First Clinical Trial for Genetically Engineered Stem Cell Treatment for Pulmonary Arterial Hypertension


A Canadian research team has published the results of the world’s first clinical trial of a genetically enhanced stem cell therapy for pulmonary arterial hypertension (PAH).

PAH is a rare and deadly disease that mainly affects young women, and is characterized by very high blood pressure in those arteries that supply blood to the lungs. Some cases of PAH are caused by mutations in the BMPR2 gene, but in many cases the cause remains unknown. Currently, PAH patients are treated with combination of various drug and oxygen. Drug treatments include blood vessel dilators, such as epoprosternol (Flolan or the inhaled form known as iloprost or Ventavis), endothelin receptor antagonists, such as bosentan (Tracleer) or ambrisentan (Letaris), sildenafil (Viagra) or tadalafil (Cialis), high doses of calcium channel blockers, anticoagulants, and diuretics. Such treatments can improve symptoms and exercise capacity (at best), but they cannot repair the blood vessel damage to the lungs or cure the disease.

This new study, entitled “Endothelial NO-Synthase Gene-Enhanced Progenitor Cell Therapy for Pulmonary Arterial Hypertension: the PHACeT Trial“ was published in the journal Circulation Research, and was coauthored lead investigator Duncan J. Stewart of the Ottawa Hospital Research Institute, and his collaborators.

The paper describes PAH as a progressive and eventually lethal disease that is characterized by eventual loss of functional lung microvasculature. This paper also argues that cell-based therapies offer the possibility of repairing and regenerating the lung microcirculation. The paper also reports that stem-cell therapy has shown promise in a pre-clinical evaluation that utilized experimental models of PAH.

This trial was a phase 1, dose-escalating clinical study whose goal was to test the tolerability, feasibility, and side-effects of a genetically-enhanced stem cell therapy to repair and regenerate lung blood vessels in PAH patients. Seven PAH patients who volunteered for this study underwent a blood cell selection process known as apheresis in order to harvest a certain population of white blood cells from their blood. These white blood cells were grown in the laboratory under special conditions that specifically selected for stem-like cells called endothelial progenitor cells (EPCs). These EPCs were genetically engineered to produce greater amounts of nitric oxide synthase, which makes the signaling molecule, nitric oxide (NO), a natural substance that widens blood vessels and is essential for efficient vascular repair and regeneration. These genetically enhanced cells were then injected directly into the lung circulation of the patient from whom there were originally harvested.

Of these seven patients, five were female and two were male, and all seven patients received treatment from December 2006 to March 2010. Continued observation and follow-up exams of these patients showed that the cell infusion procedure was well tolerated, and, on the whole, these patients showed a trend towards improvement in total pulmonary resistance (TPR) over the three-day delivery period. However, there was one serious adverse event (death) that occurred immediately after discharge in a patient who had severe, end-stage disease.

These investigators concluded that delivery of EPCs overexpressing eNOS was tolerated in PAH patients, and also produced evidence of short-term improvements, associated with long-term benefits in functional and quality-of-life assessments. However, they caution that future studies will be needed in order to further establish the efficacy of this therapy.

It must be noted that this study was not designed to rigorously assess the benefits the stem cell therapy versus a placebo. However, this research group observed improved blood flow in the lungs of patients during days following the therapy, and enhanced ability to exercise and better quality of life for up to six months after the therapy. Once again, I must provide the caveat that since this was not a double-blinded, placebo-controlled study, it is no possible to determine for sure if these observed effects were due to the cells or to psychological effects.

The therapy was generally well-tolerated, but one patient who had very severe and disease and signs of poor prognosis died one day after treatment. As unfortunate as this is, it is an expected outcome, given how sick the patient was and given their declining condition prior to treatment.

“Pulmonary arterial hypertension is a deadly and incurable disease that often strikes people in the prime of their life,” says the Circulation Research paper’s senior author Dr. Duncan Stewart, a practicing cardiologist and Executive Vice-President of Research at The Ottawa Hospital, and a professor of medicine at the University of Ottawa. “We desperately need new therapies for this disease, and regenerative medicine approaches have shown great promise in laboratory models and in clinical trials for other conditions.”

