Fibroblasts of black-bone sheep (collected 15 days after birth) were seeded in a gelatinized T75 culture flask and cultured in M10 medium. Cultured cells were dissociated using 0.5% trypsin-EDTA (Gibco, 15400-054) and harvested for electroporation at 70% confluence (1.0 × 10⁶ cells per experiment). The M10 medium formulation used was as follows: knockout DMEM (Gibco, 10829-018), 10% FBS (BI, 04-002-1A), 1× MEM non-essential amino acids (Gibco, 10370-021), 1× GlutaMAX (Gibco, 35050-061), and 1× penicillin–streptomycin (Gibco, 11140-050). Transfections were performed using the Amaxa Nucleofector device (Lonza) according to the manufacturer’s protocol (Basic Nucleofector^® Kit for Primary Mammalian Fibroblasts, VPI-1001, program U-23), with 6.0 μg DNA: 2.0 μg PB-TRE-bOMSK (bovine OCT4, cMYC, SOX2, and KLF4), 1.0 μg PB-TRE-pNhL (porcine NANOG and human LIN28), 1.0 μg PB-TRE-sLhT (SV40 large T antigen and human TERT), 1.0 μg PB-EF1a-transposase, and 1.0 μg PB-EF1a-rTTA. The PB-TRE-pNhL, PB-EF1a-transposase, and PB-EF1a-rTTA plasmids were provided by Professor Pentao Liu (Gao et al., 2019), and Dr. Lixia Zhao constructed the PB-TRE-bOMSK plasmid (Zhao et al., 2021). The human TERT and SV40 large T antigen cDNA sequences were purchased from Addgene (pBABE-hygro-hTERT, 1773; pSG5 Large T, 9053). The construction of PB-TRE-sLhT was based on PB-TRE-pNhL backbone transposons and was confirmed by sequencing. After transfection, a quarter of the electroconversion product was seeded on STO feeders in M15 medium in 10-cm plates. M15 medium formulation was as follows: knockout DMEM, 15% FBS, 1× penicillin–streptomycin, 1× GlutaMAX, 1× MEM non-essential amino acids, and 2-mercaptoethanol (0.1 mM, Sigma, M6250); LIF (10 ng/ml, Millipore, LIF1001); vitamin C (10 μg/ml, Sigma, 49752); bFGF (10 ng/ml, R&D, 233-FB-025); and doxycycline (DOX) (1.0 μg/ml, Clontech, 631311). The culture medium was changed on alternate days, and colonies were selected on days 7–10 and maintained in M15 medium supplemented with DOX. Colonies with endogenous core pluripotent markers OCT4, SOX2, and NANOG were detected by quantitative reverse transcription PCR (RT-qPCR) assay.

The sheep iPSCs were maintained on STO feeder layers and enzymatically passaged every 2–3 days using TrypLE™ Select (Gibco, 12563-029). The cells were dissociated and centrifuged (300×g for 3 min) in K10 medium. K10 medium composition was as follows: DMEM/F12 (Gibco, 11320-033), 10% KSR (Gibco, 10,828-028), 1× penicillin–streptomycin, and 1× MEM non-essential amino acids. After the supernatant was removed, the sheep iPSCs were re-suspended and seeded in M15 medium. All cell cultures used in the study were carried out under conditions of 38.5°C and 5% CO₂ unless otherwise noted. The sheep iPSCs were frozen once they were ∼80% confluent using cryopreservation medium, which contains 90% FBS and 10% (vol/vol) DMSO (Sigma, D2650).

DOX-dependent sheep iPSCs at passage 14 were dissociated in TrypLE™ Select and seeded in a 24-well plate with STO feeder. Cells were cultured in M15 medium supplemented with DOX and then switched to a candidate medium, which removes DOX after 24 h. The candidates included 2i/LIF (Ying et al., 2008), t2iL + Gӧ (Takashima et al., 2014), 4i/L/A (Irie et al., 2015), 5i/L/A (Theunissen et al., 2014), and mouse EPSC medium (mEPSCM) (Yang et al., 2017). Small molecules and cytokines were supplemented as indicated at the following final concentrations: CHIR99021, 0.2 µM (GSK3i, Selleck Chemicals, S2924); PD0325901, 0.2 µM (MEKi, Selleck Chemicals, S1036); Gӧ6983, 5.0 µM (PKCi, Selleck Chemicals, S2911); SP600125, 4.0 µM (JNKi, Selleck Chemicals, S1460); SB203580, 10 µM (p38i, Selleck Chemicals, S1076); Y27632, 10 µM (ROCKi, Selleck Chemicals, S1049); SB590885, 0.25 µM (BRAFi, Selleck Chemicals, S2220); WH4-023, 10 µM (SRCi, Selleck Chemicals, S7565); XAV939, 2.5 µM (WNTi, Selleck Chemicals, S1180); LIF, 10 ng/ml; bFGF, 10–100 ng/ml; and Activin A, 20.0 ng/ml. The medium was refreshed daily. Knockout DMEM, DMEM/F12 (Gibco, 11320-033), and mTeSR™1 (STEM CELL, 85,850) were used as basal medium for culture screening.

