| Human embryonic stem (hES) cells are capable of unlimited self-renewal and differentiation into all cells of the body, holding great promise for regenerative medicine. However, the key to this therapeutic power begins with understanding how these cells maintain their stem cell state. Growth and expansion of undifferentiated hES cells requires integration of survival and self-renewal signals along with maintenance of pluripotency. To date few genes have been identified as key regulators of survival, self-renewal and pluripotency (i.e., Oct4, Nanog, Rho-associated kinase) With the exception of these few genes, little is known about the molecular mechanisms that maintain the hES cell state.;In order to study pathways regulating the hES cell state, we first developed methods for high-efficiency genetic manipulation of hES cells. The technique combines nucleofection of single hES cells with improved methods to select cells at clonal density in order to create transient and stable genetic modifications. As validation, we reduced Oct4 and Nanog expression using siRNAs/shRNA vectors and derived many clones with stably reduced alkaline phosphatase activity or stable overexpression of GFP. These studies have demonstrated out ability to interrogate gene function in hES cells.;We next used nucleofection to analyze the function of Sox2 in maintaining hES cell pluripotency. Reduction of Sox2 expression by RNA interference resulted in a loss of the hES cell state as demonstrated by altered morphology, loss of stem cell antigen expression, and differentiation into trophectoderm. Downregulation of Sox2 also decreased Oct4/Nanog expression, suggesting a coordinated role for these factors in regulating pluripotency. These results demonstrate that Sox2 is required to preserve the hES cell state.;In an effort to identify novel factors that affect the hES cell phenotype, we then used nucleofection to perform a cell-based phenotypic screen using a dsRNA library targeting human phosphatases. Analysis of 237 phosphatases implicated several key signaling pathways involved in the maintenance of the hES cell state, including the PI3K/Akt and p53 pathways. Overall these findings describe the use of genetic modification to study hES cell fate determination and will help define the parameters that control growth and differentiation of hES cells in the future. |
