Current porcine embryo culture relies on Porcine Zygote Medium 3 (PZM3) supplemented with fetal bovine serum (FBS), which causes batch-to-batch variability due to undefined components. Here, we evaluated N2B27-a chemically defined serum-free medium-as an alternative and compared the developmental outcomes of culture in PZM3 (PZM), PZM3 with 10% (v/v) FBS (PZM + FBS), and N2B27 in parthenogenetic embryos. N2B27 increased the total cell number, blastocyst and hatched blastocyst formation rates, and epiblast, primitive endoderm, and trophectoderm cell counts compared to PZM and PZM + FBS. Epiblast allocation increased relative to PZM but decreased relative to PZM + FBS, whereas primitive endoderm allocation increased relative to PZM and did not differ from PZM + FBS. Ungulates undergo rapid expansion of extraembryonic lineages during pre-implantation; thus we examined the effects of fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4) on extraembryonic lineage specification. FGF2 increased primitive endoderm cells and decreased epiblast cells, and their allocation. BMP4 increased the number of primitive endodermal cells and their allocation. FGF2 and BMP4 demonstrated combined effects, reducing the epiblast and increasing the primitive endoderm in both cell number and allocation. These results suggest that N2B27 can serve as a serum-free alternative for porcine parthenogenetic in vitro culture and that FGF2 and BMP4 can influence lineage specification toward the primitive endoderm.

Given the value of pigs as a large-animal model bridging rodents and humans, a porcine in vitro system for skeletal muscle development is a prerequisite for advancing both fundamental and translational research. Here, we establish a stepwise and high-resolution in vitro fate-map of pig skeletal muscle development from embryonic stem cells (ESCs). Single-cell analysis revealed three “myogenic states” faithfully recapitulating the conserved in vivo developmental trajectory: (1) specification of posterior presomitic mesoderm (PSM), (2) PSM patterning and somitogenesis, and (3) myogenesis from somite. Furthermore, we uncover porcine molecular signatures by identifying CDH15 and ADAM22 as markers for pig ESC-derived myogenic progenitors capable of generating multinucleated myotubes. Altogether, we present a comprehensive in vitro roadmap of pig skeletal muscle development by constructing previously uncharacterized developmental dynamics as a continuum. These findings offer a powerful resource for comparative developmental biology and translational preclinical research.

Porcine in vitro embryo production often relies on serum-supplemented media, yet fetal bovine serum (FBS) is chemically undefined and introduces batch-to-batch variability. Here, we tested whether the relatively defined N2B27 formulation improves porcine parthenogenetic embryo development compared with FBS-containing culture media and whether any developmental effects are associated with coordinated changes in oxidative stress, mitochondrial status, and apoptosis. After parthenogenetic activation, embryos were cultured in PZM-3 to the morula stage (96 h) and then transferred to PZM-3 (PZM), PZM-3 supplemented with 10% (v/v) FBS (PZM + FBS), or N2B27 for an additional 72 h. At 168 h, we assessed intracellular ROS and mitochondrial superoxide, ΔΨm and biogenesis-related indices, and apoptosis. N2B27 significantly increased blastocyst formation and hatching rates compared with both PZM conditions. In parallel, N2B27-derived blastocysts exhibited markedly lower intracellular ROS (-66.0% vs PZM; -45.9% vs PZM + FBS) and mitochondrial superoxide, higher ΔΨm, and increased expression of PPARGC1A, POLG, and TFAM. Although the number of TUNEL-positive nuclei did not differ among groups, total cell numbers increased and the apoptotic index decreased (-70.5% vs PZM; -53.5% vs PZM + FBS). These findings support N2B27 as a defined alternative to FBS-supplemented culture for mechanistic and applied embryo production, in association with reduced oxidative stress, improved mitochondrial status/biogenesis-related signatures, and attenuated apoptosis.

