Human Amniotic Fluid Stem Cells Can Act Like Heart Cells, Sort of

Human amniotic fluid-derived stem cells (AFSC) have a demonstrated ability to differentiate into several different adult cell types, and they also fail to form tumors in laboratory animals.

A previous study of AFSCs showed that if these stem cells were grown in culture with heart muscle cells from newly born rats, the AFSCs began to express heart-specific genes. While the AFSCs did not become full-fledged heart muscle cells, they began to differentiate in that direction.

Yang Gao and others in the laboratory of Jeffrey G. Jacot at Rice University tried this same experiment with human heart cells. They used a specific set of cell culture conditions that prevent the AFSCs from fusing with the heart cells, because the fusion of two cells can deceive researchers into thinking that the stem cells have actually become heart cells when in fact they have not.

Jacot and his coworkers discovered that when human AFSC made contact with human heart cells, they began to express proteins normally found in heart muscle that help them contract. One of these proteins, cardiac troponin T (cTnT), was definitely expressed in human AFSCs, even though this protein is rather specific to heart muscle cells. cTnT is also one of the proteins released into the bloodstream after a heart attack.  Further investigation uncovered absolutely no evidence of cell fusion. Thus when AFSCs touch human heart cells, they begin to make some heart-specific proteins.

Cardiac Troponin

Jacot and his group did an additional experiment. They tried culturing the human AFSCs on one side of the porous membrane and human heart cells on the other side. These conditions allow minimal contact between cells, but still exposes them the anything the cells might be secreting. Under these culture conditions, human AFSCs still showed a statistically significant increase in cTnT expression compared to culture conditions that without contact between the two cell types.  However, human AFSCs grown in the present of human heart cells still did not express the calcium modulating proteins that are so important for regulating heart muscle contraction. Additionally, the cells and did not have functional or morphological characteristics of mature heart muscle cells.

These data suggest that contact between heart cells and human AFSCs is a necessary but not sufficient condition to drive AFSCs to differentiate into heart cells. However, touching heart cells gets AFSCs part of the way. Maybe further research will provide other cues that will push these remarkable cells the rest of the way.

Do Stem Cells from Bone Outdo Those from the Heart in Regenerating Cardiac Tissue?

Scientists at Tulane University in New Orleans, La. (US) have completed a study that suggests that stem cells derived from cortical, or compact bone do a better job of regenerating heart tissue than do the heart’s own stem cells.

The study, led by Steven R. Houser, Ph.D., FAHA, director of Tulane’s School of Medicine’s Cardiovascular Research Center (CVRC), could potentially lead to an “off the rack” source of stem cells for regenerating cardiac tissue following a heart attack.

Cortical bone stem cells (CBSCs) are considered some of the most pluripotent cells in the adult body. These cells are naïve and ready to differentiate into just about any cell type. However, even though CBSCs and similar pluripotent stem cells retain the ability to develop into any cell type required by the body, they have the potential to wander off course and land in unintended tissues. Cardiac stem cells, on the other hand, are more likely to stay in their resident tissue.

Bone cross-section

To determine how CBSCs might behave in the heart, Houser’s team, led by Temple graduate student Jason Duran, collected the cells from mouse tibias (shin bones), expanded them in the lab and then injected them into back the mice after they had undergone a heart attack.

The cells triggered the growth of new blood vessels in the injured tissue and six weeks after injection had differentiated into heart muscle cells. While generally smaller than native heart cells, the new cells had the same functional capabilities and overall improved survival and heart function.

Similar improvements were not observed in mice treated with cardiac stem cells, nor did those cells show evidence of differentiation.

“What we did generates as many questions as it does answers,” Dr. Houser said. “Cell therapy attempts to repopulate the heart with new heart cells. But which cells should be used, and when they should be put into the heart are among many unanswered questions.”

The next step will be to test the cells in larger animal models. The current study was published in the Aug. 16 issue of Circulation Research.

Transformation of Non-Beating Human Cells into Heart Muscle Cells Lays Foundation for Regenerating Damaged Hearts

After a heart attack, the cells within the damaged part of the heart stop beating and become ensconced in scar tissue. Not only does this region not beat, it does not conduct the signal to beat either and that can not only lead to a slow, sluggish heartbeat, it can also cause irregular heart rates or arrhythmias.

Now, however, scientists have demonstrated that this damage to the heart muscle need not be permanent. Instead there is a way to transform those cells that form the human scar tissue into cells that closely resemble beating heart cells.

