Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naive-like conversion, based on a naive medium (NM). The results showed successful conversion of the primed bovine iPSCs to naive-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naive-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naive-like iPSCs. We further analyzed the naive markers and differentiation po-tential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naive-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naive-like bovine iPSCs, and improve the conversion efficiency.(c) 2022 Elsevier Inc. All rights reserved.

Objective: Sertoli cells (SCs) are important sustentacular cells in the seminiferous tubules of the testis. Isolation and identification of SCs are the premise for studying their functions. Since New Zealand rabbit is a stable strain which is widely used for biomedical research and animal farming, this study aimed to develop a simple and effective protocol for SC isolation in New Zealand rabbits. Materials and Methods: The SCs of three 30-day-old New Zealand rabbits were isolated by incubation with enzymatic digestion I (Dulbecco’s modified Eagle medium supplemented with 1 mg/ml collagenase IV and 50 μg/ml DNase I) and digestion II (digestion I + 1 mg/ml hyaluronidase + 1 mg/ml trypsin), as well as differential plating. The cells were enriched and identified by using immunocytochemical staining and reverse transcription polymerase chain reaction analysis. Results: Homogeneous cells were obtained. They presented the typical large cell body and an irregular pyramidal shape after differential plating and passaging. These cells expressed mRNA of the SC marker sex-determining region Y-box 9 (SOX9) instead of the Leydig cell marker StAR. Immunocytochemically, they are positive of SOX9, GATA binding protein 4, and androgen-binding protein. Conclusion: The SCs were enriched from the testicular tissues of prepubertal New Zealand rabbits by a simple and effective protocol, which provides a basis for further theoretical researches and practical applications.

Schisandrin A and B (Sch A and B) are the important components of Asian dietary supplement and phytomedicine Schisandra chinensis (S. chinensis). They can enhance adult neumgenesis in vivo; however, these effects still need to be verified. Here NE-4 C neural stem cells (NSCs) were employed as the in vitro model and treated with Sch A and B at 0.1 mu g/mL. EdU (5-Ethynyl-2'-deoxyuridine) labeling showed that both Sch A and B treatments enhanced NSC proliferation. Real-time PCR analysis showed the mRNA abundances of telomerase gene Tert and cell cycle gene Cyclin D1 were significantly up-regulated after the treatments. During the neumsphere induction, Sch B enhanced the neumsphere formation and neuronal differentiation, and increased the neurosphere semi-diameters. Detection of the neuron differentiation marker Mapt indicates that both Sch A and B, especially Sch B, benefits the induced neuronal differentiation. Sch B treatment also enhanced mRNA expressions of the neumsphere-specific adhesion molecule Cdh2 and Wnt pathway-related genes including Mmp9, Cyclin D1 and beta-catenin. Together, Sch A especially Sch B, promotes the proliferation, affects the survival, differentiation and neumgenesis of NSCs, which is consistent with their in vivo effects. This study provides further clue on the potential neumpharmacological effects of S. chinensis.

Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.

Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3β (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3β signalling, evidencing successful cryopreservation and expansion of bovine germplasm.

Although induced pluripotent stem cells (iPSCs) had been generated from several somatic cell types in cattle, their pluripotency and differentiation capacities after freezing/thawing, and the dysregulated transcripts involved in pathways critical for reprogramming were not investigated. Additionally, selection of proper source cells is critical for iPSC derivation because the residual influence of the somatic origin may variegate their differentiation propensity. Sertoli cells (SCs) have special properties suitable for iPSCs derivation. Herein bovine SCs were enriched from the cryopreserved testicular tissues and reprogrammed into iPSCs using lentivirus carrying yamanaka factors (OSKM). These iPSCs have typical morphology resembling human iPSCs and remain normal karyotypes. They can express alkaline phosphatase activity and common pluripotency markers with a low methylation in the promoter region of Nanog. They can also form embryoid bodies and teratomas that give rise to cells/tissues from three embryonic germ layers. Transcriptome profiling showed that the exogenous OSKM were silenced and 8009 dysregulated mRNAs were identified. The pluripotency, methyldioxygenase and anti-apoptosis genes were all upregulated but the apoptotic gene downregulated in these iPSCs. Bunch of pathways related to the reprogramming, inflammation and viral infection pathways were upregulated, while pathways associated with the differentiation, senescence, metabolism and apoptosis were downregulated in these cells. After cryopreservation/thawing, the recovered iPSCs remain strong pluripotency and differentiation capabilities. Together, iPSCs were derived from the bovine SCs isolated from the cryopreserved neonatal bull testis, pluripotency and differentiation capacities verified, iPSCs cryopreserved, cultured and again reverified for pluripotency and differentiation capacities.

