Biological relevance of microRNAs in hESC differentiation to endocrine pancreas

Biological relevance of microRNAs in hESC differentiation to endocrine pancreas

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02266
Award Value: 
$1,313,649
Disease Focus: 
Diabetes
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
There remains an urgent and critical need for a cell-based cure of diabetes, one of the most costly diseases in California. 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. Human ES 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. Significant challenges, including the lack of chemical defined conditions for reproducibly differentiating hESCs into endocrine precursors (EPs), lack of strategy to purify these EPs to avoid teratoma risk, and destruction of engrafted islets by allogeneic and autoimmune rejection despite persistent immune suppression, hinder clinic development of this promising hESC based therapy. Ongoing research in our laboratories is directed at developing novel strategies to derive β-cells from hESCs. Of the several genetic factors that contribute to stem cells differentiation, miRs (microRNAs) are emerging as important determinants. We hypothesize that identification and validation of the temporal expression of miRs at discrete, functionally defined and genetically marked stages of hESC differentiation to insulin-producing cells, when combined with a computational/systems biology approach, will create a population of cells of significant therapeutic impact. The proposed studies will translate basic large-scale analysis of miR and mRNA from pancreatic precursors derived from hESC into a fundamental understanding of differentiation. This in turn will ultimately lead to novel treatments for T1D. In this project we will elucidate the importance of miRs in pancreatic cell differentiation through functional testing, genetic marking, deep sequencing, computational analysis, and validation. Within the context of the above-stated general aims the sequencing studies will be initiated for 3 reasons: 1) to establish on site the most powerful approaches currently available for measuring gene identity and expression 2) to ensure that novel and established miRs are evaluated for changes in expression during hESC differentiation 3) to validate targets of miR action. Application of this emerging technology to β-cell genesis will allow the generation of miR and mRNA profiles from uniform cell populations and validation through functional assays. Together, this information will help to better understand, describe, and ultimately optimize hESC differentiation. Basic research from this project has 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.
Statement of Benefit to California: 
Diabetes has devastating consequences on both those afflicted and on State/National healthcare costs, and, given the staggering rise in both occurrence and costs, diabetes alone possesses the potential to completely overwhelm our healthcare system. There remains an urgent and critical need for a cell-based cure. In 2007, diabetes directly affected 1 in 10 Californians (2.7 million), costing the state $24.5B annually. There have been documented, significant increases in the occurrence of both type 1 and type 2 diabetes in youths under 18 years of age (0.16% of youth <18 yr have type 1 diabetes nationally). There are more than 7,000 diabetic children within [REDACTED] alone. The following alarming statistics are provided by the California Department of Public Health, California Diabetes Control Program, CDC and NIH/NIDDK: • In the U.S., diabetes is the most costly chronic disease, costing $132B annually. This is predicted to rise to $192B by 2020. • Nearly 1 in 3 Medicare dollars and 1 in 10 of U.S. healthcare dollars are spent treating diabetes. • Diabetics average $13,243/year in health care costs, 2.4 times more than non-diabetics. • 7% of the US population has diabetes. • Every 24 hours, 4,100 Americans are diagnosed with diabetes, 613 American diabetics die of the disease and another 55 go blind. • Worldwide, every 10 seconds a diabetic dies and two new people develop diabetes. • Worldwide expenditures on insulin alone are estimated to be $15 billion annually and growing. This research would benefit the State of California and its citizens on multiple fronts. First and foremost, positive results will create a new development candidate for cell-based therapy for type 1 diabetes with the potential for avoiding the risk of tumor formation - a consequence that hinders the development of any human ES cell based therapy. Second, the application of new technologies would enhance the prospects for new biological agents that will require scale up efforts not available to academics. The creation of progenitor cells for any chronic disease, diabetes in our case, will enhance the prospects for the increase in personnel at the scientific and technical level for both academic labs and biotech companies. Finally, this work may obviate the need for immune suppression therapy that today carries serious side effects including propensity to infections and cancer, abnormalities in lipid metabolism and hypertension, and even damage to the transplanted cells as it occurs following islet transplantation procedures, the only available therapy nowadays for insulin-dependent diabetes. Avoidance of these complications represents a significant positive step in the reduction of health care expenses directly attributed to diabetes and its complications.
Progress Report: 

Year 1

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.

Year 2

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.

Year 3

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.

© 2013 California Institute for Regenerative Medicine