“This trial shows that genetically-enhanced stem cell therapy is a promising treatment approach for pulmonary arterial hypertension,” observes Dr. Stewart. “Although this is an important start, we will need to do larger studies to establish whether this therapy can produce important and durable benefits for people suffering from this challenging disease.”

Dr. Stewart is also the lead researcher of the first clinical trial in the world of a genetically-enhanced stem cell therapy for heart attack.

Rat forelimbs grown in the lab


The moving pictures of American soldiers who lost limbs while serving their country come across our computer screens with some regularity. However, while we celebrate the courage of these young men and women, we should also be amazed at the technological advances that provide artificial limbs for these soldiers. What if, we could grow replacement limbs in culture? Is this science fiction? Maybe not.

Biolimb from Ott lab
Biolimb from Ott lab

The photo above comes from work done in the laboratory of Harald Ott who is at the Massachusetts General Hospital in Boston has succeeded in growing rodent forelimbs in the laboratory. “We’re focusing on the forearm and hand to use it as a model system and proof of principle,” said Ott. “But the techniques would apply equally to legs, arms and other extremities.”

“This is science fiction coming to life,” says Daniel Weiss at the University of Vermont College of Medicine in Burlington, who works on lung regeneration. “It’s a very exciting development, but the challenge will be to create a functioning limb.”

Modern amputees are often fitted with prosthetic limbs that have an excellent cosmetic look, but these artificial limbs don’t function as well as real limbs. Bionic replacement limbs that work well are now being made, but they look quite unnatural. Hand transplants have also been successful, but these surgeries are extremely expensive, and the recipient needs lifelong immunosuppressive drugs to prevent their body rejecting the transplanted hand.

Tissue engineered “biolimbs” would get round many of these obstacles as it only contains cells from the recipient and would, therefore, avoid the need for immunosuppression. Biolimbs would also look and behave naturally.

“This is the first attempt to make a biolimb, and I’m not aware of any other technology able to generate a composite tissue of this complexity,” says Ott.

To grow rat forelimbs in the laboratory, the so-called “decel/recel” technique was used. This same technique was previously been used to build hearts, lungs and kidneys in the lab. In fact, simpler organs such as windpipes and voice box tissue have been built and transplanted into people with varying levels of success, but not without controversy.

Decel stands for decellularization is the first step. In the decel step, organs from dead donors are treated with detergents that strips the soft tissue and leaves just the “scaffold” of the organ, which consists mainly from the inert protein collagen. This retains all the intricate architecture of the original organ. In the case of the rat forearm, these collagen structures include blood vessels, tendons, muscles and bones.

The second step, the recel step, recellularizes the flesh of the organ by seeding the scaffold with the relevant cells extracted from the recipient. This scaffold is then nourished in a bioreactor, which enables the new tissue to grow and colonize the scaffold. Because none of the donor’s soft tissue remains, this bioengineered limb, or biolimb, will not be recognized as foreign and rejected by the recipient’s immune system.

As tissue engineered organs go, the forearm is much more difficult to grow that a windpipe. It has a far greater number of cell types that need to be grown. Ott began by suspending the decellularized forelimb in a bioreactor, and then plumbing the collagen artery into an artificial circulatory system to provide nutrients, oxygen and electrical stimulation to the limb. Next, Ott and his colleagues injected human endothelial cells into the collagen structures of blood vessels to recolonize the surfaces of blood vessels. This was important, because, according to Ott, this made the blood vessels more robust and prevented them from rupturing as fluids circulated through them.

Next, he injected a mixture of cells from mice that included myoblasts or muscle forming cells that would grow into muscle in the cavities of the scaffold normally occupied by muscle. In two to three weeks, the blood vessels and muscles had been rebuilt. Ott then finished off the limb by coating the forelimbs with skin grafts.

