Local Immunosuppression in Wuzhishan Pig to Rhesus Monkey Descemet's Stripping Automated Endothelial Keratoplasty: An Innovative Method to Promote the Survival of Xenografts.

INTRODUCTION: Corneal xenotransplantation is an effective solution for human corneal shortage. We investigated the feasibility and efficacy of different postoperative protocols on xeno-Descemet's stripping automated endothelial keratoplasty (DSAEK) grafts. METHODS: Thirty rhesus monkeys were randomly divided into three groups: control group (C) and only Descemet's membrane (DM) stripping, DSAEK 1 (D1) and DSAEK 2 (D2) groups, DM stripping followed by endothelial keratoplasty. Betamethasone 3.5 mg was subconjunctivally injected in groups control and D1 postoperatively, whereas rhesus monkeys in group D2 received topical 0.1% tacrolimus and topical steroids. All groups were evaluated by slit lamp, anterior segment optical coherence tomography, and laser scanning confocal microscopy for at least 9 months. RESULTS: Twenty-four monkeys met the inclusion criteria. Nine months after the DSAEK surgery, most corneas were transparent. Graft rejection was observed in 25% and 28.57% of the cases in group D1 and group D2 (p > 0.05), respectively. Corneal endothelium densities in DSAEK groups were 2,715.83 ± 516.20/mm2 (D1) and 2,220.00 ± 565.13/mm2 (D2) (p > 0.05). CONCLUSIONS: Xenogeneic corneal endothelial grafts can survive and function in rhesus monkey eyes for a prolonged period of time with subconjunctival steroid or topical tacrolimus and steroid treatment. Furthermore, topical drugs are more suitable for clinical use.

Screening and Identification of the First Non-CRISPR/Cas9-Treated Chinese Miniature Pig With Defective Porcine Endogenous Retrovirus pol Genes.

Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most significant pathogen in xenotransplantation. We screened for pigs that consistently did not transmit human-tropic replication competent PERVs (HTRC PERVs), namely, non-transmitting pigs. Then, we conducted whole-genome resequencing and full-length transcriptome sequencing to further investigate the sequence characteristics of one non-transmitting pig. Using in vitro transmission assays, we found 5 (out of 105) pigs of the Chinese Wuzhishan minipig inbred line that did not transmit PERV to human cells, i.e., non-transmitting pigs. Whole-genome resequencing and full-length transcriptome sequencing of one non-transmitting pig showed that all of the pol genes were defective at both the genome and transcript levels. We speculate that the defective PERV pol genes in this pig might be attributable to the long-term inbreeding process. This discovery is promising for the development of a strain of highly homozygous and genetically stable pigs with defective PERV pol genes as a source animal species for xenotransplantation.

Proteomic analysis of porcine mesenchymal stem cells derived from bone marrow and umbilical cord: implication of the proteins involved in the higher migration capability of bone marrow mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) have the ability to proliferate in vivo with a large variety of differentiation potentials and therefore are widely used as an ideal material for cell therapy. MSCs derived from pig and human sources are similar in many aspects, such as cell immunophenotype and functional characteristics. However, differences in proteomics and the molecular mechanisms of cell functions between porcine bone marrow MSCs (BM-MSCs) and umbilical cord MSCs (UC-MSCs) are largely unknown. To the best of our knowledge, MSCs collected from different tissue have specific phenotype and differentiation ability in response to microenvironment, known as a niche. METHODS: Porcine BM-MSCs and UC-MSCs were evaluated with flow cytometric and adipogenic and osteogenic differentiation analyses. We used isobaric tagging for relative and absolute quantitation (iTRAQ), combined with liquid chromatography-tandem mass spectrometry, to identify differentially expressed proteins (DEPs) between these two types of MSCs. Kyoto Encyclopedia of Genes and Genomes pathway and phenotype analyses were used to understand the links between cell migration ability and DEPs. RESULTS: Two separate iTRAQ experiments were conducted, identifying 95 DEPs (95% confidence interval). Five of these proteins were verified by Western blotting. These 95 DEPs were classified in terms of biological regulation, metabolic process, developmental process, immune system process, reproduction, death, growth, signaling, localization, response to stimulus, biological adhesion, and cellular component organization. Our study is the first to show results indicating that porcine BM-MSCs have a higher migration capability than UC-MSCs. Finally, one of the DEPs, Vimentin, was verified to have a positive role in MSC migration. CONCLUSIONS: These results represent the first attempt to use proteomics specifically targeted to porcine MSCs of different tissues. The identified components should help reveal a variety of tissue-specific functions in tissue-derived MSC populations and could serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.

Generation of chimeric piglets by injection of embryonic germ cells from inbred Wuzhishan miniature pigs into blastocysts.

