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Sertoli cell-mediated differentiation of male germ cell-like cells from human umbilical cord Wharton's jelly-derived mesenchymal stem cells in an in vitro co-culture system.

Xie L, Lin L, Tang Q, Li W, Huang T, Huo X, Liu X, Jiang J, He G, Ma L - Eur. J. Med. Res. (2015)

Bottom Line: Differentiated cells formed round colonies that share the morphological features of spermatogonial colonies.RT-PCR, immunofluorescence, confocal microscopy, and Western blot analyses revealed the expression of early germ cell markers STELLA and VASA and male germ cell-specific marker DAZL in differentiated HUMSCs, confirming the presence of cells with characteristics of male germ cells.Male germ cells derived from HUMSCs may be used in the therapy for male infertility.

View Article: PubMed Central - PubMed

Affiliation: Women's and Children's Hospital of Shenzhen University, Shenzhen, 518000, China. shirleyxie_2008@yahoo.cn.

ABSTRACT

Background: Microenvironment signals play a critical role in directing the differentiation of stem cells. Sertoli cells (SCs) provide a unique microenvironment that is essential for germ cell differentiation.

Methods: Our previous study has demonstrated that human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSCs) could differentiate towards male germ cells in vitro, but HUMSC-derived germ-like cells expressed only few germ cell markers. The aim of this study was to investigate the effect of SCs on the differentiation of HUMSCs towards male germ cells using a co-culture system that mimicked the in vivo male germ cell microenvironment.

Results: HUMSCs formed clump-like features on SC monolayers after seeding for 3 weeks. Differentiated cells formed round colonies that share the morphological features of spermatogonial colonies. RT-PCR, immunofluorescence, confocal microscopy, and Western blot analyses revealed the expression of early germ cell markers STELLA and VASA and male germ cell-specific marker DAZL in differentiated HUMSCs, confirming the presence of cells with characteristics of male germ cells.

Conclusion: The HUMSC-SC co-culture system mimics a native microenvironment for germ cell colonization without any in vitro artificial manipulation and can be used to explore the mechanisms controlling the differentiation of male germ cells from HUMSCs. Male germ cells derived from HUMSCs may be used in the therapy for male infertility.

No MeSH data available.


Related in: MedlinePlus

Morphology of cultured Sertoli cells (SCs). (A) Primary SCs at 4 hours after plating (magnification 100×). The white arrow indicates a SC, and the black arrow indicates a germ cell. (B) Passage 2 SCs with residual germ cells removed (magnification 100×). (C) SC monolayer on day 3 after culture (magnification 100×). (D) Passage 2 SCs stained with hematoxylin (magnification 200×). The black arrow indicates a SC.
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Fig2: Morphology of cultured Sertoli cells (SCs). (A) Primary SCs at 4 hours after plating (magnification 100×). The white arrow indicates a SC, and the black arrow indicates a germ cell. (B) Passage 2 SCs with residual germ cells removed (magnification 100×). (C) SC monolayer on day 3 after culture (magnification 100×). (D) Passage 2 SCs stained with hematoxylin (magnification 200×). The black arrow indicates a SC.

Mentions: We cultured primary SCs to more than 95% purity by repeated washing, transfer and hypotonic shock. Cell morphology was closely monitored by phase contrast microscopy. Maximal cell adhesion was reached 4 hours after plating (Figure 2A). Passage 2 SCs formed a monolayer on day 3 after plating (Figure 2B). The SCs firmly attached to the bottom of the dish, were irregularly shaped, and had cytoplasmic droplets. These features are in agreement with previously reported features of SCs [27]. After 1 week of culturing, they produced extensions, flattened and attempted to make contact with other cells (Figure 2C). Contamination of SCs by other cell types was also examined. Staining of cells with hematoxylin showed that SCs had larger nuclei than peritubular myoid cells (Figure 2D). The presence of germ cells was not detected after 1 week of culturing.Figure 2


Sertoli cell-mediated differentiation of male germ cell-like cells from human umbilical cord Wharton's jelly-derived mesenchymal stem cells in an in vitro co-culture system.

