Inhibition of TGF-ß pathway improved the pluripotency of porcine pluripotent stem cells.

Porcine pluripotent stem cells had been derived from different culture systems. PeNK6 is a porcine pluripotent stem cell line that we established from an E5.5 embryo in a defined culture system. Signaling pathways related with pluripotency had been assessed in this cell line, and TGF-ß signaling pathway-related genes were found upregulated significantly. In this study, we elucidated the role of the TGF-ß signaling pathway in PeNK6 through adding small molecule inhibitors, SB431542 (KOSB) or A83-01 (KOA), into the original culture medium (KO) and analyzing the expression and activity of key factors involved in the TGF-ß signaling pathway. In KOSB/KOA medium, the morphology of PeNK6 became compact and the nuclear-to-cytoplasm ratio was increased. The expression of the core transcription factor SOX2 was significantly upregulated compared with cell lines in the control KO medium, and the differentiation potential became balanced among three germ layers rather than bias to neuroectoderm/endoderm as the original PeNK6 did. The results indicated that inhibition of TGF-ß has positive effects on the porcine pluripotency. Based on these results, we established a pluripotent cell line (PeWKSB) from E5.5 blastocyst by employing TGF-ß inhibitors, and the cell line showed improved pluripotency.

Production of artificial synthetic spidroin gene 4S-transgenic cloned sheep embryos using somatic cell nuclear transfer.

Spider silk, which has remarkable characteristics, has wide application prospects in many fields. Many researchers have explored potential methods for directly producing spider silk proteins and spidroins with mechanical properties or obtaining recombinant spider silk fibers by genetic engineering methods. However, there are still some shortcomings with these methods, such as inability to simulate the fibrosis process of spider silk. In this study, a high glycine/tyrosine protein gene (HGT) promoter originate from sheep was first cloned by PCR. The HGT promoter was ligated into pcDNA3.1 and pcDNA3.1-HGT was obtained. After linking with the synthesized and polymerized gene 4S, a eukaryotic expression vector pcDNA3.1-HGT-4S was constructed using a series of molecular methods. Sheep fibroblasts transfected with the linearized plasmid using a liposome-mediated method were screened with G418 and a transgenic cell line was established. Cells from the transgenic line were used as nuclear donors to construct embryos with somatic cell nuclear transfer (SCNT). Reconstructed embryos derived from transgenic cells were able to develop in vitro successfully. PCR was carried out and results demonstrated that the synthetic spidroin gene 4S had integrated into the embryo genome. In summary, we explored a method and successfully obtained artificial synthetic spidroin gene transgenic sheep cloned embryos with a hair follicle specific promoter by SCNT. Further research is necessary on transgenic sheep with synthetic spidroin genes expressed in hair follicles.

Copper promotes sheep pancreatic duct organoid growth by activation of an antioxidant protein 1-dependent MEK-ERK pathway.

Proper amounts of copper supplemented in livestock feed improve the physical growth and traits of farm animals. The pancreas is an important organ with both exocrine and endocrine portions. To investigate the role and mechanism of copper in the sheep pancreas, we first established sheep pancreatic duct organoids (sPDOs). We found that an appropriate amount of copper benefited the formation and growth of sPDOs, whereas excess or deficient copper damaged sPDOs. We found that the proliferation-stimulating effect of copper was related to the copper chaperone antioxidant protein 1 (ATOX1)-dependent activation of MEK-ERK1/2 signaling. Atox1 knockdown suppressed the cell proliferation of sPDOs, even in the presence of the MEK activator. These results indicate that moderate concentrations of copper promote sPDO growth through ATOX1-regulated cell proliferation by activation of MEK-ERK. Moreover, our study indicates that organoids may be a useful model to study organ growth mechanisms in livestock.

A hydrogen sulfide-releasing alginate dressing for effective wound healing.

