a–f, Successful reprogramming of genetically corrected primary dermal fibroblasts (a) derived from patient FA90. b, Colony of iPS cells from the cFA90-44-14 line grown on Matrigel-coated plates showing human-ES-cell-like morphology. c–f, The same iPS cell line shows strong alkaline phosphatase staining (c) and expression of the transcription factors OCT4 (d), SOX2 (e) and NANOG (f) and the surface markers SSEA3 (d, e) and SSEA4 (f). g, Genetically corrected fibroblasts from patient FA404. h, Colony of iPS cells from the cFA404-FiPS4F1 line grown on feeder cells displaying typical human ES cell morphology. i–l, The same iPS cell line shows strong alkaline phosphatase staining (i) and expression of the pluripotency-associated transcription factors OCT4 (j), SOX2 (k) and NANOG (l) and surface markers SSEA3 (j), SSEA4 (k) and TRA1-80 (l). Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in d–f and j–l. Scale bars, 100 µm (a, c–g, i–l) and 250 µm (b, h).

a–c, In vitro differentiation experiments of cFA404-FiPS4F2 iPS cells reveal their potential to generate cell derivatives of all three primary germ cell layers. Immunofluorescence analyses show expression of markers of a, endoderm (α-fetoprotein, green; FOXA2, red), b, neuroectoderm (TuJ1, green; GFAP, red), and, c, mesoderm (α-actinin, red). d–f, Injection of cFA90-44-14 iPS cells into the testes of immunocompromised mice results in the formation of teratomas containing structures that represent the three main embryonic germ layers. Endoderm derivatives (d, e) include glandular structures that stain positive for endoderm markers (α-fetoprotein, green); ectoderm derivatives (e) include structures that stain positive for neuroectoderm markers (TuJ1, red); mesoderm derivatives (f) include structures that stain positive for muscle markers (α-actinin, red). All images are from the same tumour. Scale bars, 100 µm (a, b, d, e) and 25 µm (c, f).

a, Expression of CD34 and CD45 markers in FA-iPS cells subjected to haematopoietic differentiation. b, c, Representative erythroid (BFU-E) and myeloid (CFU-GM) colonies generated 14 days after the incubation of iPS-derived CD34⁺ cells in semisolid cultures. d, The myeloid nature of CFU-GM colonies was confirmed by the co-expression of the CD33 and CD45 markers in CFU-GM colonies. e, Total number of CFCs generated in the absence and the presence of 10 nM mitomycin C (MMC) from CD34⁺ cells derived from the indicated FA-iPS cell lines. For comparison, clonogenic assays were also performed using haematopoietic progenitors from healthy donors (purified CD34⁺ cord blood cells from two independent donors, CB CD34⁺; and mononuclear bone marrow cells, BM MNC), from a FA patient and from CD34⁺ cells derived from control human pluripotent stem cells, including ES[2] cells (hES) and KiPS4F1 (KiPS) cells. f, Immunofluorescence analysis showing FANCD2 foci in mitomycin-C-treated CD34⁺ cells derived from FA-iPS cells (line cFA90-44-14).

a, PCR of genomic DNA to detect integration of the indicated retroviral transgenes in FA-iPS cell lines cFA90-44-14 (cFA90) and cFA404-FiPS4F1 (cFA404). Genetically corrected fibroblasts (Fibr.) from patient FA404 before reprogramming were used as negative control. b, c, Quantitative PCR with reverse transcription (RT–PCR) analyses of the expression levels of retroviral-derived reprogramming factors (b) and of total expression levels of reprogramming factors and pluripotency-associated transcription factors (c) in the indicated patients’ fibroblasts (fibr.) and FA-iPS cell lines. Human ES cells (ES[4]) and partially silenced iPS cells (KiPS4F3) are included as controls. Transcript expression levels are plotted relative to GAPDH expression. d–g, Colony of cFA90-44-14 iPS cells showing high levels of endogenous NANOG expression (e, green channel in d) and absence of Flag immunoreactivity (f, red channel in d). Cell nuclei were counterstained with DAPI (g, blue channel in d). h, Bisulphite genomic sequencing of the OCT4 and NANOG promoters showing demethylation in FA-iPS cell lines cFA90-44-14 and cFA404-KiPS4F3, compared to patient’s fibroblasts. Open and closed circles represent unmethylated and methylated CpGs, respectively, at the indicated promoter positions. Scale bar, 100 µm.

a, Western blot analysis of FANCA in protein extracts from the indicated cell lines, showing expression of FANCA in FA-iPS cells. The expression of vinculin was used as a loading control. hES, human ES. b, FANCD2 (red channel) fails to relocate to UVC-radiation-induced stalled replication forks, visualized by immunofluorescence with antibodies against cyclobutane pyrimidine dimers (CPD, green channel), in fibroblasts from patient FA404, whereas it shows normal accumulation to damaged sites in wild-type fibroblasts (control), corrected fibroblasts (cFA404) or FA-iPS-derived cells (cFA404-FiPS4F2). c, Western blot analysis of FANCA in protein extracts from untransduced cFA404-KiPS4F3 cells or 6 days after transduction with lentiviruses expressing scramble shRNA (control) or the indicated FANCA shRNAs. The expression of vinculin was used as a loading control. Values at the bottom represent FANCA expression levels measured by densitometry quantification normalized by vinculin expression and referred to untransduced cFA404-KiPS4F3 cells. d, Alkaline phosphatase staining of cFA404-KiPS4F3 cells one passage after being transduced with lentiviruses expressing scramble shRNA (control) or the indicated FANCA shRNAs, one week after seeding. e, Mitotic index values in cFA404-FiPS4F2-derived cells transfected with scramble (control) or FANCA siRNAs and incubated in the absence or in the presence of diepoxybutane (DEB). The inset shows FANCA depletion induced by FANCA siRNAs in these experiments, as visualized by western blot using vinculin as a loading control. Data are presented as mean ± s.d.