Trials Of Stem Cell Therapy Research Articles

Neuroprotection for Amyotrophic Lateral Sclerosis: Role of Stem Cells, Growth Factors, and Gene Therapy

Various molecular mechanisms including apoptosis, inflammation, oxidative stress, mitochondrial dysfunction and excitotoxicity have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), though the exact mechanisms have yet to be specified. Furthermore, the underlying restorative molecular mechanisms resulting in neuronal and/or non-neuronal regeneration have to be yet elucidated. Therapeutic agents targeting one or more of these mechanisms to combat either initiation or progression of the disease are under research. Novel treatments including stem cell therapy, growth factors, and gene therapy might prolong survival and delay progression of symptoms. Harnessing the regenerative potential of the central nervous system would be a novel approach for the treatment of motor neuron death resulting from ALS. Endogenous neural replacement, if augmented with administration of exogenous growth factors or with pharmaceuticals that increase the rate of neural progenitor formation, neural migration, and neural maturation could slow the rate of cell loss enough to result in clinical improvement. In this review, we discuss the impact of therapeutic treatment involving stem cell therapy, growth factors, gene therapy, and combination therapy on disease onset and progression of ALS. In addition, we summarize human clinical trials of stem cell therapy, growth factor therapy, and gene therapy in individuals with ALS.

Challenges for heart disease stem cell therapy

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The use of stem cells to improve recovery of the injured heart after myocardial infarction (MI) is an important emerging therapeutic strategy. However, recent reviews of clinical trials of stem cell therapy for MI and ischemic heart disease recovery report that less than half of the trials found only small improvements in cardiac function. In clinical trials, bone marrow, peripheral blood, or umbilical cord blood cells were used as the source of stem cells delivered by intracoronary infusion. Some trials administered only a stem cell mobilizing agent that recruits endogenous sources of stem cells. Important challenges to improve the effectiveness of stem cell therapy for CVD include: (1) improved identification, recruitment, and expansion of autologous stem cells; (2) identification of mobilizing and homing agents that increase recruitment; and (3) development of strategies to improve stem cell survival and engraftment of both endogenous and exogenous sources of stem cells. This review is an overview of stem cell therapy for CVD and discusses the challenges these three areas present for maximum optimization of the efficacy of stem cell therapy for heart disease, and new strategies in progress.

New directions in cardiac stem cell therapy: An update for clinicians

The emergence of cardiac stem cell therapy can be traced to late 2001, when studies in small animal models of myocardial infarction suggested that stem cells could engraft, proliferate, and regenerate myo-cardium. Subsequent animal laboratory studies showed improved cardiac function, perfusion and survival compared to controls (Figure 1). Within two years, the first clinical trials of stem cell therapy began to appear, and we now have several trials of intracoronary infusion of bone marrow cells with more than one year follow-up. Although this clinical therapy has proven to be safe, the magnitude of improvement in objective measures like ejection fraction has been modest, and the therapy has not entered clinical practice. In the absence of a large prospective randomized trial, the field has moved back to the laboratory. This manuscript aims to provide clinicians with a broad overview of this complex field by briefly reviewing the existing status of clinical myocardial regeneration therapy, then describing selected examples from the laboratory research approaches that may provide a platform for new and potentially increasingly effective clinical strategies.

Shedding New Light on the Mechanism Underlying Stem Cell Therapy for the Heart

It is widely accepted that the adult mammalian heart is a terminally differentiated organ with little capacity to regenerate after damage, such as that caused by a myocardial infarction (MI). Nevertheless, mounting evidence suggests that mature cardiomyocytes do slowly turn over (up to 1% per year).1,2 In addition, there are many claims that the heart contains cell populations expressing stem cell markers that could in principle contribute to repair.3,4 However, whether proliferating cardiomyocytes or endogenous stem or progenitor cells actually contribute to cardiac repair after injury has remained unclear – until now. In a study recently published in Cell Stem Cell,5 Lee and colleagues used a clever lineage-tracing approach in mice1 to identify two potential sources of new cardiomyocytes in the adult heart: those arising from the division of existing cardiomyocytes and those formed by differentiation of the endogenous progenitors. Using their transgenic mouse model, they were able to distinguish these two sources of cardiomyocytes. More strikingly, the authors show that some stem cells currently being tested for the ability to improve heart function in cell transplantation trials can enhance the differentiation of endogenous progenitors, whereas others cannot. The study provides new insight into how cell therapy for the heart might work and suggests ways in which optimal cell types for therapy might be chosen – as well as how the controversial outcomes of many clinical trials might be resolved.

