (Sca-1, Abcg-2, Flk-1) and transcription factors (Isl-1, Nkx2.5, GATA4) have been employed to identify and characterize these cells in the embryonic and adult life.[7] eCSCs are clonogenic, self renewing and multipotent in vitro and in vivo,[8] capable of generating the 3 major cell types of the myocardium: myocytes, smooth muscle and endothelial vascular cells.[9] They express several markers of stemness (i.e. Oct3/4, Bmi-1, Nanog) and have significant regenerative potential in vivo.[10] When cloned in suspension they form cardiospheres,[11] which when cultured in a myogenic differentiation medium, attach and differentiate into beating cardiomyocytes.

In 2012, it was proposed that Isl-1 is not a marker for endogenous cardiac stem cells.[12] That same year, a different group demonstrated that Isl-1 is not restricted to second heart field progenitors in the developing heart, but also labels cardiac neural crest.[13] It has also been reported that Flk-1 is not a specific marker for endogenous and mouse ESC-derived Isl1+ CPCs. While some eCSC discoveries have been brought into question, there has been success with other membrane markers. For instance, it was demonstrated that the combination of Flt1+/Flt4+ membrane markers identifies an Isl1+/Nkx2.5+ cell population in the developing heart. It was also shown that endogenous Flt1+/Flt4+ cells could be expanded in vitro and displayed trilineage differentiation potential. Flt1+/Flt4+ CPCs derived from iPSCs were shown to engraft into the adult myocardium and robustly differentiate into cardiomyocytes with phenotypic and electrophysiologic characteristics of adult cardiomyocytes.[14]

With the myocardium now recognized as a tissue with limited regenerating potential,[15] harbouring eCSCs that can be isolated and amplified in vitro [16] for regenerative protocols of cell transplantation or stimulated to replicate and differentiate in situ in response to growth factors,[17] it has become reasonable to exploit this endogenous regenerative potential to replace lost/damaged cardiac muscle with autologous functional myocardium.

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Heart Stem Cell Trial: Interview With Researcher Roberto …

An interview with Roberto Bolli, MD.

University of Louisville cardiologist Roberto Bolli, MD, led the stem cell study that tested using patients' own heart stem cells to help their hearts recover from heart failure. Though that trial was preliminary, the results look promising -- and may one day lead to a cure for heart failure.

Here, Bolli talks about what this work means and when it might become an option for patients.

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"Realistically, this will not come... for another three or four years, at least," Bolli says. "It may be longer, depending on the results of the next trial, of course."

Larger studies are needed to confirm the procedure's safety and effectiveness. If those succeed, it could be "the biggest advance in cardiovascular medicine in my lifetime," Bolli says.

A total of 20 patients took part in the initial study.

All of them experienced significant improvement in their heart failure and now function better in daily life, according to Bolli. "The patients can do more, there's more ability to exercise, and the quality of life improves markedly," Bolli says.

Bolli's team published its findings on how the patients were doing one year after stem cell treatment in November 2011 in the Lancet, a British medical journal.

Each patient was infused with about 1 million of his or her own cardiac stem cells, which could eventually produce an estimated 4 trillion new cardiac cells, Bolli says. His team plans to follow each patient for two years after their stem cell procedure.

Keep in mind that this was a phase I study. Those focus on safety more than effectiveness.

The results were "much more striking" than past stem cell trials to heal the heart, Bolli says.

This trial was the first in the world to use stem cells derived from the heart. Earlier studies used stem cells gleaned from different bodily sources, including bone marrow, adipose (fat) tissue, and circulating blood. Those showed either no improvement or only modest gains in a patient's left ventricular ejection fraction, a measure of the heart's pumping ability.

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Cardiac muscle – Wikipedia, the free encyclopedia

An isolated cardiac muscle cell, beating

Cardiac muscle (heart muscle) is involuntary striated muscle that is found in the walls and histological foundation of the heart, specifically the myocardium. Cardiac muscle is one of three major types of muscle, the others being skeletal and smooth muscle. These three types of muscle all form in the process of myogenesis. The cells that constitute cardiac muscle, called cardiomyocytes or myocardiocytes, contain only three nuclei.[1][2][pageneeded] The myocardium is the muscle tissue of the heart, and forms a thick middle layer between the outer epicardium layer and the inner endocardium layer.

Coordinated contractions of cardiac muscle cells in the heart propel blood out of the atria and ventricles to the blood vessels of the left/body/systemic and right/lungs/pulmonary circulatory systems. This complex mechanism illustrates systole of the heart.

Cardiac muscle cells, unlike most other tissues in the body, rely on an available blood and electrical supply to deliver oxygen and nutrients and remove waste products such as carbon dioxide. The coronary arteries help fulfill this function.

Cardiac muscle has cross striations formed by rotating segments of thick and thin protein filaments. Like skeletal muscle, the primary structural proteins of cardiac muscle are myosin and actin. The actin filaments are thin, causing the lighter appearance of the I bands in striated muscle, whereas the myosin filament is thicker, lending a darker appearance to the alternating A bands as observed with electron microscopy. However, in contrast to skeletal muscle, cardiac muscle cells are typically branch-like instead of linear.

