Widespread clinical application of cell-based therapies for type 1 diabetes is limited by a serious shortage of islet tissue for engraftment. Therefore, alternative approaches to beta (β) cell generation are required. Presently, much of the promise of regenerative and restorative medicine lies in stem cells. Understanding the molecular events that drive the genesis of functional insulin-producing cells from undifferentiated stem cells is critical to devise strategies that will help restore and/or maintain β cell mass. One avenue of research directed towards this goal is the in vitro expansion of human embryonic stem cells (hESC). Realizing this potential requires a better understanding of the mechanisms that occur as hESCs differentiate into endoderm and subsequently into pancreatic lineage. Embryonic stem cells face several key developmental decisions in the pathway to forming a mature organ. In pancreatic development, the earliest critical steps are gastrulation and subsequent formation of definitive endoderm. Recently, a method to generate definitive endoderm was described (1); however the process is associated with massive cell loss that occurs by the induction of apoptosis, or programmed cell death. The net result of the differentiation protocol is a small population of endodermal cells that limits the experimental and applicable use of these cells. In the proposed studies, we have assembled a multi-disciplined team to explore the mechanisms of apoptosis during differentiation. We will specifically examine the role of the chemokine/chemokine receptor SDF-1/CXCR4 in this process. The appearance of the CXCR4 receptor is a marker for endodermal development. The receptor plays a plays a critical role initiating cell survival or cell death pathways. We hypothesize that inadequate levels of the CXCR4 ligand, SDF-1α, during endodermal development initiates apoptosis and we will test whether the addition of recombinant SDF-1 protects the cells from death. Additionally, we will study the mechanism by which apoptosis is induced during differentiation. We have identified a novel protein kinase A interacting protein, AKIP, that we hypothesize plays a critical role in initiating apoptotic program. Taken together, these studies will help elucidate the molecular mechanisms associated with hESC differentiation and survival.
1. K. A. D'Amour et al., Nat Biotechnol 23, 1534 (Dec, 2005).
Although Type 1 diabetes can be managed with daily injections of insulin, no cure for this disease exists. Islet transplants offer hope for many with type 1 diabetes, however, a lack of sufficient tissue for transplantation has restricted the number of patients who can receive therapy. Furthermore, a recent report found that more than 75% of islet transplant patients required insulin treatment within 2 years following the transplant. This underscores the need for a renewable and sustainable source for insulin-producing beta (β) cells. To address this problem, we have initiated studies on the mechanisms of islet growth and function in the hopes of generating a renewable cell-based system for transplant by directing differentiation of human embryonic stem cells (hESC) or pancreatic progenitor cells into insulin producing β cells. Before hESC can be routinely used as therapeutic agents, we must unravel the complicated molecular processes underlying differentiation. A critcal first step in differentiation is the transition of hESC from a pluripotent state to a cell of endodermal lineage. An effective protocol for endodermal differentiation was recently developed, however during differentiation many of the cells initiated a cell suicide program, termed apoptosis. The work proposed here will elucidate the molecular mechanisms that drive endodermal apoptosis. Identification of the key enzymes and proteins in this process will ultimately result in our ability to control differentiation, which will in turn generate larger pools of islet precursor cells.
The phenomenon of apoptosis is not unique to endodermal formation. Massive apoptosis has been documented in many differentiation protocols. Therefore, this study has broad implications for all aspects of hESC research. By answering a fundamental question about how hESCs survive during the differentiation process, the state and citizens of California will benefit and its citizens because more laboratories will have greater access to a larger population of cells to study various diseases.