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Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction.

Jiang MH, Cai B, Tuo Y, Wang J, Zang ZJ, Tu X, Gao Y, Su Z, Li W, Li G, Zhang M, Jiao J, Wan Z, Deng C, Lahn BT, Xiang AP - Cell Res. (2014)

Bottom Line: We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers.We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions.In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin.

View Article: PubMed Central - PubMed

Affiliation: 1] Cell-gene Therapy Translational Medicine Research Center, The Third Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510630, China [2] Key Laboratory for Stem Cells and Tissue Engineering, Center for Stem Cell Biology and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong 510080, China [3] Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China.

ABSTRACT
The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency.

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Transplanted Nes-GFP+ cells differentiated into LCs that are capable of producing testosterone in the testes of EDS-treated 3-month-old mice. (A) Schematic of the experimental procedure used for cell transplantation. (B) Immunofluorescence staining showed the accumulation of cells positive for PKH26 (red) and P450scc (green) or LHR (green) in the interstitial area of the testis of EDS-treated mice 10 days after implantation with Nes-GFP+ cells. In the control mice, the number of P450scc- and LHR-positive cells decreased after EDS injection. The bottom panels showed higher-magnification images of the dotted boxes in the lower-magnification images of the Nes-GFP+ cell-transplanted mice. Scale bar, 50 μm. Normal/Saline (+), 3-month-old mice received saline injection; EDS(+)/Saline (+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells (+), EDS-treated mice receiving Nes-GFP+ cells 4 days later. (C-D) qRT-PCR analysis showed the expression of 17β-HSD (C) and 3β-HSD (D) in the testes of or EDS- or EDS+Nes-GFP+ cells-treated groups at the indicated experimental time points. Expression levels of each gene were compared to normal mice (before treatment; defined as 1). Data are shown as the mean ± SEM. n = 6. (E) The serum testosterone concentration was measured at the indicated time points in each animal. The level of testosterone was significantly increased in the Nes-GFP+ cell-treated group compared to the control mice (treated with EDS alone) after cell transplantation (*P < 0.05, **P < 0.01).
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fig7: Transplanted Nes-GFP+ cells differentiated into LCs that are capable of producing testosterone in the testes of EDS-treated 3-month-old mice. (A) Schematic of the experimental procedure used for cell transplantation. (B) Immunofluorescence staining showed the accumulation of cells positive for PKH26 (red) and P450scc (green) or LHR (green) in the interstitial area of the testis of EDS-treated mice 10 days after implantation with Nes-GFP+ cells. In the control mice, the number of P450scc- and LHR-positive cells decreased after EDS injection. The bottom panels showed higher-magnification images of the dotted boxes in the lower-magnification images of the Nes-GFP+ cell-transplanted mice. Scale bar, 50 μm. Normal/Saline (+), 3-month-old mice received saline injection; EDS(+)/Saline (+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells (+), EDS-treated mice receiving Nes-GFP+ cells 4 days later. (C-D) qRT-PCR analysis showed the expression of 17β-HSD (C) and 3β-HSD (D) in the testes of or EDS- or EDS+Nes-GFP+ cells-treated groups at the indicated experimental time points. Expression levels of each gene were compared to normal mice (before treatment; defined as 1). Data are shown as the mean ± SEM. n = 6. (E) The serum testosterone concentration was measured at the indicated time points in each animal. The level of testosterone was significantly increased in the Nes-GFP+ cell-treated group compared to the control mice (treated with EDS alone) after cell transplantation (*P < 0.05, **P < 0.01).

Mentions: The ability to regenerate damaged tissues in vivo is an important property of stem cells. We therefore investigated whether the Nes-GFP+ cells could differentiate into the LCs and increase the testosterone levels in an LC-chemically disrupted model. Adult mice were treated with EDS (160 mg/kg), and then Nes-GFP+ cells were injected into the parenchyma at the cranial pole of the testis 4 days later. We observed that Nestin expression was significantly downregulated and GFP signals were also decreased dramatically after differentiation, which was in accordance with previous studies12,23 (Supplementary information, Figure S2A and S2B). Therefore, the Nes-GFP+ cells were labeled with PKH26 (a red fluorescent lipophilic dye), and implanted into recipient animals. At 0, 4, 7, 10, 12, and 14 days after EDS treatment, the serum and the testes were collected for analyses (Figure 7A). The transplanted PKH26-labeled Nes-GFP+ cells were localized exclusively to the interstitium of the testis, and expressed the LC-specific markers P450scc and LHR 10 days after transplantation (Figure 7B). Moreover, after EDS treatment, the expression of 17β-HSD and 3β-HSD, and the concentration of testosterone were decreased significantly on day 4 and recovered gradually (Figure 7C-7E). However, EDS treatment had no obvious effect on LC morphology (Figure 7B). Furthermore, the transplantation of Nes-GFP+ cells significantly increased the mRNA expression levels of LC-specific markers, 3β-HSD and 17β-HSD, and the production of testosterone in EDS-treated mice at different time points (Figure 7C-7E). On day 14 (day 10 after cell transplantation), the testosterone concentration was even higher than that of saline-injected normal mice (Figure 7E). We then isolated PKH26-labeled cells by FACS from the testis 10 days after transplantation and performed RT-PCR analysis, which revealed that these cells expressed LC lineage-specific markers including LHR, 17β-HSD and 3β-HSD (Supplementary information, Figure S3A). Although GFP gene was not expressed at the transcriptional level after differentiation, it should be still located in the genomic DNA as an exogenous gene. Indeed, we found that most PKH26-labeled cells from the mouse testis carried the GFP gene (Supplementary information, Figure S3B).


Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction.

