Abstract Background Generating expanded potential stem cells from cloned porcine embryos (pEPSCs^NT) represents a notable advancement in regenerative medicine and agricultural biotechnology. However, challenges, including low derivation efficiency, limited understanding of transcriptomic features, and unknown feasibility of culturing under feeder-free conditions, remain. This study aimed to generate pEPSCs using blastocysts derived from parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer (SCNT) using a modified culture system. Methods We derived pEPSC^NT lines using an optimized culture system. We characterized the pEPSC lines from all three origins by analyzing pluripotent marker expression, performing karyotyping, and assessing their differentiation potential into the three germ layers. Furthermore, we performed a comparative transcriptomic analysis using in vivo and cloned embryo data, with a major focus on cell lines derived from SCNT (pEPSCs^NT). We optimized feeder-free culture conditions for the pEPSC^NT line and derived the pEPSC^NT lines using an optimized culture system with an efficiency of ~ 14%. Results The cells were closely correlated with 8-cell to morula-stage embryos and exhibited significant enrichment of EPSC signature genes, suggesting a unique pluripotent state relatively close to the naïve state, specifically within a formative state. The pEPSCs^NT possessed broad differentiation capacity, indicative of Hippo signaling pathway enrichment, blastocyst-like structure formation ability, and potential differentiation into trophoblast lineage cells. Conclusions Our modified culture medium combined with the 2× Matrigel coating system facilitated the transition to feeder-independent culture conditions. These findings facilitate the establishment of a feeder-free culture system while preserving pluripotency and differentiation potential. Graphical abstract [34]graphic file with name 13287_2025_4627_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s13287-025-04627-5. Keywords: Expanded pluripotent stem cell, Porcine embryos, Somatic cell nuclear transfer, Feeder-free culture Background Pluripotent stem cells (PSCs) are versatile and invaluable tools for various research applications because of their intrinsic ability to self-renew and differentiate into multiple cell types [[35]1, [36]2, [37]3]. Among the various PSC types, those derived from domestic pigs (pPSCs) are the most promising. The physiological, genetic, and immunological similarities between pigs and humans render pigs an ideal large-animal model for comparative biology, disease modeling, and regenerative medicine [[38]4, [39]5, [40]6, [41]7]. Despite this considerable potential, establishing bona fide pPSCs has been challenging over the past few decades, often resulting in the generation of cells referred to as embryonic stem (ES)-like cells instead [[42]8, [43]9, [44]10, [45]11, [46]12]. Since 2019, important breakthroughs have shown promise in generating stable pPSCs [[47]13, [48]14, [49]15, [50]16, [51]17]. Similar to those from other species, the characteristics and status of pPSCs in terms of transcriptomic expression vary depending on the established strategies employed [[52]13, [53]14, [54]15, [55]16, [56]17]. For instance, Choi et al. [[57]14] demonstrated that the activation of the fibroblast growth factor 2 (FGF2), ACTIVIN/NODAL, and WNT signaling pathways enabled the derivation of authentic pPSCs. These cells exhibited a transcriptome developmentally similar to that of late epiblasts rather than that of an inner cell mass (ICM), suggesting a primed pluripotent state achieved by seeding hatched blastocysts under feeder cells [[58]14]. These primed pPSCs formed morphologically flat colonies, maintained stability for over 50 passages, exhibited teratoma formation, and retained two active X chromosomes [[59]14]. In the same year, Gao et al. [[60]18] demonstrated the establishment of more advanced cell lines, known as porcine-expanded potential stem cells (pEPSCs). These cells exhibited a remarkable ability to differentiate into embryonic and extra-embryonic lineages. This was achieved by inhibiting GSK3, SRC, and Tankyrase, activating the Activin A and TGFβ pathways, and adding vitamin C [[61]18]. These pEPSCs formed compact colonies with smooth edges, remained genetically stable over multiple passages, showed extensive DNA demethylation, and exhibited transcriptomic