For wounds with heavy exudate levels, a dressing that can help to absorb wound exudate and improve the wound healing process is highly desired. Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter that can improve angiogenesis which is crucial for wound healing. In this study, a functional sodium alginate (SA) dressing with H2S-releasing property (SA/JK-1) was fabricated by incorporating JK-1 molecule, a pH-dependent H2S donor, into SA sponge. The resultant SA/JK-1 sponge provided a moist and protective healing environment and was capable of releasing H2S consistently under acidic pH condition by absorbing exudate at the wound interface. The H2S release of JK-1 donor was prolonged by the SA sponge compared with JK-1 in solution. Cell study in vitro indicated that SA/JK-1 not only exhibited good cyto-compatibility, but also improved fibroblast proliferation and migration. In addition, the effects of the SA/JK-1 dressing on wound healing was evaluated using an in vivo full thickness dermal defect model, which revealed that SA/JK-1 can significantly improve wound healing process with enhanced granulation tissue formation, re-epithelialization, collagen deposition and angiogenesis, due to the H2S released from JK-1. Taken together, our results showed that SA dressing doped with H2S donor could potentially serves as an effective wound healing strategy. STATEMENT OF SIGNIFICANCE: The gasotransmitter H2S has been proven to improve the wound healing process in nanofibrous dressing due to its biological functions on angiogenesis. However, for non-healing wounds with heavy exudates, a wound dressing that can absorb wound exudates and controlled gasotransmitter release to improve the wound healing process is still in urgent need. Here we fabricated a sodium alginate (SA) sponge incorporated with H2S donor JK-1 (SA/JK-1), which showed strong water uptake capability, and released H2S under acidic condition. The SA/JK-1 sponge exhibited biocompatibility to fibroblasts and promoted cell migration in vitro, and exhibited obviously positive influence on wound healing in vivo. This H2S donor doped alginate wound dressing represents a promising strategy for treatment of non-healing wound.

Endo-siRNA deficiency results in oocyte maturation failure and apoptosis in porcine oocytes.

Both microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs) play key regulatory roles in gene expression. Some studies have demonstrated that the function of miRNA is suppressed in mouse oocytes, suggesting that endo-siRNA, not miRNA, is essential for female meiosis. This finding has yet to be confirmed in other species. In this study, by knockdown of DICER1, DROSHA and its cofactor DiGeorge syndrome critical region 8 (DGCR8) in porcine oocytes, we found that the proportion of oocytes with DICER1 deficiency that developed to meiosis II (MII) stage was significantly lower than oocytes with DROSHA and DGCR8 deficiency (39.23 versus 68.71 and 71.25% respectively; P<0.05). Oocytes lacking DROSHA and DGCR8 formed a barrel-shaped metaphase I spindle, with chromosomes tightly aligned at the metaphase plate whereas most oocytes (87%) lacking DICER1 showed spindle abnormalities during oocyte in vitro maturation. Furthermore, DICER1 deficiency also resulted in oocyte apoptosis. These results indicate that endo-siRNAs are essential for oocyte maturation in pigs.

Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer.

Many transgenes are silenced in mammalian cells (donor cells used for somatic cell nuclear transfer [SCNT]). Silencing correlated with a repressed chromatin structure or suppressed promoter, and it impeded the production of transgenic animals. Gene transcription studies in live cells are challenging because of the drawbacks of reverse-transcription polymerase chain reaction and fluorescence in situ hybridization. Nano-flare probes provide an effective approach to detect RNA in living cells. We used 18S RNA, a housekeeping gene, as a reference gene. This study aimed to establish a platform to detect RNA in single living donor cells using a Nano-flare probe prior to SCNT and to verify the safety and validity of the Nano-flare probe in order to provide a technical foundation for rescuing silenced transgenes in transgenic cloned embryos. We investigated cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts, characterized the distribution of the 18S RNA-Nano-flare probe in living cells and investigated the effect of the 18S RNA-Nano-flare probe on the development of cloned embryos after SCNT. The cytotoxic effect of the 18S RNA-Nano-flare probe on porcine fetal fibroblasts was dose-dependent, and 18S RNA was detected using the 18S RNA-Nano-flare probe. In addition, treating donor cells with 500 pM 18S RNA-Nano-flare probe did not have adverse effects on the development of SCNT embryos at the pre-implantation stage. In conclusion, we established a preliminary platform to detect RNA in live donor cells using a Nano-flare probe prior to SCNT.

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities.

The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (-) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB- oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

Discovery of porcine maternal factors related to nuclear reprogramming and early embryo development by proteomic analysis.

