Enrollment Completed in Phase 2 ALLSTAR Cardiac Clinical Trial


Capricor Therapeutics Inc. has announced the completion of patient enrollment in their Phase 2 ALLSTAR clinical trial.  ALLSTAR stands for ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration, and this trial will test Capricor’s CAP-1002 product in patients suffering from cardiac dysfunction following a heart attack.

CAP-1002 cells are cardiosphere derived cells (CDCs) that were isolated from donors.  This investigational therapy is an off-the-shelf “ready to use” cardiac cell therapy that comes from donor heart tissue.  CAP-1002 cells are made to be directly infused into a patient’s coronary artery during a catheterization procedure.

These CDCs were tested in the CADUCEUS clinical trial, in which they were shown to decrease scar size and increase viable heart tissue when implanted into the hearts of heart attack patients.  One-year follow-up examinations of these confirmed the earlier results.

ALLSTAR will study a population similar to the one in the CADUCEUS study (patients who had experienced a heart attack 30-90 days earlier), except that ALLSTAR will treat patients 91-365 days after suffering a heart attack.  The extension of the patient pool was to see if the indication window for CAD-1002 could be extended.

The Capricor CEO Linda Marbàn said, “With the last patient in ALLSTAR having been dosed on September 30, we expect to report top-line 12-month primary efficacy outcome results in the fourth quarter of 2017.”

ALLSTAR is being sponsored by Capricor and is led by Drs. Timothy Henry and Rajendra Makkar of the Cedars-Sinai Heart Institute.  The trial is being conducted at approximately 25-40 sites across the U.S.

The Phase I portion of the trial was funded in part by the National Institutes of Health and completed enrollment in December 2013, and the Phase II portion of the trial is supported in large part by the California Institute for Regenerative Medicine (CIRM).

Adult Directly Reprogrammed With Proteins into Cardiac Progenitor Cells Heal Heart After a Heart Attack and Make New Heart Muscle


Jianjun Wang from Wayne State School of Medicine in Detroit, Michigan and Xi-Yong Yu from Guangzhou Medical University and a host of graduate students and postdoctoral research fellows in their two laboratories have teamed up to make human cardiac progenitor cells (CPCs) from human skin fibroblasts through direct reprogramming. Direct reprogramming does not go through a pluripotent intermediate, and, therefore, produces cells that have a low chance of generating tumors.

To begin their study, Wang, and Yu and their colleagues isolated fibroblasts from the lower regions of the skin (dermis) and grew them in culture. Then they reprogrammed these cells in a relatively novel manner. This is a little complicated, but I will try to keep it simple.

Reprogramming cells usually requires scientists to infect cells with recombinant viruses that have been genetically engineered to express particular genes in cells or force cells to take up large foreign DNA. Both of these techniques can work relatively well in the laboratory, but you are left with cells that are filled with foreign DNA or recombinant viruses. It turns out that directly reprogramming cells only requires transient expression of specific genes, and once the cells have recommitted to a different cell fate, the expression of the genes used to get them there can be diminished.

To that end, some enterprising scientists have discovered that inducing cells to up modified proteins can also reprogram cells. Recently a new reagent called the QQ-reagent system can escort proteins across the cell membrane. The QQ-reagent has been patented and can sweep proteins into mammalian cells with high-efficiency and low toxicity (see Li Q, et al (2008) Methods Cell Biol 90:287–325).

Wang and Yu and their coworkers used genetically engineered bacteria to overexpress large quantities of four different proteins: Gata4, Hand2, Mef2c, and Tbx5. Then they mixed these proteins with their cultured human fibroblasts in the presence of the QQ reagent. This reagent drew the proteins into the cells and the fibroblasts were reprogrammed into cardiac progenitor cells (CPCs). Appropriate control experiments showed that cells that were treated with QQ reagent without these proteins were not reprogrammed. Wang and Yu and they research groups also exposed the cells to three growth factors, BMP4 and activin A, to drive the cells to become heart-specific cells, and basic fibroblast growth factor to turn the cells towards a progenitor cell fate.

The next set of experiment was intended to show that their newly reprogrammed were of a cardiac nature. First, the cells clearly expressed heart-specific genes. Flk-1 and Isl-1 are genes that earmark cardiac progenitor cells, and by the eighth day of induction, the vast majority of cells expressed both these genes.

