Date of Award
Master of Science in Biomedical Engineering - (M.S.)
Cheul H. Cho
Bryan J. Pfister
Many central nervous system (CNS) disorders are attributable to damaged oligodendrocytes, the cells that form the myelin sheath of axons. An effective source of human oligodendrocytes is crucial, making pluripotent stem cells, such as embryonic stem (ES) cells, an attractive source for nerve tissue repair and regeneration due to their unlimited capacity for self-renewal and their ability to differentiate into all major cell lineages. Bioengineered 3-D hydrogel culture systems have the potential to elucidate mechanisms required for regeneration of CNS cells. The goals of this study are: (1) to investigate the effects of retinoic acid (RA), sonic hedgehog (shh) agonist, and glucose levels on neural differentiation of mouse embryonic stem cells (mESCs), and (2) to employ micropatterned agarose hydrogels for nerve guidance. A protocol utilizing embryoid body (EB) formation, under both high and low glucose conditions, was devised for RA-induced neural lineage differentiation and RA/shh-induced oligodendrocyte differentiation. EBs exposed to lower glucose concentrations with agonists exemplified a more rapid differentiation. After 8 days in culture, there was a higher expression level of oligodendrocyte precursor cell markers in low glucose conditions. These results illustrate that physiologic glucose concentrations are more conducive to the development of EBs and favor the differentiation of mESCs into neural lineages. In order to fabricate 3-D microchannel hydrogel structures for nerve guidance, agarose solution was poured onto the plastic mold master with gridded microchannels and peeled off after hydrogel formation. An extracellular matrix (ECM) coating, such as collagen, was used to promote cell adhesion and neurite outgrowth limited to the channels. Exploitation of various cell types, including fibroblasts and PC12 cells, to optimize the hydrogel conditions, exhibited good cell and ECM patterns on the microchannels. Further studies using dorsal root ganglion (DRG) neurons, Schwann cells, and differentiating ES cells demonstrated good cell growth and nerve guidance through the micropatterned channels. Micropatterned hydrogels can be used as an in vitro model to replicate complex tissue organization, and to investigate nerve signal propagation and myelination.
Gittens, Jamila S., "Neural differentiation of pluripotent stem cells and applications to micropatterned agarose hydrogel for nerve guidance" (2012). Theses. 109.