SYNOPSIS: The first goal of this proposal is to exhaustively generate and hierarchically characterize hundreds of independent human embryonic stem cell (hESC)-derived progenitor cells, with the ultimate goal of achieving a complete characterization of in vitro derivable cell lineages. Such a “catalog” of cell lines is proposed to be a necessary first step in defining the universe of therapeutic tissues that might be derived from hESC, and in providing the raw materials for such research. The second goal is to transform progenitor hESC lines with an inducible temperature sensitive SV40 large T antigen that has been used to promote long-term growth of primary cell lines, in order to be able to expand those hESC-derived cell lines. The third goal is to use expression profiling, as well as comparative genomic hybridization and epigenomic analyses to monitor the genetic stability of hESC-derived differentiated cell lines. IMPACT AND SIGNIFICANCE: Little is known about the growth requirements, gene expression, or genetic stability of early embryonic cell types. This proposal is the first systematic attempt to clone in vitro differentiated hESCs combined with their genomic characterization. If a variety of homogeneous clonal progenitor cells with clearly different potentialities existed, they would be perfect candidates to identify broad gene expression profiles with genomic methodology. If such bona fide progenitor cell lines existed these would be valuable reagents for the study of early embryonic development. In spite of the potentially high impact and significance of this, the cell lines which are proposed to be the starting material have not been well characterized and do not yet meet the criteria expected for progenitor cells. These criteria include some proliferative capacity and the ability to differentiate into one of more differentiated cell types. The proposal is innovative in that it deals with putative hESC-derived progenitor cell populations; however, microarray profiling of gene expression is not innovative. QUALITY OF THE RESEARCH PLAN: The main focus of the research plan is to characterize numerous hESC-derived progenitor cells produced by Advanced Cell Technology through in vitro differentiation. This is essentially a brute force approach to isolating new cell lines at different stages of pluripotency. The applicants plan to establish a clonal cell bank, which may define a universe of therapeutic cell types and accompanying transcriptome profiles which define them. They claim that they already have 180 cell lines already profiled and several hundred more in the pathway. As described in the preliminary data, the progenitor cell lines are obtained both by using a non-registered hESC line, MAO3, as well as the registered line WA09. The several hundred progenitor lines are derived from differentiation of the hESC lines in five different growth media, followed by a two-step clonal isolation technique. Although the company collaborators/investigators are clearly able to work with hESCs and analysis methods such as gene expression profiling are in place, it is evident that the progenitor clones are not well characterized. A systematic approach to defining the developmental fate space using profiling is ongoing and they claim will provide bona fide biologic utility for defining differentiated progenitor lines. Specific aim 2, i.e. using SV40 T-antigen to promote long-term growth of primary hESC cell lines, is more problematic (which the applicants realize). The use of SV40 T-antigen is fraught with biologic and regulatory difficulties. It is unclear, for example, how the expression of these transforming oncogenes will influence differentiation pathways and resulting transcription profiles. Such problems may make the data almost uninterpretable. Specific aim 3, looking at genomic stability, is useful in determining the fraction of clones that can be stably propagated and should be done. They propose to sample cell populations during prolonged growth in vitro to determine the stability of the gene expression profile. They also propose to do whole genome analyses of loss of heterozygosity through comparative genomic hybridization and assessment of epigenomic stability and DNA methylation patterns as a sensitive method to measure chromosomal aberrations. As this technology is just coming on line, and the PI does not have any experience with this, it is likely that there will be a steep learning curve. In summary, this is a highly cataloging approach that will probably generate eventually useful data on the developmental fate space of single-cell clones derived from hESC cells. The high throughput isolation of new hESC lines and their high throughput characterization at the transcriptome and genome level is state-of-the-art. However, there is little hypothesis driven research in this proposal. If it works, however, accomplishing this task will benefit many stem cell researchers. STRENGTHS: The importance of isolating and characterizing new hESC cell lines by an experienced investigator in stem cell biology is one of the main strenths of the proposal. The collaboration between the PI and a well-established company with a reasonable plan to divide the work between them is an additional strength. The collaboration has already yielded voluminous preliminary data, along with the demonstration of feasibility for high throughput isolation and analysis. Many potentially useful progenitor cell lines will be produced with a careful assessment of their stability. WEAKNESSES: The first part of the proposal suffers from the fact that it is a brute force cataloguing approach that is not hypothesis-driven. The progenitor cell clones isolated so far are not well-characterized nor has their homogeneity been established. Although they are reportedly clones, the proposed gene expression methodologies used at the population level will not establish their homogeneity. The interpretation of unique transcription signatures may also be difficult as the expression of a particular set of lineage-restricted transcription factors may, in fact, represent multiple cell types along one lineage path. In the second part of the proposal, the applicants will transiently transform progenitor cell lines that are difficult to expand in culture with T-Ag. While this would allow the expansion of cells and a variety of assays to be performed, it is unclear if the results could be extrapolated to untransformed cells. Moreover, the feasibility of genetically modifying hESCs with SV40Tag constructs has not been demonstrated. These experiments are incompletely developed. The PI has no experience with genetic modification of hESCs with GFP-lineage promoter reporter constructs, which are quite challenging techniques. No real rationale or background for using the SV40 large T antigen and their relevance in hESCs or their progenitors is provided. Further, no preliminary data or experience is provided for the proposed array-based epigenomic profiling tools. There is also no clear advantage to using the non-NIH sanctioned cell line, therefore the rationale to seek alternate funding appears to be weak. It is possible that there may be poor cryopreservation efficiency of many of the putative progenitor cell lines that have been isolated which may preclude the ability to carry out the proposed experiments or expand the clones. Finally, the proposal lacks functional assessment of cell lines, which would be most useful. For instance transplantable cells could be assessed in animal transplantation studies. However, this “follow-through” research probably goes beyond the scope of this already ambitious proposal. DISCUSSION: There was a lengthy discussion of what the starting cell lines really represent. They are not well characterized in that the transcriptional profiling was done at a population level, where heterogeneity likely existed in the population. The claim of homogeneity was not well supported, and there is no preliminary data to describe a committed endoderm progenitor line. Morphology was used to see if the differentiated lines looked the same. From there, the transcriptional profile of different lines was tested. Thus, in calling these “progenitors” lines, it seems that they are just slightly differentiated from the two parent lines. With respect to the “mesoderm progenitor” line, maybe it differentiated further downstream than another line, which was judged by having a different transcriptional profile. Is this partial differentiation or a true progenitor? The profiling approach may be strength given that it is unbiased, but you would need to analyze enough lines to know that you were capturing the full range of transcript profiles (i.e., having only one member of a profile group may mean that you haven’t fully-defined the differentiation space.) Reviewers recommended focusing on a subset of the 380 lines and characterizing them better, and concurrently performing bioassays to analyze what they’re getting. The lack of functional biological assays was viewed as a real weakness of the proposal. At the end of the day, you have to prove what the profiles are, and prove what the cells are to know what you have.