Inhibition of BMP-mediated SMAD pathway supports the pluripotency of pig embryonic stem cells in the absence of feeder cells.

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.

Development of Reproducible and Scalable Culture Conditions for In Vitro Maintenance of Pig Embryonic Stem Cells Using the Sandoz Inbred Swiss Mouse Thioguanine-Resistant Ouabain-Resistant Cell Line as a Feeder Layer.

Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm2) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm2), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture.

NANOG expression in parthenogenetic porcine blastocysts is required for intact lineage specification and pluripotency.

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.

Unlocking the potential of stem cells: Their crucial role in the production of cultivated meat.

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.

Comparison data of transcriptomes from blastocyst seeding samples and cultured cell lines from pigs.

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.

The number of primitive endoderm cells in the inner cell mass is regulated by platelet-derived growth factor signaling in porcine preimplantation embryos.

OBJECTIVE: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. METHODS: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. RESULTS: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. CONCLUSION: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.

Characterization of multitype colonies originating from porcine blastocysts produced in vitro.

Many types of embryonic stem cells have been induced from pre-implantation blastocysts to study the specification of early lineages. Various cell lines have been established using chemicals, including excessive inhibitory molecules. Previous studies have also aimed to purify cell populations representing a single embryonic lineage from a protocol. In this study, we used a novel culture condition to induce cells from blastocyst seeding and analyzed their characteristics. Next, signaling inhibitors were introduced during the cell culture period. Furthermore, we investigated the cell types using RNA sequencing. Each type of cell population showed a distinct morphology and reactivity with alkaline phosphatase. Marker proteins enabled each cell type to be distinguished by immunocytochemistry, and genes such as Sox17, Gata4, Gata6, T, and Cdx2 showed applicability for the discrimination of cell types. Signaling inhibitors suppressed the production of some cell types, and gene expression and marker protein patterns were collapsed. RNA-sequencing suggested cell-type-specific marker genes and the correlation among samples. In conclusion, four types of cells could be induced from porcine embryos using a single protocol, and they could be isolated manually. Our data will help promote the study of lineage segregation based on embryonic cells.

Changes in OCT4 expression play a crucial role in the lineage specification and proliferation of preimplantation porcine blastocysts.

OBJECTIVES: Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a species-specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions. MATERIALS AND METHODS: To determine the functions of OCT4 in lineage specification and embryo proliferation, loss- and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes. RESULTS: In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased. CONCLUSION: Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation.

Species-Specific Enhancer Activity of OCT4 in Porcine Pluripotency: The Porcine OCT4 Reporter System Could Monitor Pluripotency in Porcine Embryo Development and Embryonic Stem Cells.

The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

Linoleic acid reduces apoptosis via NF-κB during the in vitro development of induced parthenogenic porcine embryos.

Fatty acid has a various role in preimplantation embryo development. Especially, Linoleic acid, polyunsaturated fatty acid, has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B. But to date, the function of NF-κB has not been demonstrated in porcine preimplantation embryos. We demonstrated that linoleic acid had a positive effect on embryo development at a certain concentration(25 µM), but developmental failure was observed at higher concentration. Furthermore, the expression level of NF-κB increased, unlike that of IL-6, as the concentration of linoleic acid increased. Interestingly, the concentration of NF-κB was found to increase even at the concentration of linoleic acid at which embryo development decreased. We found that pro-apoptotic gene expression was downregulated in the linoleic acid-treated group. It was also found that MCL-1, an anti-apoptotic gene known to be unaffected by IL-6, was found to be increased at the mRNA level in the linoleic acid-treated group. As the concentration of NF-kB increased, the nuclear translocation of C-JUN gradually increased dependent on the linoleic acid concentration. It was confirmed that NF-κB is an important factor in porcine embryos by treated ammonium pyrrolidinedithiocarbamate (APDC 0.1 µM, an inhibitor of NF-κB) affected NF-κB protein expression, IL-6 expression, and blastocyst production. These data supported porcine embryos can use exogenous linoleic acid as a metabolic energy source via NF-κB.

Pig embryonic stem cell line with porcine-specific OCT4 upstream region based dual reporter system.

Here, we report pig embryonic stem cells (ESCs) originating from parthenogenetic blastocysts which were developed from 2-cell embryos micro-injected with porcine OCT4 reporter system cultured in previous reported chemically defined culture media. The ESCs with reporter system expressed pluripotency markers and fluorescent signals produced by OCT4 reporter system. Also, they were capable of forming teratomas following subcutaneous injection into nude mice. Since reporter system enables the non-destructive classification of the condition of live pluripotent stem cells, this reporter cell line could be a useful resource for research on species-specific pluripotency.

SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development.

OBJECTIVES: Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species. MATERIALS AND METHODS: To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR. RESULTS: Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. CONCLUSIONS: Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis.

Combination of cell signaling molecules can facilitate MYOD1-mediated myogenic transdifferentiation of pig fibroblasts.

