Objective: Human adipose-derived mesenchymal stromal/stem cells (hASC) constitute an attractive source of stem cells for cell-based therapies in regenerative medicine and tissue engineering as they are easy to acquire from lipoaspirate, expansion, and genetic modification ex vivo. The combination of Pdx-1, MafA, and NeuroD1 has been indicated to possess the ability to reprogram various types of cells into insulin-producing cells. The aim of this study is to investigate whether MafA and NeuroD1 would cooperate with Pdx-1 in the differentiation of hASC into insulin-producing cells.
Materials and methods: In this experimental study, we generated polycistronic expression vectors expressing Pdx1 and MafA/NeuroD1 with a reporter from a human EF-1α promoter using 2A peptides in a single tet-off lentiviral vector system. Briefly, hASC were transduced with the lentiviral vectors and allowed to differentiate into insulin-producing cells in vitro and in vivo. Thereafter, RNA expression, dithizone staining, and immunofluorescent analysis were conducted.
Results: Cleaved transcriptional factors from a single tet-off lentiviral vector were functionally equivalent to their native proteins and strictly regulated by doxycycline (Dox). Insulin gene expression in hASC transduced with Pdx1, Pdx1/ MafA, and Pdx1/NeuroD1 in differentiation medium were successfully increased by 1.89 ± 0.39, 4.81 ± 0.98, 5.51 ± 0.63, respectively, compared to venus-transduced, control hASC. These cells could form dithizone-positive cell clusters in vitro and were found to express insulin in vivo.
Conclusion: Using our single tet-off lentiviral vector system, Pdx-1 and MafA/NeuroD1 could be simultaneously expressed in the absence of Dox. Further, this system allowed the differentiation of hASC into insulin-producing cells.
Keywords: Doxycycline; Gene Expression Regulation; Insulin-producing cells; adipose-derived mesenchymal stromal/stem cells; genetic vectors.