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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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Clonal-derived Nes-GFP+ cells exhibited multilineage differentiation capacity. (A) Schematic of the protocol for clonal Nes-GFP+ cell differentiation. (B) Images of the Nes-GFP+ clones under undifferentiated conditions (lower panels) and under osteogenic or adipogenic conditions (upper panels; left, alizarin red; right, oil red). (C-G) Single clones differentiated into osteocytes (alizarin red) (C), adipocytes (oil red) (D), chondrocytes (toluidine blue) (E), neurons (Tuj-1) (F), and astrocytes (GFAP) (G). Scale bar, C-E,100 μm; F-G, 25 μm.
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fig5: Clonal-derived Nes-GFP+ cells exhibited multilineage differentiation capacity. (A) Schematic of the protocol for clonal Nes-GFP+ cell differentiation. (B) Images of the Nes-GFP+ clones under undifferentiated conditions (lower panels) and under osteogenic or adipogenic conditions (upper panels; left, alizarin red; right, oil red). (C-G) Single clones differentiated into osteocytes (alizarin red) (C), adipocytes (oil red) (D), chondrocytes (toluidine blue) (E), neurons (Tuj-1) (F), and astrocytes (GFAP) (G). Scale bar, C-E,100 μm; F-G, 25 μm.

Mentions: To further assess the plasticity of the clonal (single cell-derived) Nes-GFP+ cells, we performed the multilineage differentiation assay with colonies derived from a single Nes-GFP+ cell (Figure 5A). The clonal Nes-GFP+ single cells were plated into 96-well plates by serial dilution (one cell/well) and were expanded under non-adherent conditions. After treating the clonal Nes-GFP+ cells with differentiation media, these cells exhibited robust plasticity. Under the mesenchymal lineage differentiation conditions, these cells showed tri-lineage differentiation potential and exhibited osteogenic (Figure 5B and 5C), adipogenic (Figure 5B and 5D), and chondrogenic (Figure 5E) lineage phenotypes during the 4-week differentiation period. Interestingly, such clones also underwent neurogenic differentiation, as shown by the staining for the neuronal and astroglial proteins Tuj-1 and GFAP, respectively (Figure 5F and 5G). Moreover, these clonogenic experiments further suggest that these cells possess self-renewal potential, which was consistent with previous reports in which extensive proliferation of a single cell with the retention of multipotency over a long period of time has been used to demonstrate stem cells in the skin21.


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)

Clonal-derived Nes-GFP+ cells exhibited multilineage differentiation capacity. (A) Schematic of the protocol for clonal Nes-GFP+ cell differentiation. (B) Images of the Nes-GFP+ clones under undifferentiated conditions (lower panels) and under osteogenic or adipogenic conditions (upper panels; left, alizarin red; right, oil red). (C-G) Single clones differentiated into osteocytes (alizarin red) (C), adipocytes (oil red) (D), chondrocytes (toluidine blue) (E), neurons (Tuj-1) (F), and astrocytes (GFAP) (G). Scale bar, C-E,100 μm; F-G, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Clonal-derived Nes-GFP+ cells exhibited multilineage differentiation capacity. (A) Schematic of the protocol for clonal Nes-GFP+ cell differentiation. (B) Images of the Nes-GFP+ clones under undifferentiated conditions (lower panels) and under osteogenic or adipogenic conditions (upper panels; left, alizarin red; right, oil red). (C-G) Single clones differentiated into osteocytes (alizarin red) (C), adipocytes (oil red) (D), chondrocytes (toluidine blue) (E), neurons (Tuj-1) (F), and astrocytes (GFAP) (G). Scale bar, C-E,100 μm; F-G, 25 μm.
Mentions: To further assess the plasticity of the clonal (single cell-derived) Nes-GFP+ cells, we performed the multilineage differentiation assay with colonies derived from a single Nes-GFP+ cell (Figure 5A). The clonal Nes-GFP+ single cells were plated into 96-well plates by serial dilution (one cell/well) and were expanded under non-adherent conditions. After treating the clonal Nes-GFP+ cells with differentiation media, these cells exhibited robust plasticity. Under the mesenchymal lineage differentiation conditions, these cells showed tri-lineage differentiation potential and exhibited osteogenic (Figure 5B and 5C), adipogenic (Figure 5B and 5D), and chondrogenic (Figure 5E) lineage phenotypes during the 4-week differentiation period. Interestingly, such clones also underwent neurogenic differentiation, as shown by the staining for the neuronal and astroglial proteins Tuj-1 and GFAP, respectively (Figure 5F and 5G). Moreover, these clonogenic experiments further suggest that these cells possess self-renewal potential, which was consistent with previous reports in which extensive proliferation of a single cell with the retention of multipotency over a long period of time has been used to demonstrate stem cells in the skin21.

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