features similar to those of 8-cell to morula-stage blastomeres [[62]19]. The derivation of pEPSCs marks a pivotal achievement in stem cell research, suggesting the possibility of overcoming longstanding challenges associated with maintaining stable and pluripotent pPSCs. In addition, owing to their unique capabilities, including differentiation into trophoblast lineage cells, EPSCs have the potential to generate blastoid structures that mimic the developmental timing and cell lineage specifications of natural blastocysts [[63]20, [64]21]. They also provide a valuable cell source for understanding pregnancy and associated complications [[65]22]. Despite these advancements and the potential of pEPSCs, several critical limitations persist that must be addressed to fully realize their utility: (1) the inefficiency and instability of current in vitro culture systems present a major challenge, particularly for cell lines derived from cloned embryos; (2) the features that define the pluripotent state of pEPSCs at the transcriptomic level are yet to be completely identified and characterized, hindering our understanding of the molecular mechanisms underlying pluripotency in these cells; and (3) the lack of an optimal feeder-free culture system for the expansion of pEPSCs is a major bottleneck. In this study, we aimed to generate pEPSCs using blastocysts derived from parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) using a modified culture system. We characterized pEPSC lines from all three origins by analyzing pluripotent marker expression, performing karyotyping, and assessing their differentiation potential into the three germ layers. Furthermore, we performed a comparative transcriptomic analysis using in vivo [[66]23] and cloned embryo [[67]24] data, focusing primarily on cell lines derived from SCNT (pEPSCs^NT) due to the limited understanding of these cell lines, despite recent demonstrations by Ruan et al. [[68]19]. Finally, we optimized feeder-free culture conditions for the pEPSCs^NT line, as feeder cells often exhibit batch-to-batch variability and can introduce unwanted variables that interfere with downstream analysis. By addressing these aspects, we aimed to advance the understanding and application of pEPSCs; to this end, our results contribute to the broader field of stem cell research and its translational potential in regenerative medicine and developmental biology. Methods Chemicals Unless otherwise indicated, all chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Porcine oocyte collection and in vitro maturation Porcine ovaries (mixed Yorkshire, approximately 6-month-old gilts) were obtained from a local slaughterhouse (Dong-A Food, Republic of Korea) and transported to the laboratory within 2 h in physiological saline at 37–39 °C. After washing the ovaries twice with physiological saline, cumulus–oocyte complexes (COCs) were aspirated from the antral follicles (3–6 mm) using an 18 G needle attached to a 10 mL disposable syringe and then collected in a 15 mL conical centrifuge tube. After 10 min settling at 37 °C, the supernatant was removed, and the precipitate was resuspended in HEPES-buffered Tyrode’s medium containing 0.05% (w/v) polyvinyl alcohol (TLH-PVA). COCs with more than three layers of compact cumulus cells and a homogenous cytoplasm were selected for in vitro maturation. Groups of 60 randomly selected COCs were transferred into each well of a four-well plate containing 480 µL of maturation medium comprising TCM199 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth factor, 75 µg/mL kanamycin, 1 µg/mL insulin, and 10% (v/v) porcine follicular fluid. Thereafter, the COCs were incubated for 40–42 h at 39 °C in an atmosphere of 5% CO[2] with humidified air. For the first 22 h, the COCs were cultured in maturation medium containing hormones (10 IU/mL equine chorionic gonadotropin and 10 IU/mL human chorionic gonadotropin; Daesung Microbiological Labs, Uiwang, Korea). After this period, the hormones were removed, and the COCs were cultured for an additional 18–20 h in hormone-free maturation medium. Next, COCs were denuded using 0.1% hyaluronidase by gentle pipetting and washed thrice in TLH-PVA medium. Oocytes with visible first polar bodies and a uniform ooplasm were selected for embryo production. Generation of porcine embryos To establish pEPSCs, IVF, PA, and SCNT were performed as previously described [[69]47, [70]48, [71]49]. Briefly, weekly