CD34 negative non-hematopoietic human umbilical cord blood stem cell amelioration of ischemic brain injury: mechanisms of action

Abstract

Background: Previous work in the laboratory has demonstrated that a novel cell population of CD34 negative non-hematopoietic umbilical cord blood stem cells (nh-UCBCs) can be isolated from the human umbilical cord blood (UCB). These cells when intravenously delivered into a transient or permanent middle cerebral artery occlusion (MCAO) rat model of stroke, 2 days after injury, helped alleviate the stroke induced behavioral deficits and reduced the infarct size. In addition, injection of nh-UCBCs directly into the brain near the site of ischemic injury also ameliorated limb placement deficits. Purpose: The central goal of this project was to explore the mechanism(s) by which CD34 negative nh-UCBCs help ameliorate the neurological deficits when injected systemically into a rat model 48hrs after inducing stroke (acute period), and when directly injected into the brain at 3 months after ischemic injury (chronic period). The improvement in behavior of the rat model was evaluated based on the neurological severity score (NSS) derived from performing various functional tests. The first aim of the project was to check whether the nh-UCBCs has an elevated level of anti-inflammatory cytokines which helps in reducing the inflammation that results from stroke and thus helps in the behavioral improvement of the animal model during the acute period after an ischemic stroke. In order to test this aim, the cytokine levels in the nh-UCBCs and nh-UCBS cell conditioned media were tested using qRT-PCR and cytokine protein array, respectively. The next aim was to check whether the nh-UCBCs that are injected intravenously into the rat model can modulate the phenotype of immune cells in the brain. To test this aim we analyzed the expression of various M1 and M2 macrophage/microglia markers in the BV2 cells (murine microglial cell line) cultured in different conditions by qRT-PCR. We expect to see a shift from M1 type macrophages/microglia phenotype to M2 type macrophage/microglia phenotype which is responsible for eliminating inflammation and help in wound healing. During the chronic period after a stroke, the inflammatory environment has most likely subsided, and the restorative effects of injecting nh-UCBCs directly into the brain parenchyma is most likely not due to the modulation of the inflammatory environment. We postulate that it might be due to the induction of fiber sprouting by neurons from the non-damaged hemisphere towards the site of injury. In order to test this trophic action of the neurons and the sprouting of fibers in the presence of the non-hematopoietic population of human umbilical cord blood, an in vitro tissue culture model is used. The plausible mechanism in this model is that the nh-UCBCs can induce neural plasticity and therefore enhance the connectivity between the interhemispheric neuronal connections.