The aim of this project is to screen large collections of small molecules to identify molecules that allow one to propagate human embryonic stem cells (hESCs) in cell culture under defined conditions in an undifferentiated, pluripotent state. The chemical structures of any biologically active small molecules will be optimized with respect to potency, selectivity and biological stability. The ability of hESCs proliferated in the presence of such small molecules to be differentiated into specific cell lineages both in cell culture and in vivo will also be assessed. And finally, we will determine the mechanism of action of active small molecules by a variety of biochemical and genomic methods. The demonstration that one can identify synthetic drug-like molecules that allow one to control the self-renewal and/or differentiation of hESCs will represent an important step in the ultimate therapeutic application of hESCs to human disease. In addition, biological studies of such molecules should provide new insights into the processes that control stem cell biology.
Historically, small molecules have been more useful than genetic approaches in the treatment of human disease. However, much of our ability to control embryonic stem cell self-renewal and directed differentiation currently involves genetic manipulation of these cells or complex mixtures of protein factors. The demonstration that one can systematically identify, optimize and characterize the mechanism of action of small drug-like molecules that selectively control stem cell biology both in vitro and in vivo will: (1) provide important tools to manipulate stem cells in the lab; (2) provide new insights into the complex biology that regulates stem cell differentiation; and (3) provide an important first step which may ultimately lead to drugs that facilitate the clinical application of stem cells.
SYNOPSIS: The goal of this proposal is to identify small molecules that control the self-renewal of human embryonic stem cells (hESCs). Based on the applicants' success in their identification of a molecule, pluripotin that is involved in the expansion of mouse ESCs, they will use similar approaches on hESCs. High through-put screening will be used to identify molecules that induce self-renewal of hESCs in chemically defined media. Such molecules will be used to expand cells into the three primary germ lines and test whether these cells form teratomas in mice. They will examine whether the signalling pathways involved are similar to their studies on mESCs. If these studies are successful, they will then screen for molecules involved in differentiation to a neural lineage.
SIGNIFICANCE AND INNOVATION: Most of the biological molecules involved in controlling cell fate are large, complex proteins that are difficult and expensive to use in a therapeutic context. The aim to identify small molecules that can control the self-renewal properties of hESCs is very significant as it will provide an important tool for maintaining human stem cells for subsequent differentiation into useful cell populations. This could greatly enhance work on human stem cells by simplifying culture conditions and eliminating the potential of contamination from feeder cell lines. Perhaps more importantly, the application of chemical biology analysis of the mechanism and molecular target(s) will reveal basic information about mechanisms of stemness of hESCs. Also, the compounds identified could be used in in vivo therapeutic applications where maintenance of self-renewal might be desired.
Given the prior work from the PI's lab on the same approach with mouse ESCs, the innovation is primarily in the application to the more difficult problem of hESC self-renewal. As noted by the PI, however, the NIH is usually reluctant to fund such an open-ended screening project without proof of feasibility. Thus this study - with high significance but perhaps more modest conceptual innovation – is clearly appropriate for such a seed grant mechanism. As noted by the investigators, the experience gained in the present study will serve as a bridge to similar approaches to lineage-specific differentiation – i.e. both identification of small molecules and characterization of their mechanism and molecular target(s).
One of the reviewers found these to be extremely important studies; while they follow on from their work on mESCs, they may be critical to the discovery of molecules involved in self-renewal of hESC’s and their differentiation toward individual germ layers and subsequent lineage fate.
STRENGTHS: The strengths of the proposal include the fact that the PI is a world class scientist and expert in the field of proteomics working in state-of-the-art facilities. In addition, the PI has prior experiences with mESCs and, using this technology has identified of a key molecular pluripotin which is involved in self-renewal of these cells.
A reviewer noted that this proposal takes good advantage of the prior experience and resources of the Schultz lab in the analysis of mechanisms and molecular targets of small molecules acting on complex biological systems. Specifically, the recent publication on pluripotin – a compound able to maintain mouse ESC self-renewal – illustrates the feasibility of the general approach. While it is likely that hESC studies will be more difficult, the substantial libraries and screening resources available provide encouragement that success is achievable. The proposed screen is quite simple – measuring maintenance of alkaline phosphatase activity in tissue culture without feeder cells – and the “gain of function” nature of the screen improves the chances of finding biologically interesting molecules (as opposed to false positive hits). The rapid move to chemical optimization of primary hits is a substantial strength as primary screening hits are often not sufficiently potent or selective to be used in follow-up biological studies.
WEAKNESSES: One weakness pointed out by a reviewer is that the investigators don’t discuss the generality of actions of the compounds that they identify (i.e. comparison of action on different human stem cell lines). The rationale for the choice of the two lines used is not explained. Also, no direct data are shown on studies of hESCs in the PI's lab raising some concerns about feasibility. The reviewer suggested that the range of hESC lines to be studied with the identified compounds should be expanded to ensure that the activity is general.
Another minor weakness: the primary screen proposed involves two components (AP enzyme activity measurements and colony morphology analysis). It is not clear how these two readouts will be integrated – i.e. will the screen be done first for AP then morphology evaluated? If morphology is to be used as a primary readout – either in parallel or simultaneously with AP, how will this be accomplished?
DISCUSSION: This proposal aims to identify small molecules with roles in self-renewal of hESCs. Small molecules will be queried in chemically-defined media with an emphasis on analysing and screening for signalling pathways that can affect differentiation to neural lineage. While this approach may not be terribly innovative, as mentioned by the PI, there are critical strenghts. First, self-renewal is of key importance in the field; second, the facilities are state of the art; third, there is considerable experience with mouse ESCs although it was noted that hESCs will be harder than mouse ESCs. Both reviewers had high enthusiasm for this strong proposal, but a number of minor weakness were noted. First, the PI doesn't show any experience with hESCs; second, there was no discussion of the choice of cell lines or the generality of findings from lines; third the proposal doesn't highlight the selection of screening parameters; finally, no details were provided of integration of alkaline phosphatase and colony morphology tests. Since mouse and human cell responses are quite different, one discussant noted the need to retest compounds previously tested on mouse cells using hESCs to see what works for humans. The library was believed to be sufficiently diverse, the approach considered risky but there was a chance of success. Although this project is fundable by NIH, it was also noted that this approach had been rejected three times by the NIH.