Supplementary Fig. 1. A, Upper panel shows schematic molecular representation of mutation- and SNP-replacement with FANCA-c-HDAdV in FANCA-corrected

Transcription

1 Supplementary Fig. 1. A, Upper panel shows schematic molecular representation of mutation- and SNP-replacement with FANCA-c-HDAdV in FANCA-corrected FA-iPSC line (C-FA-iPSCs). Lower panels show the sequencing results of the upstream SNP site (5 SNP, rs ) and downstream SNP site (3 SNP, rs ) relative to the C295T mutation. A spontaneous reversion clone with a back mutation in one of the FANCA alleles was disregarded based on the absence of the two HDAdV-specific SNPs located in intron 3 and 4. B, Sequencing results showing that the neomycin-resistance cassette (neo) was successfully removed from intron 4 of FANCA in the gene-corrected ipscs by FLPo recombinase. Schematic demonstration of a 57

2 bp sequence (including FRT and NcoI) left in intron 4 of the FANCA locus after neo removal. Supplementary Fig. 2. Absence of expression of CD34, CD31, and CD43 markers on MSCs differentiated from control-, FA-, and C-FA-iPSCs were determined by FACS analysis.

3 Supplementary Fig. 3. A, RT-qPCR analysis of Pax6 in ipscs and their NSC derivatives. Data are shown as mean±s.d. n=3. B, Western analysis of Tuj1 expression in a spontaneous neuronal differentiation assay with the indicated NSC lines (left) and quantifications (right). GAPDH was included as a loading control. NSCs derived from two independent FA-iPSC clones were used. Data are shown as mean±s.d. n=3. *p<0.05 (t-test). Also see Figure 7E. C, Hierarchical clustering on genome-wide DNA methylation profiles based on DNA methylation levels at CpG sites characterized in all samples (Ctrl-NSC, FA-NSC, and C-FA-NSC).

4 Supplementary Fig. 4. Array comparative genomic hybridization (array CGH) for genome wide detection of DNA copy number changes in FA-fibroblasts, FA-iPSCs#5, FA-iPSCs#8, C-FA-iPSCs and C-FA-MSCs. The ipsc samples were collected at passage 40. Gains are drawn on the right and losses on the left side of the diagram. Red boxes also highlight the gains and losses. List of regions between FA-fibroblasts and the control sample are shown in Supplementary Data 2.

5 Supplementary Fig. 5. A, Dendrograms of RNA-seq data of two randomly selected FA-iPSC clones (FA-iPSC#5 and FA-iPSC#8) and two FA-iPSC clones corrected by HR (C-FA-iPSC#1) or lentiviral FANCA gene complementation (C-FA-iPSC#2). B, Dendrograms of H3K4me3 ChIP-seq data of the indicated lines.

6 Supplementary Fig. 6. A, Comparison of the yield of CFU-GM from FA-iPSC derived CD34 + cells that are cultured in Methocult in the presence of vehicle (DMSO) or doramapimod. Data are shown as mean±s.d. n=3, * p<0.05 (t-test). B, Quantification of accumulated doubling population of FA-MSCs with doramapimod or tremulacin treatments at passage 5. The drug effects are normalized by the number of DMSO treated cells. Data are shown as mean±s.d. n=3. * p<0.05 (t-test). C, Quantification of TNFα levels in cell culture supernatant of FANCC -/- HSC536N cells after 6-hour treatment with vehicle (DMSO), doramapimod (500 nm) or tremulacin (5 nm). Data are shown as mean±s.d. n=3, ** p<0.01 (t-test). D, RT-qPCR analysis of TNF in FANCC -/- HSC536N cells after 6-hour treatment with vehicle (DMSO), doramapimod (500 nm) or tremulacin (5 nm). Data are shown as mean±s.d. n=3, * p<0.05 (t-test).

7 Supplementary Fig. 7. A, Immunofluorescence analysis of neural progenitor markers in ESC-FA +/+ derived NSC (NSC-FA +/+ ) and ESC-FA -/- derived NSC (NSC-FA -/- ). Bar, 50 m. B, FACS analysis of apoptosis in CD34 + /CD43 + HPCs differentiated from ESC-FA+/+ and ESC-FA-/-. The upper panels show the gates and frequencies of the CD34 + /CD43 + population. The lower panels show the frequency of apoptotic cells (Annexin V positive) within the CD34 + /CD43 + cells. C, Cell cycle analysis of CD34 + /CD43 + HPCs differentiated from ESC-FA+/+ and ESC-FA-/-. D, Quantification of the percentage of CD34 + /CD43 + cells derived from ESC-FA+/+ and ESC-FA-/- that were treated with doramapimod or tremulacin. The values are normalized against DMSO treatment.