But would the limb’s muscles work? In order to work, the muscles must be connected to motor nerves from the central nervous system. To try this out, Ott’s team used electrical pulses to activate the muscles and found that the rat’s paw could clench and unclench. This experiment “showed we could flex and extend the hand,” says Ott. They also attached the biolimbs to anaesthetized healthy rats and saw that blood from the rat circulated in the new limb. However, they didn’t test for muscle movement or rejection.

While they have decellularized around 100 rat forelimbs, recellularizing at least half of them, there is still a great deal of work to do, said Ott. First they need to seed the limb with bone, cartilage and other cells to see whether these structures can be grown in the biolimb. Then they must demonstrate that a nervous system will develop in these cells. Results of hand transplants have shown the re-enervation occurs by means of the recipient’s nerve tissue growing into the transplanted hand and penetrating it. These growing nerves then make connections with the appropriate muscles. Thus, Ott believes that this would enable the recipients of a transplanted biolimb to control of their new organ. However, whether this also works in regenerated limbs remains to be seen.

Ott and his colleagues have also shown that forearms from nonhuman primates can be successfully decellularized. His team has begun recolonizing the primate scaffolds with human cells that line blood vessels, which is the first step towards human-scale biolimb development. They have also started experiments using human myoblasts in rats instead of the mouse myoblasts. Considerable work is needed to perfect this technology and it will be at least a decade before the first biolimbs are ready for human testing, says Ott, which is probably an optimistic estimate.

Nonhuman primate limb
Nonhuman primate limb

“It’s a notable step forward, and based on sound science, but there are some technical challenges that Harald’s group has to tackle,” says Steve Badylak of the University of Pittsburgh in Pennsylvania, who has used grafts built on scaffolds made from pig muscle to rebuild damaged leg muscles in 13 people. “Of these, the circulation is probably the biggest challenge, and making sure even the tiniest capillaries are successfully lined with endothelial cells so that they don’t collapse and cause clots,” he says. “But this is really an engineering approach, taking known fundamental principles of biology and applying them as an engineer would.”

Others are more critical. “For a complex organ like the hand, there are so many tissues and compartments that this definitely will not be a feasible protocol,” says Oskar Aszmann of the Medical University of Vienna in Austria, inventor of a bionic hand that people can control through their own thoughts. “Also, the hand must be innervated by thousands of nerves to have meaningful function, and that is at this point an insurmountable problem. So although this is a worthy endeavor, it must at this stage remain in the academic arena, not as a clinical scenario.”

In humans, Ott envisages organ donation schemes being extended to include transplantation of biolimbs. Cells for regenerating blood vessels could come from minor vessels supplied by the recipient, while muscle cells could come from biopsies from large muscles, such as in the thigh. “If you took about 5 grams, the size of a finger, you could grow it into human skeletal myoblasts,” he says.

With 1.5 million amputees in the US alone, this regeneration work is important, says Ott. “At present, if you lose an arm, a leg or soft tissue as part of cancer treatment or burns, you have very limited options.”

Stem Cells from Bone Marrow Help Heal Hard-to-Heal Bone Fractures


A new study that has appeared in the journal STEM CELLS Translational Medicine demonstrates the potential of a subset of stem cells called CD34+ in treating stubborn bone fractures that prove hard to heal.

The body has mechanisms for the repair of broken bones. Consequently, most patients recover from broken bones with little or no complication. However, up to 10 percent of all fracture patients experience fractures that refuse to heal. Such heard to heal fractures can lead to several debilitating side effects that include infection and bone loss, and the healing of hard to heal fractures often requires extensive treatment that includes multiple operations and prolonged hospitalization as well as long-term disability.

Regenerating broken bones with stem cells could offer an answer to this medical conundrum. Adult human peripheral blood CD34+ cells have been shown to contain a robust population of endothelial progenitor cells (EPCs) and hematopoietic stem cells, which give rise to all types of blood cells. These two types of stem cells might be good candidates for this therapy.

