SUPPLEMENTARY INFORMATION

Transcription

1 Supplementary Figure S1 Colony-forming efficiencies using transposition of inducible mouse factors. (a) Morphologically distinct growth foci appeared from rtta-mefs 6-8 days following PB-TET-mFx cocktail transfection. For this study, colonies were picked on d12 post-induction. Only those cell lines with typical ES cell morphology (shown here) were capable of generating stable lines in culture. PB ase: piggybac transposase. Scale bars are 50µm. (c) Induction cocktails were prepared containing 100ng of each of the four transposons and 100ng transposase plasmid. The dox concentration was varied in the media as indicated. Colonies were counted on the indicated days post-dox treatment. Total colony number per 10cm 2 is indicated on the Y-axis. Transfections were performed and analyzed in triplicate. Error bars represent the standard error. 1

2 Supplementary Figure S2 Reprogramming is a stochastic process. PB-TET-mFx generated cell lines picked as early as d10 were split 1:3 on d12 and two replica wells were subjected to dox withdrawal. (a) Line 5C was used to represent clones where 24hrs of dox removal was sufficient to revert colony morphology and slow growth (middle panel). Many of these cells were still in an intermediate reprogramming state as a certain proportion may be rapidly rescued (within 48hrs) upon re-addition of dox (right panel). (b) lacz staining under such culture conditions revealed that these cells, which still retain residual lacz activity, have reverted to a fibroblast-like morphology (middle panel). (c) RT-PCR analysis of dox-independent PB-TET-mFx reprogrammed clones reveals additional pluripotency marker gene expression. R1 ES cells and parental rtta-mefs serve as positive and negative controls, respectively. Amplification of the GAPDH housekeeping gene was used as an internal loading control. 2

3 Supplementary Figure S3 PB transposon copy number and mobilization in reprogrammed cells. (a) Diagnostic Southern analysis of seven clonal PB-TET-mFx induced lines using BamHI digestion and a neo probe to determine the number of transposon integrations. The neo probe detects all PB-TET transgene insertions, regardless of the mfx transgene delivered at the insertion site. The estimated copy number for each line is indicated below the image. Note that no fragmented transgene constructs (banding below the predicted lower limit of ~1.5kb) were detected. Asterisk (*) indicates the neo band resulting from inactivated G418-resistant mouse embryonic fibroblast feeder DNA contamination (this band was not detected in G418-sensitive parental rtta-mefs). (b) Individual mobilization frequency of the nine transposase insertions in 1B, determined by analyzing subclones derived after transient expression of the transposase. Southern blot analysis of 38 subclones (Supplementary Fig. S4) revealed high frequency mobilization and loss of transposons. 3

4 Supplementary Figure S4 Southern blot for detecting transposon insertions in 38 1B subclones that underwent transient transposase expression. 12 remained unchanged regarding the parental transposons (12/38=32%; ex. G2 and H2). From the total number of transposon insertions (38x9=351), 52 (15%) underwent mobilization (cut) of which only 24 were transposed (pasted). Therefore the probability loss after cut is 0.54 (28/52) and the absolute transposon removal rate is 7%. One particular transposon insertion (band #8 in Supplementary Fig. 3b) was transposed only once (1/38=2.5%) while another was cut 11 times (band #9; 11/38=29%). 4

5 Supplementary Figure S5 Doxycycline inducibility of single-copy transposon ips cell lines; scb1 and scc5. Expression of the lacz reporter in the presence of dox is lost in the factor/transposon-removed sublines. Scale bar is 100µm. 5

6 Supplementary Figure S6 Pluripotency of reprogrammed cell lines. (a) Contribution of reprogrammed cell (1B) derivatives (GFP+) to the germ cells (Vasa+) in the genital ridges of 12.5 dpc chimaeric embryo shown by immunohistochemistry. (b) PB-TET clones were capable of differentiating in teratoma formation assays, generating tissues derived from all three embryonic germ layers. Sections from clone 1B are shown as representatives. 6

7 Supplementary Figure S7 Reprogramming induction by PB-TET-mFx inducible factors in normal human embryonic fibroblasts by co-transfection with PB-CAG-rtTA. (a) lacz activity in the presence and absence (two weeks) of dox revealed tight regulation the reprogramming transgenes in all the four human ips cell lines. Scale bars are 200 µm. (b) RT-PCR on individual transgenes and the corresponding endogenous gene expression in the presence and absence of dox further specifies the tight regulation of transgenes and proves the activation of the endogenous counterparts. CA1 and CA2 human ES cell lines1 served as positive controls, while parental human fibroblasts, R1 mouse ES cells and mouse fibroblasts acted as additional controls. (c) RT-PCR demonstrates the activation of endogenous pluripotency marker genes using human-specific primers. 1. Adewumi, O. et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25, (2007). 7

