Basic Biology V
There is an urgent need for a cure of diabetes. Islet transplantation has shown promise in curing type 1 diabetes; however, an obstacle toward implementation of this therapy is the shortage of engraftable islets. Human embryonic stem cells (hESCs) can differentiate into all cell types and are potentially an unlimited source of pancreatic β cells. Unfortunately, the lack of defined conditions for reproducibly differentiating hESCs into endocrine precursors (EPs), effective strategies to purify these EPs to avoid teratoma risk, and the destruction of engrafted islets by immune rejection despite immune suppression greatly hinder clinical development of this promising therapy. Our research is directed at identifying factors necessary to derive β-cells from hESCs. Our recent identification of a novel family of secretory kinases has led us to investigate the roles of these enzymes in hESC differentiation. However, their roles in hESC differentiation remain poorly understood. A myriad of secreted proteins are phosphorylated, including growth factors, cell surface proteins, and extracellular matrix proteins that are critical for stem cell renewal and differentiation. This research will allow us to clarify the role of secreted kinases in hESC differentiation and define a secreted kinase phosphoproteome for pancreatic endocrine cells to aid in identifying specific factors that may facilitate the development of tractable methods to generate islet cells for transplantation.
Statement of Benefit to California:
Diabetes has devastating consequences on those afflicted and on State healthcare costs. Given the staggering rise in both occurrence and cost, diabetes possesses the potential to overwhelm our healthcare system. In 2007, diabetes directly affected 1 in 10 Californians (2.7 million), costing the state $24.5B annually. Current stem cell therapies for type 1 diabetes mellitus (T1DM) suffer from deficiencies that render them ineffective for long-term treatment. As an Exploratory Concepts application, this research would benefit the State of California and its citizens on multiple fronts. First, positive results will provide a foundation to facilitate and expedite the development of improved cell-based therapies for T1DM. Second, fostering the successful creation of progenitor cells for chronic diseases such as diabetes will enhance prospects for increasing the numbers of research personnel in both academia and biotech companies. Finally, this work may obviate the need for immune suppression therapy that carries serious side effects including propensity to infections, hypertension, and damage to the transplanted cells, which can occur following islet transplantation procedures. The research outlined in this proposal will provide a better understanding of the stem cell biology in order to create tractable stem cell therapies for treatment of T1DM that minimize these complications and reduce health care expenses directly attributed to diabetes and its complications.
The main objective of this proposal is to elucidate the role of secretory pathway kinases in the development of hESCs into endocrine lineage cells. The applicant will determine the effect of a relevant gene disruption on endocrine lineage differentiation and will identify the potential targets using global approaches that compare control and kinase-deficient hESCs. This research will elucidate the role of secreted kinases in hESC differentiation and define a secreted kinase phosphoproteome for pancreatic endocrine cells to aid in identifying specific factors that may facilitate the development of tractable methods to generate islet cel Novelty and Transformative Potential - The applicant’s stated hypothesis is that secretory pathway kinases will play a direct and critical role in human embryonic stem cell biology. If that were to be tested, the research would be both novel and perhaps transformative. Unfortunately, the applicant proposes to focus on a specific gene which may not play a role in this process and thus the proposed experiments may not yield much information. - While the families of secretory kinases may have a role in pancreatic endocrine differentiation, the absence of any substantial phenotype in the homozygous null mouse of the putative kinase leads to concerns for a critical role. - While little is currently known about these enzymes and their role in differentiation, there is no sufficient to support a transformative role of these enzymes in differentiation of pancreatic endocrine cells or any other lineage. Due to these concerns, the project is viewed as having limited impact. - The project is viewed as significant and if successful, could provide the broader scientific community with the first database for secreted phosphoproteins involved in hESC differentiation as well as determine whether the relevant gene to be studied is critical for the differentiation of hESC into the pancreatic endodermal lineage. Feasibility and Experimental Design - The proposal read like two proposals that were merged together—one with a very ambitious but potentially transformative goal of understanding the role of secretory pathway kinases in human pluripotent cell differentiation and maintenance, and the other a more focused look at a single secretory kinase in the differentiation of beta cells from hPSCs. The proposal that is spelled out in greatest detail, to test a single secretory kinase in endocrine differentiation, does not have the same potential to inform the field as to the role of these kinases in hPSC biology. - The preliminary data on the function of this specific kinase in endocrine differentiation is not compelling. - Concerns were expressed over the role of the identified secretory kinase in pancreatic endocrine differentiation, based on the absence of any substantial phenotype in the homozygous null mouse. Additional concerns with experimental rationale were also noted, including the lack of homogeneity of the hESC cultures and the use of different hESC lines for different experimental protocols. Thus, it is unclear how successful the PI will be in differentiating hESCs to endoderm and to pancreatic endocrine cells. - Reviewers were also concerned that Aim 2 will likely yield a large number of targets of these extracellular kinases, though no strategy was proposed for identifying which ones are important in ES cell biology. Principal Investigator (PI) and Research Team - The PI is a world leader in assessing the function of kinases and phosphatases in a diverse range of biological roles and is highly qualified to answer the question of whether secretory kinases play a role in hPSC biology. - The co-investigator has a less distinguished research record, but appears capable of completing the proposed experiments. - The PI and assembled team have committed considerable effort, increasing the likelihood of the project's success. Responsiveness to the RFA - The proposal is responsive to the RFA. Little is currently known about these enzymes, their role in differentiation and their targets, so it is innovative. However, it there is little to support a transformative role of these kinases in differentiation of pancreatic endocrine cells or any other lineage, which somewhat diminishes the relevance of the work.