Jiang MH, Cai B, Tuo Y, Wang J, Zang ZJ, Tu X, Gao Y, Su Z, Li W, Li G, Zhang M, Jiao J, Wan Z, Deng C, Lahn BT, Xiang AP - Cell Res. (2014)

Transplanted Nes-GFP+ cells differentiated into LCs that are capable of producing testosterone in the testes of EDS-treated 3-month-old mice. (A) Schematic of the experimental procedure used for cell transplantation. (B) Immunofluorescence staining showed the accumulation of cells positive for PKH26 (red) and P450scc (green) or LHR (green) in the interstitial area of the testis of EDS-treated mice 10 days after implantation with Nes-GFP+ cells. In the control mice, the number of P450scc- and LHR-positive cells decreased after EDS injection. The bottom panels showed higher-magnification images of the dotted boxes in the lower-magnification images of the Nes-GFP+ cell-transplanted mice. Scale bar, 50 μm. Normal/Saline (+), 3-month-old mice received saline injection; EDS(+)/Saline (+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells (+), EDS-treated mice receiving Nes-GFP+ cells 4 days later. (C-D) qRT-PCR analysis showed the expression of 17β-HSD (C) and 3β-HSD (D) in the testes of or EDS- or EDS+Nes-GFP+ cells-treated groups at the indicated experimental time points. Expression levels of each gene were compared to normal mice (before treatment; defined as 1). Data are shown as the mean ± SEM. n = 6. (E) The serum testosterone concentration was measured at the indicated time points in each animal. The level of testosterone was significantly increased in the Nes-GFP+ cell-treated group compared to the control mice (treated with EDS alone) after cell transplantation (*P < 0.05, **P < 0.01).
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fig7: Transplanted Nes-GFP+ cells differentiated into LCs that are capable of producing testosterone in the testes of EDS-treated 3-month-old mice. (A) Schematic of the experimental procedure used for cell transplantation. (B) Immunofluorescence staining showed the accumulation of cells positive for PKH26 (red) and P450scc (green) or LHR (green) in the interstitial area of the testis of EDS-treated mice 10 days after implantation with Nes-GFP+ cells. In the control mice, the number of P450scc- and LHR-positive cells decreased after EDS injection. The bottom panels showed higher-magnification images of the dotted boxes in the lower-magnification images of the Nes-GFP+ cell-transplanted mice. Scale bar, 50 μm. Normal/Saline (+), 3-month-old mice received saline injection; EDS(+)/Saline (+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells (+), EDS-treated mice receiving Nes-GFP+ cells 4 days later. (C-D) qRT-PCR analysis showed the expression of 17β-HSD (C) and 3β-HSD (D) in the testes of or EDS- or EDS+Nes-GFP+ cells-treated groups at the indicated experimental time points. Expression levels of each gene were compared to normal mice (before treatment; defined as 1). Data are shown as the mean ± SEM. n = 6. (E) The serum testosterone concentration was measured at the indicated time points in each animal. The level of testosterone was significantly increased in the Nes-GFP+ cell-treated group compared to the control mice (treated with EDS alone) after cell transplantation (*P < 0.05, **P < 0.01).
Mentions: The ability to regenerate damaged tissues in vivo is an important property of stem cells. We therefore investigated whether the Nes-GFP+ cells could differentiate into the LCs and increase the testosterone levels in an LC-chemically disrupted model. Adult mice were treated with EDS (160 mg/kg), and then Nes-GFP+ cells were injected into the parenchyma at the cranial pole of the testis 4 days later. We observed that Nestin expression was significantly downregulated and GFP signals were also decreased dramatically after differentiation, which was in accordance with previous studies12,23 (Supplementary information, Figure S2A and S2B). Therefore, the Nes-GFP+ cells were labeled with PKH26 (a red fluorescent lipophilic dye), and implanted into recipient animals. At 0, 4, 7, 10, 12, and 14 days after EDS treatment, the serum and the testes were collected for analyses (Figure 7A). The transplanted PKH26-labeled Nes-GFP+ cells were localized exclusively to the interstitium of the testis, and expressed the LC-specific markers P450scc and LHR 10 days after transplantation (Figure 7B). Moreover, after EDS treatment, the expression of 17β-HSD and 3β-HSD, and the concentration of testosterone were decreased significantly on day 4 and recovered gradually (Figure 7C-7E). However, EDS treatment had no obvious effect on LC morphology (Figure 7B). Furthermore, the transplantation of Nes-GFP+ cells significantly increased the mRNA expression levels of LC-specific markers, 3β-HSD and 17β-HSD, and the production of testosterone in EDS-treated mice at different time points (Figure 7C-7E). On day 14 (day 10 after cell transplantation), the testosterone concentration was even higher than that of saline-injected normal mice (Figure 7E). We then isolated PKH26-labeled cells by FACS from the testis 10 days after transplantation and performed RT-PCR analysis, which revealed that these cells expressed LC lineage-specific markers including LHR, 17β-HSD and 3β-HSD (Supplementary information, Figure S3A). Although GFP gene was not expressed at the transcriptional level after differentiation, it should be still located in the genomic DNA as an exogenous gene. Indeed, we found that most PKH26-labeled cells from the mouse testis carried the GFP gene (Supplementary information, Figure S3B).

Bottom Line: We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers.We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions.In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin.

View Article: PubMed Central - PubMed

Affiliation: 1] Cell-gene Therapy Translational Medicine Research Center, The Third Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510630, China [2] Key Laboratory for Stem Cells and Tissue Engineering, Center for Stem Cell Biology and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong 510080, China [3] Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China.

ABSTRACT
The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency.

Show MeSH
Related in: MedlinePlus