基于四环素诱导型Hnf1β和Foxa3表达载体的小鼠胚胎成纤维细胞转分化为肝干细胞的实验体系研究

doi:10.3969/j.issn.1004-616x.2021.03.002

Abstract

Abstract: OBJECTIVE： To induce trans-differentiation of mouse embryonic fibroblasts (MEF) into hepatic stem (iHepSCs)-like cells using a tetracycline-controlled Hnf1β and Foxa3 expression system，and to provide a model for investigations into molecular mechanisms involved in the reprogramming process. METHODS： TetO-Hnf1β-EGFP and TetO-Foxa3-mCherry tetracyclic-control vectors were constructed. 293FT cells were used to determine the optimal dosages of doxycycline (Dox) in the trans- differentiation system of MEF. After vectors were transfected into 293FT cells，expression of Hnf1β and Foxa3 genes under different dosages of Dox were detected by real-time fluorescence quantification PCR (qPCR). After 20 days of induction and then amplification of the treated cells，an iHepSCs-Dox cell line was obtained. Biological characteristics of the cell line were documented using the CCK-8 method，colon formation assay，alkaline phosphatase staining，in vitro induced differentiation and reverse transcription-PCR (RT-PCR)，etc. In addition，the induction effects of tetracyclic-controlled reprogramming system were evaluated. RESULTS： The qPCR results show that expressions of exogenous Hnf1β and Foxa3 genes were turned on at 24-h after treatment with 100 ng/mL of Dox and were turned off at 48-h after Dox was removed. After treatment with Dox，markers for bile duct cells (CK19)，common marker of liver and bile (CK18)，and liver stem/precursor cell markers (Dlk1，Sox9 and EpCAM) were detected. Positive alkaline phosphatase staining and cell clones were observed. Furthermore， iHepSCs-Dox cells could be induced into hepatic cells which were similar to iHepSCs. With cessation of Hnf1β and Foxa3 expressions after Dox was removed from the medium， proliferation rates of iHepSCs- Dox cells and expressions of CK18 and EpCAM decreased significantly. In addition， cell clones formation and inductive differentiation to hepatic cells failed as well. CONCLUSION： The trans-differentiation system of MEF which was controlled by tetracyclic has been successfully established. This model can be used for investigations into molecular mechanisms involved in cellular reprogramming processes. Additionally，our results indicate that continuous expressions of exogenous Hnf1β and Foxa3 were necessary for maintenance of iHepSCsDox cell stemness.

Key words: tetracycline-controlled expression vectors, transdifferentiation, mouse embryonic fibroblast cells, induced hepatic stem cells, hepatocyte nuclear factor 1-β, forkhead box A3

CLC Number:

R329.2+8

YU Xinlu, YU Bing, WANG Chen, ZHANG Hongxia, ZHU Haiying. Induction of hepatic stem-like cells from mouse embryonic fibroblasts using tetracycline-controlled Hnf1β and Foxa3 expression vectors[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(3): 163-171.

References

[1] GRAF T, ENVER T. Forcing cells to change lineages[J]. Nature, 2009, 462(7273): 587-594.
[2] TANI H, SADAHIRO T, IEDA M. Direct cardiac reprogramming: a novel approach for heart regeneration[J]. Int J Mol Sci, 2018, 19(9): E2629.
[3] FUKUDA T, ISHIZAWA Y, DONAI K, et al. The polycistronic expression of four transcriptional factors (CRX, RAX, NEURO-D, OTX2) in fibroblasts via retro-or Lentivirus causes partial reprogramming into photoreceptor cells[J]. Cell Biol Int, 2018, 42(5): 608-614.
[4] NOLBRANT S, GIACOMONI J, HOBAN D B, et al. Direct reprogramming of human fetal- and stem cell-derived glial progenitor cells into midbrain dopaminergic neurons[J]. Stem Cell Reports, 2020, 15(4): 869-882.
[5] GALIVO F, BENEDETTI E, WANG Y, et al. Reprogramming human gallbladder cells into insulin-producing β-like cells[J]. PLoS One, 2017, 12(8): e0181812.
[6] YU B, HE Z Y, YOU P, et al. Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors[J]. Cell Stem Cell, 2013, 13(3): 328-340.
[7] BRAMBRINK T, FOREMAN R, WELSTEAD G G, et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells[J]. Cell Stem Cell, 2008, 2(2): 151-159.
[8] STADTFELD M, MAHERALI N, BREAULT D T, et al. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse[J]. Cell Stem Cell, 2008, 2(3): 230-240.
[9] MENDEL D B, HANSEN L P, GRAVES M K, et al. HNF-1 alpha and HNF-1 beta (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro[J]. Genes Dev, 1991, 5(6): 1042-1056.
[10] LE LAY J, KAESTNER K H. The Fox genes in the liver: from organogenesis to functional integration[J]. Physiol Rev, 2010, 90(1): 1-22.
[11] POLL A V, PIERREUX C E, LOKMANE L, et al. A vHNF1/ TCF2-HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells[J]. Diabetes, 2006, 55(1): 61-69.
[12] YOSHIOKA K, KUNITOMO M, YANAI K, et al. Hepatocyte nuclear factor 1β induced by chemical stress accelerates cell proliferation and increases genomic instability in mouse liver [J]. J Recept Signal Transduct Res, 2011, 31(2): 132-138.
[13] WANGENSTEEN K J, ZHANG S, GREENBAUM L E, et al. A genetic screen reveals Foxa3 and TNFR1 as key regulators of liver repopulation[J]. Genes Dev, 2015, 29(9): 904-909.
[14] MIURA T, SUGAWARA T, FUKUDA A, et al. Generation of primitive neural stem cells from human fibroblasts using a defined set of factors[J]. Biol Open, 2015, 4(11): 1595-1607.

Induction of hepatic stem-like cells from mouse embryonic fibroblasts using tetracycline-controlled Hnf1β and Foxa3 expression vectors

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 2

Recommended Articles

Metrics

Comments

[1]	JIN Cai-xia1;2;LI Wen-lin1;LI Yuan-jie2;XU Fang2;HU Yi-ping1. Utilization of insulin promoter in differentiation of liver progenitor cells [J]. , 2010, 22(3): 171-174.
[2]	JIN Cai-xia1,2， LI Wen-lin1， ZHU JI1， ZHANG Nan1， TIAN Di1， HU Yi-ping1. Utilization of Pancreatic Duodenal Homeobox-1 Promoter in Transdifferentiation of Liver Progenitor Cells [J]. , 2006, 18(5): 337-339.