However, while other types of stem cells have been tested for their bone regeneration potential, the ability of CD34+ stem cells to facilitate bone healing has not been examined; that is until now. A phase I/II clinical study that evaluated the capacity of CD34+ to stimulate bone regeneration was published in the current edition of STEM CELLS Translational Medicine. This study was conducted by researchers at Kobe University Graduate School of Medicine, led by Tomoyuki Matsumoto, M.D., and Ryosuke Kuroda, M.D., members of the university’s department of orthopedic surgery and its Institute of Biomedical Research and Innovation (IBRI).

Matsumoto’s and Kuroda’s study was designed to evaluate the safety, feasibility and efficacy of autologous and G-CSF-mobilized CD34+cells in patients with non-healing leg bone breaks that had not healed in nine months. Seven patients were treated with CD34+ stem cells after receiving bone grafts.

In case you were wondering, G-CSF is a drug that releases stem cells from the bone marrow into the blood. It is given by injection or intravenously, and works rather well to mobilize bone marrow stem cells into the peripheral circulation.  It has clinical uses for patients recovering from chemotherapy.  Filgrastim (Neupogen) and PEG-filgrastim (Neulasta) are two commercially-available forms of recombinant G-CSF.

“Bone union was successfully achieved in every case, confirmed as early as 16.4 weeks on average after treatment,” Dr. Kuroda said.

Dr. Matsumoto added, “Neither deaths nor life-threatening adverse events were observed during the one year follow-up after the cell therapy. These results suggest feasibility, safety and potential effectiveness of CD34+ cell therapy in patients with nonunion.”

Atsuhiko Kawamoto, MD, Ph.D., a collaborator in IBRI, said, “Our team has been conducting translational research of CD34+ cell-based vascular regeneration therapy mainly in cardiovascular diseases. This promising outcome in bone fracture opens a new gate of the bone marrow-derived stem cell application to other fields of medicine.”

Although the study documents a relatively small number of patients, the results suggest the feasibility, safety and potential effectiveness of CD34+ cell therapy in patients with non-healing breaks,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

Stem Cell Treatments to Improve Blood Flow in Angina Patients


Angina pectoris is defined as chest pain or discomfort that results from poor blood flow through the blood vessels in the heart and is usually activated by activity or stress.

In Los Angeles, California, physicians have initiated a double-blind, multicenter Phase III clinical trial that uses a patient’s own blood-derived stem cells to restore circulation to the heart of angina patients.

This procedure utilizes state-of-the-art imaging technology to map the heart and generate a three-dimensional image of the heart. These sophisticated images will guide the physicians as they inject stem cells into targeted sites in the heart.

This is a double-blinded study, which means that neither the patients nor the researcher will know who is receiving stem-cell injections and who is receiving the placebo.

The institution at which this study is being conducted, University of Los Angeles (UCLA), is attempting to establish evidence for a stem cell treatment that might be approved by the US Food and Drug Administration for patients with refractory angina. The subjects in this study had received the standard types of care but did not receive relief. Therefore by enrolling in this trial, these patients had nothing to lose.

Dr. Ali Nasir, assistant professor of cardiology at the David Geffen School of Medicine and co-principal investigator of this study, said: “We’re hoping to offer patients who have no other options a treatment that will alleviate their severe chest pain and improve their quality of life.”

Before injecting the stem cells or the placebo, the team examined the three-dimensional image of the heart and ascertained the health of the heart muscle and voltage it generated. Damaged areas of the heart fail to produce adequate quantities of voltage and show low levels of energy.

Jonathan Tobis, clinical professor of cardiology and director of interventional cardiology research at Geffen School of Medicine, said: “We are able to tell by the voltage levels and motion which area of the [heart] muscle is scarred or abnormal and not getting enough blood and oxygen. We then targeted the injections to the areas just adjacent to the scarred and abnormal heart muscle to try to restore some of the blood flow.”