Review Article: Current Status of Myocardial Regeneration: New Cell Sources and New Strategies

Clinical trials of stem cell therapy in cardiology are based upon a reasonably solid foundation in animal laboratory research. The most widely used cell source in clinical trials has been the patient's own reconstituted bone marrow cell (BMC) aspirate. Cell sources in human bone marrow include hematopoietic stem cells, mesenchymal progenitor cells, and other cell types with many desirable characteristics. In vitro, they can be induced to become typical sarcomeres with centrally positioned nuclei and abundant mitochondria and to express atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and contractile proteins including myosin heavy chain, myosin light chain, and alpha actin. Intracoronary BMC infusion significantly decreases infarct size, increases myocardial perfusion, and improves regional and global cardiac function. Meta-analyses of clinical trials of intracoronary autologous BMC infusion following acute myocardial infarction (MI) report that the mean absolute increase in ejection fraction (EF) is approximately 3% to 4%. This modest improvement in function appears to persist for 1 year. Some trials have shown that clinical events are reduced at 12 months, but others have reported no long-term clinical benefit, and the only 5-year follow-up suggests persistent benefit with decreased mortality, but also little evidence of significant myocardial regeneration in humans. These results have led to efforts to identify better cell sources and to create more conducive myocardial environment for cell proliferation. Among the cell types are skeletal myoblasts, cardiac stem cells, and induced pluripotent stem cells. Environmental modifiers are designed to increase cell survival, persistence, and proliferation.

Host tissue response in stem cell therapy

Preclinical and clinical trials of stem cell therapy have been carried out for treating a broad spectrum of diseases using several types of adult stem cells. While encouraging therapeutic results have been obtained, much remains to be investigated regarding the best cell type to use, cell dosage, delivery route, long-term safety, clinical feasibility, and ultimately treatment cost. Logistic aspects of stem cell therapeutics remain an area that requires urgent attention from the medical community. Recent cardiovascular trial studies have demonstrated that growth factors and cytokines derived from the injected stem cells and host tissue appear to contribute largely to the observed therapeutic benefits, indicating that trophic actions rather than the multilineage potential (or stemness) of the administered stem cells may provide the underlying tissue healing power. However, the capacity for trophic factor production can be aberrantly downregulated as seen in human heart disease. Skeletal muscle is a dynamic tissue with an impressive ability to continuously respond to environmental stimuli. Indeed, a relation exists between active skeletal muscle and low cardiovascular risk, highlighting the critical link between the skeletal muscle and cardiovascular systems. Adding to this notion are recent studies showing that stem cells injected into skeletal muscle can rescue the failing rodent heart through activation of the muscle trophic factor network and mobilization of bone marrow multilineage progenitor cells. However, aging and disease can adversely affect the host tissue into which stem cells are injected. A better understanding of the host tissue response in stem cell therapy is necessary to advance the field and bridge the gap between preclinical and clinical findings.

Cell therapy--success does not come easy

More than 6 years ago the initial report on clinical application of bone marrow-derived mononuclear cells (BMMNCs) in patients with acute myocardial infarction (AMI) opened up a new era of regenerative cardiology and brought with it great enthusiasm and expectations.1 Since then, numerous clinical trials have been carried out with the aim of assessing the efficacy and safety of stem cell therapy. In all cases the safety of the therapy was proved, but its effectiveness still remains a matter of controversy and fierce debate.2 Since stem cell therapy trials in patients with AMI began, the primary outcome measure has been the change in left ventricular (LV) ejection fraction (EF). The LV volumes have also been analysed to assess more accurately the cause of the changes in LVEF after bone marrow cell (BMC) therapy.1,3–6 So far, the data on the effectiveness of BMC infusion in improving the global LV contractility remain equivocal. For example, the ASTAMI trial showed no improvement of LVEF and no reduction of end-diastolic volume in patients treated with intracoronary transfer of BMCs. In this trial, a significant improvement of LVEF was observed not only in patients treated with BMCs, but also in the control group.7 The BOOST trial showed an initial improvement in LVEF 6 months after BMC infusion, but the difference between the BMC arm and the control group was no longer significant at 18 months.8 On the other hand, REPAIR-AMI, so far the largest randomized placebo-controlled study, showed a significant … *Corresponding author. Tel: +48 32 353 9543, Fax: +48 32 353 9543, Email: michal.tendera{at}gmail.com or michal.tendera{at}gcm.pl