The sheep iPSCs on feeder cells were switched to plates coated with 20 ug/ml fibronectin (Millipore, FC010) and maintained in M15 medium supplemented with DOX. The siPSCs were passaged every 2–3 days and split at a ratio of 1:4. The siPSCs were collected in different passages for the detection of core pluripotent markers by RT-qPCR.

Sheep iPSCs were detached from culture plates using TrypLE™ Select, resuspended in M10 medium, and then seeded in a 96-U bottom well plates with ultra-low cell attachment (Corning, 7007). After 3 days in suspension culture, the embryoid bodies (EBs) were transferred and cultured in gelatin-coated plates for an additional 3 days to detect line gene expression by RT-qPCR and an additional 7 days for immunostaining.

Sheep iPSCs for immunofluorescence (IF) staining were fixed in 4% paraformaldehyde for 15 min at room temperature after washing with PBS and then permeabilized with IF buffer for 30 min. IF buffer composition was as follows: 0.1% Triton X-100 (Sigma) and 1% BSA in PBS. Cells were incubated with primary antibody at 4°C overnight. After the cells were washed three times with IF buffer for 5 min per wash, secondary antibodies were used to incubate for 1 h at room temperature in the dark and then washed once for 5 min in IF buffer and twice for 5 min in PBS. The cells were then mounted on VECTASHIELD with DAPI (Vector Laboratories). The anti-bodies used are listed in Supplementary Table S1.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, 74104). Complementary DNA (cDNA) was prepared using HiScript Q RT SuperMix for qPCR (Vazyme Biotech Co.,ltd, R223-01). RT-qPCR primers are listed in the Key Resource Table. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co.,ltd, Q711-02) was used for RT-qPCR assays. All RT-qPCR reactions were performed on a Veriti 96-Well Thermal Cycler (Applied Biosystems, United States). To evaluate the expression of the exogenous reprogramming factors, primers were designed to detect the across two adjacent genes in bOSMK, pNhL, and sLhT. The primers used are listed in Supplementary Table S2. Gene expression was determined relative to GAPDH using the ΔΔCt method.

The H2B-CAG-tdTomato plasmid was transfected into sheep iPSCs using Lipofectamine 2000 (Gibco, 11668027), then screened with hygromycin (100 μg/ml, Gibco, 10687010) after transfection for 48 h. For chimeric contribution to sheep, 10–15 tdTomato⁺ siPSC-4 were injected into sheep blastocysts as described above to detect chimeric contribution in vitro and in vivo. Half of the injected sheep embryos were cultured in vitro in SOF medium and M15 with DOX mixture medium (1:1) at 38.5°C in a 5% CO₂ atmosphere for 46–48 h for the evaluation of chimerism. The other embryos were transferred to the uteri of pseudopregnant sheep at 7 days post coitus (dpc). After the transplantation for 30 days, pregnancy was diagnosed by ultrasonography. The fetuses were isolated at embryonic stage of days 18–60 to check chimeric contribution.

For chimeric contribution to mice, first, 10–15 tdTomato⁺ siPSC-4 were carefully injected into the early blastocysts of ICR mice using a piezo-assisted micromanipulator attached to an inverted microscope (Zeiss, Eppendorf). One-third of the injected embryos were cultured in KSOM medium (Millipore, MR-020P-5F) and M15 medium combined with the DOX mixture medium (1:1) in vitro at 37°C for 16 h to check chimeric contribution in vitro. Other embryos were transplanted into the uterus of pseudopregnant ICR female mice at 2.5 dpc. Chimeric embryos were collected at E6.5 to check chimeric contribution in vivo. E6.5 mouse embryos (ICR) were dissected from the decidua under stereomicroscopy. Secondly, 10–15 tdTomato⁺ siPSC-4 were injected into the middle post-posterior of E6.5 mouse embryos (ICR) in the same way as above and then cultured in vitro in commercial rat serum and M15 with DOX mixture medium (1:1) at 37°C in a 5% CO₂ atmosphere for 48 h for the evaluation of E8.5 chimerism.