What did they inject? The UCLA team extracted bone marrow from the pelvic bones and isolated CD34+ cells. CD34 refers to a cell surface protein that is found on bone marrow stem cells and mediates the adhesion of bone marrow stem cells to the bone marrow matrix. It is found on the surfaces of hematopoietic stem cells, placental cells, a subset of mesenchymal stem cells, endothelial progenitor cells, and endothelial cells of blood vessels. These are not the only cells that express this cell surface protein, but it does list the important cells for our purposes. Once the CD34+ cells were isolated, the were injected into the heart through a catheter that was inserted into a vein in the groin.

CD34

The team hopes that these cells (a mixture of mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitor cells) will stimulate the growth of new blood vessels (angiogenesis) in the heart, and improve blood flow and oxygen delivery to the heart muscle.

“We will be tracking patients to see how they’re doing,” said William Suh MD, assistant clinical professor of medicine in the division of cardiology at Geffen School of Medicine.

The goal of this study is to enroll 444 patients nation-wide, of which 222 will receive the stem cell treatment, 111 will receive the placebo, and 111 who will be given standard heart care.

Adult Stem Cells Help Build Human Blood Vessels in Engineered Tissues


University of Illinois researchers have identified a protein expressed by human bone marrow stem cells that guides and stimulates the construction of blood vessels.

Jalees Rehman, associate professor of cardiology and pharmacology at the University of Illinois at Chicago College of Medicine and lead author of this paper, said: “Some stem cells actually have multiple jobs.”

As an example, stem cells from bone marrow known as mesenchymal stem cells can form bone, cartilage, or fat, but they also have a secondary role in that they support other cells in bone marrow.

Rehman and others have worked on developing engineered tissues for use in cardiac patients, and they noticed that mesenchymal stem cells were crucial for organizing other cells into functional stem cells.

Workers from Rehman’s laboratory mixed mesenchymal stem cells from human bone marrow with endothelial cells that line the inside of blood vessels. The mesenchymal stem cells elongated and formed a kind of scaffold upon which the endothelial cells adhered and organized to form tubes.

“But without the stem cells, the endothelial cells just sat there,” said Rehman.

When the cell mixtures were implanted into mice, blood vessels formed that were able to support the flow of blood. Then Rehman and his colleagues examined the genes expressed when their stem cells and endothelial cells were combined. They were aided in this venture by two different bone marrow stem cell lines, one of which supported the formation of blood vessels, and the other of which did.

Their microarray experiments showed that the vessel-supporting mesenchymal stem cells expressed high levels of the SLIT3 protein. SLIT3 is a blood vessel-guidance protein that directs blood vessel-making cells to particular places and induces them to make blood vessels. The cell line that do not stimulate blood vessel production made little to no SLIT3.

Rehman commented, “This means that not all stem cells are created alike in terms of their SLIT3 production and their ability to encourage blood vessel formation.”

Rehman continued: “While using a person’s own stem cells for making blood vessels is ideal because it eliminates the problem of immune rejection, it might be a good idea to test a patient’s stem cells to make sure they are good producers of SLIT3. If they aren’t, the engineered vessels may not thrive or even fail to grow.

Mesenchymal stem cells injections are being evaluated in clinical trials to see if their can help grow blood vessels and improve heart function in patients who have suffered heart attacks.

So far, the benefits of stem cell injection have been modest, according to Rehman. Evaluating the gene and protein signatures of stem cells from each patient may allow for a more individualized approach so that every patient receives mesenchymal stem cells that are most likely to promote blood vessel growth and cardiac repair. Such pre-testing might substantially improve the efficacy of stem cell treatments for heart patients.

Stem Cell Therapy Repairs Brain Damage Hours After Stroke Occurs


According to the Center for Disease Control, stroke is a leading cause of death in the United States. Fortunately stroke has been the subject of significant research efforts, but unfortunately, developing treatments that ensure complete recovery for stroke patients is extremely challenging. The challenge increase when more than a few hours have passed between onset of the stroke and administration of treatment.