Another histological difference between cardiac muscle and skeletal muscle is that the T-tubules in the cardiac muscle are bigger and wider and track laterally to the Z-discs. There are fewer T-tubules in comparison with skeletal muscle. The diad is a structure in the cardiac myocyte located at the sarcomere Z-line. It is composed of a single T-tubule paired with a terminal cisterna of the sarcoplasmic reticulum. The diad plays an important role in excitation-contraction coupling by juxtaposing an inlet for the action potential near a source of Ca2+ ions. This way, the wave of depolarization can be coupled to calcium-mediated cardiac muscle contraction via the sliding filament mechanism. Cardiac muscle forms these instead of the triads formed between the sarcoplasmic reticulum in skeletal muscle and T-tubules. T-tubules play critical role in excitation-contraction coupling (ECC). Recently, the action potentials of T-tubules were recorded optically by Guixue Bu et al.[3]

The cardiac syncytium is a network of cardiomyocytes connected to each other by intercalated discs that enable the rapid transmission of electrical impulses through the network, enabling the syncytium to act in a coordinated contraction of the myocardium. There is an atrial syncytium and a ventricular syncytium that are connected by cardiac connection fibres.[4] Electrical resistance through intercalated discs is very low, thus allowing free diffusion of ions. The ease of ion movement along cardiac muscle fibers axes is such that action potentials are able to travel from one cardiac muscle cell to the next, facing only slight resistance. Each syncyntium obeys the all or none law.[5]

Intercalated discs are complex adhering structures that connect the single cardiomyocytes to an electrochemical syncytium (in contrast to the skeletal muscle, which becomes a multicellular syncytium during mammalian embryonic development). The discs are responsible mainly for force transmission during muscle contraction. Intercalated discs are described to consist of three different types of cell-cell junctions: the actin filament anchoring adherens junctions, the intermediate filament anchoring desmosomes , and gap junctions. They allow action potentials to spread between cardiac cells by permitting the passage of ions between cells, producing depolarization of the heart muscle. However, novel molecular biological and comprehensive studies unequivocally showed that intercalated discs consist for the most part of mixed-type adhering junctions named area composita (pl. areae compositae) representing an amalgamation of typical desmosomal and fascia adhaerens proteins (in contrast to various epithelia).[6][7][8] The authors discuss the high importance of these findings for the understanding of inherited cardiomyopathies (such as arrhythmogenic right ventricular cardiomyopathy).

Under light microscopy, intercalated discs appear as thin, typically dark-staining lines dividing adjacent cardiac muscle cells. The intercalated discs run perpendicular to the direction of muscle fibers. Under electron microscopy, an intercalated disc's path appears more complex. At low magnification, this may appear as a convoluted electron dense structure overlying the location of the obscured Z-line. At high magnification, the intercalated disc's path appears even more convoluted, with both longitudinal and transverse areas appearing in longitudinal section.[9]

In contrast to skeletal muscle, cardiac muscle requires extracellular calcium ions for contraction to occur. Like skeletal muscle, the initiation and upshoot of the action potential in ventricular muscle cells is derived from the entry of sodium ions across the sarcolemma in a regenerative process. However, an inward flux of extracellular calcium ions through L-type calcium channels sustains the depolarization of cardiac muscle cells for a longer duration. The reason for the calcium dependence is due to the mechanism of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum that must occur under normal excitation-contraction (EC) coupling to cause contraction. Once the intracellular concentration of calcium increases, calcium ions bind to the protein troponin, which initiate extracellular fluid and intracellular stores, and skeletal muscle, which is only activated by calcium stored in the sarcoplasmic reticulum.

Until recently, it was commonly believed that cardiac muscle cells could not be regenerated. However, a study reported in the April 3, 2009 issue of Science contradicts that belief.[10] Olaf Bergmann and his colleagues at the Karolinska Institute in Stockholm tested samples of heart muscle from people born before 1955 who had very little cardiac muscle around their heart, many showing with disabilities from this abnormality. By using DNA samples from many hearts, the researchers estimated that a 20-year-old renews about 1% of heart muscle cells per year, and about 45 percent of the heart muscle cells of a 50-year-old were generated after he or she was born.

One way that cardiomyocyte regeneration occurs is through the division of pre-existing cardiomyocytes during the normal aging process.[11] The division process of pre-existing cardiomyocytes has also been shown to increase in areas adjacent to sites of myocardial injury. In addition, certain growth factors promote the self-renewal of endogenous cardiomyocytes and cardiac stem cells. For example, insulin-like growth factor 1, hepatocyte growth factor, and high-mobility group protein B1 increase cardiac stem cell migration to the affected area, as well as the proliferation and survival of these cells.[12] Some members of the fibroblast growth factor family also induce cell-cycle re-entry of small cardiomyocytes. Vascular endothelial growth factor also plays an important role in the recruitment of native cardiac cells to an infarct site in addition to its angiogenic effect.

