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Long-term culture and significant expansion of human Sertoli cells whilst maintaining stable global phenotype and AKT and SMAD1/5 activation.

Guo Y, Hai Y, Yao C, Chen Z, Hou J, Li Z, He Z - Cell Commun. Signal (2015)

Bottom Line: Morphology, phenotypic characteristics, and the signaling pathways of adult human Sertoli cells from different passages were compared.This study demonstrates that adult human Sertoli cells can be cultured for a long period and expanded with remarkable increase of cell numbers whilst maintaining their primary morphology, phenotype and signaling pathways.This study could provide adequate human Sertoli cells for reproductive and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China. kaoyanyong1@126.com.

ABSTRACT

Background: Sertoli cells play key roles in regulating spermatogenesis and testis development by providing structural and nutritional supports. Recent studies demonstrate that Sertoli cells can be converted into functional neural stem cells. Adult Sertoli cells have previously been considered the terminally differentiated cells with a fixed and unmodifiable population after puberty. However, this concept has been challenged. Since the number of adult human Sertoli cells is limited, it is essential to culture these cells for a long period and expand them to obtain sufficient cells for their basic research and clinic applications. Nevertheless, the studies on human Sertoli cells are restricted, because it is difficult to get access to human testis tissues.

Results: Here we isolated adult human Sertoli cells with a high purity and viability from obstructive azoospermia patients with normal spermatogenesis. Adult human Sertoli cells were cultured with DMEM/F12 and fetal bovine serum for 2 months, and they could be expanded with a 59,049-fold increase of cell numbers. Morphology, phenotypic characteristics, and the signaling pathways of adult human Sertoli cells from different passages were compared. Significantly, adult human Sertoli cells assumed similar morphological features, stable global gene expression profiles and numerous proteins, and activation of AKT and SMAD1/5 during long-period culture.

Conclusions: This study demonstrates that adult human Sertoli cells can be cultured for a long period and expanded with remarkable increase of cell numbers whilst maintaining their primary morphology, phenotype and signaling pathways. This study could provide adequate human Sertoli cells for reproductive and regenerative medicine.

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Gene and protein characterization of the freshly isolated human Sertoli cells. (A) RT-PCR showed the expression of numerous genes, including WT1, GATA4, GATA1, GDNF, BMP4, SCF, FGF2, EGF, FHSR, AR and ABP. ACTB was used as a loading control, and RNA without reverse transcriptase enzyme but with PCR of ACTB primers was used as a negative control (NC). (B-I) Immunofluorescence revealed the expression of WT1 (B), GDNF (C), SCF (D), BMP4 (E), VIM (F), PCNA and GATA4 (G), SMA (H), and CYP11A1 (I) in isolated human Sertoli cells. Scale bars in B, C, D, F, H =50 μm; scale bars in E, G, I =20 μm.
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Fig2: Gene and protein characterization of the freshly isolated human Sertoli cells. (A) RT-PCR showed the expression of numerous genes, including WT1, GATA4, GATA1, GDNF, BMP4, SCF, FGF2, EGF, FHSR, AR and ABP. ACTB was used as a loading control, and RNA without reverse transcriptase enzyme but with PCR of ACTB primers was used as a negative control (NC). (B-I) Immunofluorescence revealed the expression of WT1 (B), GDNF (C), SCF (D), BMP4 (E), VIM (F), PCNA and GATA4 (G), SMA (H), and CYP11A1 (I) in isolated human Sertoli cells. Scale bars in B, C, D, F, H =50 μm; scale bars in E, G, I =20 μm.

Mentions: The freshly isolated human Sertoli cells were identified using a number of markers of Sertoli cells. RT-PCR showed that the transcripts of WT1, GATA4, GATA1, GDNF, BMP4, SCF, FGF2, EGF, FHSR, AR and ABP were expressed in the isolated Sertoli cells (Figure 2A). Immunocytochemistry further revealed that primary human Sertoli cells were positive for WT1 (Figure 2B), GDNF (Figure 2C), SCF (Figure 2D), BMP4 (Figure 2E), VIM (Figure 2F), and PCNA and GATA4 (Figure 2G). No positive staining was seen when primary antibodies were replaced with isotype rabbit or goat IgGs (Additional file 1: Figure S1) or in human male germ cells with these antibodies (Additional file 2: Figure S2), confirming the specific expression of these proteins in freshly isolated human Sertoli cells. The purity of isolated Sertoli cells was more than 95% as showed by our immunostaining results that less than 5% of the cells were positive for antibodies against SMA (Figure 2H) or CYP11A1 (Figure 2I), markers for myoid cells and Leydig cells, respectively. To assess the proliferation ability of human Sertoli cells, PCNA expression was measured and almost of the cells were observed to be positive for both PCNA and GATA4 (Figure 2G), reflecting that human Sertoli cells have a high level of proliferative potential.Figure 2


Long-term culture and significant expansion of human Sertoli cells whilst maintaining stable global phenotype and AKT and SMAD1/5 activation.

