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.
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 goal of this project is to isolate new human ESC lines that are pluripotent (PSC) and represent an earlier ICM-like stage, more similar to conventional mESC.
Reviewer One Comments
Dr. Sheng Ding proposes to develop a new and potentially better pluripotent human stem cell line with better properties such as ease of culture and manipulation. Dr. Ding has already identified some small molecules that could block the further differentiation and therefore promote self-renewal of stem cells by using mouse embryonic stem cells in the absence of feeders, LIF, BMP, and serum. One of the molecules identified inhibits both RasGAP and ERK. The inhibition of these two proteins blocks the cells’ differentiation. This result suggests that this small molecule (“pluripotin”) may act also at an earlier stage, such as the ICM-stage of pluripotent stem cells, to capture the cells and block any differentiation and provide a novel cell type. This is the strength of this proposal. Pluripotin is just one of seven molecules that regulate cell fate that now should be studied for its use in deriving new and better human pluripotent stem cell lines.
The resources asked for this project include 20% of the PI’s time, plus 5% of co-PI (no salary), 1% of a consultant, and 4 post-docs at 100% of time. Aim 1 is to derive human ICM-stage pluripotent stem cells from IVF blastocysts using their small molecules. The hope is that this procedure will produce an improved pluripotent human stem cell line. Aim 2 is to screen 100,000 small molecules for compounds that will reprogram conventional hESCs to ICM-stage pluripotent stem cells. This was similar to a previous screen that was done with mouse cells. Aim 3 is to characterize the newly derived human ICM-stage pluripotent stem cells. One possibility is that one of the molecules that have already been identified may be effective in the human ICM-stage cells. This data isn’t provided. Unfortunately, it isn’t practical from a cell number point a view, to use IVF embryos for the screen so instead the screen will be for small molecules that can reprogram the conventional hESCs to ICM-stage pluripotent stem cells. The weakness is that this may well be different and not translate to the cells from IVF embryos. It would be more compelling to know whether the compound they describe that was identified in the mouse screen, pluripotin, prevents differentiation in human IVF embryos similar to the mouse ESC effects. As far as the post-docs, one will focus on aim 1, two on aim 2, and the fourth on aim 3. The design of the research plan is excellent with the distribution of effort between the three aims. The aim 2 will take most of the effort and has two post-docs assigned to it. My only concern is the compound library that was produced through a combinatorial scaffold approach. The advantage of this design is the ease for follow up libraries and analogs that are already within the library. The disadvantage of this type of library is limited general diversity of the structures. They say there are over 50 distinct chemical classes, so that is 2000 compounds around each structural class. It has been used for other screening projects and has provided a source of active compounds in the past. Follow-up strategies? Medicinal chemists?
Responsiveness to RFA:
This proposal is right in line with the RFA, they propose to develop new pluripotent human stem cell lines that could offer an improvement over what is currently available. Their strategy is a high throughput screening approach that they have done on a more limited scale to identify seven compounds including one that may already have the activity they are looking for.
Reviewer Two Comments
A number of studies support the concept that conventional hESC represent a relatively late epiblast (post-implantation) stage. For example, murine epiblast-derived stem cells are more similar to conventional hESC than mESC. The idea is to generate hPSC by using small molecules that might block or capture the human cells at an earlier, pre-implantation stage. The PI provides good rationale for doing so. ICM-stage hESC lines 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.
The PI is an expert in design of HT assays for screening diverse small molecule libraries, having trained with Peter Schultz, and over the past few years has applied this toward stem cell biology. He recently identified the molecule pluripotin as an inhibitor of RasGAP and ERK, which facilitates culture and self-renewal of mES cells in the absence of feeders or LIF. The idea is to use pluripotin or other differentiation-blockers to capture hES cells at the ICM stage.
The first Aim is to derive new lines using blocking conditions. Human blastocysts will be plated onto MEFs in culture with pluripotin +/- specific inhibitors of FGFR, MEK, GSK3. This is a perfectly reasonable approach, albeit limited in scope and it is a bit surprising that it was not already attempted.
