Generation of iPSC-like cells from differentiated cells of mouse, birds, fish, and Drosophila using the mouse transcription factors.

(A) Non-transduced mouse, avian and zebrafish embryonic fibroblasts, and Drosophila S2 cell line. (B) Transformed cells (colonies) after 20 days (first passage), using optimal titers (Figure 2—figure supplement 1). (C) Non-transduced cells labeled for ALP activity. (D) Colonies formed by transformed cells labeled for ALP activity after the first passages (10^th passage staining can be seen in Figure 2—figure supplement 2). (E) Non-transduced cells and F, transduced cells after colony formation reacted with a Stage Specific Embryonic Antigen-1 (SSEA-1; green fluorescence) antibody. (G) Colonies of embryonic stem cells (positive controls). (H) Embryonic stem cells labeled for ALP activity (positive controls). Black scale bars, 100 µm; green and red bars, 50 µm.

Karyotyping and in vitro pluripotency of iPSC-like cells.

(A) Embryoid bodies (EB) from iPSC-like cells in differentiation media. (B) qRT-PCR gene expression analyses of Nestin (ectoderm marker), Brachyury (mesoderm), and Gata-4 (endoderm) homologs in undifferentiated iPSC-like cells (green) and in EBs (yellow) from mouse, bird and fish relative to their control fibroblasts (normalized; blue). Error bars, S.E.M. (n = 5 replicates of independently generated cell lines or EBs). Statistics in Supplementary file 1D. (C–F) Karyotypes of macro chromosomal arrangements of the chicken iPSC-like (C), chicken control fibroblasts (D), zebra finch iPSC-like cells (E) and zebra finch control fibroblasts (F), exhibiting 18 normal chromosomes. ZZ is female and ZW is male in birds. Black scale bar, 100 µm.

In-vivo pluripotency of iPSC-like cells from chicken and fish.

(A and C) 4 day old chicken embryos that had been injected with GFP-labeled chicken fibroblasts (A) or GFP-labeled iPSC-like cells (C) 3 days earlier (GFP labeled cells in Figure 6—figure supplement 1). Incorporated GFP-labeled cells (green) are spread throughout the body for the iPSC cells but, not fibrobloast. (B and D) Histological sections stained with antibodies to GFP (brown) confirming absence of label in chicken fibroblast injected animals (B), and presence of label in multiple tissue types in the iPSC-like injected animals (D). (E and G) 3-day old (72hpf) zebrafish embryos injected with GFP-labeled zebrafish fibroblasts (E) or GFP-labelled iPSC-like cells (G), respectively. (F and H) Histological sections stained with antibodies to GFP (brown) confirming absence in controls (F) and presence of labeled cells in iPSC-like injected animals (H). Arrows in all images point to GFP-labeled cells; P.duct = pronephric duct (P. duct). 1 day old fish embryo is shown in Figure 5—figure supplement 2. Black bars, 30µm; white scale bar, 3000 µm for the chicken and 350 µm for the fish. 1 day old post fertilization zebrafish embryos (Figure 6—figure supplement 2), and chicken embryos with partial incorporation (Figure 6—figure supplement 3).

iPSC-like cells for (A) chicken, (B) zebrafish, and (C) Drosophila, transfected with a GFP expressing lentivirus. Post induction to iPSC-like state.

1 day old post fertilization zebrafish embryos.

(A) Embryos generated with control fibroblast cells exhibiting some localized flourescent cells. (B) Generated with iPSC-like GFP cells distributed in several parts of the embryo. (C) A double axis embryo, generated with iPSC-like GFP cells, showning one axis with high gfp flourescence, and none on the other. Explanation of histology sections is the same as in Figure 5.