PAX7 promotes CD49f-positive dairy goat spermatogonial stem cells' self-renewal.

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs.

The Regulatory Functionality of Exosomes Derived from hUMSCs in 3D Culture for Alzheimer's Disease Therapy.

Reducing amyloid-ß (Aß) accumulation could be a potential therapeutic approach for Alzheimer's disease (AD). Particular functional biomolecules in exosomes vested by the microenvironment in which the original cells resided can be transferred to recipient cells to improve pathological conditions. However, there are few reports addressing whether exosomes derived from cells cultured on scaffolds with varying dimension can reduce Aß deposition or ameliorate cognitive decline for AD therapy. Herein, both 3D graphene scaffold and 2D graphene film are used as the matrix for human umbilical cord mesenchymal stem cell culture, from which the supernatants are obtained to isolate exosomes. The levels of 195 kinds of miRNAs and proteins, including neprilysin, insulin-degrading enzyme and heat shock protein 70, in 3D-cultured exosomes (3D-Exo) are dramatically different from those obtained from 2D culture. Hence, 3D-Exo could up-regulate the expression of α-secretase and down-regulate the ß-secretase to reduce Aß production in both AD pathology cells and transgenic mice, through their special cargo. With rescuing Aß pathology, 3D-Exo exerts enhanced therapeutic effects on ameliorating the memory and cognitive deficits in AD mice. These findings provide a novel clinical application for scaffold materials and functional exosomes derived from stem cells.

miR-19b-3p induces cell proliferation and reduces heterochromatin-mediated senescence through PLZF in goat male germline stem cells.

Promyelocytic leukemia zinc finger PLZF, known as ZBTB16 or ZFP145 is a critical zinc finger protein of male germline stem cells (mGSCs), it's an essential transcriptional factor for goat testis development and spermatogenesis. Loss of PLZF results in progressive depletion of SSCs after the first wave of spermatogenesis leading to eventual spermatogenic arrest, apparently the result of a shift in the balance in SSC fate away from self-renewal and toward differentiation. Cumulating evidences have demonstrated that microRNAs are expressed in a cell-specific or stage-specific manner during spermatogenesis and acts as regulators on specific makers such as Stra8, ETV5, and PLZF. However, the post transcriptional function of PLZF still poorly elucidate in mGSCs. Bioinformatic analysis and dual luciferase reporter assay showed that miR-19b-3p binds the 3'UTR of PLZF, suggesting that PLZF is a direct target of miR-19b-3p. The profile of miR-19b-3p and PLZF analyzed in dairy goat testis at different age showed that miR-19b-3p was significantly up-regulated in goat testis at 1, 3, 6 months and downregulated at 12, 18, and 24 months which was inversely correlated with PLZF in the same testis. Focusing on the role of miR-19b-3p, we found that miR-19b-3p changes c-KIT and mTOR signaling through PLZF to promote proliferation in goat nGSCs and infertile mice testes. Over-expression of PLZF significantly reversed miR-19b-3p-mediated proliferation in mice testes. We found also that miR-19b-3p reduced heterochromatin-mediated senescence through PLZF localized on HP1α. Taken together, our findings indicate that miR-19b-3p promotes proliferation and reduces heterochromatin-mediated senescence through PLZF in mGSCs.

Melatonin Relieves Busulfan-Induced Spermatogonial Stem Cell Apoptosis of Mouse Testis by Inhibiting Endoplasmic Reticulum Stress.