Based on the natural role of stem cells in cardiomyocyte regeneration, researchers and clinicians are increasingly interested in using these cells to induce regeneration of damaged tissue. Various stem cell lineages have been shown to be able to differentiate into cardiomyocytes, including bone marrow stem cells. For example, in one study, researchers transplanted bone marrow cells, which included a population of stem cells, adjacent to an infarct site in a mouse model. Nine days after surgery, the researchers found a new band of regenerating myocardium.[13] However, this regeneration was not observed when the injected population of cells was devoid of stem cells, which strongly suggests that it was the stem cell population that contributed to the myocardium regeneration. Other clinical trials have shown that autologous bone marrow cell transplants delivered via the infarct-related artery decreases the infarct area compared to patients not given the cell therapy.[14]

Occlusion (blockage) of the coronary arteries by atherosclerosis and/or thrombosis can lead to myocardial infarction (heart attack), where part of the myocardium is injured due to ischemia (not receiving enough oxygen). This occurs because coronary arteries are functional end arteries - i.e. there is almost no overlap in the areas supplied by different arteries (anastomoses) so that if one fails, others cannot adequately perfuse the region, unlike in other tissues.

Certain viruses lead to myocarditis (inflammation of the myocardium). Cardiomyopathies are inherent diseases of the myocardium, many of which are caused by genetic mutations.

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Injecting the Heart With Stem Cells Helps Chest Pain – ABC …

George Reed's heart wasn't doing so well: He's 71, and after suffering a heart attack years earlier, Reed had undergone open heart surgery and was put on multiple medications. But nothing seemed to help the dizziness and chest pain he experienced daily.

"I'd get dizzy and just fall over -- sometimes twice a day. I would run my head into the concrete. I was a bloody mess," the Perry, Ohio, native says. Despite his doctor's best efforts, Reed continued to experience angina, a type of chest pain that occurs when the heart doesn't get enough oxygen-rich blood; it can be accompanied by dizziness. So when he was recommended for an experimental study that would inject his own stem cells into his damaged heart, Perry signed on. "I needed something to change," he says.

Researchers gave Reed a drug commonly used in bone marrow transplants that stimulates the marrow to make more stem cells. Then they removed some of Reed's blood, isolated the stem cells and injected them into and around the damaged areas of his heart.

"The goal was to grow new blood vessels with stem cells from the patient's own body," says Dr. Tim Henry, a co-author of the study and director of research at the Minneapolis Heart Institute Foundation.

Within a few months, Reed, along with many of the other 100 or so patients at 26 hospital centers who'd received this stem cell treatment, reported feeling better than he had in years.

"When it started kicking in, I felt like a kid. I felt good," Reed says. He wasn't passing out and falling down anymore.

For Jay Homstad, 49, who was part of the Minnesota branch of the study, he felt the changes most in his ability to walk and be active.

"My activity level increased tenfold. Before, I struggled with chest pain every day. My activity level was about as close to zero as you could get. Now I can participate ... just in life. It may sound silly, but the best part is that in the wintertime I could go out and walk with my dog along the Red River. When you're walking through snow that is waist deep, you can tell there's a difference," Homstad says.

Homstad had had about a dozen surgeries and nine stents put in before he enrolled in the study, but he still struggled with angina daily. Within a few months of the stem cell shots, he could walk farther, and his chest pain subsided and was kept at bay for nearly four years.

"These are people for whom other treatment hasn't worked. They're debilitated by their chest pain, but their other options are really limited, that's why we picked them," says Henry. If the positive results seen in this study hold up in the next phase of the study, which is set to begin enrollment in the fall, this type of cardiac stem cell injection could be added to the arsenal of weapons against angina. The upcoming phase three trial has already been approved by the Food and Drug Administration.

Shot to the Heart, Before It's too Late

While several smaller studies have suggested that injecting stem cells into damaged heart tissue might be effective, this study, in its scope and rigor, was the first of its kind. A total of 167 patients were recruited and randomly assigned to receive a lower dose of stem cells, a higher dose or a placebo. The patients didn't know who got what treatment, and neither did the doctors treating them.

When tracked for a year after the injection, patients who received the lower dose of stem cells could last longer during a treadmill exercise than those who had received the placebo, and they averaged seven fewer episodes of chest pain in a week. To put this in perspective, a popular drug to treat angina, Ranolazine, reduced chest pain by fewer than two episodes a week in clinical trials.

Although the goal of the stem cell shots was to grow new blood vessels, it's impossible to tell if these stem cells were actually growing into blood vessels or if they were just triggering some other kind of healing process in the body, Henry says. Tests in animal models, however, do suggest that new blood vessels are forming, says Dr. Marco Costa, a co-author of the study and George Reed's doctor at UH Case Medical Center in Cleveland.

For now, the only gauge of the injections is improvement in symptoms.

Despite the positive results of the study, cardiologists remain "cautiously optimistic" about stem cells as a treatment for angina.

"The number of patients is relatively small, so this trial would probably not carry much scientific weight," says Dr. Jeff Brinker, a professor of cardiology at Johns Hopkins University. The results did justify the next, larger trial, he says, which would offer more answers as to whether this treatment is actually working the way researchers suspect.

The fact that lower doses of stem cells were puzzlingly more effective than larger ones is cause for caution, says Dr. Steve Nissen, chairman of the department of cardiovascular medicine at the Cleveland Clinic.

"The jury is still out for stem cell therapies to treat heart disease," says Dr. Cam Paterson, a cardiologist at the University of North Carolina at Chapel Hill.

