| The overall goal of the thesis was to develop tools to advance investigations of stem cells in tissue engineering therapeutics for neurodegenerative disease and spinal cord injury. Two tools to characterize cell fate and a tool to separate a subset of neural cells were developed and evaluated. In the first study, a digital PCR technology, called polonies, was applied to measure mRNA from several key stem cell genes in small numbers of ES cells. Due to its properties, we hypothesized that polonies would be uniquely poised to profile stem cells. Polonies were counted for Oct3 in a sample of 10 ES cells and from three pluripotency genes: Oct3, Nanog, and Rex1 from a single blastocyst, containing 30 ICM cells. The polony method is sensitive, can be applied to most genes, and allows for a degree of multiplexing. Second, DNA methylation was explored as a tool to measure cell fate as ES cells are differentiated into neural cells. We tested the hypothesis that promoter DNA methylation correlates to gene silencing. Promoter methylation of a pluripotency and neural fate determining genes in ES cells, ES derived neural cells, and non-neural tissues was measured by direct bisulfite sequencing. As expected Oct3, was methylated in differentiated cells and tissues. Unexpectedly, neural genes: Sox1, Olig1, and Olig2 were unmethylated in non-neural cells and tissues. The correlation between methylation and silencing was not universal; it was gene specific. In the third study, ES cells were genetically engineered to permit drug selection of subset of ES derived neural cells. We hypothesized that engineering ES cells with puromycin acetyltransferase gene (PAC) under the control of the Olig2 promoter would allow for selection of Olig2 expressing neural cells with puromycin. Two targeted ES cell lines were generated with PAC inserted into the Olig2 gene. Both lines have the expected functional properties and enable purification of Olig2 expressing cells from ES derived neural cells by puromycin selection. Overall, the tools developed in this thesis are a small step toward generating well-defined cell populations from stem cells needed to advance tissue engineering therapeutics for neurodegenerative disease and injury. |
