Generation of zebra fish iPS-like cells from embryonic and fin fibroblasts. (A) Workflow to generate zebra fish iPS cells (ziPSCs). (B) zSEF (left two panels) and zFF (right two panels) cells grown in ZF medium after transduction with TetO-FUW-GFP. (C) The time course and cell morphology of reprogramming in primary transduced zSEF (left four panels) and zFF (right four panels) cells with an optimized reprogramming system. (D) Zebra fish iPS-like cell line (right) and iPS-like colony with AP positive staining (left) derived from zSEF (E-ziPSCs, left two panels) and zFF (F-ziPSCs, right two panels) cells. (E) The number of colonies at day 17 (black columns) or day 22 (white columns) after transduction. The results were obtained from at least three independent experiments. Scale bars represent 200 μm.

Characterization of zebra fish iPS-like cells. (A) RT-PCR analysis of exogenous rTTA, oct4, sox2, klf4 and c-myc in zSEFs and zFFs at 7-day post-transduction. At least three independent experiments were conducted for these results). (B) RT-PCR analysis of endogenous oct4, sox2, lin28, and nanog in the E-ziPSCs (passage 20) and F-ziPSCs (passage 18) (At least three independent experiments were conducted for these results). (C) Immunofluorescence staining of Oct4, Nanog, and Sox2 in E-ziPSCs (passage 20) (Scale bars represent 20 μm). (D) Immunofluorescence staining of Oct4, Nanog, and Sox2 in F-ziPSCs (passage 18) (Scale bars represent 20 μm). (E) Distribution of chromosome numbers among 100 F-ziPSCs metaphases (passage 18-30). (F) A metaphase plate of the chromosomes of a diploid F-ziPSC (2n = 50) after Giemsa staining (Scale bar represents 10 μm). (G) Diploid karyotype of an F-ziPSC (homologous chromosomes were paired according to their sizes).

Differentiation potential of zebra fish iPS-like cells in vitro. (A) Embryoid bodies (EBs) derived from F-ziPSCs (passage 15). (B) Differentiated cell types from the EBs after attaching to a culture plate, showing a flat cell (arrow) and a neuron-like cell (asterisk). (C) Star-shaped cells. (D) Results by RT-PCR analyses of the three differentiation markers (nestin, brachyury, and gata4) for the three germ layers (At least three independent experiments were conducted for these results). (E) Nestin expression in the EBs formed from F-ziPSCs (passage 15) determined by immunofluorescence. The scale bars in (A-C) represent 50 μm and that in (E) represents 200 μm).

Differentiation potential of zebra fish iPS-like cells in vivo. PKH26-labeled F-ziPSCs (passage 22) and PKH26-labeled zFF cells (passage 10) were transplanted with approximately 300 cells to host blastulae. The resultant chimeras were analyzed by microscopy. For the PKH26-labeled F-ziPSCs (red): (A) bright field micrograph, (B) red fluorescent micrograph, (C) merge between bright and fluorescent optical fields, and (D-H) different distributions of PKH26-labeled donor cells in different stages, in which the chimeras were analyzed by microscopy at 2 (D-G) and 5 days post-fertilization (H) (ey, eye; ht, heart). For the PKH26-labeled zFF cells (red): (I-L) the zFF cells did not migrate. The scale bars in A-E and I-L represent 200 μm and those in F-H represent 300 μm.

Teratoma formation in vivo. PKH26-labeled F-ziPSCs (passage 22, red) were transplanted with approximately 1000 cells to host adult male pearl danios (D. albolineatus), and the resultant teratomas were analyzed by microscopy at four weeks after injection (A-C). After 10 weeks post-injection, the teratomas were photographed (D, E), and histological analysis was conducted with hematoxylin and eosin staining (F, G). Of the 10 injected fish, teratomas were observed in two fish. (A) Red fluorescent micrograph. (B) Merge between bright and fluorescent optical fields. (C) A close-up of the boxed area in (A) to show the possible proliferation of PKH26-labeled cells. (D) Photograph of adult male pearl danio at 10 weeks after injection to show the teratomas (arrow). (E) Magnification of the boxed area in (D). (F) Various tissues in the ziPSC teratomas. (G) Histological section of teratoma including muscle for mesoderm (asterisk) and epithelium for ectoderm (arrow). The scale bars in (A-E) represent 500 μm and those in (F, G) represent 20 μm.