METTL3 promotes human amniotic epithelial stem cells differentiation into insulin-producing cells by regulation of MaFA expression

Abstract

Objective: Generating mature β-cells from stem cells remains a significant challenge in diabetes cell therapy. Human amniotic epithelial stem cells (hAESCs) have made their mark in regenerative medicine, and provide several advantages compared to other stem cells. Methyltransferase-like 3 (METTL3), an essential RNA methyltransferase participating in N6-methyladenosine (m6A) mRNA methylation, plays a critical role in the normal development of β-cells, yet its deletion in β-cells leads to β-cell dysfunction and hyperglycemia.

Methods: In this study, we isolated and characterized hAESCs from human amniotic membranes, differentiated these hAESCs into insulin-producing cells (IPCs), and explored the role of METTL3 in such differentiation. We examined the expression of METTL3 and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2, a decodes m6A methylation "reader") in the generated IPCs. Subsequently, we suppressed METTL3 using an inhibitor (STM2457) and overexpressed METTL3 via plasmid transfection (METTL3-OE). The differentiated STM2457 and METTL3-OE IPCs were compared to normal induction (WT) IPCs regarding the expression of β-cell markers by RT-qPCR and western blotting, immunofluorescence, C-peptide release, and glucose-stimulated insulin secretion (GSIS). Methylated RNA immunoprecipitation (MeRIP)-qPCR was used to examine the molecular mechanism underlying METTL3/m6A signaling axis in MaFA (endocrine pancreatic β-cells marker) expression. We examined the potential therapeutic uses and efficacy of IPCs through streptozotocin (STZ)-induced C57BL/6 DM.

Results: Isolated hAESCs displayed all characteristics of ESCs and could generate IPCs. METTL3 and IGF2BP2 were elevated during differentiation. Overexpressing METTL3 improved the expression of β-cell markers in the final differentiated IPCs, improved C-peptide release, and demonstrated increased insulin secretion upon challenging with high glucose conditions, whereas inhibiting METTL3 attenuated these effects. Moreover, METTL3 modulated the MaFA expression in an m6A-dependent manner.

Conclusions: These findings suggest METTL3 as a promoting factor of IPCs generation, with its up-regulation potentially generating more mature IPCs for hAESCs therapy of diabetes mellitus.

Keywords: Diabetes; Human amniotic epithelial stem cells; Insulin-producing cells; METTL3.