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.

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.

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.

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.

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.

Porcine OCT4 Reporter System Can Monitor Species-Specific Pluripotency During Somatic Cell Reprogramming.

This study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in fibroblast growth factor (FGF)- or leukemia inhibitory factor (LIF)-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. Porcine embryonic fibroblasts were coinfected with the pOCT4-ΔPE-eGFP (distal enhancer [DE]-green fluorescent protein [GFP]) and pOCT4-ΔDE-DsRed2 (proximal enhancer [PE]-red fluorescent protein [RFP]) vectors, and GFP and RFP expression were verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 DE and PE were activated in different expression patterns simultaneously as changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems confirmed that porcine iPSCs are in primed state after reprogramming in FGF2- or LIF-containing media. This work demonstrates the applicability of 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.

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.

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.

Pluripotent pig embryonic stem cell lines originating from in vitro-fertilized and parthenogenetic embryos.

Here, we report in vivo developmental competent pig embryonic stem cells (ESCs) originating from in vitro-fertilized and parthenogenetic embryos cultured in chemically defined culture media. The pig ESC lines expressed pluripotency markers and were capable of forming teratomas following subcutaneous injection into nude mice. These cell lines would be a useful resource for comparative developmental biology and agricultural biotechnology.

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.

Transcriptome profiling of pluripotent pig embryonic stem cells originating from uni- and biparental embryos.

OBJECTIVES: Pig pluripotent stem cells have tremendous potential because the pig is a valuable animal as both an agricultural resource and as a preclinical model of human therapy. To date, a lack of understanding of pig pluripotency has inhibited the derivation of embryonic stem cells (ESCs) and transgene-free induced pluripotent stem cells. Therefore, there has been no accessible or reliable transcriptome data for researching the genuine pig pluripotency network. Our previous study isolated authentic pig ESCs, which had teratoma-forming and direct differentiation ability, that were derived by activating the FGF2, ACTIVIN A, and WNT pathways. Here, we aimed to provide detailed information on transcriptome data of the newly derived pig ESCs and perform a comparative analysis with pig preimplantation embryo transcriptomes in a public database. DATA DESCRIPTION: The transcriptome data of ESCs derived from in vitro fertilized and parthenogenetic embryos were generated by HiSeq 2500. Then, differentially expressed genes (DEGs) from each sample were compared with fibroblasts, and gene expression profiling was carried out for comparative analysis. Our data, as the first transcriptome dataset for genuine pig pluripotent cells, could be a general reference for explaining the molecular mechanism of species-specific pluripotency and improving understanding of the embryo development of domestic animals.

Progesterone receptor membrane component 1 (PGRMC1)-mediated progesterone effect on preimplantation development of in vitro produced porcine embryos.

Progesterone is a steroid hormone well known for its significant role in the reproduction process of mammals. Numerous studies have reported on the regulation of progesterone during implantation, pregnancy and parturition, but there are fewer studies on progesterone in relation to the early stages of embryo development. In the present study, we investigated the effects of progesterone during the development of in vitro produced porcine embryos. First, gene expression of various progesterone receptors in the in vitro produced porcine embryos were analyzed. PGRMC1 and PGRMC2 (progesterone receptor membrane component 1 and 2) showed distinct expression. Next, the embryos were treated with two concentrations of progesterone (10 nM and 100 nM) for two different durations (from day 0 and from day 4) to compare the developmental rates, cell numbers, and apoptosis rates of day 7 blastocysts. The experimental groups in both durations showed similarly increased blastocyst cell numbers and decreased apoptosis rates when treated with 100 nM progesterone. Furthermore, the expression levels of PGRMC1, PGRMC2, PAIRBP1 (plasminogen activator inhibitor RNA-binding protein 1), and apoptosis-related genes were examined in blastocysts and showed significant increases in the 100 nM treatment group compared to the control group. Subsequently, the embryos were treated with the PGRMC1 inhibitor, AG-205, and developmental rates, cell numbers, and apoptosis rates of day 7 blastocysts were compared. In addition, 100 nM progesterone was treated simultaneously with AG-205 to test if the inhibition effect is relieved by progesterone. Groups treated with 1 µM and 2 µM AG-205 showed decreased cell numbers and increased apoptosis rates in day 7 blastocysts compared to the control group. We also confirmed the recovery of inhibition by 100 nM progesterone. In conclusion, the present study indicated that progesterone positively affects the development of in vitro produced preimplantation porcine embryos by increasing cell proliferation and decreasing apoptosis via PGRMC1-involved actions. However, the detailed mechanisms of PGRMC1 need further elucidation.

Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa.

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5' upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.