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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

CD51+ cells exhibited the characteristics of SLCs. Primary Nes-GFP+ cells were isolated from postnatal 7-day-old Nes-GFP transgenic mice and the expression of the indicated cell surface markers was analyzed by flow cytometry. (B) Representative flow cytometric profiles of CD51+ cells in testis of postnatal 7-day-old C57BL/6 mice. Phase-contrast micrographs showed that primary (P0, 4 days after plating) and P1 (7 days after plating) cells in both CD51+ and CD51− cells. Scale bar, 25 μm. (C) Cultured CD51+ spheres expressed CD51, Nestin, PDGFR-α, LIFR, GATA4 and PH3, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (D) Representative images showing clonal sphere growth derived from single cells, which were observed by bright field microscopy. Scale bar, 10 μm (days 1 and 2) and 25 μm (days 4 and 10). (E) Frequency of sphere formation from single cell-derived CD51+ cells was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (F-H) Representative images showed that clonally expanded CD51+ cells differentiated into adipocytes (oil red) (F), osteocytes (alizarin red) (G), and chondrocytes (toluidine blue) (H). Scale bar, 50 μm. (I) CD51+ cells were tested by immunofluorescence staining with 3β-HSD, P450c17, P450scc, LHR, StAR, SF-1 and GATA4 7days after differentiation in DIM. BF, bright field. Scale bar, 25 μm. (J) Differentiated CD51+ cells were examined by RT-PCR analysis for expression of LC-related genes, 3β-HSD, StAR and LHR. (K) Testosterone production was measured in culture medium 0, 3, 5, 7 days after LC differentiation. Data are expressed as the mean ± SEM. n = 6.
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fig10: CD51+ cells exhibited the characteristics of SLCs. Primary Nes-GFP+ cells were isolated from postnatal 7-day-old Nes-GFP transgenic mice and the expression of the indicated cell surface markers was analyzed by flow cytometry. (B) Representative flow cytometric profiles of CD51+ cells in testis of postnatal 7-day-old C57BL/6 mice. Phase-contrast micrographs showed that primary (P0, 4 days after plating) and P1 (7 days after plating) cells in both CD51+ and CD51− cells. Scale bar, 25 μm. (C) Cultured CD51+ spheres expressed CD51, Nestin, PDGFR-α, LIFR, GATA4 and PH3, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (D) Representative images showing clonal sphere growth derived from single cells, which were observed by bright field microscopy. Scale bar, 10 μm (days 1 and 2) and 25 μm (days 4 and 10). (E) Frequency of sphere formation from single cell-derived CD51+ cells was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (F-H) Representative images showed that clonally expanded CD51+ cells differentiated into adipocytes (oil red) (F), osteocytes (alizarin red) (G), and chondrocytes (toluidine blue) (H). Scale bar, 50 μm. (I) CD51+ cells were tested by immunofluorescence staining with 3β-HSD, P450c17, P450scc, LHR, StAR, SF-1 and GATA4 7days after differentiation in DIM. BF, bright field. Scale bar, 25 μm. (J) Differentiated CD51+ cells were examined by RT-PCR analysis for expression of LC-related genes, 3β-HSD, StAR and LHR. (K) Testosterone production was measured in culture medium 0, 3, 5, 7 days after LC differentiation. Data are expressed as the mean ± SEM. n = 6.

Mentions: As Nestin is an intracellular protein, it is useful to identify cell surface markers specific for Nes+ SLCs. We screened the expression of the previously reported neural and mesenchymal stem cell surface markers in Nes-GFP+ cells derived from testes of postnatal 7-day-old mice, including CD51, p75 (CD271), PDGFR-α, NG2, CD146, CD106, CD73, CD44, CD90, Sca-1, CD117, CD119, CD11b, and CD45 through flow cytometry. CD51 and p75 were highly expressed in Nes-GFP+ cells (93.53% ± 3.10% and 74.73% ± 1.89%, respectively). Another putative SLC marker, PDGFR-α was expressed by 67.89% ± 2.83% of Nes-GFP+ cells. Moreover, two known perivascular pericyte markers, NG233 and CD14634, were expressed in 64.63% ± 3.16% and 40.38% ± 2.76% of Nes-GFP+ population, respectively. Furthermore, the expression of other conventional mesenchymal lineage markers (CD106, CD73, CD44, CD90, Sca-1, and CD117) was detected in a low percentage of Nes-GFP+ cells (< 40%). Moreover, Nes-GFP+ cells lacked the expression of hematopoietic cell markers, CD11b and CD45 (Figure 10A and Supplementary information, Figure S8).