8 Supplementary Fig. 8. Original images of Western blots.

9 Supplementary Table 1. Primer list. Primer Sequence (5 to 3 ) Application bs-oct4-5-f AATAGATTTTGAAGGGGAGTTTAGG Bisufite sequencing bs-oct4-5-r TTCCTCCTTCCTCTAAAAAACTCA Bisufite sequencing h-pax6-f CCAGAAAGGATGCCTCATAAA qpcr h-pax6-r TCTGCGCGCCCCTAGTTA qpcr EBNA1-F ATCAGGGCCAAGACATAGAGATG qpcr EBNA1-R GCCAATGCAACTTGGACGTT qpcr h-oct4-end-f GGGTTTTTGGGATTAAGTTCTTCA qpcr h-oct4-end-r GCCCCCACCCTTTGTGTT qpcr h-sox2-end-f CAAAAATGGCCATGCAGGTT qpcr h-sox2-end-r AGTTGGGATCGAACAAAAGCTATT qpcr h-nanog-f ACAACTGGCCGAAGAATAGCA qpcr h-nanog-r GGTTCCCAGTCGGGTTCAC qpcr h-lin28-f AGCCATATGGTAGCCTCATGTCCGC qpcr h-lin28-r TCAATTCTGTGCCTCCGGGAGCAGGGTAGG qpcr h-gapdh-f TCGGAGTCAACGGATTTGGT qpcr h-gapdh-r TTGCCATGGGTGGAATCATA qpcr h-p16-f GAGCAGCATGGAGCCTTCG qpcr h-p16-r CATCATCATGACCTGGATCG qpcr h-p21-f GGAAGACCATGTGGACCTGT qpcr h-p21-r GGCGTTTGGAGTGGTAGAAA qpcr h-ho-1-f ATGGCCTCCCTGTACCACATC qpcr h-ho-1-r TGTTGCGCTCAATCTCCTCCT qpcr h-cd44-f CTGCCGCTTTGCAGGTGTA qpcr h-cd44-r CATTGTGGGCAAGGTGCTATT qpcr h-dppa4-f TCCTGGGCGAGAATTTCAGC qpcr h-dppa4-r GCAGGTGAACCCAACCATCT qpcr h-cd29-f CCTACTTCTGCACGATGTGATG qpcr h-cd29-r CCTTTGCTACGGTTGGTTACATT qpcr h-hoxa9-f TACGTGGACTCGTTCCTGCT qpcr h-hoxa9-r CGTCGCCTTGGACTGGAAG qpcr h-hoxc9-f ACTCGCTCATCTCTCACGACA qpcr h-hoxc9-f TACGTGGACTCGTTCCTGCT qpcr h-magea12-f GGAGACGAGCTTCCAAGTAGC qpcr h-magea12-r GCACGATCTGATTGTCGCC qpcr h-csag1-f GAGACCAGGTGGACTGGAGTA qpcr h-csag1-r TCTTGGGAACCTCTTTGGTGT qpcr h-csag2-f GGCCTCATCCAATTAGTTGAAGG qpcr h-csag2-r GGCATGAAACTCACAGTGCAT qpcr h-postn-f CTCATAGTCGTATCAGGGGTCG qpcr h-postn-r ACACAGTCGTTTTCTGTCCAC qpcr h-etv4-f CAGTGCCTTTACTCCAGTGCC qpcr h-etv4-r CTCAGGAAATTCCGTTGCTCT qpcr h-hhip-f TCTCAAAGCCTGTTCCACTCA qpcr h-hhip-r GCCTCGGCAAGTGTAAAAGAA qpcr h-has2-f CTCTTTTGGACTGTATGGTGCC qpcr h-has2-r AGGGTAGGTTAGCCTTTTCACA qpcr h-adm-f ATGAAGCTGGTTTCCGTCG qpcr h-adm-r GACATCCGCAGTTCCCTCTT qpcr