 

Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.
Generation of protein-induced cardiac progenitor cells by modified transcript proteins. (A): Strategy of protein-induced cardiac progenitor cell (piCPC) generation. (B): Cell colonies were initially observed around days 4–8 and could be passaged to many small colonies around day 12. Representative phase contrast images are shown. The control was untreated human dermal fibroblasts in vehicle medium after 8 days. Scale bars = 100 μm. (C): quantitative polymerase chain reaction analysis of cardiac progenitor genes Flk-1 and Isl-1 in piCPCs. Fibroblast markers Col1a2 and FSP1 were also detected (∗, p < .05; ∗∗, p < .01 vs. day 0 control; error bars indicate SD; n = 3). (D): Representative fluorescent images are shown with typical cardiac progenitor markers Flk-1 (red) and Isl-1 (green) and fibroblast markers ColI (green) and FSP-1 (S100A4) (green) before and after reprogramming at day 8. DAPI staining was performed to visualize nuclei (blue) and all images were merged. Scale bars, 100 μm. (E): Flow cytometry analysis demonstrated Flk-1 and Isl-1 expressions were increased from d0 to d8 separately. Abbreviations: bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; ColI, collagen I; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FSP1, fibroblast-specific protein 1; mGHMT, modified Gata4/Hand2/Mef2c/Tbx5.

Second, cardiac cells can differentiate into three different cell types: heart muscle cells, blood vessels cells, and smooth muscle cells that surround the blood vessels. In mesoderm progenitors made from embryonic stem cells, inhibition of the Wnt signaling pathway can drive such cells to become heart muscle cells (see Chen, et al Nat Chem Biol 5:100–107; Willems E, et al Circ Res 109:360–364; Hudson J, et al Stem Cells Dev 21:1513–1523). However, Wang, Yu and company showed that treating the cells with a small molecule called IWR-1 that inhibits Wnt signaling drove their cells to differentiate into, not only heart muscle cells, but also endothelial (blood vessel) cells and smooth muscle cells when the cells were grown on gelatin coated dishes. When left to differentiate in culture, the cells beat synchronously and released calcium in a wave-like fashion that spread from one cell to another, suggesting that some cells were acting as pacemakers and setting the beat.

 

Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.
Protein-induced cardiac progenitor cells (piCPCs) differentiated into three cardiac lineages: cardiomyocytes, endothelial cells, and smooth muscle cells. (A): Schematic representation of the strategy to differentiate piCPCs in differentiation medium with IWR1 factor. (B): Quantitative data of mRNA expression of cardiac lineage marker genes (∗, p < .05; ∗∗, p < .01; and ∗∗∗, p < .001 vs. day 0 control; error bars indicate SD; n = 3). (C): Immunofluorescent staining for MHC, MYL2, CD31, CD34, smMHC, and αSMA. The combination of the four factors, GHMT, induces abundant MHC and Myl2, and some expression of CD31 and smMHC 28 days after transduction. Nuclei were counter stained with DAPI. Scale bars = 100 μm. (D): Flow cytometry analysis for cTnI, CD31, and smMHC. mGHMT plus IWR1 significantly enhances cTnI expression, and, to a lesser extent, CD31 and smMHC expression. Abbreviations: αSMA, α-smooth muscle actin; BMP4, bone morphogenetic protein 4; cTnI, cardiac troponin I; cTnT, cardiac troponin T; d, day; DAPI, 4′,6-diamidino-2-phenylindole; GHMT, Gata4/Hand2/Mef2c/Tbx5; mGHMT, modified GHMT; MHC, myosin heavy chain; MYL2, myosin light chain 2; smMHC, smooth muscle myosin heavy chain.

Then these cells were transplanted into the heart of mice that had suffered heart attacks. When compared to control hearts that received fluid, but no cells, the hearts of the animals that received protein-induced CPCs showed decreased scarring by 4 weeks after the transplantations. They also showed the growth of new heart muscle. A variety of staining experiments established that the engrafted protein-induced CPCs positive for heart muscle- and endothelial-specific cell markers. These experiments showed that transplantation of cardiac progenitor cells can not only help attenuate remodeling of the left ventricular after a heart attack, but that the protein-induced CPCs (piCPCs) can develop into cells of the cardiac lineage.

In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.
In vivo delivery of protein-induced cardiac progenitor cells improves cardiac function after myocardial infarction. (A): EF, FS, LVDd, and LVDs were analyzed by echocardiography (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001 vs. relevant 1 week; all data are presented as mean ± SD, n = 8). (B): Transplanted cells were detected by magnetic resonance imaging 4 weeks after myocardial infarction (MI). Red arrow points to the signal loss due to SPIO-labeled cells. (C): Masson trichrome staining on heart sections 4 weeks after MI injection in sham, vehicle, and piCPC groups. Scale bar = 0.5 cm. (D): Immunofluorescent staining for cTnI (red), CD31 (red), and anti-dextran (SPIO, green) of heart sections after piCPCs were transplanted 4 weeks after MI. White arrows point to transplanted cells or colocalization of cTnI or CD31 with SPIO. Scale bars = 100 μm. Abbreviations: cTnI, cardiac troponin I; DAPI, 4′,6-diamidino-2-phenylindole; EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular internal diameter at end-diastole; LVDs, left ventricular internal diameter at end-systole; piCPCs, protein-induced cardiac progenitor cell; SPIO, superparamagnetic iron oxide; W, week.