Last year, researchers from the laboratory of Deepak Srivastava, MD, the director of Cardiovascular and Stem Cell Research at the Gladstone Institute, transformed scar-forming heart cells (fibroblasts) into beating heart-muscle cells in live mice. Now they report doing the same to human cells in a culture dishes.

“Fibroblasts make up about 50 percent of all cells in the heart and therefore represent a vast pool of cells that could one day be harnessed and reprogrammed to create new muscle,” said Dr. Srivastava, who is also a professor at the University of California, San Francisco. “Our findings here serve as a proof of concept that human fibroblasts can be reprogrammed successfully into beating heart cells.”

In 2012, Srivastava and his team reported that fibroblasts could be reprogrammed into beating heart cells by injecting just three genes (collectively known as GMT, which is short for Gata4, Mef2c, and Tbx5), into the hearts of live mice that had been damaged by a heart attack (Qian L, et al., Nature. 2012 31;485(7400):593-8). From this work, they reasonably concluded that the same three genes could have the same effect on human cells.

“When we injected GMT into each of the three types of human fibroblasts (fetal heart cells, embryonic stem cells and neonatal skin cells) nothing happened—they never transformed—so we went back to the drawing board to look for additional genes that would help initiate the transformation,” said Gladstone staff scientist Ji-dong Fu, Ph.D., the study’s lead author. “We narrowed our search to just 16 potential genes, which we then screened alongside GMT, in the hopes that we could find the right combination.”

The research team began by injecting all candidate genes into the human fibroblasts. They then systematically removed each one to see which were necessary for reprogramming and which were dispensable. In the end, they found that injecting a cocktail of five genes—the 3-gene GMT mix plus the genes ESRRG and MESP1—were sufficient to reprogram the fibroblasts into heart-like cells. They then found that with the addition of two more genes, called MYOCD and ZFPM2, the transformation was even more complete.

To help things along, the team used a growth factor known as Transforming Growth Factor-Beta (TGF-Beta) to induce a signaling pathway during the early stages of reprogramming that further improved reprogramming success rates.

“While almost all the cells in our study exhibited at least a partial transformation, about 20 percent of them were capable of transmitting electrical signals—a key feature of beating heart cells,” said Dr. Fu. “Clearly, there are some yet-to-be-determined barriers preventing a more complete transformation for many of the cells. For example, success rates might be improved by transforming the fibroblasts within living hearts rather than in a dish—something we also observed during our initial experiments in mice.”

The immediate next steps are to test the five-gene cocktail in hearts of larger mammals. Eventually, the team hopes that a combination of small, drug-like molecules could be developed to replace the cocktail, which would offer a safer and easier method of delivery.

This latest study was published online August 22 in Stem Cell Reports.

Drug Developers Increase Their Use of Stem Cells

Industries have increased their use of stem cells in research and development and product testing and the industrial use of stem cells will almost certainly increase in the future.

Despite the image of stem cells in the popular imagination as the stalwarts of regenerative medicine, stem cells have revolutionized drug development and testing. James Thomson, director of regenerative biology at the Morgridge Institute for Research in Madison, Wisconsin, and one of the founders of Cellular Dynamics International, also in Madison, said, “I think there are tremendous parallels to the early days of recombinant DNA in this field. I don’t think people appreciated what a broad-ranging tool recombinant DNA was in the middle ’70s.” Thomson also thinks that people also seriously underestimate the tremendous number of hurdles that must be overcome in order to use such technologies in clinical treatments. Stem cells, according to Thomson, are in a similar situation. While the therapeutic use of these cells might eventually come to fruition, “people underappreciate how broadly enabling a research tool it is.”

About two years ago, drug companies began to investigate the use of stem cells in testing and evaluating new drugs. Today, the pharmaceutical industries all over the world are increasingly using stem cell lines to test drug toxicity and identify and evaluate potential new therapies. For example, Thomson’s company, Cellular Dynamics, sells human heart cells called cardiomyocytes, which are made from induced pluripotent stem (iPS) cells. Thomson says that “essentially all the major pharma companies” have purchased these cells for use in their laboratories. The company also produces brain cells and cells that line blood vessels, and is about to release a line of human liver cells.

Cellular Dynamics is not the only company that makes stem cell lines for drug testing. Three years ago, a stem-cell biologist named Stephen Minger left his job in at a United Kingdom university to be the head of General Electric Healthcare’s push into stem cells. This medical-technology company, which is headquartered in Chalfont St. Giles, UK, has been selling human heart cells made from embryonic stem (ES) cells for well over a year, and is due to start selling ES cell-derived liver cells soon.

