Year 3 + NCE
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. Islet transplantation with persistent immune suppression has shown promise in curing type 1 diabetes (TID). However, one major obstacle towards large scale implementation of this approach is the shortage of engraftable islets. 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.
Published studies. A) “sRNA-seq analysis of human embryonic stem cells and definitive endoderm reveal differentially expressed microRNAs and novel isomiRs with distinct targets”
Submitted studies. Recently, we submitted a manuscript entitled “MicroRNA Dynamics During Human Embryonic Stem Cell Differentiation to Pancreatic Endoderm” to Stem Cells and Development. This work is a comprehensive microRNA study that details our deep sequencing experiments. In the study, we describe changes in microRNA expression that occur during the first 10 days of pancreatic progenitor formation, confirm expression of selected miRNAs at various time points, correlate expression with known mRNA targets, and integrate protein expression with data from our quantitative LC/MS experiments. This work provides a rare, simultaneous and comprehensive look at hESC for the microRNA, mRNA, and protein level.
Work in progress.
A) Transcriptional Regulation by Ago2 and miRNAs.
microRNAs and hESC differentiation. MicroRNAs (miRs) regulate post-transcriptional gene networks and function in a manner analogous to transcription factors. Mature miRNAs are partially complementary to one or more messenger RNAs (mRNA), and function primarily to down regulate gene expression. The importance of miRNA activity in hESC during mammalian development has been established by deleting genes necessary for global miRNA biogenesis. Our recent work has found that miRNA expression in hESC is dynamic, indicating that miRNAs drive differentiation. We have been working under the hypothesis that transcription of miRNA loci are driven by mature miRNA and Ago2, and by manipulating Ago2, we can effect levels of precursor and mature miRNA expression and ultimately regulate hESC differentiation.
B) PKCβ isozyme expression is regulated by miR-653 during Definitive Endoderm Formation.
Recently, we have employed deep sequencing to generate temporal maps of changes in microRNA expression during the first ten days of hESC differentiation towards endocrine cell development. The results from the miRNA/mRNA expression studies identified the beta isozyme of PKC as a potential target of miR-653. We found that miR-653 is specifically associated with DE fate and contains a strong consensus binding site in the 3’ UTR of PKCβ. In both Cyt49 and H9 hESC lines, the log2 value for mRNA expression of PKCβ dramatically dropped during DE formation. This was unique to the beta PKC isoform. Western blot analysis of endogenous PKCβII expression confirmed loss at the protein level as cells left pluripotency for DE.