Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

Development of LIBRA-seq for the guinea pig model system as a tool for the evaluation of antibody responses to multivalent HIV-1 vaccines.

Consistent elicitation of serum antibody responses that neutralize diverse clades of HIV-1 remains a primary goal of HIV-1 vaccine research. Prior work has defined key features of soluble HIV-1 Envelope (Env) immunogen cocktails that influence the neutralization breadth and potency of multivalent vaccine-elicited antibody responses including the number of Env strains in the regimen. We designed immunization groups that consisted of different numbers of SOSIP Env strains to be used in a cocktail immunization strategy: the smallest cocktail (group 2) consisted of a set of two Env strains, which were a subset of the three Env strains that made up group 3, which, in turn, were a subset of the six Env strains that made up group 4. Serum neutralizing titers were modestly broader in guinea pigs that were immunized with a cocktail of three Envs compared to cocktails of two and six, suggesting that multivalent Env immunization could provide a benefit but may be detrimental when the cocktail size is too large. We then adapted the LIBRA-seq platform for antibody discovery to be compatible with guinea pigs, and isolated several tier 2 neutralizing monoclonal antibodies. Three antibodies isolated from two separate guinea pigs were similar in their gene usage and CDR3s, establishing evidence for a guinea pig public clonotype elicited through vaccination. Taken together, this work investigated multivalent HIV-1 Env immunization strategies and provides a novel methodology for screening guinea pig B cell receptor antigen specificity at a high-throughput level using LIBRA-seq.IMPORTANCEMultivalent vaccination with soluble Env immunogens is at the forefront of HIV-1 vaccination strategies but little is known about the influence of the number of Env strains included in vaccine cocktails. Our results suggest that adding more strains is sometimes beneficial but may be detrimental when the number of strains is too high. In addition, we adapted the LIBRA-seq platform to be compatible with guinea pig samples and isolated several tier 2 neutralizing monoclonal antibodies, some of which share V and J gene usage and >70% CDR3 identity, thus establishing the existence of public clonotypes in guinea pigs elicited through vaccination.

Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge.

African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.

The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions.

Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.

Preliminary construction of non-coding RNAs and ceRNA regulatory networks mediated by exosomes in porcine follicular fluid.

BACKGROUND: Follicles are fundamental units of the ovary, regulated intricately during development. Exosomes and ovarian granulosa cells (OGCs) play pivotal roles in follicular development, yet the regulatory mechanisms governing exosomes remain elusive. RESULTS: High-throughput sequencing was employed to evaluate the complete transcript expression profiles of six samples (three porcine ovarian granulosa cells-exosome co-culture samples (GCE) and three porcine ovarian granulosa cells (POGCs) samples). Differential expression analysis revealed 924 lncRNAs, 35 circRNAs, 49 miRNAs, and 9823 mRNAs in the GCE group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated enrichment of differentially expressed transcripts in pathways related to cell proliferation and apoptosis. Furthermore, a ceRNA regulatory network comprising 43 lncRNAs, 6 circRNAs, 11 miRNAs, and 126 mRNAs was constructed based on intergene co-expression correlations. Seven miRNAs associated with cell proliferation and apoptosis regulation were identified within this network, encompassing 92 subnet pairs as candidate genes for further exploration of exosome regulatory mechanisms. Additionally, preliminary verification at the cellular level demonstrated that exosomal miR-200b enhances the viability of POGCs. CONCLUSIONS: Transcriptome analysis unveiled a pivotal candidate ceRNA network potentially implicated in exosome-mediated regulation of granulosa cell proliferation and apoptosis, thereby influencing porcine follicular development. These findings offer insights into the molecular mechanisms of follicular fluid exosome regulation, encompassing both coding and non-coding RNA perspectives.

Cell specification and functional interactions in the pig blastocyst inferred from single-cell transcriptomics and uterine fluids proteomics.

The embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mice and humans. During this peri-implantation period, a complex series of paracrine signals establishes an intimate dialogue between the embryo and the uterus. To better understand the biology of the pig blastocyst during this period, we generated a large dataset of single-cell RNAseq from early and hatched blastocysts, spheroid and ovoid conceptus and proteomic datasets from corresponding uterine fluids. Our results confirm the molecular specificity and functionality of the three main cell populations. We also discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent progenitor cells, and the other, expressing pro-apoptotic markers, which could correspond to the Rauber's layer. Our work provides new insights into the biology of these populations, their reciprocal functional interactions, and the molecular dialogue with the maternal uterine environment.

