Date of Award
Doctor of Philosophy (PhD)
Embryonic stem cells hold great potential for cell replacement strategies in the central nervous system. Pre-differentiation into various neural cell types can help generate tissue-specific cell populations that can replace cells and tissue lost due to do injury or disease. A small number of undifferentiated pluripotent stem cells persist in most transplant populations even after pre-differentiation. Given the right environment, i.e. biomaterial scaffolds, these cells can lead to tumor formation thereby eliminating any potential therapeutic benefit. This dissertation focused on the development of high purity embryonic stem cell-derived cell populations devoid of pluripotent stem cells for transplantation into the central nervous system, in particular the injured spinal cord. In the first study, transgenic expression of the puromycin resistance enzyme, puromycin N-acetyltransferase, is driven by the gene regulatory elements of the progenitor motor neuron associated transcription factor Olig2. Selection by puromycin exposure resulted in an enriched population of progenitor motor neurons, as well as recent progeny of progenitor motor neurons. Furthermore, undifferentiated stem cells were removed by puromycin selection. The efficacy of these enriched populations was evaluated in tissue engineered fibrin scaffolds containing a heparin-based delivery system for controlled delivery of two growth factor combinations. Greater differentiation into oligodendrocytes in vitro was observed in selected cell groups compared to unselected controls in fibrin scaffolds delivery neurotrophin-3 and glial derived neurotrophic factor. Enriched progenitor motor neurons survived and differentiated into oligodendrocytes, astrocytes and motoneurons in a two week sub-acute dorsal hemisection model of spinal cord injury. Encapsulating the transplant population in the tissue engineered fibrin scaffold with growth factors did not enhance proliferation or survival suggesting that tumorgenic cell populations were not present. In the final study, high purity mature cholinergic motoneurons were generated by driving puromycin resistance under control of two highly conserved enhancers for the motoneuron transcription factor Hb9. Puromycin selection resulted in a uniform group of post-mitotic immature motoneurons. Purity was observed through maturation and no proliferating glia were observed at any time point. Selected motoneurons maintained appropriate electrophysiological characteristics. Through this work, antibiotic selection appears to be a suitable method for generating high purity ES-cell derived neural populations.
Dennis Barbour, Donald Elbert, James Huettner, Daniel Moran, Paul S. Stein