Sha Peng

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.

GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)

Background The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present. Results To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis. Conclusions These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs.

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.

Reversine promotes porcine muscle derived stem cells (PMDSCs) differentiation into female germ‐like cells

A small molecular chemical‐Reversine has been shown to promote cell reprogramming and induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, and then differentiate into other cell types within mesodermal lineages as well as neuroectodermal. However, the possibilities of these cells to give rise to germ cell lineages have not been examined. The objective of the current study was to detect the effect of Reversine on PMDSCs differentiation into germ cells. PMDSCs from fetal porcine skeletal muscle and their potential of differentiation into germ cells in vitro were investigated. The phenotype, proliferation potential, characteristic markers of the first adhesion cells (pp1), and the purified 2 times cells (pp3) were analyzed by growth curve, FACS, and RT‐PCR, respectively. Then, the purified cells were induced with 10% or 20% bovine follicular fluid (FF), the results showed that some of the induced pp3 cells were similar as porcine oocyte, and expressed germ cell and oocyte markers analyzed by semi‐quantitative RT‐PCR and immunofluorescent staining. Reversine clearly increased the potentiality of PMDSCs differentiation into large round germ‐like cells in FF induction medium analyzed by morpholgogy, QRT‐PCR and immunofluoresce. The BrdU labeled PMDSCs might differentiate into female germ‐like cells in recipients kidney capsule, which were positive for germ cell and meiotic markers (Dazl, Vasa, Figla, Stra8, Scp3) and oocyte markers (Zp2, Zp3). These findings provided an efficient model to study the mechanism of cell proliferation and germ cell differentiation in livestock promoted by Reversine. J. Cell. Biochem. 113: 3629–3642, 2012.

Platelet-derived growth factor promotes the proliferation of human umbilical cord-derived mesenchymal stem cells

This study was designed to investigate the effect of platelet‐derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC‐MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC‐MSCs. The human UC‐MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT‐PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real‐time (QRT‐PCR). The results showed that PDGF could promote the proliferation of UC‐MSCs in vitro in a dose‐dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC‐MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC‐MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF‐treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation‐related genes C‐MYC, PCNA and TERT and cell cycle–related genes cyclin A, cyclin 1 and CDK2 were up‐regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT‐PCR. The PDGF could promote the proliferation of human UC‐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.

miR-375 controls porcine pancreatic stem cell fate by targeting 3-phosphoinositide–dependent protein kinase-1 (Pdk1)

miR‐375 is one of the highly expressed microRNAs (miRNAs) found in pancreatic islets of both humans and mice. In this study, we investigated functions of miRNA miR‐375 in porcine pancreatic stem cells (PSC).

Canonical Wnt signaling pathway contributes to the proliferation and survival in porcine pancreatic stem cells (PSCs)

Pancreatic stem cells (PSCs) transplantation is a potential therapeutic approach to type 1 diabetes mellitus (D1M). However, before clinical use, there are some major hurdles to be faced that need to be comprehensively considered and given some potential solutions in vitro. Human PSCs are difficult to obtain and have a short replicative senescence. As an alternative, we instead established porcine PSCs; as insulin is highly conserved and physiological glucose levels are similar between human and porcine. In order to solve the problems during transplantation therapy, such as the need for an enormous amount of PSCs and good cell survival in overactive autoimmunity induced by reactive oxygen cpecies (ROS) in D1M patients, we utilized Wnt3a overexpression to activate the canonical Wnt signaling pathway in PSCs. We found that the expression of proliferation genes, such as c-Myc, was up-regulated as the downstream of β-catenin, which promoted the PSCs proliferation and made cell numbers to meet the transplantation needs. We also showed that activation of the Wnt pathway made cells more readily tolerate ROS-caused mitochondria injury and cell apoptosis, thus making cells survive in autoimmune patients. The present study provides a theoretical basis for cell transplantation therapy of diabetes.