Integrated analysis of lncRNA, miRNA and mRNA expression profiles reveals regulatory pathways associated with pig testis function.

Long noncoding RNA (lncRNA) and microRNA (miRNA) are known to play pivotal roles in mammalian testicular function and spermatogenesis. However, their impact on porcine male reproduction has yet to be well unraveled. Here, we sequenced and identified lncRNA and miRNA expressed in the testes of Chinese indigenous Banna mini-pig inbred line (BMI) and introduced Western Duroc (DU) and Large White (LW) pigs. By pairwise comparison (BMI vs DU, BMI vs LW, and DU vs LW), we found the gene expression differences in the testes between Chinese local pigs and introduced Western commercial breeds were more striking than those between introduced commercial breeds. Furthermore, we found 1622 co-differentially expressed genes (co-DEGs), 122 co-differentially expressed lncRNAs (co-DELs), 39 co-differentially expressed miRNAs (co-DEMs) in BMI vs introduced commercial breeds (DU and LW). Functional analysis revealed that these co-DEGs and co-DELs/co-DEMs target genes were enriched in male sexual function pathways, including MAPK, AMPK, TGF-ß/Smad, Hippo, NF-kappa B, and PI3K/Akt signaling pathways. Additionally, we established 10,536 lncRNA-mRNA, 11,248 miRNA-mRNA pairs, and 62 ceRNA (lncRNA-miRNA-mRNA) networks. The ssc-miR-1343 had the most interactive factors in the ceRNA network, including 20 mRNAs and 3 lncRNAs, consisting of 56 ceRNA pairs. These factors played extremely important roles in the regulation of testis function as key nodes in the interactive regulatory network. Our results provide insight into the functional roles of lncRNAs and miRNAs in porcine testis and offer a valuable resource for understanding the differences between Chinese indigenous and introduced Western pigs.

Epigenomic analysis of the myometrium during late implantation revealed regulatory elements in genes related to the cellular zinc homeostasis pathway in pigs.

The myometrium, composed of the inner circular muscle (CM) and outer longitudinal muscle (LM), is crucial in establishing and maintaining early pregnancy. However, the molecular mechanisms involved are not well understood. In this study, we identified the transcriptomic features of the CM and LM collected from the mesometrial (M) and anti-mesometrial (AM) sides of the pig uterus on day 18 of pregnancy during the placentation initiation phase. Some genes in the cellular zinc ion level regulatory pathways (MT-1A, MT-1D, MT-2B, SLC30A2, and SLC39A2) were spatially and highly enriched in uterine CM at the mesometrial side. In addition, the histone modification profiles of H3K27ac and H3K4me3 in uterine CM and LM collected from the mesometrial side were characterized. Genomic regions associated with the expression of genes regulating the cellular zinc ion level were detected. Moreover, six highly linked variants in the H3K27ac-enriched region of the pig SLC30A2 gene were identified and found to be significantly associated with the total number born at the second parity (P < 0.05). In conclusion, the genes in the pathways of cellular zinc homeostasis and their regulatory elements identified have implications for pig reproduction trait improvement and warrant further investigations.

Assembly of novel sequences for Chinese domestic pigs reveals new genes and regulatory variants providing new insights into their diversity.

There is an increasing understanding that a reference genome representing an individual cannot capture all the gene repertoire of a species. Here, we conduct a population-scale missing sequences detection of Chinese domestic pigs using whole-genome sequencing data from 534 individuals. We identify 132.41 Mb of sequences absent in the reference assembly, including eight novel genes. In particular, the breeds spread in Chinese high-altitude regions perform significantly different frequencies of new sequences in promoters than other breeds. Furthermore, we dissect the role of non-coding variants and identify a novel sequence inserted in the 3'UTR of the FMO3 gene, which may be associated with the intramuscular fat phenotype. This novel sequence could be a candidate marker for meat quality. Our study provides a comprehensive overview of the missing sequences in Chinese domestic pigs and indicates that this dataset is a valuable resource for understanding the diversity and biology of pigs.

Definition of regulatory elements and transcription factors controlling porcine immune cell gene expression at single cell resolution using single nucleus ATAC-seq.