These are exciting results. It shows that direct reprogramming can occur without introducing genes into cells by means that can complicate the safety of the implanted cells. Also, because the cells are differentiated into progenitor cells, they still have the ability to proliferate and expand their numbers, which is essential for proper regeneration of a damaged tissue.

After a heart attack, the ventricle wall scars over and can become thin. However, piCPCs that have been directly reprogrammed from mature, adult cells can be used to replace dead heart muscle in a living animal.

Despite these exciting advances, further questions remain. For example, are the physiological properties of cells made from piCPCs similar enough to match the functional parameters of the heart into which they are inserting themselves? More work is necessary to answer that question. Functional equivalence is important, since a heart that does not function similarly from one end to the other can become arrhythmic, which is clinically dangerous. Further work is also required to precisely determine how well cells derived from piCPCs mature and coupling with neighboring cells. Therefore, larger animal studies and further studies in culture dishes will be necessary before this technique can come to the clinic. Nevertheless, this is a tremendous start to what will hopefully be a powerful and fruitful technique for healing damaged hearts.

Combining Umbilical Cord Cells with Hyaluronic Acid Improves Heart Repair After a Heart Attack


Umbilical cord blood cells have an advantage over bone marrow or peripheral blood cells in that aging, systemic inflammation, and stress or damage caused by cell processing procedures can potentially compromise and diminish the regenerative capability of these cells. This problem is particularly acute in the case of treating patients who have recently suffered a heart attack, since transplanted cells experience a rather hostile environment that kills off most cells. Additionally, blood flow through the heart tends to wash out infused cells, which further decreases any regenerative activities the cells might have otherwise exerted.

With this in mind, Patrick Hsieh and his colleagues at the Academia Sinica, in Taipei, Taiwan tested if ability of human cord blood mononuclear cells (CB-MNCs) injected into the heart in combination with a hyaluronan (HA) hydrogel could extend the regenerative abilities of these cells in a pig model. HA is a common component of connective tissue, and, in general, it is very well tolerated by patients and implanted cells. Furthermore, it has the added bonus of shielding cells from a hostile environment and preventing them from being washed out of the heart.

Hsieh used a total of 34 minipigs and divided them into five different groups. One group was the sham operation group in which minipigs received surgical incisions but no heart attack was induced. The second group had heart attacks surgically induced and received infusions of normal saline solutions. The third group of minipigs also experienced heart attacks, and had HA injected into the heart walls. The fourth group also suffered heart attacks and received injections of human umbilical cord stem cells into their heart walls. The fifth group experienced heart attacks and received injections of both HA and human umbilical cord blood cells. The animals were kept and examined two months after surgery.

Two months after the surgery, the minipigs that received injections of human umbilical cord blood cells plus HA showed the highest left ventricle ejection fraction (51.32% ± 0.81%). This is significant when compared to 42.87% ± 0.97%, for the group that received injections of normal saline, 44.2% ± 0.63% for the group that received injections of HA alone, and 46.17% ± 0.39% for the group that received injections of umbilical cord blood cells only. Additionally, hearts from minipigs that received cord blood cells plus HA improved the systolic and diastolic function significantly better than the other experimental groups. Injections of either cord blood cells alone or in combination with HA significantly decreased the scar area and promoted the formation of new blood vessels in the infarcted region. In general, this study suggests that combined infusion of umbilical cord blood cells and HA improves the function of the heart after a heart attack and might prove to be a promising treatment option of heart attack patients.

This is a preclinical study, but it is a preclinical study in a larger animal model system. Umbilical cord blood cells have a demonstrated ability to induce healing in the heart after a heart attack. However, the combination of these cells with HA almost certainly significantly increases cell retention in the heart, thereby significantly improving cardiac performance, and preventing cardiac remodeling. Therefore, using healthy cells donated from another source to replace damaged or moribund cells may be a better option to treat a heart patient and repair their sick heart.

