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Cultured meat (CM) production has emerged as a breakthrough technology for the global food industry, with the potential to reduce challenges associated with environmental sustainability, global public health, animal welfare, and competition for food between humans and animals. The muscle stem cell lines currently used for CM production cannot be passaged in vitro for extended periods of time. Here, we successfully developed a directional differentiation system of porcine pregastrulation epiblast stem cells (pgEpiSCs) with stable cellular features and achieved serum-free myogenic differentiation of the pgEpiSCs. The pgEpiSCs-derived skeletal muscle progenitor cells and skeletal muscle fibers show typical muscle cell characteristics and display skeletal muscle transcriptional features during myogenic differentiation. Importantly, we further established a three-dimensional differentiation system for shaping cultured tissue by screening plant-based edible scaffolds of non-animal origin, followed by the generation of pgEpiSCs-derived CM. These advances provide a novel technical approach for the development of CM.

Pluripotent stem cells (PSCs) harbor the capacity of unlimited self-renewal and multilineage differentiation potential, which are crucial for basic research and biomedical science. Establishment of PSCs with defined features was previously reported from mice and humans, while generation of stable large animal PSCs has experienced a relatively long trial stage and only recently has made breakthroughs. Pigs are regarded as ideal animal models for their similarities in physiology and anatomy to humans. Generation of porcine PSCs would provide cell resources for basic research, genetic engineering, animal breeding, and cultured meat. In this review, we summarize the progress on the derivation of porcine PSCs and reprogramed cells and elucidate the mechanisms of pluripotency changes during pig embryo development. This will be beneficial for understanding the divergence and conservation between different species involved in embryo development and the pluripotent-regulated signaling pathways. Finally, we also discuss the promising future applications of stable porcine PSCs. Even though challenges remain in the field of porcine stem cells, these progress and viewpoints would provide guidance in future research direction.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Summary The pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells., Graphical Abstract, Highlights • Mthfd2 improves complete reprogramming of mouse iPSCs • Mthfd2 regulates pluripotency of mESCs by enzyme-independent mechanisms • MTHFD2 directly interacts with CDK1 to modulates DNA repair in mESCs • Mthfd2 regulates both mitochondrial function and genomic stability in mESCs, In this article, Han and colleagues show that Mthfd2 is a powerful bifunctional regulator for pluripotency of pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair.

Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis., The precise cellular patterning and decisions of early embryogenesis have been hard to mimic in vitro. Here, the authors culture murine embryonic and trophoblast stem cells together with extra-embryonic endoderm stem cells to form embryo-like structures (ETX-embryoids), which can initiate an implantation response.

Background Despite years of research, porcine-induced pluripotent stem cells (piPSCs) with germline chimeric capacity have not been established. Furthermore, the key transcription factors (TFs) defining the naïve state in piPSCs also remain elusive, even though TFs in the inner cell mass (ICM) are believed to be key molecular determinants of naïve pluripotency. In this study, interferon regulatory factor 1 (IRF-1) was screened to express higher in ICM than trophectoderm (TE). But the impact of IRF-1 on maintenance of pluripotency in piPSCs was not determined. Methods Transcriptome profiles of the early ICM were analyzed to determine highly interconnected TFs. Cells carrying these TFs’ reporter were used to as donor cells for somatic cell nuclear transfer to detect expression patterns in blastocysts. Next, IRF1-Flag was overexpressed in DOX-hLIF-2i piPSCs and AP staining, qRT-PCR, and RNA-seq were conducted to examine the effect of IRF-1 on pluripotency. Then, the expression of IRF-1 in DOX-hLIF-2i piPSCs was labeled by GFP and qRT-PCR was conducted to determine the difference between GFP-positive and GFP-negative cells. Next, ChIP-Seq was conducted to identify genes target by IRF-1. Treatment with IL7 in wild-type piPSCs and STAT3 phosphorylation inhibitor in IRF-1 overexpressing piPSCs was conducted to confirm the roles of JAK-STAT3 signaling pathway in IRF-1’s regulation of pluripotency. Moreover, during reprogramming, IRF-1 was overexpressed and knocked down to determine the change of reprogramming efficiency. Results IRF-1 was screened to be expressed higher in porcine ICM than TE of d6~7 SCNT blastocysts. First, overexpression of IRF-1 in the piPSCs was observed to promote the morphology, AP staining, and expression profiles of pluripotency genes as would be expected when cells approach the naïve state. Genes, KEGG pathways, and GO terms related to the process of differentiation were also downregulated. Next, in the wild-type piPSCs, high-level fluorescence activated by the IRF-1 promoter was associated with higher expression of naïve related genes in piPSCs. Analysis by ChIP-Seq indicated that genes related to the JAK-STAT pathway, and expression of IL7 and STAT3 were activated by IRF-1. The inhibitor of STAT3 phosphorylation was observed could revert the expression of primed genes in IRF-1 overexpressing cells, but the addition of IL7 in culture medium had no apparent change in the cell morphology, AP staining results, or expression of pluripotency related genes. In addition, knockdown of IRF-1 during reprogramming appeared to reduce reprogramming efficiency, whereas overexpression exerted the converse effect. Conclusion The IRF-1 expressed in the ICM of pigs’ early blastocyst enhances the pluripotency of piPSCs, in part through promoting the JAK-STAT pathway.