The second Aim is to set up a screen to find new molecules that might reprogram hESC to PSC. This assay is proposed, since it would be impractical to screen using human embryos. However, the rationale is therefore not quite so compelling, since the screen requires reversal or reprogramming, rather than simply blocking forward differentiation programs, and if this requires hitting more than one pathway it will likely not work. Two criteria will be used 1) expression of SSEA1 (by IHC) and growth in domed colonies; 2) expression of GFP in an Oct4:gfp KI line, in the presence of MAPK inhibition. While this block would normally enhance hESC differentiation, it would promote self-renewal in cells that are more equivalent to conventional mESC. This (albeit an alternative approach) would seem to be a particularly tricky assay, since reprogramming might require time and even cell division, while presumably the MAPK inhibitors and library components are added together. The PI is set up to carry out such a screen with robotics for compound distribution, confocal imaging, etc.
Candidate lead molecules discovered here would be put back into the derivation strategy in Aim 1, yet it is unclear if they should function similarly in derivation as they did in reprogramming.
Experiments in the final Aim will characterize any new cell lines in terms of ICM-stage phenotype, including analysis of markers, response to Gfs, EB formation, teratomas. A comparison of hESCs and hPSCs will be carried out for transcripts, histone modifications, and promoter methylation. Of course, this is dependent on isolation of such lines.
The PI has preliminary data that such a HT screen can be used to identify a molecule to facilitate chemically defined media for hESC (pluripotin) and also rat ESC that may be more primitive compared to rat epiblast-derived stem cells (unpublished). He is funded by 2 R21 grants to screen molecules that direct ES cells to cardiac or pancreatic fate. It should be noted that he is funded already by CIRM to carry out a similar screen on somatic cells. He has recruited Evan Snyder who is Director of the Burnham Stem Cell Program and the IVF embryo bank for embryo and ESC assistance. Dr. Belmonte is also an expert in hESC biology.
Responsiveness to RFA:
The idea of isolating new and “earlier” hESC lines is well justified and highly responsive. There are however several weaknesses that lessen enthusiasm. Experiments for the first aim are highly feasible and can be carried out. However, if this fails, it is unclear if the experiments in Aim 2, to identify molecules that reprogram will be applicable to the Aim1 strategy, and the inhibitors may need to act “instantly” to block differentiation in the presence of MAPK inhibitors. Aim 3 depends entirely on success of Aim 1.
Reviewer Three Comments
This is a very innovative and potentially very exciting application designed to develop new human ES cell lines that represent a more primitive type of human ES cell that is equivalent to the inner cell mass stage of development compared to the epiblast type cells. The applicants propose to use novel small molecules to look for factors that promote pluripotency and maintenance of pluripotency, and then to subsequently use these to derive new human ES cell lines using small molecule differentiation inhibitors.
It is the contention of the applicants that current human embryonic stem cell lines represent a later developmental stage of cells within the embryo compared to mouse ES cell lines which are more representative of the inner cell mass. The major goal of this project is to generate new human ES cell lines that are more representative of the human inner cell mass stage of development compared to the epiblast stage of current ES cell lines. The applicants thus propose to use small molecules as the means of generating tools which will allow for the generation of ICM-like cells rather than epiblast-like hES cells.
The workflow is to derive new human embryonic stem cells using already published small molecules and then, in parallel, to use high throughput imaging to screen molecule libraries against current ES cell lines with the goal of identifying molecules which promote conversion of current ES cells from an epiblast to an ICM-like phenotype. Once individual molecules that convert already existing hES cell lines to ICM-like cells are identified, these molecules will be subjected to structure-function relationship studies to optimize the potency and specificity of these molecules. This would allow for, in future studies, the use of these small molecules to specifically derive hES cell lines with an ICM-like phenotype.
Responsiveness to RFA:
The applicants have a well-defined program to analyze the pluripotency of all new cell lines using conventional molecular biochemical and in vivo assays. This characterization will also include a transcriptional comparison of ICM-like ES cells with conventional ES cells, as well as a global epigenetic analysis of both types of cells.