BACKGROUND/AIMS: Busulfan is commonly used for cancer chemotherapy. Although it has the advantage of increasing the survival rate of patients, it can cause male infertility via damaging the testes and reducing sperm counts. Therefore, the underlying mechanism should be explored, and new agents should be developed to protect the male reproductive system from busulfan-induced damage. Endoplasmic reticulum stress (ERS) is considered a key contributor to numerous pathologies. Despite several studies linking ERS to toxicants, studies have yet to determine whether ERS is a contributing factor to busulfan-induced testicular damage. Melatonin is a well-known broad-spectrum antioxidant, anti-inflammatory and antitumour agent, but the effects of melatonin on busulfan-induced ERS in mouse testes damage are less documented. METHODS: The effects of melatonin were measured by immunofluorescence staining, Western blot, qRT-PCR analysis and flow cytometry assay. The underlying mechanism was investigated by measuring ERS. RESULTS: We found that ERS was strongly activated in mouse testes (in vivo) and the C18-4 cell line (in vitro) after busulfan administration. ERS-related apoptosis proteins such as caspase-12, CHOP and caspase-3 were activated, and the expression of apoptotic proteins such as P53 and PUMA were upregulated. Furthermore, we investigated whether melatonin reduced the extent of damage to mouse testes and improved the survival rates of busulfan-treated mice. When exploring the underlying mechanisms, we found melatonin could counteract ERS by decreasing the expression levels of the ERS markers GRP78, ATF6, pIRE1 and XBP1 in mouse testes and mouse SSCs (C18-4 cells). Moreover, it blocked the activation of ERS-related apoptosis proteins caspase-12, CHOP and caspase-3 and suppressed P53 and PUMA expression stimulated by busulfan both in vivo and in vitro. CONCLUSION: Our results demonstrate that ERS is an important mediator for busulfan-induced apoptosis. The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage. Thus, this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment.

PLZF-Induced Upregulation of CXCR4 Promotes Dairy Goat Male Germline Stem Cell Proliferation by Targeting Mir146a.

Previous studies have shown that promyelocytic leukemia zinc finger (PLZF), chemokine (C-X-C motif) receptor 4 (CXCR4) and mir146a were associated with the self-renewal of mouse spermatogonial stem cells (SSCs); however, there is little information on their effects on the fate of livestock SSCs. Here, we have identified a regulatory pathway in dairy goat mGSCs, involving PLZF, mir146a and the SDF-1 receptor CXCR4. PLZF overexpression downregulated mir146a and simultaneously upregulated the expression of CXCR4 protein, whereas PLZF knockdown (siPLZF) induced the specifically opposite effects. The in vitro assays demonstrated that PLZF specifically interacts with and suppresses the mir146a promoter, and mir146a targets CXCR4 to impede its translation. The levels of ERK1/2 phosphorylation in the mGSCs overexpressed CXCR4 and PLZF were upregulated, respectively, whereas mir146a expression was decreased and CXCR4 protein was increased. Mir146a overexpression and siPLZF impaired mGSC proliferation and differentiation, however, Mir146a knockdown induced the opposite effects. The effects of PLZF and mir146a were mediated regulation by mir146a and CXCR4, respectively. Overexpression of CXCR4 or addition of CXCL12 in cultures of dairy goat mGSCs resulted in the upregulation of their signaling, and the phosphorylation of ERK1/2 was increased. Collectively, these findings indicate that PLZF is an important transcription factor in the regulation of the expression of CXCR4 to promote dairy goat mGSC proliferation by targeting mir146a.

miR-544 Regulates Dairy Goat Male Germline Stem Cell Self-Renewal via Targeting PLZF.

The balance between the self-renewal and differentiation of male germline stem cells (mGSCs) is critical for the initiation and maintenance of mammalian spermatogenesis. The promyelocytic leukemia zinc finger (PLZF), a zinc finger protein, is a critical factor for maintaining the self-renewal of mGSCs, so, evaluation of the PLZF pathway in mGSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for the self-renewal and differentiation of mGSCs; however, there is currently no data indicating that which miRNA regulate the self-renewal and differentiation of mGSCs via PLZF. Here, we predicted the prospective miRNA targeting to PLZF using the online Bioinformatics database-Targetscan, and performed an analysis of the dual-luciferase recombinant vector, psiCHCEKTM-2-PLZF-3'UTR. miR-544 mimics (miR-544m), miR-544 inhibitors (miR-544i), Control (NC, scrambled oligonucleotides transfection), pPLZF-IRES2-EGFP or PLZF siRNA were transfected into mGSCs; the cells proliferation was evaluated by BRDU incorporation assay and flow cytometry, and the mGSC marker, GFRa1, PLZF, KIT, DAZL, and VASA expression were analyzed by RT-qPCR, immunofluorescence and Western blot. The results showed that miR-544 regulates dairy goat male germline stem cell self-renewal via targeting PLZF. Our study identifies a new regulatory pathway for PLZF and expands upon the PLZF regulatory network in mGSCs.

Amorphous curcumin-based hydrogels to reduce the incidence of post-surgical intrauterine adhesions.