Stem Cell Therapy Trials: A Call for Standardization

There are currently 25 new clinical cellular therapy trials in progress for cardiovascular disease in the US, and a similar number ongoing in Europe. The lack of standardization in cell isolation, preparation, storage, and time and localization of delivery, present clear hurdles to this growing field. This emphasizes the need for an organized task force to work closely with the FDA to agree upon standardized methods with the end result being improvements in patient care and enhanced knowledge in the field.

Clinical trials update from the American Heart Association 2007: CORONA, RethinQ, MASCOT, AF‐CHF, HART, MASTER, POISE and stem cell therapy

This article provides information and a commentary on trials relevant to the pathophysiology, prevention and treatment of heart failure, presented at the American Heart Association 2007. These should be considered as preliminary data, as analyses may change in the final publication. Rosuvastatin did not reduce mortality compared to placebo in patients with heart failure and left ventricular systolic dysfunction due to ischaemic heart disease in the CORONA study. Results of RethinQ provide equivocal evidence of benefit from CRT in patients with heart failure, echocardiographic dyssynchrony and QRS interval <130 ms. In the MASCOT study, the addition of atrial overdrive pacing did not reduce the incidence of permanent atrial fibrillation in patients receiving CRT. The AF-CHF study failed to show a benefit of rhythm control over rate control in patients with heart failure and atrial fibrillation. Self-management skills training and education had no benefit on the combined outcome of death or heart failure hospitalisation, compared with education alone in heart failure patients in the HART study. Microvolt T-wave alternans testing failed to identify patients at increased risk of life-threatening ventricular arrhythmias in the MASTER study. POISE suggests that initiating metoprolol therapy shortly prior to non-cardiac surgery increases the risk of hypotension, stroke and death, despite reducing the risk of myocardial infarction. Three trials of stem cell therapy in post-MI patients gave conflicting results.

Cellular transplantation: future therapeutic options

Cardiac transplantation is a complex undertaking and an imperfect solution to end-stage heart failure. Cellular transplantation has been proposed as an alternative solution; however, clinical trials at present are small and show variable results. The mechanisms behind stem cell therapy have not yet been elucidated. Several large trials have been presented that address the question of bone marrow stem cells as therapy for acute myocardial infarction, and also the possible benefits of therapy with granulocyte colony-stimulating factor. Although some trials show a modest improvement in ejection fraction or reduction of infarct size, other trials show no change with treatment. Fewer clinical data are available on the treatment of chronic left ventricular systolic function. Many questions remain such as what cell type to use, dosing, the ideal timing for therapy, and the technique of cell delivery. Finally, further research continues on the cellular milieu, enhancement of cell engraftment, proliferation, and survival. This review briefly examines the background for stem cell therapy, as well as the larger clinical trials of stem cell therapy for acute myocardial infarction and chronic left ventricular systolic dysfunction, and possible pharmacologic enhancement options.

REPAIR-AMI: Stem Cells for Acute Myocardial Infarction

Evaluation of: Schachinger V, Erbs S, Elsasser A et al.: Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N. Engl. J. Med. 355, 1210-1221 (2006). The Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial, the largest randomized, placebo-controlled trial of stem cell therapy in acute myocardial infarction, studied the efficacy of the intracoronary delivery of bone marrow mononuclear cells (BMCs) versus placebo in patients with acute ST-segment elevation myocardial infarction following successful percutaneous coronary intervention. At 4 month follow-up, patients treated with BMCs had a significant improvement in left ventricular ejection fraction compared with placebo (+5.5 vs +3.0%, absolute difference +2.5%). In addition, treatment with BMCs was associated with a statistically significant reduction in adverse clinical events at 1 year follow-up. Despite these promising findings, other studies have shown mixed results and several unresolved clinical and physiological issues remain. Key findings from ongoing basic and clinical research will define the future role of stem cell therapy for acute myocardial infarction.