shipped liquid semen (Darby Genetics Inc., Gyeonggi-do, Republic of Korea) was used for IVF. After washing twice with Dulbecco’s phosphate-buffered saline (dPBS) containing 0.1% bovine serum albumin (BSA) via centrifugation, sperm pellets were resuspended in modified Tris-buffered medium (mTBM) [[72]50]. Fifteen mature oocytes were placed into each mTBM droplet (40 µL), followed by sperm introduction to achieve a final concentration of 5 × 10^5 sperm/mL. After 20 min of co-incubation, loosely attached sperm cells were carefully removed from the zona pellucida of the oocytes using precise pipetting techniques. After washing twice, the oocytes were incubated in fresh mTBM for 5–6 h. For PA, mature oocytes were initially placed in calcium-free HEPES-buffer Tyrode’s medium supplemented with 0.2% BSA (TLH-BSA). Subsequently, the mature oocytes were rinsed twice with an activation solution (pH: 7.0–7.4; osmolarity: 280 mOsm/L; composition: 280 mM mannitol, 0.001 mM CaCl[2]•2H[2]O, and 0.05 mM MgCl[2]•6H[2]O). Subsequently, the oocytes were placed between two electrodes in a chamber overlaid with activation solution and subjected to two direct-current pulses of 120 V/mm for 60 µs using a LF101 Electro Cell Fusion Generator (Nepa Gene Co., Ltd. Chiba, Japan). These activated oocytes were immediately transferred into porcine zygotic medium-3 (PZM-3) [[73]51] supplemented with 7.5 µg/mL cytochalasin B (CB; C6762) for 3 h of incubation. For SCNT, mature oocytes were incubated for 5 min in TLH-BSA medium supplemented with 5 µg/mL CB and 5 µg/mL Hoechst 33342 (B2261). After washing several times, oocytes were placed into a drop of TLH-BSA with 5 µg/mL CB, after which they were enucleated by aspirating the polar body and adjacent metaphase II spindle-containing ooplasm with a glasses pipette (16 μm diameter) (Humagen, Charlottesville, VA, USA). After trypsinization, donor cells with smooth surfaces were selected for transfer into the perivitelline space of the enucleated oocytes using a fine injection pipette. The couplets were rinsed twice with activation solution, placed between two electrodes in a chamber overlaid with fusion solution (pH: 7.0–7.4; osmolarity: 260 mOsm/L; composition: 260 mM mannitol, 0.1 mM CaCl[2]•2H[2]O and 0.05 mM MgCl[2]•6H[2]O), and subjected to direct-current pulses of 160 V/mm for 60 µs using a LF101 Electro Cell Fusion Generator. After 30 min, the membrane fusion status of oocytes in the TLH-BSA solution was confirmed by stereomicroscopy. Fully fused oocytes were transferred to PZM-3 medium, which was supplemented with 2 mM 6-dimethylaminopurine and 0.4 mg/mL demecolcine for 4 h of incubation. Embryos obtained via IVF, PA, and SCNT were then transferred into 30 µL PZM-3 droplets (10 gametes/drop) covered with pre-warmed mineral oil. Thereafter, the embryos were incubated at 39 °C in a humidified atmosphere comprising 5% O[2], 5% CO[2], and 90% N[2] for a developmental period of 6 or 7 days. The PZM-3 droplets were replaced on days 2 and 4 of culture. Cultivation of donor cells for SCNT Porcine fetal fibroblasts derived from a fetus (Landrace × Duroc crossbreed) on embryonic day 40 were prepared as donor cells following the methodology outlined in previous studies [[74]52, [75]53]. Before SCNT, the cells, which were stored in liquid nitrogen (− 150 °C), were thawed and cultured in cell culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1× non-essential amino acids (NEAA), 1× glutamine, 1× ß-mercaptoethanol, and 1× antibiotic–antimycotic (Anti-anti) at 37 °C under 5% CO[2] in air. After cultivating for 3–4 days to achieve approximately 100% confluence, the donor cells were subjected to trypsinization for 1 min to dissociate the monolayer into single cells for subsequent use in nuclear transfer. All media and reagents used in this culture were purchased from Gibco. Feeder cell preparation Mouse embryonic fibroblast (MEFs) derived from embryonic day-13.5 ICR mouse, purchased from DBL (Seoul, Republic of Korea), were utilized as feeder cells. The mouse was euthanized by cervical dislocation, and all efforts were made to minimize suffering. The preparation procedure involved removing the fetal heads, internal organs, and limbs. The remaining tissues were minced, washed in dPBS, and centrifuged at 2,000 rpm for 3 min at least twice. Thereafter, the MEFs were cultured in the aforementioned cell culture medium and maintained at 37 °C under 5% CO[2] in air. To inactivate the MEFs, they were treated with 10 µg/mL mitomycin C (Roche, Basel, Switzerland) at passage 1–2 for 2 h. Subsequently, the cells were plated at a density of 5 × 10^5 cells/mL in a four-well dish pre-coated with 0.5% gelatin (MilliporeSigma, Burlington, MA, USA) in culture medium before their utilization for pEPSC seeding. Derivation and culture of pEPSCs To remove the zona pellucida, porcine IVF, PA, and SCNT blastocysts were incubated in a 0.5% protease solution for 1 min. For plating, intact day-6 or -7 blastocysts were washed and plated directly onto feeder layers under a microscope (SMZ645; Nikon, Tokyo, Japan) and cultured in modified pEPSCs medium (pEPSCM). The modified pEPSCM was prepared by incorporation using basal medium in a 1:1 mixture of mTeSR^TM1 (100–0276; STEMCELL Technologies, Vancouver, BC, Canada) and DMEM/F-12 (Gibco) supplemented with 1× N2 supplement (Thermo Fisher Scientific), 1× B27 supplement (Thermo Fisher Scientific), 1× NEAA, 1× GlutaMAX supplement (Gibco), 1 × β-mercaptoethanol, 0.5 µM CHIR99021 (S1263; Selleck Chemicals, Houston, TX, USA), 0.3 µM WH-4-023 (S7565; Selleck Chemicals), 2.5 µM XAV939 (X3004), 65.0 µg/mL vitamin C (50-81-7), 10.0 ng/mL leukemia inhibitory factor (LIF) (250-02; PeproTech, Cranbury, NJ, USA), 20.0 ng/mL Activin A (78001; STEMCELL Technologies), 0.3% FBS, and 0.5× Anti-anti (Gibco). After 48 h, the attachment efficiency of primary cultures was determined by scoring the number of attached colonies. After 5–7 days of culture, the primary outgrowth of EPSC-like cells was mechanically dissociated into several clumps using pulled-glass pipettes under a stereomicroscope (Nikon). The clumps were then re-seeded on fresh feeder cells, and subsequent EPSC lines were passaged mechanically every 3–4 days using TrypLE Express (12605010; Gibco) after washing briefly in Hanks’ balanced salt solution (14170-112; Gibco). The medium was replaced daily, and all cells were cultured at 37 °C with 5% CO[2] under humidified conditions. Cultivation of porcine induced pluripotent stem cells (iPSCs) The porcine iPSCs used in this study were kindly supplied by Professor Jongpil Kim from Dongguk University, Republic of Korea. These cells were generated by transfecting porcine embryonic fibroblasts with five doxycycline-inducible human factors: FUW-tetO-hOct4, FUW-tetO-hSox2, FUW-tetO-hKlf4, FUW-tetO-hc-Myc, and FUW-M2rtTA, all acquired from Addgene (Watertown, MA, USA) and delivered via lentiviral vectors. iPSCs were cultured at 37 °C in an atmosphere of 5% O[2] and 95% air on feeder layers using stem cell basal medium [a 1:1 mixture of DMEM (Gibco) and F10 (Gibco), supplemented with 15% FBS, 1× GlutaMAX, 1× ß-mercaptoethanol, 1× MEM-NEAA, and 1× Anti-anti] containing 10.0 ng/mL LIF (250-02; PeproTech) and 2 µg/mL doxycycline (D9891). The culture medium was refreshed daily, and the cells were passaged every 3 days. Passaging was performed using a 0.04% trypsin treatment, which allowed the cells to maintain a stable morphology for over 50 passages. Derivation and culture of porcine NT-derived embryonic stem cells (ESCs) using cloned embryos To plate the cloned blastocysts, the procedure outlined above for deriving pEPSCs was performed. Following a culture period of 10–14 days, ES-like primary colonies were obtained and subsequently mechanically dissociated into multiple clumps using pulled-glass pipettes. These clumps were then re-seeded onto a fresh feeder layer, and the resulting ES cell lines were passaged mechanically at 5–9 days intervals without enzymatic treatment. Upon reaching a size of 3–4 mm, the colony was carefully detached from the feeder layer and transferred into a drop of ESC culture medium comprising stem cell basal medium, as mentioned above, supplemented with 4 ng/mL basic fibroblast growth factor (Invitrogen, Carlsbad, CA, USA), where it was dissociated into smaller clumps. The clumps were subsequently placed in a new feeder layer supplemented with fresh ESC culture medium. The medium was refreshed daily, and the cells were cultured in a humidified atmosphere maintained with 5% CO[2] at 37 °C. Gene expression analysis by qPCR For gene expression analysis, stem cells or embryoid bodies (EBs) were washed twice in dPBS (Gibco) and stored at − 80 °C until analysis. Total RNA was extracted using RNAiso Plus reagent (TaKaRa Bio Inc., Otsu, Japan), followed by complementary DNA (cDNA) synthesis using Reverse Transcription 5× Master Mix (Elpis Bio, Inc., Daejeon, Republic of Korea) in accordance with the manufacturer’s instructions. Next, 1 µL of the synthesized cDNA was mixed with 10 µL 2× SYBR Premix Ex Taq (TaKaRa Bio Inc.) and 10 pmol of specific primers (Macrogen, Daejeon) (Additional file 1) and subjected to qPCR using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Reactions were performed as follows: 40 cycles of denaturation at 95 °C for 15 s, annealing at 57 °C for 15 s, and extension at 72 °C for 15 s. Relative quantification was performed using threshold cycle (Ct)-based methodologies, maintaining a constant fluorescence intensity. The relative mRNA expression (R) was calculated using the equation R = 2^−[∆Ct sample − ∆Ct control]. The R-values obtained for each gene were quantified relative to those of GAPDH. Immunofluorescence analysis After washing twice with dPBS containing calcium and magnesium ions (LB 001–01; Welgene Biotech, Taipei City, Taiwan), cells grown in 8-well chamber slides (154534; Thermo Fisher Scientific) were fixed with 4% paraformaldehyde (PFA) for 10 min. Subsequently, the cells were incubated with blocking buffer (12411 S; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature (RT) and labeled with primary antibodies (shown in Additional file 2) overnight at 4 °C. Afterward, the cells were washed thrice with dPBS containing 0.2% Tween-20 and incubated with the appropriate secondary antibodies for 1 h at RT. After three washes, nuclei were stained with Hoechst 33342 and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA). Stained cells were imaged using a confocal microscope (Carl Zeiss, Oberkochen, Germany) and ZEN 2009 Light Edition software (Carl Zeiss). Alkaline phosphatase (AP) staining For AP activity detection, pEPSCs were fixed in 4% PFA for 5–10 min. After washing with 0.1 M Tris-HCl (pH 9.5) solution twice, they were stained with a solution containing nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt stock solution (Roche) for 30 min. The stained cells were analyzed under a microscope (Nikon). Staining was performed at RT. Karyotyping To induce metaphase, confluent monolayer pEPSCs were exposed to 10 µg/mL colcemid (Gibco) for 3–5 h. Subsequently, karyotyping analysis was performed as described previously [[76]32]. Finally, the chromosomes on the slides were counted and observed under a light microscope (Nikon) to assess cytogenetic abnormalities. BLS and EB formation via spontaneous differentiation Colonies of the pEPSCs^NT line were dissociated into single cells using TrypLE Express Reagent (Gibco) and subsequently cultured in suspension on a 35 mm low-attachment plate containing the aforementioned cell culture medium to promote spontaneous differentiation. For the initial 24 h of culture, the medium was supplemented with 10 µM ROCK inhibitor (Y-27632; ROCKi); thereafter, the medium was replaced with ROCKi-free medium, with subsequent medium changes every 2 days. After a culture period of 7 days, the blastocyst-like structures (BLSs) were fixed directly using 4% (v/v) PFA solution for 10 min, following a wash with dPBS for subsequent immunofluorescence staining. Concurrently, a subset of EBs was collected and plated onto eight-well chamber slides (Thermo Fisher Scientific) coated with 0.1% (v/v) gelatin for adherent culture in cell culture medium. After an additional 10 days of culture, the adherent cells were fixed with 4% (v/v) PFA for subsequent immunofluorescence staining. The remaining EBs were cultured for an additional period of up to 46 days before being fixed for immunohistochemical staining. Cryosectioning and immunohistochemistry using day-46 EBs The fixed EBs were washed at least thrice with dPBS and subsequently immersed in a 30% sucrose–PBS solution within a microcentrifuge tube for incubation overnight at 4 °C. Following an additional 2–3 washes, the EBs were placed in a microcentrifuge tube with dPBS and dispatched to OBEN Bio Inc. (Suwon, Republic of Korea) for cryosectioning. The slide-affixed EB sections were stained according to the procedure outlined in the section titled “Immunofluorescence analysis.” During staining, the section slides were circumscribed with a PAP Pen to create a hydrophobic barrier (ImmEdge™; Vector Laboratories) and placed in a humid chamber. All subsequent procedures were performed at RT in a humid environment. Transcriptome analysis