Thus a new study released in STEM CELLS Translational Medicine has generated more than a little excitement. This study indicates that indicates that endothelial precursor cells (EPCs), which are found in the bone marrow, umbilical cord blood, and rarely in peripheral blood, can make a significant difference for these patients’ recovery. The contribution of EPCs even extends to the later stages of stroke. In animal studies, EPC implantation into the brain after a stroke minimized the initial brain injury and helped repair the stroke damage.

“Previous studies indicated that stem/progenitor cells derived from human umbilical cord blood (hUCB) improved functional recovery in stroke models,” noted Branislava Janic, Ph.D., a member of Henry Ford Health System’s Cellular and Molecular Imaging Laboratory in Detroit and lead author of the study. “We wanted to examine the effect of hUCB-derived AC133+ endothelial progenitor cells (EPCs) on stroke development and resolution in rats.”

Dr. Janic and his team injected EPCs into the brains of rats that had suffered strokes. When they later examined the animals using MRI, they found that the transplanted EPCs had selectively migrated to the injured area, stopped the tissue damage from spreading, initiated regeneration, and affected the time course for stroke resolution. The lesion size in the brain was significantly decreased at a dose of 10 million cells, if the cells were given as early as seven days after the onset of the stroke.

“This led us to conclude that cord blood-derived EPCs can significantly contribute to developing more effective treatments that allow broader time period for intervention, minimize the initial brain injury and help repair the damage in later post-stroke phases,” Dr. Janic said.

“The early signs of stroke are often unrecognized, and many patients cannot take advantage of clot-busting treatments within the required few hours after stroke onset,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “In this animal study, a combination of stem cells shows promise for healing stroke damage when administered 24 hours after the stroke.”

Tissue Kallikrein-Modified Human EPCs Improve Cardiac Function


When cells are implanted into the heart after a heart attack, the vast majority of them succumb to the hostile environment in the heart and die. Twenty-four hours after implantation there is a significant loss of cells (see Wu et al Circulation 2003 108:1302-1305). That fact that implanted bone marrow or fat-based stem cells benefit the heart despite their evanescence is a remarkable testimony to their healing power.

To mitigate this problem, stem cell scientists have used a variety of different strategies to increase the heartiness and survival of implanted stem cells. Two main strategies have emerged: preconditioning cells and genetically engineering cells. Both strategies increase the survival of implanted stem cells (see here, and here).

When it comes to genetically engineering stem cells, Lee and Julie Chao from the Medical University of South Carolina in Charleston, South Carolina have used endothelial progenitor cells (EPCs) from human umbilical cord blood to treat mice that had suffered heart attacks, except that these cells were genetically engineered to express “Tissue Kallikrein” or TK. TK is encoded by a gene called KLKB1, which is on chromosome 4 at region q34-35 (in human genetics, the long arm of a chromosome is the “q” arm and the small arm is the “p” or petite arm). TK is initially synthesized as an inactive precursor called prekallikrein. Prekallikrein must be clipped in order to be activated and the proteases (proteases are protein enzymes that cut other proteins into smaller fragment) that do so are either clotting factor XII, which plays a role in blood clotting, and PRCP, which is also known as Lysosomal Pro-X carboxypeptidase.

TK is a protease that degrades a larger protein called kininogen in two smaller peptides called bradykinin and kallidin, both of which are active signaling molecules. Bradykinin and kallidin cause relaxation of smooth muscles, thus lowering blood pressure, TK can also degrade plasminogen to form the active enzyme plasmin.

So why engineer EPCs to express TK? As it turns out, TK activates an internal protein in cells called Akt, and activated Akt causes cells to survive and prevents them from dying (see Krankel et al., Circulation Research 2008 103:1335-1343; Yao YY, et al., Cardiovascular Research 2008 80: 354-364; Yin H et a., J Biological Chem 2005 280: 8022-8030).