This work appeared in Stem Cells Trans Med November 2015, doi: 10.5966/sctm.2015-0092

Umbilical Cord Stem Cells Improve Heart Function after a Heart Attack


The umbilical cord connects the baby to the placenta and contains umbilical arteries, umbilical veins, and a gooey material between the umbilical vessels called Warton’s jelly. Warton’s Jelly (WJ), besides being rich in extracellular matrix molecules also contains a mesenchymal stem cell population that is rather primitive. These WJ mesenchymal stem cells or WJMSCs have excellent potential for therapeutic strategies.

Lian Gao and her colleagues from the Navy General Hospital in Beijing, China, in collaboration with coworkers from the Shenzhen Beike Cell Engineering Research Institute in Shenzhen, China conducted a clinical trial that examined the use of these WJMSCs in human patients who had suffered a heart attack.  The results are as interesting as they are suggestive and were published in the journal BMC Medicine.

First we must consider the design of the study. Gao and others recruited 160 heart attack patients who were no younger than 18 and no older than 80-years old. All patients had to be free of liver or kidney disease, cancer or some other terminal illness. They were admitted to 11 hospitals in China between February 2011 and January 2012 and had suffered from a documented heart attack as defined by symptoms and their EKG (ST elevation). All patients has also been treated with the implantation of a stent within 12 hours of their heart attack and still retained a respectable amount of movement of the heart wall in the left ventricle. If patients were outside these parameters, they were excluded from the study.

Of the 160 patients who were recruited for the study, 44 were excluded, either because they did not fit within the exclusion criteria, did not wish to participate in the trial, or opted out for undisclosed reasons. This left 116 patients who were randomly assigned to the placebo group or the experimental group (58 in each group). Of these two groups, the placebo group had one patient discontinue the study because of a bout with stomach cancer. The experimental group had one patient die ten days into the trial, another was lost because they moved and a third patients withdrew because of leukemia. This left 57 subjects for the placebo group and 55 for the experiment group who went through all 18 months of follow-up after their respective procedures.

There were two end points for this clinical trial after patients were observed for 18 months after the procedure. The first was safety and this was measured by examining the number of adverse effects (AEs) within these 18 months. Such AEs include things like death, hospitalization for worsening heart function, severe arrhythmias, repeated coronary intervention, blood clots forming in the stents (stent thrombosis), coronary artery obstruction, and the growth of extra tissue in the heart that does not belong there, disorders of the immune system and so on. The second end pointy was efficacy of the implanted cells. To ascertain this, the function of the heart was measured using positron emission computer tomography (PET), and single-photo-emission computer tomography or SPECT. These imaging procedures allow cardiologists to take very precise snapshots of the heart and determine with a good deal of accuracy the performance of the heart.

The WJMSCs were acquired from umbilical cords that were donated from healthy mothers who had delivered healthy babies by means of Caesarian section. 21 of these umbilical cords had their blood vessels removed and then the gelatinous tissue surrounding the vessels was removed, sliced up, and cultured. The MSCs in the gelatinous tissue, which is Warton’s Jelly, migrated from the WJ to the culture dishes. After three passages in the culture dishes, he cells were harvested, concentrated, and tested for viruses, toxins, and cell viability. All cells were negative for viruses and toxins and other contaminants, and were also clearly MSCs, based on the ensemble of cell surface proteins that presented on their membranes, and showed high degrees of viability.

In infuse the cells into the hearts of the patients, six million WJMSCs were delivered into the coronary arteries using the usual over-the-wire techniques that are used to place stents, except that instead of placing stents, WJMSCs were slowly released into the coronary arteries. The cells will home to the damaged heart tissue and are able to pass through the blood vessels into the area of the infarct. Patients receiving the placebo, only received infusions of physiological saline solution, which was used to resuspend the WJMSCs.

The results are very encouraging. With respect to safety, the number of AEs was approximately the same for both groups. In the words of the study, “The groups did not differ in occurrences of MACEs (major adverse cardiac events), including death, recurrences of AMIs (acute myocardial infarctions) and re-hospitalization due to heart failure, during the course of treatment and the 18-month follow-up period.” There were no indications of cancer or the increase in tumor-specific molecules in the blood of the patients from either group. No biochemical or immune abnormalities were observed in any pf the patients either. The stomach cancer in one patient in the placebo group and leukemia in a patient from the experimental group were shown to be unrelated to the procedures. Therefore, at 18 months after the procedure, the infusion of these cells appears to be safe.

