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.

Nonhematopoietic Stem Cells from Umbilical Cord Blood Improve Heart Function After a Heart Attack


Xin Yu, who has dual appointments at Case Western Reserve University, in Cleveland, Ohio and the University of Minnesota Medical School in Minneapolis, Minnesota, has published a remarkable paper in the journal Cell Transplantation that describes the use of a stem cell population from umbilical cord blood to treat mice that had suffered heart attacks. The non-invasive way in which these cells were administered and the tremendous healing qualities of these cells makes paper unique.

In 2005, Water Low at the University of Minnesota Medical School described the isolation and characterization of a unique stem cell population from umbilical cord blood that he called nonhematopoietic umbilical cord blood stem cells or nh-UCBSCs. These cells were used to treat animals with strokes and they induced the growth of new brain cells in the brains of treated mice (see J Xiao, Z Nan, Y Motooka, and WC Low, Stem Cells Dev. 2005 Dec;14(6):722-33).

Yu used these cells to treat male Lewis rats that had suffered heart attacks. In all cases, the rats were subjected to open-heart surgery and the left anterior descending artery was tied off to induce a heart attack. One group of rats were operated on but no heart attacks were induced. A second group was given heart attacks, and then two days later were given intravenous saline infusions. The third group was given a heart attack and then two days later, were injected with one million nonhematopoietic umbilical cord stem cells into their tail veins.

Ten months after the surgery, the heart structure and function of animals from all three groups was assessed with tensor diffusion magnetic imaging, and a pressure‐volume conductance catheter. The hearts were also extirpated from the animals and structurally assessed by means of staining and 3-D imaging.

The stem cell-treated animals were compared with the sham-operated animals and the saline-treated animals. In almost all categories, the stem cell-treated animals had better function. Also, the overall structure of the heart was preserved and looked more like the normal heart than the saline-treated hearts. For example, in the saline-treated group, the heart wall thickness in the infarct zone was reduced by 50% compared to the control rats, and wall thickness at the border zone was also significantly
decreased. However, there were no statistical difference in wall thickness between the stem cell-treated group and the control group.

Additional finds were that the stem cell-treated group had significantly smaller areas of dead cells, more blood vessels, and better heart muscle fiber structure that contracted better.

These data show that the long-term effects of nh-UCBSC administration was to preserve the structure, and, consequently, the function of the heart after a heart attack.

However, the added bonus to this work is that the animals were injected with these cells into the tail vein. The animals did not have to have their chests cracked, or have over-the-wire stent technology to implant these cells; they merely introduced them intravenously. Apparently, the nh-UCBSCs homed to the damaged heart and mediated its healing. If such healing can be translated to human patients, this could truly be a revolutionary find.

Making Heart Muscle from Skeletal Muscle Stem Cells


Several experiments in animals and a few clinical trials in human patients have shown that implanting skeletal muscle cells isolated from muscle biopsies into the heart after a heart attack can help the heart to some degree, but the implanted skeletal muscle cells do not integrate into the existing heart muscle mass and the skeletal muscle cells do not differentiate into heart muscle cells.

Experiments like those mentioned above utilized muscle satellite cells. Muscle satellite cells are a resident stem cell population that respond to muscle damage and divide to form skeletal muscle cells form new muscle. Satellite cells are a perfect example of a unipotent stem cell, which is to say a cell that makes one type of terminally differentiated cell type.

Skeletal muscles, however, have another cell population called muscle-derived stem cells or MDSCs. MDSCs express an entirely different set of cell surface proteins than satellite cells, and have the capacity to differentiate into skeletal muscle, smooth muscle, bone, tendon, nerve, endothelial and hematopoietic cells. MDSCs grow well in culture, tolerate low oxygen conditions quite well, and show excellent regenerative potential.

Other laboratories have managed to culture MDSCs in collagen and produce beating heart muscle cells. Others have observed MDSCs forming a proper myocardium under certain conditions. Several studies have established the ability to MDSCs to treat laboratory animals that have suffered a heart attack. The most recent work from Sekiya and others has established that cell sheets made from MDSCs can reduce dilation of the left ventricle, increased capillary density, and promoted recovery without causing erratic heat beat patterns.

Despite their obvious efficacy. MDSCs remain difficult to isolate in high enough numbers to therapeutic purposes. None of the cell surface molecules sported by MDSCs are unique to those cells. Therefore, getting clean cultures of MDSCs remains a challenge. Still, these cells represent some of the best hopes for regenerative medicine in the heart. These cells do form heart muscle cells and heal ailing hearts. They can be grown in bioreactors to high numbers and can also be combined with engineered materials to shore up a damaged heart and mediate its regeneration. While the use of MDSCs is still in its infancy, the promise certainly is there.

New US Phase IIa Trial and Phase III Trial in Kazakhstan Examine CardioCell’s itMSC Therapy to Treat Heart Attack Patients


The regenerative medicine company CardioCell LLC has announced two new clinical trials in two different countries that utilize its allogeneic stem-cell therapy to treat subjects with acute myocardial infarction (AMI), which is a problem that faces more than 1.26 million Americans annually. The United States-based trial is a Phase IIa AMI clinical trial that is designed to evaluate the clinical safety and efficacy of the CardioCell Ischemia-Tolerant Mesenchymal Stem Cells or itMSCs. The second clinical trial in collaboration with the Ministry of Health in Kazakhstan is a Phase III AMI clinical trial on the intravenous administration of CardioCell’s itMSCs. This clinical trial is proceeding on the strength of the efficacy and safety of itMSCs showed in previous Phase II clinical trials.