The incidence of intrauterine adhesions (IUA) has increased with the rising utilization of intrauterine surgery. The postoperative physical barrier methods commonly used, such as balloons and other fillers, have limited effectiveness and may even cause further damage to the remaining endometrial tissue. Herein, we developed an injectable thermosensitive hydrogel using Pluronic F127/F68 as pharmaceutical excipients and curcumin as a natural active molecule. The hydrogel effectively addresses solubility and low bioavailability issues associated with curcumin. In vitro, drug release assays revealed that the amorphous curcumin hydrogel promotes dissolution and sustained release of curcumin. In vitro experiments reveal high biocompatibility of the hydrogel and its ability to enhance vascular formation while inhibiting the expression of fibrotic factor TGF-ß1. To assess the effectiveness of preventing IUAs, in vivo experiments were conducted using IUA rats and compared with a class III medical device, a new-crosslinked hyaluronic acid (NCHA) gel. According to the study, curcumin hydrogel is more effective than the NCHA group in improving the regeneration of the endometrium, increasing the blood supply to the endometrium and reducing the abnormal deposition of fibrin, thus preventing IUA more effectively. This study provides a promising strategy for treating and preventing IUA.

PLZF protein forms a complex with protein TET1 to target TCF7L2 in undifferentiated spermatogonia.

The transcription factor promyelocytic leukemia zinc finger (PLZF, also known as ZBTB16) is critical for the self-renewal of spermatogonial stem cells (SSCs). However, the function of PLZF in SSCs is not clear. Here, we found that PLZF acted as an epigenetic regulator of stem cell maintenance and self-renewal of germ cells. The PLZF protein interacts with the ten-eleven translocation 1 (TET1) protein and subsequently acts as a modulator to regulate the expression of self-renewal-related genes. Furthermore, Transcription Factor 7-like 2 (TCF7L2) is promoted by the coordination of PLZF and Tri-methylation of lysine 4 on histone H3 (H3K4me3). In addition, testicular single-cell sequencing indicated that TCF7L2 is commonly expressed in the PLZF cluster. We demonstrated that PLZF directly targets TCF7L2 and alters the landscape of histone methylation in the SSCs nucleus. Meanwhile, the RD domain and Zn finger domain of PLZF synergize with H3K4me3 and directly upregulate TCF7L2 expression at the transcriptional level. Additionally, we identified a new association between PLZF and the histone methyltransferase EZH2 at the genomic level. Our study identified a new association between PLZF and H3K4me3, established the novel PLZF&TET1-H3K4me3-TCF7L2 axis at the genomic level which regulates undifferentiated spermatogonia, and provided a platform for studying germ cell development in male domestic animals.

A precise design strategy for a cell-derived extracellular matrix based on CRISPR/Cas9 for regulating neural stem cell function.

The extracellular matrix (ECM) is a natural microenvironment pivotal for stem cell survival, as well as proliferation, differentiation and metastasis, composed of a variety of biological molecular complexes secreted by resident cells in tissues and organs. Heparan sulfate proteoglycan (HSPG) is a type of ECM protein that contains one or more covalently attached heparan sulfate chains. Heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains of great importance in development and normal physiology. Herein, we constructed endogenous HSPG2 overexpression in mouse embryonic fibroblasts based on the CRISPR/Cas9 synergistic activation mediator system and then fabricated a cell-derived HSPG2 functional ECM (ECMHSPG2). The ECMHSPG2 is capable of enriching basic fibroblast growth factor (bFGF), which binds more strongly than the negative control ECM. With a growing bFGF concentration, ECMHSPG2 could better maintain neural stem cell (NSCs) stemness and promote NSC proliferation and differentiation in culture. These findings provide a precise design strategy for producing a specific cell-derived ECM for biomaterials in research and regenerative medicine.

High-efficiency brain-targeted intranasal delivery of BDNF mediated by engineered exosomes to promote remyelination.

The regeneration of myelin sheaths is the ultimate goal of the treatment of demyelination disease, including multiple sclerosis (MS). However, current drugs for MS mainly target the immune system and can only slow down the disease development and do not promote the differentiation of oligodendrocyte precursor cells (OPCs) abundant in the myelin injury region into mature oligodendrocytes to form a new myelin sheath. Brain-derived neurotrophic factor (BDNF) plays an important role in the regulation of OPC proliferation and differentiation into mature oligodendrocytes. Exosomes, a kind of nanoscale membrane vesicle secreted by cells, can be used as potential therapeutic drug delivery vectors for central nervous system diseases. Here, brain-targeted modification and BDNF intracellular-loaded exosomes were produced through engineering HEK293T cells, which can promote the differentiation of OPCs into mature oligodendrocytes in vitro. The intranasal administration of the brain-targeted engineered exosome-mediated BDNF was a highly effective delivery route to the brain and had a significant therapeutic effect on remyelination and motor coordination ability improvement in demyelination model mice. The combination of intranasal administration with brain-targeted and BDNF-loaded designed exosomes provides a strategy for efficient drug delivery and treatment of central nervous system diseases.

