Phase-contrast images of biPSCs established in two different culture conditions.

<p>(A) bADCs. (B) Primary colonies appearing in primed cell-culture medium. (C) Established primed-type biPSCs. (D) Colonies converted from the primed to naïve state. (E) Primary colonies appearing in niPSCs medium. (F) Established naïve-type biPSCs. (A)–(C), (E), scale bars = 500 μm. (D), (F), scale bars = 100 μm.</p

Aggregation of pnbiPSCs into <i>in vitro</i> fertilized embryos, and their development <i>in vitro</i>.

<p>*RFP vector was introduced</p><p>Aggregation of pnbiPSCs into <i>in vitro</i> fertilized embryos, and their development <i>in vitro</i>.</p

Differentiation potential of biPSCs in culture.

<p>(A) Embryoid body formation of pbiPSCs grown for 6 days in low cell-adhesion dishes. Immunocytochemical staining for markers for the three germ-layer in differentiated cells derived from pbiPSCs. α-fetoprotein (B, endoderm), actin smooth muscle (C, mesoderm), and glial fibrillary acidic protein (D, ectoderm) were used as markers. (E) Embryoid body formation by pnbiPSCs. Immunocytochemical staining for α-fetoprotein (F), actin smooth muscle (G), glial fibrillary acidic protein (H). (A), (E), scale bars = 500 μm. (B)–(D), (F)–(H), scale bars = 100 μm.</p

Naïve-type features of iPSCs.

<p>(A) pnbiPSCs cultured for 4 days in niPSC medium in the presence of JAK inhibitor. (B) pnbiPSCs cultured in the presence of DMSO. (C) The number of cells cultured in the presence of JAK inhibitor or DMSO (*p < 0.05). (D) <i>XIST</i> expression evaluated in pbiPSCs, but not in pnbiPSCs. Immunocytochemistry images of methylation status at H3K27me3 sites (E, pbiPSCs; F, Hoechst staining; G, pnbiPSCs; H, Hoechst staining). Arrowheads indicate puncta of H3K27me3. (A), (B), scale bars = 500 μm.</p

Endogenous and exogenous expression of genes specific to undifferentiated ESCs in biPSCs.

<p>mRNA expression was evaluated by reverse-transcription polymerase chain reaction (RT-PCR). pbiPSCs (P5), primed-type iPSCs at passage 5; pbiPSCs (P50), primed-type iPSCs at passage 50; pnbiPSCs, naïve-type iPSCs at passage 3 converted from primed-type iPSCs at passage 48; nbiPSCs, naïve-type iPSCs cultured under naïve medium from primary culture; bADCs, bovine amnion-derived cells; SNL feeder, SNL feeder cells; vector, plasmid DNA of PB vectors; bACT, bovine β-ACTIN specific for cattle; Uni-ACT, universal β-ACTIN that reacts with both cattle and mice.</p

Reprogramming of bovine amnion-derived cells (bADCs) into iPSCs using Dox-inducible PB vectors.

<p>(A) Timeline for the establishment of primed-type biPSC lines. (B) Timeline for the establishment of naïve-type biPSC lines.</p

Characterization of biPSCs.

<p>(A) Alkaline phosphatase activity in primed-type iPSCs (pbiPSCs). (B) Alkaline phosphatase activity in naïve-type iPSCs derived from pbiPSCs (pnbiPSCs). (C) Karyotyping image of pbiPSCs at passage 65. (D) Karyotyping image of nbiPSCs at passage 10. (E) Proportion of cells with the indicated number of chromosomes (n = 20). (F)–(I) OCT3⁄4 (F, OCT3⁄4 staining; G, Hoechst staining) and NANOG (H, NANOG staining; I, Hoechst staining) expression in pbiPSCs. (J)–(M) OCT3⁄4 (J, OCT3⁄4 staining; K, Hoechst staining) and NANOG (L, NANOG staining; M, Hoechst staining) expression in pnbiPSCs. (A), (B), (J)–(M), scale bars = 100 μm. (F)–(G), scale bars = 500 μm.</p

Production of chimeric fetuses from bovine embryos using the aggregation method.

<p>(A) Naïve-type biPSCs expressing Tag-RFP were aggregated with host at the 8- to 16-cell stage of <i>in vitro</i> fertilized embryos. (B) Chimeric fetuses at day 90 of gestation derived from aggregated embryos. (C) PCR analysis using transgene-specific primers for genomically integrated Oct3/4-2A-Klf4 sequences in 14 tissues. Genomic DNA isolated from pnbiPSCs was used as a positive control. H₂O was used as a negative control (buffer alone for RT-PCR). Immunofluorescence analysis showing the distribution of pnbiPSC-derived cells (RFP-positive with red signals) in the small intestine (D), placenta (E), gonad (F, VASA-positive cells with green signals; arrowheads indicate the portion that is double-positive for RFP and VASA), and kidney (G) of the chimeric fetus. Nuclei were stained with DAPI (blue).</p

Generation of bovine-induced trophoblastic cells (biTBCs) using Dox-inducible PB vectors.

<p>(A) Timeline for the establishment of biTBCs in culture medium used for human iPSCs. (B) Timeline for the establishment of biTBCs in culture medium used for bovine trophoblast cells (TBM).</p

Characterization of bovine-induced trophoblastic cells (biTBCs).

<p>(A) Alkaline phosphatase activity in biTBCs. (B) CDX2 expression in biTBCs. (C) OCT3/4 expression in biTBCs. (D) NANOG expression in biTBCs. (A) scale bars = 500 μm. (B)-(D) scale bars = 100 μm.</p