Minger’s team at GE Healthcare assessed their ES-derived heart muscle cells in a blind trial against a set of unnamed drug compounds to determine if they could determine which compounds were toxic. Once the tests were completed, Minger said that they found that the cells had been affected by those compounds that are known to be toxic. However, the stem cells also identified a problem that had only been discovered after the drugs had reached the market (after they had been approved by the US Food and Drug Administration). According to Minger, “These are compounds which went all the way through animal testing, then went through phase I, II, III and then were licensed in many cases by the FDA.”

Stem cell lines can do more than identify drugs with dangerous side effects’ they can save the industry millions of dollars in wasted development costs. However, they might also be tools for drug development. Cellular Dynamics and GE Healthcare even market their cells from this very purpose. Adam Rosenthal, senior director for strategic and corporate development at iPierian, a biopharmaceutical company based in San Francisco, California, said, “Many of the animal models out there are poor, demonstrating great efficacy in the mouse, but not repeating in man during late-stage clinical trials. Therefore having an in vitro model years before, which can actually recapitulate human disease, would be a huge advantage.

iPierian has a different strategy than other stem cell companies, since it has its own proprietary in-house stem cell lines that it uses. It does not sell those cell lines, but uses them to develop treatments for neurodegenerative diseases; e.g., Alzheimer’s. This same company has recently announced that they are going to move forward with their development of monoclonal antibodies that target the tau proteins thought to be important in the onset of Alzhiemer’s disease. iPierian made this decision based on information that came from stem-cell work.

Lee Rubin, co-founder of iPierian and director of translational medicine at the Harvard Stem Cell Institute in Cambridge, Massachusetts, says that there is debate within industry if stem cells serve as appropriate model systems to study certain diseases. This is particularly the case with particularly non-genetic or late-onset disorders or conditions that result from pathological interactions between different tissues. Rubin has used stem cells in his research to model a disease called spinal muscular atrophy, which is actually a group of early onset genetic disorders. Rubin makes it clear that the only way to definitively demonstrate that stem cells are a superior model system from drug discovery is to show that the drugs developed from stem cell-based models works in people. Rubin put it this way, “That’s a long-term project. That’s the ultimate test.”

Thomson notes that stem cells will almost certainly find even wider uses than drug-development work. “What human ES cells and iPS cells now do is give you access to the basic building blocks of the human body, just for basic study. We will understand the human body at a much greater detail because of these cells.” How stem cells will be used are not clear, but Thomson added, “But I do think it will profoundly change human medicine.”

Different Kinds of Stem Cells in the Heart

For almost a century, the sciences of human physiology, cardiology, and medicine have believed that the heart is a terminally differentiated organ whose cells do not undergo further cell division. Essentially however many heart cells you were born with persisted throughout your own personal lifespan. Any increases in the size of the heart were thought to result from expansion of the size of the heart muscle cells .

Work from several labs over the last 15 years have shown that this dogma does not stand further scrutiny. In 1995, Peiro Anversa and his colleagues at New York Medical College in Valhalla, NY examined the differences in heart size between men and women at various ages and found that heart mass was stable in women, but in men, loss of heart mass was due to cell loss and not a decrease in cell size. Also, cell size was stable in women, but tended to increase in men. This increase in cell volume compensated for the loss of heart muscle cells and kept the thickness of the heart walls the same in older and younger men. However, the mass of the heart still decreased in men as they age. This finding does not support the assumption that heart muscle cells are born during development and stay with you throughout your life (for this study see Giorgio Olivetti, et al., Gender Differences and Aging: Effects on the Human Heart. JACC Vol. 26, No. 4 (1995): 1068-79).

Other work that contradicted the commonly accepted dogma examined hearts of people who had experienced “acromegalic cardiomyopathy.”  In a nutshell, individuals with acromegalic cardiomyopathy had a problem with too much growth hormone.  This growth hormone imbalance caused the patient to be really tall, and suffer from bone abnormalities.  This also causes enlargement of the heart.  This patient died at the age of 65, and had a heart that weighed 800 grams.  This is six times the weight of a normal heart.  However, when the size of the heart muscle cells from the man who had died of acromegaly were compared with that of a 99-year old women who had died of pneumonia, the volume of their cells was similar (Leri, Kajstura and Anversa, Role of Cardiac Stem Cells in Cardiac Pathophysiology: A Paradigm SHift in Human Myocardial Biology. Circulation Research 109 (2011): 941-61).  This strongly calls into question the notion that heart enlargement is due to an increase in cell size.