The objective of this study was to investigate the combined role of IGF-1, MEK inhibitor (Mi), and Notch inhibitor (Ni) in promoting myogenic differentiation of porcine skeletal muscle stem cells (pMuSCs). To determine the optimal supplementation, dose-response assays were performed for Mi, Ni, and IGF-1, considering both differentiation efficiency and cell viability. The selected concentrations were applied under single, binary, and ternary conditions, and differentiation was evaluated by immunofluorescence staining, qPCR, Western blotting, and assays of viability and oxidative stress. Compared to single or dual treatments the ternary combination (MNI treatment) most effectively increased differentiation, fusion, and maturation indices, as well as myotube diameter. Importantly, MNI strongly induced secondary myogenesis-related isoforms (MYH1, MYH2, MYH4). Western blot analysis revealed that MNI markedly increased phosphorylation of Akt and S6K1, while simultaneously suppressing ERK phosphorylation and reducing Hes1 expression, a key effector of Notch signaling. These findings indicate that simultaneous modulation of IGF-1, MEK, and Notch signaling provides an effective strategy to accelerate myogenic progression. In conclusion, MNI treatment represents a promising approach for designing culture conditions that promote muscle maturation in vitro, offering a mechanistic framework for optimizing muscle cell culture systems.

Extraembryonic endoderm (XEN) cells can be derived from blastocyst primitive endoderm (PrE), becoming a useful tool for studying mammalian development, including early lineage segregation and embryo patterning. Establishment of stem cells representing the respective lineages in blastocysts has been robustly attempted in domestic animals, especially pigs, to reconstitute embryogenesis in vitro for comparative studies. Therefore, we developed a serum-free culture system for pig XEN cells by dissecting the signals governing the core gene network of the PrE lineage. The FGF, LIF and WNT signaling pathways and B27 supplements are essential for maintaining a rapid proliferation rate in pig XEN cells. These cells recapitulated the molecular features and differentiation capacity of the PrE lineage. Especially, the XEN cells incorporated into normal development, retaining cellular identity and contributing to the PrE lineage when injected into in vitro-produced porcine blastocysts. In addition, species-specific characteristics of pigs were observed, including the involvement of lipid metabolism and NANOG/GATA co-expression in XEN cells. Taken together, our findings can contribute to the expansion of the understanding of developmental biology and its biomedical applications by enabling reproducible and homogeneous porcine XEN cell culture.

Pig embryonic stem cells were introduced with an MSGN1 upstream region-based reporter system, composed of EGFP sequence under porcine-specific MSGN1 promoter (pMSGN1), through electroporation. The engineered cell line maintained pluripotency similar to that of the parental cell line, and reporter activity was confirmed upon the induction of the paraxial mesoderm. The pMSGN1-EGFP reporter system is a valuable tool for monitoring the differentiation process of the paraxial mesoderm and analyzing induction efficiency, providing insight into developmental mechanisms.

Optimizing the metabolic environment during porcine skeletal muscle stem cells (PSCs) proliferation and differentiation is essential for efficient muscle cell cultivation. This study investigated metabolic shifts during myogenic progression using cells at P1, P3, D1, and D2 for characterization and metabolomic analysis. Cell number increased until P3, and myotubes formed by D2 but detached by D3 in 2D culture. Gene and protein expression analyses confirmed myogenic progression, including increased MYOD and MHC expression. Metabolomic profiling identified 94 intracellular metabolites, with 24 and 17 differentially abundant metabolites (DAMs) in proliferation and differentiation, respectively. Pathway analysis showed that energy production via amino acid metabolism was key to progression. Notably, PSCs relied on glycolysis during proliferation and shifted to mitochondrial oxidative phosphorylation during differentiation. These results highlight stage-specific metabolic reprogramming and provide insights for optimizing culture conditions, offering foundational knowledge to improve the efficiency and quality of cultured meat production.