Specific anti-glioma targeted-delivery strategy of engineered small extracellular vesicles dual-functionalised by Angiopep-2 and TAT peptides.

Glioma is one of the primary malignant brain tumours in adults, with a poor prognosis. Pharmacological reagents targeting glioma are limited to achieve the desired therapeutic effect due to the presence of blood-brain barrier (BBB). Effectively crossing the BBB and specifically targeting to the brain tumour are the major challenge for the glioma treatments. Here, we demonstrate that the well-defined small extracellular vesicles (sEVs) with dual-targeting drug delivery and cell-penetrating functions, modified by Angiopep-2 and trans-activator of transcription peptides, enable efficient and specific chemotherapy for glioma. The high efficiency of engineered sEVs in targeting BBB and glioma was assessed in both monolayer culture cells and BBB model in vitro, respectively. The observed high targeting efficiency was re-validated in subcutaneous tumour and orthotopic glioma mice models. After loading the doxorubicin into dual-modified functional sEVs, this specific dual-targeting delivery system could cross the BBB, reach the glioma, and penetrate the tumour. Such a mode of drug delivery significantly improved more than 2-fold survival time of glioma mice with very few side effects. In conclusion, utilization of the dual-modified sEVs represents a unique and efficient strategy for drug delivery, holding great promise for the treatments of central nervous system diseases.

Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis.

Enhancing neurogenesis of neural stem cells (NSCs) is crucial in stem cell therapy for neurodegenerative diseases. Within the extracellular microenvironment, extracellular matrix (ECM) plays a pivotal role in modulating cell behaviors. However, a single ECM biomaterial is not sufficient to establish an ideal microenvironment. As multifunctional nanocarriers, exosomes display tremendous advantages for the treatments of various diseases. Herein, collagen binding domain peptide-modified exosomes (CBD-Exo) were obtained from the SH-SY5Y cell line infected with lentivirus particles encoding CBD-lysosome associated membrane glycoprotein 2b (CBD-Lamp2b) to improve the binding efficiency of exosomes and ECM. An exosomes-functionalized ECM (CBD-Exo/ECM) was then constructed via the interaction between CBD and collagen in ECM. Then, CBD-Exo/ECM was employed as a carrier for NSCs culture. The results showed that CBD-Exo/ECM can support the neurogenesis of NSCs with the percentage of proliferation marker EdU-positive (35.8% ± 0.47% vs 21.9% ± 2.32%) and neuron maker Tuj-1-positive (55.8% ± 0.47% vs 30.6% ± 2.62%) were both significantly increased in the exosomes-functionalized ECM system. This exosomes-functionalized ECM was capable to promote the cell proliferation and accelerate neuronal differentiation of NSCs, providing a potential biomedical material for stem cell application in tissue engineering and regenerative medicine.

Strategy for Designing a Cell Scaffold to Enable Wireless Electrical Stimulation for Enhanced Neuronal Differentiation of Stem Cells.

Electrical stimulation (ES) offers significant advantages in modulating the behavior of stem cells on conductive scaffolds for neural tissue engineering. However, it is necessary to realize wireless ES to avoid the use of external wires in tissues. Thus, herein, a strategy is reported to develop a stem cell scaffold that allows wireless ES. A conductive annular graphene substrate is designed and grown by chemical vapor deposition; this substrate is used as a secondary coil to achieve wireless ES via electromagnetic induction in the presence of a primary coil. The substrate shows excellent biocompatibility for the culture of neural stem cells (NSCs). The results indicate that the applied wireless ES enhances neuronal differentiation, facilitates the formation of neurites, and does not substantially affect the viability and stemness maintenance of NSCs. Collectively, this system provides a strategy for achieving synergy between wireless ES and conductive scaffolds for neural regenerative medicine, which can be further utilized for the regeneration of other tissues.

