Right Column

RL1-00642-1: Derivation of New ICM-stage hESCs

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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.

Statement of Benefit to California (provided by applicant)

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.

Review

The applicant proposes to develop new pluripotent human stem cell lines with improved properties such as ease of culture and manipulation. The proposed approach is based on the assumption that current human embryonic stem cell (hESC) lines represent a later developmental stage of the embryo, the epiblast stage, compared to mouse (mESC) lines which are more representative of the earlier inner cell mass (ICM) stage. The major goal of this project is to generate new hESC lines that are more representative of the human ICM stage of development, cells which the applicant terms hPSC. The applicant proposes to generate hPSC by two different methods. In Aim 1, s/he will attempt to derive hPSC from in vitro fertilization (IVF) blastocysts by using small molecules previously identified by him/herself and molecules identified by others that promote self-renewal in mESC. In Aim 2, s/he will screen a small molecule library for compounds that will reprogram conventional hESCs to hPSCs. Finally, a detailed characterization of any newly derived cell lines is proposed.

The principal investigator (PI) provides a good rationale for hPSC derivation. ICM-stage hESC lines, when compared to conventional hESC, may facilitate culture and manipulation, they may allow easier comparison to mouse systems, and they would provide a novel cell type for studying stem cell biology. One reviewer felt that this is an innovative and potentially exciting application. However, reviewers raised several concerns about this application. With regard to Aim 1, they were uncertain whether the compound that was previously identified in the mouse screen also acts to maintain pluripotency in human cells, and they felt that although Aim 1 describes a perfectly reasonable approach, it is limited in scope. Furthermore, concerns were raised about Aim 2, in which a screen would be employed to identify new small molecules that might reprogram hESC to hPSC. This assay is proposed, since it would be impractical to screen for such a compound using human embryos. However, reviewers were concerned about the rationale and complexity of the screen, since it requires reversal or reprogramming of cells, rather than simply blocking forward differentiation programs during stem cell derivation from blastocysts. If reprogramming requires affecting more than one pathway, the screen is unlikely to work. Furthermore, the PI proposes that candidate lead molecules discovered in Aim 2 would be put back into the derivation strategy in Aim 1, yet it is unclear if these compounds would function similarly in the derivation process as they did in reprogramming.

Reviewers were further concerned about the choice of a specific marker for successful reprogramming, since that marker is known to be expressed following differentiation of hESC. Furthermore, during the discussion, the panelists questioned the PI’s hypothesis that hESC represent a later developmental stage than mESC. Some investigators claim that hESC appear to be epiblast-like (e.g. murine epiblast-derived stem cells are more similar to conventional hESC than to mESC by some criteria); however, a reviewer stated that hESC can produce trophoblast cells, and therefore may represent a very early developmental phenotype.

The PI is an expert in the design of high throughput assays for screening diverse small molecule libraries, and has applied this toward stem cell biology over the past few years. S/he trained with a prominent investigator. S/he is set up to carry out such a screen with robotics for compound distribution and confocal microscopic imaging. The proposed compound library has been successfully used for other screening projects in the past, and the PI provides relevant preliminary data. However, one reviewer was concerned that the library has limited general diversity of structures. Furthermore, follow-up strategies for the screening experiments were lacking, and the inclusion of a medicinal chemist as a collaborator would have strengthened the proposal.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Cowan, Chad