Systematic generation and molecular characterization of hES-derived cell strains utilizing combinatorial cloning.
Tools and Technologies I
The promise of regenerative medicine is the use of human embryonic stem cells (hESC) 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 regenerative medicine, it is not currently clear how many different types of cell lines derive 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 many refer to the estimate of 200 such specialized cell types in the human body, it is probable 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 clinical-grade cell lines for use . 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 derived from single cells that have begun specialization. As a result, it is anticipated that the resulting bank of cells will possess a diversity and purity never yet achieved in the field. 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 diverse cell lines that are known to be expandable into known cell types and manufactured in a way that they could be shared among researchers and used in humans to treat disease. The proposed research 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 to be shared among researchers so that they can compare their results with the same cell types. 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.
This proposal is focused on the generation of new human embryonic progenitor cell lines (hEP) from pluripotent hESC using a combinatorial cloning technology. hEP lines will be expanded, banked, documented and quality control assays performed under GMP conditions. Lines would be characterized by gene expression patterns, and a subset of the hEP lines characterized for both gene expression and genomic stability upon isolation and after extended passage. hEP lines would be exposed to multiple differentiating conditions and the differentiated phenotype(s) characterized by gene expression and some immunophenotyping. The output would be a number of hEP lines and a map of hESC-derivable cell lineages. The new lines and associated data will be made available to researchers. The availability of well-characterized, stable, clinical grade reporter embryonic progenitor cell lines derived from hESC and the associated data set could be of benefit to basic and applied research. Overall however, reviewers questioned the value to the community of the generation and characterization of hundreds more hEP lines in addition to the over one hundred hEP lines already generated using similar methods. They expressed concern that this was essentially a shotgun approach that will generate many more hEP lines from only a single hESC line, and a huge amount of data without focus and clarity of goals. They also considered the achievement of the project goals overly ambitious given the limited personnel effort proposed for the project. The reviewers noted that following strengths of the application: A large amount of preliminary data that indicates that the combinatorial cloning approach and molecular characterization can yield a wide diversity of hEP with different cell fates. The reviewers note that over one hundred hEP lines have been isolated and characterized. They were enthusiastic about the plans to evaluate the stability of the in vitro passaged lines, and felt this work is critical to characterization of new line isolates. All reviewers commented that the PI and the research team are well qualified to conduct the proposed research. However, these strengths were offset by weaknesses. Reviewers viewed the proposal as a shotgun approach lacking focus and direction. They expressed concern that a single hESC line was to be used to generate all the hEP lines, noting the potential for bias from use of a single line contrasted with the desirability of using more than one line to evaluate the reproducibility and general applicability of the combinatorial approach. The reviewers expressed concern about the clonality of the lines and the lack of tests for clonality. They commented that it was unfortunate that the reproducibility of and stability of gene expression profiles and differentiation restrictions of the lines will not be well addressed given the minimal repeat testing proposed (justified on the basis of cost). All reviewers expressed concern that insufficient effort (five persons with no one person devoting more than 15% time) was allocated to conduct a project of this scope and magnitude. Overall, the enthusiasm of the reviewers was reduced by their concern about the value to the community for so many new lines as well as their concern about the feasibility of achieving the goals of the project given the limited personnel effort.