The long term goal of our research is to understand the biochemical processes that regulate differentiation of human embryonic stem cells (hESCs) into pancreatic progenitor cells, and ultimately, glucose-responsive, insulin producing (beta) β cells. hESCs, which can undergo unlimited self-renewal and differentiate into all cell types in the body, have the potential to become an unlimited source of pancreatic β cells, however, significant challenges have hindered clinic development of this promising hESC based therapy.
Ongoing research in our laboratory is directed at deriving β-cells from hESCs. Of the several genetic factors that contribute to stem cells differentiation, miRNAs (microRNAs) are emerging as important determinants. miRNAs are noncoding, regulatory RNAs expressed dynamically during differentiation of hESC. Mapping developmental expression of miRNAs during transition from pluripotency to pancreatic progenitors will help clarify the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. Specifically, the objectives of this CIRM grant are to elucidate the role miRNAs play in the development of hESC into cells of endocrine lineage and to provide crucial details on the molecular architecture of endocrine precursor populations, lineage specification, and β-cell maturation.
The central hypothesis driving the research is that miRNAs are essential regulators of endocrine cell development. We are working under the postulate that miRNAs are logical targets for in vitro experimentation because of their role in mediating pancreatic cell development. Our aims are as follow:
Aim 1 – Generate miR expression profiles using deep sequencing for defined stages of development from pluripotent to endocrine cells and select candidate miRs for manipulations involving silencing and overexpression.
Aim 2 – Identify miRs targets through deep sequencing of RNA induced silencing complexes (RISC) in defined cell populations and assessment of their roles in differentiation in vitro and after experimental transplantation.
During the current funding period, progress has been made on both specific aims originally proposed.
Published studies. “A) “Imaging human fetal pancreas.” was published in Journal of Visualized Experiments.
Provisional Patents Filed. A) “Novel combinations of transcriptional gene regulators”. B) “Assay to detect onco-miRs circulating in serum or cells”
Work in progress.
A) Paired microRNA expression and development of a reporter system for lineage fate.
We have made a cell line that reports the expression of PDX1, a marker for both pancreatic precursors and for mature beta cells, in order to select and purify the target cells in late differentiation. miR-375 was previously described by our lab to be the most abundant miRNA in definitive endoderm (DE). Other labs have shown that miR-375 is also expressed in pancreatic during development, and specifically in beta cells in mature islets, where it regulates insulin secretion. Conversely, miR-122 is the most highly expressed miRNA in liver, which arises in the region of endoderm that is closest to the pancreatic buds. In our hESC differentiation protocol, only miR-375 is expressed at the DE stage, which is typically about 98% pure. miR-122 is not expressed in DE, but increases in levels to coincide with lower miR-375 expression as a more heterogeneous mixture of cells form as DE differentiates into multiple lineages. We have generated a reporter cell lines that can distinguish pancreatic cells from liver cells in post-DE differentiation, and possibly mature beta cells from other endocrine cells.
B) Deep sequence purified hESC populations from selected time points during hESC differentiation and develop algorithms to analyze change in miRNA expression in complex systems.
In our previous approach, we applied pattern filters to the data to see which miRs matched a particular filter. This was valuable as it helped us determine which miRs had similar expression patterns. However there was still a lot of variance. Therefore, we wre-did the analysis using a new clustering methods developed in conjunction with NSI. The latest approach has given us very valuable insight into the data. We have found that master regulators of miRs exist and/or are being regulated by another regulator but that regulator is biasing them over a large time scale (weeks). The day-to-day fluctuations that most investigators focus upon are not found in this latest group, suggesting that short-term and long-term miR regulation are differentially regulated.
Together, the information generated in this study is helping us to better understand, describe, and ultimately optimize hESC differentiation. We believe that the results from this project have the potential to create a paradigm shift in understanding the cellular ontogeny of the pancreas and help identify which cell types can be used for transplantation therapy in T1D.