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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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Nes-GFP+ cells showed self-renewal property. (A) Cultured Nes-GFP+ spheres expressed PDGFR-α, LIFR, PH3, SSEA-1, SSEA-4, and GATA-4, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (B) Schematic of the experimental procedure used to generate the Nes-GFP+ clonal sphere cultures. (C) Representative images showed clonal sphere growth from single cells observed using a bright field (top panel) or fluorescence (bottom panel) microscope. Scale bar, 10 μm (days 1 and 2) and 25 m (days 4 and 10). (D) A comparison of the sphere formation in wells containing a single cell showed that the frequency of sphere formation was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (E) The proliferation rates of the Nes-GFP+ cells at different passages (P7, P15 and P30) were assessed using the CCK8 assay and were shown to be similar (n = 6). Data are expressed as the mean ± SEM.
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fig3: Nes-GFP+ cells showed self-renewal property. (A) Cultured Nes-GFP+ spheres expressed PDGFR-α, LIFR, PH3, SSEA-1, SSEA-4, and GATA-4, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (B) Schematic of the experimental procedure used to generate the Nes-GFP+ clonal sphere cultures. (C) Representative images showed clonal sphere growth from single cells observed using a bright field (top panel) or fluorescence (bottom panel) microscope. Scale bar, 10 μm (days 1 and 2) and 25 m (days 4 and 10). (D) A comparison of the sphere formation in wells containing a single cell showed that the frequency of sphere formation was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (E) The proliferation rates of the Nes-GFP+ cells at different passages (P7, P15 and P30) were assessed using the CCK8 assay and were shown to be similar (n = 6). Data are expressed as the mean ± SEM.

Mentions: We characterized the cells of the Nes-GFP+ spheres at P6 by immunofluorescence staining and examined the expression levels of PDGFR-α and LIFR, and embryonic stem cell markers, such as NANOG, OCT4, SOX2, SSEA-1 and SSEA-4. Interestingly, the Nes-GFP+ sphere cells highly expressed Nestin and also expressed PDGFR-α, LIFR, SSEA-1, and SSEA-4 (Figure 3A); however, they did not express OCT4 or NANOG and exhibited only negligible expression of SOX2 (data not shown). In addition, these isolated cells were negative for the LC markers LHR and 3β-HSD, indicating that the culture conditions maintained the SLCs in their undifferentiated state. A majority of these cells were also positive for GATA4, which had been implicated as a key transcription factor in LC development18,19. Moreover, the Nes-GFP+ cells were positive for the proliferation marker PH3 (Figure 3A). These results strongly suggest that Nes-GFP+ cells have the characteristics of SLCs in the testis.


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 showed self-renewal property. (A) Cultured Nes-GFP+ spheres expressed PDGFR-α, LIFR, PH3, SSEA-1, SSEA-4, and GATA-4, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (B) Schematic of the experimental procedure used to generate the Nes-GFP+ clonal sphere cultures. (C) Representative images showed clonal sphere growth from single cells observed using a bright field (top panel) or fluorescence (bottom panel) microscope. Scale bar, 10 μm (days 1 and 2) and 25 m (days 4 and 10). (D) A comparison of the sphere formation in wells containing a single cell showed that the frequency of sphere formation was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (E) The proliferation rates of the Nes-GFP+ cells at different passages (P7, P15 and P30) were assessed using the CCK8 assay and were shown to be similar (n = 6). Data are expressed as the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260348&req=5

fig3: Nes-GFP+ cells showed self-renewal property. (A) Cultured Nes-GFP+ spheres expressed PDGFR-α, LIFR, PH3, SSEA-1, SSEA-4, and GATA-4, but did not express 3β-HSD or LHR. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. (B) Schematic of the experimental procedure used to generate the Nes-GFP+ clonal sphere cultures. (C) Representative images showed clonal sphere growth from single cells observed using a bright field (top panel) or fluorescence (bottom panel) microscope. Scale bar, 10 μm (days 1 and 2) and 25 m (days 4 and 10). (D) A comparison of the sphere formation in wells containing a single cell showed that the frequency of sphere formation was equivalent in the primary and secondary spheres (n = 4). Data are expressed as the mean ± SEM. (E) The proliferation rates of the Nes-GFP+ cells at different passages (P7, P15 and P30) were assessed using the CCK8 assay and were shown to be similar (n = 6). Data are expressed as the mean ± SEM.
Mentions: We characterized the cells of the Nes-GFP+ spheres at P6 by immunofluorescence staining and examined the expression levels of PDGFR-α and LIFR, and embryonic stem cell markers, such as NANOG, OCT4, SOX2, SSEA-1 and SSEA-4. Interestingly, the Nes-GFP+ sphere cells highly expressed Nestin and also expressed PDGFR-α, LIFR, SSEA-1, and SSEA-4 (Figure 3A); however, they did not express OCT4 or NANOG and exhibited only negligible expression of SOX2 (data not shown). In addition, these isolated cells were negative for the LC markers LHR and 3β-HSD, indicating that the culture conditions maintained the SLCs in their undifferentiated state. A majority of these cells were also positive for GATA4, which had been implicated as a key transcription factor in LC development18,19. Moreover, the Nes-GFP+ cells were positive for the proliferation marker PH3 (Figure 3A). These results strongly suggest that Nes-GFP+ cells have the characteristics of SLCs in the testis.

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