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Abstract
Chemicals that regulate cell epigenetic states by inhibiting DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) have been used to improve somatic cell reprogramming during somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPSC) generation. However, reprogramming induced by fusion with embryonic stem cells (ESCs) and by exposure to pluripotent cell extracts has not been well studied, especially in human cells.
Data presented in this thesis first optimized hESC-somatic cell fusion condition by applying three conventional fusion methods and the results provide direct evidence that hESCs have the ability to reprogram human somatic cells. Using a combination of immunoblotting and immunocytochemistry, I also demonstrated that hESC extracts can reprogram human somatic cells towards pluripotency. Reprogramming was confirmed by morphological changes and quantitative PCR (qPCR) analysis on pluripotency related genes. When somatic cells were pre-treated with DNMT and HDAC inhibitors before exposing to hESC extracts, changes in global epigenetic states were observed and morphological analysis revealed a higher rate of hESC-like colony formation than without pre-treatment. qPCR demonstrated that pluripotency genes were upregulated to higher levels as compared to those of somatic cells treated with hESC extracts alone. Thus, pre-modifying the epigenetics of somatic cells could be used to improve the efficiency of reprogramming process. These reprogrammed cells could not be maintained for a long time due to the up-regulation of apoptotic genes in the culture conditions used here. However, under directed differentiation conditions, the reprogrammed cells exhibited differentiation ability into neuronal cells suggesting that, although complete reprogramming was not achieved, the cells could be transdifferentiated after partial reprogramming.
In summary, this study showed that both hESC-somatic cell fusion and hESC extracts could induce partial reprogramming of human somatic cells. The latter was improved by pre-treating of the somatic cells with epigenetic modifying agents. The resulting reprogrammed cells could be differentiated into neuronal cells. Thus, the combination of epigenetic and hESC extract treatment for inducing human somatic cell reprogramming provides a new way for in vitro reprogramming and generating patient-specific cell lines.