Construction of a graphene/polypyrrole composite electrode as an electrochemically controlled release system.

A new class of stimuli responsive drug delivery systems is emerging to establish new paradigms for enhancing therapeutic efficacy. To date, most electro-responsive systems rely on noble metal electrodes that likely cause the limitations for implantation applications. Herein, a graphene/polypyrrole composite electrode (GN-PPy-FL) was fabricated based on two-dimensional (2D) graphene (GN) film and conductive and biocompatible polypyrrole (PPy) nanoparticles loaded with a negative drug model of fluorescein sodium (FL) via chemical oxidation polymerization. The conductive composite electrode was utilized as a drug carrier to realize the electrically controlled release of the FL. The release rate from conductive nanoparticles can be controlled by the applied voltages. The study provides a multi-stimuli responsive drug release system, demonstrating the potential applications of the controlled release of various drugs, peptides or proteins.

Incorporation of Laminarin-Based Hydrogel with Graphene Foam To Enhance the Toughness of Scaffold and Regulate the Stem Cell Behavior.

Three-dimensional (3D) carbon-based scaffolds have rapidly risen in tissue engineering due to the excellent conductivity and unique topological structures. For specific in vivo application, it is desirable to maintain the rigidity and improve the toughness of scaffolds in response to the compression force from surrounding tissues. In light of the combined advantages of graphene and hydrogels, we here construct a 3D composite scaffold consisting of a graphene foam (GF) and a laminarin hydrogel (LAgel). The composite scaffold was fabricated by immersing the GF in an LA hydrogel precursor followed by exposure to ultraviolet (UV) radiation to form a photocross-linked LAgel surrounding the GF. This composite scaffold exhibited the improved toughness compared with the GF or LAgel. The 3D GF can support cell attachment and cell spreading of human mesenchymal stem cells (hMSCs), while the in situ-formed LAgel with cell adhesive peptide arginine-glycine-aspartic acid (RGD) conjugated can induce the cell migration. The results suggest that the approach to incorporate the LAgel with the 3D GF not only enhances the toughness of the scaffold but also offers a carrier to realize the cargo of biosignals to regulate cell behaviors, showing the potential of this composite scaffold for tissue regeneration.

A fully degradable and photocrosslinked polysaccharide-polyphosphate hydrogel for tissue engineering.

Extracellular matrix degradability meditates cell behaviors and gains increasing importance in the development of implantation materials for tissue engineering. Here, we developed a fully biodegradable hydrogel combining the unique features of synthetic polyphosphate polymer and natural polysaccharide polymer. Polyphosphate copolymer poly(butynyl phospholane)-random-poly(ethylethylene phosphate) (PBYP-r-PEEP) bearing pendent alkynes was synthesized through a facile one-pot reaction. Subsequently, thiol-yne "click" reaction was employed to fabricate the fully degradable and photocrosslinked hydrogel by mixing PBYP-r-PEEP with thiolated biodegradable hyaluronic acid (HA-SH). The generated HA/PPE hydrogels show viscoelastic properties and enzymatic biodegradability, supporting the growth of human mesenchymal stem cells (hMSCs). HA/PPE hydrogel is permissive to the covalent conjugation of cell-adhesive peptide RGD, which can enhance the cell-cell interactions. This HA/PPE hydrogel system provides a fully biodegradable platform that can support hMSCs growth and facilitate the formation of cell clustering, expanding the range of fully degradable materials for tissue engineering and regenerative medicine.

The Modification of Tet1 in Male Germline Stem Cells and Interact with PCNA, HDAC1 to promote their Self-renewal and Proliferation.

Epigenetic modification plays key roles in spermatogenesis, especially DNA methylation dynamic is important in sustaining normal spermatogenesis. Ten-eleven translocation 1 (Tet1) is not only a key demethylase, which works in specific gene regions, but also crosstalks with partners to regulate epigenetic progress as protein complexes. Dairy goat is an important livestock in China, while the unstable culture system in vitro inhibits optimization of new dairy goat species. The study of epigenetic modification in male germline stem cells (mGSCs) is beneficial to the optimization of adult stem cell culture system in vitro, and the improvement of sperm quality and breeding of selected livestock. In our study, we not only analyzed the morphology, gene expression, DNA methylation and histone methylation dynamic in mouse Tet1 (mTet1) modified mGSCs, we also analyzed the stemness ability by in vivo transplantation and explored the functional mechanism of Tet1 in dairy goat mGSCs. The results showed mTet1 modified mGSCs had better self-renewal and proliferation ability than wild-type mGSCs, mTet1 could also up-regulate JMJD3 to decrease H3K27me3, which also showed to suppress the MEK-ERK pathway. Furthermore, Co-IP analysis demonstrated that TET1 interact with PCNA and HDAC1 by forming protein complexes to comprehensively regulate dairy goat mGSCs and spermatogenesis.