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)

CD51+ cells exhibited the characteristics of SLCs. Primary Nes-GFP+ cells were isolated from postnatal 7-day-old Nes-GFP transgenic mice and the expression of the indicated cell surface markers was analyzed by flow cytometry. (B) Representative flow cytometric profiles of CD51+ cells in testis of postnatal 7-day-old C57BL/6 mice. Phase-contrast micrographs showed that primary (P0, 4 days after plating) and P1 (7 days after plating) cells in both CD51+ and CD51− cells. Scale bar, 25 μm. (C) Cultured CD51+ spheres expressed CD51, Nestin, PDGFR-α, LIFR, GATA4 and PH3, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (D) Representative images showing clonal sphere growth derived from single cells, which were observed by bright field microscopy. Scale bar, 10 μm (days 1 and 2) and 25 μm (days 4 and 10). (E) Frequency of sphere formation from single cell-derived CD51+ cells was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (F-H) Representative images showed that clonally expanded CD51+ cells differentiated into adipocytes (oil red) (F), osteocytes (alizarin red) (G), and chondrocytes (toluidine blue) (H). Scale bar, 50 μm. (I) CD51+ cells were tested by immunofluorescence staining with 3β-HSD, P450c17, P450scc, LHR, StAR, SF-1 and GATA4 7days after differentiation in DIM. BF, bright field. Scale bar, 25 μm. (J) Differentiated CD51+ cells were examined by RT-PCR analysis for expression of LC-related genes, 3β-HSD, StAR and LHR. (K) Testosterone production was measured in culture medium 0, 3, 5, 7 days after LC differentiation. Data are expressed as the mean ± SEM. n = 6.
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Related In: Results  -  Collection

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fig10: CD51+ cells exhibited the characteristics of SLCs. Primary Nes-GFP+ cells were isolated from postnatal 7-day-old Nes-GFP transgenic mice and the expression of the indicated cell surface markers was analyzed by flow cytometry. (B) Representative flow cytometric profiles of CD51+ cells in testis of postnatal 7-day-old C57BL/6 mice. Phase-contrast micrographs showed that primary (P0, 4 days after plating) and P1 (7 days after plating) cells in both CD51+ and CD51− cells. Scale bar, 25 μm. (C) Cultured CD51+ spheres expressed CD51, Nestin, PDGFR-α, LIFR, GATA4 and PH3, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (D) Representative images showing clonal sphere growth derived from single cells, which were observed by bright field microscopy. Scale bar, 10 μm (days 1 and 2) and 25 μm (days 4 and 10). (E) Frequency of sphere formation from single cell-derived CD51+ cells was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (F-H) Representative images showed that clonally expanded CD51+ cells differentiated into adipocytes (oil red) (F), osteocytes (alizarin red) (G), and chondrocytes (toluidine blue) (H). Scale bar, 50 μm. (I) CD51+ cells were tested by immunofluorescence staining with 3β-HSD, P450c17, P450scc, LHR, StAR, SF-1 and GATA4 7days after differentiation in DIM. BF, bright field. Scale bar, 25 μm. (J) Differentiated CD51+ cells were examined by RT-PCR analysis for expression of LC-related genes, 3β-HSD, StAR and LHR. (K) Testosterone production was measured in culture medium 0, 3, 5, 7 days after LC differentiation. Data are expressed as the mean ± SEM. n = 6.
Mentions: As Nestin is an intracellular protein, it is useful to identify cell surface markers specific for Nes+ SLCs. We screened the expression of the previously reported neural and mesenchymal stem cell surface markers in Nes-GFP+ cells derived from testes of postnatal 7-day-old mice, including CD51, p75 (CD271), PDGFR-α, NG2, CD146, CD106, CD73, CD44, CD90, Sca-1, CD117, CD119, CD11b, and CD45 through flow cytometry. CD51 and p75 were highly expressed in Nes-GFP+ cells (93.53% ± 3.10% and 74.73% ± 1.89%, respectively). Another putative SLC marker, PDGFR-α was expressed by 67.89% ± 2.83% of Nes-GFP+ cells. Moreover, two known perivascular pericyte markers, NG233 and CD14634, were expressed in 64.63% ± 3.16% and 40.38% ± 2.76% of Nes-GFP+ population, respectively. Furthermore, the expression of other conventional mesenchymal lineage markers (CD106, CD73, CD44, CD90, Sca-1, and CD117) was detected in a low percentage of Nes-GFP+ cells (< 40%). Moreover, Nes-GFP+ cells lacked the expression of hematopoietic cell markers, CD11b and CD45 (Figure 10A and Supplementary information, Figure S8).

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