As to the efficacy of the procedure, there were significant improvements in the heart function of patients who had received the WJMSCs over those who had received placebo. First of all, the baseline heart function of patients in both groups was approximately the same on the average, except that the patients in the experimental group had slightly better heart parameter than those in the placebo group. Therefore, the efficacy of this procedure was determined by measuring the change in heart performance after the procedure. Patients who had received the placebo had about a 3% increase in the uptake of the F18-labeled sugar molecule after 4 months. The uptake of this marker indicates the presence of live cells. An increase in uptake of the modified sugar molecule shows that some new heart tissue has been produced, probably by the resident stem cell population in the heart. The experimental group, however, after 4 months showed an approximate 7% increase in PET signal intensity. This shows that a good deal more heart cells are being formed in the WJMSC-treated hearts that in the placebo-treated hearts. The SPECT imaging assays the “perfusion” of the heart tissue or the degree to which the heart tissue is being fed by blood vessels. After a heart attack, the dead area of the heart lacks blood vessels and its poor perfusion can affect nearby areas. The placebo-treated patients had a roughly 4% increase in SPECT signal, whereas the WJMSC-treated group had a 7% increase. Thus, the WJMSC-treated hearts had more blood vessels to feed the blood, oxygen and nutrients to the heart muscle and therefore, better perfusion.

Finally, the percentage of blood ejected by the heart during each contraction increase about 3% in the placebo group, but increase by about 8% in the WJMSC-treated group after 18 months. This parameter of heart function, the ejection fraction, is a very important measure of heart function and the fact that it significantly increased in the WJMSC-treated patients over the placebo-treated patients is an important finding.

This was a double-blinded, placebo-controlled study that determined the safety and efficacy of infusions of WJMSCs into the hearts of patients who had recently suffered from a heart attack. In animal experiments, these cells have been shown to increase heart function, increase blood vessel density in the hearts of animals, and increase resident heart-specific stem cell activity in the heart (see Lupu and others, Cell Physiol Biochem 2011; 28:63-76; Gao and others, Cell Transplant 2013; 22:1883-1900; Lopez Y, and others, Current Stem Cell Res Ther 2013;8:46-59). This clinical trial suggests that those benefits documented in laboratory animals might translate to human patients.

This is not a perfect study. These patients will need to be followed for several years to establish that these benefits are long-term and not short-term. Also, there is no indication that patients were given a 6-minute walking test to determine if the improvements in cardiac function translated to improvements in basic activities. However, it is an interesting study and it suggests that banking WJMSCs in addition to cord blood might be a good idea for use in trials like this one and maybe, someday for treatments of heart attack patients.

Heart Muscle Cells Produced from Induced Pluripotent Stem Cells Repair Heart Attacks in Pigs


When heart muscle cells are made from embryonic stem cells, they integrate into the heart and form proper connections with other heart muscle cells. Such experiments have been conducted in mice, guinea pigs, and nonhuman primates (i.e. monkeys). Chong and others earlier this year (Nature (2014) 510, 273-277) implanted heart muscle cells produced from embryonic stem cells into the hearts of nonhuman primates that had suffered from heart attacks. There was extensive evidence of engraftment of these cells, remuscularization of the heart, and electrical synchronization 2 to 7 weeks after transplantation. However, despite these successes, the hearts of some of these animals also showed abnormal heart beat patterns (known as arrhythmias). Such a problem has also been observed in other laboratory animals as well (see my book The Stem Cell Epistles), and this problem has to be addressed before derivatives of pluripotent stem cells can be used to treat damaged hearts (pluripotent means capable of differentiating into all the mature adult cell types).

Jianyi Zhang and his colleagues at the University of Minnesota have used induced pluripotent stem cells made from human skin cells to produce heart muscle cells that were used to treat pigs that had suffered from induced heart attacks.  Their results differed slightly from those of Chong and others.

Zhang and others noted that implanted heart muscle cells typically survive better if they are implanted with blood vessel cells (endothelial cells or ECs).  This was first shown in culture by Xiong and others in 2012 (Circulation Research 111, 455-468), but other work has confirmed this.  That is, Zhang’s coworkers in his laboratory co-transplanted heart muscle cells made from induced pluripotent stem cells with endothelial cells and smooth muscle cells (which are also a part of blood vessels), and saw that the co-transplanted cells survived much better than heart muscle cells that were transplanted without these other cell types.

On the basis of these experiments, Zhang and his crew decided that implanted heart muscle cells would do much better if they were implanted into pig hearts if they were implanted with endothelial and smooth muscle cells.  This was the hypothesis that Zhang and others wanted to test in this paper (which was published in Cell Stem Cell, Dec 4, 2014, 750-761).

