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Genome-wide analysis of hormonal systems important for human embryonic stem cell derivation

Funding Type: 
Comprehensive Grant
Grant Number: 
Funds requested: 
$2 470 247
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Mammalian development requires the differentiation of a single fertilized egg into more than 200 unique cell types in the body. Human embryonic stem cells (hESCs) are derived from preimplantation embryos and these cells can be cultured in the laboratory in an undifferentiated state while maintaining the potential to become almost any cell type the body may require. Although limited success has been achieved in generating hESCs, no therapeutic success in regenerative medicine has been reported. Earlier studies have indicated that embryos cultured as a group show superior growth and less cell death as compared to those cultured alone. These findings suggest that development of embryos is facilitated by hormonal factors, and that culturing multiple embryos in a small droplet maintained these secreted factors in sufficient concentration for optimal development. Indeed, several factors have been discovered to be secreted by embryos and exert their actions on embryonic cells. Our results further demonstrated that ovarian cells surrounding the eggs secrete hormones that are essential for the optimal development of eggs into early embryos. Although advances have been made to fertilize and culture human eggs for development into preimplantation embryos, only a small fraction of cultured eggs reach the preimplantation stage and the success ratio in hESC line derivation is poor. This low success is due primarily to the routine use of simple culture media lacking essential hormonal factors in which to culture human early embryos and to derive hESC lines from the inner cell mass of preimplantation embryos. We have identified hormonal factors secreted by ovarian cells surrounding the egg as well as those secreted by the embryonic cells themselves, and demonstrated their importance in the development of eggs into preimplantation embryos in mice. Using this animal model, we propose to formulate hormone-enriched culture media for in vitro fertilization and embryo cultures. Because these hormonal factors are likely to be essential for human embryo development, we will determine the presence of receptors for these hormonal factors in surplus blastocysts and zygotes donated by in vitro fertilization patients. After confirming the existence of key hormonal receptors in human embryos, we will thaw the surplus zygotes and 4- to 8-cell embryos and culture them in hormone-enriched media for development into preimplantation embryos. Once reaching the preimplantation stage, the inner cell mass of these embryos will be used to derive hESCs following culturing with selective hormonal factors. Our goal is to substantially increase the efficiency of the development of human eggs into preimplantation embryos and enhance success in hESC derivation. An added benefit of the present project would be improvement in the in vitro fertilization procedure for embryo transfer that is presently used for an estimated one million treatment cycles per year worldwide.
Statement of Benefit to California: 
The present application proposes to establish optimal conditions for efficient derivation of human embryonic stem cells. After identifying key ovarian paracrine and embryonic autocrine factors important for the derivation of human embryonic stem cells, it will be possible to formulate a hormone-enriched culture media for the routine generation of human embryonic stem cell lines. Future adaptation of this procedure for somatic cell nuclear transfer will allow the generation of patient-compatible embryonic stem cell lines. Because embryonic stem cells can be induced to differentiate into diverse cell types of the body, establishment of optimal culture conditions for the derivation of these cells would benefit medical research and allow new treatment modalities for diverse degenerative diseases. We will submit patent applications on our findings to protect intellectual property rights according to {REDACTED} guidelines. In addition to application in embryonic stem cell fields, the present findings also are expected to substantially improve the success rate of in vitro fertilization procedures that are presently used for an estimated one million treatment cycles per year worldwide. We anticipate that successful completion of the present proposal could benefit patients with degenerative diseases and infertility in California and throughout the world. The P.I. currently has a patent application pending on the use of brain-derived neurotrophic factor in the promotion of early embryo development which is provisionally licensed by a pharmaceutical company. We expect future findings will lead to similar patent applications and licensing agreements that would benefit the State of California.