CardioCell’s itMSCs are exclusively licensed from CardioCell’s parent company Stemedica Cell Technologies Inc. Normally, when mesenchymal stem cells from fat, bone marrow, or some other tissue source are grown in the laboratory, the cells are provided with normal concentrations of oxygen. However, CardioCell itMSCs are grown under low oxygen or hypoxic conditions. Such growth conditions more closely mimic the environment in which these stem cells normally live in the body. By growing these MSCs under these low-oxygen conditions, the cells become tolerant to low-oxygen conditions (ischemia-tolerant), and if transplanted into other low-oxygen environments, they will flourish rather than die.

Another advantage of itMSCs for regenerative treatments over other types of MSCs is that itMSCs secrete higher levels of growth factors that induce the formation of new blood vessels and promote tissue healing. These clinical trials have been designed to help determine if CardioCell’s itMSC-based therapies stimulate a regenerative response in acute heart attack patients.

“CardioCell’s new Phase IIa AMI study is built on the excellent safety data reported in previous Phase I clinical trials using our unique, hypoxically grown stem cells,” says Dr. Sergey Sikora, Ph.D., CardioCell’s president and CEO. “We are also pleased to report that the Ministry of Health in Kazakhstan is proceeding with a Phase III CardioCell-therapy study following its Phase II study that was highly promising in terms of efficacy and safety. Our studies target AMI patients who have depressed left ventricular ejection fraction (LVEF), which makes them prone to developing extensive scarring and therefore to the development of chronic heart failure. CardioCell hopes our itMSC therapies will inhibit the development of extensive scarring and, thus, the occurrence of chronic heart failure in these patients.”

The United States-based Phase IIa clinical trial will take place at Emory University, Sanford Health and Mercy Gilbert Medical Center. The CardioCell Phase IIa AMI trial is a double-blinded, multicenter, randomized study designed to assess the safety, tolerability and preliminary clinical efficacy of a single, intravenous dose of allogeneic mesenchymal bone-marrow cells infused into subjects with ST segment-elevation myocardial infarction (STEMI).

“While stem-cell therapy for cardiovascular disease is nothing new, CardioCell is bringing to the field a new, unique type of stem-cell technology that has the possibility of being more effective than other AMI treatments,” says MedStar Heart Institute’s Director of Translational and Vascular Biology Research and CardioCell’s Scientific Advisory Board Chair Dr. Stephen Epstein. “Evidence exists demonstrating that MSCs grown under hypoxic conditions express higher levels of molecules associated with angiogenesis and healing processes. There is also evidence indicating they migrate with greater avidity to various cytokines and growth factors and, most importantly, home more robustly to ischemic tissue. Studies like those underway using CardioCell’s technology are designed to determine if we can evoke a more potent healing response that will reduce the extent of myocardial cell death occurring during AMI and thereby decrease the amount of scar tissue resulting from the infarct. A therapy that could achieve this would have a major beneficial impact in reducing the occurrence of chronic heart failure.”

Kazakhstan’s National Scientific Medical Center is conducting a Phase III AMI clinical trial using CardioCell’s itMSCs, which are sponsored by local licensee Altaco. This clinical trial is entitled, “Intravenous Administration of itMSCs for AMI Patients,” and is proceeding based on a completed Phase II efficacy and safety study. However, the results of this previous Phase II study are preliminary because the sample group was so small. Despite these limitations, the findings demonstrated statistically significant elevation (more than 12 percent over the control group) in the ejection fraction of the left ventricle of the heart in patients who had received itMSCs. Also, a significant reduction in inflammation was also observed, as ascertained by lower CRP (C-reactive protein) levels in the blood of treated patients in comparison to control groups. Thus, Dr. Daniyar Jumaniyazov, M.D., Ph.D., principal investigator in Kazakhstan clinical trials states: “In our clinical Phase II trial for patients with AMI, treatment using itMSCs improved global and local myocardial function and normalized systolic and diastolic left ventricular filling, as compared to the control group. We are encouraged by these results and look forward to confirming them in a Phase III study.”

CardioCell’s treatment is the first to apply itMSC therapies for cardiovascular indications like AMI, chronic heart failure and peripheral artery disease. Manufactured by CardioCell’s parent company Stemedica and approved for use in clinical trials, itMSCs are manufactured under Stemedica’s patented, continuous-low-oxygen conditions and proprietary media, which provide itMSCs’ unique benefits: increased potency, safety and scalability. itMSCs differ from competing MSCs in two key areas. itMSCs demonstrate increased migratory ability towards the place of injury, and they show increased secretion of growth and transcription factors (e.g., VEGF, FGF and HIF-1), as demonstrated in a peer-reviewed publication (Vertelov et al., 2013). This can potentially lead to improved regenerative abilities of itMSCs. In addition, itMSCs have significantly fewer HLA-DR receptors on the cell surface than normal MSCs, which might reduce the propensity to cause immune responses. As another benefit, itMSCs are highly scalable. A single donor specimen can currently yield about 1 million patient treatments, and this number is expected to grow to 10 million once full robotization of Stemedica’s facility is complete.

