Alison E. Willing

Infusion of Human Umbilical Cord Blood Cells in a Rat Model of Stroke Dose-Dependently Rescues Behavioral Deficits and Reduces Infarct Volume

Background and Purpose— Intravenously delivered human umbilical cord blood cells (HUCBC) have been previously shown to improve functional recovery of stroked rats. To extend these findings, we examined the behavioral recovery and stroke infarct volume in the presence of increasing doses of HUCBC after permanent middle cerebral artery occlusion (MCAO). Methods— Rats were subjected to MCAO and allowed to recover for 24 hours before intravenous infusion of 104 up to 3 to 5×107 HUCBC. Behavioral tests (spontaneous activity, step test, elevated body swing test) were performed 1 week before MCAO and at 2 and 4 weeks after HUCBC infusion. On completion of behavioral testing, animals were euthanized and brain infarct volumes quantified. HUCBC were identified by immunofluorescence for human nuclei and by polymerase chain reaction (PCR) using primers specific for human glycerol 3-phosphate dehydrogenase. Results— At 4 weeks after infusion, there was a significant recovery in behavioral performance when 106 or more HUCBC were delivered (p=0.001 to p=0.05). Infarct volume measurements revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 cells, p<0.01; 3 to 5×107 cells, p<0.05). Moreover, HUCBC were localized by immunohistochemistry and PCR analysis only in the injured brain hemisphere and spleen. Conclusions— These results extend previous observations of HUCBC infusion in the MCAO rat stroke model by demonstrating a dose relationship between HUCBC, behavioral improvement, and neuronal sparing.

Intravenous Administration of Human Umbilical Cord Blood Cells in a Mouse Model of Amyotrophic Lateral Sclerosis: Distribution, Migration, and Differentiation

Amyotrophic lateral sclerosis (ALS), a multifactorial disease characterized by diffuse motor neuron degeneration, has proven to be a difficult target for stem cell therapy. The primary aim of this study was to determine the long-term effects of intravenous mononuclear human umbilical cord blood cells on disease progression in a well-defined mouse model of ALS. In addition, we rigorously examined the distribution of transplanted cells inside and outside the central nervous system (CNS), migration of transplanted cells to degenerating areas in the brain and spinal cord, and their immunophenotype. Human umbilical cord blood (hUCB) cells (10(6)) were delivered intravenously into presymptomatic G93A mice. The major findings in our study were that cord blood transfusion into the systemic circulation of G93A mice delayed disease progression at least 2-3 weeks and increased lifespan of diseased mice. In addition, transplanted cells survived 10-12 weeks after infusion while they entered regions of motor neuron degeneration in the brain and spinal cord. There, the cells migrated into the parenchyma of the brain and spinal cord and expressed neural markers [Nestin, III Beta-Tubulin (TuJ1), and glial fibrillary acidic protein (GFAP)]. Infused cord blood cells were also widely distributed in peripheral organs, mainly the spleen. Transplanted cells also were recovered in the peripheral circulation, possibly providing an additional cell supply. Our results indicate that cord blood may have therapeutic potential in this noninvasive cell-based treatment of ALS by providing cell replacement and protection of motor neurons. Replacement of damaged neurons by progeny of cord blood stem cells is probably not the only mechanism by which hUCB exert their effect, since low numbers of cells expressed neural antigens. Most likely, cord blood efficacy is partially due to neuroprotection by modulation of the autoimmune process.

Expression of neural markers in human umbilical cord blood.

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.

Cord blood rescues stroke-induced changes in splenocyte phenotype and function.