The transcriptome of porcine peripheral blood mononuclear cells (PBMC) at single cell (sc) resolution is well described, but little is understood about the cis-regulatory mechanism behind scPBMC gene expression. Here, we profiled the open chromatin landscape of porcine PBMC that define cis-regulatory elements and mechanism contributing to the transcription using single nucleus ATAC sequencing (snATAC-seq). Approximately 22 % of the identified peaks overlapped with annotated transcription start sites (TSS). Using clustering based on open chromatin pattern similarity, we demonstrate that cell type annotations using snATAC-seq are highly concordant to that reported for sc RNA sequencing (scRNA-seq). The differentially accessible peaks (DAPs) for each cell type were characterized and the pattern of accessibility of the DAPs near cell type markers across cell types was similar to that of the average gene expression level of corresponding marker genes. Additionally, we found that peaks identified in snATAC-seq have the potential power to predict the cell type specific transcription starting site (TSS). We identified both transcription factors (TFs) whose binding motif were enriched in cell type DAPs of multiple cell types and cell type specific TFs by conducting transcription factor binding motif (TFBM) analysis. Furthermore, we identified the putative enhancer or promoter regions bound by TFs for each differentially expressed gene (DEG) with a DAP that overlapped with its TSS by generating cis-co-accessibility networks (CCAN). To predict the regulators of such DEGs, TFBM analysis was performed for each CCAN. The regulator TF-target DEG pairs predicted in this way were largely consistent with the results reported in the ENCODE Transcription Factor Targets Dataset (TFTD). This snATAC-seq approach provides insights into the regulation of chromatin accessibility landscape of porcine PBMCs and enables discovery of TFs predicted to control DEG through binding regulatory elements whose chromatin accessibility correlates with the DEG promoter region.

A network of transient domains for breaking symmetry during anterior-posterior axis formation in the porcine embryo.

Breaking radial symmetry for anterior-posterior axis formation is one of the key developmental steps of vertebrate gastrulation and is established through a succession of transient domains defined by morphology or gene expression. Three such domains were interpreted recently in the rabbit to be part of a "three-anchor-point model" for axis formation. To answer the question as to whether the model is generally applicable to mammals, the dynamic expression patterns of four marker genes were analyzed in the pig, where gastrulating epiblast forms from half the inner cell mass: EOMES and PKDCC transcripts display decreasing expression intensities in the anterior hypoblast and-together with WNT3-increasing intensity in the anterior streak domain and the node; TBX6 expression changes from an initial central expression to exclusive expression in the posterior extremity of the primitive streak. The anterior streak domain has thus a molecular footprint similar to the one in the rabbit, the end node shares TBX6 between the species, while the anterior hypoblast-mirroring specific porcine epiblast derivation and fate-is marked by PKDCC instead of WNT3. The molecular similarities in transient domains point to conserved mechanisms for establishing the mammalian anterior-posterior axis and, possibly, breaking radial symmetry.

Diabetic retinopathy from the vitreous proteome perspective: The INSC94Y transgenic pig model study.

INSC94Y transgenic pigs represent a model for mutant insulin gene-induced diabetes of youth, with impaired insulin secretion and beta cell loss, leading to elevated fasting blood glucose levels. A key complication of diabetes mellitus is diabetic retinopathy (DR), characterized by hyperglycemia-induced abnormalities in the retina. Adjacent to the retina lies the vitreous, a gelatinous matrix vital for ocular function. It harbors proteins and signaling molecules, offering insights into vitreous biology and ocular health. Moreover, as a reservoir for secreted molecules, the vitreous illuminates molecular processes within intraocular structures, especially under pathological conditions. To uncover the proteomic profile of porcine vitreous and explore its relevance to DR, we employed discovery proteomics to compare vitreous samples from INSC94Y transgenic pigs and wild-type controls. Our analysis identified 1404 proteins, with 266 showing differential abundance in INSC94Y vitreous. Notably, the abundances of ITGB1, COX2, and GRIFIN were significantly elevated in INSC94Y vitreous. Gene Set Enrichment Analysis unveiled heightened MYC and mTORC1 signaling in INSC94Y vitreous, shedding light on its biological significance in diabetes-associated ocular pathophysiology. These findings deepen our understanding of vitreous involvement in DR and provide valuable insights into potential therapeutic targets. Raw data are accessible via ProteomeXchange (PXD038198).

Integrated Proteomics Analysis of Baseline Protein Expression in Pig Tissues.