Taiwanese Group Identifies Stem Cell-Based Drug to Rejuvenate Aged Hearts


A southern Taiwan-based National Cheng Kung University research team led by Patrick Ching-Ho Hsieh has discovered that a molecule called prostaglandin E2 can regenerate aged hearts in rodents.

This discovery provides a useful new perspective on heart regeneration and presents an effective option for heart disease patients other than heart transplant.

According to Hsieh, congestive heart disease and other cardiovascular diseases are a leading cause of morbidity and mortality throughout the world. There are some six million patients with congestive heart failure in the US alone and some 400,000 in Taiwan. Despite intensive drug, surgical and other medical interventions, 80 percent of all heart patients die within 8 years of diagnosis.

Even though several experiments and clinical trials have established that heart regeneration can take place, the means by which the heart regenerates is still not completely clear, and there are also no drugs to stimulate heart regeneration by the resident stem cell population in the heart.

Now, after seven years of hard work, Hsieh’s team has identified the critical time period and the essential player that directs heart repair.

Hsieh and his colleagues used genetically engineered mice that Hsieh had developed as a postdoctoral research fellow at Harvard Medical School. By using this transgenic mouse strain, Hsieh and others showed that the self-repair process of the heart begins 7 days after injury and peaks at 10 days after injury.

The “director” of this self-repair process is the molecule PGE2. PGE2 regulates heart-specific stem cell activities.

PGE2

“More importantly, both young and old mice have significant improvements for cardiac remodeling if you treat both of them [with] PGE2,” said Hsieh.

Hsieh’s team also established that PGE2 decreases expression of a gene associated with aging, TGF-beta1. PGE2 also rejuvenates the micro-environment of the aged cells, according to Hsieh.

When Is the Best Time to Treat Heart Attack Patients With Stem Cells?


Several preclinical trials in laboratory animals and clinical trials have definitively demonstrated the efficacy of stem cell treatments after a heart attack. However, these same studies have left several question largely unresolved. For example, when is the best time to treat acute heart attack patients? What is the appropriate stem cell dose? What is the best way to administer these stem cells? Is it better to use a patient’s own stem cells or stem cells from someone else?

A recent clinical trial from Soochow University in Suzhou, China has addressed the question of when to treat heart attack patients. Published in the Life Sciences section of the journal Science China, Yi Huan Chen and Xiao Mei Teng and their colleagues in the laboratory of Zen Ya Shen administered bone marrow-derived mesenchymal stromal cells at different times after a heart attack. Their study also examined the effects of mesenchymal stem cells transplants at different times after a heart attack in Taihu Meishan pigs. This combination of preclinical and clinical studies makes this paper a very powerful piece of research indeed.

The results of the clinical trial came from 42 heart attack patients who were treated 3 hours after suffering a heart attack, or 1 day, 3 days, 2 weeks or 4 weeks after a heart attack. The patients were evaluated with echocardiogram to ascertain heart function and magnetic resonance imaging of the heart to determine the size of the heart scar, the thickness of the heart wall, and the amount of blood pumped per heart beat (stroke volume).

When the data were complied and analyzed, patients who received their stem cell transplants 2-4 weeks after their heart attacks fared better than the other groups. The heart function improved substantially and the size of the infarct shrank the most. 4 weeks was better than 2 weeks,

The animal studies showed very similar results.

Eight patients were selected to receive additional stem cell transplants. These patients showed even greater improvements in heart function (ejection fraction improved to an average of 51.9% s opposed to 39.3% for the controls).

These results show that 2-4 weeks constitutes the optimal window for stem cell transplantation. If the transplant is given too early, then the environment of he heart is simply too hostile to support the survival of the stem cells. However, if the transplant is performed too late, the heart has already experiences a large amount of cell death, and a stem cell treatment might be superfluous. Instead 2-4 weeks appears to be the “sweet spot” when the heart is hospitable enough to support the survival of the transplanted stem cells and benefit from their healing properties. Also, this paper shows that multiple stem cell transplants a two different times to convey additional benefits, and should be considered under certain conditions.

Umbilical Cord Stem Cells Preserve Heart Function After a Heart Attack in Mice


A consortium of Portuguese scientists have conducted an extensive examination of the effects of mesenchymal stromal cells from umbilical cord on the heart of mice that have suffered a massive heart attack. Even more remarkable is that these workers used a proprietary technique to harvest, process, and prepare the umbilical cord stem cells in the hopes that this technique would give rise to a commercial product that will be tested in human clinical trials,

Human umbilical cord tissue-derived Mesenchymal Stromal Cells (MSCs) were obtained by means of a proprietary technology that was developed by a biomedical company called ECBio. Their product,, UCX®, consists of clean, high-quality, umbilical cord stem cells that are collected under Good Manufacturing Practices. The use of Good Manufacturing Practice means that UCX is potentially a clinical-grade product. Thus, this paper represents a preclinical evaluation of UCX.

This experiments in this paper used standard methods to give mice heart attacks that were later received injections of UCX into their heart muscle. The same UCX cells were used in experiments with cultured cells to determine their effects under more controlled conditions.

The mice that received the UCX injections into their heart muscles after suffering from a large heart attack showed preservation of heart function. Also, measurements of the numbers of dead cells in the heart muscle of heart-sick mice that did and did not receive injections of umbilical cord cells into their hearts showed that the umbilical cord stem cells preserved heart muscle cells and prevented them from dying. Additionally, the implanted umbilical cord MSCs induced the growth and formation of many small blood vessels in the infarcted area of the heart. This prevented the heart from undergoing remodeling (enlargement), and preserved heart structure and function.

