Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture.

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Super-enhancers conserved within placental mammals maintain stem cell pluripotency.

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

Comparative analysis of porcine iPSCs derived from Sertoli cells and fibroblasts.

Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor ß signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming.

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network.

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.

Peptide-coating 2D and small chemical molecules prolong the passage of porcine spermatogonia stem cells.

Porcine spermatogonia stem cells (pSSCs) are the only type of somatic stem cell that can pass genetic information to the successive generations. Little is known about pSSCs vitality in vitro, and due to their increasing importance in stem cell research, here, we optimized a protocol to culture pSSCs and explored their potential fate in vitro. Utilizing a feeder-free culture system with a 2D peptide-coating and small chemical molecules (including CHIR99021, Repsox, vitamin C, folic acid, and CD lipid concentrate), we were able to prolong the culture time of pSSCs by at least three months compared with previous methods. Moreover, we found that pSSCs could proliferate and self-renew in the seminiferous tubules of infertile mice. However, they could not perform meiosis. Our study shows that this feeder-free culture system optimizes cell culture and may facilitate advanced research on SSC biology and genetic manipulation of germ cells.

KDM4C Contributes to Trophoblast-like Stem Cell Conversion from Porcine-Induced Pluripotent Stem Cells (piPSCs) via Regulating CDX2.

Studies on ESRRB-regulating porcine-induced pluripotent stem cells (piPSCs) converted to trophoblast-like stem cells (TLSCs) contribute to the understanding of early embryo development. However, the epigenetic modification regulation network during the conversion is poorly understood. Here, the global change in histone H3 Lysine 4, 9, 27, 36 methylation and Lysine 27 acetylation was investigated in piPSCs and TLSCs. We found a high modification profile of H3K36me2 in TLSCs compared to that of piPSCs, whereas the profiles of other modifications remained constant. KDM4C, a H3K36me3/2 demethylase, whose gene body region was combined with ESRRB, was upregulated in TLSCs. Moreover, KDM4 inhibitor supplementation rescued the AP-negative phenotype observed in TLSCs, confirming that KDM4C could regulate the pluripotency of TLSCs. Subsequently, KDM4C replenishment results show the significantly repressed proliferation and AP-positive staining of TLSCs. The expressions of CDX2 and KRT8 were also upregulated after KDM4C overexpression. In summary, these results show that KDM4C replaced the function of ESRRB. These findings reveal the unique and crucial role of KDM4C-mediated epigenetic chromatin modifications in determination of piPSCs' fate and expand the understanding of the connection between piPSCs and TSCs.

AXIN2 Reduces the Survival of Porcine Induced Pluripotent Stem Cells (piPSCs).

The establishment of porcine pluripotent stem cells (piPSCs) is critical but remains challenging. All piPSCs are extremely sensitive to minor perturbations of culture conditions and signaling network. Inhibitors, such as CHIR99021 and XAV939 targeting the WNT signaling pathway, have been added in a culture medium to modify the cell regulatory network. However, potential side effects of inhibitors could confine the pluripotency and practicability of piPSCs. This study aimed to investigate the roles of AXIN, one component of the WNT pathway in piPSCs. Here, porcine AXIN1 and AXIN2 genes were knocked-down or overexpressed. Digital RNA-seq was performed to explore the mechanism of cell proliferation and apoptosis. We found that (1) overexpression of the porcine AXIN2 gene significantly reduced survival and negatively impacted the pluripotency of piPSCs, and (2) knockdown of AXIN2, a negative effector of the WNT signaling pathway, enhanced the expression of genes involved in cell cycle but reduced the expression of genes related to cell differentiation, death, and apoptosis.

The ACE2 expression in Sertoli cells and germ cells may cause male reproductive disorder after SARS-CoV-2 infection.

The serious coronavirus disease-2019 (COVID-19) was first reported in December 2019 in Wuhan, China. COVID-19 is an infectious disease caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Angiotensin converting enzyme 2(ACE2) is the cellular receptor for SARS-CoV-2. Considering the critical roles of testicular cells for the transmission of genetic information between generations, we analyzed single-cell RNA-sequencing (scRNA-seq) data of adult human testis. The mRNA expression of ACE2 was expressed in both germ cells and somatic cells. Moreover, the positive rate of ACE2 in testes of infertile men was higher than normal, which indicates that SARS-CoV-2 may cause reproductive disorders through pathway activated by ACE2 and the men with reproductive disorder may easily to be infected by SARS-CoV-2. The expression level of ACE2 was related to the age, and the mid-aged with higher positive rate than young men testicular cells. Taken together, this research provides a biological background of the potential route for infection of SARS-CoV-2 and may enable rapid deciphering male-related reproductive disorders induced by COVID-19.