Skin biopsies from human volunteers were used as a source of skin cells that were then genetically engineered and then cultured to form human induced pluripotent stem cells (hiPSCs).  These cultured hiPSCs were differentiated into heart muscle cells by means of the “Sandwich method,” which yielded beating heart muscle cells in about 30 days.  Additionally, their hiPSC lines were differentiated into smooth muscle and endothelial cells as well.

Next, Zhang and his colleagues and collaborators used 92 pigs and subjected them to experimentally-induced heart attacks.  Why pigs?  Pigs are a larger animal than rodents, and their hearts are larger and beat much slower than the hearts of rats and mice.  Therefore, they are a more expensive, but better experimental model system for the human heart.  Nevertheless, these pigs were divided into six different groups (3 pigs died from the procedure, so there were 89 pigs involved in this experiment).  Animals in the first group or SHAM group underwent the surgery to induce a heart attack, but no heart attack was induced.  The second group was called the MI group and this group received no other interventions after surgery.  The Patch group received a fibrin patch over the site of injury, but no cells.  The CM + EC + SMC group received injections of 2 million heart muscle cells, two million endothelial cells, and two million smooth muscle cells directly into the injured portion of the heart.  The Cell + Patch group received all three cell types in a fibrin patched that was imbued with a growth factor called Insulin-like growth Factor-1 (IGF-1) that had been loaded into microspheres.  This causes the growth factor to be released gradually and exert its effects over a much greater period of time.

That’s a lot of information so let’s review – six groups: 1) SHAM (no heart attack; 2) MI (heart attack and no treatment); 3) Patch (just the fibrin patch); 4) Cells + Patch (fibrin patch with the three cell types); 5) Cells (cells, but no patch), and a final group cells Patch + CM (just heart muscle cells in the patch).

Animals were evaluated one week after the heart attack and four weeks after their heart attacks. I am uncertain how soon after the heart attack the treatments were given, but in the paper it reads to me as though the treatments were given right after the heart attacks had been induced.  Because all implanted cells were engineered to glow in the dark, the number of surviving cells could be counted and tracked.

Only 4.2% of the cell survived in the Cells group, up to 9% of the cells in the Cell + Patch group survived.  32% of the cells in the CM + Patch group survived.  Thus, it seemed as though the presence of the other cell types did increase the survival of the heart muscle cells and the patch also increased cell survival rates.  Secondly, the heart function of all the treated groups was better than the MI group, but the hearts treated with Cells + Patch were clearly superior to all the others, with the exception of the SHAM group.  The hiPSC-derived heart muscle cells also clearly engrafted into the hearts of the pigs, but the big surprise in this paper is that THERE WERE NO INDICATIONS OF ARRHYTHMIAS!!!  Apparently the manner in which these hiPSC-derived heart muscle cells integrated and adapted to the native heart in such as way as to preclude irregular electrical activity.  Another indicator measured was ratio of phosphocreatine to ATP.  If that sounds like a language from outer space, it simply means a measurement of the efficiency of muscle mitochondria (the part of the cell that makes all the energy).  Again the Cells + Patch hearts had significantly more efficient mitochondria, and, hence, better energy production than the other hearts.  Damage to mitochondria also tends cause cells to up and die, which means that these cells were in better health that those from the MI group.

This paper shows that an ingenious tissue engineering innovation that uses a fibrin patch and a a combination of cells, not just heart muscle cells can significantly increase the healing after a heart attack.  Also, even though neither embryonic stem cell-derived cells nor iPSC-derived cells are ready for clinical trials, this paper shows that iPSCs are not as far behind iPSCs as some authors have suggested.  Furthermore, because iPSCs would not be subject to immunological rejection, they have an inherent superiority over embryonic stem cells.  The problem comes with the time required to make iPSCs and then derived heart muscle cells from them, which might put it outside the time window for treat of an acute heart attack.

Conditioning Stem Cells to Survive in the Heart


After a heart attack, the heart is a very inhospitable place for implanted stem cells. These cells have to deal with low oxygen levels, marauding white blood cells, toxins released from dead or nearly-dead cells, and other nasty things.

Getting cells to survive in this place is essential if the cells are going to provide any healing to he heart. Fortunately, a Chinese group has discovered that growing cells in inhospitable conditions before implantation greatly improves their survival. Now, this same group from Emory University School of Medicine in Atlanta, Georgia has shown that a small molecule can do the same thing.