The neuroprotective mechanism of human umbilical cord blood cells (HUCBC) in the rat middle cerebral artery occlusion (MCAO) stroke model remains uncertain. Given the inflammatory sequelae that occur following stroke, we investigated whether HUCBC protection could be derived from the modulation of this immuno-inflammatory event, suggested by the attraction of the HUCBC to the spleen. We found that, following MCAO, rat spleen size was reduced concomitantly with their CD8+ T-cell counts. Interestingly, MCAO-induced spleen size reduction correlated with the extent of ischemic damage, however, HUCBC treatment rescued the spleen weight, splenic CD8+ T-cell counts, as well as the amount of brain injury. Additionally, splenocyte proliferation assays demonstrated that HUCBC treatment opposed MCAO-associated T-cell proliferation by increasing the production of IL-10 while decreasing IFN-gamma. Taken together, these results suggest a novel immunomodulatory mechanism by which HUCBC mediate protection in the rat MCAO model of stroke.

Human Umbilical Cord Blood Stem Cells Infusion in Spinal Cord Injury: Engraftment and Beneficial Influence on Behavior

The use of human umbilical cord blood (hUCB)--a rich source of nonembryonic or adult stem cells--has recently been reported to ameliorate behavioral consequences of stroke. In this study, we tested whether human cord blood leukocytes also ameliorate behavioral impairments of spinal cord injury. Rats were divided into five groups: (1) laminectomy (without spinal cord injury) only; (2) laminectomy + cord blood infusion; (3) spinal cord injury + cord blood infused 1 day post injury; (4) spinal cord injury + cord blood infused 5 days post injury; and (5) spinal cord injury only. Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. Open-field behavior was assessed 1, 2, and 3 weeks after intravenous injection of prelabeled human cord blood cells. Open-field test scores of spinal cord injured rats treated with human cord blood at 5 days were significantly improved as compared to scores of rats similarly injured but treated at day 1 as well as the otherwise untreated injured group. The results suggest that cord blood stem cells are beneficial in reversing the behavioral effects of spinal cord injury, even when infused 5 days after injury. Human cord blood-derived cells were observed in injured areas, but not in noninjured areas, of rat spinal cords, and were never seen in corresponding areas of spinal cord of noninjured animals. The results are consistent with the hypothesis that cord blood-derived stem cells migrate to and participate in the healing of neurological defects caused by traumatic assault.

The spleen contributes to stroke-induced neurodegeneration

Stroke, a cerebrovascular injury, is the leading cause of disability and third leading cause of death in the world. Recent reports indicate that inhibiting the inflammatory response to stroke enhances neurosurvival and limits expansion of the infarction. The immune response that is initiated in the spleen has been linked to the systemic inflammatory response to stroke, contributing to neurodegeneration. Here we show that removal of the spleen significantly reduces neurodegeneration after ischemic insult. Rats splenectomized 2 weeks before permanent middle cerebral artery occlusion had a >80% decrease in infarction volume in the brain compared with those rats that were subjected to the stroke surgery alone. Splenectomy also resulted in decreased numbers of activated microglia, macrophages, and neutrophils present in the brain tissue. Our results demonstrate that the peripheral immune response as mediated by the spleen is a major contributor to the inflammation that enhances neurodegeneration after stroke.

Timing of cord blood treatment after experimental stroke determines therapeutic efficacy.

Embolic stroke is thought to cause irreparable damage in the brain immediately adjacent to the region of reduced blood perfusion. Therefore, much of the current research focuses on treatments such as anti-inflammatory, neuroprotective, and cell replacement strategies to minimize behavioral and physiological consequences. In the present study, intravenous delivery of human umbilical cord blood cells (HUCBC) 48 h after a middle cerebral artery occlusion (MCAo) in a rat resulted in both behavioral and physiological recovery. Nissl and TUNEL staining demonstrated that many of the neurons in the core were rescued, indicating that while both necrotic and apoptotic cell death occur in ischemia, it is clear that apoptosis plays a larger role than first anticipated. Further, immunohistochemical and histochemical analysis showed a diminished and/or lack of granulocyte and monocyte infiltration and astrocytic and microglial activation in the parenchyma in animals treated with HUCBC 48 h poststroke. Successful treatment at this time point should offer encouragement to clinicians that a therapy with a broader window of efficacy may soon be available to treat stroke.

Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain.

Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III β-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain.

Human umbilical cord blood mononuclear cells for the treatment of acute myocardial infarction.

Cell transplantation is a new treatment to improve cardiac function in hearts that have been damaged by myocardial infarction. We have investigated the use of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction. The control group consisted of 24 normal rats with no interventions. The infarct + vehicle group consisted of 33 rats that underwent left anterior descending coronary artery (LAD) ligation and after 1 h were given Isolyte in the border of the infarction. The infarct + HUCBC group consisted of 38 rats that underwent LAD ligation and after 1 h were given 106 HUCBC in Isolyte directly into the infarct border. Immunosuppression was not given to any rat. Measurements of left ventricular (LV) ejection fraction, LV pressure, dP/dt, and infarct size were determined at baseline and 1, 2, 3, and 4 months. The ejection fraction in the controls decreased from 88 ± 3% to 78 ± 4% at 4 months (p = 0.03) as a result of normal aging. Following infarction in the infarct + vehicle group, the ejection fraction decreased from 87 ± 4% to 51 ± 3% between 1 and 4 months (p < 0.01). In contrast, the ejection fraction of the infarcted + HUCBC-treated rat hearts decreased from 87 ± 4% to 63 ± 3% at 1 month, but progressively increased to 69 ± 6% at 3 and 4 months, which was different from infarct + vehicle group rats (p < 0.02) but similar to the controls. At 4 months, anteroseptal wall thickening in infarct + HUCBC group was 57.9 ± 11.6%, which was nearly identical to the control anteroseptal thickening of 59.2 ± 8.9%, but was significantly greater than the infarct + vehicle group, which was 27.8 ± 7% (p < 0.02). dP/dtmax increased by 130% in controls with 5.0 μg of phenylephrine (PE)/min (p < 0.001). In the infarct + vehicle group, dP/dtmax increased by 91% with PE (p = 0.01). In contrast, in the infarct + HUCBC group, dP/dtmax increased with PE by 182% (p < 0.001), which was significantly greater than the increase in dP/dtmax in the infarct + vehicle group (p = 0.03) and similar to the increase in the controls. Infarct sizes in the infarct + HUCBC group were smaller than the infarct + vehicle group and averaged 3.0 ± 2.8% for the infarct + HUCBC group versus 22.1 ± 5.6% for infarct + vehicle group at 3 months (p < 0.01); at 4 months they averaged 9.2 ± 2.0% for infarct + HUCBC group versus 40.0 ± 9.2% for the infarct + vehicle group (p < 0.001). The present experiments demonstrate that HUCBC substantially reduce infarction size in rats without requirements for immunosuppression. As a consequence, LV function measurements, determined by LV ejection fraction, wall thickening, and dP/dt, are significantly greater than the same measurements in rats with untreated infarctions.

Umbilical Cord Blood‐Derived Stem Cells and Brain Repair

Abstract: Human umbilical cord blood (HUCB) is now considered a valuable source for stem cell‐based therapies. HUCB cells are enriched for stem cells that have the potential to initiate and maintain tissue repair. This potential is especially attractive in neural diseases for which no current cure is available. Furthermore, HUCB cells are easily available and less immunogenic compared to other sources for stem cell therapy such as bone marrow. Accordingly, the number of cord blood transplants has doubled in the last year alone, especially in the pediatric population. The therapeutic potential of HUCB cells may be attributed to inherent ability of stem cell populations to replace damaged tissues. Alternatively, various cell types within the graft may promote neural repair by delivering neural protection and secretion of neurotrophic factors. In this review, we evaluate the preclinical studies in which HUCB was applied for treatment of neurodegenerative diseases and for traumatic and ischemic brain damage. We discuss how transplantation of HUCB cells affects these disorders and we present recent clinical studies with promising outcome.