Modification of Tet1 and histone methylation dynamics in dairy goat male germline stem cells.

OBJECTIVES: Tet (ten-eleven translocation) protein 1 is a key enzyme for DNA demethylation, which modulates DNA methylation and gene transcription. DNA methylation and histone methylation are critical elements in self-renewal of male germline stem cells (mGSCs) and spermatogenesis. mGSCs are the only type of adult stem cells able to achieve intergenerational transfer of genetic information, which is accomplished through differentiated sperm cells. However, numerous epigenetic obstacles including incomplete DNA methylation and histone methylation dynamics make establishment of stable livestock mGSC cell lines difficult. The present study was conducted to detect effects of DNA methylation and histone methylation dynamics in dairy goat mGSCs self-renewal and proliferation, through overexpression of Tet1. MATERIALS AND METHODS: An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened and characteristics of the cells were assayed by quantitative real-time PCR (qRT-PCR), immunofluorescence assay, western blotting, fluorescence activated cell sorting (FACS) and use of the cell counting kit (CCK8) assay. RESULTS: The screened immortalized dairy goat mGSC cell line bearing mTet1, called mGSC-mTet1 cells was treated with optimal doxycycline (Dox) concentration to maintain Tet1 gene expression. mGSC-mTet1 cells proliferated at a significantly greater rate than wild-type mGSCs, and mGSCs-specific markers such as proliferating cell nuclear antigen (PCNA), cyclinD1 (CCND1), GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1, Tet2 were upregulated. The cells exhibited not only reduction in level of histone methylation but also changes in nuclear location of that methylation marker. While H3K9me3 was uniformly distributed throughout the nucleus of mGSC-mTet1 cells, it was present in only particular locations in mGSCs. H3K27me3 was distributed surrounding the edges of nuclei of mGSC-mTet1 cells, while it was uniformly distributed throughout nuclei of mGSCs. Our results conclusively demonstrate that modification of mGSCs with mTet1 affected mGSC maintenance and seemed to promote establishment of stable goat mGSC cell lines. CONCLUSIONS: Taken together, our data suggest that Tet1 had novel and dynamic roles for regulating maintenance of pluripotency and proliferation of mGSCs by forming complexes with PCNA and histone methylation dynamics. This may provide new solutions for mGSCs stability and livestock mGSC cell line establishment.

CD49f promotes proliferation of male dairy goat germline stem cells.

OBJECTIVES: CD49f enhances multipotency and maintains stemness in embryonic stem cells (ESCs), however, whether it would be effective in mGSCs has remained unclear. Moreover, better standards for mGSC enrichment and purification are necessary. The present study was conducted to determine roles of CD49f in mGSC enrichment and regulation. MATERIALS AND METHODS: CD49f expression patterns were investigated in dairy goats. CD49f positive cells were purified and enriched using magnetic-activated cell sorting (MACS), and characteristics of the cultured cells were assayed using alkaline phosphatase (AP) analysis, quantitative real-time PCR (QRT-PCR) and immunofluorescence analysis. Furthermore, the exogenous CD49f gene was transfected into mGSCs and its effects were analysed. RESULTS: CD49f was found to be conserved in both mRNA and amino acid sequences and that it was an efficient marker for dairy goat mGSC identification, enrichment and purification. CD49f positive cells expressed higher levels of mGSC-specific markers, and proliferated faster than CD49f negative cells. Overexpression CD49f promoted proliferation of dairy goat mGSCs, and Oct4 expression was upregulated; histone H3-lysine 9 dimethylation (H3K9me2) was reduced. CONCLUSIONS: Taken together, our data suggest that CD49f plays novel and dynamic roles in regulating maintenance of pluripotency in mGSCs via Oct4 crosstalk and histone methylation dynamics,which may provide new solutions for mGSCs stability in vitro.