Long-Term Survival of Transplanted Human Neural Stem Cells in Primate Brains

A Korean research consortium has transplanted human neural stem cells (hNSCs) into the brains of nonhuman primates and ascertained the fate of these cells after being inside the brains of these animals for 22 and 24 months. They discovered that the implanted hNSCs had not only survived, but differentiated into neurons and never caused any tumors.

This important study is slated to be published in the journal Cell Transplantation.

To properly label the hNSCs so that they were detectable inside the brains of the animals, Lee and others loaded them with magnetic nanoparticles to enable them to be followed by magnetic resonance imaging (MRI). Also, they did not use immunosuppressants when they transplanted their hNSCs into the animals. This study is the first to examine the long-term survival and differentiation of hNSCs without the need for immunosuppression.

“Stroke is the fourth major cause of death in the US behind heart failure, cancer, and lower respiratory disease,” said study co-author Dr. Seung U. Kim of University of British Columbia Hospital’s department of neurology in Canada. “While tissue plasminogen activator (tPA) treatment within three hours after a stroke has shown good outcomes, stem cell therapy has the potential to address the treatment needs of those stroke patients for whom tPA treatment was unavailable or did not help.”

Based on the ability of hNSCs to differentiate into a variety of types of nerves cells, Lee and his colleagues thought that these cells have remarkable potential to treat damaged brain tissue and replace what was lost after a stroke, head injury or other type of brain trauma. Cell regeneration therapy can potentially treat brain-specific diseases like Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinal cord injury and stroke.

Dr. Kim and colleagues in Korea grafted magnetic particle-labeled hNSCs into the brains of laboratory primates and evaluated their performance to assess their survival and differentiation over 24 months. Of particular interest was determining their ability to differentiate into neurons and to determine whether the cells caused tumors.

“We injected hNSCs into the frontal lobe and the putamen of the monkey brain because they are included in the middle cerebral artery (MCA) territory, which is the main target in the development of the ischemic lesion in animal stroke models,” commented Dr. Kim. “Thus, research on survival and differentiation of hNSCs in the MCA territory should provide more meaningful information to cell transplantation in the MCA occlusion stroke model.”

Lee’s team said that they chose NSCs for transplantation because the existence of multipotent NSCs “has been known in developing rodents and in the human brain with the properties of indefinite growth and multipotent potential to differentiate” into the three major CNS cell types – neurons, astrocytes and oligodendrocytes.

“The results of this study serve as a proof-of-principle and provide evidence that hNSCs transplanted into the non-human primate brain in the absence of immunosuppressants can survive and differentiate into neurons,” wrote the researchers. “The study also serves as a preliminary study in our planned preclinical studies of hNSC transplantation in non-human primate stroke models.”

“The absence of tumors and differentiation of the transplanted cells into neurons in the absence of immunosuppression after transplantation into non-human primates provides hope that such a therapy could be applicable for use in humans.” said Dr. Cesar V. Borlongan, Prof. of Neurosurgery and Director of the Center of Excellence for Aging & Brain Repair at the University of South Florida. “This is an encouraging study towards the use of NSCs to treat neurodegenerative disorders”.

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

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

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

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

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

The animal studies showed very similar results.

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

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

The Benefits of Repeated Mesenchymal Stem Cell Treatments to the Heart

Mesenchymal stem cells have the ability to improve the heart after a heart attack. However can repeated administrations of mesenchymal stem cells cause an increased benefit to the heart after a heart attack?

A collaborative research project between the Royal Adelaide Hospital, the University of Adelaide in South Australia, and the Mayo Clinic in Rochester, Minnesota has administered mesenchymal stem cells multiple times to rodents after a heart attack to determine if administering these stem cells multiple times after a heart attack increases the performance of the heart.

