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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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Nestin expression in the testis of Nes-GFP transgenic mice. (A) Nestin (green) expression in the testis of Nes-GFP transgenic mice on postnatal days 0, 7, 14, 28, and 90. Scale bar, 50 μm. (B) Quantification of Nes-GFP+ cells in the testis sections at the indicated time points. The percentage of Nes-GFP+ cells in the testis interstitum was counted. Three sections per slide and three slides per mouse testis were counted. (C) Immunohistochemical analyses showed that the Nes-GFP+ cells in the testis expressed PDGFR-α and LIFR, but did not express LHR or 3β-HSD on postnatal day 7. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (D) qRT-PCR analysis of Nestin, PDGFR-α and LIFR gene expression in testes of normal mice on postnatal days 7, 14, 28, 90 (the value on postnatal day 7 was defined as 1). n = 5. Data are expressed as the mean ± SEM.
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fig1: Nestin expression in the testis of Nes-GFP transgenic mice. (A) Nestin (green) expression in the testis of Nes-GFP transgenic mice on postnatal days 0, 7, 14, 28, and 90. Scale bar, 50 μm. (B) Quantification of Nes-GFP+ cells in the testis sections at the indicated time points. The percentage of Nes-GFP+ cells in the testis interstitum was counted. Three sections per slide and three slides per mouse testis were counted. (C) Immunohistochemical analyses showed that the Nes-GFP+ cells in the testis expressed PDGFR-α and LIFR, but did not express LHR or 3β-HSD on postnatal day 7. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (D) qRT-PCR analysis of Nestin, PDGFR-α and LIFR gene expression in testes of normal mice on postnatal days 7, 14, 28, 90 (the value on postnatal day 7 was defined as 1). n = 5. Data are expressed as the mean ± SEM.

Mentions: To systemically evaluate the expression patterns of Nestin during postnatal development in the mouse testes, we assessed GFP expression in the Nes-GFP transgenic mouse model (expressing GFP driven by the Nestin (Nes) promoter) using confocal microscopy. Consistent with previous reports14,15, we detected GFP expression in the interstitial or peritubular cells of the mouse testes on postnatal days 0, 7, 14, 28 and 90, but not within the seminiferous tubules (Figure 1A). Quantitative analysis showed a significant peak of the number of Nes-GFP+ cells on postnatal day 7, which then gradually decreased with age in the total population of the interstitial compartment; by day 90, Nes-GFP+ cells were barely seen in the testis interstitium (Figure 1B). Additionally, using specific Nestin antibodies yielded similar staining patterns in these tissues (data not shown). No staining was found in the negative controls.


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)

Nestin expression in the testis of Nes-GFP transgenic mice. (A) Nestin (green) expression in the testis of Nes-GFP transgenic mice on postnatal days 0, 7, 14, 28, and 90. Scale bar, 50 μm. (B) Quantification of Nes-GFP+ cells in the testis sections at the indicated time points. The percentage of Nes-GFP+ cells in the testis interstitum was counted. Three sections per slide and three slides per mouse testis were counted. (C) Immunohistochemical analyses showed that the Nes-GFP+ cells in the testis expressed PDGFR-α and LIFR, but did not express LHR or 3β-HSD on postnatal day 7. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (D) qRT-PCR analysis of Nestin, PDGFR-α and LIFR gene expression in testes of normal mice on postnatal days 7, 14, 28, 90 (the value on postnatal day 7 was defined as 1). n = 5. 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

fig1: Nestin expression in the testis of Nes-GFP transgenic mice. (A) Nestin (green) expression in the testis of Nes-GFP transgenic mice on postnatal days 0, 7, 14, 28, and 90. Scale bar, 50 μm. (B) Quantification of Nes-GFP+ cells in the testis sections at the indicated time points. The percentage of Nes-GFP+ cells in the testis interstitum was counted. Three sections per slide and three slides per mouse testis were counted. (C) Immunohistochemical analyses showed that the Nes-GFP+ cells in the testis expressed PDGFR-α and LIFR, but did not express LHR or 3β-HSD on postnatal day 7. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (D) qRT-PCR analysis of Nestin, PDGFR-α and LIFR gene expression in testes of normal mice on postnatal days 7, 14, 28, 90 (the value on postnatal day 7 was defined as 1). n = 5. Data are expressed as the mean ± SEM.
Mentions: To systemically evaluate the expression patterns of Nestin during postnatal development in the mouse testes, we assessed GFP expression in the Nes-GFP transgenic mouse model (expressing GFP driven by the Nestin (Nes) promoter) using confocal microscopy. Consistent with previous reports14,15, we detected GFP expression in the interstitial or peritubular cells of the mouse testes on postnatal days 0, 7, 14, 28 and 90, but not within the seminiferous tubules (Figure 1A). Quantitative analysis showed a significant peak of the number of Nes-GFP+ cells on postnatal day 7, which then gradually decreased with age in the total population of the interstitial compartment; by day 90, Nes-GFP+ cells were barely seen in the testis interstitium (Figure 1B). Additionally, using specific Nestin antibodies yielded similar staining patterns in these tissues (data not shown). No staining was found in the negative controls.

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