Cyndy D. Sanberg

Central Nervous System Entry of Peripherally Injected Umbilical Cord Blood Cells Is Not Required for Neuroprotection in Stroke

Background and Purpose— To date, stem cell graft-mediated neuroprotection is equated with graft survival and secretion of neurotrophic factors in the brain. Here, we examined whether neuroprotection by systemically delivered human umbilical cord blood (HUCB) cells was dependent on their entry into the central nervous system in a rodent model of acute stroke. Methods— Adult male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion for 60 minutes. During the 1-hour occlusion, animals were randomly assigned to 1 of the following treatments: intravenous injection of HUCB (a subtherapeutic dose of 200 000 cells in 10 μL) with blood–brain barrier (BBB) permeabilizer (1.1 mol/L mannitol at 4°C) or vehicle, intravenous vehicle alone, or intravenous mannitol alone. Behavioral tests, using elevated body swing test and passive avoidance test, were conducted at day 3 poststroke, and thereafter, animals were euthanized for: (1) immunohistochemical examination of HUCB, which were lentivirally labeled with green fluorescent protein; (2) cerebral infarction analysis using 2,3,5-triphenyl-tetrazolium chloride; and (3) enzyme-linked immunosorbent assay of trophic factors within the striatal region. Results— We did not detect intravenously administered low dose of HUCB cells in the brains of animals at day 3 after stroke even when cells were coinfused with a BBB permeabilizer (mannitol). However, HUCB–mannitol treatment significantly increased brain levels of neurotrophic factors, which correlated positively with reduced cerebral infarcts and improved behavioral functions. Conclusions— Our data show that central nervous system availability of grafted cells is not a prerequisite for acute neuroprotection provided that therapeutic molecules secreted by these cells could cross the BBB.

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.

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.

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.

Menstrual Blood Cells Display Stem Cell–Like Phenotypic Markers and Exert Neuroprotection Following Transplantation in Experimental Stroke

Cell therapy remains an experimental treatment for neurological disorders. A major obstacle in pursuing the clinical application of this therapy is finding the optimal cell type that will allow benefit to a large patient population with minimal complications. A cell type that is a complete match of the transplant recipient appears as an optimal scenario. Here, we report that menstrual blood may be an important source of autologous stem cells. Immunocytochemical assays of cultured menstrual blood reveal that they express embryonic-like stem cell phenotypic markers (Oct4, SSEA, Nanog), and when grown in appropriate conditioned media, express neuronal phenotypic markers (Nestin, MAP2). In order to test the therapeutic potential of these cells, we used the in vitro stroke model of oxygen glucose deprivation (OGD) and found that OGD-exposed primary rat neurons that were co-cultured with menstrual blood-derived stem cells or exposed to the media collected from cultured menstrual blood exhibited significantly reduced cell death. Trophic factors, such as VEGF, BDNF, and NT-3, were up-regulated in the media of OGD-exposed cultured menstrual blood-derived stem cells. Transplantation of menstrual blood-derived stem cells, either intracerebrally or intravenously and without immunosuppression, after experimentally induced ischemic stroke in adult rats also significantly reduced behavioral and histological impairments compared to vehicle-infused rats. Menstrual blood-derived cells exemplify a source of individually tailored donor cells that completely match the transplant recipient, at least in women. The present neurostructural and behavioral benefits afforded by transplanted menstrual blood-derived cells support their use as a stem cell source for cell therapy in stroke.

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.

Cytokines produced by cultured human umbilical cord blood (HUCB) cells: Implications for brain repair

The potential therapeutic benefits from human umbilical cord blood (HUCB) cells for the treatment of injuries, diseases, and neurodegeneration are becoming increasingly recognized. The transplantation or infusion of cord blood cells in various animal models, such as ischemia/stroke, traumatic brain injury, myocardial infarction, Parkinsons disease, and amyotropic lateral sclerosis, has resulted in amelioration of behavioral deficits, and with some diseases, a prolonged lifespan decreased neuropathology. Previously, we reported the migration of HUCB cells to ischemic brain supernatant (tissue extracts) is time-dependent, and the expression of specific chemokines responds to this migration pattern. The mechanism(s) responsible for these effects are unknown. The expression of cytokines and chemokines produced by HUCB cells (under various culturing conditions) was investigated in this study. IL-8, MCP-1, and IL-1alpha were consistently expressed by the HUCB mononuclear cells regardless of the culture condition. These results provide insights to factors that may be partially responsible for the functional improvements seen in the animal models of injury investigating the therapeutic use of HUCB cells.

Infusion of human umbilical cord blood ameliorates neurologic deficits in rats with hemorrhagic brain injury

Abstract: Umbilical cord blood is a rich source of hematopoietic stem cells. It is routinely used for transplantation to repopulate cells of the immune system. Recent studies, however, have demonstrated that intravenous infusions of umbilical cord blood can ameliorate neurologic deficits associated with ischemic brain injury in rodents. Moreover, the infused cells penetrate into the parenchyma of the brain and adopt phenotypic characteristics typical of neural cells. In the present study we tested the hypothesis that the administration of umbilical cord blood can also diminish neurologic deficits caused by intracerebral hemorrhage (ICH). Intracerebral hemorrhage is a major cause of morbidity and mortality, and at the present time there are no adequate therapies that can minimize the consequences of this cerebrovascular event. ICH was induced in rats by intrastriatal injections of collagenase to cause bleeding in the striatum. Twenty‐four hours after the induction of ICH rats received intravenous saphenous vein infusions of human umbilical cord blood (2.4 × 106 to 3.2 to 106 cells). Animals were evaluated using a battery of tests at day 1 after ICH, but before the administration of umbilical cord blood, and at days 7, and 14 after ICH (days 6 and 13, respectively, after cord blood administration). These tests included a neurological severity test, a stepping test, and an elevated body‐swing test. Animals with umbilical cord blood infusions exhibited significant improvements in (1) the neurologic severity test at 6 and 13 days after cord blood infusion in comparison to saline‐treated animals (P < 0.05); (2) the stepping test at day 6 (P < 0.05); and (3) the elevated body‐swing test at day 13 (P< 0.05). These results demonstrate that the administration of human umbilical cord blood cells can ameliorate neurologic deficits associated with intracerebral hemorrhage.

Peripherally Administered Human Umbilical Cord Blood Cells Reduce Parenchymal and Vascular β-Amyloid Deposits in Alzheimer Mice

Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimers disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.

Mannitol facilitates neurotrophic factor up‐regulation and behavioural recovery in neonatal hypoxic‐ischaemic rats with human umbilical cord blood grafts

We recently demonstrated that blood–brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood–brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic‐ischaemic (HI) injury model. Seven‐day‐old Sprague–Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post‐transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme‐linked immunosorbent assay at day 3 post‐transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up‐regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor–mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood–brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.