The slides were imaged with a FluoView FV1000 confocal microscope (Olympus). First, E8.5 chimeric embryos were fixed for immunofluorescence assays in 4% PFA for 2 h at room temperature, then dehydrated in 30% sucrose (Sigma) until embryos deposited to the bottom. The E8.5 chimeric embryos were then embedded in a mixture of Thermo O.C.T. compound (SAKURA, 4583) and 30% sucrose overnight at 4°C, then snap frozen in liquid nitrogen. The frozen OCT blocks were sectioned with CryoStar NX50 (Thermo) at 10 µm each section. Sections were first permeabilized with 0.1% Triton and blocked with 5% donkey serum plus 1% BSA followed by incubation with primary antibodies overnight at 4°C. Fluorescence-conjugated secondary antibodies were used to incubate the slides at room temperature for 1 h. Antibodies used are listed in Supplementary Table S1.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. After quality control using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, United States), 1–2 µg of RNA was used to extract mRNA according to the NEB Next Poly(A) mRNA Magnetic Isolation Module. The RNA sequencing libraries were then constructed according to the manufacturer’s instructions for the Illumina NEBNext Ultra RNA Library Prep Kit (NEB). The generated libraries were pooled and sequenced on an Illumina NovaSeq™ 6000 following the vendor’s recommended protocol platforms with a 150-bp paired-end mode (sequenced by LC-Bio Technology Co., Ltd. Hangzhou, China).

We generated sheep iPSCs by expressing eight DOX-inducible exogenous reprogramming factors, bOMSK (bovine OCT4, cMYC, SOX2, and KLF4), pNhL (porcine NANOG and human LIN28), and sLhT (SV40 large T antigen and human TERT) in fibroblasts of black-bone sheep, delivered via piggyBac transposon system (Figure 1A). DOX induction reprogrammed ∼0.1% of the transfected sheep original fibroblasts to primary colonies (Figure 1B). The sheep iPSC colonies were harvested on days 7–10 and passaged in single-cell suspension in serum-containing medium (M15) (Supplementary Figure S1A). The expression of endogenous OCT4 was examined in the second passaged cells by RT-qPCR, and the cells with the highest expression were selected for further study (Supplementary Figure S1B). Then, the cells expressed the eight exogenous reprogramming factors when maintained in M15 in the presence of DOX (Figure 1C and Supplementary Figure S1C). The colonies of cells were dense, with clear boundaries and a high nucleocytoplasmic ratio (Figure 1D). In addition, some cells in the colonies were positive for alkaline phosphatase (AP) (Figure 1E). These cells showed a normal 54 XY karyotype, and the percentage of normal karyotype was 77.5% (31/40) at passage 25 (Figure 1F). The selected cells expressed the endogenous key pluripotent genes OCT4 (POU5F1), SOX2, and NANOG and could be maintained undifferentiated for at least 32 passages when cultured in M15 medium supplemented with DOX (Figure 1G).

Endogenous pluripotent markers, including OCT4, NANOG, SOX2, KLF17, KLF4, SALL4, and TFCP2L1, were detected in sheep iPSCs by RT-qPCR (Figure 2A), and core pluripotent markers such as OCT4, NANOG, and SOX2 were positive in sheep iPSCs by IF staining (Figure 2B). When cultured without STO feeders, sheep iPSCs still maintained the previous stem cell morphology for at least 10 passages (Figure 2C); however, the cells lost the expression of OCT4 (Figure 2D), suggesting differentiation. We also tested the culture conditions for mouse or human PSCs, including 2i/LIF (Ying et al., 2008), t2iL + Gӧ (Takashima et al., 2014), 4i/L/A (Irie et al., 2015), 5i/L/A (Theunissen et al., 2014), and mEPSCM (Yang et al., 2017). The sheep iPSCs at passage 14 were cultured under these conditions, and the gene expression was examined. We found that upon DOX removal, sheep iPSCs cultured in 2i/LIF, t2iL + Gӧ, 4i/L/A, and mEPSCM were dead after 96 h, and cells cultured in 5i/L/A were dead at 48 h after one passage, concomitant with the loss of pluripotency gene expression (Figure 2E and Supplementary Figure S2A). The results indicated that pluripotency in sheep iPSCs depended on the expression of the DOX-induced exogenous factors in the serum-containing medium and the feeder STO.

