Development of high-throughput screening platform for directing hESCs into pancreatic progenitor cells and mature pancreatic beta cells
New Faculty II
Human embryonic stem cells (hESCs) have been recognized as an inexhaustible source for regenerative medicine, a promising platform for developing and testing new drugs as well as an invaluable tool to study human development. However, the mechanisms about how hESCs maintain its multiple lineage development potential (also known as pluripotency) and differentiate into different adult cell types in responding to exogenous stimuli are far from clear. The ability to direct controlled differentiations of hESCsinto specific cell types, e.g. pancreaticÔÄ†ÔÅ¢-cells, will significantly advance the hESCs research and translation into medical and pharmaceutical applications. We propose to develop critical basic tools of establishing three-dimensional hydrogel-based cultural technique. We will then use this technology platform as cell-based high-throughput assays for hESCs directly differentiation into pancreatic progenitors and pancreatic ÔÅ¢-cells. We will use multiple reporter genes at the same time for each lineage/stage differentiation in order to ensure the fully differentiation program in responding to stimuli. We will first generate multiple reporter gene constructscontaining the EGFP/ECFP/ERFP, driven by multiple promoters of established pluripotency and differentiation genes. We will then establish hESCs clones stably expressing these reporter genes and culture these engineered hESCs in 3-D hydrogel environment in order to develop into high throughput screening platform. To develop cell-based high-throughput assays for detecting directed hESCs differentiation, we will test these reporter cell lines in 3-D culture environment by applying established multiple step dfferentiation procedure. We will then perform high throughput screening for hESCs differentiations into pancreatic progenitors, as well mature pancreatic ÔÅ¢-cells. We will exam the expression of endogenous lineage differentiation markers of hESCs by immunofluroescence staining. Finally, we will elucidate the targets of screening hits by combinatorial approaches to elucidate the underlining mechanisms. The host university is a center of excellence for chemical genomics with CIRM-funded stem cell faciliy and a NSF-supported chemical genomics platform, and committed to support stem cell research. Technologies to make the stem cell manipulation easier in culture and to more efficiently direct their maturation into adult cell types are vital to further developments in the field. Stem cell technology provides a paradise of cell research, where rapid advances will make immediate impact on cell replacement therapy and drug discovery/development. This proposal will lead to new technologies and products developments and the outcomes of the project will enhance our capability of manipulating hESCs for medcal and pharmaceutical applications and particularly facilitate the eventually development of treatments for degenerate diseases like diabetes.
Statement of Benefit to California:
The disability and loss of earning power and personal ability resulting from a disease or disorder are devastating and create a financial burden for California in addition to the suffering caused to patients and their families. Although it is only in its early stage, human embryonic stem cells (hESCs) have already been shown to be capable of long-term self-renewal in culture and have remarkable potential to develop into many different cell types in the body. They therefore represent an infinite source of precursor cells to treat degenerative, genetic diseases, or injury, and trauma. Technology and products resulting from this project will enhance our capability of manipulating hESCs for pharmaceutical applications and particularly facilitate the eventual development of treatments for degenerate diseases like diabetes. Improved function in patients afflicted with these diseases will greatly promote the public health and result in tremendous savings to California in healthcare costs, particularly in the areas of long-term care. Federal constraints on stem cell research create a critical need for non-federal funds to achieve these goals. Funding by the California Institute for Regenerative Medicine improve California’s stem cell infrastructure and speed the translation of basic university research into medical products that change lives. The improved ability to maintain viable stems cells as individual cell in culture and to better control their maturation by exogenous stimuli will unlock whole new techniques for working with these valuable but sophisticated resources. The proposed project, when successful and scaled up, eventually will serve to improve the source, storage, price, availability, quality, purity, and diversity of a wide variety of stem cell types as used in many downstream medical, biotechnology and pharmaceutical projects. A ready source of conveniently available stem cells of all types, in turn, will act as a catalyst for new fundamental discoveries in stem cell biology, and will provide enabling reagents for many future products and methods. These discoveries, products, and methods will improve the tax base, create many new jobs, and save billions in healthcare costs in California. The 3-D culture, high throughput screening technologies and small molecular modulator(s) produced in this program will have broad implications for the advancement of medical and life science related to human stem cells, including replacement tissue treatments and drug discovery. Stem cell technology is currently a very strong research area where rapid advances are possible and the research in developing new products and unlocking basic understanding of how human cells function is of very high value. California is viewed as a world leader in stem cell research, biotechnology and pharmaceutical research and development, and the advances in the field made here will contribute to the California’s leading position in these industries immediately.