When subjected to a battery of tests on cultured cells, UCX activated cardiac stem cells, which are the resident stem cell population in the heart. Implanted UCX cells activated the proliferation of cardiac stem cells and their differentiation into heart muscle cells. There was no evidence that umbilical cord MSCs differentiated into heart muscle cells and engrafted into the heart. Rather UCX seems to help the heart by means of paracrine mechanisms, which simply means that they secrete healing molecules in the heart and help the heart heal itself.

In conclusion, Diana Santos Nascimento, the lead author of this work, and her colleagues state that, “the method of UCX® extraction and subsequent processing has been recently adapted to advanced therapy medicinal product (ATMP) standards, as defined by the guideline on the minimum quality data for certification of ATMP. Given that our work constitutes a proof-of-principle for the cardioprotective effects UCX® exert in the context of MI, a future clinical usage of this off-the-shelf cellular product can be envisaged.”

Preclinical trials with larger animals should come next, and after that, hopefully, the first human clinical trials will begin.

New Tool for Stem Cell Transplantation into the Heart


Researchers from the famed Mayo Clinic, in collaboration with scientists at a biopharmaceutical biotechnology company in Belgium have invented a specialized catheter for transplanting stem cells into a beating heart.

This new device contains a curved needle with graded openings along the shaft of the needle. The cells are released into the needle and out through the openings in the side of the needle shaft. This results in maximum retention of implanted stem cells to repair the heart.

“Although biotherapies are increasingly more sophisticated, the tools for delivering regenerative therapies demonstrate a limited capacity in achieving high cell retention in the heart,” said Atta Behfar, the lead author of this study and a cardiologist. “Retention of cells is, of course, crucial to an effective, practical therapy.”

Researchers from the Mayo Clinic Center for Regenerative Medicine in Rochester, MN and Cardio3 Biosciences in Mont-Saint-Guibert, Belgium, collaborated to develop the device. Development of this technology began by modeling the dynamic motions of the heart in a computer model. Once the Belgium group had refined this computer model, the model was tested in North America for safety and retention efficiency.

These experiments showed that the new, curved design of the catheter eliminates backflow and minimizes cell loss. The graded holes that go from small to large diameters decrease the pressures in the heart and this helps properly target the cells. This new design works well in healthy and damaged hearts.

Clinical trials are already testing this new catheter. In Europe, the CHART-1 clinical trial is presently underway, and this is the first phase 3 trial to examine the regeneration of heart muscle in heart attack patients.

These particular studies are the culmination of years of basic science research at Mayo Clinic and earlier clinical studies with Cardio3 BioSciences and Cardiovascular Centre in Aalst, Belgium, which were conducted between 2009 and 2010.  This study, the C-CURE or Cardiopoietic stem Cell therapy in heart failURE study examined 47 patients, (15 control and 32 experimental) who received injections of bone marrow-derived mesenchymal stem cells from their own bone marrow into their heart muscle.  Control patients only received standard care.  After six months, those patients who received the stem cell treatment showed an increase in heart function and the distance they could walk in six minutes.   No adverse effects were observed in the stem cell recipients.

This study established the efficacy of mesenchymal stem cell treatments in heart attack patients.  However, other animal and computer studies established the efficacy of this new catheter for injecting heart muscle with stem cells.  Hopefully, the results of the CHART-1 study will be available soon.

Postscript:  The CHART-2 clinical trial is also starting.  See this video about it.

Nanotubules Link Damaged Heart Cells With Mesenchymal Stem Cells to Both of Their Benefit


Mesenchymal stem cells are found throughout the body in bone marrow, fat, tendons, muscle, skin, umbilical cord, and many other tissues. These cells have the capacity to readily differentiate into bone, fat, and cartilage, and can also form smooth muscles under particular conditions.

Several animal studies and clinical trials have demonstrated that mesenchymal stem cells can help heal the heart after a heart attack. Mesenchymal stem cells (MSCs) tend to help the heart by secreting a variety of particular molecules that stimulate heart muscle survival, proliferation, and healing.

Given these mechanisms of healing, is there a better way to get these healing molecules to the heart muscle cells?

A research group from INSERM in Creteil, France has examined the use of tunneling nanotubes to connect MSCs with heart muscle cells. These experiments have revealed something remarkable about MSCs.

Florence Figeac and her colleagues in the laboratory of Ann-Marie Rodriguez used a culture system that grew fat-derived MSCs and with mouse heart muscle cells. They induced damage in the heart muscle cells and then used tunneling nanotubes to connect the fat-based MSCs.

They discovered two things. First of all, the MSCs secreted a variety of healing molecules regardless of their culture situation. However, when the MSCs were co-cultured with damaged heart muscle cells with tunneling nanotubes, the secretion of healing molecules increased. The tunneling nanotubes somehow passed signals from the damaged heart muscle cells to the MSCs and these signals jacked up secretion of healing molecules by the MSCs.

The authors referred to this as “crosstalk” between the fat-derived MSCs and heart muscle cells through the tunneling nanotubes and it altered the secretion of heart protective soluble factors (e.g., VEGF, HGF, SDF-1α, and MCP-3). The increased secretion of these molecules also maximized the ability of these stem cells to promote the growth and formation of new blood vessels and recruit bone marrow stem cells.