Clinical trials in stem cell therapy: pitfalls and lessons for the future

The first human trial of stem cell therapy for cardiovascular disease was performed 4 years ago. Since that time, almost a dozen studies have reported the early and late clinical effects of cell therapy in acute myocardial infarction and chronic ischemic cardiomyopathy. Initial nonrandomized trials universally showed slight improvement in the left ventricular ejection fraction. Later randomized, controlled trials, however, suggested a less significant effect. They showed either no difference between cell therapy and control treatment or a slight treatment benefit with cell therapy that is lost by 12 months' follow-up. These results have dampened the enthusiasm of some members of the scientific community for the continuation of clinical trials. Because early phase I trials should not be judged on issues other than safety, however, research is unlikely to be hindered. Indeed, the clinical studies reported so far have already taught us a lot about the biology of myocardial repair. Achieving clinical success will, however, probably require much more investment in basic and experimental research. Here, we address some of the current pitfalls in clinical cell therapy trials and lessons that should be learned as we face the challenges of the future.

Intracoronary infusion of the mobilized peripheral blood stem cell by G-CSF is better than mobilization alone by G-CSF for improvement of cardiac function and remodeling: 2-Year follow-up results of the Myocardial Regeneration and Angiogenesis in Myocardial Infarction with G-CSF and Intra-Coronary Stem Cell Infusion (MAGIC Cell) 1 trial

The results of stem cell therapy trials in myocardial infarction using granulocyte colony-stimulating factor (G-CSF) are inconsistent among trials, and the long-term outcome of G-CSF-based stem cell therapy remains unknown. We reported 2 years of follow-up results of 2 different strategies of G-CSF-based stem cell therapy. We compared outcomes of intracoronary infusion of the mobilized peripheral blood stem cells (PBSCs) with G-CSF, mobilization alone with G-CSF, and the control PCI alone in patients with myocardial infarction. At 2 years of follow-up evaluation, cell infusion improved left ventricular systolic function and remodeling compared to baseline, but G-CSF alone did not. Cell infusion group showed better improvements of left ventricular ejection fraction (+6.2% +/- 3.6% vs -4.3% +/- 10.1%, P = .004) and end-systolic volume (-15.7 +/- 13.0 vs +0.3 +/- 16.7 mL, P = .075) compared to G-CSF alone at 6 months of follow-up, and these trends were maintained till 2 years of follow-up (P = .094 and .046, respectively). Improvements in cell infusion group are not significantly better than that of control group because of small sample size. Patients who received G-CSF administration showed a tendency of modest increase of binary restenosis (50% vs 30%, P > .05) and a greater late loss of minimal luminal diameter (P > .05) at 6 months of follow-up, compared to the control group. Till 2 years follow-up, intracoronary cell infusion with mobilized PBSCs by G-CSF is better than G-CSF alone but not significantly better than control. Efficacy and safety of intracoronary infusion of mobilized PBSCs by G-CSF should be evaluated in a large randomized controlled trial.

Placental umbilical cord blood transfusion in acute ischaemic stroke

Stroke is a major cause of neurological disability across all countries in the world. Functional recovery after stroke is limited because of neuronal death and degeneration. Although early reperfusion therapy may improve outcome, thrombolysis does not reverse ischaemic neuronal death and carries the risk of cerebral haemorrhage. Early trials of stem cell therapy in promoting functional recovery after completed stroke remain inconclusive. Based on experimental data, we propose human umbilical cord blood transfusion as a possible therapy for ischaemic cerebral stroke to aid functional recovery.

