Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System

Moriyama, Hiroyuki; Moriyama, Mariko; Ozawa, Toshiyuki; Tsuruta, Daisuke; Hayakawa, Takao

doi:10.22074/cellj.2022.557533.1063

Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System

Document Type : Original Article

Authors

¹ Pharmaceutical Research and Technology Institute, Kindai university

² Department of Dermatology, Graduate School of Medicine, Osaka Metropolita University, 1-4-3 Asahimachi, Abeno-Ku, Osaka 545-8585, Japan

10.22074/cellj.2022.557533.1063

Abstract

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

20.1001.1.22285806.2022.24.12.1.8

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