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Abstract

Objectives: Diabetes results from inadequate insulin production from pancreatic ß-cells. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all diabetic patients. Thus, finding a new source with effective maturation of ß-cells is the major goal of many studies. MicroRNAs are a class of small noncoding ribonucleic acid that regulate gene expression through posttranscriptional mechanisms. MicroRNA-7 has high expression level during pancreatic islet development in humans, thereby playing a critical role in pancreatic β-cell function. We study aimed to develop a protocol to differentiate human-induced pluripotent stem cells efficiently into isletlike cell clusters in vitro by using microRNA-7. Materials and Methods: Human-induced pluripotent stem cell colonies were transfected with hsamicroRNA-7 by using siPORT NeoFX transfection agent. Total ribonucleic acid was extracted 24 and 48 hours after transfection. The expression of transcription factors which were important during pancreases development was also performed. On the third day, the potency of the clusters was assessed in response to high glucose levels. Diphenylthiocarbazone was used to identify the existence of the ß-cells. The presence of insulin and Neurogenin-3 proteins was investigated by immunocytochemistry. Results: Morphologic changes were observed on the first day after chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The isletlike cell clusters were positive for insulin and Neurogenin-3 proteins in immunocytochemistry. The clusters were stained with Diphenylthiocarbazone and secreted insulin in a glucose challenge test. Conclusions: MicroRNA-7 transcription factor net-work is important in pancreatic endocrine differe-ntiation. Chemical transfection with microRNA-7 can differentiate human induced pluripotent stem cells into functional isletlike cell clusters in a short time.

MiRNAs in β-Cell Development, Identity, and Disease

“…Conversely, overexpression of miR-186 and miR-375 by chemical transfection of human iPSCs promoted islet-like cell cluster formation associated with the induction of markers found in endocrine progenitors (Ngn3) and mature β-cells (Insulin, Pdx1, Pax4/6, Nkx6-1, Glut2, and Kir6.2) (Shaer et al, 2014a). Interestingly, the same group obtained similar results when miR-7 was overexpressed (Shaer et al, 2016). Virus-mediated overexpression of miR-375 in human skin fibroblast-derived iPSCs is sufficient to trigger their differentiation into insulin-expressing cells and allow glucose-dependent insulin secretion in vitro (Lahmy et al, 2014).…”

iPSC-Derived Pancreatic Progenitors Lacking FOXA2 Reveal Alterations in miRNA Expression Targeting Key Pancreatic Genes

“…Previous studies reported that several miRNAs play crucial roles in regulating the development and function of pancreatic β-cells [ 12 , 13 , 18 ] and glucose homeostasis [ 19 ]. Examples of those miRNAs are miR-26, miR-24, and miR-148 [ 20 ], miR-375 [ 13 ], miR-21 [ 21 ], miR-30d [ 22 , 23 ], let-7 [ 24 ], miR-34a and miR-34c [ 25 ], miR-9 [ 26 ], and miR-7 [ 27 ]. Furthermore, miRNAs have been found to be involved in maintaining β-cell identity [ 28 , 29 ].…”