Thesis

Differentiating Mouse Embryonic Stem Cells to Use for Implantation as a Therapy for Spinal Cord Injury

In the United States, an estimated 6 million individuals live with paralysis, 23% of these incidents being due to spinal cord injury. Following an injury to the central nervous system there are events that occur to segregate inflammation from the surrounding healthy tissue, this is characterized by the formation of the astrocyte scar. Once the astrocyte scar has formed, regrowth of axon across the injured area is greatly impeded if not impossible. Using mouse embryonic stem cells we aim to generate a population of neural progenitor following a differentiation protocol using retinoic acid and sonic hedgehog. Following differentiation in vitro we fixed cells to cover slips and stained using immunohistochemistry to identify if we cell markers designating the cell lineage. Our data show that embryonic stem cells that were exposed to the differentiation protocol were positive for the neural progenitor marker Nestin. We also wanted to determine that we could influence the development of these progenitors toward either astrocyte or neuron lineages using small molecules. Following the differentiation protocol we further treated cells with FGF or SUN11602, an FGF mimetic, to generate astrocytes or DAPT, a Notch inhibitor, to generate neurons. From our initial results, FGF was capable of generating a large population of astrocytes although not homogeneous. When using the SUN11602 depending on the concentration of retinoic acid used in the differentiation protocol we either saw no differentiation for the low concentration of retinoic acid or we saw the generation of mostly neurons for the higher concentration of retinoic acid used. A similar result was observed for cells that were treated with DAPT, when differentiating with lower concentrations of retinoic acid we saw very little generation of astrocytes or neurons and when higher concentrations were used we saw some generation of neurons. We will continue in designing a more robust differentiation protocol, which will eventually lead us to our next goal of implanting these cells in vivo using a mouse model for spinal cord injury.

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