Resveratrol Enhances Self-Renewal of Mouse Embryonic Stem Cells.

Resveratrol (RSV) has been shown to affect the differentiation of several types of stem cells, while the detailed mechanism is elusive. Here, we aim to investigate the function of RSV in self-renewal of mouse embryonic stem cells (ESCs) and the related mechanisms. In contrast with its reported roles, we found unexpectedly that differentiated ESCs or iPSCs treated by RSV would not show further differentiation, but regained a naïve pluripotency state with higher expressions of core transcriptional factors and with the ability to differentiate into all three germ layers when transplanted in vivo. In accordance with these findings, RSV also enhanced cell cycle progression of ESCs via regulating cell cycle-related proteins. Finally, enhanced activation of JAK/STAT3 signaling pathway and suppressed activation of mTOR were found essential in enhancing the self-renewal of ESCs by RSV. Our finding discovered a novel function of RSV in enhancing the self-renewal of ESCs, and suggested that the timing of treatment and concentration of RSV determined the final effect of it. Our work may contribute to understanding of RSV in the self-renewal maintenance of pluripotent stem cells, and may also provide help to the generation and maintenance of iPSCs in vitro. J. Cell. Biochem. 118: 1928-1935, 2017. © 2017 Wiley Periodicals, Inc.

[Development of porcine induced pluripotent stem cells with a CD163 reporter system].

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation.

As a transcription factor of the Pit-Oct-Unc (POU) domain family, octamer-binding transcription factor 6 ( OCT6) participates in various aspects of stem cell development and differentiation. At present, however, its role in porcine-induced pluripotent stem cells (piPSCs) remains unclear. Here, we explored the function of OCT6 in piPSCs. We found that piPSCs overexpressing OCT6 maintained colony morphology and pluripotency under differentiation conditions, with a similar gene expression pattern to that of non-differentiated piPSCs. Functional analysis revealed that OCT6 attenuated the adverse effects of extracellular signal-regulated kinase (ERK) signaling pathway inhibition on piPSC pluripotency by activating phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling activity. Our research sheds new light on the mechanism by which OCT6 promotes PSC maintenance.

Melatonin treatment improves human umbilical cord mesenchymal stem cell therapy in a mouse model of type II diabetes mellitus via the PI3K/AKT signaling pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of MSCs, resulting in failure of MSC therapy. This study investigated whether the combination of melatonin and human umbilical cord mesenchymal stem cells (hUC-MSCs) was superior to hUC-MSCs alone in ameliorating high-fat diet and streptozocin (STZ)-induced type II diabetes mellitus (T2DM) in a mouse model. METHODS: Mice were divided into four groups: normal control (NC) group; T2DM group; hUC-MSCs treatment alone (UCMSC) group and pretreatment of hUC-MSCs with melatonin (UCMSC/Mel) group. RESULTS: RNA sequence analysis showed that certain pathways, including the signaling pathway involved in the regulation of cell proliferation signaling pathway, were regulated by melatonin. The blood glucose levels of the mice in the UCMSC and UCMSC/Mel treatment groups were significantly reduced compared with the T2DM group without treatment (P < 0.05). Furthermore, hUC-MSCs enhance the key factor in the activation of the PI3K/Akt pathway in T2DM mouse hepatocytes. CONCLUSION: The pretreatment of hUC-MSCs with melatonin partly boosted cell efficiency and thereby alleviated impaired glycemic control and insulin resistance. This study provides a practical strategy to improve the application of hUC-MSCs in diabetes mellitus and cytotherapy. Overview of the PI3K/AKT signaling pathway. (A) Underlying mechanism of UCMSC/Mel inhibition of hyperglycemia and insulin resistance T2DM mice via regulation of PI3K/AKT pathway. hUC-MSCs stimulates glucose uptake and improves insulin action thus should inhibition the clinical signs of T2DM, through activation of the p-PI3K/Akt signaling pathway and then regulates glucose transport through activating AS160. UCMSC/Mel increases p53-dependent expression of BCL2, and inhibit BAX and Capase3 protein activation. Leading to the decrease in apoptosis. (B) Melatonin modulated PI3K/AKT signaling pathway. Melatonin activated PI3K/AKT response pathway through binding to MT1and MT2 receptor. Leading to the increase in hUC-MSCs proliferation, migration and differentiation. → (Direct stimulatory modification); ┴ ( Direct Inhibitory modification); → ┤ (Multistep inhibitory modification); ↑ (Up regulate); ↓ (Down regulate); PI3K (Phosphoinositide 3-Kinase); AKT ( protein kinase B); PDK1 (Phosphoinositide-dependent protein kinase 1); IR, insulin receptor; GLUT4 ( glucose transporter type 4); ROS (reactive oxygen species); BCL-2 (B-cell lymphoma-2); PDK1 (phosphoinositide-dependent kinase 1) BAX (B-cell lymphoma-2-associated X protein); PCNA (Proliferating cell nuclear antigen); Cell cycle-associated proteins (KI67, cyclin A, cyclin E).