The availability of an increasingly large amount of public proteomics data sets presents an opportunity for performing combined analyses to generate comprehensive organism-wide protein expression maps across different organisms and biological conditions. Sus scrofa, a domestic pig, is a model organism relevant for food production and for human biomedical research. Here, we reanalyzed 14 public proteomics data sets from the PRIDE database coming from pig tissues to assess baseline (without any biological perturbation) protein abundance in 14 organs, encompassing a total of 20 healthy tissues from 128 samples. The analysis involved the quantification of protein abundance in 599 mass spectrometry runs. We compared protein expression patterns among different pig organs and examined the distribution of proteins across these organs. Then, we studied how protein abundances were compared across different data sets and studied the tissue specificity of the detected proteins. Of particular interest, we conducted a comparative analysis of protein expression between pig and human tissues, revealing a high degree of correlation in protein expression among orthologs, particularly in brain, kidney, heart, and liver samples. We have integrated the protein expression results into the Expression Atlas resource for easy access and visualization of the protein expression data individually or alongside gene expression data.

Protein Dynamic Landscape of Pig Embryos during Peri-Implantation Development.

Properly developed embryos are critical for successful embryo implantation. The dynamic landscape of proteins as executors of biological processes in pig peri-implantation embryos has not been reported so far. In this study, we collected pig embryos from days 9, 12, and 15 of pregnancy during the peri-implantation stage for a PASEF-based quantitative proteomic analysis. In total, approximately 8000 proteins were identified. These proteins were classified as stage-exclusive proteins and stage-specific proteins, respectively, based on their presence and dynamic abundance changes at each stage. Functional analysis showed that their roles are consistent with the physiological processes of corresponding stages, such as the biosynthesis of amino acids and peptides at P09, the regulation of actin cytoskeletal organization and complement activation at P12, and the vesicular transport at P15. Correlation analysis between mRNAs and proteins showed a general positive correlation between pig peri-implantation embryonic mRNAs and proteins. Cross-species comparisons with human early embryos identified some conserved proteins that may be important in regulating embryonic development, such as STAT3, AP2A1, and PFAS. Our study provides a comprehensive overview of the pig embryo proteome during implantation, fills gaps in relevant developmental studies, and identifies some important proteins that may serve as potential targets for future research.

Short-term CaMKII inhibition with tatCN19o does not erase pre-formed memory in mice and is neuroprotective in pigs.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

Leukotriene B4 is elevated in diabetes and promotes ventricular arrhythmogenesis in guinea pig.

Diabetes (DM) patients have an increased risk (~50%) for sudden cardiac death (SCD), mostly as a result of ventricular arrhythmias. The molecular mechanisms involved remain partially defined. The potent proinflammatory lipid mediator leukotriene (LT) B4, is pathologically elevated in DM compared to nondiabetic patients, resulting in increased LTB4 accumulation in heart, leading to an increased risk for life-threatening proarrhythmic signatures. We used electrophysiology, immunofluorescence, and confocal microscopy approaches to evaluate LTB4 cellular effects in guinea pig heart and ventricular myocytes. We have observed that LTB4 is increased in multiple mouse models (C57BL/6 J/Lepob/ob and PANIC-ATTAC) of DM, promotes profound cellular arrhythmogenesis (spontaneous beats and early after depolarizations, EADs), and severely depresses the rapidly activating delayed rectifier K current (hERG1/IKr) density in HEK293 cells and guinea pig ventricular myocytes. We have further found that guinea pigs challenged with LTB4 displayed a significantly prolonged QT interval, and that this can be prevented with LTB4R inhibition, suggesting that preventing such LTB4R effects may be therapeutically beneficial in DM. Our data suggests that a further elucidation of LTB4 vulnerable substrates, and how this leads to ventricular arrhythmias, is likely to lead to continued improvements in management options, and the development of new therapies for prevention of SCD in DM patients.

Building in vitro tools for livestock genomics: chromosomal variation within the PK15 cell line.

BACKGROUND: Cultured porcine cell lines are powerful tools for functional genomics and in vitro phenotypic testing of candidate causal variants. However, to be utilised for genomic or variant interrogation assays, the genome sequence and structure of cultured cell lines must be realised. In this work, we called variants and used read coverage in combination with within-sample allele frequency to detect potential aneuploidy in two immortalised porcine kidney epithelial (PK15) cell lines and in a pig embryonic fibroblast line. RESULTS: We compared two PK15 cultured cells samples: a new American Type Culture Collection (ATCC) sample and one that has been utilised and passaged within the laboratory for an extended period (> 10 years). Read coverage and within-sample allele frequencies showed that several chromosomes are fully or partially aneuploid in both PK15 lines, including potential trisomy of chromosome 4 and tetrasomy of chromosome 17. The older PK15 line showed evidence of additional structural variation and potentially clonal variation. By comparison, the pig embryonic fibroblast line was free from the gross aneuploidies seen in the PK15s. CONCLUSIONS: Our results show that the PK15 cell lines examined have aneuploidies and complex structural variants in their genomes. We propose that screening for aneuploidy should be considered for cell lines, and discuss implications for livestock genomics.