But the results so far provide cautious hope for heart patients like George Reed and Jay Homstad.

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Stem Cell Therapy for Heart Disease – Cleveland Clinic

Stem Cell Therapy: Helping the Body Heal Itself

Stem cells are natures own transformers. When the body is injured, stem cells travel the scene of the accident. Some come from the bone marrow, a modest number of others, from the heart itself. Additionally, theyre not all the same. There, they may help heal damaged tissue. They do this by secreting local hormones to rescue damaged heart cells and occasionally turning into heart muscle cells themselves. Stem cells do a fairly good job. But they could do better for some reason, the heart stops signaling for heart cells after only a week or so after the damage has occurred, leaving the repair job mostly undone. The partially repaired tissue becomes a burden to the heart, forcing it to work harder and less efficiently, leading to heart failure.

Initial research used a patients own stem cells, derived from the bone marrow, mainly because they were readily available and had worked in animal studies. Careful study revealed only a very modest benefit, so researchers have moved on to evaluate more promising approaches, including:

No matter what you may read, stem cell therapy for damaged hearts has yet to be proven fully safe and beneficial. It is important to know that many patients are not receiving the most current and optimal therapies available for their heart failure. If you have heart failure, and wondering about treatment options, an evaluation or a second opinion at a Center of Excellence can be worthwhile.

Randomized clinical trials evaluating these different approaches typically allow enrollment of only a few patients from each hospital, and hence what may be available at the Cleveland Clinic varies from time to time. To inquire about current trials, please call 866-289-6911 and speak to our Resource Nurses.

Cleveland Clinic is a large referral center for advanced heart disease and heart failure we offer a wide range of therapies including medications, devices and surgery. Patients will be evaluated for the treatments that best address their condition. Whether patients meet the criteria for stem cell therapy or not, they will be offered the most advanced array of treatment options.

Allogenic: from one person to another (for example: organ transplant)

Autogenic: use of one's own tissue

Myoblasts: immature muscle cells, may be able to change into functioning heart muscle cells

Stem Cells: cells that have the ability to reproduce, generate new cells, and send signals to promote healing

Transgenic: Use of tissue from another species. (for example: some heart valves from porcine or bovine tissue)

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Stem Cell Therapy for Heart Disease - Cleveland Clinic

Heart Stem Cell Therapy | University of Utah Health Care

Keeping in tradition with the Us commitment to advance the fields of medicine and surgery, our physicians are focusing on regenerative medicine as the next frontier in treating cardiovascular disease. Researchers within the Cardiovascular Center estimate cell therapy will be FDA-approved within three years. The goal of this therapy is to give cells back to the heart in order for it to grow stronger, work harder, and function more like a younger heart. Currently, studies include the potentiality of injecting cardiac repair cells into patients hearts to improve function.

This is the first trial of its kind in the United States, providing heart patients who have limited or no other options with a viable treatment. Using some of the best imaging technology, researchers have been able to see improvements in patients within six months after injecting their own cells directly into the left ventricle of the heart during minimally invasive surgery.

To contact us, please use the contact number provided.

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Heart Stem Cell Therapy | University of Utah Health Care

Stem Cells Show Promise in Heart Failure Treatment

A new method for delivering stem cells to damaged heart muscle has shown early promise in treating severe heart failure, researchers report.

In a preliminary study, they found the tactic was safe and feasible for the 48 heart failure patients they treated. And after a year, the patients showed a modest improvement in the heart's pumping ability, on average.

It's not clear yet whether those improvements could be meaningful, said lead researcher Dr. Amit Patel, director of cardiovascular regenerative medicine at the University of Utah.

He said larger clinical trials are underway to see whether the approach could be an option for advanced heart failure.

Other experts stressed the bigger picture: Researchers have long studied stem cells as a potential therapy for heart failure -- with limited success so far.

"There's been a lot of promise, but not much of a clinical benefit yet," said Dr. Lee Goldberg, who specializes in treating heart failure at the University of Pennsylvania.

Researchers are still sorting through complicated questions, including how to best get stem cells to damaged heart muscle, said Goldberg, who was not involved in the new study.

What's "novel" in this research, he said, is the technique Patel's team used to deliver stem cells to the heart. They took stem cells from patients' bone marrow and infused them into the heart through a large vein called the coronary sinus.

Patel agreed that the technique is the advance.

"Most other techniques have infused stem cells through the arteries," Patel explained. One obstacle, he said, is that people with heart failure generally have hardened, narrowed coronary arteries, and the infused stem cells "don't always go to where they should."

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Stem Cells Show Promise in Heart Failure Treatment

Cardiac Stem Cells (CSCs) | University of Maryland Medical …

For immediate release: September 10, 2012

Baltimore, MD --Researchers at the University of Maryland School of Medicine, who are exploring novel ways to treat serious heart problems in children, have conducted the first direct comparison of the regenerative abilities of neonatal and adult-derived human cardiac stem cells. Among their findings: cardiac stem cells (CSCs) from newborns have a three-fold ability to restore heart function to nearly normal levels compared with adult CSCs. Further, in animal models of heart attack, hearts treated with neonatal stem cells pumped stronger than those given adult cells. The study is published in the September 11, 2012, issue of Circulation.

