Dopaminergic neurons derived from porcine induced pluripotent stem cell like cells function in the Lanyu pig model of Parkinson's disease.

The induced pluripotent stem cells (iPSCs) are able to differentiate into dopaminergic neurons and execute the therapeutic effects for Parkinson's disease (PD). Here, we established a animal model of PD in Lanyu pigs by injecting 5 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP). Next, the porcine iPSC-like cells (piPSC-like cells) were differentiated into D18 neuronal progenitors (D18 NPs) that were transplanted into the striatum to evaluate their therapeutic effects of PD. We showed that after 8 weeks of cell transplantation, the behavior score was significantly ameliorated and fully recovered at the 14th week of cell transplantation. The number of dopaminergic neurons was also significantly improved at the end of the experiment although the number was still about 50% lower than that in the control group. Our findings suggest that piPSC-like cell-derived D18 NPs exhibit a potential for the treatment of PD in the Lanyu pig model.

Gamma-oryzanol supplemented in extender enhances the quality of semen cryopreservation and alters proteomic profile in Thai swamp buffalo.

Reactive oxygen species (ROS) exert an adverse effect on sperm quality during the freezing process. Gamma-oryzanol is an effective antioxidant and has the ability to inhibit lipoperoxidation in various cells. Therefore, this study aims to investigate the effect of gamma-oryzanol supplementation in extender on post-thawed motility and proteomic profiles of swamp buffalo spermatozoa. Each ejaculate of an individual bull was divided into four equal aliquots. Gamma-oryzanol was supplemented at 0 (control), 0.1, 0.25, and 0.5 mM in tris-citrate egg yolk extender. The parameters of sperm motility were evaluated using computer assisted semen analyzer (CASA). The results showed that the progressive motility was significantly higher in 0.5 mM of gamma-oryzanol supplementation group when compared with the control group (p < 0.05), but no significant differences were observed among the treatments. In addition, a proteomic approach was applied to analyze the differentially expressed proteins in post-thawed sperm with or without gamma-oryzanol supplementation in extender. We confirmed that 2-phospho-d-glycerate hydro-lyase (ENO1), glutathione s-transferase mu 1 (GSTM1), phospholipid hydroperoxide glutathione peroxidase (GPX4), outer dense fiber protein 2 (ODF2), tektin-4 (TEKT4), tubulin beta-4B chain (TUBB4B), and ATP synthase subunit beta (ATP5B) were up-regulated in 0.5 mM of gamma-oryzanol supplementation group, which might be associated with the improved post-thawed motility observed in this treatment group. These results demonstrate the beneficial effect of gamma-oryzanol on post-thawed survival of swamp buffalo spermatozoa and help advance the understanding about molecular metabolism of sperm in this species.

The replacement of fresh egg yolk by lyophilized egg yolk in Tris-base extender in cryopreserved Boer and Saanen semen.

Egg yolk is a common cryoprotectant that can be used as a semen extender to protect the spermatozoa from damage during cryopreservation. Therefore, the aim of this study was to investigate the efficacy of fresh and lyophilized egg yolk, as a Tris-base extender, on the quality of cryopreserved goat semen. Semen from 10 rams of two different breeds (Boer and Saanen) was collected using an artificial vagina. Each ejaculate sample was divided into four equal aliquots, which contained 20% of the fresh egg yolk (a control group), and then 10%, 15%, and 20% of the lyophilized egg yolk as a Tris-base extender. Sperm motility and kinetic parameters were determined using a computer-assisted semen analyser. The results showed that the addition of 20% of the fresh egg yolk in Tris-base extender exhibited significantly higher progressive motility, progressive fast motility, distance curve line, and beat-cross frequency parameters in the post-thaw Boer and Saanen goat sperm when compared with the addition of 10%, 15%, and 20% of the lyophilized egg yolk. The percentage of total motility and immotile parameters in the post-thaw Boer and Saanen goat sperm were not significantly different between the control and 10%, 15% as well as 20% of the lyophilized egg yolk groups. Moreover, the percentage of viability parameter in the Boer and Saanen goat sperm was not significantly different between the control and 10% of the lyophilized egg yolk group but showed significant difference between the control group and 15% and 20% of the lyophilized egg yolk groups. Furthermore, the interaction between the two breeds was significantly different in terms of head activity and straightness parameter. In conclusion, the treatment with 20% of fresh egg yolk in Tris-base extender is superior to the lyophilized egg yolk. However, an addition of 10% of the lyophilized egg yolk in Tris-base extender presented the percentage of total motility and viability parameters showing no difference with 20% of fresh egg yolk. Therefore, 10% of the lyophilized egg yolk in Tris-base extender provided detail of the lyophilized egg yolk protocol in cryopreserved goat semen as an example of an alternative extender to 20% of fresh egg yolk for situations where an animal's origin represents a microbiological risk.

