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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 differentiated into LCs in vitro. (A) Seven days after differentiation, immunofluorescence staining showed that the Nes-GFP+ cells clearly expressed LC lineage-specific markers. The representative images showed the expression of 3β-HSD, P450scc, LHR, SF-1, GATA4, StAR and P450c17 in these cells. BF, bright field. Scale bar, 25 μm. (B) RT-PCR analysis confirmed that the expression of the Leydig lineage-specific markers StAR, 3β-HSD, and LHR in differentiated cells (DIFF) was significantly increased while the undifferentiated controls (UNDIFF) exhibited undetectable levels of these markers. (C) Testosterone production progressively increased during culture in DIM. Data are expressed as the mean ± SEM (n = 6).
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fig6: Nes-GFP+ cells differentiated into LCs in vitro. (A) Seven days after differentiation, immunofluorescence staining showed that the Nes-GFP+ cells clearly expressed LC lineage-specific markers. The representative images showed the expression of 3β-HSD, P450scc, LHR, SF-1, GATA4, StAR and P450c17 in these cells. BF, bright field. Scale bar, 25 μm. (B) RT-PCR analysis confirmed that the expression of the Leydig lineage-specific markers StAR, 3β-HSD, and LHR in differentiated cells (DIFF) was significantly increased while the undifferentiated controls (UNDIFF) exhibited undetectable levels of these markers. (C) Testosterone production progressively increased during culture in DIM. Data are expressed as the mean ± SEM (n = 6).

Mentions: To investigate the potential of the Nes-GFP+ cells to differentiate into LCs in vitro, we placed the cells in the differentiation-inducing medium (DIM) previously reported by Ge et al.9. After 7 days of culture in DIM, numerous cells exhibited signs of LC differentiation, including the expression of proteins and enzymes involved in androgen biosynthesis. Immunohistochemical analysis showed that most of the differentiated Nes-GFP+ cells expressed LC steroidogenic enzymes, including 3β-HSD, P450scc and P450c17, as well as LHR and steroidogenic acute regulatory protein (StAR). In addition, these cells were positively stained for the nuclear transcription factors, steroidogenic factor-1 (SF-1)22 and GATA418, which were known to be involved in LC development (Figure 6A). Moreover, RT-PCR demonstrated the upregulated expression of the LC lineage genes 3β-HSD, StAR and LHR after 7 days of induction (Figure 6B). Next, to determine whether the differentiated Nes-GFP+ cells could synthesize testosterone, we collected the supernatants at different time points and found that testosterone synthesis gradually increased, peaking 7 days after differentiation (Figure 6C). We also tested the steroidogenic potential of the clonal Nes-GFP+ cell lines derived from the Nes-GFP+ single cells. Indeed, these cells successfully differentiated into LCs as demonstrated by the expression of LC lineage-specific markers as described above and the production of testosterone after differentiation (Supplementary information, Figure S1A and S1B). Taken together, these results suggest that Nes-GFP+ cells can differentiate into mature LCs capable of testosterone synthesis.


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 differentiated into LCs in vitro. (A) Seven days after differentiation, immunofluorescence staining showed that the Nes-GFP+ cells clearly expressed LC lineage-specific markers. The representative images showed the expression of 3β-HSD, P450scc, LHR, SF-1, GATA4, StAR and P450c17 in these cells. BF, bright field. Scale bar, 25 μm. (B) RT-PCR analysis confirmed that the expression of the Leydig lineage-specific markers StAR, 3β-HSD, and LHR in differentiated cells (DIFF) was significantly increased while the undifferentiated controls (UNDIFF) exhibited undetectable levels of these markers. (C) Testosterone production progressively increased during culture in DIM. Data are expressed as the mean ± SEM (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Nes-GFP+ cells differentiated into LCs in vitro. (A) Seven days after differentiation, immunofluorescence staining showed that the Nes-GFP+ cells clearly expressed LC lineage-specific markers. The representative images showed the expression of 3β-HSD, P450scc, LHR, SF-1, GATA4, StAR and P450c17 in these cells. BF, bright field. Scale bar, 25 μm. (B) RT-PCR analysis confirmed that the expression of the Leydig lineage-specific markers StAR, 3β-HSD, and LHR in differentiated cells (DIFF) was significantly increased while the undifferentiated controls (UNDIFF) exhibited undetectable levels of these markers. (C) Testosterone production progressively increased during culture in DIM. Data are expressed as the mean ± SEM (n = 6).
Mentions: To investigate the potential of the Nes-GFP+ cells to differentiate into LCs in vitro, we placed the cells in the differentiation-inducing medium (DIM) previously reported by Ge et al.9. After 7 days of culture in DIM, numerous cells exhibited signs of LC differentiation, including the expression of proteins and enzymes involved in androgen biosynthesis. Immunohistochemical analysis showed that most of the differentiated Nes-GFP+ cells expressed LC steroidogenic enzymes, including 3β-HSD, P450scc and P450c17, as well as LHR and steroidogenic acute regulatory protein (StAR). In addition, these cells were positively stained for the nuclear transcription factors, steroidogenic factor-1 (SF-1)22 and GATA418, which were known to be involved in LC development (Figure 6A). Moreover, RT-PCR demonstrated the upregulated expression of the LC lineage genes 3β-HSD, StAR and LHR after 7 days of induction (Figure 6B). Next, to determine whether the differentiated Nes-GFP+ cells could synthesize testosterone, we collected the supernatants at different time points and found that testosterone synthesis gradually increased, peaking 7 days after differentiation (Figure 6C). We also tested the steroidogenic potential of the clonal Nes-GFP+ cell lines derived from the Nes-GFP+ single cells. Indeed, these cells successfully differentiated into LCs as demonstrated by the expression of LC lineage-specific markers as described above and the production of testosterone after differentiation (Supplementary information, Figure S1A and S1B). Taken together, these results suggest that Nes-GFP+ cells can differentiate into mature LCs capable of testosterone synthesis.

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