Schisandrin A and B (Sch A and B) are the main effective components of Schisandra chinensis (S. chinensis), which is traditionally used to enhance mental and intellectual functions in eastern Asia. Previously, we reported Sch A and B remarkably affect adult neurogenesis in the subventricular zone of mouse lateral ventricle. Since the neurogenesis in the hippocampal dentate gyrus (DG) is more important to learning, memory and cognition, here we further examined their effects on the adult DG neurogenesis. Phosphohistone H3 (PHH3) immunostaining showed that Sch B significantly enhanced the cell proliferation in the DG. Glial fibrillary acidic protein (GFAP, mostly labels astrocytes and some stem cells) staining was used to further identify the proliferating cell type. Dramatically, increases of GFAP(+) cells in both Sch A and B treated groups were observed. What's more, the total numbers of the mature neurons labeled by neuron-specific nuclear protein (NeuN) were also increased in both Sch A and B treated groups compared with the controls. Together, Sch A and B enhance the adult DG neurogenesis by increasing astrocytes/stem cells and improving the survival and maturation of DG neurons. Our study shed a new light on the neuropharmacological functions of the herbal medicine S. chinensis.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me2SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-1 (GFR alpha-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me2SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFR alpha-1 typically. Collectively, supplementation of 5-10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.

Acetaminophen (APAP) overdose has become the most common cause of drug-induced acute liver failure. Angiogenesis and redox homeostasis play an important role in liver protection and repair of APAP-induced acute liver injury (AILI). Hypoxia inducible factor-1 (HIF-1) is a transcription factor that plays a crucial role in regulating the expression of genes associated with angiogenesis, redox homeostasis and energy balance. Prolyl hydroxylase 2 (PHD2) predominantly hydroxylates proline residues in HIF-1α to promote its degradation. In our previous study, we reported an intrabody against PHD2 (ER-INP) that enhances angiogenesis by blocking PHD2 activity to increase HIF-1α abundance and activity. The present study was designed to explore the role and possible mechanisms of ER-INP in AILI in mice. Mice were pretreated intravenously with ER-INP before intraperitoneal injection of APAP to induce AILI. The results showed that pretreatment with ER-INP dramatically decreased the high ALT and AST activities and significantly ameliorated the centrilobular necrosis induced by APAP administration. ER-INP expression promoted angiogenesis in vivo by upregulating the mRNA and protein levels of HIF-1α target genes. Meanwhile, ER-INP pretreatment restored redox homeostasis, verified by reinforcement of PRDX4 activity and suppression of GSH depletion. This study demonstrated that ER-INP protects against AILI in part by increasing angiogenesis and maintaining redox homeostasis. These results indicate that ER-INP may provide a potential liver protection strategy against AILI in the future.

Thrombotic diseases have become a global burden due to morbidity, mortality, and disability. Traditional Chinese medicine has been proven effective in removing blood stasis and promoting blood circulation, but the exact mechanisms remain unclear. Plasminogen activator inhibitor-1 (PAI-1) is a natural inhibitor of tissue-type and urokinase-type plasminogen activators. In this study, we screened four fractions of Resina Draconis (a traditional Chinese medicine) extract for PAI-1 inhibitory activity. Bioactivity-guided purification and chromogenic substrate-based assay led to the identification of loureirin B as the major PAI-1 inhibitor, with an IC50 value of 26.10 μM. SDS-PAGE analysis showed that formation of the PAI-1/uPA complex was inhibited by loureirin B, and the inhibitory effect of loureirin B on PAI-1 was also confirmed by clot lysis assay. In vivo studies showed that loureirin B significantly prolonged the tail bleeding time and reduced the weight and size of arterial thrombus, reduced hydroxyproline level, and partly cured liver fibrosis in mice. Taken together, the results revealed loureirin B as a PAI-1 inhibitor, adding a new pharmacological target for loureirin B and uncovering a novel mechanism underlying the antithrombotic property of Resina Draconis, which might be useful in the treatment of cardiovascular diseases such as thrombosis and fibrosis.