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Overexpression of CD61 promotes hUC-MSC differentiation into male germ-like cells.

ABSTRACT: OBJECTIVES:Previous studies have shown that germ-like cells can be induced from human umbilical cord mesenchymal stem cell (hUC-MSCs) in vitro. However, induction efficiency was low and a stable system had not been built. CD61, also called integrin-?3, plays a significant role in cell differentiation, in that CD61-positive-cell-derived pluripotent stem cells easily differentiate into primordial germ-like cells (PGC). Here, we have explored whether overexpression of CD61 would promote hUC-MSC differentiation into PGC and male germ-like cells. MATERIALS AND METHODS:hUC-MSCs were cultured and transduced using pCD61-CAGG-TRIP-pur (oCD61) and pTRIP-CAGG plasmid (Control), and hUC-MSCs overexpressed CD61 were induced by bone morphogenetic protein 4 (BMP4, 12.5 ng/ml), to differentiate into PGC and male germ cells. Quantitative real-time PCR (RT-qPCR), western blotting and immunofluorescence staining were used to examine PGC- and germ cell-specific markers. RESULTS:High expression levels of PGC-specific markers were detected in oCD61 hUC-MSCs compared to controls. After BMP4 induction, expression levels of male germ cell markers such as Acrosin (ACR), Prm1 and meiotic markers including Stra8, Scp3 in oCD61 were significantly higher than those of the Control group. CONCLUSIONS:Under induction of BMP4, CD61-overexpressing hUC-MSCs, which had turned into PGC-like cells, could be further differentiated into male germ-like cells. Thus, a simple and efficient approach to study male germ cell development by using hUC-MSCs has been established.

SUBMITTER: Li B

PROVIDER: S-EPMC6496844 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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BMP4 promotes SSEA-1(+) hUC-MSC differentiation into male germ-like cells in vitro.

Project description:ObjectivesRecent studies have demonstrated that primordial germ cells (PGC) can be differentiated from human umbilical cord mesenchymal stem cells (hUC-MSCs), and embryonic stem cells (ESCs) in vitro. Nevertheless, efficiencies were low and unstable. Here, whether hUC-MSCs can be induced to differentiate into germ-like cells with the aid of bone morphogenetic protein (BMP4) was investigated.Materials and methodsHuman umbilical cord mesenchymal stem cells were freshly isolated and cultured with BMP4. SSEA-1(+/-) cells were purified using magnetic-activated cell sorting (MACS) from the hUC-MSCs, and further induced with BMP4. Quantitative real-time PCR (qRT-PCR) and immunofluorescence analysis were used to determine PGC and germ-like cell-specific markers.ResultsHuman umbilical cord mesenchymal stem cells differentiated into SSEA-1(+) spherical PGC-like cells efficiently with 12.5 ng/ml BMP4. qRT-PCR and immunofluorescence analysis demonstrated that SSEA-1(+) cells expressed higher levels of PGC-specific markers than SSEA-1(-) cells. Furthermore, SSEA-1(+) cells were induced with BMP4 to differentiate into STRA8, SCP3, DMRT1 and PLZF-positive male germ-like cells, and some sperm-like cells were obtained by 7-14 days after induction.ConclusionThese results suggest that SSEA-1(+) hUC-MSCs can differentiate into male germ-like cells in the presence of BMP4. This study provides an efficient protocol to study germ-cell development using hUC-MSCs.

| S-EPMC6495181 | biostudies-literature

CD61 promotes the differentiation of canine ADMSCs into PGC-like cells through modulation of TGF-β signaling.