This work, published in Current Stem Cell Research and Therapy, centers upon a pathway in cells controlled by a protein called the hypoxia-inducible factor or HIF. This protein regulates those genes that allow cells to withstand low-oxygen and other stressful conditions. HIF is composed of two parts: an oxygen-sensitive inducible HIF-1α subunit and a constitutive HIF-1β subunit. During nonstressful conditions, the alpha subunit is constantly being degraded after it is made because it is modified by a enzymes called prolyl hydroxylase (PHD) enzymes. In the presence of low oxygen conditions, PHD enzymes are inhibited and HIF-1α increases in concentration. The HIFα/β heterodimer forms and is stabilized, and translocates to the nucleus where it activates target genes.

nrd1199-f1

It turns out that small molecules can inhibit PHD enzymes and induce the low-oxygen status in cells without subjecting them to rigorous culture conditions. For example, dimethyloxalylglycine (DMOG) can inhibit PHD enzymes and produce in cells the types of responses normally observed under low-oxygen conditions.

In this paper, Ling Wei and colleagues cultured mesenchymal stem cells from bone marrow with or without 1 mM DMOG for 24 hours in complete culture medium before transplantation. These cells were then transplanted into the hearts of rats 30 minutes after those rats had suffered an experimentally-induced heart attack. They then measured the rates of cell death 24 hours after engraftment, and heart function, new blood vessel formation and infarct size 4 weeks later.

In DMOG-preconditioned bone marrow MSCs (DMOG-BMSCs), the expression of survival and blood-vessel-making factors were significantly increased. In comparison with control cells.  DMOG-BMSCs also survived better and enhanced the formation of new blood vessels in culture and when implanted into the heart of a living animal.
C to H , Angiogenesis was inspected using vWF staining (red) in heart sections from MI, C-BMSC and DMOG-BMSC groups 4 weeks after MI. Hoechst staining (blue) s hows the total cells. I. Summary of total tube length measured in experiments A and B. The t otal tube length in C- BMSC group was arbitrarily presented as 1. N = 3 independent measure ments. J , Summary of total vessel density in different groups of in vivo experiments. N = 8 animals in each group. * P <0.05 compared with C-BMSC group; # P <0.05 compared with MI control group.
C to H, Angiogenesis was inspected using vWF staining (red) in heart sections from MI, C-BMSC
and DMOG-BMSC groups 4 weeks after MI. Hoechst staining (blue) shows the total cells. I. Summary of total tube length measured in experiments A and B. The total tube length in C-BMSC group was arbitrarily presented as 1. N = 3 independent measurements. J, Summary of total vessel density in different groups of in vivo experiments. N = 8 animals in each group.
Transplantation of DMOG-BMSCs also reduced heart infarct size and promoted functional benefits of the cell therapy.
Effect of BMSCs transplantation on ischemia-induced infarct formation. Heart infarct area and scar formation were determined using Masson’s Trichrome staining 4 weeks after MI. A to C . Images of representative infarcted hearts from a MI control rat, a MI rat received C-BMSCs, and a MI rat received DMOG-BMSCs. D. Transplantation of BMSCs reduced heart infarction formation, the protective effects were significantly greater with transplantation of DMOG-BMSCs. N = 5 rats in each group. * P <0.05 compared with MI group; # P <0.05 compared with C-BMSC group.
Effect of BMSCs transplantation on ischemia-induced infarct formation. Heart infarct area and scar formation were determined using Masson’s
Trichrome staining 4 weeks after MI. A to C. Images of representative infarcted hearts from a MI control
rat, a MI rat received C-BMSCs, and a MI rat received DMOG-BMSCs. D. Transplantation of BMSCs
reduced heart infarction formation, the protective effects were significantly greater with transplantation of DMOG-BMSCs. N = 5 rats in each group.
Thus, this paper shows that targeting an oxygen sensing system in stem cells such as PHD enzymes (prolyl hydroxylase) provides a new promising pharmacological approach for enhanced survival of BMSCs.  This procedure also increases paracrine signaling, augments the regenerative activities of these cells, and, ultimately, and improves functional recovery of the heart as a result of cell transplantation therapy for the heart after a heart attack.  This is only a preclinical study, but the data is strong, and hopefully new clinical trials will bear this out.

Stem Cell Therapy Replaces Dead Heart Muscle in Primates


The laboratory of Charles Murry at the University of Washington has used embryonic stem cells to make heart muscle cells that were then used to regenerate damaged hearts in non-human primates. This experiment demonstrates the possibility of using heart muscle cells derived from pluripotent stem cells, but it also underscores the many challenges that still must be overcome.