To test the differentiation capacity of sheep iPSCs in vitro, we evaluated EB formation using siPSC-4. Cells formed EBs in 3 days (Figure 3A) and underwent differentiation in M10 medium for 3 days, before analyzing the expression of three germ layer markers. The RT-qPCR results showed that sheep iPSCs were able to differentiate into the three germ layers and expressed the ectoderm (KRT8, NEUROD, and NESTIN), mesoderm (NANOS3 and RENIN), and endoderm markers (GATA4, DCN, and GATA6) (Figure 3B). We then examined the differentiation ability of sheep iPSCs after the EBs were transferred to gelatin-coated plates for continued culture in vitro. The attached cells exhibited various types of morphologies and were positive for GATA6 (endoderm), smooth muscle actin (SMA; mesoderm), and β-tubulin (ectoderm) (Figure 3C).

To characterize the molecular mechanism of somatic reprogramming and insights into sheep iPSCs, gene expression was determined by transcriptome analyses (RNA-seq). The volcano plot showed that there were 12,740 differentially expressed genes (DEGs) between sheep fibroblasts and sheep iPSCs (p-value <0.05, fold change >10), of which 10,270 genes were up-regulated and 2,470 genes were down-regulated (Figure 4A). Compared to sheep fibroblasts, sheep iPSCs presented a significantly higher expression of genes involved in pyruvate metabolism, oxidative phosphorylation, and the cell cycle, whereas genes involved in WNT signaling, TNF signaling, P53 signaling, and PI3K-AKT signaling were significantly overexpressed in the gene set enrichment analysis (GSEA) (Figure 4B). Next, we evaluated the expression of naïve and primed pluripotency markers in sheep iPSCs; some naïve markers, such as KLF4, NR0B1, OCT4, and PRDM14, and primed markers, including DNMT3B, MEIS2, MYC, and ZIC1, were expressed in sheep iPSCs (Figure 4C), suggesting the pluripotency of sheep iPSCs. Corresponding to the results of RT-qPCR, we also found the expression of key pluripotency genes, such as OCT4, SOX2, and SALL4 (Figure 3A; Figure 4D). The DNA methyltransferase genes, DNMT1, DNMT3A, and DNMT3B, were expressed at higher levels in sheep iPSCs (Figure 4E). Sheep iPSCs appeared to have enriched transcriptomic characteristics of CTFR-sESCs, compared to fibroblasts and ICM (Vilarino et al., 2020) in principal component analysis (PCA) (Figure 4F), indicating that sheep iPSCs were reprogrammed in a different state from ICM.

To further investigate the developmental capacity of sheep iPSCs, chimera experiments were performed (Figure 5A). First, the sheep iPSCs were transfected with the H2B-CAG-tdTomato plasmid (Supplementary Figure S2B). We then injected 10–15 tdTomato⁺ siPSCs into the sheep early blastocysts (n = 24) and allowed them to develop further for 46 h in vitro. tdTomato⁺ cells were detected in ICM at 26% (6 out of 24) (Figure 5B), and some tdTomato⁺ cells were positive for ICM-specific antibodies (OCT4) (Figure 5C). We also injected sheep early blastocysts (n = 25) with tdTomato⁺ siPSCs and transferred them to pseudopregnant recipient sheep (n = 6). Four recipients became pregnant, and the embryos were harvested on day 11 (n = 2) and day 59 (n = 1), and we did not detect any siPSCs with tdTomato in the samples (Supplementary Figures S2B, C).

In addition to sheep blastocyst chimera experiments, we also injected tdTomato⁺ siPSCs into mouse early blastocysts to further evaluate the developmental potential of sheep iPSCs. Some of the injected embryos (n = 27) were allowed to develop in vitro, and others (n = 37) were transplanted into 2.5 dpc pseudopregnancy female mice to generate chimeras. tdTomato⁺ cells were detected in both the trophectoderm and the ICM of mouse blastocysts that developed for 16 h in vitro (Figure 5D). However, for post-implantation embryos, we did not detect positive tdTomato signals in the E6.5 chimeras (n = 27) (Supplementary Figure S2D).

To further assess the incorporation sheep iPSCs in mouse post-implantation epiblasts, we injected 10–15 tdTomato⁺ siPSCs in mid-posterior of E6.5 mouse embryos (Huang et al., 2012; Wu et al., 2015). The embryos were cultured to further assess the incorporation of sheep iPSCs into mouse post-implantation epiblasts in vitro, and we identified the descendants of donor siPSCs in chimeric embryos at E8.5 (Figure 5E). We sectioned the cultured embryos and performed IF analysis to detect the expression of the lineage marker of tdTomato⁺ cells. The results revealed that tdTomato⁺ cells could contribute to the ectoderm and mesoderm and expressed markers of NESTIN (ectoderm) and SMA (mesoderm) (Figure 5F).

Collectively, the differentiation data in vitro and our siPSCs showed a developmental potential in both sheep and mouse early embryos, but they could not synchronize with the developmental stages of recipient embryo after being transferred in vivo.