ESRRB Facilitates the Conversion of Trophoblast-Like Stem Cells From Induced Pluripotent Stem Cells by Directly Regulating CDX2.

Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.

Mir-34c affects the proliferation and pluripotency of porcine induced pluripotent stem cell (piPSC)-like cells by targeting c-Myc.

MicroRNAs are important regulators in stem cells, which involve in gene regulation, including cell proliferation, differentiation and apoptosis. As an important one, miR-34c participates in various processes by targeting protein-coding genes. It is generally considered as a tumor suppressor and cell adhesion inhibitor. However, whether miR-34c has effects on pluripotent stem cells is not clear. Here, by mir-34c mimics transfection, the function of miR-34c on porcine induced pluripotent stem cell (piPSC)-like cells was investigated. Bioinformatics analyses showed that c-Myc is miR-34c's candidate target, which was confirmed by dual Luciferase assay. The knockout of miR-34c indicated that mir-34c affects the proliferation and pluripotency of piPSC-like cells by targeting c-Myc. Our study explored the regulatory mechanism of miR-34c on piPSC-like cells, providing a reference for the establishment of true porcine PSCs.

BCL2 enhances survival of porcine pluripotent stem cells through promoting FGFR2.

OBJECTIVES: The establishment of porcine pluripotent stem cells (pPSCs) is still a critical topic. However, all pPSCs were failed to contribute to efficient chimeric pig and were extremely sensitive to changes of culture conditions. This study aimed to investigate the role of BCL2 in pPSCs and further explain the mechanism. MATERIALS AND METHODS: Porcine BCL2 gene was cloned and overexpressed in porcine induce pluripotent stem cells (piPSCs). Digital RNA-seq was performed to explain the mechanism of anti-apoptosis. Finally, the cells carrying BCL2 were injected into mouse early embryo to evaluate its chimeric ability. RESULTS: Here, we found that overexpression of porcine BCL2 gene significantly improved the survivability of piPSCs and the efficiency of embryonic chimerism, and did not wreck the pluripotency of piPSCs. Furthermore, the Digital RNA-seq analysis revealed that BCL2, as a downstream gene of the PI3K signal pathway, enhanced the expression of PI3K signal pathway receptors, such as FGFR2, and further promoted oxidoreductases activity and lipid metabolism, thus maintaining the survival and pluripotency of piPSCs. CONCLUSION: Our data not only suggested that porcine BCL2 gene could enhance the survivability and chimeric ability of pPSCs, but also explained the positive feedback mechanism in this process, providing strong support for the chimeric experiment of pPSCs.

LIN28A inhibits DUSP family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells.

LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.

Single-cell RNA sequencing reveals atlas of dairy goat testis cells.

Single-cell RNA sequencing (scRNA-seq) is useful for exploring cell heterogeneity. For large animals, however, little is known regarding spermatogonial stem cell (SSC) self-renewal regulation, especially in dairy goats. In this study, we described a high-resolution scRNA-seq atlas derived from a dairy goat. We identified six somatic cell and five spermatogenic cell subtypes. During spermatogenesis, genes with significantly changed expression were mainly enriched in the Notch, TGF-ß, and Hippo signaling pathways as well as the signaling pathway involved in the regulation of stem cell pluripotency. We detected and screened specific candidate marker genes ( TKTL1 and AES) for spermatogonia. Our study provides new insights into goat spermatogenesis and the development of testicular somatic cells.

Reconstitution of male germline cell specification from mouse embryonic stem cells using defined factors in vitro.

In vitro induction of functional haploid cells from embryonic stem cells (ESCs) has been reported by several groups. However, these reports either involve complex induction process with undefined induction factors or show low-induction efficiency. Here, we report complete meiosis in vitro from ESCs with defined induction factors. ESCs were first induced into primordial germ cell-like cells, which were further induced into male germline cells, including spermatogonial stem cell-like cells (SSCLCs) and spermatid-like cells. Importantly, the obtained SSCLCs were functional as infertile male mice sired healthy offspring via SSCLC transplantation. Further, we found that eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) was essential for spermatogenesis. Eif2s3y-overexpressing ESCs showed enhanced spermatogenesis in vitro, as demonstrated by higher expression levels of SSC-specific markers during SSCLC induction process, improved reproductive ability recovery of infertile male mice, and increased efficiency of haploid cell induction. Our work provides a convenient and efficient approach to obtain functional male germline cells.

Characterization of porcine extraembryonic endoderm cells.

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.