After these experiments in cell culture, Figeac and her colleagues used these cells in a living animal. They discovered that the fat-based MSCs did a better job at healing the heart if they were previously co-cultured with heart muscle cells.

Exposure of the MSCs to damaged heart muscle cells jacked up the expression of healing molecules, and therefore, these previous exposures made these MSCs better at healing hearts in comparison to naive MSCs that were not previously exposed to damaged heart muscle.

Thus, these experiments show that crosstalk between MSCs and heart muscle cells, mediated by nanotubes, can optimize heart-based stem cells therapies.

Priming Cocktail for Cardiac Stem Cell Grafts


Approximately 700,000 Americans suffer a heart attack every year and stem cells have the potential to heal the damage wrought by a heart attack. Stem cells therapy has tried to take stem cells cultured in the laboratory and apply them to damaged tissues.

In the case of the heart, transplanted stem cells do not always integrate into the heart tissue. In the words of Jeffrey Spees, Associate Professor of Medicine at the University of Vermont, “many grafts simply didn’t take. The cells would stick or would die.”

To solve this problem, Spees and his colleagues examined ways to increase the efficiency of stem cell engraftment. In his experiments, Spees and others used mesenchymal stem cells from bone marrow. Mesenchymal stem cells are also called stromal cells because they help compose the spider web-like filigree within the bone marrow known as “stroma.” Even though the stroma does not make blood cells, it supports the hematopoietic stem cells that do make all blood cells.  Here is a picture of bone marrow stroma to give you an idea of what it looks like:

Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Stromal cells are known to secrete a host of molecules that protect injured tissue, promote tissue repair, and support the growth and proliferation of stem cells.

Spees suspected that some of the molecules made by bone marrow stromal cells could enhance the engraftment of stem cells patches in the heart. To test this idea, Spees and others isolated proteins from the culture medium of bone marrow stem cells grown in the laboratory and tested their ability to improve the survival and tissue integration of stem cell patches in the heart.

Spees tenacity paid off when he and his team discovered that a protein called “Connective tissue growth factor” or CTGF plus the hormone insulin were in the culture medium of these stem cells. Furthermore, when this culture medium was injected into the heart prior to treating them with stem cells, the stem cell patches engrafted at a higher rate.

“We broke the record for engraftment,” said Spees. Spees and his co-workers called their culture medium from the bone marrow stem cells “Cell-Kro.” Cell-Kro significantly increases cell adhesion, proliferation, survival, and migration.

Spees is convinced that the presence of CTGF and insulin in Cell-Kro have something to do with its ability to enhance stem cell engraftment. “Both CTGF and insulin are protective,” said Spees. “Together they have a synergistic effect.”

Spees is continuing to examine Cell-Kro in rats, but he wants to take his work into human trials next. His goal is to use cardiac stem cells (CSCs) from humans, which already have a documented ability to heal the heart after a heart attack. See here, here, and here.

“There are about 650,000 bypass surgeries annually,” said Spees. “These patients could have cells harvested at their first surgery and banked for future application. If they return for another procedure, they could then receive a graft of their own cardiac progenitor cells, primed in Cell-Kro, and potentially re-build part of their injured heart.”

Grafted Stem Cell Derivatives Restore Normal Heart Rhythms in Mice


American researchers, in collaboration with technicians from Fujifilm VisualSonics, Inc., have used advanced ultrasonic software to document microscopic, regenerative improvements to heart muscle that has suffered from previous damage.

High-frequency ultrasound and special cardiac-assessment software was developed by FujiFilm VisualSonics, Inc of Toronto, Canada. Scientists from Mayo Clinic implanted engineered cells into the damaged hearts of mice and then used the special software and ultrasound imaging to observe the regeneration of the heart so that it began to contract with normal cardiac rhythms.

After a heart attack, dead heart tissue is replaced with a cardiac scar that consists of scar tissue that neither contracts nor conducts the signals to contract. Depending of the size of the heart scar, the heart can beat abnormally. An abnormal heart beat is known as arrhythmia. Arrhthymias come in three different categories: a heart that beats too fast (tachycardia), a heart that beats too slowly (bradycardia), and a heart that beats erratically. Arrhythmias after a heart attack can be life-threatening, and restoring normal heart rhythm to the heart after a heart attack is very important.

In this experiment, mice were given heart attacks, and then undifferentiated induced pluripotent stem cells (iPSCs) were implanted into these hearts. Those mice that received induced pluripotent stem cells gradually normalized, their heart beat. The resynchronization of the heart beat of these mice was imaged with high-resolution ultrasound.

Satsuki Yamada, first author of this paper, said, “A high-resolution ultrasound revealed harmonized pumping [of the heart] where iPS cells were introduced to be the previously damaged heart tissue.” Yamada also noted that Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, and achieves resynchronization of the heart beat after a heart attack.

This experiment shows, for the first time that undifferentiated iPSCs have the potential to stabilize a patient’s heart after a heart attack. The healing of the heart was documented by ultrasound imaging and by “speckle-tracking echocardiogram.,” Speckle-tracking echocardiography was designed by VevoStrain Advanced Cardiac Analysis Software, which was manufactured by VisualSonics.

This software package provides advanced imaging and quantification capabilities for studying sensitive movements in heart muscles and it is also the only commercial cardiac-strain package optimized for assessing cardiovascular function preclinical rodent studies.

