Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

Abstract

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine.

Keywords: Sertoli cell line; human; human telomerase; phenotypic characteristics; proliferation.

Figure 1. Immortalization of human Sertoli cells

(A) Human Sertoli cells were freshly isolated from human testis tissues derived from OA patients by a two-step enzymatic digestion and differential plating. (B) The diagram showed the structure of lentivirus vector namely Lv-EF1A-hTERT-IRES-EGFP. (C) The isolated human Sertoli cells adhered to the culture dish. (D) EGFP-positive Sertoli cells infected with Lv-EF1A-hTERT-IRES-EGFP were sorted by FACS. Scale bars in A, C, D = 10 μm. (E) The expression of hTERT in the immortalized Sertoli cells at passage 10, 15, 20 and primary human Sertoli cells (the control). ACTB was used as a loading control of the proteins.

Figure 2. Phenotypic feature of the immortalized human Sertoli cells

(A–B) RT-PCR showed the expression of AR, BMP4, BMP6, GATA4, GDNF, SCF, SOX9, WT1, hTERT, 3β-HSD, SMA, and VASA in the immortalized human Sertoli cells (A) and primary human Sertoli cell (B). GAPDH was used as a loading control of total RNA, and RNA sample without RT (RT-) but with PCR of GAPDH primers served as a negative control. (C) Western blot revealed the proteins of FSHR, AR, GDNF, SCF, BMP4, WT1, SOX9, PCNA, 3β-HSD, VASA, and SMA in the immortalized human Sertoli cells (hS1) and primary Sertoli cells (Con). ACTB was used as a loading control of proteins, while replacement of primary antibodies with PBS served as negative controls (NC).

Figure 3. Phenotypic characteristics of the immortalized human Sertoli cells

(A–E) Immunocytochemistry demonstrated the expression of SOX9 (A), WT1 (B), OCLN (C), VIM (D), and SCF (E) in the immortalized human Sertoli cells. Scale bars in A–E = 10 μm.

Figure 4. Phenotypic characterization of the immortalized human Sertoli cells

(A–F) Immunocytochemistry displayed the expression of BMP4 (A), GDNF (B), 3β-HSD (C), SMA (D), and VASA (E) in the immortalized human Sertoli cells. Replacement of primary antibodies with isotype IgGs (F) served as negative controls (NC). Scale bars in A–F = 10 μm.

Figure 5. The quality assessment of total RNA of the immortalized human Sertoli cells and primary human Sertoli cells

(A) Total RNA was quantified by the NanoDrop ND-2000 and the RNA integrity was assessed using Agilent Bioanalyzer 2100. (B-E) Electropherogram by Agilent bioanalyzer indicated the concentrations and nucleotides (nt) of RNA isolated from the immortalized human Sertoli cells (C) and primary human Sertoli cells (E). B and D were RNA ladders.

Figure 6. Expression levels of numerous genes between the immortalized human Sertoli cells and primary human Sertoli cells

Real-time PCR demonstrated that the expression of GATA4, GDNF, SOX9, GATA1, WT1, LIF, BMP4, BMP6, and FGF2 in the immortalized human Sertoli cells and primary human Sertoli cells. * indicated statistically significant differences (p < 0.05) between the immortalized human Sertoli cells and primary human Sertoli cells.

Figure 7. Proliferation capacity of the immortalized human Sertoli cells in vitro

(A) Immunocytochemistry revealed the expression of Ki-67 in human Sertoli cell line. Scale bar in A = 20 μm. (B) CCK-8 assays were utilized to compare the effect of various concentrations of FBS ranging from 0.5% to 15% on the growth of human Sertoli cell line. (C–E) CCK-8 assays were employed to compare the proliferation of human Sertoli cell line and primary human Sertoli cells when they were cultured with 2% FBS (C), 5% FBS (D), and 10% FBS (E).

Figure 8. Karyotype analysis of the immortalized human Sertoli cells

(A–F) Cytogenetic assay revealed normal karyotype (A–D) and abnormal karyotype (E–F) in the immortalized human Sertoli cells. The data were calculated from more than 100 cells of human Sertoli cell line.

Figure 9. Y chromosome microdeletion analysis of the immortalized human Sertoli cells

(A–C) Multiplex real-time PCR displayed the expression of all eight specific STS markers from AZFa, AZFb and AZFc regions, containing ZFX/ZFY, SRY, sY254, sY127, sY86, sY134, sY84 and sY255, in the immortalized human Sertoli cells (A). DNA from normal human blood served as a positive control (C) and water substituted for DNA as a negative control (B).

Figure 10. Tumorgenesis of the immortalized human Sertoli cells by nude mouse xenografting assays

(A-F) Digital camera and H&E staining revealed no tumor formation in recipient mice with transplantation of the immortalized human Sertoli cells (A, B) or without cell transplantation (C, D). Tumor formation was observed in the recipient mice with transplantation of PC3 cells (E, F). Scale bars in B, D, F= 20 μm.