pEPSCs^NT, iPSCs, and cloned blastocysts that developed to day 7 were washed twice with dPBS, pelleted by centrifugation, and immediately frozen in liquid nitrogen. The samples were then sent to Theragen Etex, Inc. (Seongnam, Republic of Korea) for RNA extraction, cDNA library preparation, and whole-transcriptome sequencing using an Illumina NovaSeq6000 platform (Illumina, San Diego, CA, USA). For porcine data, Ensembl build Sscrofa11.1 (GCA_000003025.6) was used. Libraries were constructed for 151 bp paired-end sequencing using a SMARTer Stranded Total RNA-Seq Kit V2-Pico Input (Takara Bio, Inc.). Before generating single-stranded cDNA, rRNA was removed. The integrity of the total RNA was evaluated using an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA; Additional file 3), and cDNA libraries were quantified using a KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA). RNA-seq data of porcine in vivo and cloned embryos at various developmental stages, as published by He et al. [[77]24], were downloaded from the Gene Expression Omnibus (GEO) database (Accession no. [78]GSE125706). The data were used to perform principal component analysis and Pearson’s correlation coefficient analysis, facilitating a comparative assessment of our sample data. The expression of the specific genes of interest was extracted from the expression matrix and visualized as a heatmap using GraphPad Prism 10.1 (GraphPad Software, La Jolla, CA, USA). After assessing the differentially expressed genes (DEGs), volcano plots were generated by Theragen Etex, Inc., and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were evaluated using the DAVID database ([79]https://david.ncifcrf.gov). Plate coating with various ECMs In this study, we tested several ECM products, including Matrigel (354277; Corning Life Sciences, Corning, NY, USA), fibronectin (33010-018; Thermo Fisher Scientific), Laminin-521 (A29248; Gibco), and vitronectin (VTN)-N (A14700; Gibco), to optimize feeder-free culture conditions for pEPSCs^NT line. The coating process was performed according to each manufacturer’s instructions. After plating the extracellular matrix (ECM)-coated plates, pEPSCs^NT were dissociated using TrypLE Express (Gibco) and assessed at 1.5, 24, and 72 h to analyze the adherence rate and survival index by cell counting. Differentiation of pEPSCs^NT cultured under feeder-free conditions to trophoblast lineages After dissociation with TrypLE Express (Gibco), pEPSCs^NT cultured under feeder-free conditions (FF-pEPSCs^NT) were plated onto a Matrigel-coated plate with DMEM/F12 (Gibco) supplemented with 20% (v/v) knockout serum replacement (10828028; Gibco) and 10 µM ROCKi for a day. On the second day, the medium was replaced by 20% (v/v) knockout serum replacement basal media without ROCKi containing 10 µM SB431542 (S4317), 50 ng/mL BMP4, and 0.1 µM [80]PD032590 (FGF receptor inhibitor) to induce trophoblast differentiation. On day six of differentiation, the cells were collected for analysis. Statistical analysis Statistical analyses were performed using GraphPad Prism 10.1 (GraphPad Software). The results are presented as means ± standard error of the mean. Each experiment was repeated at least thrice unless a different number of replicates is stated in the legend. Statistical evaluations were performed using an unpaired two-tailed Student’s t-test or ANOVA, as detailed in the figure legends. P < 0.05 was considered statistically significant. The statistical methods, P-values, and sample sizes are provided in the figure legends. The work has been reported in line with the ARRIVE guidelines 2.0. Results Derivation of pEPSCs from pre-implantation embryos of various origins Most attempts to derive pEPSC lines from pre-implantation embryos from IVF, PA, and SCNT were conducted using day-6 or -7 porcine embryos under pEPSCM conditions, with minor modifications (Fig. [81]1A) [[82]18]. As shown in Fig. [83]1B, four pEPSC^IVF lines were established from 37 fertilized blastocysts, five pEPSC^PA lines from 39 parthenogenetic blastocysts, and five pEPSC^NT lines from 35 cloned blastocysts. The efficiencies of attachment (71–85%) and primary outgrowth (25–30%) were similar between pre-implantation embryos of different origins. The pEPSCs that formed primary colonies with outgrowth were passaged on day 12 after seeding; thereafter, pEPSCs^PA were routinely sub-cultured every 3–4 days at a ratio of 1:8, pEPSCs^IVF