The surprising finding is that the cells from neonates are extremely regenerative and perform better than adult stem cells, says the study's senor author, Sunjay Kaushal, M.D., Ph.D., associate professor of surgery at the University of Maryland School of Medicine and director, pediatric cardiac surgery at the University of Maryland Medical Center. We are extremely excited and hopeful that this new cell-based therapy can play an important role in the treatment of children with congenital heart disease, many of whom don't have other options.

Dr. Kaushal envisions cellular therapy as either a stand-alone therapy for children with heart failure or an adjunct to medical and surgical treatments. While surgery can provide structural relief for some patients with congenital heart disease and medicine can boost heart function up to two percent, he says cellular therapy may improve heart function even more dramatically. We're looking at this type of therapy to improve heart function in children by 10, 12, or 15 percent. This will be a quantum leap in heart function improvement.

Heart failure in children, as in adults, has been on the rise in the past decade and the prognosis for patients hospitalized with heart failure remains poor. In contrast to adults, Dr. Kaushal says heart failure in children is typically the result of a constellation of problems: reduced cardiac blood flow; weakening and enlargement of the heart; and various congenital malformations. Recent research has shown that several types of cardiac stem cells can help the heart repair itself, essentially reversing the theory that a broken heart cannot be mended.

Stem cells are unspecialized cells that can become tissue- or organ-specific cells with a particular function. In a process called differentiation, cardiac stem cells may develop into rhythmically contracting muscle cells, smooth muscle cells or endothelial cells. Stem cells in the heart may also secrete growth factors conducive to forming heart muscle and keeping the muscle from dying.

To conduct the study, researchers obtained a small amount of heart tissue during normal cardiac surgery from 43 neonates and 13 adults. The cells were expanded in a growth medium yielding millions of cells. The researchers developed a consistent way to isolate and grow neonatal stem cells from as little as 20 milligrams of heart tissue. Adult and neonate stem cell activity was observed both in the laboratory and in animal models. In addition, the animal models were compared to controls that were not given the stem cells.

Dr. Kaushal says it is not clear why the neonatal stem cells performed so well. One explanation hinges on sheer numbers: there are many more stem cells in a baby's heart than in the adult heart. Another explanation: neonate-derived cells release more growth factors that trigger blood vessel development and/or preservation than adult cells.

This research provides an important link in our quest to understand how stem cells function and how they can best be applied to cure disease and correct medical deficiencies, says E. Albert Reece, M.D., Ph.D., M.B.A., vice president for medical affairs, University of Maryland; the John Z. and Akiko K. Bowers Distinguished Professor; and dean, University of Maryland School of Medicine. Sometimes simple science is the best science. In this case, a basic, comparative study has revealed in stark terms the powerful regenerative qualities of neonatal cardiac stem cells, heretofore unknown.

Insights gained through this research may provide new treatment options for a life-threatening congenital heart syndrome called hypoplastic left heart syndrome (HLHS). Dr. Kaushal and his team will soon begin the first clinical trial in the United States to determine whether the damage to hearts of babies with HLHS can be reversed with stem cell therapy. HLHS limits the heart's ability to pump blood from the left side of the heart to the body. Current treatment options include either a heart transplant or a series of reconstructive surgical procedures. Nevertheless, only 50-60 percent of children who have had those procedures survive to age five.

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Cardiac Stem Cells (CSCs) | University of Maryland Medical ...

Cardiovascular Stem Cell Therapy

Stem Cell Clinical Research & Deployment Cardiovascular & Pulmonary Conditions

The Manhattan Regenerative Medicine Medical Group is proud to be part of the only Institutional Review Board (IRB)-based stem cell treatment network in the United States that utilizes fat-transfer surgical technology. The Manhattan Regenerative Medicine Medical Group offers IRB approved protocols and investigational use ofAdult Autologous Adipose-derived Stem Cells (ADSCs) for clinical research and deployment for numerous Cardiovascular and Pulmonary disorders, inclusive of:

Cardiovascular conditions include medical problems involving the heart and vascular system (the arterial and venous blood vessels). The most common cardiovascular condition is atherosclerotic coronary artery disease (ASCVD), which especially affects the coronary arteries and is the leading cause of heart attacks and death worldwide; and Congestive Heart Failure (CHF).

Other common cardiovascular conditions involve the cardiac muscle (CHF), cardiac valves, and heart rhythm. Many patients are typically treated with a multitude of medications; many patients require surgical interventions such as coronary angioplasty, coronary artery bypass, or other surgeries. Often patients, despite maximum therapy with medications and surgery, continue to suffer pain, discomfort, disability and have marked restrictions in their normal daily living activities.

The Manhattan Regenerative Medicine Medical Group is proud to be part of the only Institutional Review Board (IRB)-based stem cell treatment network in the United States that utilizes fat-transfer surgical technology. We have an array of ongoing IRB-approved protocols, andwe provide care for patients with a wide variety of disorders that may be treated with adult stem cell-based regenerative therapy.