BACKGROUND: Differentiated cell nuclei can be reprogrammed to a pluripotent state in several ways, including incubation with oocyte extracts, transfer into enucleated oocytes, and induced pluripotent stem cell technology. Nuclear transfer-mediated reprogramming has been proven to be the most efficient method. Maternal factors stored in oocytes have critical roles on nuclear reprogramming and early embryo development, but remain elusive. RESULTS: In this study, we showed most of porcine oocytes became nuclear matured at 33 h of IVM and the rate had no significant difference with oocytes at 42 h of IVM (p > 0.05). Moreover, the cleavage and blastocyst rates of SCNT and PA embryos derived from 42O were significantly higher than that of 33O (p < 0.05). But 33O could sustain IVF embryo development with higher cleavage and blastocyst rates comparing to 42O (p < 0.05). To clarify the development potential difference between 33O and 42O, 18 differentially expressed proteins were identified by proteomic analysis, and randomly selected proteins were confirmed by Western blot. Bioinformatic analysis of these proteins revealed that 33O highly synthesized proteins related to fertilization, and 42O was rich in nuclear reprogramming factors. CONCLUSIONS: These results present a unique insight into maternal factors related to nuclear reprogramming and early embryo development.

Development of phenylboronic acid-functionalized nanoparticles for emodin delivery.

Stable and monodisperse phenylboronic acid-functionalized nanoparticles (PBA-NPs) were fabricated using 3-((acrylamido)methyl)phenylboronic acid homopolymer (PBAH) via solvent displacement technique. The effect of operating parameters, including stirring time, initial polymer concentration and the proportion of methanol on the self-assembly process were systematically investigated. The diameters of the PBA-NPs were increased as increasing the initial PBAH concentration and the proportion of methanol. Likewise, there was a linear dependence between the size of self-assembled nanoparticles and the polymer concentration. Moreover, the dissipative particle dynamics (DPD) simulation technique was used to investigate the mechanism of self-assembly behavior of PBAH, which indicated that the interior of PBA-NPs was hydrophobic and compact, and the boronic acid groups were displayed on both the outermost and interior of PBA-NPs. The resulting PBA-NPs could successfully encapsulate emodin through PBA-diol interaction and the encapsulation efficiency (EE%) and drug loading content (DLC%) of drug-loaded PBA-NPs were 78% and 2.1%, respectively. Owing to the acid-labile feature of the boronate linkage, a reduction in environmental pH from pH 7.4 to 5.0 could trigger the disassociation of the boronate ester bonds, which could accelerate the drug release from PBA-Emodin-NPs. Besides, PBA-Emodin-NPs showed a much higher cytotoxicity to HepG2 cells (cancer cells) than that to MC-3T3-E1 cells (normal cells). These results imply that PBA-NPs would be a promising scaffold for the delivery of polyphenolic drugs.

Evolution of mammalian and avian bornaviruses.

Recently, Avian Bornavirus (ABV) was identified to be a new member of the Bornaviridae family consisting solely of the mammal-infecting Borna disease virus (BDV). Here, to gain more insights into the evolution of these bornaviruses, the time-stamped N gene sequences of BDV genotype 1 (BDV1) and ABV were subjected to Bayesian coalescent analyses. The nucleotide substitution rates and the divergence times were estimated. Age calculations suggested that the first diversification event of the analyzed BDV1 isolates might have taken place about 300years ago, and revealed that ABV was an old virus newly recognized. Great differences were observed in the rate of nucleotide substitution and the pattern of codon usage bias between BDV1 and ABV. Moreover, the analyzed bornaviruses might be descended from an AT-rich ancestor.

[Role of microRNA in induced pluripotent stem cell].

MicroRNAs are ~22 nt long small noncoding RNA molecules that silence post-transcription gene expression. It has proven that microRNAs are widely expressed in eukaryotes and play an important role in the regulation of cell differentiation and development, growth metabolism, and many other cell activities. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cells reprogrammed from somatic cells and exhibit the essential characteristics of embryonic stem cells. iPS technology has been widely applied in the biological and medical fields, and the key of it is reprogramming of somatic epigenetic state. Therefore, it is of important theoretical and practical significance to study the mechanisms of somatic reprogramming for establishment of an improved iPS technology. The methods of transfection of defined exogenetic stem factors, such as Oct4, Sox2, Klf4, and c-Myc into somatic cells through viral vectors have been continuously improving, but the genome integration and reactivation of the oncogenic gene increase the tumorigenicity of induced cells. The integration-free ways, such as adenovirus, plasmid, recombinant proteins, and L-myc replacement used in iPS technology significantly reduce the risk of cancer. However, the inducing mechanisms are still unclear. Recent studies showed that microRNA affect the process of somatic cell reprogramming, especially embryonic stem cell regulating (ESCC) family of microRNAs (miR302/367, miR200, miR-34, and miR290/295) enhances the reprogramming of embryonic fibroblasts to iPS. This article reviews the recent progresses of roles of microRNA in iPS.