at 1:6, and pEPSCs^NT at 1:4, as single cells. Each pEPSC line exhibited morphological changes during derivation and maintenance (Fig. [84]1C). Two days after seeding, two cell morphologies were observed in the attached colony: small round cells and giant cells originating from the trophectoderm. After 5–6 days of culture, the giant cells gradually disappeared, and the small, round inner cells remained in the primary colonies. After sub-culturing under our modified pEPSCM conditions, cells from the three origins consistently displayed compact colonies with smooth edges, showing similar morphological changes during derivation. They stabilized without overt differentiation after more than 10 passages, as indicated by chromosomal profile normality with karyotyping analyses (Fig. [85]1D, pEPSCs^IVF 36 + XY, pEPSCs^PA 36 + XX, pEPSCs^NT36+XY) and AP-positive activity after staining (Fig. [86]1E). Fig. 1. [87]Fig. 1 [88]Open in a new tab Derivation of pEPSCs from pre-implantation embryos of various origins. A Schematic diagram of the experimental workflow for establishing pEPSC lines. B Information on established putative pEPSC lines from porcine IVF, PA, and NT embryos. C Morphological changes during pEPSC induction from porcine IVF, PA, and NT embryos. Representative morphologies of attached porcine blastocysts 2 days after seeding (Attached), expanded outgrowth 7 days after seeding (Primary colony; P0), and sub-cultured colonies (P2) in pEPSC medium. D Karyotyping analysis of pEPSC lines. Normal chromosomes were observed in all pEPSC lines (EPSCs^IVF:36 + XY, EPSCs^PA: 36 + XX, EPSCs^NT:36 + XY). E. Identification of AP activity in pEPSC lines of various origins. Scar bars = 100 μm. AP alkaline phosphatase, IVF in vitro fertilization, NT nuclear transfer, PA parthenogenetic activation, pEPSC porcine expanded potential stem cell Derivation and characterization of pEPSC^NT lines reconstructed with porcine cloned eGFP donor cells To track the cells derived from the cloned embryos, we used eGFP-tagged cells as donors when performing SCNT. Blastocysts that developed until day 6 or 7 were seeded onto MEF feeder cells and cultured in modified pEPSCM (Fig. [89]2A). Under UV illumination, green fluorescence originating from donor cells was consistently visualized throughout the cloned blastocysts, pEPSC^NT lines, and EBs (Fig. [90]2B). Notably, a significantly higher primary outgrowth probability of dome-shaped colonies was observed in blastocysts cultured until day 6 (P < 0.05) than in those cultured until day 7. However, the attachment efficiency between the groups did not differ (Fig. [91]2C). As expected, the core transcription factors of pluripotency (SOX2, OCT4, and NANOG) and surface markers (SSEA4) were clearly detectable in these pEPSC^NT lines using immunofluorescence staining (Fig. [92]2D and E). Fig. 2. [93]Fig. 2 [94]Open in a new tab Derivation and characterization of pEPSCs from NT blastocysts. A Representative images of porcine NT-derived blastocysts (i), primary outgrowth (ii), and established EPSC^NT lines (iii and iv). Scale bars = 100 μm. B Representative bright field and fluorescent images of the donor cells, NT-derived day-6 blastocysts, pEPSCs^NT, and EBs. pEPSCs^NT lines were established from cloned blastocysts reconstructed with porcine cloud eGFP cell lines as donors. C Quantified attachment and primary outgrowth rates of pESPCs^NT from day-6 or -7 blastocysts explanted whole on MEF feeder cells. ns: not significant. ^*P < 0.05. D Expression of core pluripotency markers in pEPSC^NT lines, including SOX2, OCT4, SSEA4, and NANOG, as assessed by immunofluorescence analysis. Scale bars = 100 μm. E Three-dimensional fluorescence image of detection of the core pluripotency markers in pEPSC^NT lines. F Gene expression analysis of porcine EBs differentiated from pEPSCs^NT. Genes related to embryonic lineage were assessed by qPCR. The relative expression was normalized to GAPDH. Data represent the mean ± SEM; n = 3 independent experiments. The P-values were calculated using a two-tailed Student’s t-test. G Expression of the differentiation markers Desmin (mesoderm), Vimentin (ectoderm), and Cytokeratin 17 (endoderm) in differentiated cells from the porcine EPSC^NT line determined by immunofluorescence analysis. Scale bars = 100 μm. EB embryoid body, MEF mouse embryonic fibroblast, NT nuclear transfer, pEPSC porcine expanded potential stem cell Next, we