The first experiments were test tube experiments in which TK EPCs were incubated with cultured heart muscle cells to determine their ability to prevent cell death. When cultured heart muscle cells were exposed to hydrogen peroxide, they died left and right, but when they were incubated with the TK-EPCs and hydrogen peroxide, far fewer of them died.

Upper panel consists of cells stained with a TUNEL stain, which designates those cells that are dead or dying.  The bottom panel are DAPI stained cells, which is a nuclear stain that marks all available cells dead or live. From left to right, normal cells, cell exposed to hydrogen peroxide, cells exposed to hydrogen peroxide plus the genes for TK, and finally, cells exposed to hydrogen peroxide and TK-EPCs.
Upper panel consists of cells stained with a TUNEL stain, which designates those cells that are dead or dying. The bottom panel are DAPI stained cells, which is a nuclear stain that marks all available cells dead or live.
From left to right, normal cells, cell exposed to hydrogen peroxide, cells exposed to hydrogen peroxide plus the genes for TK, and finally, cells exposed to hydrogen peroxide and TK-EPCs.

When these cells were exposed to low levels of oxygen, a similar result was observed, expect that the cells co-incubated with TK-EPCs showed significantly less cell death.

When TK-EPCs were injected into the infarct border zones of the heart just after they had heart attacks, the results seven days after the heart attacks were striking. The heart function of the control mice was lousy to say the least. The heart walls had thinned, their ejection fractions were in the tank (~23%) and their echocardiograms were far from normal. However, the TK-EPC-injected mice had a relatively normal echocardiogram, thick heart wall, pretty good ejection fractions (52% and oppose to the 76% of mice that had never had a heart attack), and good heart function in general. Also, the size of the infarcts was reduced in those animals whose hearts had been injected with TK-EPCs.

Representative Masson’s trichrome staining. Original magnification is 10. (f) Echocardiographic measurements for determination of LV function from M-mode measurements. (g) MDA in the ischemic mouse heart at day 7 after MI. Values are expressed as mean±s.e.m. (n¼6, *Po0.05 vs Ad.Null-hEPC- and medium-treated group; #Po0.05 vs medium-treated group).
Representative Masson’s trichrome staining. Original magnification is 10. (f) Echocardiographic measurements for determination of LV function from M-mode measurements. (g) MDA in the ischemic mouse heart at day 7 after MI. Values are expressed as mean±s.e.m. (n¼6, *Po0.05 vs Ad.Null-hEPC- and medium-treated group; #Po0.05 vs medium-treated group).

There were two other bonuses to using TK-EPCs. First, as expected, the density of new blood vessels was substantially higher in hearts that received injections of TK-EPCs. Secondly, the TK-EPCs definitely survived better than their non-genetically engineered counterparts.

Ex-vivo optical imaging study. (a, b) Representative NIR fluorescent images in explanted organs at days 2 or 7 following implantation of DiDlabeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (a: 2 days after cell delivery; b: 7 days after cell delivery). (c) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean±s.e.m. (n¼3–4, *Po0.01 vs control group).
Ex-vivo optical imaging study. (a, b) Representative NIR fluorescent images in explanted organs at days 2 or 7 following implantation of DiDlabeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (a: 2 days after cell delivery; b: 7 days after cell delivery). (c) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean±s.e.m. (n¼3–4, *Po0.01 vs control group).

These results also confirm that TK works in heart muscle cells by activating the Akt protein inside the cells.  This establishes that TK works through the Akt pathway.

Once again, we see that transplantation of stem cells after a heart attack can improve the function and structure of the heart after a heart attack.  Indeed this strategy seems to work again and again.  These experiments were done in mice and therefore, they must be successful in a larger animal, like a pig before they can be deemed efficacious and safe for use in human clinical trials.  Even so, these results are hopeful.

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.