8 Supplementary Figure S8 Immunohistochemistry results reveal the expression of SSEA4, NANOG, TRA-1-160, and Tra-1-81 in ips cells induced from human embryonic fibroblasts by PB-TET-mFx transfection. Cells staining positive only for DAPI are fibroblast feeders. Scale bars are 50 µm. 8

9 Supplementary Figure S9 Immunohistochemistry to detect in vitro differentiated human ips cell-derivatives positive for markers representing each of the three embryonic germ layers. -SMA: alpha smooth muscle actin; vwf: von Willebrand Factor; GFAP: glial fibrillary acidic protein; AFP: alpha-fetoprotein. Scale bars are 50 µm. 9

10 Supplementary Figure S10 (a) Contribution of 2ºF/1B and /6C derived cells to the chimaeric cell population in the function of factor induction time. The data was derived from Q2+Q4 of the FACS in Fig. 4. (b) Ratio of SSEA1 positive cells in the 2ºF/1B and /6C reprogramming fibroblasts. The data was obtained from 100*Q2/(Q2+Q4) in the FACS in Fig. 4. (c) Semi-quantitative RT-PCR to compare the level of exogenous reprogramming factor expression in 2ºF on d2 and 12 in three different mouse ips cell lines. 10

11 Supplementary Figure S11 Onset of Nanog expression during the process of reprogramming of secondary fibroblast (2ºF). 2ºF/1B and /6C were cultured in the presence of dox for 5, 9 and 13 days and then stained for Nanog (red). Many of the 2ºF/1B cells expressed Nanog at d9, while 2ºF/6C only showed robust Nanog expression at day13. The bottom row shows magnified images of the inserts in the upper panels. Scale bars are 100 µm. 11

