Dynamic Expression of microRNA in hiPSCs During Differentiation into Insulin-Producing Cells

JIANG Jinxing¹, JIANG Doukou², ZHANG Wen³, OUYANG Zhibin¹, REN Lili¹, LI Ning¹*

(¹Cytotherapy Laboratory , Shenzhen People′s Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; ²Department of Pathophysiology, Shenzhen People’s Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China; ³Emergency Department, Shenzhen People′s Hospital (the Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology), Shenzhen 518020, China)

Abstract:

This study aimed to analyze the dynamic expression changes of microRNAs during the differentiation of human induced pluripotent stem cells into insulin-secreting cells. First, the research synthesized the insulin gene promoter sequence and downstream of the enhanced EGFP (green fluorescent protein) gene sequence, infected the hiPSCs by the lentiviral vector carrying the above sequence, to obtain INS-promoter-hiPSCs cell line, and they were induced into insulin-secreting cells by the five-step method. Definitive endoderm cells, pancreatic progenitor cells, and insulin-secreting cells were collected. miRNAs associated with pancreas development were predicted and the S-Poly(T) plus assay was used to detect their expression levels. The results showed that INSpromoter-hiPSCs can report insulin expression, which can be differentiated into insulin-secreting cells by induction scheme, and accumulate about 16% of insulin-secreting cells by flow sorting. Further verification showed that the expression levels of miR-495-3p, miR-199-5p and miR-369-5p were extremely low. miR-152-3p, miR-133A3P, miR-181C-5p, miR-410-3p, miR-487a-3p, miR-338-3p, miR-30-5p, miR-655-3p, miR-2655-3p, miR-182-5p, miR210-3P, miR-342-3p, and miR-589-5p were continuously up-regulated during the differentiation. The expression levels of miR-302A-3P, miR-222-3p and miR-802 were significantly down-regulated during differentiation. Therefore, the stable INS-promoter-hiPSCs cell line can be successfully induced to differentiate into insulin-secreting cells and enrich by flow sorting. By verifying and analying the dynamic expression changes of 19 miRNAs that might be involved in regulating the directional differentiation of islet cells, the research reveals new clues for the mechanism of the directional differentiation of islet cells from pluripotent stem cells.