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. 2017 Nov 30;7:16675. doi: 10.1038/s41598-017-16856-7

Figure 1.

Figure 1

Generation of human induced hepatocyte-like cells (hiHeps) from human fetal fibroblasts. (A) Human fetal fibroblasts (MRC-5 cells) were transduced with 5,000 VP/cell/each vector of nine transcription factors (9TFs)-expressing LV vectors (LV-9TFs) for 12 hr, and cultured until day 28. The hepatic gene (ALB, AAT, and CYP3A4) expression levels were measured by real-time RT-PCR. ND: Not detected. (B) MRC-5 cells were transduced with LV-9TFs or LV-8TFs (9TFs-ATF5, 9TFs-CEBPA, 9TFs-PROX1, 9TFs-FOXA2, 9TFs-FOXA3, 9TFs-HNF1A, 9TFs-HNF4A, 9TFs-HNF6, or 9TFs-GATA4) for 12 hr, and cultured until day 28. In the case of combination transduction of multiple LV vectors, 5,000 VP/cell of each LV-TF were transduced. The hepatic gene (ALB, AAT, and CYP3A4) and fetal-specific hepatic gene (AFP and CYP3A7) expression levels were then measured by real-time RT-PCR. The gene expression levels in LV-9TF-transduced MRC5 cells were taken as 1.0. *p < 0.05; **p < 0.01 (vs LV-9TFs). (C) MRC5 cells were transduced with LV-6TFs or LV-5TFs (6TFs-ATF5, 6TFs-PROX1, 6TFs-FOXA2, 6TFs-FOXA3, 6TFs-HNF1A, or 6TFs-HNF4A) for 12 hr, and cultured until day 28. In the case of combination transduction of multiple LV vectors, 5,000 VP/cell of each LV-TF were transduced. The hepatic gene (ALB, AAT, and CYP3A4) and fetal-specific hepatic gene (AFP and CYP3A7) expression levels were then measured by real-time RT-PCR. The gene expression levels in LV-6TF-transduced MRC5 cells were taken as 1.0. *p < 0.05; **p < 0.01 (vs LV-6TFs). (D) MRC5 cells were transduced with LV-5TFs or LV-HNF4A for 12 hr, and cultured until day 28. In the case of combination transduction of multiple LV vectors, 5,000 VP/cell of each LV-TF were transduced. The hepatic gene (ALB, AAT, and CYP3A4) and fetal-specific hepatic gene (AFP and CYP3A7) expression levels were then measured by real-time RT-PCR. The gene expression levels in LV-5TF-transduced MRC5 cells were taken as 1.0. *p < 0.05; **p < 0.01 (vs LV-5TFs). (E) MRC5 cells were transduced with 5,000, 25,000, or 50,000 VP/cell of each LV-TF for 12 hr, and cultured until day 28. The hepatic gene (ALB, AAT, and CYP3A4) and fetal-specific hepatic gene (AFP and CYP3A7) expression levels were then measured by real-time RT-PCR. The gene expression levels in LV-5TF-transduced MRC5 cells were taken as 1.0. *p < 0.05; **p < 0.01 (vs 500 VP/cell). All data are represented as means ± SD (n = 3). PHH 48 hr: PHH cultured for 48 hr after plating; PHH 0 hr: PHH collected immediately after thawing.