The experimental procedure was relatively straight-forward. Three groups of mice were evaluated by means of cardiac magnetic resonance imaging (MRI). Then all three were given heart attacks by tying off the left anterior descending artery. Immediately after the heart attack, two groups were injected with one million mesenchymal stem cells into the heart. The third group was injected with ProFreeze (a cryopreservation solution). One week later, a second set of heart MRIs were taken, and the first and third group of mice received injections of ProFreeze and the third group received another one million mesenchymal stem cells. All animals were given two more heart MRIs one week later and two weeks after that. One month after the initial heart attacks, the mice were euthanized and their hearts were sectioned and examined.

Those mice that did not receive injections of mesenchymal stem cells showed a precipitous drop in their heart performance. The ejection fraction (average percent of blood pumped from the heart) dropped from around 60% to about 20% and then stayed there. Those mice treated with one round of mesenchymal stem cells (MSCs) after their ejection fractions drop from 60% to about 35% after one week, and then stayed there. Those animals that received two shots of MSCs have their ejection fractions drop from around 60% to about 41%. Thus the administration of a second round of MSCs did significantly increase the performance of the heart.

The heart also shows tremendous structural improvements as a result of MSC transplantation. These improvements are even more dramatic in those mice that received two doses of MSCs. The mass of the heart and the thickness of the walls of the heart are greater in those animals that received two MSC doses, than those that received only one dose. Secondly, the size of the heart scar is smallest in those animals that received two doses of MSCs. Third, the density of blood vessels was MUCH higher in the animals that received two MSC doses. Also, the tissue far from the infarction in those animals that had received two doses of MSCs showed twice the density of blood vessels per cubic millimeter of heart tissue than those animals that had only received one injection of MSCs. Therefore, additional transplantations of MSCs increase blood vessel density, decrease the size of the heart scar and increase the thickness of the walls of the heart.

MSCs have the capacity to heal the heart after a heart attack. The degree to which they heal the heart differs from patient to patient, but additional treatments have the capacity to augment the healing capacities of these cells.  Also, in this experiment, the mice received someone else’s MSCs.  This is known as “allogeneic” transplantation, and it is an important concept, since older patients, diabetic patients, or those who have had a heart attack typically have MSCs that do not perform well.  Therefore to receive MSCs from a donor is a way around this problem.

The problem with this experiment is that it was done in mice, and they were injected directly into the heart tissue. Such a procedure is almost certainly impractical for human patients. Instead, intracoronary delivery is probably more practical, but here again, repeated releasing cells into the coronary arteries increases the risk of clogging them. Therefore, it is probably necessary to administer the second dose of MSCs some time after the first dose. To calibrate when to administer the second dose, large animal experiments will be required.

Thus, while this experiment looks interesting and hopeful, more work is required to make this usable in humans.  It does, however, establish the efficacy of repeated allogeneic MSC transplantations, which is an important feature of these experiments.

Cardiosphere-Derived Stem Cells Improve Function in the Infarcted Rat Heart for 16 Weeks

Stem cells in the heart were first identified in 2003 and since then there has been a great deal of excitement about their potential for use in regenerative therapy. Two clinical trials have been conducted with heart-specific stem cells, SCIPIO and CADUCEUS. Both of these clinical trials were very successful, and further trials might, hopefully, bring this work to the operating room. In the meantime, further work with cardiac stem cells in animals models is necessary in order to clarify the mechanism by which these cells improve the function of an ailing heart.

In light of these findings, Carolyn Carr in Kieran Clarke’s lab at the University of Oxford has published an interesting paper in collaboration with Georgina Ellison at the University La Sapienza in Rome. This paper used magnetic resonance imaging (MRI) to examine rats hearts that had suffered a heart attack, and then had been implanted with cardiosphere-derived stem cells (CDCs). The study showed that CDCs not only improve the function of the heart, but do so for an extended period of time.

Cardiospheres are small balls of cells that have been grown from stem cells that are harvested from the heart. The cells are easily isolated from the upper chambers of the heart (atria; see Messina et al., Circulation Research 95 (2004): 911-24), and when the cells grow, they beat in synchrony and strongly attach to each other. The strong attachment of the cells for each other causes them to grow into small balls of cells that can be implanted into a living heart.