10 h-grem1-f CGGAGCGCAAATACCTGAAG qpcr h-grem1-r GGTTGATGATGGTGCGACTGT qpcr h-etv5-f TCAGCAAGTCCCTTTTATGGTC qpcr h-etv5-r GCTCTTCAGAATCGTGAGCCA qpcr h-id3-f GAGAGGCACTCAGCTTAGCC qpcr h-id3-r TCCTTTTGTCGTTGGAGATGAC qpcr h-osr2-f TCCGCCTAAGATGGGAGACC qpcr h-osr2-r GGTAAAGTGTCTGCCGCAAAA qpcr h-nrg1-f CGGTGTCCATGCCTTCCAT qpcr h-nrg1-r GGGAGGCTGTTACTGTCATGC qpcr h-kcnma1-f TCTTTGCTCTCAGCATCGGTG qpcr h-kcnma1-r CCGCAAGCCGAAGTAGAGAAG qpcr h-tmeff2-f CTGCCCGTCATGCTACTCATC qpcr h-tmeff2-r AATTTACAGGTGTTGGTGTCACA qpcr h-egr1-f GGTCAGTGGCCTAGTGAGC qpcr h-egr1-r GTGCCGCTGAGTAAATGGGA qpcr h-kiaa1804-f CGTGGCTTATGGGGTAGCAG qpcr h-kiaa1804-r TCTTGTTGCCAGCATTCTTTCA qpcr h-ptprn2-f CCGAGAGCATCCTGACCTATG qpcr h-ptprn2-r CCACTGTCCACCTTAGGGC qpcr h-cnr1-f GTGTTCCACCGCAAAGATAGC qpcr h-cnr1-r GGGGCCTGTGAATGGATATGT qpcr h-stmn2-f GCTCTTGCTTTTACCCGGAAC qpcr h-stmn2-r AGGCACGTTTGTTGATTTGCT qpcr h-dbx2-f TTCCCCTAAAGCTGTGCCC qpcr h-dbx2-r CAGCACGCCGAGTAGAATG qpcr h-elavl4-f AACCTCTATGTTAGCGGCCTT qpcr h-elavl4-r TGGACACTCCTGTGACTTGAT qpcr h-bhlhe22-f CTGCGGCTTAACATCAATGCC qpcr h-bhlhe22-r TGGTTGAGGTAGGCGACTAGG qpcr h-col1a2-f GTTGCTGCTTGCAGTAACCTT qpcr h-col1a2-r AGGGCCAAGTCCAACTCCTT qpcr h-nrn1-f CCCCTCGTCCTTTTACCTCC qpcr h-nrn1-r GCGAAGCCCTGGTATATGGTG qpcr h-syt4-f ATGGGATACCCTACACCCAAAT qpcr h-syt4-r TCCCGAGAGAGGAATTAGAACTT qpcr h-dcx-f GACTCGCCACTTGTGTGATG qpcr h-dcx-r GACAGCTTCAGACTAAATGGGG qpcr h-neurod1-f ATGACCAAATCGTACAGCGAG qpcr h-neurod1-r GTTCATGGCTTCGAGGTCGT qpcr h-bace1-f ACCAACCTTCGTTTGCCCAA qpcr h-bace1-r TCTCCTAGCCAGAAACCATCAG qpcr h-cav1-f GCGACCCTAAACACCTCAAC qpcr h-cav1-r ATGCCGTCAAAACTGTGTGTC qpcr h-esrrg-f GCCCTCACTACACTGTGTGAC qpcr h-esrrg-r CCTGCTAATTTGGACTGGTCTT qpcr h-cdh6-f AGAACTTACCGCTACTTCTTGC qpcr h-cdh6-r TGCCCACATACTGATAATCGGA qpcr h-crabp1-f ACGCAAGTGCAGGAGTTTAG qpcr h-crabp1-r CGGGTCCAGTAGGTTTTGGG qpcr h-sulf1-f TGGCGAGAATGGCTTGGATTA qpcr h-sulf1-r TAACGGGCCTATGGGGATACA qpcr

11 h-st18-f GATAAAACGCTGCGTACTCGC qpcr h-st18-r GGAGCAATCATAGGCAACACT qpcr h-ptpro-f CCCAATGTGGTAGTGATCTCCG qpcr h-ptpro-r AGCTTTCCATCCCTCTCTAAGG qpcr h-ebf3-f GGGGACGACCATGAAGGAG qpcr h-ebf3-r CCCCTGCCTATCGTAGAGC qpcr h-chchd2-f ACACATTGGGTCACGCCATTA qpcr h-chchd2-r GCACCTCATTGAAACCCTCACA qpcr h-otp-f GCACAGCTCAACGAGTTGGA qpcr h-otp-r GTCAGCCCGATACGCAGTG qpcr h-tnf-f GCCCCCAGAGGGAAGAGTTC qpcr h-tnf-r TCAGCTTGAGGGTTTGCTACA qpcr h-infg-f TCAGCCATCACTTGGATGAG qpcr h-infg-r CGAGATGACTTCGAAAAGCTG qpcr h-il6-f GTCAGGGGTGGTTATTGCAT qpcr h-il6-r AGTGAGGAACAAGCCAGAGC qpcr h-hoxb4-f GTGAGCACGGTAAACCCCAAT qpcr h-hoxb4-r CGAGCGGATCTTGGTGTTG qpcr h-cd45-f ACAGCCAGCACCTTTCCTAC qpcr h-cd45-r GTGCAGGTAAGGCAGCAGA qpcr h-cd34-f GCGCTTTGCTTGCTGAGTTT qpcr h-cd34-r GCCATGTTGAGACACAGGGT qpcr h-cd31-f AACAGTGTTGACATGAAGAGCC qpcr h-cd31-r TGTAAAACAGCACGTCATCCTT qpcr h-fanca-f TGTGGCATCTTCACGTACAAGG qpcr h-fanca-r CCTGAAGAGCCACGATCCC qpcr h- Tuj1-F GGAGCGGATCAGCGTCTACTACAAC qpcr h- Tuj1-R ACTGTCCATGGTTCCGGGTTC qpcr