Tools and Technologies I
According to the American Association of Diabetes, 20.8 million children and adults in the United States, or 7% of the population, suffer from diabetes. In addition to those already having diabetes, 54 million Americans have pre-diabetes. Diabetes results from the disruption in either production or utilization of insulin, a hormone that is needed to convert food into energy needed for daily life. Insulin is produced in the pancreas by specific endocrine cells, known as β-cells. Overt diabetes occurs when the β-cells are unable to secrete a sufficient amount of insulin to maintain normal levels of sugars in the blood. Reduced insulin levels may be due to pure β-cell loss (as in type I diabetes), pure β-cell dysfunction (as with some monogenic disorders affecting the β-cells), or to a mixed picture with both β-cell secretory dysfunction and β-cell loss, as in classic type II diabetes. Although the data are quite limited, there is evidence that β-cell regeneration can occur early in both type I and type II diabetes, but this early regenerative response is lost over time. Understanding the biology of β-cell regeneration has a high potential to provide new approaches to therapeutic intervention in diabetes. In this proposal we aim to test the hypothesis that there is a subset of cells within the non-endocrine pancreatic cell population that serve as stem/progenitor cells, which under certain conditions are capable of endocrine differentiation to supply lost β-cells. Answering this question will have tremendous importance for developing treatment strategies for diabetes. We will address this by developing two sets of tools that will be useful not only for answering this particular question, but can be applied for further research on pancreatic cells. To this end, we propose to develop two specific tools. First tool will include a set of biomarkers that will uniquely identify the stem/progenitor cells within the non-endocrine pancreatic cell population. Significantly, instead of a highly labor intensive analysis process involving a high degree of subjectivity, we will develop a second tool, an automatic software package, that will be capable of precise and detailed analysis of the pancreatic tissue sections to assess the presence of β-cells differentiated from the stem/progenitor precursors and robustness of the differentiation. Conclusion. Identifying the cells within that have the potential to exhibit endocrine differentiation is of enormous significance in defining the nature of those progenitors in the adult pancreas. In the long term, this will lead to a large increase in the supply of β-cells available for transplantation for diabetic patients and should also guide efforts to induce β-cell neogenesis in vivo.
Statement of Benefit to California:
Diabetes is a very significant health problem in California. Some of the ethnic groups known to be high risk for diabetes, such as Hispanic Americans, Native Americans and Asian Americans, comprise a very significant segment of our population. Research aimed at understanding the biology of β-cells and their progenitors in the adult pancreas is of tremendous importance for increasing the supply of β-cells available for transplantation for diabetic patients, as well as for developing novel treatment strategies.
This proposal focuses on the development of technology for the identification of non-endocrine pancreatic epithelial cells (NEPECs) capable of differentiation to endocrine cell types, specifically pancreatic beta-cells. The Principal Investigator (PI) proposes to determine the differentiation potential of duct versus acinar cells using lineage studies. The research is aimed primarily at producing biomarkers for identification of NEPECs capable of beta-cell differentiation. In addition, the PI proposes to optimize existing image analysis software to allow for automated analysis of differentiating progenitor cells into beta cells in pancreatic tissue slices. The reviewers agreed that while the goal of identifying NEPECs capable of differentiation into beta-cells is an important one, the potential impact of the approach described in this proposal is low. The identification of a novel source of stem/progenitor cells for the production of pancreatic endocrine cells, notably insulin-producing beta-cells, may have value for therapy of diabetes mellitus, especially Type 1. There remains considerable confusion and debate in the field regarding the existence and nature of stem/progenitor cells in the mature pancreas that can give rise to endocrine cells. The proposed research is likely to contribute to clarification of the nature of pancreatic cells that can yield new endocrine cells. It is not geared to explore the underlying developmental pathways, but mainly to provide pragmatic tools for the isolation of candidate progenitors of endocrine cells from within the NEPEC population of human pancreas, using a limited number of available monoclonal antibodies. One reviewer noted that the utility of this approach for clinical application does not appear to be high because the essential starting material, donated human pancreatic tissue, is in short supply. Unless the relevant cell types prove to be capable of extensive expansion in culture, they are not likely to be of great significance for cell therapy. Another reviewer echoed this criticism by noting that the levels of beta-cell differentiation reported from NEPECs are quite small, so even if effective, this technology would not provide significant numbers of beta cells for clinical work. Despite these limitations, reviewers recognized that biomarkers for screening NEPEC precursor/stem cells could be clinically relevant and that their targets, identified by this proposal could provide targets for therapies to drive the expansion and differentiation of endogenous endocrine progenitors in patients with diabetes. One reviewer commented that the potential clinical significance of the proposed work is diminished by the recent report from Doug Melton and colleagues that genetic reprogramming with three transcription factors can drive acinar cells of the pancreas to convert to a beta-cell phenotype with rather high efficiency in mice. Overall, reviewers felt that because the antibodies proposed in this study are available, no novel biomarker targets would be identified, and the number of beta cells produced would be small, the overall impact of the proposal would be low. The reviewers agreed that the proposal is feasible while raising some concerns about the experimental design. The use of proposed reporter appears reasonable, and the preliminary data are convincing that the NEPECs can be transduced readily. The evidence for generation of insulin-positive cells with a beta-like phenotype from non-endocrine cells also seems strong. The survey of candidate markers to fractionate potential progenitors of endocrine cells from NEPECs is reasonable but shows limited novelty. One reviewer was concerned by the use of a proposed reporter construct to label cells expressing duct- and acinar-specific genes. This reviewer noted that once these cells differentiate into insulin-positive cells, they will stop expressing the reporter gene. Thus, to unequivocally demonstrate that ductal/acinar cells differentiate into insulin-positive cells using this method, they would have to ensure 100% purity in their starting population of ductal/acinar cells. Alternatively, they would need to demonstrate the presence of the integrated reporter in differentiated insulin-positive cells. Multiple reviewers raised questions about the proposed aim to optimize automated image analysis software for use with pancreatic tissue slices. The goals of this software development are stated generally with little specificity regarding the current state-of-the-art or the deliverables. A reviewer noted that the PI did not convey sufficient information to judge the actual scope or significance of the aim. The reviewers agreed that the research team is qualified to carry out the work described in the proposal. They noted that PI is relatively junior but has a record of publication in good journals and the necessary expertise for the project. Reviewers also noted the strong credentials of the senior consultant, a leading investigator in the beta-cell field, and considered this collaboration a major strength of the proposal. One reviewer noted that the PI is listed at 65% effort, which would be appropriate assuming that the PI will play an active role in the laboratory research. But the statement that a postdoctoral associate "will be the primary person conducting the proposed studies" raised some concern. Furthermore, qualifications of this postdoctoral associate do not appear to be described. Overall, while this proposal addresses an important problem in the field, the reviewers felt that its potential impact was severely limited by its approach.