Systematic generation and molecular characterization of hES-derived cell strains utilizing combinatorial cloning.
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.
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.