Recent studies in the derivation of rodent pluripotent epiblast stem cells and their molecular characterizations have provided strong evidence that the conventional human embryonic stem cells may represent a distinct, later developmental stage, i.e. late epiblast stage, than the conventional murine embryonic stem cells, which is a “capture” of the ICM stage. Those two stages (i.e. ICM/pre-implantation stage vs. epiblast/post-implantation stage) of pluripotent stem cells are typically maintained in their self-renewal state by different sets of exogenous signaling molecules. Meanwhile, other studies have suggested that rather than exogenously activating multiple additional pathways to achieve a fine balanced self-renewal state, a more fundamental approach to main self-renewal of stem cells is to inhibit endogenously expressed differentiation-inducing protein activity. In addition, cell-permeable small molecules have the unique advantage of acting intracellularly to inhibit differentiation without requirement of expression of the desirable membrane receptors by cells for transducing differentiation-inhibiting signals by the desirable exogenous growth factors in the culture media. Those studies together suggested the possibility that an earlier stage (i.e. ICM-stage) of human pluripotent stem cells than the conventional human embryonic stem cells, which would represent an equivalent counterpart of the conventional murine embryonic stem cells, could be derived with helps of small molecules that could block further differentiation and capture the state of human ICM-stage of pluripotent stem cells. Here we propose to screen chemical libraries for small molecules that can facilitate derivation of the above hypothesized, new, earlier developmental state of human pluripotent stem cells from donated human IVF blastocysts. Such new human pluripotent stem cells may have better properties than the conventional human embryonic stem cells (e.g. ease of culture and manipulation), facilitate ready transfer of knowledge/techniques learn from murine embryonic stem cells to human pluripotent stem cells, and perhaps provide a new cell type for studying fundamental biology.
The putative human pluripotent stem cells proposed here may have better properties than the conventional human embryonic stem cells (e.g. ease of culture and manipulation), facilitate ready transfer of knowledge/techniques learn from murine embryonic stem cells to human pluripotent stem cells, and perhaps provide a new cell type for studying fundamental biology. In addition, small molecules have been more useful than genetic approaches in the treatment of human disease. The demonstration that one can systematically identify, optimize and characterize the mechanism of action of small drug-like molecules that selectively control cell fate and reprogramming will: (1) provide important tools to manipulate cell fate in the lab; (2) provide new insights into the complex biology that regulates (stem) cell fate; and (3) provide an important first step which may ultimately lead to drugs that facilitate the clinical application of stem cells.
During the reporting period, we have made significant progress toward the following research aims: (1) Screened small molecules and identified a robust and specific condition that can convert murine epiblast-stage pluripotent cells (that correspond to the conventional human embryonic stem/hES cells) to murine embryonic stem cell-like cells that exhibit similar cellular behaviors to various signaling pathway modulations and most importantly contribute to chimerism in vivo as mES cells. This validated the concept of and a chemically defined condition for converting later developmental stage (i.e., post-implantation, late epiblast) of pluripotency to earlier developmental stage (i.e., pre-implantation, inner cell mass) of pluripotency. (2). Established a series of reliable assays to examine hES cell conversion, including markers and cell behaviors under various signaling conditions. (3) Validated the concept that human cells could exhibit a different pluripotency state that is similar to mESC by combining the small molecules and the genetic reprogramming approach. Those novel human pluripotent cells generated from somatic cells share similar colony morphology and responses to signaling modulations as mESCs. This establishes a basis for identifying new small molecules and fine-tuning the converting as well as maintenance conditions for ultimately achieving derivation of ICM-stage, mESC-like hES cells.