Biological ageing can be defined as a gradual loss of homeostasis across various aspects of molecular and cellular function(1,2). Mammalian brains consist of thousands of cell types(3), which may be differentially susceptible or resilient to ageing. Here we present a comprehensive single-cell RNA sequencing dataset containing roughly 1.2million high-quality single-cell transcriptomes of brain cells from young adult and aged mice of both sexes, from regions spanning the forebrain, midbrain and hindbrain. High-resolution clustering of all cells results in 847 cell clusters and reveals at least 14 age-biased clusters that are mostly glial types. At the broader cell subclass and supertype levels, we find age-associated gene expression signatures and provide a list of 2,449 unique differentially expressed genes (age-DE genes) for many neuronal and non-neuronal cell types. Whereas most age-DE genes are unique to specific cell types, we observe common signatures with ageing across cell types, including a decrease in expression of genes related to neuronal structure and function in many neuron types, major astrocyte types and mature oligodendrocytes, and an increase in expression of genes related to immune function, antigen presentation, inflammation, and cell motility in immune cell types and some vascular cell types. Finally, we observe that some of the cell types that demonstrate the greatest sensitivity to ageing are concentrated around the third ventricle in the hypothalamus, including tanycytes, ependymal cells, and certain neuron types in the arcuate nucleus, dorsomedial nucleus and paraventricular nucleus that express genes canonically related to energy homeostasis. Many of these types demonstrate both a decrease in neuronal function and an increase in immune response. These findings suggest that the third ventricle in the hypothalamus may be a hub for ageing in the mouse brain. Overall, this study systematically delineates a dynamic landscape of cell-type-specific transcriptomic changes in the brain associated with normal ageing that will serve as a foundation for the investigation of functional changes in ageing and the interaction of ageing and disease.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

We evaluated the myogenic potential of muscle stem cells (MuSCs) derived from three distinct chicken purebreds—Rhode Island Red (RIR), White Leghorn (WL), and Cornish (CN). Chicken MuSCs were isolated from the breast muscles of chicken embryos on embryonic day 18 using a pre-plating method. Subsequently, the cells underwent a three-day proliferation period, followed by a three-day differentiation phase. WL MuSCs exhibited higher myogenic potential initially compared to RIR and CN. Despite a relatively lower proliferation rate, WL demonstrated a higher proportion of PAX7-positive cells and showed higher expression of myogenic regulatory factor genes than the other breeds. During differentiation, MuSCs from WL formed thicker muscle fibers and showed the highest proportion of myosin-heavy chain-positive cells than MuSCs from other breeds. Additionally, the expression of MYH1, associated with muscle maturation, was highest in WL. Overall, this finding suggests that the myogenic potential of MuSCs from WL surpasses those of RIR and CN. Given the fact that WL is primarily used for egg laying with a low growth rate in the traditional poultry industry, the present study highlights the crucial distinction between selecting production characteristics in conventional animal husbandry and those desirable for cultured meat production.

The mammalian cortex is composed of a highly diverse set of cell types and develops through a series of temporally regulated events that build out the cell type and circuit foundation for cortical function. The mechanisms underlying the development of different cell types remain elusive. Single-cell transcriptomics provides the capacity to systematically study cell types across the entire temporal range of cortical development. Here, we present a comprehensive and high-resolution transcriptomic and epigenomic cell type atlas of the developing mouse visual cortex. The atlas was built from a single-cell RNA-sequencing dataset of 568,674 high-quality single-cell transcriptomes and a single-nucleus Multiome dataset of 194,545 high-quality nuclei providing both transcriptomic and chromatin accessibility profiles, densely sampled throughout the embryonic and postnatal developmental stages from E11.5 to P56. We computationally reconstructed a transcriptomic developmental trajectory map of all excitatory, inhibitory, and non-neuronal cell types in the visual cortex, identifying branching points marking the emergence of new cell types at specific developmental ages and defining molecular signatures of cellular diversification. In addition to neurogenesis, gliogenesis and early postmitotic maturation in the embryonic stage which gives rise to all the cell classes and nearly all subclasses, we find that increasingly refined cell types emerge throughout the postnatal differentiation process, including the late emergence of many cell types during the eye-opening stage (P11-P14) and the onset of critical period (P21), suggesting continuous cell type diversification at different stages of cortical development. Throughout development, we find cooperative dynamic changes in gene expression and chromatin accessibility in specific cell types, identifying both chromatin peaks potentially regulating the expression of specific genes and transcription factors potentially regulating specific peaks. Furthermore, a single gene can be regulated by multiple peaks associated with different cell types and/or different developmental stages. Collectively, our study provides the most detailed dynamic molecular map directly associated with individual cell types and specific developmental events that reveals the molecular logic underlying the continuous refinement of cell type identities in the developing visual cortex.