Why was the acromegalic heart larger?  The answer seems to be that it contained far more cells, and recent work has demonstrated that hearts have a stem cell population that can divide and generate new heart cells.  At all ages, the heart contains heart muscle cells that are dividing and expressing a host of genes found only in dividing cells (CDC6, Ki67, MCM5, Phospho-H3, aurora B kinase).  When the heart enlarges for pathological reasons, the proportion of heart muscle cells that expresses these genes increases (see Levi P, Kajstura J and Anversa P, Cardiac Stem Cells and Mechanisms of Myocardial Regeneration. Physio Rev 85 (2005): 1373-1416).

There is not one population of heart stem cells, but four of them, and they all possess different characteristics, and, possibly, different embryological origins.  The first group is “side population cells.”  Side population cells (SPCs) are identified by their ability to expel toxic compounds and dyes (Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett.2002 Oct 23;530(1-3):239-43).  SPCs have a membrane protein that pumps such molecules from the cell, and when cultured in a semisolid medium, they will differentiate into heart muscle cells.  The exact genes expressed by SPCs is uncertain, since there seem to be, at least in rodents, a few subclasses of SPCs.  In mice, 2% of all heart cells are SPCs, and they have an expression pattern that looks like this: Sca1[high], c-kit[low], CD34[low], and CD45[low].  Cells that express Sca1 normally form blood vessels, but SPCs do not seem to form blood vessels.  This is the conclusion of cell tracing experiments that marks cells and then places them into damaged hearts.  Once the stem cells have divided and integrated into the heart, the animals are sacrificed and their hearts are stained for the marker that characteristic of the implanted stem cells.  Such experiments show that SPCs do not make blood vessels in mice (Tara L. Rasmussen, et al., Getting to the Heart of Myocardial Stem Cells and Cell Therapy. Circulation. 2011; 123: 1771-1779).

In rats, the data is less clearly interpretable, because rat SPCs express a gene called Bcpr1, but the Bcpr1-positive cells either express CD31 and can form blood vessels or do not express CD31 and cannot form blood vessels.  It appears that only the Sca-1[positive] CD31[negative] cells have a pronounced ability to form heart muscle cells.  SCPs might come from neural crest cells, and this hypothesis comes from their behavior in culture (Oyama et al., Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo.  J Cell Biol 176 (2007): 329-41).

The second population of cardiac stem cells is the Sca-1 cells.  Sca1 cells do not possess the functional properties of stem cells, but they represent 2% of all heart cells.  While they can be grown in culture, only a very small percentage of the cells express any heart muscle-specific proteins (3%-4%), and when delivered to a damaged heart, Sca1 cells will fuse with existing cells to modestly improve heart function (Matsuura K., et al., JBC 279 (2004): 11384-91).  When injected into damaged hearts, Sca1 cells form blood vessels but their ability to survive in the heart is very poor (Li Z., et al., JACC 53 (2009): 1229-40).  Also, Sca1 cells seem to secrete molecules that help the heart and its other stem cell populations to work better.  Most of the heart muscle turnover seems to result from c-kit[positive] cells and the role of SPCs and Sca1 cells is, to date, uncertain.

On the cardiac surface, a third heart stem cells exists, the epicardial progenitors.  There are several different subtypes of epicardial progenitors; Flk1-expressing cells form blood vessels, WT1- and Tbx18-expressing cells make heart muscle (Zhou B., et al., Nature 454 (2008): 109-13 and Cai CL., et al., Nature 454 (2008): 104-108).  There is also a pool of c-kit[positive] cells in the human heart that can differentiate into heart muscle cells and blood vessels (Castaldo C, et al., Stem Cells 26(7), 2008: 1723-31).

The final heart stem cell population is the cardiosphere derived cell (CDC) population.  “Cardiospheres” are balls of cells formed by CDCs in culture.  While in these spheres, a variety of cells form around a core of primitive, c-kit[positive] cells.  Cardiospheres do not consist of a uniform mass of cells, but a pastiche of cells.  Some of these cells have gap junction proteins that are found in mature heart muscle cells that allow them to connect with each other and pass ions from one cell to another.  Others are highly uncommitted and have tremendous growth potential.  These cells express c-kit at high levels.  CDCs are the stem cells that have been used in the recent CADUCEUS and SCIPIO clinical trials.  They are capable of forming heart muscle cells and blood vessels.  They are also easily extracted from hearts by biopsies that are out-patient procedures.

Thus even though there are several different types of heart stem cells, they play a role in repair, and pathology.  They can also be exploited to heal hearts, shrink heart scars, and make a denser collection of blood vessels in the heart.  Further work on them will increase the ability of cardiologists to heal the hearts of patients with failing hearts.