BACKGROUND: Human embryonic stem/germ (ES/EG) cell research poses ethical dilemma, it is therefore critical to establish alternative sources of cells for relevant studies. Considering the similarities between the inbred miniature Wuzhishan pigs (WZSP) and humans, ES/EG from these pigs can serve as potential substitutes in human research. In this study, we reported our results that successfully established stable EG cell lines from the WZSP. METHODS: Primordial germ cells (PGCs) were isolated from the genital ridges of pig fetuses at 25 to 28 days of pregnancy. To obtain stable EG cell line, PGCs were maintained on STO cells in DMEM containing multiple essential growth factors. RESULTS: Two EG cell lines were established and characterized by positive alkaline phosphatase staining (AKP), expressions of Oct-4, SSEA-1, SSEA-3, SSEA-4, ability to differentiate into cells of all three germ layers in vitro, and generation of chimeric offsprings after microinjection and embryo transfer. Transmission electron microscopy demonstrated that the cytoplasmic structure of pig EG cells were rather simple and had a higher nuclear-to-cytoplasm ratio. Scanning electron microscopy showed the sizes of pig EG cells were similar to mouse EG cells. Both EG cell lines showed normal karyotypes. The EG cells were propagated for more than 20 passages and underwent multiple cycles of freezing and thawing, without losing their pluripotency (as distinguished by AKP staining). CONCLUSIONS: Both in vitro and in vivo evidence strongly demonstrated that EG cells harvested from the inbred miniature WZSP were pluripotent and can be used for relevant pig or human studies.

The sequence and analysis of a Chinese pig genome.

BACKGROUND: The pig is an economically important food source, amounting to approximately 40% of all meat consumed worldwide. Pigs also serve as an important model organism because of their similarity to humans at the anatomical, physiological and genetic level, making them very useful for studying a variety of human diseases. A pig strain of particular interest is the miniature pig, specifically the Wuzhishan pig (WZSP), as it has been extensively inbred. Its high level of homozygosity offers increased ease for selective breeding for specific traits and a more straightforward understanding of the genetic changes that underlie its biological characteristics. WZSP also serves as a promising means for applications in surgery, tissue engineering, and xenotransplantation. Here, we report the sequencing and analysis of an inbreeding WZSP genome. RESULTS: Our results reveal some unique genomic features, including a relatively high level of homozygosity in the diploid genome, an unusual distribution of heterozygosity, an over-representation of tRNA-derived transposable elements, a small amount of porcine endogenous retrovirus, and a lack of type C retroviruses. In addition, we carried out systematic research on gene evolution, together with a detailed investigation of the counterparts of human drug target genes. CONCLUSION: Our results provide the opportunity to more clearly define the genomic character of pig, which could enhance our ability to create more useful pig models.

[DNA methylation patterns of mouse tetraploid embryos].

In the present study, the DNA methylation patterns of in vitro-derived mouse tetraploid embryos were investigated by immunofluorescence staining with an antibody against 5-methylcytosine (5MeC). Tetraploid embryos could be produced by electrofusion at the stage of two-cell embryos, which could develop to blastocysts followed by fusion of cytoplasm and nucleus and cleavage in vitro. During the fusion of cytoplasm, the DNA methylation levels of the fused embryos are as high as these of two-cell diploid embryos in vivo Then the embryos are rapidly demethylated when the nucleus begin to fuse, resulting in the lowest DNA methylation levels when the nucleus are fused completely. After that, the DNA methylation levels of the fused embryos are gradually increased until the morula stage. However, whereas an asymmetric distribution of DNA methylation is established in vivo-derived blastocysts with a higher methylation level in the inner cell mass (ICM) than that in the trophectoderm, we can not detect the asymmetric distribution in most in vitro-derived tetraploid blastocysts. So the DNA methylation patterns of mouse tetraploid embryos are aberrant, which may lead to subsequent developmental failure and embryo death. This is the first report on the methylation patterns of in vitro-derived mouse tetraploid embryos.

Characterization of porcine EPLIN gene revealed distinct expression patterns for the two isoforms.

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein that is down-regulated in transformed cells. Two EPLIN isoforms (alpha and beta) are generated by alternative promoter usage from a single gene. In pigs EPLIN was preferentially expressed in the muscle of splay-legged piglets, but not in the healthy ones, suggesting that it plays an important role in muscle development. A precise mRNA expression analysis through muscle development could shed some light on the EPLIN function associated with splayed legs. This article describes the isolation of the two alternative splice variants of EPLIN mRNA in pigs. The chromosome assignment and several polymorphism sites were also identified to lay a foundation for potential breeding applications. Gene expression analysis by real-time polymerase chain reaction (PCR) showed that both of transcripts were expressed in almost all tissues examined but in variable amounts in adult pigs. The temporal expression analysis indicated that they are not coexpressional through muscle development: EPLIN-alpha was detected in developing skeletal muscle, but EPLIN-beta was not.