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.
During the current funding period, progress has been made on both specific aims originally proposed. From this work, one manuscript and one review article have been published and two other research articles are submitted/in review.
Published studies. A) “The SDF-1α/CXCR4 axis is required for proliferation and maturation of human fetal pancreatic endocrine progenitor cells.” was published in PLoSONE. B) “From pluripotency to islets: miRNAs as critical regulators of human cellular differentiation” was published in Advances in Genetics.
Submitted studies. A) “sRNA-seq analysis of human embryonic stem cells and definitive endoderm reveal differentially expressed microRNAs and novel isomiRs with distinct targets” is in revision at Stem Cells. B) “Jak/Stat and MAP kinase signaling regulate human embryonic stem cell pluripotency” will be re-submitted to Cell Stem Cell in early October.
Work in progress. A) Deep sequencing of miRNAs from a purified population of PDX-1+ cells derived from hESC. Towards our goal of understanding the role miRNAs play in driving differentiation of insulin producing cells from pluripotent hESC, we have sequenced miRNAs from a heterogeneous population of hESC that have been directed towards endocrine lineage. B) Deep sequencing of miRNAs at 24 hour intervals during hESC differentiation towards pancreatic precursors. A major undertaking during the first year of funding is to sequence the changes in miRNA expression at selected intervals during the differentiation process. This information is critical for us to develop algorithms to determine how miRNAs drive differentiation and for identification of miRNA/mRNA targets. C) Development of algorithms to analyze change in miR expression in complex systems. During the first phases of the CIRM project, Natural Selection Inc. focused on algorithms to analyze change in microRNA expression over multiple data sets. D) Generation of a population of PDX1+ cells using zinc finger nuclease technology. One critical goal of the proposed studies is to generate a purified population of endocrine precursor cells. Although some technical problems with construction of the vector arose, we believe that we have overcome the major obstacles and will have these cells for microRNA analysis during the next funding period.
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.