Major Clinical Trial Finds Bone Marrow Stem Cell Treatments Provide No Benefits After a Heart Attack


A large and very well designed and carefully controlled clinical trial known as TIME has failed to demonstrate any benefit for infusions of bone marrow stem cells into the heart 3-7 days after a heart attack.  This study comes on the heals of a similar clinical study known as LateTIME, which stands for Late Timing In Myocardial infarction Evaluation, and tested the effects of bone marrow stem cells infusions into the heart of heat attack patients 2-3 weeks after a heart attack.

LateTIME enrolled 87 heart attack patients, and harvested their bone marrow stem cells.  The stem cells were delivered into the hearts through the coronary arteries, but some received a placebo.  All patients had their ejection fractions measured, their heart wall motions in the damaged areas of the heart and outside the damaged areas and the size of their infarcts.  There were no significant changes in any of these characteristics after six months. Because another large clinical study known as the REPAIR-AMI study showed significant differences between heart attack patients that had received the placebo and those that had received bone marrow stem cells 3-7 days after a heart attack, this research group, known as the Cardiovascular Cell Therapy Research Network (CCTRN), sponsored by the National Institutes of Health, decided the test their bone marrow infusions at this same time frame.

TIME was similar in design to LateTIME.  This study enrolled 120 patients that had suffered a heart attack and all patients received either an infusion of 150 million bone marrow stem cells or a placebo within 12 hours of bone marrow aspiration and cell processing either 3 days after the heart attack to 7 days.  The researchers examined the changes in ejection fraction, movement of the heart wall, and the number of major adverse cardiovascular events plus the changes in the infarct size.

The results were resoundingly negative.  At 6 months after stem cell infusion, there was no significant increase in ejection fractions versus the placebo and no significant treatment effect on the function of the left ventricle in either the infarct or the border zones.  These findings were the same for those patients that received bone marrow stem cell infusions 3 days after their heart attack or 7 days after their heart attacks.  Fortunately, the incidence of major adverse events were rare among all treatment groups.

Despite the negative results for these clinical trials, there are a few silver linings.  First of all, the highly controlled nature of this trial sets a standard for all clinical trials to come.  A constant number of stem cells were delivered in every patient, and because the stem cells were delivered soon after they were harvested, there were no potential issues about bone marrow storage.

Jay Traverse, the lead author of this study, made this point about this trial:  “With this baseline now set, we can start to adjust some of the components of the protocol to grow and administer stem cell [sic] to find cases where the procedure may improve function.  For example, this therapy may work better in different population groups, or we might need to use new cell types or new methods of delivery.”

When one examines the data for this study, it is clear that some patients definitely improved dramatically, whereas others did not.  Below is a figure from the Traverse et al paper that shows individual patient’s heart function data 6 months after the stem cell infusions.

BMC indicates bone marrow mononuclear cell; MI, myocardial infarction.

From examining these data even cursorily, it is clear that some patients improved dramatically while others tanked.  Traverse is convinced that bone marrow stem cell infusions help some people, but not others (just like any other treatment).  He is convinced that by mining these data, he can begin to understand who these patients are who are helped by bone marrow stem cell transplants and who are not.  Also, the stem cells of these patients have been stored.  Hopefully, further work with them will help Traverse and his colleagues clarify what, if anything, about the bone marrow of these patients makes them more likely to help their patients and so on.

There are some possible explanations for these negative results.  Whereas the positive REPAIR-AMI used the rather labor-intensive Ficoll gradient protocols for isolating mononculear cells from bone marrow aspirates, the TIME trials used and automated system for collecting the bone marrow mononuclear cells.  Cells isolated by the automated system have neither been tested in an animal model of heart attacks, nor established as efficacious in a human study of heart disease.  Therefore, it is possible that the bone marrow used in this study was largely dead.  Secondly, the cell products were kept in a solution that had a heparin concentration that is known to inhibit the migratory properties of mononuclear cells (See Seeger et al., Circ Res 2012 111(7): 1385-94).  Therefore, there is a possibility that the bone marrow used in this study was no good.  Until the bone marrow stem cells collected by this method are confirmed to be efficacious, judgment must be suspended.