When the CDCs are grown in culture, they generate a mixed population of cells that include progenitor cells that express a gene called “c-kit,” and heart-specific mesenchymal cells that express a surface proteins called “CD90.” Additionally, there are other cells in cultured cardiac cells called explant derived cells (EDCs), which are round and refractory. There is a fair amount of debate as to the nature of these EDCs. Three main papers have demonstrated that EDCs neither form heart muscle nor survive when they are implanted into a living heart (see Shenje LT,et al., Lineage tracing of cardiac explant derived cells. PLoS One. 3(4), 2008:e1929; & Li Z, et al., Imaging survival and function of transplanted cardiac resident stem cells. J Am Coll Cardiol. 53(14), 2009 :1229-40; & Andersen DC,et al., Murine “cardiospheres” are not a source of stem cells with cardiomyogenic potential, Stem Cells. 27(7) 2009, 1571-81). However, other papers have established that EDCs and CDCs can grow in culture and renew themselves (Davis et al., PLoS ONE 4 (2009): e7195; & Chimenti et al., Circulation Research 106 (2010): 971-80; & Davis et al., Journal of Molecular and Cellular Cardiology 49 (2010): 312-21).  Other studies have even shown that implantation of EDCs can improve the function of the heart after a heart attack (Shintani, et al., Journal of Molecular and Cell Cardiology 47 (2009): 288-295).

If you are confused by all these conflicting data, join the club because I am too.  The clinical studies say that CDCs work to fix a heart, but these animals studies leave us asking if the whole thing is not just a ponzi scheme.  This shows us why more animal studies are necessary.  Just because something works, does not mean that we know how it works.

In the Carr study, CDCs were isolated from rats and cultured.  The cultured tissue yielded CDCs and EDCs that grew well in culture.  The CDCs were then labeled with magnetic microspheres to make them easier to detect after transplantation.  The CDCs were able to differentiate into heart muscle in culture as determined by their expression of proteins and genes that are specific to heart muscle.  Then the rats were given heart attacks, and seven of them received CDC infusions and another seven rats did not.  All rats underwent perfusion of the heart, and two days after perfusion, all rats were given CDCs in the tail vein (4 x 10[6] cells).  MRI was used to view the heart and assess its function, and to view the CDCs in the heart.

EDCs in the hands of these researchers not only grew and renewed themselves in culture, but they also formed cardiospheres.  Most of the cells were mesenchymal stem cells, but others were fibroblasts and a few others were stem cells (3%).  Cardiosphere-derived cells expressed several heart-specific genes.  It was also clear that labeling the cells with iron microspheres did not affect the cells.

MRI of the heart showed that the untreated hearts were in functional decline, but those that had been treated with CDCs had better functional readings, an a much smaller scar, and had less problems with heart wall that did not move.  MRI also showed that the CDCs had integrated into the heart muscle and were a part of the heart muscle wall.  They did not have the maturity of adult heart muscle cells in that they were poorly connected with the neighboring cells.  However, they did not die but were maintained in the heart for at least 16 weeks. Blood vessel density was also increased in the hearts of the rats that had received the CDC implantations.

Thus, this CDC population, which far more CD90-expressing mesenchymal cells (30%) and fewer c-Kit cells (12%) homed to the site of heart damage and retained for at least 16 weeks.  Furthermore, a large proportion of CDCs formed heart muscle, reduced scarring and increased blood vessel synthesis.  These features improved the function of the rat hearts and prevented them from deteriorating further.  These cells also survived in the heart and did not die.  This shows that the EDCs can differentiate into heart muscle cells and preserve long-term function in the infarcted heart.  Therefore, we have evidence that the cells in cardiac stem cells can contribute to a damaged heart and help regenerate dead heart muscle.

