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RNA-seq analysis of Myt3 suppression and over-expression in mouse pancreatic islets

ABSTRACT: Purpose: Myt3 is a putative pro-survival factor in pancreatic islets and is expressed from E18.5 onwards. The genes that Myt3 regulates and its role in islet-cell development, function and survival are not fully known. The purpose of this study was to determine the changes in gene expression following the suppression or over-expression of Myt3 to identify possible pathways that may be regulated by Myt3. Methods: Pancreatic islets were isolated from both male and female C57/B6 mice by collagenase digestion and were picked by hand. Hand-picked islets were plated onto 804G, a complete extracellular-matrix (ECM) produced by a rat bladder carcinoma cell line, treated tissue culture dishes and transduced with 1x106 pfu virus overnight. The next day media was changed to fresh RPMI and islets were cultured for 7 days. At the end of the culture period islets were dispersed and FACs sorted for GFP+ cells. Sorted cells from at least 5 experiments were pooled and RNA was isolated using Trizol reagent. 800ng of RNA was used in the Illumina TruSeq kit for library preparation. Results: Comparison of gene expression levels between control-transduced islets and either shMyt3- or Myt3-transduced islets resulted in the identification of 51 and 89 significantly altered genes respectively. Conclusions: Our study represents the first comprehensive analysis of genes that are potentially regulated by Myt3 and highlight potential pathways that are likely important for mediating Myt3's effects of islet development, function and survival.

ORGANISM(S): Mus musculus

PROVIDER: GSE70699 | GEO | 2015/07/10

SECONDARY ACCESSION(S): PRJNA289390

REPOSITORIES: GEO

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Project description:Pancreatic islet transplantation as a cure for type 1 diabetes (T1D) cannot be scaled up due to a scarcity of human pancreas donors. In vitro expansion of beta cells from mature human pancreatic islets provides an alternative source of insulin-producing cells. The exact nature of the expanded cells produced by diverse expansion protocols, and their potential for differentiation into functional beta cells, remain elusive. We performed a large-scale meta-analysis of gene expression in human pancreatic islet cells, which were processed using three different previously described protocols for expansion and attempted re-differentiation. All three expansion protocols induced dramatic changes in the expression profiles of pancreatic islets; many of these changes are shared among the three protocols. Attempts at re-differentiation of expanded cells induce a limited number of gene expression changes. Nevertheless, these fail to restore a pancreatic islet-like gene expression pattern. Comparison with a collection of public microarray datasets confirmed that expanded cells are highly comparable to mesenchymal stem cells. Genes induced in expanded cells are also enriched for targets of transcription factors important for pluripotency induction. The present data increases our understanding of the active pathways in expanded and re-differentiated islets. Knowledge of the mesenchymal stem cell potential may help development of drug therapeutics to restore beta cell mass in T1D patients. Experiment Overall Design: In this study, we have tested three different protocols to expand human pancreatic islets in monolayer and after attempted maneuvers to re-differentiate the expanded cells back to islets. We have characterized the resulting cells in detail by performing microarray analyses with fresh pancreatic islets, expanded islet cells and re-differentiated cells. Genes modified by either of three protocols have 70 to 80% overlap with the genes changed by the other two protocols. Although there are promising changes in the right direction, none of the three protocols could achieve a return to a functional islet state. The expanded cells highly resemble Mesenchymal Stem Cells (MSC), and similar gene regulatory networks seem to be active in both cell types. On the other hand, the expanded islet cells are different from MSC in that they seem to retain activity of some islet gene modules. The current results highlight the importance of designing new strategies that take into account the MSC potential of expanded cells.

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RNA-seq analysis of Myt3 suppression and over-expression in mouse pancreatic islets

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