Jinlian Hua

miR‐34c works downstream of p53 leading to dairy goat male germline stem‐cell (mGSCs) apoptosis

Recent lines of evidence have indicated that miR‐34c can play important roles in regulation of the cell cycle, cell senescence and apoptosis of mouse and human tumour cells, spermatogenesis, and male germ‐cell apoptosis. However, there is little information on the effects of miR‐34c on proliferation and apoptosis of livestock male germ cells. The dairy goat is a convenient domestic species for biological investigation and application. The purpose of this study was to investigate the effects of miR‐34c on apoptosis and proliferation of dairy goat male germline stem cells (mGSCs), as well as to determine the relationship between p53 and miR‐34c in this species.

Characterization of immortalized mesenchymal stem cells derived from foetal porcine pancreas

Islet replacement therapy is limited by shortage of donor islet cells. Usage of islet cells derived from porcine pancreatic stem cells (PSCs) is currently viewed as the most promising alternative for human islet transplantation. However, PSCs are rare and have a finite proliferative lifespan. In this study, we isolated and established an immortalized mesenchymal stem cell (MSC) line derived from foetal porcine pancreas, by transfecting human telomerase reverse transcriptase (hTERT) and called these immortalized pancreatic mesenchymal stem cells (iPMSCs). The iPMSCs have been cultured for more than 80 passages and have capacity to differentiate into neurons, cardiomyocytes, germ cells and islet‐like cells, analysed by morphology, RT‐PCR, western blotting, immunofluorescence, immunocytochemistry and transplantation assay. Islets derived from iPMSCs reversed hyperglycaemia in streptozotocin‐induced diabetic mice and secreted insulin and C‐peptide in vitro. These results demonstrated that iPMSCs might provide unlimited resources for islet replacement therapy and models for functional cell differentiation.

Characterization of female germ‐like cells derived from mouse embryonic stem cells through expression of GFP under the control of Figla promoter

Previous studies have demonstrated that germ cells can be derived from mouse embryonic stem cells (ESCs). However, there is still no efficient system, which can visualize the stage of germ cell specification in vitro, and further to identify and enrich germ cells derived from ESCs. Figla (factor in the germline, alpha) gene encodes a germ cell specific transcription factor that coordinates the expression of the oocyte‐specific zona pellucida (Zp) genes and is essential for folliculogenesis in mouse. Here, we first constructed a pFigla‐EGFP recombinant plasmid that expressed enhanced green fluorescent protein (EGFP) under the control of Figla promoter, and generated and characterized an ESC line stably carrying this pFigla‐EGFP reporter construct. Then the ESCs were induced to differentiate into female germ‐like cells by culturing adherent embryoid bodies (EBs) in retinoic acid (RA) induction medium or transplanting ESCs under the kidney capsule with ovarian cells. A population of differentiated ESCs expressed GFP, and these cells were analyzed by RT‐PCR and immunofluorescence. The GFP positive cells showed the expression of germ cell markers Vasa, meiotic specific gene Stra8, Scp3, oocyte markers Gdf9, Zp3 and Figla, indicating that this method could be used for the purification and selection of female germ cells. Our study establishes a new selective system of female germ‐like cell derivation and offers an approach for further research on the development and the differentiation of germ cells derived from stem cells. J. Cell. Biochem. 113: 1111–1121, 2012.

GSK3 inhibitor-BIO regulates proliferation of female germline stem cells from the postnatal mouse ovary

It is widely believed that in most female mammalian neonates, all germ cells enter meiosis to form the primary oocyte at the end of foetal development, and as a result, the postnatal mammalian ovary harbours only a limited supply of oocytes that cannot be regenerated. However, this idea has been challenged by the discovery of the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries.

Retinol (vitamin A) maintains self‐renewal of pluripotent male germline stem cells (mGSCs) from adult mouse testis

Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum‐free and feeder‐free) system for the successful culture of adult germline stem cells from adult Kunming mice (8–24 weeks) testis. The isolated mGSCs cultured in RE serum‐free and feeder‐free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT‐PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self‐renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum‐free and feeder‐free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility. J. Cell. Biochem. 112: 1009–1021, 2011.

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.

Rapamycin inhibits acrolein-induced apoptosis by alleviating ROS-driven mitochondrial dysfunction in male germ cells.

Acrolein (Acr) is a highly reactive α, β‐unsaturated aldehyde, which can induce reactive oxygen species (ROS) generation. Several factors, including lipid peroxidation, clinical use of cyclophosphamide, fried foods, automobile exhausts, smoking and aging can increase its concentration in blood serum. Mounting evidence has suggested that Acr‐induced ROS might reduce quality of sperm. Thus, the aim of this study was to examine reproductive toxicity of Acr‐caused ROS in vitro and find a means to alleviate it.

CD49f-Positive Testicular Cells in Saanen Dairy Goat Were Identified as Spermatogonia-Like Cells by miRNA Profiling Analysis

miRNAs, a type of small RNA, play critical roles in mammalian spermatogenesis. Spermatogonia are the foundation of spermatogenesis and are valuable for the study of spermatogenesis. However, the expression profiling of the miRNAs in spermatogonia of dairy goats remains unclear. CD49f has been one of the surface markers used for spermatogonia enrichment by magnetic activated cell sorting (MACS). Therefore, we used a CD49f microbead antibody to purify CD49f‐positive and ‐negative cells of dairy goat testicular cells by MACS and then analysed the miRNA expression in these cells in depth using Illumina sequencing technology. The results of miRNA expression profiling in purified CD49f‐positive and ‐negative testicular cells showed that 933 miRNAs were upregulated in CD49f‐positive cells and 916 miRNAs were upregulated in CD49f‐negative cells with a twofold increase, respectively; several miRNAs and marker genes specific for spermatogonial stem cells (SSCs) in testis had a higher expression level in CD49f‐positive testicular cells, including miR‐221, miR‐23a, miR‐29b, miR‐24, miR‐29a, miR‐199b, miR‐199a, miR‐27a, and miR‐21 and CD90, Gfra1, and Plzf. The bioinformatics analysis of differently expressed miRNAs indicated that the target genes of these miRNAs in CD49f‐positive cells were involved in cell‐cycle biological processes and the cell‐cycle KEGG pathway. In conclusion, our comparative miRNAome data provide useful miRNA profiling data of dairy goat spermatogonia cells and suggest that CD49f could be used to enrich dairy goat spermatogonia‐like cells, including SSCs. J. Cell. Biochem. 115: 1712–1723, 2014.

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.

PRMT5 enhances generation of induced pluripotent stem cells from dairy goat embryonic fibroblasts via down‐regulation of p53

Protein arginine methyltransferase 5 (PRMT5), is thought to play a role in epigenetic reprogramming of mouse germ cells. However, up to now there has been little information concerning its expression profile and effects on generation of induced pluripotent stem cells (iPSCs) from somatic cells, in livestock. Here, we have explored PRMT5 expression profiles in dairy goats and its consequences to derivation of iPSCs from dairy goat embryonic fibroblasts (GEFs).