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.


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.