CADUCEUS Clinical Trial Shows that Cardiosphere-Derived Stem Cells Can Regrow Heart Muscle After a Heart Attack

Cedars-Sinai Heart Institute is home to the CADUCEUS clinical trial. CADUCEUS stands for cardiosphere-derived autologous stem cells to reverse ventricular dysfunction. In this clinical trial, patients who had experienced a heart attack (and had left ventricular ejection fractions between 25% – 45%) were split into two groups. One group was given standard medical care for a heart attack patient and the other group was given standard care plus heart-based stem cells known as CDCs, which is short for “Cardiosphere-Derived Cells.” Patients assigned to receive CDC infusions of 12-25 million cells into the infarct-related artery 1.5 – 3 months after the heart attack.

The results 6 months after the stem cell infusion revealed that none of the patients in either group had died, developed tumors in their hearts or had experienced any major adverse heart-related event. Also Magnetic Resonance Imaging analysis of patients from both groups showed that those patients treated with CDCs displayed reductions in the mass of the heart scar, and increases in living, heart muscle mass. Additionally, the ability of the region of the heart that had experienced the heart attack contracted better in those patients who had received the CDC infusion. Also, the thickness of the wall of the heart was thicker in those patients who had received CDC infusions. Unfortunately, changes in other heart-specific functions such as EDV (end-diastolic volume), ESV (end-systolic volume), and LVEF (left ventricular ejection fraction) did not differ between the two groups by 6 months, which is difficult to reconcile with the structural changes in the hearts. .

Inventor of the procedures and technology used in this study, Eduardo Marbán, MD, PhD, who is also the director of the Cedars-Sinai Heart Institute, noted, “While the primary goal of our study was to verify safety, we also looked for evidence that the treatment might dissolve scar and regrow lost heart muscle. This has never been accomplished before, despite a decade of cell therapy trials for patients with heart attacks. Now we have done it. The effects are substantial, and surprisingly larger in humans than they were in animal tests.”

Shlomo Melmed, MD, dean of the Cedars-Sinai medical faculty and the Helene A. and Philip E. Hixon Chair in Investigative Medicine added, “These results signal an approaching paradigm shift in the care of heart attack patients. In the past, all we could do was to try to minimize heart damage by promptly opening up an occluded artery. Now, this study shows there is a regenerative therapy that may actually reverse the damage caused by a heart attack.”

An initial part of this study was conducted in 2009. In that study, Marbán and his colleagues used a patient’s own heart tissue to grow specialized heart stem cells. These specialized stem cells were injected back into the patient’s heart in an effort to repair and re-grow healthy muscle in a heart that had been injured by a heart attack. This experiment, at that time, was the first of its kind.

The results of that initial study were quite encouraging. The 25 patients, who participated in the study, had an average age of 57 and had suffered heart attacks that left them with damaged heart muscle. Each patient underwent extensive imaging scans to precisely locate the exact location and severity of the scars generated by the heart attack. Patients were treated at Cedars-Sinai Heart Institute and at Johns Hopkins Hospital in Baltimore.

Of these patients, eight received conventional medical care for heart attack survivors (prescription medicine, exercise recommendations and dietary advice) and were the control patients in this study. The remaining 17 patients were randomized to receive the stem cells underwent a minimally invasive heart biopsy, under local anesthesia that utilized a catheter inserted through a vein in the patient’s neck. From this catheter, doctors removed small pieces of heart tissue, about half the size of a raisin, that were taken to Marbán’s laboratory at Cedars-Sinai, where they were subjected to culture methods invented by Marbán to grow and expand the heart-based stem cells.

During a second, minimally invasive [catheter] procedure, the expanded heart-derived cells were reintroduced into the patient’s coronary arteries. Patients who received stem cell treatments experienced an average of 50 percent reduction in their heart attack scars 12 months after infusion while patients who received standard medical management did not experience shrinkage in the damaged tissue.

Marbán explained, “This discovery challenges the conventional wisdom that, once established, scar is permanent and that, once lost, healthy heart muscle cannot be restored.”

This phase I study definitely shows that the CDC infusion procedure is safe, which warrants the expansion of this procedure to a phase 2 study.