Yamada and her co-researchers utilized this software during the implantation and observation of the iPSCs within the hearts of mice. This software package the motion of the heart wall both at the regional and global levels and from several different perspectives, measurements of these movements, the changes in dimension in the left ventricle during the heart cycle.

The software definitely showed that homogeneous wall movement was restored in those mice that had received implants of iPSCs.

When iPSCs were implanted into mice that had dysfunctional immune systems, they produced tumors, but in mice with normal immune systems, the implanted iPSCs did not produce tumors. What became of those cells is uncertain, but they clearly helped heal the heart and did not cause tumors.

Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.
Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.

This paper is interesting and suggests that undifferentiated cells can also exert healing effects on the heart.

Encapsulation of Cardiac Stem Cells and Their Effect on the Heart


Earlier I blogged about an experiment that encapsulated mesenchymal stem cells into alginate hydrogels and implanted them into the hearts of rodents after a heart attack. The encapsulated mesenchymal stem cells showed much better retention in the heart and survival and elicited better healing and recovery of cardiac function than their non-encapsulated counterparts.

This idea seems to be catching on because another paper reports doing the same thing with cardiac stem cells extracted from heart biopsies. Audrey Mayfield and colleagues in the laboratory of Darryl Davis at the University of Ottawa Heart Institute and in collaboration with Duncan Steward and his colleagues from the Ottawa Hospital Research Institute used cardiac stem cells extracted from human patients that were encased in agarose hydrogels to treat mice that had suffered heart attacks. These experiments were reported in the journal Biomaterials (2013).

Cardiac stem cells (CSCs) were extracted from human patients who were already undergoing open heart procedures. Small biopsies were taken from the “atrial appendages” and cultured in cardiac explants medium for seven days.

atrial appendage

Migrating cells in the culture were harvested and encased in low melt agarose supplemented with human fibrinogen. To form a proper hydrogel, the cells/agarose mixture was added drop-wise to dimethylpolysiloxane (say that fast five times) and filtered. Filtration guaranteed that only small spheres (100 microns) were left. All the larger spheres were not used.

Those CSCs that were not encased in hydrogels were used for gene profiling studies. These studies showed that cultured CSCs expressed a series of cell adhesion molecules known as “integrins.” Integrins are 2-part proteins that are embedded in the cell membrane and consist of an “alpha” and “beta” subunit. Integrin subunits, however, come in many forms, and there are multiple alpha subunits and multiple beta subunits.

integrin-actin2

This mixing and matching of integrin subunits allows integrins to bind many different types of substrates. Consequently it is possible to know what kinds of molecules these cells will stick to based on the types of integrins they express. The gene prolifing experiments showed that CSC expressed integrin alpha-5 and the beta 1 and 3 subunits, which shows that CSC can adhere to fibronectin and fibrinogen.

fibronectin

fibrinogen-cleave

When encapsulated CSCs were supplemented with fibrinogen and fibronectin, CSCs showed better survival than their unencapsulated counterparts, and grew just as fast ans unencapsulated CSCs. Other experiments showed that the encapsulated CSCs made just as many healing molecules as the unencapsulated CSCs, and were able to attract circulating angiogenic (blood vessel making) cells. Also, the culture medium of the encapsulated cells was also just as potent as culture medium from suspended CSCs.

With these laboratory successes, encapsulated CSCs were used to treat non-obese diabetic mice with dysfunctional immune systems that had suffered a heart attack. The CSCs were injected into the heart, and some mice received encapsulated CSCs, other non-encapsulated CSCs, and others only buffer.

The encapsulated CSCs showed better retention in the heart; 2.5 times as many encapsulated CSCs were retained in the heart in comparison to the non-encapsulated CSCs. Also, the ejection fraction of the hearts that received the encapsulated CSCs increased from about 35% to almost 50%. Those hearts that had received the non-encapsulated CSCs showed an ejection fraction that increased from around 33% to about 39-40%. Those mice that had received buffer only showed deterioration of heart function (ejection fraction decreased from 36% to 28%). Also, the heart scar was much smaller in the hearts that had received encapsulated CSCs. Less than 10% of the heart tissue was scarred in those mice that received encapsulated CSCs, but 16% of the heart was scarred in the mice that received free CSCs. Those mice that received buffer had 20% of their hearts scarred.

Finally, did encapsulated CSCs engraft into the heart muscle? CSCs have been shown to differentiate into heart-specific tissues such as heart muscle, blood vessels, and heart connective tissue. Encapsulation might prevent CSCs from differentiating into heart-specific cell types and connecting to other heart tissues and integrating into the existing tissues. However, at this point, w have a problem with this paper. The text states that “encapsulated CSCs provided a two-fold increase in the number of engrafted human CSCs as compared transplant of non-encapsulated CSCs.” The problem is that the bar graft shown in the paper shows that the non-encapsulated CSCs have twice the engraftment of the capsulated CSCs. I think the reviewers might have missed this one. Nevertheless, the other data seem to show that encapsulation did not affect engraftment of the CSCs.

The conclusion of this paper is that “CSC capsulation provides an easy, fast and non-toxic way to treat the cells prior to injection through a clinically acceptable process.”

Hopefully large-animal tests will come next. If these are successful, then maybe human trials should be on the menu.

