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.

Biowire Technology Matures Stem Cell-Derived Heart Cells


Heart research has taken yet another step forward with the invention of a new technique for maturing human heart cells in culture.

Researchers from the University of Toronto have created a fast and reliable method of creating mature human heart muscle patches in a variety of sizes. This technique applies pulsed electric current to the cells that mimics the heart rate of fetal humans.

Milica Radisic, an associate professor at the Institute of Biomaterials and Biomedical Engineering (IBBME), explained the significance of her new discovery: “You cannot obtain human cardiomyocytes (heart cells) from human patients.” However heart cells are vitally important for testing the safety and efficacy of heart drugs, and because human heart muscle cells do not normally divide robustly and form large swaths of heart tissue in culture, finding enough human heart tissue for pharmacological and toxicological test tests has been rather difficult. Tho circumvent this problem, researchers have been using heart muscle cells made from induced pluripotent stem cells (iPSCs). Unfortunately, once these cells are differentiated into heart muscle cells, they form highly immature heart muscle cells that beat too fast to work as a proper model system for adult human heart cells.

As Radisic put it: “The question is, if you want to test drugs or treat adult patients, do you want to use cells that look and function like fetal cardiomyocytes? Can we mature these cells to become more like adult cells?”

Radisic and her co-workers designed the “biowire” culture system for stem cell-derived cardiomyocytes. This system can mature heart muscle cells in culture in a reliable and reproducible manner.

The technique seeds human heart muscle cells along a silk suture, much like the kind used to sew up patients after surgery. The suture directs cells to grow along its length, after which they a treated to cycles of electric pulses. The biowire provides the pulses and acts like a stripped-down pacemaker. The biowire induces the heart muscle cells to increase in size and beat like more mature heart tissue. However the manner in shich the pulses are applied turns out to be very important. Radisic and her team discovered that if the cells were ramped up from zero pulses to 180 pulses per minute to 360 beats per minute, it mimicked the conditions that occur naturally in the developing heart. The fetal heart increases its heart rate prior to birth, and by ramping up the rate at which the pulses were delivered, Radisic and her team exposed the heart cells to the same kind of environment they would have experienced in the fetal heart.

“We found that pushing the cells to their limits over the course of a week derived the best effect,” said Radisic.

Growing the cells on sutures brings an added bonus: They can be sewn directly into a patient, which makes the biowires fully transplantable. Also, the cells can be grown on biodegradable sutures as well, which has practical implications for health care.

“With this discovery we can reduce the costs on the health care system by creating more accurate drug screening.” This discovery brings heart research one step closer to viable heart patches for replacing dead areas of the heart.

The paper’s first author, Sarah Nunes, said this: “One of the greatest challenges of tgransplanting these patches is getting the cells to survive, and for that they need blood vessels. Our next challenge is to put the vascularization together with cardiac cells.” Nunes is a cardiac and a vascular specialist.

Radisic enthusiastically labeled the new technique as a “game changer” in the field of cardiac medicine and it is a sign of how far the field has come in a very short time.

“In 2006 science saw the first derivation of induced pluripotent stem cells from mice. Now we can turn stem cells into cardiac cells and make relatively mature tissue from human samples, without ethical concerns.”

The vascularization part of this should be rather easy, since bone marrow-derived endothelial progenitor cells (EPCs) have been shown to make blood vessels in the heart. Putting these together with the heart patch should provide a winning combination

Inching toward human trials, but definitely making progress!!

Adult Stem Cells to Cure Diabetes?


Type 1 diabetics must inject themselves with insulin on a daily basis in order to survive. Without these shots, they would die.

Insulin injection

In most cases, type 1 diabetics have diabetes because their immune systems have attacked their insulin-producing cells and have destroyed them. However, a recent study at the University of Missouri has revealed that the immune system-dependent damage to the pancreas in type 1 diabetics goes beyond direct damage to the insulin-producing cells in the pancreas, The immune response also destroys blood vessels that feed tissues within the pancreas. This finding could provide the impetus for a cure that includes a combination of drugs and stem cells.

Habib Zaghouani and his research team at the University of Missouri School of Medicine discovered that “type 1 diabetes destroys not only insulin-producing cells but also blood vessels that support them,” explained Zaghouani. “When we realized how important the blood vessels were to insulin production, we developed a cure that combines a drug we created with adult stem cells from bone marrow. The drug stop the immune system attack, and the stem cells generate new blood vessels that help insulin-producing cells to multiply and thrive.”