Effect of metabolically divergent pig breeds and tissues on mesenchymal stem cell expression patterns during adipogenesis.

BACKGROUND: Unraveling the intricate and tightly regulated process of adipogenesis, involving coordinated activation of transcription factors and signaling pathways, is essential for addressing obesity and related metabolic disorders. The molecular pathways recruited by mesenchymal stem cells (MSCs) during adipogenesis are also dependent on the different sources of the cells and genetic backgrounds of donors, which contribute to the functional heterogeneity of the stem cells and consequently affect the developmental features and fate of the cells. METHODS: In this study, the alteration of transcripts during differentiation of synovial mesenchymal stem cells (SMSCs) derived from fibrous synovium (FS) and adipose synovial tissue (FP) of two pig breeds differing in growth performance (German Landrace (DL)) and fat deposition (Angeln Saddleback (AS)) was investigated. SMSCs from both tissues and breeds were stimulated to differentiate into adipocytes in vitro and sampled at four time points (day 1, day 4, day 7 and day 14) to obtain transcriptomic data. RESULTS: We observed numerous signaling pathways related to the cell cycle, cell division, cell migration, or cell proliferation during early stages of adipogenesis. As the differentiation process progresses, cells begin to accumulate intracellular lipid droplets and changes in gene expression patterns in particular of adipocyte-specific markers occur. PI3K-Akt signaling and metabolic pathways changed most during adipogenesis, while p53 signaling and ferroptosis were affected late in adipogenesis. When comparing MSCs from FS and FP, only a limited number of differentially expressed genes (DEGs) and enriched signaling pathways were identified. Metabolic pathways, including fat, energy or amino acid metabolism, were highly enriched in the AS breed SMSCs compared to those of the DL breed, especially at day 7 of adipogenesis, suggesting retention of the characteristic metabolic features of their original source, demonstrating donor memory in culture. In contrast, the DL SMSCs were more enriched in immune signaling pathways. CONCLUSIONS: Our study has provided important insights into the dynamics of adipogenesis and revealed metabolic shifts in SMSCs associated with different cell sources and genetic backgrounds of donors. This emphasises the critical role of metabolic and genetic factors as important indications and criteria for donor stem cell selection.

Differential chromatin accessibility and Gene Expression Associated with Backfat Deposition in pigs.

BACKGROUND: Backfat serves as a vital fat reservoir in pigs, and its excessive accumulation will adversely impact pig growth performance, farming efficiency, and pork quality. The aim of this research is to integrate assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to explore the molecular mechanisms underlying porcine backfat deposition. RESULTS: ATAC-seq analysis identified 568 genes originating from 698 regions exhibiting differential accessibility, which were significantly enriched in pathways pertinent to adipocyte differentiation and lipid metabolism. Besides, a total of 283 transcription factors (TFs) were identified by motif analysis. RNA-seq analysis revealed 978 differentially expressed genes (DEGs), which were enriched in pathways related to energy metabolism, cell cycle and signal transduction. The integration of ATAC-seq and RNA-seq data indicates that DEG expression levels are associated with chromatin accessibility. This comprehensive study highlights the involvement of critical pathways, including the Wnt signaling pathway, Jak-STAT signaling pathway, and fatty acid degradation, in the regulation of backfat deposition. Through rigorous analysis, we identified several candidate genes (LEP, CTBP2, EHHADH, OSMR, TCF7L2, BCL2, FGF1, UCP2, CCND1, TIMP1, and VDR) as potentially significant contributors to backfat deposition. Additionally, we constructed TF-TF and TF-target gene regulatory networks and identified a series of potential TFs related to backfat deposition (FOS, STAT3, SMAD3, and ESR1). CONCLUSIONS: This study represents the first application of ATAC-seq and RNA-seq, affording a novel perspective into the mechanisms underlying backfat deposition and providing invaluable resources for the enhancement of pig breeding programs.