The Manhattan Regenerative Medicine Medical Group offers IRB approved protocols and investigational use of Autologous Adult Adipose Derived Stem Cells (ADSCs) for clinical research and deployment for numerous cardiovascular conditions. These ADSCs cells are derived from fat an exceptionally abundant source of stem cells that has been removed during our mini-liposuction office procedure. The source of the regenerative stem cells actually comes from stromal vascular fraction (SVF) a protein rich segment from processed adipose tissue. SVF contains a mononuclear cell line (predominantly autologous mesenchymal stem cells), macrophage cells, endothelial cells, red blood cells, and important growth factors that facilitate the stem cell process and promote their activity. Our technology allows us to isolate high numbers of viable cells that we can deploy during the same surgical setting.

The SVF and stem cells are then deployed back into the patients body via injection or IV infusion on an outpatient basis; the total procedure takes less than two hours; and only local anesthesia is used. Not all cardiovascular problems respond to stem cell therapy, and each patient must be assessed individually to determine the potential for optimal results from this regenerative medicine process.

The Manhattan Regenerative Medicine Medical Group is committed not only to providing a high degree of quality care for our patients with cardiovascular problems but we are also highly committed to clinical stem cell research and the advancement of regenerative medicine. At the Miami Stem Cell Treatment Center we exploit anti-inflammatory, immuno-modulatory and regenerative properties of adult stem cells to mitigate cardiovascular conditions which are otherwise lethal to our bodies.

Myocardial infarction (heart attack) is responsible for significant cardiac muscle destruction and impairment due to ischemia (lack of blood flow). This can lead to further or recurrent restriction of blood flow thereby causing re-current infarct and pain on exertion (or even rest) known as chronic angina. Chronic angina causes restriction of daily activities of everyday living and is plagued with chest pain, chest pressure, and depression. This problem is caused most commonly by coronary artery disease which is very common in the United States and associated with significant morbidity and mortality.

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Cardiovascular Stem Cell Therapy

Stem Cells for Heart Cell Therapies – National Center for …

Abstract

Myocardial infarctioninduced heart failure is a prevailing cause of death in the United States and most developed countries. The cardiac tissue has extremely limited regenerative potential, and heart transplantation for reconstituting the function of damaged heart is severely hindered mainly due to the scarcity of donor organs. To that end, stem cells with their extensive proliferative capacity and their ability to differentiate toward functional cardiomyocytes may serve as a renewable cellular source for repairing the damaged myocardium. Here, we review recent studies regarding the cardiogenic potential of adult progenitor cells and embryonic stem cells. Although large strides have been made toward the engineering of cardiac tissues using stem cells, important issues remain to be addressed to enable the translation of such technologies to the clinical setting.

Heart disease is a significant cause of morbidity and mortality worldwide. In the United States, heart failure is ranked number one as a cause of death, affecting over 5 million people and with more than 500,000 new cases diagnosed each year.1 The health care expenditures associated with heart failure were $26.7 billion in 2004 and are estimated to $33.2 billion in 2007. Although significant progress has been made in mechanical devices and pharmacological interventions, more than half of the patients with heart failure die within 5 years of initial diagnosis. Wide application of heart transplantation is severely hindered by the limited availability of donor organs. To this end, cardiac cell therapy may be an appealing alternative to current treatments for heart failure.

Recent investigations focusing on engineering cells and tissues to repair or regenerate damaged hearts in animal models and in clinical trials have yielded promising results. Considering the limited regenerative capacity of the heart muscle, renewable sources of cardiomyocytes are highly sought. Cells suitable for myocardial engineering should be nonimmunogenic, should be easy to expand to large quantities, and should differentiate into mature, fully functional cardiomyocytes capable of integrating to the host tissue. Adult progenitor cells (APCs) and embryonic stem cells (ESCs) have extensive proliferative potential and can adopt different cell fates, including that of heart cells. The recent advances in the fields of stem cell biology and heart tissue engineering have intensified efforts toward the development of regenerative cardiac therapies. In this article, we review findings pertaining to the cardiogenic potential of major APC populations and of ESCs (). We also discuss significant challenges in the way of realizing stem cellbased therapies aiming to reconstitute the normal function of heart.

Potential sources of stem/progenitor cells for cardiac repair. ESCs derived from the inner cell mass of a blastocyst can be manipulated ex vivo to differentiate toward heart cells. APCs residing in various tissues such as the BM and skeletal muscle may ...

Bone marrow (BM) is a heterogeneous tissue comprising of multiple cell types, including minute fractions of mesenchymal stem cells (MSCs; 0.0010.01% of total cells2) and hematopoietic stem cells (HSCs; 0.71.5cells/108 nucleated marrow cells3). The heterogeneity of BM makes challenging the identification of a subpopulation of cells capable of cardiogenesis, and studies of BM celltocardiac cell transdifferentiation should be examined through this prism.

The notion that BM-derived cells may contribute to the regeneration of the heart was first illustrated when dystrophic (mdx) female mice received BM cells from male wild-type mice.4 More than 2 months after the transplantation, tissues of the recipient mice were histologically examined for the presence of Y-chromosome+ donor cells. Besides the skeletal muscle, donor cells were identified in the cardiac region, suggesting that circulating BM cells contribute to the regeneration of cardiomyocytes.