Melatonin supplementation improved cryopreserved Thai swamp buffalo semen.

The production of reactive oxygen species (ROS) during cryopreservation process impairs the sperm characteristics and fertilizing ability. However, melatonin, an antioxidant, could protect spermatozoa against this cell damage during cryopreservation. Therefore, we attempted to evaluate whether the melatonin supplementing in the semen extender could improve the sperm quality of swamp buffalo during cryopreservation. The semen collected from six swamp buffalo bulls were diluted with tris-citrate egg yolk extender supplementing with 0, 0.1, 0.5, 1.0, 2.0 and 3.0 mM of melatonin. The parameters of sperm viability and motility were evaluated using computer-assisted semen analyser (CASA) after cryopreservation on days 1, 7, 15 and 30. The group supplemented with 1.0 mM melatonin exhibited the higher viability after cryopreservation on days 1, 7, 15 and 30 with 58.346 ± 2.1a , 57.586 ± 2.0a , 55.082 ± 1.8a and 55.714 ± 1.8a , respectively, and showed the best results of motility parameters. However, higher concentration of melatonin at 3.0 mM impaired all the parameters. In conclusion, the addition of melatonin at 1 mM to semen extender could exert the best protection against sperm damage in swamp buffalo bull during cryopreservation.

A protocol for differential staining of cartilages and ossified bones in fetal and adult mouse skeletons using alcian blue and alizarin red S.

The technique for clearing and staining whole specimens consists of many steps. This study discusses the alcian blue/alizarin red S staining method and aims to provide a useful reference and review for users who intend to do this staining. To specifically address the influences of tissue removal on staining results, the mouse fetuses at embryonic stage E18.5 and adult mice at 12 weeks of age were used in this study. The fetuses were divided into three groups: Group 1 skin, muscle, and viscera removed, Group 2 skin and muscle removed, and Group 3 viscera removed. For successful skeletal staining, it was concluded that (1) skin removal from fetuses was necessary for alcian blue staining but unnecessary for alizarin red S staining, (2) removal of muscle surrounding thorax and neck of fetuses could improve transparency effects, (3) retaining fetal viscera would not significantly affect transparency but might avoid the tissue damage, and (4) complete skin, muscle, and viscera removal were essential for good staining of adult mice. The representative images and detailed staining procedures might be good for researchers presently using alcian blue and alizarin red S staining to differentiate cartilages and ossified bones.

Porcine-induced pluripotent stem cell-derived osteoblast-like cells ameliorate trabecular bone mass of osteoporotic rats.

AIM: We created rat models of osteoporosis and verified a novel idea to recover bone mass via local cell transplantation. MATERIALS & METHODS: The rats were treated with ovariectomy, 0.1% calcium diet or 3 mg/kg body weight/day of prednisolone and porcine-induced pluripotent stem cell (piPSC)-derived osteoblast-like cells were transplanted into the medullary cavity of the left femurs. RESULTS: The piPSC-derived osteoblast-like cells exerted therapeutic potential on prednisolone treatment group, which confirmed by improvements in trabecular bone volume (15.93 ± 2.20%), bone surface/volume ratio (27.82 ± 1.40 1/mm), thickness (1.40 ± 0.01 mm), separation (0.99 ± 0.10 mm), number (1.13 ± 0.13 1/mm) and total porosity (84.06 ± 2.20%). CONCLUSION: These results first uncovered therapeutic potential of xenotransplantation with piPSCs for glucocorticoid-induced osteoporosis treatment in the rat models.

Porcine induced pluripotent stem cell-derived osteoblast-like cells prevent glucocorticoid-induced bone loss in Lanyu pigs.

