Evaluating Environmental Cues that Affect Schwann Cell Gene Expression to Enhance Peripheral Nerve Regeneration

The goal of this thesis was to evaluate the effects of different environmental cues on Schwann cell (SC) differentiation and phenotype maintenance to design better SC transplantation therapies for peripheral nerve repair. First, a set of markers, specific for motor or sensory-derived SCs were identified from the literature and gene chips. After 30 days, gene expression patterns of SCs, after expansion in culture, were dysregulated. Cues that have been hypothesized to re-differentiate the SCs in vitro are extracellular matrix (ECM) molecules, growth factors (GFs), and acetylcholine (Ach). To test the effects of ECM, SCs were transplanted into acellular nerve grafts (ANGs), which have an intact ECM, and were used to repair a 14 mm rat sciatic nerve injury. After 2 weeks, the RNA was analyzed for expression levels of GFs (nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and glial cell derived neurotrophic factor (GDNF)) and also phenotypic markers. The phenotype-specific SCs expressed higher levels of NGF, BDNF, and GDNF compared to levels in the injuries repaired with an isograft. The expression patterns of the phenotypic markers were still disrupted at 2 weeks post transplant suggesting that other cues (GFs or Ach) are necessary to promote native marker expression. The addition of GFs NGF and GDNF to SCs in culture promoted increased mature marker expression (S100) over a period of 7 days. Evaluation of expression patterns showed sensory-derived SCs treated with NGF had increased expression of sensory markers and motor-derived SCs had no detectable expression of sensory markers. GDNF promoted the correct phenotypic marker expression in both sets of SCs treated. Finally, Ach was added to motor-derived SC cultures to determine if it had an effect on the phenotypic maintenance of motor-derived SCs. In addition, Ach receptors were blocked with gallamine to test the specificity of the Ach effect. Gene expression analysis showed that Ach promoted increased expression of motor markers in motor-derived SCs, and that gallamine blocked the effects of Ach. Overall, this work has shown that environmental cues, such as ECM, GFs, and Ach affect SC phenotype and differentiation. iii To my loving husband Bastin and my wonderful family for all their wonderful support and letting me be the person I am meant to be.