Further supporting evidence was provided by Jackson et al.5 in studies using a side population (SP) of cells characterized by their intrinsic capacity to efflux Hoechst 33342 dye through the ATP-binding Bcrp1/ABCG2 transporter. The cells were isolated from the BM fraction of HSCs of Rosa26 mice constitutively expressing the -galactosidase reporter gene (LacZ). After SP cells were injected into mice with coronary occlusioninduced ischemia, cells coexpressing LacZ and cardiac -actinin were identified around the infarct region with a frequency of 0.02%. Endothelial engraftment was more prevalent (3.3%). The observed improvement in myocardial function may thus be attributed to the potential of BM cells to give rise to a rather endothelial progeny. This may be a parallel to cardiovascular progenitors from differentiating ESCs giving rise to cardiomyocytes, and endothelial and vascular smooth muscle lineages.6,7

Orlic et al.8 also reported the regeneration of infarcted myocardium after transplantation of lineage-negative (LIN)/C-KIT+ BM cells from transgenic mice constitutively expressing enhanced green fluorescent protein (eGFP). Cells were injected in the contracting wall close to the infarct area. Nine days after transplantation, an impressive 68% of the infarct was occupied by newly formed myocardium with eGFP+ cells displaying cardiomyocyte markers such as troponin, MEF2, NKX2.5, cardiac myosin, GATA-4, and -sarcomeric actin. Similar outcomes were reported by the same group9 when mouse C-KIT+ (but not screened for LIN) BM cells were transplanted.

Although these findings led to the conclusion that BM cells can repopulate a damaged heart, work by other investigators has casted doubt on this assertion. Balsam et al.10 noted that mice with infarcts receiving BM LIN/C-KIT+, C-KIT-enriched or THY1.1low/LIN/stem cell antigen-1 (SCA-1+) cells exhibited improved ventricular function. However, donor cells expressed granulocyte but not heart cell markers 1 month after injection. In another study,11 HSCs carrying a nuclear-localized LacZ gene flanked by the cardiac -myosin heavy chain promoter were delivered into the periinfarct zone of mice 5h after coronary artery occlusion. One to 4 weeks later, LacZ+ cells were absent in heart tissue sections from 117 mice that received HSCs. Similarly, no eGFP+ cells were detected in the infarcted hearts of mice infused with BM cells constitutively expressing eGFP. Finally, Nygren et al.12 in similar transplantation experiments observed only blood cells (mainly leukocytes) originating from BM HSCs in the infarcted myocardium without evidence of transdifferentiation of donor cells to cardiomyocytes.

Continued here:
Stem Cells for Heart Cell Therapies - National Center for ...

cardiovascular disease :: Cardiac stem cells | Britannica.com

Cardiac stem cells, which have the ability to differentiate (specialize) into mature heart cells and therefore could be used to repair damaged or diseased heart tissue, have garnered significant interest in the development of treatments for heart disease and cardiac defects. Cardiac stem cells can be derived from mature cardiomyocytes through the process of dedifferentiation, in which mature heart cells are stimulated to revert to a stem cell state. The stem cells can then be stimulated to redifferentiate into myocytes or endothelial cells. This approach enables millions of cardiac stem cells to be produced in the laboratory.

In 2009 a team of doctors at Cedars-Sinai Heart Institute in Los Angeles, California, reported the first attempted use of cardiac stem cell transplantation to repair damaged heart tissue. The team removed a small section of tissue from the heart of a patient who had suffered a heart attack, and the tissue was cultured in a laboratory. Cells that had been stimulated to dedifferentiate were then used to produce millions of cardiac stem cells, which were later reinfused directly into the heart of the patient through a catheter in a coronary artery. A similar approach was used in a subsequent clinical trial reported in 2011; this trial involved 14 patients suffering from heart failure who were scheduled to undergo cardiac bypass surgery. More than three months after treatment, there was slight but detectable improvement over cardiac bypass surgery alone in left ventricle ejection fraction (the percentage of the left ventricular volume of blood that is ejected from the heart with each ventricular contraction).

Stem cells derived from bone marrow, the collection of which is considerably less invasive than heart surgery, are also of interest in the development of regenerative heart therapies. The collection and reinfusion into the heart of bone marrow-derived stem cells within hours of a heart attack may limit the amount of damage incurred by the muscle.

There are many types of arterial diseases. Some are generalized and affect arteries throughout the body, though often there is variation in the degree they are affected. Others are localized. These diseases are frequently divided into those that result in arterial occlusion (blockage) and those that are nonocclusive in their manifestations.

Atherosclerosis, the most common form of arteriosclerosis, is a disease found in large and medium-sized arteries. It is characterized by the deposition of fatty substances, such as cholesterol, in the innermost layer of the artery (the intima). As the fat deposits become larger, inflammatory white blood cells called macrophages try to remove the lipid deposition from the wall of the artery. However, lipid-filled macrophages, called foam cells, grow increasingly inefficient at lipid removal and undergo cell death, accumulating at the site of lipid deposition. As these focal lipid deposits grow larger, they become known as atherosclerotic plaques and may be of variable distribution and thickness. Under most conditions the incorporation of cholesterol-rich lipoproteins is the predominant factor in determining whether or not plaques progressively develop. The endothelial injury that results (or that may occur independently) leads to the involvement of two cell types that circulate in the bloodplatelets and monocytes (a type of white blood cell). Platelets adhere to areas of endothelial injury and to themselves. They trap fibrinogen, a plasma protein, leading to the development of platelet-fibrinogen thrombi. Platelets deposit pro-inflammatory factors, called chemokines, on the vessel walls. Observations of infants and young children suggest that atherosclerosis can begin at an early age as streaks of fat deposition (fatty streaks).

