Fol. Biol. 2019, 65, 246-255
γ-Aminobutyric Acid (GABA) Induced in Vitro Differentiation of Rat Pancreatic Ductal Stem Cells into Insulin-Secreting Islet-Like Cell Clusters
In vitro produced β-like cells can provide promising cell therapy for curing the epidemic of diabetes. In this context, we aimed to investigate the effects of different concentrations of γ-aminobutyric acid (GABA) on the differentiation of rat pancreatic ductal epithelial-like stem cells (PDESCs) into β-like cells. The PDESC line cells were cultured in the basal media (DMEM/F12 + 10% FBS + 1% penicillinstreptomycin) supplemented with 0 μM, 5 μM, 50 μM, 500 μM, and 5 mM of GABA for 28 days to induce their differentiation. The differentiated cells were detected by cell morphology, dithizone (DTZ) staining, immunofluorescence staining, real-time polymerase chain reaction (qPCR), and glucose-stimulated insulin secretion (GSIS) assay to validate their identity. At the end of 28 days, compared with the control group, enrichment of induced cells was high among the 5 μM, 50 μM, 500 μM, and 5 mM GABA induction groups. The formation of islet-like cell clusters (ICCs) began at 14 days, and the cell clusters showed a growth trend with the culture time. The induced ICCs were positive for DTZ staining, while the control group showed negative results for DTZ staining and the differentiated cells were also positive for β-cell-specific markers (Ins1 and Pdx1). GSIS assay of 50 μM induction group cells at 28 days showed significantly higher levels of C-peptide and insulin secretion than the control, 5 μM, 500 μM, and 5 mM GABA-treated groups (P < 0.01). At the same time, the 50 μM induction group cells also showed significantly higher levels of Ins1, Pdx1 and Nkx6.1 mRNA as compared to the 5 μM, 500 μM and 5 mM GABA groups (P < 0.01). Thus, the addition of GABA to the basal medium effectively induced differentiation of adult rat PDESCs into insulin-secreting β-like cells, and 50 μM was the most effective concentration for the induction.
Keywords
γ-aminobutyric acid, pancreatic ductal stem cells, differentiation, insulin-secreting cells.
Funding
This work was supported by the Natural Science Foundation of Guangdong Province (No. 10152408801000023), the Scientific Research Foundation of the Education Ministry for Returned Chinese Scholars (No. [2010]1561), the Innovation and Develop University Program of Guangdong Province (No. [2014]97 and [2017]54) and the National Innovative Project for University Student (No. 201310566013 and 201310566005).
References
Copyright
This is an open-access article distributed under the terms of the Creative Commons Attribution License.
