Regenerative medicine holds the promise that tissues can be engineered in vitro and then transplanted into patients to treat debilitating diseases. Human Embryonic Stem Cells differentiate into a wide array of adult tissue types and are thought to be the best hope for future regenerative therapies. This grant has three main goals: 1. The creation of new human embryonic stem cells in animal free conditions which will allow for future therapeutic uses. 2. The creation of human embryonic stem cell that contain mutations in their genomes that cause diseases, including cystic fibrosis, muscular dystrophy, Downs Syndrome and many others. These lines can be used to study these diseases and to test potential therapies 3. A close biological assessment of one of the first tissues to arise during differentiation of human embryonic stem cells – the endoderm. Since the endoderm eventually, during many days of development, becomes the pancreas, liver, and gut. It is critically important that we know everything about this very specialized tissue if we are to attempt to engineer these organs in the laboratory. Our overwhelming goal is to provide tools that clinician can use to treat disease whether it is to establish new and improved human embryonic stem cell lines or to find new ways of creating endodermal tissues within the laboratory for future therapeutic uses.
Statement of Benefit to California:
This grant will provide to the state of California new human embryonic stem cell lines that could be used in future therapeutic uses. It will also provide disease specific human embryonic stem cell lines that can be used to study disease and as models to test pharmacological compounds to treat disease. We will also provide a characterization of tissues generated from the new human embryonic stem cells which we hope will someday aid in the formation of liver, lung and pancreas.
SYNOPSIS:In this proposal, the investigator proposes the following three aims. The first aim is the derivation of hESCs in chemically defined conditions from wildtype and PGD (prenatal genetic diagnosis) embryos. The investigator has already established hESC lines in chemically defined conditions that passed all the tests to be bonafide human embryonic stem cell lines. The applicant proposes to build on these experiments by following two parallel approaches. One is to generate four additional hESC lines, using 300 cryopreserved embryos. The second is to generate hESC lines that have been diagnosed by PGD for mutations with respect to cystic fibrosis (CF), Duchenne multiple dystrophy (DMD) and Downs Syndrome. Four lines from each condition will be derived, so at the end of specific aim one, 16 additional lines will be generated, 12 from diseased backgrounds. The second specific aim is the systematic comparison of hESCs derived in chemically defined conditions and standard hESC lines. The first part of Aim 2 will test the population doubling time and karyotype stability in hESC lines derived in chemically defined medium, and harvested at two different timepoints: at 15 to 20 passages and at 45 to 50 passages. The second part of the Aim uses DNA obtained from the same harvested cells to examine methylation changes in prolonged culture in different media. The third part of Aim 2 will examine gene expression in the harvested cells using Affymetrix gene chip assays. The third specific aim concerns the functional genomics of definitive endoderm formation from new hESCs. Here, the objective is to test whether hESCs in defined chemical conditions give rise to definitive endoderm in vitro, and to define the global molecular mechanisms in early fate specification. This specific aim is divided into four parts, one targeting the cellular analysis of differentiation in a comparative platform. Second, targeting global RNA expression in endodermal differentiation. Third targets the analysis of methylation. The fourth part provides a global assessment of which genes are repressed by looking at members of the Phc complex and and H3K27me3 as probed for the state of of their repression. IMPACT AND SIGNIFICANCE: This proposal targets a set of the highest priority experiments currently needed in the HESC field, both for basic research as well as clinical application. Although the work is largely descriptive, it has high significance and impact and will advance our understanding of basic hESC biology. The correlation of epigenetic regulation of the genome with endoderm formation seems particularly promising. In addition, since PGD is currently perhaps the only way to obtain hESC lines harboring mutations for specific diseases, this work should have high priority. QUALITY OF THE RESEARCH PLAN: The investigator brings extensive training in both molecular biology and experimental embryology to the dissection of the molecular events underlying hESC pluripotency and differentiation. The proposal is clearly written with each of the basic elements of the specific aims clearly expressed, including the rationale, experimental approach, and an outcome analysis that includes possible pitfalls and successes. All experiments are controlled appropriately with the appropriate statistical analysis of the high-throughput information generated by this type of approach. STRENGTHS: The applicant is experienced in derivation of hESC lines and will be generating disease-specific lines, which will be shared. The project will also address basic questions in hESC biology, especially with respect to those of endoderm differentiation. There is an excellent infrastructure for this work. Finally, the grant is technically sound and extremely well-written - a pleasure to read. WEAKNESSES: The major weakness is that the proposal is over-ambitious in scope and the relation of the specific aims is unclear. Thus there is no plan for how the disease-specific lines will be used. Also, while the preliminary result establishes that the investigator has the ability to derive hESCs from a variety of sources, none of them have been published and undergone peer review. Finally, much of the grant is descriptive in nature. DISCUSSION: There was discussion about the relative priority of deriving disease-specific cell lines versus "normal" lines. Also the question of why the focus on endoderm? One discussant attributed this to the applicant's training in embryology and pointed out that we know less about this germ layer than the other two. There was general agreement that the proposal was exceptionally well-written and that this was a young, but already accomplished investigator with much future promise. Although there was some concern about the over-ambitious nature of the proposal, the applicant's record of achievement mitigated this concern.