Derivation of New ICM-stage hESCs
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) Established two screening assay conditions that allowed us to carry out high throughput screens of small molecules for enhancing conversion of conventional human ESCs to the earlier developmental naive state like murine embryonic stem cells. A couple of hit compounds were identified from the screen, and validated. (2). Established and characterized naive state human pluripotent cell lines converted from HUES7, HUES10 and classic H1. Human pluripotent stem cells in such naive state exhibit similar cellular behaviors, e.g., cell survival, proliferation, and responses to various signaling pathway modulations as mES cells. (3) Identified a new fundamental mechanism in such reprogramming/conversion process.
During the reporting period, we have made significant progress toward discovering new molecules and conditions that can induce the generation of the earlier developmental, naïve state of pluripotency in human cells. Specifically, we have carried out additional high throughput screening of chemical libraries for enhancing conversion of conventional human embryonic stem cells to the earlier developmental, naive pluripotency state. Novel hit compounds were identified from the screen, and validated. To further improve and characterize the molecules, we established the synthetic chemistry to generate analogs of one compound series, have conducted structure-activity-relationship study, and found activity improved compounds. This also allowed generation of affinity chromatography probes for target identification. Furthermore, mechanistic studies suggested two distinct pathways that affect the conversion to naïve state.
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