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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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Related in: MedlinePlus

Nes-GFP+ cells promoted spermatogenesis 10 days after transplantation. (A) Representative images showed PH3-positive (green) cells in seminiferous tubules of testes in each experimental mouse. Ten days after transplantation, the Nes-GFP+ cell-treated animals showed a significantly higher number of proliferating cells in the seminiferous tubules compared to mice with saline injection. Scale bar, 50 μm. (B) Quantitative analysis showing the percentage of PH3-positive cells in seminiferous tubules of testes. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). (C, E) The meiotic spermatocytes were observed by immunofluorescence staining with anti-SYCP1 (green) (C) and anti-SYCP3 (green) (E) antibodies. Nucleus was detected by DAPI staining (blue). The SYCP1- or SYCP3-positive cells were located at the pachytene stage in each testis. Scale bar, 50 μm. (D, F) Quantitative analysis showing the percentage of SYCP1- or SYCP3-positive cells in seminiferous tubules of testis. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). Normal/Saline (+), 3-month-old mice receiving saline injection; EDS(+)/Saline(+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells(+), EDS-treated mice receiving Nes-GFP+ cells 4 days later; Old/Saline (+), 22-month-old mice receiving saline injection; Old/Cells (+), 22-month-old mice receiving Nes-GFP+ cells. n = 6.
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fig9: Nes-GFP+ cells promoted spermatogenesis 10 days after transplantation. (A) Representative images showed PH3-positive (green) cells in seminiferous tubules of testes in each experimental mouse. Ten days after transplantation, the Nes-GFP+ cell-treated animals showed a significantly higher number of proliferating cells in the seminiferous tubules compared to mice with saline injection. Scale bar, 50 μm. (B) Quantitative analysis showing the percentage of PH3-positive cells in seminiferous tubules of testes. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). (C, E) The meiotic spermatocytes were observed by immunofluorescence staining with anti-SYCP1 (green) (C) and anti-SYCP3 (green) (E) antibodies. Nucleus was detected by DAPI staining (blue). The SYCP1- or SYCP3-positive cells were located at the pachytene stage in each testis. Scale bar, 50 μm. (D, F) Quantitative analysis showing the percentage of SYCP1- or SYCP3-positive cells in seminiferous tubules of testis. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). Normal/Saline (+), 3-month-old mice receiving saline injection; EDS(+)/Saline(+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells(+), EDS-treated mice receiving Nes-GFP+ cells 4 days later; Old/Saline (+), 22-month-old mice receiving saline injection; Old/Cells (+), 22-month-old mice receiving Nes-GFP+ cells. n = 6.

Mentions: It has been reported that spermatogenic dysfunction is often associated with impaired LC function27. Furthermore, it has been established that testosterone is essential for spermatogenesis28. To further verify whether the transplanted Nes-GFP+ cells played a role in the maintenance of spermatogenesis, we examined the proliferative capacity of spermatogonial cells in the seminiferous tubules. PH3-positive cells virtually disappeared in the EDS-treated mice (Figure 9A), whereas the number of PH3-positive proliferating cells was increased dramatically in the seminiferous tubules 10 days after transplantation with the Nes-GFP+ cells (0.33% ± 0.11% versus 21.97% ± 2.49%, P < 0.01, Figure 9B). More interestingly, in the testes of aging mice, PH3 expression was rarely detected in the seminiferous tubules (2.40% ± 0.58%), whereas the number of proliferating spermatogenic cells was obviously increased after Nes-GFP+ cell transplantation (2.40% ± 0.58% versus 10.87% ± 1.01%, P < 0.01, Figure 9B).


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)

Nes-GFP+ cells promoted spermatogenesis 10 days after transplantation. (A) Representative images showed PH3-positive (green) cells in seminiferous tubules of testes in each experimental mouse. Ten days after transplantation, the Nes-GFP+ cell-treated animals showed a significantly higher number of proliferating cells in the seminiferous tubules compared to mice with saline injection. Scale bar, 50 μm. (B) Quantitative analysis showing the percentage of PH3-positive cells in seminiferous tubules of testes. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). (C, E) The meiotic spermatocytes were observed by immunofluorescence staining with anti-SYCP1 (green) (C) and anti-SYCP3 (green) (E) antibodies. Nucleus was detected by DAPI staining (blue). The SYCP1- or SYCP3-positive cells were located at the pachytene stage in each testis. Scale bar, 50 μm. (D, F) Quantitative analysis showing the percentage of SYCP1- or SYCP3-positive cells in seminiferous tubules of testis. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). Normal/Saline (+), 3-month-old mice receiving saline injection; EDS(+)/Saline(+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells(+), EDS-treated mice receiving Nes-GFP+ cells 4 days later; Old/Saline (+), 22-month-old mice receiving saline injection; Old/Cells (+), 22-month-old mice receiving Nes-GFP+ cells. n = 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig9: Nes-GFP+ cells promoted spermatogenesis 10 days after transplantation. (A) Representative images showed PH3-positive (green) cells in seminiferous tubules of testes in each experimental mouse. Ten days after transplantation, the Nes-GFP+ cell-treated animals showed a significantly higher number of proliferating cells in the seminiferous tubules compared to mice with saline injection. Scale bar, 50 μm. (B) Quantitative analysis showing the percentage of PH3-positive cells in seminiferous tubules of testes. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). (C, E) The meiotic spermatocytes were observed by immunofluorescence staining with anti-SYCP1 (green) (C) and anti-SYCP3 (green) (E) antibodies. Nucleus was detected by DAPI staining (blue). The SYCP1- or SYCP3-positive cells were located at the pachytene stage in each testis. Scale bar, 50 μm. (D, F) Quantitative analysis showing the percentage of SYCP1- or SYCP3-positive cells in seminiferous tubules of testis. Three sections per slide and three slides per mouse testis were counted (**P < 0.01). Normal/Saline (+), 3-month-old mice receiving saline injection; EDS(+)/Saline(+), EDS-treated mice receiving saline 4 days later; EDS(+)/Cells(+), EDS-treated mice receiving Nes-GFP+ cells 4 days later; Old/Saline (+), 22-month-old mice receiving saline injection; Old/Cells (+), 22-month-old mice receiving Nes-GFP+ cells. n = 6.
Mentions: It has been reported that spermatogenic dysfunction is often associated with impaired LC function27. Furthermore, it has been established that testosterone is essential for spermatogenesis28. To further verify whether the transplanted Nes-GFP+ cells played a role in the maintenance of spermatogenesis, we examined the proliferative capacity of spermatogonial cells in the seminiferous tubules. PH3-positive cells virtually disappeared in the EDS-treated mice (Figure 9A), whereas the number of PH3-positive proliferating cells was increased dramatically in the seminiferous tubules 10 days after transplantation with the Nes-GFP+ cells (0.33% ± 0.11% versus 21.97% ± 2.49%, P < 0.01, Figure 9B). More interestingly, in the testes of aging mice, PH3 expression was rarely detected in the seminiferous tubules (2.40% ± 0.58%), whereas the number of proliferating spermatogenic cells was obviously increased after Nes-GFP+ cell transplantation (2.40% ± 0.58% versus 10.87% ± 1.01%, P < 0.01, Figure 9B).

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