Stem cell therapy in end-stage ischaemic heart failure: a catheter-based therapeutic strategy targeting myocardial viability

Recent evidence that the adult heart can repair itself through the proliferation of cardiac precursor cells and the recruitment and engraftment of bone marrow-derived precursor cells has stimulated interest in stem cell therapy for cardiac diseases. Although preclinical studies have shown the efficacy of stem cell treatment, numerous controversies exist, and many basic questions still await answers. Meanwhile, clinical trials of stem cell therapy continue to advance. Our group has focused on using stem cells to treat patients with end-stage ischaemic heart failure who are ineligible for surgical or percutaneous revascularization. The preliminary results of the phase I trial are encouraging but need to be reproduced before larger randomized trials can be attempted. This review covers some of the most important clinical developments in targeted, transendocardially delivered stem cell therapy for end-stage ischaemic heart failure.

Stem Cell Therapy for Cardiac Repair

Coronary heart disease and heart failure continue to be significant burdens to healthcare systems in the Western world. In the United States alone, there are 7.1 million survivors of myocardial infarction (MI) and 4.9 million people living with congestive heart failure (CHF).1 Despite recent advances in medical and device therapy for heart failure, the incidence, hospitalization, and mortality rates continue to rise. After receiving a diagnosis of CHF, 1 in 5 patients will be dead within 12 months.1 Therefore, any new treatment modality that benefits heart failure patients has the potential to result in a dramatic improvement in health outcomes and substantial cost savings for the community. Response by Oettgen p 352 The possibility of using stem cell–based therapies for people suffering an acute MI or living with CHF has captured the imagination of both the medical and popular communities. Since early reports in animal models >10 years ago,2,3 the stem cell field has made enormous advances in moving toward clinically applicable treatment options, and we now stand at the dawn of a new therapeutic era. An abundance of preclinical data demonstrate safety, feasibility, and efficacy, justifying the current entry into clinical trials of stem cell therapy in humans.4–8 This position, however, is extremely controversial, with some arguing that trials are premature because mechanistic insights are insufficiently addressed.9,10 Here, we argue that properly conducted rigorous clinical trials are a key and appropriate next step not only to start the long process of therapeutic development but also as an essential component in the process of understanding the scientific underpinnings of cardiac regeneration and its therapeutic utilization. The field of regenerative medicine will advance through the parallel conduct of in vitro/animal model studies and clinical trials, the latter frequently guiding the former. The publication of Menasche et …

Cardiac Stem Cell Therapy

Cardiovascular disease remains the number one cause of morbidity and mortality in the United States and Europe. In the United States alone, ≈1 million patients suffer a myocardial infarction every year, with an associated mortality of 25% at 3 years.1 A more sobering statistic is the fact that there are ≈5 million Americans with congestive heart failure, with an associated 20% mortality per year. This remains the case despite advances in pharmacotherapy, cardiac resynchronization therapies, and the use of implantable cardioverter-defibrillators.2 Some patients with end-stage congestive heart failure are considered for cardiac transplantation, but the demand for this therapeutic approach greatly outweighs the availability of donor hearts. Over the past few years, several animal studies and a few clinical trials have supported the use of stem cells as a potential therapeutic modality to address this unmet clinical need. Response by Boyle et al p 358 Several different types of cells have been used in both animal studies and patients to promote the repair of damaged myocardium. The 2 main sources of stem cells are adult stem and embryonic stem (ES) cells. ### ES Cells ES cells are derived from the inner mass of developing embryos during the blastocyst stage. Characteristic features of ES cells include their proliferative and self-renewing properties and their ability to differentiate into a wide variety of cell types, including cardiac myocytes.3 The major concerns with their use in human trials include the formation of teratomas when ES cells are injected into immunocompromised animals. This is particularly important because the ES cells currently available for use in humans would be of allogeneic origin and therefore would require immunosuppression. As nuclear transfer techniques improve, they will provide a way of generating an unlimited supply of histocompatible ES cells using the nuclei of cells obtained directly from the recipient …

Behavioral and Histological Characterization of Intrahippocampal Grafts of Human Bone Marrow-Derived Multipotent Progenitor Cells in Neonatal Rats with Hypoxic-Ischemic Injury