Atherosclerotic lesions are frequently found in the aorta and in large aortic branches. They are also prevalent in the coronary arteries, where they cause coronary artery disease. The distribution of lesions is concentrated in points where arterial flow gives rise to abnormal shear stress or turbulence, such as at branch points in vessels. In general the distribution in most arteries tends to be closer to the origin of the vessel, with lesions found less frequently in more distal sites. Hemodynamic forces are particularly important in the system of coronary arteries, where there are unique pressure relationships. The flow of blood through the coronary system into the heart muscle takes place during the phase of ventricular relaxation (diastole) and virtually not at all during the phase of ventricular contraction (systole). During systole the external pressure on coronary arterioles is such that blood cannot flow forward. The external pressure exerted by the contracting myocardium on coronary arteries also influences the distribution of atheromatous obstructive lesions.

Excerpt from:
cardiovascular disease :: Cardiac stem cells | Britannica.com

cardiovascular disease :: Cardiac stem cells | Britannica.com

Cardiac stem cells, which have the ability to differentiate (specialize) into mature heart cells and therefore could be used to repair damaged or diseased heart tissue, have garnered significant interest in the development of treatments for heart disease and cardiac defects. Cardiac stem cells can be derived from mature cardiomyocytes through the process of dedifferentiation, in which mature heart cells are stimulated to revert to a stem cell state. The stem cells can then be stimulated to redifferentiate into myocytes or endothelial cells. This approach enables millions of cardiac stem cells to be produced in the laboratory.

In 2009 a team of doctors at Cedars-Sinai Heart Institute in Los Angeles, California, reported the first attempted use of cardiac stem cell transplantation to repair damaged heart tissue. The team removed a small section of tissue from the heart of a patient who had suffered a heart attack, and the tissue was cultured in a laboratory. Cells that had been stimulated to dedifferentiate were then used to produce millions of cardiac stem cells, which were later reinfused directly into the heart of the patient through a catheter in a coronary artery. A similar approach was used in a subsequent clinical trial reported in 2011; this trial involved 14 patients suffering from heart failure who were scheduled to undergo cardiac bypass surgery. More than three months after treatment, there was slight but detectable improvement over cardiac bypass surgery alone in left ventricle ejection fraction (the percentage of the left ventricular volume of blood that is ejected from the heart with each ventricular contraction).

Stem cells derived from bone marrow, the collection of which is considerably less invasive than heart surgery, are also of interest in the development of regenerative heart therapies. The collection and reinfusion into the heart of bone marrow-derived stem cells within hours of a heart attack may limit the amount of damage incurred by the muscle.

There are many types of arterial diseases. Some are generalized and affect arteries throughout the body, though often there is variation in the degree they are affected. Others are localized. These diseases are frequently divided into those that result in arterial occlusion (blockage) and those that are nonocclusive in their manifestations.

Atherosclerosis, the most common form of arteriosclerosis, is a disease found in large and medium-sized arteries. It is characterized by the deposition of fatty substances, such as cholesterol, in the innermost layer of the artery (the intima). As the fat deposits become larger, inflammatory white blood cells called macrophages try to remove the lipid deposition from the wall of the artery. However, lipid-filled macrophages, called foam cells, grow increasingly inefficient at lipid removal and undergo cell death, accumulating at the site of lipid deposition. As these focal lipid deposits grow larger, they become known as atherosclerotic plaques and may be of variable distribution and thickness. Under most conditions the incorporation of cholesterol-rich lipoproteins is the predominant factor in determining whether or not plaques progressively develop. The endothelial injury that results (or that may occur independently) leads to the involvement of two cell types that circulate in the bloodplatelets and monocytes (a type of white blood cell). Platelets adhere to areas of endothelial injury and to themselves. They trap fibrinogen, a plasma protein, leading to the development of platelet-fibrinogen thrombi. Platelets deposit pro-inflammatory factors, called chemokines, on the vessel walls. Observations of infants and young children suggest that atherosclerosis can begin at an early age as streaks of fat deposition (fatty streaks).

Atherosclerotic lesions are frequently found in the aorta and in large aortic branches. They are also prevalent in the coronary arteries, where they cause coronary artery disease. The distribution of lesions is concentrated in points where arterial flow gives rise to abnormal shear stress or turbulence, such as at branch points in vessels. In general the distribution in most arteries tends to be closer to the origin of the vessel, with lesions found less frequently in more distal sites. Hemodynamic forces are particularly important in the system of coronary arteries, where there are unique pressure relationships. The flow of blood through the coronary system into the heart muscle takes place during the phase of ventricular relaxation (diastole) and virtually not at all during the phase of ventricular contraction (systole). During systole the external pressure on coronary arterioles is such that blood cannot flow forward. The external pressure exerted by the contracting myocardium on coronary arteries also influences the distribution of atheromatous obstructive lesions.

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cardiovascular disease :: Cardiac stem cells | Britannica.com

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