Date of Award

1-1-2006

Embargo Period

1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Ling Wei

Second Advisor

Demetri Spyropoulos

Third Advisor

Gary Wright

Fourth Advisor

Brad Schulte

Fifth Advisor

Shan-Ping Yu

Abstract

Embryonic stem (ES) cell transplantation therapy offers a novel therapeutic alternative for ameliorating the pathologic consequences of cerebral ischemia. The success of ES cell transplantation therapy relies not only on initial survival in the post-ischemic brain but also on whether or not these cells will reliably differentiate into the appropriate neuronal phenotypes required for cerebral repair. To address both issues, the present study investigates the neural differentiation of retinoic acid treated ES cells and how activating signaling pathways common in response to hypoxia can impact their usefulness as graft material intended to repair, replace and restore function after stroke. These studies tested the hypothesis that selective expression of Src-family tyrosine kinases is required for neuronal phenotype specific differentiation of ES cell derived neurons following retinoic acid (RA) neural induction. Neurons derived from ES cells showed significant neurite growth, increased expression of Src, Fyn and Lek, which coincided with the emergence of NMDA and kainate currents, vulnerability to excitotoxicity, and maturation of functional excitatory synapses. These developmental events were attenuated when cells were grown in the presence of Src-family kinase inhibitor, PP2. However, there was no change in the expression of GABAergic specific protein GAD67 during PP2 treatment suggesting that Src tyrosine kinases selectively function in the production of excitatory neurons following retinoic acid directed differentiation. This study tested the novel use of hypoxic preconditioning (HP) as a priming strategy for ES cell transplantation therapy. Pretreatment of neural-derived ES (ndES) cells to sublethal hypoxia increased secretion of erythropoietin (EPO), upregulated endogenous expression of bcl-2, GDNF, HIF-1α, neurofilament, synaptophysin and erythropoietin receptor (EPOR). HP induced significant protection against in vitro apoptotic stimuli and the post-ischemic environment after transplantation. The hypoxia induced protective response lasted up to 6 days and could be blocked by preventing EPOR activation. Also, direct pretreatment of ES cells with recombinant human EPO mimicked the HP effects. Finally, in vitro hypoxic preconditioning of ndES cells prior to transplantation enhanced neurological and motor function after transient cerebral ischemia. Therefore, the application of HP to transplantable ndES cells is more favorable for transplantation therapy.

Rights

All rights reserved. All rights reserved. Copyright is held by the author.

Download

Share

COinS