Keeping Implanted Stem Cells in the Heart


Globally, thousands of heart patients have been treated with stem cells from bone marrow and other sources. While many of these patients have been helped by these treatments, the results have been inconsistent, and most patients only show a modest improvement in heart function.

The reason for these sometimes underwhelming results seems to result from the fact that implanted stem cells either die soon after they are delivered to the heart or washed out. Since the heart is a pump, it is constantly contracting and having fluid (blood) wash through it. Therefore, it is one of the last places in the body we should expect implanted stem cells to stay put.

To that end, cardiology researchers a Emory University in Atlanta, Georgia have packaged stem cells into small capsules made of alginate (a molecule from seaweed) to keep them in the heart once they are implanted there.

alginate_formel

W. Robert Taylor, professor of medicine and director of the cardiology division at Emory University School of Medicine, and his group encapsulated mesenchymal stem cells in alginate and used them to male a “patch” that was applied to the hearts of rats after a heart attack. Taylor’s group compared the recovery of these animals to those rats that had suffered heart attacks, but were treated with non-encapsulated cells, or no cells at all. The rats treated with encapsulated cells not only showed a more robust recovery, but they had larger numbers of stem cells in their hearts and showed better survival.

Histological appearance of encapsulated human mesenchymal stem cells (hMSCs). Light microscopic appearance of encapsulated hMSCs at the time of implantation with approximately 200 cells within each 250 μm capsule. (Scale bar=100 μm)
Histological appearance of encapsulated human mesenchymal stem cells (hMSCs). Light microscopic appearance of encapsulated hMSCs at the time of implantation with approximately 200 cells within each 250 μm capsule. (Scale bar=100 μm)

Of this work, Taylor said, “This approach appears to be an effective way to increase cell retention and survival in the context of cardiac cell therapy. It may be a strategy applicable to many cell types for regenerative therapy in cardiovascular medicine.

Readers of this blog might remember that I have detailed before the inhospitable environment inside the heart after a heart attack. Oxygen levels are low because blood vessels have died, and roving white blood cells are gobbling up cell debris and releasing toxic molecules while they do it. Also the dying cells have released a toxic cocktail of molecules that make the infarcted area very inhospitable. Injecting stem cells into this region is an invitation for more cells to die. Previous experiments have shown that preconditioning stem cells either by genetically engineering them to withstand high stress levels of by growing them in high-stress conditions prior to implantation can increase their survival in the heart.

Taylor also pointed out that the mechanical forces of the contracting heart can squeeze them and displace them from the heart, much like pinching a watermelon seed between your fingers causes it to slip out. “These cells are social creatures and like to be together,” said Taylor. “From some studies of cell therapy after myocardial infarction, one can estimate that more than 90 percent of the cells are lost in the first hour. With numbers like that, it’s easy to make the case that retention is the first place to look to boost effectiveness.”

Encapsulation keeps the mesenchymal stem cells together in the heart and “keeps them happy.” Encapsulation, however, does not completely cut off the cells from their environment. They can still sense the cardiac milieu and release growth factors and cytokines while they are protected from marauding white blood cells and antibodies that might damage, destroy, or displace them.

Alginate already has an impressive medical pedigree as a biomaterial. It is completely non-toxic, and chefs use it to make edible molds to encase other types of tasty morsels. Dentists use alginate to take impressions of a patient’s teeth and it is also used a component of wound dressings. One of Taylor’s co-authors, an Emory University colleague named Collin Weber has used alginate to encapsulate insulin-producing islet-cells that are being tested in clinical trials with diabetics.

Encasing cells in alginate prevents them from replacing dead cells, but mesenchymal stem cells tend to do the majority of their healing by means of “paracrine” mechanisms; that is to say, mesenchymal stem cells tend to secrete growth factors, cytokines and other healing molecules rather than differentiating into heart cells. Mesenchymal stem cells can be isolated from bone marrow or fat.

One month after suffering from a heart attack, those rats that had suffered a heart attack saw their ejection fractions (a measure of how much volume the heart pumps out with every beat) fell from an average of 72% to 34%. However, rats treated with encapsulated mesenchymal stem cells saw an increase in their ejection fractions from 34% to 56%. Those treated with unencapsulated mesenchymal stem cells saw their ejection fractions rise to 39%.

Detailed cardiac functional analysis by cardiac magnetic resonance imaging (CMR) and transthoracic echocardiography (TTE) showed improvement in animals treated with encapsulated human mesenchymal stem cells (hMSCs). A, Representative short axis CMR at end systole of animals treated with encapsulated hMSCs or controls. Myocardial thinning and chamber dilation, delineated by traced endocardium (red) and epicardium (green) was reduced in the encapsulated hMSC group (arrow). Quantification of end systolic volume (B) and ejection fraction (C) by CMR at day 28 showed improved contractile function in the encapsulated hMSC treated group (n=4 per group). D, TTE comparison of untreated animals (n=9) to animals treated with encapsulated hMSCs (n=7) or hMSCs delivered by direct injection (n=7) into the infarcted myocardium showed greater benefit of treatment with encapsulated cells. Data represent mean±SEM. *P<0.05 by Dunnett's test of multiple comparisons; #P<0.05 by analysis of variance (ANOVA). LVESV indicates left ventricular end systolic volume; MI, myocardial infarction.
Detailed cardiac functional analysis by cardiac magnetic resonance imaging (CMR) and transthoracic echocardiography (TTE) showed improvement in animals treated with encapsulated human mesenchymal stem cells (hMSCs). A, Representative short axis CMR at end systole of animals treated with encapsulated hMSCs or controls. Myocardial thinning and chamber dilation, delineated by traced endocardium (red) and epicardium (green) was reduced in the encapsulated hMSC group (arrow). Quantification of end systolic volume (B) and ejection fraction (C) by CMR at day 28 showed improved contractile function in the encapsulated hMSC treated group (n=4 per group). D, TTE comparison of untreated animals (n=9) to animals treated with encapsulated hMSCs (n=7) or hMSCs delivered by direct injection (n=7) into the infarcted myocardium showed greater benefit of treatment with encapsulated cells. Data represent mean±SEM. *P