Here, we examined the effects of the BMP signaling pathway inhibitor LDN-193189 on the pluripotency of porcine embryonic stem cells (ESCs) in the absence of feeder cells using molecular and transcriptomic techniques. Additionally, the effects of some extracellular matrix components on porcine ESC pluripotency were evaluated to develop an optimized and sustainable feeder-free culture system for porcine ESCs. Feeder cells were found to play an important role in supporting the pluripotency of porcine ESCs by blocking trophoblast and mesodermal differentiation through the inhibition of the BMP pathway. Additionally, treatment with LDN-193189, an inhibitor of the BMP pathway, maintained the pluripotency and homogeneity of porcine ESCs for an extended period in the absence of feeder cells by stimulating the secretion of chemokines and suppressing differentiation, based on transcriptome analysis. Conclusively, these results suggest that LDN-193189 could be a suitable replacement for feeder cells in the maintenance of porcine ESC pluripotency during culture. Additionally, these findings contribute to the understanding of pluripotency gene networks and comparative embryogenesis.

Cellular agriculture is an emerging research field of agribiotechnology that aims to produce agricultural products using stem cells, without sacrificing animals or cultivating crops. Cultivated meat, as a representative cellular product of cellular agriculture, is being actively researched due to global food insecurity, environmental, and ethical concerns. This review focuses on the application of stem cells, which are the seeds of cellular agriculture, for the production of cultivated meat, with emphasis on deriving and culturing muscle and adipose stem cells for imitating fresh meat. Establishing standards and safety regulations for culturing stem cells is crucial for the market entry of cultured muscle tissue-based biomaterials. Understanding stem cells is a prerequisite for creating reliable cultivated meat and other cellular agricultural biomaterials. The techniques and regulations from the cultivated meat industry could pave the way for new cellular agriculture industries in the future.

OBJECTIVE:Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo.METHODS:A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers.RESULTS:In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased.CONCLUSION:Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1-3. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

Fertilized embryos develop and move freely in the reproductive tract until implantation. Subsequently, the embryos continue to develop after attachment to the uterus. Because of the absence of the uterus, in vitro culturing of embryos is limited to a period of approximately a week. Hatched blastocysts were seeded on feeder cells to extend the culture period. We cultured the colonies formed from the blastocysts for an additional 14 days. From the colonies, four types of cells were established, and each type was isolated to extract RNA. RNA sequencing was conducted using NovaSeq6000. Sequencing reads were aligned to genes and transcripts. Raw data from our previous study were used to compare these samples with the cultured cell lines. We analyzed differentially expressed genes and Gene Ontology terms between new samples and cultured cell lines. Our data can provide essential information for extending the period of embryo culture in vitro.

Biological aging can be defined as a gradual loss of homeostasis across various aspects of molecular and cellular function. Aging is a complex and dynamic process which influences distinct cell types in a myriad of ways. The cellular architecture of the mammalian brain is heterogeneous and diverse, making it challenging to identify precise areas and cell types of the brain that are more susceptible to aging than others. Here, we present a high-resolution single-cell RNA sequencing dataset containing ∼1.2 million high-quality single-cell transcriptomic profiles of brain cells from young adult and aged mice across both sexes, including areas spanning the forebrain, midbrain, and hindbrain. We find age-associated gene expression signatures across nearly all 130+ neuronal and non-neuronal cell subclasses we identified. We detect the greatest gene expression changes in non-neuronal cell types, suggesting that different cell types in the brain vary in their susceptibility to aging. We identify specific, age-enriched clusters within specific glial, vascular, and immune cell types from both cortical and subcortical regions of the brain, and specific gene expression changes associated with cell senescence, inflammation, decrease in new myelination, and decreased vasculature integrity. We also identify genes with expression changes across multiple cell subclasses, pointing to certain mechanisms of aging that may occur across wide regions or broad cell types of the brain. Finally, we discover the greatest gene expression changes in cell types localized to the third ventricle of the hypothalamus, including tanycytes, ependymal cells, and Tbx3 + neurons found in the arcuate nucleus that are part of the neuronal circuits regulating food intake and energy homeostasis. These findings suggest that the area surrounding the third ventricle in the hypothalamus may be a hub for aging in the mouse brain. Overall, we reveal a dynamic landscape of cell-type-specific transcriptomic changes in the brain associated with normal aging that will serve as a foundation for the investigation of functional changes in the aging process and the interaction of aging and diseases.