Guo Y, Hai Y, Yao C, Chen Z, Hou J, Li Z, He Z - Cell Commun. Signal (2015)

Gene and protein characterization of the freshly isolated human Sertoli cells. (A) RT-PCR showed the expression of numerous genes, including WT1, GATA4, GATA1, GDNF, BMP4, SCF, FGF2, EGF, FHSR, AR and ABP. ACTB was used as a loading control, and RNA without reverse transcriptase enzyme but with PCR of ACTB primers was used as a negative control (NC). (B-I) Immunofluorescence revealed the expression of WT1 (B), GDNF (C), SCF (D), BMP4 (E), VIM (F), PCNA and GATA4 (G), SMA (H), and CYP11A1 (I) in isolated human Sertoli cells. Scale bars in B, C, D, F, H =50 μm; scale bars in E, G, I =20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4380114&req=5

Fig2: Gene and protein characterization of the freshly isolated human Sertoli cells. (A) RT-PCR showed the expression of numerous genes, including WT1, GATA4, GATA1, GDNF, BMP4, SCF, FGF2, EGF, FHSR, AR and ABP. ACTB was used as a loading control, and RNA without reverse transcriptase enzyme but with PCR of ACTB primers was used as a negative control (NC). (B-I) Immunofluorescence revealed the expression of WT1 (B), GDNF (C), SCF (D), BMP4 (E), VIM (F), PCNA and GATA4 (G), SMA (H), and CYP11A1 (I) in isolated human Sertoli cells. Scale bars in B, C, D, F, H =50 μm; scale bars in E, G, I =20 μm.
Mentions: The freshly isolated human Sertoli cells were identified using a number of markers of Sertoli cells. RT-PCR showed that the transcripts of WT1, GATA4, GATA1, GDNF, BMP4, SCF, FGF2, EGF, FHSR, AR and ABP were expressed in the isolated Sertoli cells (Figure 2A). Immunocytochemistry further revealed that primary human Sertoli cells were positive for WT1 (Figure 2B), GDNF (Figure 2C), SCF (Figure 2D), BMP4 (Figure 2E), VIM (Figure 2F), and PCNA and GATA4 (Figure 2G). No positive staining was seen when primary antibodies were replaced with isotype rabbit or goat IgGs (Additional file 1: Figure S1) or in human male germ cells with these antibodies (Additional file 2: Figure S2), confirming the specific expression of these proteins in freshly isolated human Sertoli cells. The purity of isolated Sertoli cells was more than 95% as showed by our immunostaining results that less than 5% of the cells were positive for antibodies against SMA (Figure 2H) or CYP11A1 (Figure 2I), markers for myoid cells and Leydig cells, respectively. To assess the proliferation ability of human Sertoli cells, PCNA expression was measured and almost of the cells were observed to be positive for both PCNA and GATA4 (Figure 2G), reflecting that human Sertoli cells have a high level of proliferative potential.Figure 2

Bottom Line: Morphology, phenotypic characteristics, and the signaling pathways of adult human Sertoli cells from different passages were compared.This study demonstrates that adult human Sertoli cells can be cultured for a long period and expanded with remarkable increase of cell numbers whilst maintaining their primary morphology, phenotype and signaling pathways.This study could provide adequate human Sertoli cells for reproductive and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China. kaoyanyong1@126.com.

ABSTRACT

Background: Sertoli cells play key roles in regulating spermatogenesis and testis development by providing structural and nutritional supports. Recent studies demonstrate that Sertoli cells can be converted into functional neural stem cells. Adult Sertoli cells have previously been considered the terminally differentiated cells with a fixed and unmodifiable population after puberty. However, this concept has been challenged. Since the number of adult human Sertoli cells is limited, it is essential to culture these cells for a long period and expand them to obtain sufficient cells for their basic research and clinic applications. Nevertheless, the studies on human Sertoli cells are restricted, because it is difficult to get access to human testis tissues.

Results: Here we isolated adult human Sertoli cells with a high purity and viability from obstructive azoospermia patients with normal spermatogenesis. Adult human Sertoli cells were cultured with DMEM/F12 and fetal bovine serum for 2 months, and they could be expanded with a 59,049-fold increase of cell numbers. Morphology, phenotypic characteristics, and the signaling pathways of adult human Sertoli cells from different passages were compared. Significantly, adult human Sertoli cells assumed similar morphological features, stable global gene expression profiles and numerous proteins, and activation of AKT and SMAD1/5 during long-period culture.

Conclusions: This study demonstrates that adult human Sertoli cells can be cultured for a long period and expanded with remarkable increase of cell numbers whilst maintaining their primary morphology, phenotype and signaling pathways. This study could provide adequate human Sertoli cells for reproductive and regenerative medicine.

Show MeSH
Related in: MedlinePlus