12 Supplementary Table S1. RT-PCR and Cloning primers used in this study. RT-PCR Primers Gene Forward Reverse PB-mFx tg PB c-myc cmycrtf TCAAGCAGACGAGCACAAGC TFIBRTR CGACTAGCTAGAGCGGCCATC PB Klf4 Klf4RTF GGCGAGAAACCTTACCACTGT TFIBRTR CGACTAGCTAGAGCGGCCATC PB Oct3/4 Oct4RTF CCAACGAGAAGAGTATGAGGC TFIBRTR CGACTAGCTAGAGCGGCCATC PB Sox2 Sox2RTF TCTGTGGTCAAGTCCGAGGC TFIBRTR CGACTAGCTAGAGCGGCCATC Mouse endogenous c-myc cmycrtf TCAAGCAGACGAGCACAAGC cmycutrrtr TACAGTCCCAAAGCCCCAGC Klf4 Klf4RTF GGCGAGAAACCTTACCACTGT Klf4utrRTR TACTGAACTCTCTCTCCTGGCA Oct3/4 Oct4RTF CCAACGAGAAGAGTATGAGGC Oct4utrRTR CAAAATGATGAGTGACAGACAGG Sox2 Sox2RTF TCTGTGGTCAAGTCCGAGGC Sox2utrRTR TTCTCCAGTTCGCAGTCCAG Nanog NanogRTF CCTCCAGCAGATGCAAGAA NanogRTR GCTTGCACTTCATCCTTTGG E-Ras ErasRTF GCCCCTCATCAGACTGCTAC ErasRTR GCAGCTCAAGGAAGAGGTGT Zfp296 Zfp296RTF CCTATGCTTGTGCCCAGAGTA Zfp296RTR CTAAAGTGCCTGCCCATTTC Rex1 Rex1RTF GGAAGAAATGCTGAAGGTGGAGAC Rex1RTR AGTCCCCATCCCCTTCAATAGC Fbxo15 Fbxo15RTF TGCCAATTGTTGGGAGTACA Fbxo15RTR CAGATGAGCCTCTAACAAACTTACTTC FoxD3 FoxD3RTF AGGTCTGACCCCGAACAAG FoxD3RTR AGCGCGATGTAAGAGTAGGG Dax1 Dax1RTF TGCTGCGGTCCAGGCCATCAAGAG Dax1RTR GGGCACTGTTCAGTTCAGCGGATC GAPDH GapdhF ACCACAGTCCATGCCATCAC GapdhR TCCACCACCCTGTTGCTGTA Human endogenous hc-myc hcmycf3 CCAAGCAGAGGAGCAAAAGCTCATT hcmycutrr GGTTGTGAGGTTGCATTTGATCATGC hklf4 hklf4f1 GTCTCTTCGTGCACCCACTT hklf4utrr GTGGGTCATATCCACTGTCTGGG hoct3/4 hoct4f2 GTGAGAGGCAACCTGGAGAATT hoct4utrr CATTCCTAGAAGGGCAGGCACC hsox2 hsox2f3 TCTTGGCTCCATGGGTTCG hsox2utrr CATTTGCTGTGGGTGATGGG hnanog hnanogf3 CCTACCTACCCCAGCCTTTACTC hnanogutrr GAGGAAGGATTCAGCCAGTGTCC hlin28 hlin28f1 TCACCGGACCTGGTGGAGTA hlin28utrr AGAATAGCCCCCACCCATTGTG hrex1 hrex1f GCTGACCACCAGCACACTAGGC hrex1r TTTCTGGTGTCTTGTCTTTGCCCG hfgf4 hfgf4f CTACAACGCCTACGAGTCCTACA hfgf4r GTTGCACCAGAAAAGTCAGAGTTG htert htertf CCTGCTCAAGCTGACTCGACACCGTG htertr GGAAAAGCTGGCCCTGGGGTGGAGC hdnmt3b hdnmt3bf TGCTGCTCACAGGGCCCGATACTTC hdnmt3br TCCTTTCGAGCTCAGTGCACCACAAAAC Gateway Cloning rtta-advanced tetadvbfo ggggacaagtttgtacaaaaaagcaggcttcaccat GTCTAGACTGGACAAGAGC tetadvbrev ggggaccactttgtacaagaaagctgggtagttacccg GGGAGCATGTCAAG Genomic PCR GFP transgene GFP-F CTGACCCTGAAGTTCATCTGCACC GFP-R TGGCTGTTGTAGTTGTACTCCAGC PB-TET geo LacZ-F1 ACGGTTTCCATATGGGGATT Neo-R AGTGACAACGTCGAGCACAG Chr11 Chr11-F2 GCTGTTCCAAGGCTGAGTTC Chr11-R2 CAGAGGTTCACACGGTCTT Chr16 1 o Chr16-Left TGGGTATCACTTGGTGCAGA Chr16-Right GGTCATCTATAGAAGTGTTGTGTCC Chr16 2 o Chr16-F AACAGCAAAGCGGCAGGTTC Chr16-R CACCCAAGCCAAGGTTAAAA Chr16 3 o Chr16-F2 AAAAGAAAAAGCTGGCCTGA Chr16-R2 TCCAAAAATGCGGAGGAATA PB junction 5 TR Ch11-F2 or Ch16-F2 PB-5R2 CCGATAAAACACATGCGTCA PB junction 3 TR PB-3F ACGGATTCGCGCTATTTAGA Ch11-R2 or Ch16-R2 RT-PCR primers were designed specifically for the PB constructs in this report (tg), based on designs suggested by K. Kaji and references indicated in the main text (mouse endogenous), or drawn from alternate publications{takahashi, 2006 #38}(human endogenous). The Gateway attb1/b2 sites incorporated into the rtta-advanced primers are shown in small case. Genomic PCR primers were designed to amplify the PB insertion loci as determined from the NCBI m37 mouse genome assembly. 12

13 Supplementary Table S2. Resulting chimaeric embryos from diploid and tetraploid aggregations. Diploid Cell Line Embryos Transferred Implantations Embryos GFP Chimerism lacz Chimerism 1B (10.5dpc) Total (67%) a 38 (95%) b 6 (16%) c 28 (70%) d 1B n/d (12.5dpc) n/d n/d Total (30%) a 7 (39%) b 3 (43%) c 1B n/d (13.5dpc) n/d n/d n/d n/d Total (42) a 37 (88%) b 8 (22%) c 6C n/d (13.5dpc) n/d n/d n/d n/d Total (43%) a 30 (70%) b 12 (40%) c 6C n/d (15.5dpc) n/d Total (38%) a 10 (67%) b 1 (10%) c 3D (10.5dpc) Total (63%) a 23 (92%) b 5 (22%) c 8 (35%) d scb n/d (14.5dpc) n/d n/d Total (42%) a 18 (72%) b 6 (33%) c scc n/d (14.5dpc) n/d n/d Total (28%) a 13 (76%) b 7 (54%) c Tetraploid 1B n/d (13.5dpc) n/d n/d n/d Total (28) a 2 (9%) b 2 (100%) c PB-reprogrammed cell lines were aggregated with electro-fused tetraploid embryos or 2.5dpc diploid ICR embryos, and transferred the following day. Embryos were dissected on the indicated day post-coitus and assayed for GFP fluorescence by microscopy. To determine contribution by lacz reporter expression, embryos were treated with dox in utero for 20hrs prior to dissection. a implantations/embryos transferred b developed embryos/implantations c GFP chimaerism/developed embryos d lacz chimaerism/developed embryos n/d not determined 13