Type 1 diabetes or juvenile diabetes, can lead to numerous complications, including cardiovascular disease, kidney damage, nerve damage, osteoporosis and blindness. The immune response that leads to type 1 diabetes attacks the pancreas, and in particular, the cell clusters known as the islet of Langerhans or pancreatic islets. Pancreatic islets contain several hormone-secreting cells types, but the one cell type in particular attacked by the immune system in type 1 diabetics are the insulin-secreting beta cells.

Pancreatic islets
Pancreatic islets

Destruction of the beta cells greatly decreases the body’s capability to make insulin, and without sufficient quantities of insulin, the body’s capability to take up, utilize and store sugar decelerates drastically, leading to mobilization of fats stores, the production of acid, wasting of several organs, excessive water loss, constant hunger, thirst, urination, acidosis (acidification of the blood), and eventually coma and death if left untreated.

The immune system not only destroys the beta cells, it also causes collateral damage to small blood vessels (capillaries) that carry blood to and from the pancreatic islets. This blood vessel damage led Zaghouani to examine ways to head this off at the pass and heal the resultant damage.

In previous studies, Zaghouani and others developed a drug against type 1 diabetes called Ig-GAD2. Treatment with this drug stops the immune system from attacking beta cells, but, unfortunately too few beta cells survived the onslaught from the immune system to reverse the disease. In his newest study, Zaghouani and his colleagues treated non-obese diabetic (NOD) with Ig-GAD2 and then injected bone marrow-based stem cells into the pancreas in the hope that these stem cells would differentiate into insulin-secreting beta cells.

“The combination of Ig-GAD2 and bone marrow [stem] cells did result in production of new beta cells, but not in the way we expected,” explained Zaghouani. “We thought the bone marrow [stem] cells would evolve directly into beta cells. Instead, the bone marrow cells led to growth of new blood vessels, and it was the new blood vessels that facilitated reproduction of the new beta cells. In other words, we discovered that to cure type 1 diabetes, we need to repair the blood vessels that allow the subject’s beta cells to grow and distribute insulin throughout the body.”

Zaghouani would lie to acquire a patent for his promising treatment and hopes to translate his preclinical research discovery from mice to larger animals and then to humans. In the meantime, his research continues to be funded by the National Institutes of Health and the University of Missouri.

Mesenchymal Stem Cells Engineered to Express Tissue Kallikrein Increase Recovery After a Heart Attack


Julie Chao is from the Department of Biochemistry and Molecular Biology, at the Medical University of South Carolina. Dr. Chao and her colleagues have published a paper in Circulation Journal about genetically modified mesenchymal stem cells and their ability to help heal a heart that has just experienced a heart attack.

Several laboratories have used mesenchymal stem cells (MSCs), particularly from bone marrow, to treat the hearts of laboratory animals that have recently experienced a heart attack. However, heart muscle after a heart attack is a very hostile place, and implanted MSCs tend to pack up and die soon after injection. Therefore, such injected cells do little good.

To fix this problem, researchers have tried preconditioning cells by growing them in a harsh environment or by genetically engineering them with genes that can increase their tolerance of harsh environments. Both procedures have worked rather well. In this paper, Chao and her group engineered bone marrow-derived MSCs to express the genes that encode “tissue kallikrein” (TK). TK circulates throughout our bloodstream but several different types of cells also secrete it. It is an enzyme that degrades the protein “kininogen” into small bits that have several benefits. Earlier studies from Chao’s own laboratory showed that genetically engineering TK into the heart improved heart function after a heart attack and increased the ability of MSCs to withstand harsh conditions (see Agata J, Chao L, Chao J. Hypertension 2002; 40: 653 – 659; Yin H, Chao L, Chao J. Journal of  Biol Chem 2005; 280: 8022 – 8030). Therefore, Chao reasoned that using MSCs engineered to express TK might also increase the ability of MSCs to survive in the post-heart attack heart and heal the damaged heart.

In this paper, Chao and others made adenoviruses that expressed the TK gene. Adenoviruses place genes inside cells, but they do not integrate those genes into the genome of the host cell. Therefore, they are safer to use than retroviruses. Chao and others used these TK-expressing adenoviruses to infect tissue and MSCs.