One of the main effects of implanted stem cells is the promotion of the growth of new blood vessels.  In capsule-treated rats, the damaged area of the heart had a blood vessel density that was several times that of the hearts of control animals.  Also, the area of cell death was much lower in the hearts treated with encapsulated MSCs.

Treatment of hearts with encapsulated human mesenchymal stem cells (hMSC) post myocardial infarction reduced myocardial scarring at 28 days. A, Representative sections of infarcted hearts stained with Masson's Trichrome and treated with encapsulated hMSCs or control gels. Blue indicates fibrotic scar. ×15, scale bar=1 mm. B, Animals treated with encapsulated hMSCs showed reduced scar area (7±1%; n=6) at 28 days compared to control treated hearts (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+Empty Caps: 12±2%, n=5). Data represent mean±SEM. *P<0.05. MI indicates myocardial infarction.
Treatment of hearts with encapsulated human mesenchymal stem cells (hMSC) post myocardial infarction reduced myocardial scarring at 28 days. A, Representative sections of infarcted hearts stained with Masson’s Trichrome and treated with encapsulated hMSCs or control gels. Blue indicates fibrotic scar. ×15, scale bar=1 mm. B, Animals treated with encapsulated hMSCs showed reduced scar area (7±1%; n=6) at 28 days compared to control treated hearts (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+Empty Caps: 12±2%, n=5). Data represent mean±SEM. *P

The encapsulated stem cells seem to stay in the heart for just over ten days, which is the time is takes for the alginate hydrogels to break down.  Taylor said that he and his lab would like to test several different materials to determine how long these capsules remain bound to the patch.

The goal is to use a patient’ own stem cells as a source for stem cell therapy.  Whatever the source of stem cells, a patient’s own stem cells must be grown outside the body for several days in a stem cell laboratory, much like Emory Personalized Immunotherapy Center in order to have enough material for a therapeutic effect.

Culture Medium from Endothelial Progenitor Cells Heals Hearts


Endothelial Progenitor Cells or EPCs have the capacity to make new blood vessels but they also produce a cocktail of healing molecules. EPCs typically come from bone marrow, but they can also be isolated from circulating blood, and a few other sources.

The laboratory of Noel Caplice at the Center for Research in Vascular Biology in Dublin, Ireland, has grown EPCs in culture and shown that they make a variety of molecules useful to organ and tissue repair. For example, in 2008 Caplice published a paper in the journal Stem Cells and Development in workers in his lab showed that injection of EPCs into the hearts of pigs after a heart attack increased the mass of the heat muscle and that this increase in heart muscle was due to a molecule secreted by the EPCs called TGF-beta1 (see Doyle B, et al., Stem Cells Dev. 2008 Oct;17(5):941-51).

In other experiments, Caplice and his colleagues showed that the culture medium of EPCs grown in the laboratory contained a growth factor called “insulin-like growth factor-1” or IGF1. IGF1 is known to play an important role in the healing of the heart after a heart attack. Therefore, Caplice and his colleagues tried to determine if IGF1 was one of the main reasons EPCs heal the heart.

To test the efficacy of IGF1 from cultured EPCs, Caplice’s team grew EPCs in the laboratory and took the culture medium and tested the ability of this culture medium to stave off death in oxygen-starved heart muscle cells in culture. Sure enough, the EPC-conditioned culture medium prevented heart muscle cells from dying as a result of a lack of oxygen.

When they checked to see if IGF1 was present in the medium, it certainly was. IGF1 is known to induce the activity of a protein called “Akt” inside cells once they bind IGF1. The heart muscle cells clearly had activated their Akt proteins, thus strongly indicating the presence of IGF1 in the culture medium. Next they used an antibody that specifically binds to IGF1 and prevents it from binding to the surface of the heart muscle cells. When they added this antibody to the conditioned medium, it completely abrogated any effects of IGF1. This definitively demonstrates that IGF1 in the culture medium is responsible for its effects on heart muscle cells.

Will this conditioned medium work in a laboratory animal? The answer is yes. After inducing a heart attack, injection of the conditioned medium into the heart decreased the amount of cell death in the heart and increased the number of heart muscle cells in the infarct zone, and increased heart function when examined eight weeks after the heart attacks were induced. The density of blood vessels in the area of the infarct also increased as a result of injecting IGF1. All of these effects were abrogated by co-injection of the antibody that specifically binds IGF1.

From this study Caplice summarized that very small amounts of IGF1 (picogram quantities in fact) administered into the heart have potent acute and chronic beneficial effects when introduced into the heart after a heart attack.

These data are good enough grounds for proposing clinical studies. Hopefully we will see some in the near future.