Exosomes from β-Cells Promote Differentiation of Induced Pluripotent Stem Cells into Insulin-Producing Cells Through microRNA-Dependent Mechanisms

Abstract

Objective Exosomes have emerged as potential tools for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells (IPCs). Exosomal microRNAs are receiving increasing attention in this process. Here, we aimed at investigating the role of exosomes derived from a murine pancreatic β-cell line and identifying signature exosomal miRNAs on iPSCs differentiation. Methods Exosomes were isolated from MIN6 cells and identified with TEM, NTA and Western blot. PKH67 tracer and transwell assay were used to confirm exosome delivery into iPSCs. qRT-PCR was applied to detect key pancreatic transcription gene expression and exosome-derived miRNA expression. Insulin secretion was determined using FCM and immunofluorescence. The specific exosomal miRNAs were determined via RNA-interference of Ago2. The therapeutic effect of 21 day-exosome-induced IPCs was validated in T1D mice induced by STZ. Results iPSCs cultured in medium containing exosomes showed sustained higher expression of MAFA, Insulin1, Insulin2, Isl1, Neuroud1, Nkx6.1 and NGN3 compared to control iPSCs. In FCM analysis, approximately 52.7% of the differentiated cells displayed insulin expression at the middle stage. Consistent with the gene expression data, immunofluorescence assays showed that Nkx6.1 and insulin expression in iPSCs were significantly upregulated. Intriguingly, the expression of pancreatic markers and insulin was significantly decreased in iPSCs cultured with siAgo2 exosomes. Transplantation of 21 day-induced IPCs intoT1D mice efficiently enhanced glucose tolerance and partially controlled hyperglycemia. The therapeutic effect was significantly attenuated in T1D mice that received iPSCs cultured with siAgo2 exosomes. Of the seven exosomal microRNAs selected for validation, miR-706, miR-709, miR-466c-5p, and miR-423-5p showed dynamic expression during 21 days in culture. Conclusion These data indicate that differentiation of exosome-induced iPSCs into functional cells is crucially dependent on the specific miRNAs encased within exosomes, whose functional analysis is likely to provide insight into novel regulatory mechanisms governing iPSCs differentiation into IPCs.

Differentially expressed microRNAs during the differentiation of muscle-derived stem cells into insulin-producing cells, a promoting role of microRNA-708-5p/STK4 axis

“…Emerging data suggest that miRNAs play a pivotal role in IPCs differentiation from diverse stem cells [10]. Guo et al indicated that the differentiation of induced pluripotent stem cells into IPCs is dependent on miRNA-related mechanisms [26]. MiRNA-101a and miRNA-107 showed prominent expression during the differentiation of adipose-derived mesenchymal stem cells into IPCs [27].…”

Current status of stem cell therapy for type 1 diabetes: a critique and a prospective consideration

“…The administration of these exosomes into STZ-induced diabetic mice improved glucose tolerance, increased the insulin content, and preserved the architecture of pancreatic islets. Guo and associates cocultured exosomes derived from a mouse insulinoma cell line (MIN6) with human iPSCs for 21 days [ 95 ]. After 7 days of coculture, the proportion of insulin-positive cells by flow cytometry was 22.3% in the exosome-treated cell population, while it was only 11.9% among the controls.…”

Extracellular vesicles in human milk

“…EVs and exosomes in particular, have been found to play a crucial role in different biological processes, including: cell signaling, neuronal function [46], tissue regeneration [47], intercellular communication [48], immune function [49], development and differentiation of stem cells [50], cancer life cycle [51,52] and viral replication [53]. EVs have the ability to transfer their cargo and to be selectively taken up by specific cells, thereby reprogramming the target cell and, possibly, inducing or preventing disease.…”