FIG. 1.

Characteristics of Bengal tiger induced pluripotent stem cells (TiPSCs). (A) Morphology of TiPSCs at P1. (B) Alkaline phosphatase (AP) staining of a putative TiPSC colony at P14. (C) Confocal images of immunofluorescence staining of TiPSC at P14. OCT-4: (i) green fluorescence, (ii) 4′,6-diamidino-2-phenylindole (DAPI); NANOG: (iii) green fluorescence, (iv) DAPI; stage-specific embryonic antigen (SSEA)-4: (v) red fluorescence, (vi) DAPI. The scale bar for all confocal images represents 25 μm. (D) Reverse-transcription polymerase chain reaction (RT-PCR) gene expression analysis of endogenous cat OCT4 (286 bp), NANOG (246 bp), and GAPDH (271 bp) at P4 and P14 in Bengal tiger, jaguar, and serval iPSCs (TiPSCs, JiPSCs, and SiPSCs, respectively) and fibroblasts (TFs, JFs, and SFs), mouse and human embryonic stem cells (mESCs and hESCs), transgene (Tg), and water (H₂O). (E, F) Expression of human transgenes pMX-OCT-4, pMX-NANOG, pMX-SOX-2, pMX-KLF-4, and pMX-cMYC in TiPSCs, JiPSCs, SiPSCs, Tg, TFs, JFs, SFs, mESCs, hESCs, and H₂O at P4 and P14. (G) Karyotype of TiPSCs at P14 showing a euploid count of 18 autosomal and one XY pair of chromosomes. (H) TiPSCs differentiated in vitro to form embryoid bodies. (I) Confirmation of differentiation of embryoid bodies into the three germ layers by specific gene expression by RT-PCR (housekeeping gene: β-actin; ectoderm: NESTIN; mesoderm: CD31; and endoderm: FOXA2) on TiPSCs, JiPSCs, SiPSCs, and H₂O. (J) Cross-section of a teratoma obtained at 10 weeks after injection of TiPSCs at P14 in a severe combined immunodeficient (SCID) mouse, stained with hematoxylin and eosin with tissues representative of the three primary germ layers: (i) ectoderm: keratinizing stratified squamous epithelium; (ii) mesoderm: bone surrounded by connective tissue; (iii) endoderm: ciliated epithelium.