Temporal and spatial expression of AIF in the mouse uterus during early pregnancy.

Apoptosis-inducing factor (AIF) is a phylogenetically old, bifunctional protein with a pro-apoptotic function and redox activity. AIF regulates apoptosis and also plays a role in the defense against stress depending on its subcellular localization. Embryo implantation is a complicated process, in which an activated blastocyst interacts with a receptive uterus. The expression and regulation of AIF were investigated in this study in the mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization and under hormonal treatment using in situ hybridization, immunohistochemistry and real-time PCR. During early pregnancy, temporally and spatially regulated patterns of AIF expression were found in the mouse uterus; AIF expression in the luminal epithelium and glandular epithelium is regulated by steroid hormones; AIF mRNA expression in the stroma is influenced by the active blastocyst; and AIF protein was found to be located in the cytoplasm rather than the nucleus through confocal microscope. Our data suggest that AIF might play an important role during mouse embryo implantation and that the role of AIF might be implemented through its physiological activity rather than through its pro-apoptotic function in the mouse uterus during this period.

The type and extent of injuries in vitrified mouse oocytes.

To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws.

[miRNAs play an essential role in stem cell self-renewal and differentiation].

Stem cells and miRNAs are two of the most intriguing research topics in recent years. Stem cells with properties of self-renewal and multipotency are remarkable cells since they play key roles in animal development and tumorigenesis. microRNAs (miRNAs) are approximately 22nt non-coding RNAs, which have spatiotemporally specific expression patterns, and are highly conserved between species. Researchers have demonstrated that miRNAs can regulate gene expression at post-transcriptional level, and their action is one of the essential intracellular regulatory pathways. Recent evidence showed that miRNAs can regulate stem cells self-renewal and differentiation. The evidence mostly derived from the following research strategies: (1) deletion or mutation of the genes that encoding essential enzymes for miRNAs biogenesis, such as Dicer1, Loqs, DGCR8, and Argonaute; (2) screening of specific miRNAs from stem cells and investigation of their function. Comprehension of the miRNAs functions in stem cells is of great importance for further understanding their self-renewal and differentiation mechanism, as well as identification of stem cells. This paper reviews the potential roles of miRNAs in stem cells on the basis of recent researches.

[Progress in treating diabetes mellitus with adult stem cells].

Diabetes mellitus is a metabolic diseases, mainly including type 1 and type 2 diabetes. Treatment for type 1 and part of type 2 often involves regular insulin injection. However, this treatment neither precisely controls the blood sugar levels, nor prevents the diabetes complications. Transplantation of islets of Langerhans offers an attractive strategy for diabetes therapies, but its wide application has been limited by donor shortage and immunological rejection after transplantation. Stem cells with strong proliferation capacity and multipotential may be potential cell sources in diabetes therapies. For this, adult stem cells are interesting because of absence of teratoma formation and ethnical problems. Adult pancreatic stem cells (PSCs) really exist and could produce insulin-secreting cells both under the condition of pancreatic injury and in vitro culture, but lack of effective markers to enrich PSCs hampers the studies of exploring the expanding and differentiating conditions in vitro. Some other adult stem cells, such as hepatic stem cells, marrow stem cells or intestine stem cells, were also suggested to transdifferentiate into insulin-producing cells under special culture conditions in vitro or by genetic modifications. Moreover, transplanting these adult stem cells-derived insulin-secreting cells into the diabetic mouse could cure diabetes. Thus, adult stem cells would supply the abundant beta-cell sources for cell replacement therapy of diabetes.

[Advances in the study of stem cell niche].

A stem cell niche is defined as the microenvironment surrounding stem cells, and it is generally composed of adjacent cells, adhensive molecules, extracellular matrix, and so on. Stem cells in different tissues differ in their niches. Stem cell niches regulate stem cell self-renewal and differentiation. According to the recent studies in stem cell niches, this review summarizes on the constitution and function of germ-line stem cells niche in fruit, hematopoietic stem cell niche, intestinal stem cell niche, hair follicle epidermal stem cell niche and neural stem cell niche in mammals, and discusses the internal mechanism of niches'action on stem cells.