When TK-expressing MSCs were exposed to low-oxygen conditions, like what cells might experience in a post-heart attack heart, the TK-expressing cells were much heartier than their non-TK-expressing counterparts. When injected into rat hearts 20 minutes after a heart attack had been induced, the TK-expressing MSCs showed good survival and robust TK expression. Control hearts that had been injected with non-TK-expression MSCs or had not been given a heart attack showed no such elevation of TK expression.

There were also added bonuses to TK-expressing MSC injections. The amount of inflammation in the hearts was significantly less in the hearts injected with TK-expressing MSC injections compared to the controls. There were fewer immune cells in the heart 1 day after the heart attack and the genes normally expressed in a heart that is experiencing massive inflammation were expressed at lower levels relative to controls, if they were expressed at all.

Reduced inflammation by TK-MSC administration was determined by (C) ED-1 immunohistochemical staining, (D) monocyte/macrophage quantification, (E) neutrophil quantification, and gene expression of (F) TNF-α, (G) ICAM-1, and (H) MCP-1. ED-1-positive cells are indicated by arrows. Original magnification, ×200. Data are mean ± SEM (n=5–8). *P<0.05 vs. other MI groups; **P<0.05 vs. MI/Control group. MSC, mesenchymal stem cell.
Reduced inflammation by TK-MSC administration was determined by (C) ED-1 immunohistochemical staining, (D) monocyte/macrophage quantification, (E)
neutrophil quantification, and gene expression of (F) TNF-α, (G) ICAM-1, and (H) MCP-1. ED-1-positive cells are indicated by arrows.
Original magnification, ×200. Data are mean ± SEM (n=5–8). *P

Another major bonus to the injection of TK-expressing MSCs into the hearts of rats was that these cells protected the heart muscle cells from programmed cell death. To make sure that this was not some kind of weird artifact, Chao and her team placed the TK-expressing MSCs in culture with heart muscle cells and then exposed them to low-oxygen tension conditions. Sure enough, the heart muscle cells co-cultured with the TK-expressing MSCs survived better than those co-cultured with non-TK-expressing MSCs.

TK-MSCs protect against cardiac cell apoptosis at 1 day after myocardial infarction (MI) and in vitro. TK-MSC administration reduced apoptosis in the infarct area at 1 day after MI, as determined by (A) TUNEL staining, (B) quantification of apoptotic cells, and (C) caspase-3 activity. Original magnification, ×200. Data are mean ± SEM (n=5–8). *P<0.05 vs. other MI groups. Cultured cardiomyocytes treated with 0.5 ml of TK-MSC-conditioned medium exhibit higher tolerance to hypoxia-induced apoptosis, as evidenced by (D) Hoechst staining,
TK-MSCs protect against cardiac cell apoptosis at 1 day after myocardial infarction (MI) and in vitro. TK-MSC administration
reduced apoptosis in the infarct area at 1 day after MI, as determined by (A) TUNEL staining, (B) quantification of apoptotic
cells, and (C) caspase-3 activity. Original magnification, ×200. Data are mean ± SEM (n=5–8). *Pcardiomyocytes treated with 0.5 ml of TK-MSC-conditioned medium exhibit higher tolerance to hypoxia-induced apoptosis, as
evidenced by (D) Hoechst staining,

Finally, when the hearts of the rats were examined 2 weeks after the heart attack, it was clear that the enlargement of the heart muscle (so-called “remodeling”) occurred in animals that had received non-TK-expressing MSCs or had received no MSCs at all, but did not occur in the hearts of rats that had received injections of TK-expressing MSCs. The heart scar was also significantly smaller in the hearts of rats that had received injections of TK-expressing MSCs, and had a greater concentration of new blood vessels. Apparently, the TK-expressing MSCs induced the growth of new blood vessels by recruiting EPCs to the heart to form new blood vessels.

In conclusion, the authors write that “MSCs genetically-modified with human TK are a potential therapeutic for ischemic heart diseases.”

Getting FDA approval for genetically engineered stem cells will not be easy, but TK engineering seems much safer than some of the other modifications that have been used. Also the vascular and cardiac benefits of this gene seem clear in this rodent model. Pre-clinical trials with larger animals whose cardiac physiology is more similar to humans is definitely warranted and should be done before any talk of human clinical trials ensues.

