Weishuai Liu

Pluripotent male germline stem cells from goat fetal testis and their survival in mouse testis.

Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis, and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4, Sox2, C-myc, and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3, STRA8, and germ cell marker-VASA, and haploid marker-FE-J1, PRM1, indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However, most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice, and showed that mGSCs can colonize, self-renew, and differentiate into germ cells. Thus, we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro, providing a promising platform for generation of transgenic goat for production of specific humanized proteins.

Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs)

Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over‐expression of Boule promoted the expression of meiosis‐related genes in dairy goat mGSCs. The expression of Stra8 was up‐regulated by over‐expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up‐regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over‐expression of Boule may promote spermatogenesis and meiosis in dairy goat. J. Cell. Biochem. 114: 294–302, 2013.

Promyelocytic leukaemia zinc finger maintains self-renewal of male germline stem cells (mGSCs) and its expression pattern in dairy goat testis.

Previous studies have shown that promyelocytic leukaemia zinc finger (PLZF) is a spermatogonia‐specific transcription factor in the testis, required to regulate self‐renewal and maintenance of the spermatogonia stem cell. Up to now, expression and function of PLZF in the goat testis has not been known. The objectives of this study were to investigate PLZF expression pattern in the dairy goat and its effect on male goat germline stem cell (mGSC) self‐renewal and differentiation.

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

Recent 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.

Gender depended potentiality of differentiation of human umbilical cord mesenchymal stem cells into oocyte-Like cells in vitro.

Recent studies have demonstrated that germ‐like cells could be differentiated from human umbilical cord mesenchymal stem cells (hUC‐MSCs) in vitro. Whether the sexuality of hUC‐MSCs affects the formation efficiency of germ‐like cells derived from hUC‐MSCs is still unclear. To clearly test the formation efficiency of oocyte‐like cells from male and female hUC‐MSCs, obtained hUC‐MSCs were induced by 20% follicular fluid (FF) according to the method that has been proved by our previous studies. Results showed that hUC‐MSCs differentiated into oocyte‐like structures and expressed germ cell makers. It was noted that the presence of advanced oocyte‐like cells in male hUC‐MSCs (m‐hUC‐MSCs) was similar as that in female hUC‐MSCs (f‐hUC‐MSCs); however, the expression of germ cells specific markers in m‐hUC‐MSCs was delayed compared with that in f‐hUC‐MSCs. In addition, immunofluorescence analysis demonstrated that germ cell–specific markers, Oct4, Vasa, Dazl, ZP2, ZP3 and Stra8, were expressed on the 14th day after induction in both f‐hUC‐MSCs and m‐hUC‐MSCs. However, the size of oocyte‐like cells from f‐hUC‐MSCs was larger than that in m‐hUC‐MSCs. The level of secreted oestradiol was significantly higher in f‐hUC‐MSCs than m‐hUC‐MSCs. We sought to determine whether critical germ cells transcription factor‐Figlα will promote the development of oocyte‐like cells. Some germ cell–specific markers were increased when exogenous Figlα was transfected into hUC‐MSCs. This process implied that germ‐like cells might be produced by over‐expression of exogenous germ cell–specific gene, and this process was similar as that in production of germ cells in induced pluripotent stem cells (iPSCs). Finally, to verify the feasibility that hUC‐MSCs differentiate into germ cells, hUC‐MSCs were transplanted into seminiferous tubules and kidney capsule of mouse, respectively, and we found the transplanted cells differentiated into germ‐like cells in recipients seminiferous tubules and kidney capsule. This study will provide a simple model to study mammalian germ cell specification using hUC‐MSCs in vitro. Copyright

Expression profile of Nanos2 gene in dairy goat and its inhibitory effect on Stra8 during meiosis.

Nanos2, an RNA‐binding protein, belongs to the Nanos gene‐coding family and contains two CCHC zinc‐finger motifs. In mouse, it plays a pivotal role in male germ cell development, and self‐renewal of spermatogonial stem cells. However, little is known of its expression pattern and functions in dairy goat testis.

Location and characterization of female germline stem cells (FGSCs) in juvenile porcine ovary

Existence of germline stem cells (GSCs) in juvenile mammalian female ovaries has been drastically debated recently since reports that adult mouse ovaries still have mitotically active germ cells have been proposed. In addition, definitive location of such female germline stem cells (FGSCs) had not been demonstrated.

Transplantation of Goat Bone Marrow Mesenchymal Stem Cells (gMSCs) Help Restore Spermatogenesis in Endogenous Germ Cells-Depleted Mouse Models

Mesenchymal stem cells (MSCs) derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs (gMSCs) and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these cells were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid (RA), as analysed by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.

Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

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

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‐β3, 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.