The applicant proposes to develop a three-dimensional hydrogel–based culture assay system of human embryonic stem cells (hESC) engineered with reporter genes that will be used to carry out a high throughput screen (HTS) of chemical libraries for activity in directing differentiation of hESCs to pancreatic progenitors and beta cells. The overall goal will be the generation of tools for preclinical studies and drug discovery for diabetes. All the reviewers acknowledged the potential impact and significance of the overall goal to generate improved and reproducible culture-based methods for the generation of pancreatic beta cells from hESCs that could one day lead to cell-based therapies for diabetes. They also agreed that the proposed identification of small molecules to drive lineage specification is a valid, fairly straightforward and potentially powerful approach. However, they had a number of serious concerns. The application is rather poorly written with a highly inadequate and vague research plan. The feasibility of the experimental design depends heavily on the ability to generate key reagents, such as hESC reporter lines. The multiple transgenic hESC reporter lines have not been produced or validated, there is no preliminary data, no clear evidence that they will work as planned and no good alternative approaches are presented. The Principal Investigator (PI) does not seem to be aware of the difficulties in hESC culture and in establishing cell type-specific stable reporter lines in hESCs. It was not clear to a reviewer that the PI had a clear understanding about which reporters are eventually going to be used for the HTS assay. There is also no evidence for proper developmental understanding of the system. Reviewers were puzzled by the PI’s reliance on a 3D hydrogel culture system for the differentiation scheme. The logic for why it would improve differentiation and be superior for HTS is lacking, the PI has limited experience with this system and there are no preliminary results to suggest this approach is feasible. The PI’s scientific background in other areas relevant to the proposal (stem cell biology and pancreas development) is weak, further calling the feasibility of this proposal into question. On the positive side, the PI seems to have considerable experience in HTS outside the stem cell arena and has identified a compound that promotes insulin secretion. Given the strong interdependence of the aims, the very early stage, and the technical and expertise issues of the proposed research, the reviewers considered this a high risk proposal. The PI has trained at good institutions, in both academia and industry and has specifically trained and worked with leaders and experts in HTS before joining the faculty of the applicant institution in 2006. His/her recent publications are in respectable but not top-notch journals, and most are not directly related to the proposed research. The PI currently lacks independent extramural grant support. The career development plan describes interest and effort toward a wide range of research projects, none of which are directly relevant to the project proposed in the application, suggesting that the PI’s efforts may not be focused. It lacks milestones and a promotion timetable. The mentoring plan involves mentors with appropriate expertise in germ cells/stem cell biology and combinatorial chemistry, but they lack expertise in areas where the PI is weak, hESC and pancreatic development. The plan is only modestly developed and no supporting letters from mentors were provided. While clearly a very competent scientist, there is no indication that the PI is a future leader in the field. The institutional commitment to this young faculty member is impressive. Although the department where the PI has an appointment is relatively new and lacks a track record for mentoring young faculty toward successful academic research careers, the financial, space and equipment commitment by the institution is more than adequate. The PI’s department appears to be growing but there are no specific future plans presented to support stem cell research. Overall, the strong institutional support and the expertise of the investigator in HTS were insufficient to overcome the reviewers’ concerns over high risk of the research proposal, given the very early stage of the work, and the absence of expertise on the part of the PI and his/her mentors in several areas important for the research.