Blood Vessel-Making Stem Cells From Fat


Blood vessel obstruction deprives tissues of life-giving oxygen and leads to the death of cells. If enough cells within a tissue die, the organ in which whose tissues reside could experience organ failure.

To quote the Sound of Music, “How does one solve a problem like blood vessel obstruction?” The obvious answer is to make new blood vessels to replace the blocked ones. Scientists have identified growth factors that are important in blood vessel formation during development. Therefore, injecting these growth factors should lead to the formation of new blood vessels, right? Unfortunately, such a strategy does not work very well (see Collison and Donnelly, Eur J Vasc Endovasc Surg 2004 28:9-23). Therefore, vascular specialists have focused on the ability of stem cells make new blood vessels, and this approach has yielded some very definite successes.

During development, the same stem cell gives rise to blood vessels and blood cells. This stem cell, the hemangioblast is found in a structure known as the yolk sac (even though it never functions as a yolk sac). In the yolk sac, during the third week of development, little specs form called “blood islands. These blood islands are small clusters of hemangioblasts with the cells at the center of the cluster forming blood cells and the cells at the periphery of the blood island forming blood vessels.

In adults, blood cell-making stem cells are found in the bone marrow. Blood vessel-making stem cells are endothelial progenitor cells or EPCs can be rather easily isolated from peripheral blood, however they are thought to originate from bone marrow. EPCs are not a homogeneous group of cells. There are different types with different surface molecules found in different locations.

Recently another cell from circulating blood called an “endothelial colony forming cell” or ECFC has been discovered, and this cell can attach to uncoated plastic surfaces in a growth medium. These cells can be grown to high numbers, even though it takes a rather long time to expand them. Once the ECFC culture system is further perfected, ECFCs will be excellent candidates for therapeutic trials (Reinisch et al., Blood 2009 113: 6716-25).

Fat tissue is also a reservoir of EPCs and mesenchymal stem cells. Fat-based mesenchymal stem cells help induce blood vessel formation and stimulate fat-based EPCs form blood vessels. Because of this remarkable “one-two punch” in fat, with cells that stimulate blood vessel formation and cells that actually form blood vessels, fat is a source of blood vessel-forming cells that can be used for therapeutic purposes.

Stem cells from fat.
Stem cells from fat.

Several pre-clinical experiments and presently ongoing clinical trials have examined the ability of fat-based stems to treat patients with conditions that result from insufficient circulation to various tissues. In rodents, experimental obstruction of the blood vessels in the hindlimb create a condition called “hindlimb ischemia.” In a rodent model of hindlimb ischemia, human fat-based stem cell applications not only improve the use of the limb and decrease limb damage, but also induce the formation of new blood vessels that definitely come from the applied stem cells (Miranville, et al., Circulation 2004 110: 349-55; Planat-Bernard, et al., Circulation 2004 109: 656-63 & Moon et al., Cell Physiol Biochem 2006 17: 279-90). Several clinical trials have been conducted with bone marrow-based EPCs for limb-based ischemia in humans, and these trials have been largely successful(see Szoke and Brinchmann, Stem Cells Translational Medicine 2012: 658-67 for a list of these trials). Adding mesenchymal stem cells from fat might improve the results of these trials.

In the heart, obstructed blood vessels can cause intense chest pain, a condition known as “angina pectoris.” EPCs have been used in clinical trials to treat patients with angina pectoris, and these trials have all been successful and have all used EPCs from bone marrow. These experiments, despite their success, have used bone marrow-based cells that were not fractionated and EPCs are less than 1% of the total number of cells. Also, the vast majority of cells introduced into heart migrate into the lungs, spleen and other organs. Also, those cells that remain tend to die off. A way to improve the survival of these implanted cells might be to combine them with mesenchymal stem cells from fat with EPCs from fat. Presently, the MyStromalCell trial is underway to test the efficacy of fat-based stem cells on the heart.

Fat provides an incredible treasure-trove of healing cells that have been demonstrated in animal experiments to relieve tissue ischemia and generate new blood vessels (for a summary of pre-clinical experiments in laboratory animals, see Qayyum AA, et al., Regen Med. 2012 May;7(3):421-8). Clinical trials with these cells are also underway. We have almost certainly only begun to tap to potential of these exciting cells that can be extracted so easily for our bodies.