Review Summary: 
SYNOPSIS OF PROPOSAL: This is a proposal to examine whether autocrine and paracrine factors can be used to facilitate the development of cultured two-cell-stage mouse embryos into blastocysts, and then to use this information to develp strategies to identify similar approaches to facilitate development of human embryonic stem cell lines. The rationale is that no systematic attempt has yet been made to include hormones during cultures of human early embryos for hESC derivation. Hormonal candidates for this study have been identified by the PI using receptor DNA microarray and identification of corresponding receptor ligands, peri-ovulatory ovaries and early embryos, and include a large series of growth factors. Inseminated oocyte cultures with and without autocrine and paracrine substances will be used; outcomes include contrast microscopy, glutathione concentration, and number of cells and rate of apoptosis in blastocycts. IMPACT AND SIGNIFICANCE: The major impact that this work might have is to identify autocrine and paracrine substances that can be used in culture to facilitate the rate of stem cell replication, thereby enhancing the availability of hESCs for research and for potential therapeutic uses. In a pragmatic sense, successful completion of the work described in this proposal could provide a major boon the the field of stem cell research. Beyond this, however, no scientifically innovative discovery in the basic sense of the word will result, as there is only a modest amount of mechanistic design in the proposed experiments. The proposal is partially but not completely compliant with the RFA as the first aim does not deal with human stem cells, but the second aim does. The first aim, to optimize development of mouse oocytes into blastocyts by growth factor manipulation, may be useful, particularly for ART research, but does not seem programmatically of high priority. The second aim, derivation of hESCs from human blastocysts and zygotes is directly relevant. One limitation of the impact even of this aim is that hESCs can be generated now and the key issue may be generating lines with greater lineage predictability or adaptability, which is not addressed in the application. QUALITY OF THE RESEARCH PLAN: The overall research plan is straightforward and will be carried out by a very highly productive and well-funded group of investigators. The strategies used in this search for new substances to enhance stem cell growth will take the proven approach of receptor mRNA measurements to identify relevant ligands as well as the examination of concentration ranges of factors and use of receptor blockade to demonstrate specificity of autocrine and paracrine effects. The logical march from mouse work to hESC work is timely and well thought out. The investigators have demonstrated considerable skill in studying and exploiting changes in growth factor expression in oocyte development, and they plan to extend those studies in several ways. They will examine microarray datasets to identify ovarian autocrine and paracrine factors and test their ability to promote mouse oocyte development into blastocysts. A concern is that there would likely be dozens or hundreds of factors that could be modified, at varying levels and in varying combinations, and prioritization is unclear. Better might be a high throughput screening approach. The applicant plans to improve current techniques for hESC development by pairing with the Stanford IVF program, and the applicant is very skilled in this work. The research team is aware of the inefficiency of hESC derivation from ICM given the lack of paracrine factors produced by the trophectoderm. However, there is a good deal of guess work and limited ability for combinatorial tests given the limited number of samples. The application is also essentially empirical rather than mechanistic. STRENGTHS: The strengths of this proposal include the quality of the invesigative team. Dr. Hsueh, the PI, has published over 350 articles in superb scientific journals and has a past and current history of very strong peer-reviewed funding in this area. Dr. Behr is the Director of the IVF Program at Stanford and ensures the availability of the clinical material needed for the work described in the proposal. Dr. Baker is well funded and experienced in derivation, maintenance and characterization of hESC lines. The work itself could be of major importance to scientists searching for improved methods of stem cell line derivaion, expansion, and maintenance. The emphasis on autocrine and paracrine growth factors is a strength, as is the improvement of hESC derivation in general. The experience of the research team in hormonal systems/mechanisms and the clinical ARTs are attractive, as is the use of the mouse model in a screening capacity. WEAKNESSES: This proposal is focused primarily on oocyte in vitro maturation and embryo culture, not hESC research, and therefore does not adequately address the objectives of the CIRM Comprehensive Research Grant program announcement. An additional weakness is the exclusively pragmatic, workman-like approach to what is basically a laboratory problem, i.e. the development and expansion of stem cell lines. Virtually no mechanistic studies are incorporated in the work that might provide new scientific insights into the signal transduction pathways that might or might not be involved in action of the various autocrine and paracrine substances that will be used. The proposal also suffers from the lack of true high throughput or combinatorial screening approach to solving the problem. There is inadequate definition of the mouse system used for screening. Given that developmental capacity is mouse strain and culture media dependent, how applicable are the unpublished results of the PI to outbred or other inbreed strains? What are the blastocyt development and ESC derivation rates for in vitro versus in vivo produced embryos in the strain utilized? In vitro oocyte maturation imposes a substantial additional set of confounding factors on embryo quality and developmental potential. Critical controls are lacking: culture environments should be subjected to the measurement of hormonal factors as a control step. DISCUSSION: Great collaboration is a strength of this proposal. However, the weaknesses are signficant. Although new scientific information will be derived, the proposed research does not provide a mechanistic approach to understand resultant observations. The proposal would have been stronger if, for example, the applicant had proposed to study the signaling pathway of at least one of the paracrine factors that may be discovered. One reviewer felt that the proposal outlined research that would consume a lot of time and effort with little in the way of return. Another reviewer felt that given the objectives, some of the proposed experiments should be designed with high throughput approaches. There was considerable concern about the relevance of the proposal. The embryo research seemed irrelevant to the goal of deriving hESCs, and didn't seem to fit the program announcement adequately.

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