When the heart undergoes a heart attack or other types of damage, heart muscle cells begin to die off and these cells are not easy to replace. Heart muscle cells, also known as cardiomyocytes, do not readily replace themselves. Even though the heart has a resident stem cell population, (cardiac progenitor cells or CPCs) these heart-specific stem cells have a limited capacity to regenerate the heart. After a heart attack, as many as one billion cardiomyocytes or more die. The loss of so many beating heart muscle cells compromises heart function and can also lead to chronic heart failure and even death.

Physicians, cardiologists, and researchers have been on the lookout for new and improved procedures and technologies to replenish damaged heart tissue. Several different types of stem cells have shown promise in animal models and in human clinical trials. Stem cells from bone marrow have the ability to secrete a cocktail of molecules that stimulate heart regeneration. Whole bone marrow or isolated stem cell populations have shown variable, but statistically significant in patients who have had a recent heart attack. Unfortunately, stem cells from bone marrow do not have the ability to differentiate into heart muscle cells, and to maximize regeneration of the heart, damaged heart muscle must be replaced.

Human embryonic stem cells have proven promising in small animal models, but the long-term effects of embryonic stem cell-mediated improvements in some cases have proven to be transient. An additional problem with embryonic stem cell-derived heart muscle cells is their tendency to cause abnormal heart rates, otherwise known as arrhythmias.

Scientists in Murry’s laboratory tried to scale-up the production of cardiomyocytes from human embryonic stem cells in order to test the regenerative ability of these cells in a large animal model – non-human primates. These experiments were published online on April 30, 2014, in the journal Nature.

Murry’s team derived cardiomyocytes from genetically-engineered human embryonic stem cells that made a fluorescent calcium indicator that glowed in the presence of high calcium ion concentrations. With this fluorescent calcium indicator, Murray and his coworkers could track the calcium waves that mark the electrical activity of a beating heart. The animal subjects for this experiment were pigtail macaques (Macaca nemestrina) that had suffered heart damage and had been treated with drugs to suppress their immune systems. Five days later, the embryonic stem cell-derived cardiomyocytes were delivered in a surgical procedure to the damaged regions and surrounding border zones of the heart.

Over a 3-month period, the implanted cells infiltrated damaged heart muscle, matured, and organized themselves into muscle fibers in all the monkeys who received the treatment. An average of 40% of the damaged tissue was replaced by these grafts. Three-dimensional imaging showed that arteries and veins integrated into the grafts. Because sick hearts often contain clogged blood vessels, oxygen delivery to the damaged heart tissue was minimal. However, because these grafts contained integrated blood vessels, they would potentially be long-lasting.

Calcium activity studies showed that the heart muscle tissue within the grafts were electrically active and coupled to activity of the host heart. The grafts beat along with host muscle at rates of up to 240 beats per minute, the highest rate tested.

Cardiac cells derived from human stem cells (green) meshed and beat along with primates’ heart cells (red). Credit: Murry Lab/University of Washington.
Cardiac cells derived from human stem cells (green) meshed and beat along with primates’ heart cells (red). Credit: Murry Lab/University of Washington.

All the macaques that received the grafts showed transient arrhythmias or irregular heart rates. However, these subsided by 4 weeks post-transplantation. The animals remained conscious and in no apparent distress during periods of arrhythmia. However, this problem will need to be addressed before this approach can be tested in humans.

“Before this study, it was not known if it is possible to produce sufficient numbers of these cells and successfully use them to remuscularize damaged hearts in a large animal whose heart size and physiology is similar to that of the human heart,” Murry says.

This article shows that despite the obstacles that remain, transplantation of human cardiomyocytes derived from pluripotent stem cells may be feasible for heart patients.

There are a few caveats I would like to mention.  First of all, these animals underwent immunosuppression.  If this procedure were to be used in a human patient, the human patient would need life-long immunosuppression, which has a wide range of side effects and tends to stop working over time.  Therefore, induced pluripotent stem cells are a better choice.  Secondly, the paper admits that the implanted cells underwent “progressive but incomplete maturation over a 3-month period.”  If the implanted cells are not maturing completely, then the risk of arrhythmias still exists, even though they may have subsided in these animals after 4 weeks.  This leads me to my third point.  These animals were watched for 3 months.  How do we know that these results were not transient?  Longer-term experiments are needed to establish that this treatment actually is long-term and not transient.  It is, however, gratifying to see an experiment that was extended to 12 weeks rather than the usual 4 weeks that is usually seen in mice.

Finally, tucked away in the extended data is the statement: “The cell-treated animals showed variable responses, with some having increased function and some having decreased function. Because of small group size, no statistical effects of hESC-CM therapy can be discerned.”  In other words, the treatments worked swimmingly in some animals and not at all in others.  This was a small animal trial and better numbers will be needed if this technology is to come to the clinic.