investigated whether pEPSC^NT lines could differentiate into all embryonic lineages in vitro. EBs differentiated for 7 days were used to assess the expression of lineage marker genes via qPCR, while cells extending from attached EBs were used to determine the expression of marker proteins via immunostaining. All detected genes related to the three germ layers (mesoderm markers HAND1, SOX7, and DES; endoderm markers AFP and SDC1; ectoderm markers PAX6 and CRABP2) were dramatically upregulated after differentiation (Fig. [95]2F). Furthermore, the marker proteins for the mesoderm (Desmin), ectoderm (Vimentin), and endoderm (Cytokeratin 17) were expressed in the differentiated cells (Fig. [96]2G). Transcriptomic features of pEPSCs^NT Next, we attempted to analyze the key transcriptomic features and define the pluripotency states of our established pEPSCs through transcriptomic analysis, comparing them with typical porcine pluripotent stem cell lines (iPSCs and ESCs^NT) and pre-implantation embryos at different stages (Fig. [97]3). Here, we considered that the iPSC line [[98]25], which can be well-maintained in an LIF-based culture medium, is propagated as single cells, and displays compact dome-like colonies closer to the naïve state [[99]26]. Conversely, we considered that the ESC^NT line [[100]27], which can be established in a basic fibroblast growth factor-based culture medium, is propagated in small clumps, and forms flat colonies closer to the primed state [[101]28]. Fig. 3. [102]Fig. 3 [103]Open in a new tab Transcriptomic features of porcine pEPSCs^NT. A Genes associated with core pluripotency, naïve, formative, and primed lineages in several porcine PSC lines and porcine fetal fibroblasts. B Bulk RNA seq data and comparison of the gene expression of pEPSCs^NT1220 (n = 2), iPSCs (n = 2), and diverse stages of embryos (n = 1) fertilized in vivo or cloned in vitro. Embryo datasets were obtained from He et al. [[104]24]. The dashed arrow represents the path of maturation or differentiation. C pEPSCs^NT1220 and in vivo or cloned embryos in multiple stages. Correlation matrix clustering of the pEPSCs^NT1220 cell line with previous in vivo embryo datasets [[105]24] in (i) and with the previous cloned embryo datasets [[106]24] in (ii). D Expression dynamics of selected pEPSC signatures, DNA methylation, 2 C, 4 C, 8 C, Mo, gastrulation, and trophoblast marker genes for pEPSCs^NT1220, iPSCs, and day-7 cloned blastocysts (Blastocysts^NT_Day 7; n = 2). Data are presented as log 2 normalized counts. E Expression dynamics of the genes related to JAK/STAT3, Activin/Nodal, FGF/ERK, and Wnt/β-catenin signaling pathways for pEPSCs^NT1220, iPSCs, and Blastocysts^NT_Day 7. Data are presented as log 2 normalized counts. F Overlapping up- or downregulated genes in (i) Blastocysts^NT_Day 7 and (ii) iPSCs compared with pEPSCs^NT1220. G Differential gene expression between Blastocysts^NT_Day 7 and pEPSCs^NT1220 (upper panel) and iPSCs and pEPSCs^NT1220 (lower panel). Genes with significant differential expression are shown in red, while those without are in blue. The horizontal and vertical dashed lines represent the false discovery rate (FDR, 0.05) and log2 fold change (± 0.6) cut-offs, respectively. h KEGG pathway enrichment analysis based on the data of DEGs between Blastocysts^NT_Day 7 and pEPSCs^NT1220 (upper panel) and iPSCs and pEPSCs^NT1220 (lower panel). The statistical significance of each pathway was evaluated using adjusted p-values, and results are presented as − log 10 (adjusted p-value). A red dashed line indicates the threshold of − log 10 (adjusted p-value) = 1.3, which corresponds to an adjusted p-value of 0.05. Pathways with values above this threshold are considered statistically significant. PFF, porcine fetal fibrobalst; DEG, differentially expressed gene; iPSC, induced pluripotent stem cell; KEGG, Kyoto Encyclopedia of Genes and Genomes; pEPSC, porcine expanded potential stem cell As expected, qPCR revealed that the iPSC line exhibited strong expression of core pluripotency (OCT4, NANOG, and SOX2) and naïve-state (KLF4 and KLF17) genes but showed low expression of formative (LIN28A and DNMT3A) and primed (DUSP6, ZIL2, SALL2, ACTC1, and SOX11) genes. In contrast, the ESC^NT line displayed the opposite expression patterns (Fig. [107]3A and Additional file 4). Based on the expression profiles of these two types of pPSCs as references, our pEPSCs exhibited broad