Xie L, Lin L, Tang Q, Li W, Huang T, Huo X, Liu X, Jiang J, He G, Ma L - Eur. J. Med. Res. (2015)

Morphology of cultured Sertoli cells (SCs). (A) Primary SCs at 4 hours after plating (magnification 100×). The white arrow indicates a SC, and the black arrow indicates a germ cell. (B) Passage 2 SCs with residual germ cells removed (magnification 100×). (C) SC monolayer on day 3 after culture (magnification 100×). (D) Passage 2 SCs stained with hematoxylin (magnification 200×). The black arrow indicates a SC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389972&req=5

Fig2: Morphology of cultured Sertoli cells (SCs). (A) Primary SCs at 4 hours after plating (magnification 100×). The white arrow indicates a SC, and the black arrow indicates a germ cell. (B) Passage 2 SCs with residual germ cells removed (magnification 100×). (C) SC monolayer on day 3 after culture (magnification 100×). (D) Passage 2 SCs stained with hematoxylin (magnification 200×). The black arrow indicates a SC.
Mentions: We cultured primary SCs to more than 95% purity by repeated washing, transfer and hypotonic shock. Cell morphology was closely monitored by phase contrast microscopy. Maximal cell adhesion was reached 4 hours after plating (Figure 2A). Passage 2 SCs formed a monolayer on day 3 after plating (Figure 2B). The SCs firmly attached to the bottom of the dish, were irregularly shaped, and had cytoplasmic droplets. These features are in agreement with previously reported features of SCs [27]. After 1 week of culturing, they produced extensions, flattened and attempted to make contact with other cells (Figure 2C). Contamination of SCs by other cell types was also examined. Staining of cells with hematoxylin showed that SCs had larger nuclei than peritubular myoid cells (Figure 2D). The presence of germ cells was not detected after 1 week of culturing.Figure 2

Bottom Line: Differentiated cells formed round colonies that share the morphological features of spermatogonial colonies.RT-PCR, immunofluorescence, confocal microscopy, and Western blot analyses revealed the expression of early germ cell markers STELLA and VASA and male germ cell-specific marker DAZL in differentiated HUMSCs, confirming the presence of cells with characteristics of male germ cells.Male germ cells derived from HUMSCs may be used in the therapy for male infertility.

View Article: PubMed Central - PubMed

Affiliation: Women's and Children's Hospital of Shenzhen University, Shenzhen, 518000, China. shirleyxie_2008@yahoo.cn.

ABSTRACT

Background: Microenvironment signals play a critical role in directing the differentiation of stem cells. Sertoli cells (SCs) provide a unique microenvironment that is essential for germ cell differentiation.

Methods: Our previous study has demonstrated that human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSCs) could differentiate towards male germ cells in vitro, but HUMSC-derived germ-like cells expressed only few germ cell markers. The aim of this study was to investigate the effect of SCs on the differentiation of HUMSCs towards male germ cells using a co-culture system that mimicked the in vivo male germ cell microenvironment.

Results: HUMSCs formed clump-like features on SC monolayers after seeding for 3 weeks. Differentiated cells formed round colonies that share the morphological features of spermatogonial colonies. RT-PCR, immunofluorescence, confocal microscopy, and Western blot analyses revealed the expression of early germ cell markers STELLA and VASA and male germ cell-specific marker DAZL in differentiated HUMSCs, confirming the presence of cells with characteristics of male germ cells.

Conclusion: The HUMSC-SC co-culture system mimics a native microenvironment for germ cell colonization without any in vitro artificial manipulation and can be used to explore the mechanisms controlling the differentiation of male germ cells from HUMSCs. Male germ cells derived from HUMSCs may be used in the therapy for male infertility.

No MeSH data available.


Related in: MedlinePlus