Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:24 |
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| Enthalten in: |
Cell journal - 24(2022), 12 vom: 01. Dez., Seite 705-714 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Moriyama, Hiroyuki [VerfasserIn] |
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| Links: |
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| Themen: |
Adipose-derived mesenchymal stromal/stem cells |
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| Anmerkungen: |
Date Revised 04.01.2023 published: Electronic Citation Status PubMed-not-MEDLINE |
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| doi: |
10.22074/cellj.2022.557533.1063 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM35039184X |
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| 100 | 1 | |a Moriyama, Hiroyuki |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System |
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| 500 | |a Date Revised 04.01.2023 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status PubMed-not-MEDLINE | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Doxycycline | |
| 650 | 4 | |a Gene Expression Regulation | |
| 650 | 4 | |a Insulin-producing cells | |
| 650 | 4 | |a adipose-derived mesenchymal stromal/stem cells | |
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| 700 | 1 | |a Moriyama, Mariko |e verfasserin |4 aut | |
| 700 | 1 | |a Ozawa, Toshiyuki |e verfasserin |4 aut | |
| 700 | 1 | |a Tsuruta, Daisuke |e verfasserin |4 aut | |
| 700 | 1 | |a Hayakawa, Takao |e verfasserin |4 aut | |
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