Additional file 1: Figure S1. Screening for potential transcription factors related to porcine pluripotency, related to Fig. 1. (A) GO terms and KEGG pathways enriched from upregulated genes in ICM.(B) Analysis of ChIP-Seq data of H3K4me3. Marks indicate promoter regions of selected transcript factors. ChIP-Seq data of H3K4me3 in pEPSCs was obtained by Liu et al. [19]. The peaks from -4 k to -5 k upstream from the transcriptional start site of IRF-1 were peaks associated with other genes not IRF-1. (C) GFP fluorescence in PEFs transfected with reporter plasmid. Scale bar, 500 μm.(D) Distribution of red fluorescence in porcine reconstructed blastocysts by SCNT. GFP fluorescence represents successful integration of the reporter system. Red fluorescence represents the promoter’s activity. Scale bar, 100 μm.(E) qRT-PCR analysis of pluripotency-associated genes in BCL3 overexpressing cells and controls. Figure S2. Pluripotency characterization of DOX-hLIF-2i piPSCs, related to Fig. 2. (A) Immunofluorescence assay of SSEA-4, TRA-1-60, TRA-1-80. Scale bar, 20 μm. (B) EBs of DOX-hLIF-2i piPSCs obtained at day 6 of differentiation. Scale bar, 100 μm. (C) Fluorescence detection of OCT4-tdTomato in DOX-hLIF-2i piPSCs. Scale bar of the top figure, 100 μm. Scale bar of the bottom figure, 50 μm. (D) Cell morphology and AP staining of DOX-hLIF-2i piPSCs with DOX and without DOX. Scale bar, 200 μm. (E) RT-PCR analysis of endogenous expression of OCT4, SOX2, KLF4 and cMYC and exogenous OKSM. EF1A was used as internal control. 1#, 2# represent two lines of DOX-hLIF-2i piPSCs. Figure S3. The effect of IRF-1 overexpression on DOX-hLIF-2i piPSCS morphology, related to Fig. 3. (A) DAPI staining of IRF-1-overexpressing and negative control piPSCs in Fig. 3a. Scale bars from left to right, 200 μm, 50 μm. (B) RT-PCR analysis of endogenous expression of OCT4, SOX2, KLF4 and cMYC and exogenous OKSM. EF1A was used as internal control. OE: IRF-1 overexpressing piPSCs, WT: DOX-hLIF-2i piPSCs. Figure S4. Detection of heterogeneity stability of IRF-1 in DOX-hLIF-2i piPSCs, related to Fig. 4. (A) Fluorescence detection of GFP positive and negative cells after passage. Scale bars from left to the right, 100 μm, 200 μm. Figure S5. The effect of treatment with IL7 or Stattic treatment on pluripotency of DOX-hLIF-2i piPSCs, related to Fig. 5. (A) Cell morphology and AP staining of DOX-hLIF-2i piPSCs after treatment with IL7. Scale bars, 200 μm. (B) qRT-PCR analysis of pluripotency associated genes in piPSCS treated with IL7. *, P

After zygotic genome activation and lineage specification, zygotes develop into late blastocysts comprising three distinct cell types. The molecular mechanisms underlying this progress are largely unknown in pigs. Here, we intended to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the porcine blastocysts. Using a quantitative microfluidics approach in single cells, we detected mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency simultaneously in 480 individual cells derived from porcine preimplantation embryos. The developmental transitions can be distinguished based on distinctive gene expression profiles, and we identified paired box 6 (PAX6) and aquaporin 3 (AQP3) expressed in early and late developmental stages, respectively. Two lineages can be segregated in porcine early and late blastocysts by the expression patterns of lineage-specific genes such as DAB2, clathrin adaptor protein (DAB2) for trophectoderm (TE), platelet derived growth factor receptor alpha (PDGFRA), Nanog homeobox (NANOG), fibronectin 1 (FN1), hepatocyte nuclear factor 4 alpha (HNF4A), goosecoid homeobox (GSC), nuclear receptor subfamily 5 group A member 2 (NR5A2), and lysine acetyltransferase 6A (KAT6A; previously known as MYST3) for inner cell mass (ICM). However, the epiblast and primitive endoderm cannot be identified in late blastocysts, and those TE or ICM lineage-specific genes were low expressed in blastomeres from the morula. Our results shed light on early cell fate determination in porcine preimplantation embryos and offer theoretical support for deriving porcine embryonic stem cells.