Children born with hypoxic-ischemic (HI) brain injury account for a significant number of live births wherein no clinical treatment is available. Limited clinical trials of stem cell therapy have been initiated in a number-of neurological disorders, but the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. One major postulated mechanism underlying therapeutic benefits of stem cell therapy involves stimulation of endogenous neurogenesis via transplantation of exogenous stem cells. To this end, transplantation has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents as highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. Here, we investigated the efficacy of intrahippocampal transplantation of multipotent progenitor cells (MPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, which involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI injury, animals received stereotaxic hippocampal injections of vehicle or cryopre-served MPCs (thawed just prior to transplantation) derived either from Sprague-Dawley rats (syngeneic) or Fisher rats (allogeneic). All animals were treated with daily immunosuppression throughout the survival period. Behavioral tests were conducted on posttransplantation days 7 and 14 using the elevated body swing test and the rotarod to reveal general and coordinated motor functions. MPC transplanted animals exhibited reduced motor asymmetry and longer time spent on the rotarod than those that received the vehicle infusion. Both syngeneic and allogeneic MPC transplanted injured animals did not significantly differ in their behavioral improvements at both test periods. Immunohistochemical evaluations of graft survival after behavioral testing at day 14 posttransplantation revealed that syngeneic and allogeneic transplanted MPCs survived in the hippocampal region. These results demonstrate for the first time that transplantation of MPCs ameliorated motor deficits associated with HI injury. In view of comparable behavioral recovery produced by syngeneic and allogeneic MPC grafts, allogeneic transplantation poses as a feasible and efficacious cell replacement strategy with direct clinical application. An equally major finding is the observation lending support to the hippocampus as an excellent target brain region for stem cell therapy in treating HI injury.

Time course and outcome of recovery from stroke: Relevance to stem cell treatment

Stroke is the third leading cause of death in the United States after heart disease and cancer; it has an incidence of approximately 750,000 cases per year, and it is a leading cause of disability in adults. The factors predicting a poor outcome from stroke include severe initial neurological dysfunction, hypertension, cardiac arrhythmias, myocardial infarction, hypercholesterolemia, and diabetes mellitus. The armamentarium available for improvement of neurological function after stroke is currently limited to placement in specialized stroke units, optimal therapy for medical complications, and intense physical, occupational and speech rehabilitation. Despite many trials, no pharmacological intervention has been shown convincingly to improve neurological outcome. This review was undertaken to determine the appropriate time for new approaches to the therapy of stroke such as the infusion of stem cells into the central nervous system. The literature shows in large case series that functional recovery from stroke reaches a maximum level by 3-6 months after onset, and no further recovery occurs beyond this time. Nevertheless, about 80% of these patients reach their maximum function for activities of daily living within 6 weeks from onset. Initiation of a clinical trial of stem cell therapy would require demonstration of optimal clinical improvement by neurological evaluations, with no change over at least 4 weeks of observation. Accordingly, in subjects with first-ever ischemic stroke who remained neurologically unchanged from the second until the third month after the acute event, implementation of stem cell therapy would be appropriate at approximately 3 months after the stroke.

Transplantation of Cryopreserved Human Bone Marrowderived Multipotent Adult Progenitor Cells for Neonatal Hypoxie- Ischemic Injury: Targeting the Hippocampus

There is currently no treatment for neonatal hypoxic-ischemic (HI) injury. Although limited clinical trials of stem cell therapy have been initiated in a number of neurological disorders, the preclinical evidence of a cell-based therapy for neonatal HI injury remains in its infancy. Stem cell therapy, via stimulation of endogenous stem cells or transplantation of exogenous stem cells, has targeted neurogenic sites, such as the hippocampus, for brain protection and repair. The hippocampus has also been shown to secrete growth factors, especially during the postnatal period, suggesting that this brain region presents a highly conducive microenvironment for cell survival. Based on its neurogenic and neurotrophic factor-secreting features, the hippocampus stands as an appealing target for stem cell therapy. In the present study, we investigated the efficacy of intrahippocampal transplantation of multipotent adult progenitor cells (MAPCs), which are pluripotent progenitor cells with the ability to differentiate into a neuronal lineage. Seven-day old Sprague-Dawley rats were initially subjected to unilateral HI injury, that involved permanent ligation of the right common carotid artery and subsequent exposure to hypoxic environment. At day 7 after HI