BACKGROUND: Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis. In this study, we attempted to transdifferentiate pig embryonic fibroblasts (PEFs) myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF, TGF-ß, WNT, and cAMP signaling pathways. RESULTS: The MYOD1 overexpression vector was constructed based on comparative sequence analysis, demonstrating that pig MYOD1 has evolutionarily conserved domains across various species. Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers, transdifferentiated muscle cells from fibroblasts were not observed. Therefore, various signaling molecules, including FGF2, SB431542, CHIR99021, and forskolin, along with MYOD1 overexpression were applied to enhance the myogenic reprogramming. The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs, as determined by qPCR and immunostaining. Notably, a sarcomere-like structure was observed, indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells. CONCLUSIONS: In summary, we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules. Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.

Muscle stem cell isolation and in vitro culture for meat production: A methodological review.

Cultured muscle tissue-based protein products, also known as cultured meat, are produced through in vitro myogenesis involving muscle stem cell culture and differentiation, and mature muscle cell processing for flavor and texture. This review focuses on the in vitro myogenesis for cultured meat production. The muscle stem cell-based in vitro muscle tissue production consists of a sequential process: (1) muscle sampling for stem cell collection, (2) muscle tissue dissociation and muscle stem cell isolation, (3) primary cell culture, (4) upscaled cell culture, (5) muscle differentiation and maturation, and (6) muscle tissue harvest. Although muscle stem cell research is a well-established field, the majority of these steps remain to be underoptimized to enable the in vitro creation of edible muscle-derived meat products. The profound understanding of the process would help not only cultured meat production but also business sectors that have been seeking new biomaterials for the food industry. In this review, we discuss comprehensively and in detail each step of cutting-edge methods for cultured meat production. This would be meaningful for both academia and industry to prepare for the new era of cellular agriculture.

MicroRNA expression data of pluripotent and somatic cells and identification of cell type-specific MicroRNAs in pigs.

Typical models of pluripotency, humans and mice, have been used to analyse the characteristics of pluripotent stem cells. However, these species exhibit molecular differences in many aspects. With similar physiology and genomics as humans, pigs are promising model for the research of pluripotency. The data of porcine pluripotent cells would be helpful in understanding the molecular network of human pluripotency. Pluripotent cells of humans and mice exhibit specific MicroRNA (miRNA) expression patterns to maintain the pluripotent state. Information about miRNA expression in pig pluripotent cells is not sufficient, so we analysed miRNAs in pluripotent (blastocysts and ES-like) and somatic cell samples (PEB and PFF). We screened cell-type specific miRNAs and identified their target genes. Functional annotation of the target genes was also conducted. Our data may facilitate miRNA-based induction and maintenance of the pluripotent state of porcine cells and provide support to fill the gap between the pluripotency networks of humans and mice.

Identification of the Lineage Markers and Inhibition of DAB2 in In Vitro Fertilized Porcine Embryos.

Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.

Purification of Pig Muscle Stem Cells Using Magnetic-Activated Cell Sorting (MACS) Based on the Expression of Cluster of Differentiation 29 (CD29).

The muscle stem cells of domestic animals are of interest to researchers in the food and biotechnology industries for the production of cultured meat. For producing cultured meat, it is crucial for muscle stem cells to be efficiently isolated and stably maintained in vitro on a large scale. In the present study, we aimed to optimize the method for the enrichment of pig muscle stem cells using a magnetic-activated cell sorting (MACS) system. Pig muscle stem cells were collected from the biceps femoris muscles of 14 d-old pigs of three breeds [Landrace×Yorkshire×Duroc (LYD), Berkshire, and Korean native pigs] and cultured in skeletal muscle growth medium-2 (SkGM-2) supplemented with epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580). Approximately 30% of total cultured cells were nonmyogenic cells in the absence of purification in our system, as determined by immunostaining for cluster of differentiation 56 (CD56) and CD29, which are known markers of muscle stem cells. Interestingly, following MACS isolation using the CD29 antibody, the proportion of CD56+/CD29+ muscle stem cells was significantly increased (91.5±2.40%), and the proportion of CD56 single-positive nonmyogenic cells was dramatically decreased. Furthermore, we verified that this method worked well for purifying muscle stem cells in the three pig breeds. Accordingly, we found that CD29 is a valuable candidate among the various marker genes for the isolation of pig muscle stem cells and developed a simple sorting method based on a single antibody to this protein.

Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro.

Muscle stem cells isolated from domestic animals, including cows and pigs, were recently spotlighted as candidates for the production of alternative protein resources, so-called cultured meat or lab-grown meat. In the present study, we aimed to optimize the in vitro culture conditions for the long-term expansion of pig muscle stem cells via the screening of various signaling molecules. Pig muscle stem cells were collected from the biceps femoris muscles of 3-d-old crossbred pigs (Landrace×Yorkshire×Duroc, LYD) and cultured in minimum essential medium-based growth media. However, the pig muscle stem cells gradually lost their proliferation ability and featured morphologies during the long-term culture over two weeks. To find suitable in vitro culture conditions for an extended period, skeletal muscle growth medium-2, including epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580), was used to support the stemness of the pig muscle stem cells. Interestingly, pig muscle stem cells were stably maintained in a long-term culture without loss of the expression of myogenic marker genes as determined by PCR analysis. Immunostaining analysis showed that the stem cells were capable of myogenic differentiation after multiple passaging. Therefore, we found that basal culture conditions containing EGF, dexamethasone, and a p38 inhibitor were suitable for maintaining pig muscle stem cells during expanded culture in vitro. This culture method may be applied for the production of cultured meat and further basic research on muscle development in the pig.