The application of appropriate animal models and techniques for the study of osteoporosis is important. Lanyu pigs, a local miniature breed, have been widely used in various biomedical studies in Taiwan. This study aimed to induce bone loss in Lanyu pigs and to examine whether porcine induced pluripotent stem cell (piPSC)-derived osteoblast-like cells could recover bone mass of tibiae via local cell transplantation. piPSCs were directed to differentiate into osteoblast-like cells using osteogenic medium, and differentiated cells expressed osteogenic markers and phenotypes. Twenty mature female Lanyu pigs were divided into four groups, including control (C, 1% calcium diet), treatment 1 (T1, ovariectomy + 1% calcium diet), treatment 2 (T2, ovariectomy + 0.5% calcium diet), and treatment 3 (T3, ovariectomy + 0.5% calcium diet + 1 mg/kg of prednisolone) and were subjected to bone loss induction for twelve months. Micro-CT images revealed that the lowest trabecular bone parameters, such as trabecular bone volume, thickness, separation, number, and total porosity, were detected in the T3 group. The lowest proportions of cortical bone in the proximal metaphysis, proximal diaphysis, and distal diaphysis were also found in the T3 group. These results indicate that ovariectomy, calcium restriction, and prednisolone administration can be applied to induce proper bone loss in Lanyu pigs. After bone loss induction, pigs were subjected to cell transplantation in the left tibiae and were maintained for another six months. Results showed that transplanted piPSC-derived osteoblast-like cells significantly improved trabecular bone structures at transplanted sites and maintained cortical bone structures in the proximal metaphysis. In conclusion, the therapeutic potential of piPSC-derived osteoblast-like cells was confirmed via cell transplantation in the left tibiae of Lanyu pigs. These findings reveal the therapeutic potential of piPSCs for glucocorticoid-induced bone loss in pig models.

Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance in Porcine Induced Pluripotent Stem Cell.

OBJECTIVES: The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells. MATERIALS AND METHODS: In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. RESULTS: Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas. CONCLUSIONS: The pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

Superovulation at a specific stage of the estrous cycle determines the reproductive performance in mice.

Inconsistent reproductive performance has been reported in superovulated mice. Hence, the aim of this study was to analyze the effect and possible mechanism of superovulation timing on mouse reproductive performance. The results showed that mice superovulated at the metestrous (23.08±6.08%) and diestrous stages (33.33±11.45%) presented significantly lower pregnancy rates compared with those superovulated at the estrous stage (66.67±9.20%). After superovulation at the proestrous and estrous stages, mucin 1 (MUC1) and let-7a/let-7b microRNA (miRNA) expression levels were significantly attenuated and enhanced on embryonic day 3.5 (E3.5), respectively, whereas no significant differences in the expression level were found in mice superovulated at the other two stages. A higher number of developing and Graafian follicles was observed in the ovarian sections 48h after the administration of pregnant mare serum gonadotropin (PMSG) at the proestrous and estrous stages. The sections from mice treated at the metestrous and diestrous stages, however, presented more corpora lutea. Therefore, mice superovulated at the proestrous and estrous stages exhibited the best pregnancy rates. Furthermore, the disordered expression of MUC1 and let-7a/let-7b miRNA in mice superovulated at the metestrous and diestrous stages may impair reproduction performance.

Derivation of porcine pluripotent stem cells for biomedical research.

Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy.

The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding protein 1 (CREB1) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2, in vitro and in vivo. Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

Inhibition of fumonisin B1 cytotoxicity by nanosilicate platelets during mouse embryo development.

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

[Influence of freshwater marsh tillage on microbial biomass and dissolved organic carbon and nitrogen].

The changes in microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) were examined in order to assess the effect of surface layer soil (0 - 10 cm) under different land-use types after freshwater marshes tillage in the Sanjiang Plain Northeast China. Land uses were Deyeuxia angustifolia freshwater marshes ((DAM), cultivated land (CL), recovery freshwater marsh (RFM), constructed woodland (CW). After DAM soil tillage, MBC, MBN, DOC and DON declined strongly in agricultural surface soil layer, decreased 63.8%-80.5% (MBC), 56.3%-67.1% (MBN), 43.1%-44.3% (DOC) and 25.2%-56.1% (DON) respectively. In contrast, these C, N fraction had significant recovered in RFM and CW surface soil, increased 36.1%-59.9% (MBC), 46.7%-65.9% (MBN), 67.0%-69.3% (DOC)and 81.2%-88.3% (DON) respectively. Cultivation and land-use affected soil MBC, MBN, DOC and DON intensely. Therefore these labile C, N fractions have the significant relative under different land-use types. However DOC was more obvious controlled than DON by the land-use types. The relative between DOC and MBC, MBN have much difference than DON, the main reason of this distinction is the diverse source in available carbon and nitrogen that taken by microbial property under different land uses.