Tools and Technologies I
In the small molecule drug discovery field, the “bottom-up” approach, which is based on structural considerations of known targets, has not been as fruitful as was once promised; in 2004, only 36 new drugs were approved by the United States Food and Drug Administration, and 24 in 2005. Until recently, transformed human cell lines were used as the major cellular platform for pharmaceutical drug screening, sometimes referred to as the “top-down” approach. The major concern with the use of transformed cell lines is that compounds identified as “hits” may not have direct relevance to the “normal” biological processes that are being targeted; as a result, false leads consume resources set aside for the subsequent testing on animal models and in clinical trials. Use of normal primary human somatic cells for large-scale screening is not currently feasible due to the limited number of cells that can be obtained from biopsy and subsequent propagation in culture. Human embryonic stem cells (hESCs) offer a potential solution to this bottleneck. First established in 1998 by James Thomson and co-workers, hESCs can be grown in large numbers and maintained in a pluripotent state in culture. They can also be induced in culture to differentiate into cells in a relatively “normal” fashion that is faithful to developmental programs. Thus, three properties make hESCs an ideal platform for drug discovery: (1) hESCs can provide virtually inexhaustible quantities of target cells, which is necessary for screening of large numbers of compounds; (2) hESCs can differentiate into mature cells with phenotypes that mimic their counterparts in normal development; and (3) compared with animal cells, hESCs and their derivatives will provide a much more accurate platform for the “top-down” drug screening approach. In this proposal, we will use the differentiation of hESCs into pancreatic-like cells as a cell culture tool to test whether our culture technology can be used to successfully identify small molecule effectors that affect cellular function of this particular lineage, with the intent of potentially using these molecules for future clinical applications such as cell replacement therapy or drug treatment for diabetics. If we can demonstrate proof-of-principle that hESC-derived lineage-specific cells could be used to identify clinically applicable small molecule effectors, this will be a major step toward improving the pharmaceutical R&D productivity and lead to an increase in the number of successful small molecules that graduate from the clinical trial pipeline.
Statement of Benefit to California:
In this proposal, we will employ human embryonic stem cells (hESCs) and their derivatives in culture as cellular tools to screen for small drug-like molecules that may have effects on pancreatic beta cell progenitors, with the intent of eventually applying these small molecules for clinical therapy on diabetic patients. In Type I and some Type II diabetic patients, the endogenous pancreatic beta cells, which secrete insulin in response to elevated glucose concentrations in the blood, are insufficient. As a result, these patients require injection of exogenous insulin as a current treatment. However, insulin injection cannot match the physiological response conferred by beta cells, and complications inevitably develop over time. If small molecule effectors could be found that mediate beta cell progenitor activation, proliferation and/or maturation, these effectors would be useful for generation of large quantities of high quality transplantable insulin-secreting cells or their progenitors from hESCs in culture for cell replacement therapy. Alternatively, if proven to be safe in the body, these effectors could also be used directly in diabetic patients to encourage the remaining endogenous beta cell progenitors to proliferate or differentiate. Since diabetes is currently an endemic disease in the United States, if successful, our work may have an impact on the health of a large number of Americans, including Californians.
The overall goal of this proposal is to identify small molecules that drive the differentiation of human embryonic stem cells (hESC) into pancreatic, endocrine-like cells. Applicants propose to engineer hESC reporter lines that express a fluorescent protein under the control of the insulin promoter. By adapting a currently used, small scale assay to a high throughput format, the applicants then propose to screen a library of small molecules to identify factors that increase the expression of the fluorescent marker in the reporter cells. In the third aim, the applicants will develop an insulin-expression colony assay in order investigate the mechanisms of action of these small molecules and to assess their capacity to mediate activation, proliferation, or maturation of hESC into functional, insulin-producing cells. The reviewers agreed that if successful, the potential impact of this proposal would be very high. There is currently a dearth of healthy, functional tissues for beta cell replacement therapy, and the ability to derive insulin-producing cells from hESC could address this need directly. In addition, mechanistic insights from the small molecule studies could lead to a better understanding of endocrine cell generation and offer new strategies for producing functional beta cells from hESC, two developments that could positively impact clinical applications such as cell replacement therapy and drug treatment of diabetes. The reviewers felt that this was a well-written proposal with well-integrated aims and thoughtful consideration of potential difficulties and alternate approaches. Several concerns were raised about the feasibility of certain aspects of this effort, however. One reviewer felt the premise of Aim 1 was somewhat naïve. As the applicant points out, rodent studies have shown that insulin expression is not restricted to beta cells in the fetus, but it is unclear whether the proposed second marker to identify pancreatic cells amongst fluorescent-positive cells is not also expressed in these other cell types. Thus, more extensive analysis of the transgenic lines would be necessary to determine their validity as pancreatic cell reporters. Reviewers were also concerned about potential silencing of the transgene, which was not discussed in the proposal, although the applicant described an alternative detection method should problems arise with reporter expression. Some reviewers expressed concern that the proposed scale up of the embryoid body differentiation method may not be feasible, although this method would probably prove useful for testing smaller numbers of compounds. Furthermore, should the applicants encounter difficulties with reporter gene expression, the proposed alternative methodology is not likely to be scalable either. However, the reviewers recognized that the research team is particularly qualified to tackle such challenges, and several were confident that useful information could emerge from this effort, even if the potential is not fully realized. Reviewers appreciated the follow-up assays proposed in Aim 3 since they would move the analysis of the hit compounds beyond a mere yes/no screen. The reviewers felt that the applicants were uniquely qualified to conduct the described experiments. The principal investigator is a young scientist with a proven track record in stem cell and pancreatic progenitor cell biology, and the co-investigator currently directs the high throughput core facility at the applicant institution and has extensive experience with small molecule screens. The reviewers felt that the combined talents of the team and the excellent research environment were ideally suited to this effort. Overall, this is an interesting proposal from a talented, highly suitable team of investigators. The feasibility is uncertain, but the impact would be high if technical challenges can be met.