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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 multiple lineages. (A) Schematic of the experimental procedure used for inducing differentiation. (B-F) Representative micrographs of histological staining showing neurons (TuJ-1) (B), astrocytes (GFAP) (C), osteocytes (alizarin red) (D), adipocytes (oil red) (E), and chondrocytes (toluidine blue) (F). Scale bar, B-C, 25 μm; D-F, 100 μm. (G) RT-PCR analysis of differentiated Nes-GFP+ cells cultured for 4 weeks (DIFF) showing increased expression of neural (Tuj-1, NF200 and GFAP), osteogenic (ALP, SPARC and Runx2), adipogenic (Fabp4 and PPAR-γ), and chondrogenic (Col2A and Aggrecan) markers relative to undifferentiated SLCs (UNDIFF).
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fig4: Nes-GFP+ cells differentiated into multiple lineages. (A) Schematic of the experimental procedure used for inducing differentiation. (B-F) Representative micrographs of histological staining showing neurons (TuJ-1) (B), astrocytes (GFAP) (C), osteocytes (alizarin red) (D), adipocytes (oil red) (E), and chondrocytes (toluidine blue) (F). Scale bar, B-C, 25 μm; D-F, 100 μm. (G) RT-PCR analysis of differentiated Nes-GFP+ cells cultured for 4 weeks (DIFF) showing increased expression of neural (Tuj-1, NF200 and GFAP), osteogenic (ALP, SPARC and Runx2), adipogenic (Fabp4 and PPAR-γ), and chondrogenic (Col2A and Aggrecan) markers relative to undifferentiated SLCs (UNDIFF).

Mentions: Nestin has been identified as a marker for neural and mesenchymal stem cells, and these Nestin-expressing cells are capable of differentiating into neural20 or mesenchymal12 cell lineages, suggesting that Nestin represents a characteristic marker of multilineage progenitor cells. To characterize the multipotency of the Nes-GFP+ cells in the testis, we cultured these cells under defined differentiation conditions (Figure 4A). After induction, the neural lineage differentiation of Nes-GFP+ cells was confirmed by staining for Tuj-1 (Neuronal class III β-tubulin) and GFAP (glial fibrillary acidic protein; Figure 4B and 4C). In addition, the induction of Tuj-1, neurofilament 200 (NF200) and GFAP gene expression was observed by RT-PCR analysis (Figure 4G). Osteogenic differentiation was confirmed by the deposition of alizarin red-positive mineralized matrix after 4 weeks of induction (Figure 4D). This increased calcium deposition was associated with increased mRNA levels of the osteogenic markers runt related transcription factor 2 (Runx2), secreted protein acidic and rich in cysteine (SPARC), and alkaline phosphatase (ALP; Figure 4G). Oil red O staining, which detects the presence of lipid-rich vacuoles, was used to evaluate the adipogenic induction (Figure 4E). The differentiated cells exhibited intracellular lipid vacuoles that were absent in the undifferentiated cells. We further found that the expression levels of adipogenic markers including peroxisome proliferator activated receptor-γ (PPAR-γ) and fatty acid-binding protein 4 (Fabp4) were significantly upregulated after differentiation (Figure 4G). Four weeks after chondrogenic induction, the pellet was intensely stained with toluidine blue (Figure 4F). Furthermore, in accordance with the histochemistry results, the mRNA expression levels of the chondrogenic genes Col2A and Aggrecan were also highly upregulated (Figure 4G). It needs to be noted that the expression of these lineage-specific markers was not detected prior to differentiation (Figure 4G).


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 multiple lineages. (A) Schematic of the experimental procedure used for inducing differentiation. (B-F) Representative micrographs of histological staining showing neurons (TuJ-1) (B), astrocytes (GFAP) (C), osteocytes (alizarin red) (D), adipocytes (oil red) (E), and chondrocytes (toluidine blue) (F). Scale bar, B-C, 25 μm; D-F, 100 μm. (G) RT-PCR analysis of differentiated Nes-GFP+ cells cultured for 4 weeks (DIFF) showing increased expression of neural (Tuj-1, NF200 and GFAP), osteogenic (ALP, SPARC and Runx2), adipogenic (Fabp4 and PPAR-γ), and chondrogenic (Col2A and Aggrecan) markers relative to undifferentiated SLCs (UNDIFF).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Nes-GFP+ cells differentiated into multiple lineages. (A) Schematic of the experimental procedure used for inducing differentiation. (B-F) Representative micrographs of histological staining showing neurons (TuJ-1) (B), astrocytes (GFAP) (C), osteocytes (alizarin red) (D), adipocytes (oil red) (E), and chondrocytes (toluidine blue) (F). Scale bar, B-C, 25 μm; D-F, 100 μm. (G) RT-PCR analysis of differentiated Nes-GFP+ cells cultured for 4 weeks (DIFF) showing increased expression of neural (Tuj-1, NF200 and GFAP), osteogenic (ALP, SPARC and Runx2), adipogenic (Fabp4 and PPAR-γ), and chondrogenic (Col2A and Aggrecan) markers relative to undifferentiated SLCs (UNDIFF).
Mentions: Nestin has been identified as a marker for neural and mesenchymal stem cells, and these Nestin-expressing cells are capable of differentiating into neural20 or mesenchymal12 cell lineages, suggesting that Nestin represents a characteristic marker of multilineage progenitor cells. To characterize the multipotency of the Nes-GFP+ cells in the testis, we cultured these cells under defined differentiation conditions (Figure 4A). After induction, the neural lineage differentiation of Nes-GFP+ cells was confirmed by staining for Tuj-1 (Neuronal class III β-tubulin) and GFAP (glial fibrillary acidic protein; Figure 4B and 4C). In addition, the induction of Tuj-1, neurofilament 200 (NF200) and GFAP gene expression was observed by RT-PCR analysis (Figure 4G). Osteogenic differentiation was confirmed by the deposition of alizarin red-positive mineralized matrix after 4 weeks of induction (Figure 4D). This increased calcium deposition was associated with increased mRNA levels of the osteogenic markers runt related transcription factor 2 (Runx2), secreted protein acidic and rich in cysteine (SPARC), and alkaline phosphatase (ALP; Figure 4G). Oil red O staining, which detects the presence of lipid-rich vacuoles, was used to evaluate the adipogenic induction (Figure 4E). The differentiated cells exhibited intracellular lipid vacuoles that were absent in the undifferentiated cells. We further found that the expression levels of adipogenic markers including peroxisome proliferator activated receptor-γ (PPAR-γ) and fatty acid-binding protein 4 (Fabp4) were significantly upregulated after differentiation (Figure 4G). Four weeks after chondrogenic induction, the pellet was intensely stained with toluidine blue (Figure 4F). Furthermore, in accordance with the histochemistry results, the mRNA expression levels of the chondrogenic genes Col2A and Aggrecan were also highly upregulated (Figure 4G). It needs to be noted that the expression of these lineage-specific markers was not detected prior to differentiation (Figure 4G).

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