Generation and functional genomic characterization of human embryonic stem cell-derived progenitor cells.
$2 094 929
The promise of therapeutic cloning from human embryonic stem cells (hESC) is to provide engineered cell lines and tissues for patients whose own tissues have been damaged or lost through disease. Examples of potential therapeutic uses of hES-derived cell lines include the replacement of pancreatic beta cells in Type I diabetics, to provide insulin, and treatment of neurodegenerative disorders with hESC-derived neuronal lines. Despite the promise of such therapeutic cloning, however, it is not currently clear how many different types of cell lines are potentially derivable from hES cells, as there has not been any systematic mapping of what scientists refer to as cell lineages. A lineage is an ordered developmental pathway, a route from one definable cell type to another, ultimately leading to a terminally differentiated cell type, such as an insulin-producing pancreatic beta cell. While there are estimated to be about 200 such specialized cell types in the human body, it is conceivable that there are thousands of intermediate, transient cell types that arise during development, representing steps in such lineages. With the exception of blood cell lineages, however, there is little detailed information about the routes by which these cell types arise from stem cell progenitors and from each other. Moreover, there is not an existing repository of cell lines that are known to be expandable into known cell types. To the extent that such lines are "raw materials" for therapeutic cloning, there is an urgent need to develop such a resource. This project will provide these resources: a map of hESC-derivable cell lineages and a broad physical repository of cell lines for use as raw materials in therapeutic cloning. The technologies to be employed are straightforward in principle, but require a high degree of technical sophistication to be carried out at an appropriate scale. In this project, over one thousand hES-derived cell lines will be cloned and characterized using functional genomic profiling tools, including gene expression profiling using whole-genome microarrays and cutting-edge epigenomic profiling tools for the detection of "silenced" region of chromosomes that are known to be intimately involved in determining cell type. Additionally, the project will address a critical and relatively neglected area, namely, the genomic stability of hESC-derived cell lines. Genomic stability refers to the ability of a cell to retain its integrity, in other words, to retain its cell type identity, and to avoid the accumulations of mutations (changes in the genetic code) and epimutations (the "turning on" or "turning off" of chromosomal regions. Cells with genomic instability are poor candidates for therapeutic use, as they may be prone to losing their desired characteristics and to developing undesirable traits, such as the ability to form tumors. In this project, genomic stability will be measured in hESC-derived cell lines using genomic tools.
Statement of Benefit to California:
Through their funding of the California Institute of Regenerative Medicine, the people of California are investing in the hope that the biotechnology industry will be able to deliver stem cell therapies for currently untreatable diseases. Like any industry, a future stem cell industry will require raw materials in the from of cell lines that can be directed to the formation of specific cell types, such as the pancreatic beta cells that produce insulin, or the the motor neurons that atrophy in amyotrophic lateral sclerosis. The lack of Federal funding for human embryonic stem cell research, however, has prevented many of the basic studies that are required before such basic raw materials can be developed. There is no existing repository of cell lines that are known to be expandable into known cell types, and to the extent that such lines are "raw materials" for therapeutic cloning, there is an urgent need to develop such a resource. This project will provide these resources: a map of how cell lines develop from human embryonic cell lines and a physical repository of cell lines differentiated from human embryonic stem cells for use as raw materials in therapeutic cloning. These cell lines will be developed here in California, and will provide materials for all other CIRM stem cell researchers. We envision that the database of information, and the cell lines generated and characterized in this project, will be leveraged by others developing targeted applications.
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.