Bringing the Dysfunctional Bone Marrow of Diabetics Back to Life


One of the most insidious consequences of diabetes mellitus is its nocuous effects on the ability of the circulatory system to repair itself. The small vessels within our organ undergoes constant remodeling and repair in response to the wears and tears of life. Diabetes seriously decreases the ability of the circulatory system to execute this repair.

This day-to-day circulatory repair relies upon a group of bone marrow stem cells known as “bone marrow-derived early outgrowth cells or EOCs, and EOCs from patients with diabetes mellitus are impaired in their ability to repair the circulatory system (See Fadini GP, Miorin M, Facco M et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol 2005;45:1449–1457).

Is there are way to reverse this destructive trend? There is a protein known as SIR1, which stands for Silent Information Regulator 1. This gene product regulates aging and the formation of blood vessels, and might very well play a role in the diabetes-induced decrease in blood vessels repair and EOC impairment.

To answer this question, the laboratory of Richard E. Gilbert from the University of Toronto, Toronto, Ontario, Canada, used drugs to increase SIR1 activity in EOCs from diabetic rodents to determine if such treatments abrogated the diabetes-induced decrease in EOC function.

Gilbert’s lab isolated EOCs from normal and diabetic mice and subjected them to a variety of tests. They determined how many blood vessel-inducing molecules were made by these cells, and the EOCs from diabetic mice produced much less of such molecules and had reduced levels of SIR1.  EOCs from diabetic mice also performed poorly in blood vessel-making assays in culture dishes.

Would kicking up the levels of SIR1 in EOCs from diabetic mice improve the function of their EOCs? By using a drug to increase SIR1 activity in EOCs, GIlbert and others were able to show that increased SIR1 activity in EOCs from diabetic mice restored their production of blood-vessel-inducing molecules, and also improved their ability to make blood vessels in culture.

This extraordinary publication shows that the diminished abilities of bone marrow from diabetic or aged individuals is not irreversible. Perhaps research such as this can spur the discovery of drugs that reserve the decline of SIR1 activity in diabetics and aged patients to beef up their circulatory self-repair mechanisms.

See Darren A. Yuen, et al., “Angiogenic Dysfunction in Bone Marrow-Derived Early Outgrowth Cells from Diabetic Animals Is Attenuated by SIRT1 Activation,” Stem Cells Translational Medicine 2012;1:921–926.

A Patient-Friendly Way to Make Stem Cells


Scientists at Cambridge University in the laboratory Amer Ahmed Rana have used blood samples to isolate cells from which patient-specific stem cells were made. Because blood samples are far more routine than tissue or organ biopsies, they can provide a much more patient-friendly way to secure material for the production of patient-specific stem cells.

Induced pluripotent stem cells (iPSCs) are made from adult cells by genetic engineering techniques that introduce four specific genes into them. The adult cells then de-differentiate to a more developmentally primitive state and if these cells survive and are successfully cultured, they will form an iPSC line.

Rana and his co-workers cultured blood drawn from several heart patients to isolate a blood cells known as a “late outgrowth endothelial progenitor cell” or L-EPC. Endothelial cells are those cells that compose blood vessels, and endothelial progenitor cells or EPCs are the stem cell population that make endothelial cells. EPCs are found in bone marrow, but some are also found in the peripheral circulation.

There are two main types of EPCs: early-outgrowth and late-outgrowth EPCs. Early-outgrowth EPCs are among the first cells to form spindle-shaped clusters of cells only a few days after being placed in culture. Early-outgrowth EPCs secrete high levels of blood vessel-inducing molecules, but they have only a limited ability to proliferate. They also are able to ingest bacteria, like other white blood cells. Late outgrowth EPCs are much rarer and they grow very well in culture, but are unable to ingest bacteria. They also can form capillaries and repair damaged blood vessels when injected into laboratory animals. There is a debate as to whether or not these cells come from the bone marrow or are dislodged from blood vessels.

Rana and his colleagues have designed a protocol for converting L-EPCs into iPSCs that can then be differentiated into heart, or blood vessel cells rather easily. This practical and rather efficient method does not require tissue biopsies, which are painful and impractical in very young or very old patients, and only requires the cells available from a single, routine blood sample.

Also, because blood samples can be efficiently and safely frozen, the cells from the blood sample can be locked in time for later use, when the patient needs regenerative treatments. The ease of this procedure should, Rana hopes, push it further toward human clinical trials in the near future.