Project description:Previous studies have shown that CD61 (integrin-β3) promotes the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into germ-like cells. However, the mechanism remains unclear. In this study, we showed that overexpression of CD61 in canine adipose-derived mesenchymal stem cells (cADMSCs) promotes their differentiation into primordial germ cell (PGC)-like cells. Quantitative real-time PCR, immunocytochemistry and western blot detected higher levels of PGC-specific markers in CD61-overexpressed cADMSCs compared with those in control cells. Moreover, phosphorylation of Smad2, a downstream mediator of transforming growth factor beta (TGF-β), was increased in CD61-overexpressed cADMSCs than that in control cells. However, the expression of PGC-specific markers was downregulated in cADMSCs treated with a TGF-β inhibitor. These results suggested that CD61 could induce cADMSCs to differentiate into PGC-like cells by relying on the activation of TGF-β pathway. ADMSCs possess a considerable potential in treating the infertility of rare animal species.

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3D hUC-MSC spheroids exhibit superior resistance to autophagy and apoptosis of granulosa cells in POF rat model.

Project description:In briefThis study reveals that orthotopic transplantation of 3D hUC-MSC spheroids is more effective than monolayer-cultured hUC-MSCs in improving POF and distinctly reducing oxidative stress through the paracrine effect, thereby preventing apoptosis and autophagy of GCs.AbstractPremature ovarian failure (POF) is a common reproductive disease in women younger than 40 years old, and studies have demonstrated that the application of human umbilical cord mesenchymal stem cells (hUC-MSCs) is a promising therapy strategy for POF. Given the previously established therapeutic advantages of 3D MSC spheroids, and to evaluate their effectiveness, both 3D hUC-MSC spheroids and monolayer-cultured hUC-MSCs were employed to treat a cyclophosphamide-induced POF rat model through orthotopic transplantation. The effects of these two forms on POF were subsequently assessed by examining apoptosis, autophagy, and oxidative damage in ovarian granulosa cells (GCs). The results indicated that hUC-MSC spheroids exhibited superior treatment effects on resisting autophagy, apoptosis, and oxidative damage in GCs compared to monolayer-cultured hUC-MSCs. To further elucidate the impact of hUC-MSC spheroids in vitro, a H2O2-induced KGN cells model was established and co-cultured with both forms of hUC-MSCs. As expected, the hUC-MSC spheroids also exhibited superior effects in resisting apoptosis and autophagy caused by oxidative damage. Therefore, this study demonstrates that 3D hUC-MSC spheroids have potential advantages in POF therapy; however, the detailed mechanisms need to be further investigated. Furthermore, this study will provide a reference for the clinical treatment strategy of POF.

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Differentiation of murine male germ cells to spermatozoa in a soft agar culture system.

Project description:Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

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SPAG17 Is Required for Male Germ Cell Differentiation and Fertility.

Project description:Spag17 encodes a protein present in the axoneme central pair complex of motile cilia and flagella. A mutation in this gene has been reported to be associated with infertility caused by defects in sperm motility. Here, we report that Spag17 knockout mice are infertile because of a severe defect in spermatogenesis. The histological evaluation of testis sections from mutant mice revealed seminiferous tubules with spermatogenesis arrested at the spermatid stage and cell debris in the cauda epididymis. The few sperm collected from the cauda epididymis were immotile and displayed abnormal tail and head morphology. Immunofluorescence analysis of Spag17 knockout germ cells showed spermatids with abnormally long manchette structures and morphological defects in the head. Electron microscopy showed altered manchette microtubules, reduced chromatin condensation, irregular nuclear shape, and detached acrosomes. Additionally, the transport of proteins (Pcdp1 and IFT20) along the manchette microtubules was disrupted in the knockout elongating spermatids. Our results show for the first time that Spag17 is essential for normal manchette structure, protein transport, and formation of the sperm head and flagellum, in addition to its role in sperm motility.

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Role of ?-catenin in post-meiotic male germ cell differentiation.

Project description:Though roles of ?-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that ?-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of ?-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of ?-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that ?-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since ?-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell ?-catenin complex to ?-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.

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Selection of the Inducer for the Differentiation of Chicken Embryonic Stem Cells into Male Germ Cells In Vitro.

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| S-EPMC5065142 | biostudies-literature

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