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.
SYNOPSIS: The proposed research is based on the hypothesis that changes in the microRNA population control the differentiation of human embryonic stem cells (hESC) to restrict development potential, for example, toward endoderm from which insulin-producing beta-cells arise. The goal of this proposed research is to determine whether temporal patterns of microRNA expression can be correlated with changes in protein expression analyzed globally. The experimental aims will compare microRNA profiles of hESC prior to and after differentiation to an early definitive endoderm phenotype; clone and identify new microRNAs present during gastrulation and in early definitive endoderm; and investigate the roles of several microRNAs implicated in endoderm development through gain- and loss-of-function experiments with transfected hESC. INNOVATION AND SIGNIFICANCE: MicroRNAs are likely to play regulatory roles in early human embryonic development. An overview of the number and specificity of microRNA species present and changing during the developmental transition of gastrulation through early definitive endoderm may provide insights applicable to ongoing attempts to generate insulin-secreting cells for human transplant therapy of diabetes. The innovation is the opportunity to link changes of expressed microRNAs with changes in proteins that may be their regulatory targets during stem cell development and also to use in vitro differentiated hESC cultures that recreate, at least in part, human developmental stages (i.e., gastrulation and early definitive endoderm) that are otherwise experimentally unavailable. Another innovative aspect of this proposal is the incorporation of a large number of different fields to generate a huge amount of data. It can be difficult to obtain funding for large-scale data-generating projects that will not immediately identify target genes or produce a clinically useful differentiated cell line. The experiments outlined in this proposal could lay the groundwork for numerous future studies. STRENGTHS: Regulation of development by microRNAs is a line of research, and the PI is positioned to make seminal discoveries concerning endodermal development with potential for advancing the use of stem cells toward a treatment for diabetes. The PI has an active, productive laboratory for proteomic analysis and appears to have assembled an outstanding collaborative team with highly relevant expertise in microRNA biology, hESC and diabetes, although the precise roles of the collaborators and their commitment are not defined. Preliminary results by the PI suggest that the D’Armour protocol for hESC differentiation toward an endodermal phenotype can be applied to these feeder-cell free culture conditions. Thus, cloning of microRNAs for early stages of human embryonic development appears feasible. This is an inventive way to identify new microRNA species specific to gastrulation or enriched in early definitive endoderm, which has not been possible. The experiments in this proposal will generate a huge amount of data regarding the proteins and miRNAs that are expressed in hESC that have undergone differentiation into endoderm. It is reassuring that the author very clearly states that all data will be placed in a public database. The PI has thought about the problems of trying to identify miRNA target genes from a list of possible proteins and has proposed the use of a model system (C. elegans) as a way to further investigate the role miRNAs identified in human cells may play in cell differentiation. WEAKNESSES: The proposal is very ambitious. The amount of data that will be produced by performing these experiments is far beyond what the PI and his collaborators will be able to analyze during the two years of this proposal. The vast majority of the effort will provide a catalog of changes in the microRNAs and proteins present during in vitro differentiation of hESC cultures, and of correlations between changes of microRNA species and proteomic trends during the in vitro differentiation protocol. Although discovery research can yield unanticipated insights, the breadth and depth of the proposed proteomic and genomic analyses are unclear, as are the methods and rigor to derive meaningful correlations from the large quantity of data that might be expected and the potential to detect changes in proteins at low levels expected for many regulatory molecules controlling cell differentiation. The planned analysis of the effects of over-expression or inhibition of individual microRNAs is likely to overlook important regulatory interactions, partly because of incomplete transfection of a cell population that is also not homogeneous. Also, microRNAs appear to work through a power of convergence, so that perturbation of a single microRNA species may have only a small effect on a target mRNA, whereas major effects require the concerted action of multiple microRNAs. No provision is made to verify whether changes in proteins reflect direct or indirect by targeting microRNAs. Finally, the expression or removal of a large number of miRNAs without a clear strategy of how these the phenotypes (if any) will be analyzed may not yield useful information. The PI has suggested the use of the C. elegans model system to further identify the role of miRNAS identified in the human system. It would have been useful to briefly outline the experiments especially since the C. elegans researcher that will be performing these experiments is a Co-PI on the proposal. DISCUSSION: There was no additional discussion following reviewers' comments.