After the Edmonton protocol demonstrated the potential of islet cell transplants as a viable cell-based therapy for type 1 diabetes, research efforts intensified to develop renewable sources of ß cells. One avenue of research directed towards this goal is the differentiation of human embryonic stem cells (hESCs) towards pancreatic ß-cells. hESCs can be expanded in culture, and have the theoretical potential to differentiate into any cell type. Several recent reports have claimed to derive insulin-producing cells from animal models and hESC cultures. However, they all show low levels of insulin expression, low percentages of insulin positive cells, and a lack of co-expression of known ß cell markers, suggesting further investigation is necessary. We believe that a population of functional islets can be derived through a careful recapitulation of in vivo differentiation. The first stage of development occurs when pluripotent cells to enter the endodermal lineage. With hESCs, in vitro differentiation provides models for elucidation key signaling pathways in pancreatic development. hESCs express a unique pattern of small RNA molecules that regulate gene expression patterns, termed microRNAs (miRNAs). hESCs express a unique pattern of miRNAs that diminish upon differentiation. We hypothesize that miRNAs unique to hESCs are critical to maintain the pluripotency of the cells, and that these genes will be down-regulated during the developmental program, which will then be characterized by a new set of miRNAs. Early efforts to analyze miRNA content and changes in hESCs were hampered by the presence of feeder layers which contributed superfluous mouse RNA to the system. To circumvent this problem, our laboratory developed a feeder layer-free culture system that maintains stem cell pluripotency during cell culture. This gives us a unique platform to study the role of miRNAs as pluripotent hESC differentiate into definitive endoderm. In these studies, we will generate profiles of microRNAs expressed in undifferentiated human stem cells and follow the changes in their expression during endoderm formation. From this information, we will develop new miRNA libraries representative of gastrulation and early definitive endoderm formation. Subsequently, we will study the effect of specific miRNAs whose expression changes dramatically during differentiation on global changes in protein expression at defined intervals of endodermal development. Mapping large-scale temporal changes in protein expression, interactions, and chemical modifications, termed proteomics, in the presence or absence of specific miRNAs will help us to better understand the molecular changes that occur during development and are essential to progress towards a therapeutic use of human embryonic stem cells.
Statement of Benefit to California:
Type 1 diabetes is a devastating disease where the insulin-producing beta (β) cells of the pancreas are destroyed by the immune system. Our team of experts has been studying the mechanisms of β growth and function in the hopes of generating insulin-producing, glucose responsive cells for transplant into patients with type 1 diabetes. Recent work with pluripotent stem cells has provided hope that these cells may be transformed, or forced to differentiate into insulin producing cells. However, this initial approach did not produce the desired results, so we have determined that a much more sophisticated analysis is required. It now appears that we must recapitulate several critical stages of development to achieve our goal. The first critical step in this development process is cell differentiation into one of the three primary germ layers, endoderm, ectoderm, or mesoderm. For β cells, differentiation must proceed first through endoderm. Although an effective protocol for endodermal differentiation was recently developed, the underlying molecular mechanisms of differentiation are poorly understood. The work proposed here will describe the molecular signatures of RNA and proteins that regulate the changes that occur during the genesis of endoderm. The results will provide a molecular road map that will allow scientists to further refine and streamline the differentiation process. This will then in turn generate larger pools of islet precursor cells so that further studies may be conducted to study different aspects of differentiation. This work will benefit the state of California and its citizens by moving us a step closer to providing a long-term treatment to type 1 diabetes, a disease that affects approximately one in six hundred lives.