Direct conversion of human fibroblasts into functional Leydig-like cells by SF-1, GATA4 and NGFI-B

Abstract

The reprogramming of fibroblasts to induced pluripotent stem cells raises the possibility that a somatic cell can be reprogrammed to an alternative, differentiated fate without first becoming a stem/progenitor cell. Recent work has shown that fibroblasts can be reprogrammed to other, terminally differentiated cells with a combination of several transcription factors. Here, we report that a combination of four developmental transcription factors; GATA4, SF-1, NGFI-B, and COUP TF2; efficiently reprogrammed human foreskin fibroblasts into functional induced Leydig-like cells (iLCs). The iLCs expressed Leydig-specific markers and secreted testosterone in vitro. We found that GATA4 and SF-1 were particularly critical for Leydig-specific markers expression and that GATA4, SF-1, and NGFI-B were necessary to generate functional iLCs that secreted testosterone. These findings demonstrate that fibroblasts can be directly converted into iLCs with a few, defined factors and may provide insight into potential therapies to treat testosterone deficiency.

Keywords: Human foreskin fibroblasts; induced Leydig-like cells; reprogramming.

Figure 1

Gene expression analysis of the induced Leydig-like cells (iLCs). A: Scheme of the experimental procedures. B-E: Kinetics analysis of the expression of key genes related using quantitative RT-PCR. The copy number of the mRNA of each gene was normalized to that of the housekeeping gene GAPDH. Data represent mean ± standard deviation (SD) of triplicate experiments. **P < 0.01, ***P < 0.001 as compared with the group of HFFs transfected with GFP. F and G: Western blotting for protein expression of CYP11A1 and CYP17A1 in iLCs at 3 and 4 weeks after infection.

Figure 2

Kinetic analysis of the expression of the four transgenes. The copy number of the mRNA of each gene was normalized to that of the housekeeping gene GAPDH. Data represent mean ± standard deviation (SD) of triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the group of HFFs. (A) 1 week after transfection, (B) 2 weeks after transfection, (C) 3 weeks after transfection, and (D) 4 weeks after transfection.

Figure 3

The morphology of iLCs under the microscope and the growth rate. (A) iLCs of 2 weeks under the fluorescence microscope. (B) HFFs transfected with GFP at 2 weeks after transfection. Scale bars in (A and B) represent 200 µm. (C and D) Cell growth assay at day 3 after transfection. Data represent mean ± SD of triplicate experiments. *P < 0.05 as compared with the group of HFFs. iLCs refers to the 4TFs group; HFFs refers to the control group; and GATA4, SF-1, NGFI-B, and COUP TF2, respectively, refer to removing each of the 4 TFs.

Figure 4

Immunofluorescent staining of HSD3B1, CYP11A1, and CYP17A1. The results confirmed the expression of the Leydig steroidogenic markers at 2 weeks after infection. Nuclei were stained with DAPI (blue). Scale bars, 50 µm.

Figure 5

Oil red O staining of iLCs at 2 weeks after infection.

Figure 6

Analysis of testosterone production during culture. The line at 0.1 ng/ml represents the test sensitivity of the Access Testosterone kit. The testosterone concentration of the HFFs was under the line. All quantitative data were obtained from three independent experiments and are presented as mean ± SD.