Real-time quantitative PCR analysis of insulin-producing cells differentiation from canine adipose-derved mesenchymal stem cells

We established a useful protocol for generating insulin -producing cells (IPCs) from canine adipose mesenchymal stem cells (cAD-MSCs). IPCs were induced from cAD-MSCs with the three-stepwise protocol. For the first stage , cAD-MSCs was induced to definitive endoderm (DE) with the cooperation of Chir99021 and Activin A for 3 days. For the second stage, pancreatic endocrine (PE) progenitor was induced from DE after treatment with taurine, retinoic acid, FGF2 EGF, TGFβ inhibitor, dorsomorphin, nicotinamide, and DAPT for the next 5 days. In the last stage, the combination of taurine, nicotinamide, Glp-1, forskolin, PI3K inhibitor, and TGFβ inhibitor was used to yield efficiently functional IPCs from PE precursors for 5 days. To indentify differences in the expression pattern of genes related to pancreatic beta cell development, we performed real time quantitative polymerase chain reaction (RT-qPCR). qPCR gene expression profiling of differentiation cells at every stage of induction (day 3, day 5, and day 13) compared to undifferentiated cells.