Nature Methods A simple method to generate integration-free human ips cells Keisuke Okita, Yasuko Matsumura, Yoshiko Sato, Aki Okada, Asuka Morizane, Satoshi Okamoto, Hyenjong Hong, Masato Nakagawa, Koji Tanabe, Ken-ichi Tezuka, Toshiyuki Shibata, Takahiro Kunisada, Masayo Takahashi, Jun Takahashi, Hiroh Saji & Shinya Yamanaka Supplementary Figure Supplementary Figure Supplementary Figure Supplementary Figure 4 Supplementary Figure Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Figure 9 Supplementary Table Supplementary Table Supplementary Table Supplementary Table 4 Supplementary Table Supplementary Table 6 Supplementary Table 7 Supplementary Table 9 Supplementary Table Morphology of induced colonies. G-band staining of a pla-ipsc clone (49B-. Gene expression and copy number of the episomal plasmids in fibroblast-derived pla-ipscs. Quantitative PCR for detection of transgenic expression in DP-derived pla-pscs. Quantitative PCR for the detection of transgene expression in fibroblast-derived pla-ipscs. Comparison of the global gene expression profiles. Expression of GFP fluorescence from episomal vector. Teratoma derived from pla-ipscs. Differentiation of pla-ipscs into dopamine neurons. Comparison of integration-free methods of human ipsc generation. Summary of plasmid mixtures. Summary of the ipsc induction experiments. Karyotype of episomal vector-derived ipscs. STR analyses of pla-ipscs. Pearson correlation coefficient table. HLA Type of 7 Individuals. Pla-iPSC clone list. Primer list for quantitative PCR. Note: Supplementary Table 8 is available on the Nature Methods website.
a b Supplementary Figure. Morphology of induced colonies. (a Morphology of an ESC-like colony. Bar = mm. (b Morphology of a non-esc-like colony. Bar = mm.
4 6 7 8 9 4 6 7 8 9 X Y Supplementary Figure. G-band staining of a pla-ipsc clone (49B-.
a OCT/4 Ret-OCT SOX NANOG DPPA ZFP4 DNMTB no template HDF KhES 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- HDF-elepo GAPDH RT(- b No. of copy/cell (log - - - -4 - fibro-d6 6. x 4 (6. x 4 44C- ( 8.9 x 4 49B- (8 6. x 4 44C- (9.9 x 4 48C- ( 4.8 x 4 4C- (6. x 4 46C- ( 6. x 4 47D-4 (4 7.7 x 4 48C- (8 Supplementary Figure. Gene expression and copy number of the episomal plasmids in fibroblast-derived pla-ipscs. (a Expressions of pluripotent cell marker genes by an RT-PCR analysis. Pla-iPSCs established with the Y4 mix from various fibroblasts (44C-, 49B-, 44C-, 48C-, 4C-, 46C-, 47D-4, and 48C-, retrovirus-derived ipscs ( and, and ESCs (KhES- and were used for the analyses. Endogenous OCT/4 (OCT/4, retrovirus-derived OCT/4 (Ret-OCT, and endogenous SOX (SOX were examined. GAPDH was examined as a loading control. For the control, fibroblasts were examined 4 days after electroporation of Y4 mixture (HDF-elepo. (b Copy numbers of episomal vectors that remained in the Pla-iPSC clones. Established clones were harvested, lysed, and used for the quantitative PCR analyses. In several clones, including 44C- and 49B-, we were unable to detect any episomal vectors. The numbers in parentheses indicate the passage number. The examined cell numbers are also indicated. For the control, retrovirus-derived ipsc clone ( and fibroblasts at 6 days after electroporation of Y4 mixture (fibro-d6 were analyzed.
6 OCT/4 6 GAPDH Copy number (log 4 Copy number (log 4 DP74 KhES- 44B- 44C- 44D- 44E- 4F- 47C- 4F- DP74 KhES- 44B- 44C- 44D- 44E- 4F- 47C- 4F- 7 SOX 6 KLF4 Copy number (log 6 4 Copy number (log 4 DP74 KhES- 44B- 44C- 44D- 44E- 4F- 47C- 4F- DP74 KhES- 44B- 44C- 44D- 44E- 4F- 47C- 4F- L-MYC LIN8 Copy number (log 4 Copy number (log 4 DP74 KhES- 44B- 44C- 44D- 44E- 4F- 47C- 4F- DP74 KhES- 44B- 44C- 44D- 44E- 4F- 47C- 4F- Supplementary Figure 4. Quantitative PCR for detection of transgenic expression in DP-derived pla-pscs. Total RNA was isolated from ESCs (KhES- and, retrovirus-derived ipscs ( and, and pla-ipscs (44B- established by Y mix, 44C- by Y, 44D- by Y, 44E-, 4F-, 47C-, and 4F- by Y4, and were used for the RT-PCR analyses. White columns show the copy numbers obtained with specific primers to the episomal vector of the indicated genes. Black columns show the copy numbers obtained with primers for the coding sequence, which could detect both Nature endogenous Methods: and doi..8/nmeth.9 episomal vector-derived transcripts. For the controls, fibroblasts at 4 days after electroporation of Y4 mixture were analyzed (.
6 OCT/4 6 GAPDH Copy number (log 4 Copy number (log 4 HDF KhES- 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- HDF KhES- 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- 6 SOX KLF4 Copy number (log 4 Copy number (log 4 HDF KhES- 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- HDF KhES- 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- L-MYC LIN8 Copy number (log 4 4 Copy number (log HDF KhES- 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- HDF KhES- 44C- 49B- 44C- 48C- 4C- 46C- 47D-4 48C- Supplementary Figure. Quantitative PCR for the detection of transgene expression in fibroblast-derived pla-ipscs. Total RNA was extracted from ESCs (KhES- and, retrovirus-derived ipscs ( and, and pla-ipscs (44C-, 49B-, 44C-, 48C-, 4C-, 46C-, 47D-4, and 48C-, and were used for the RT-PCR analyses. White columns show the copy numbers obtained with specific primers against the episomal vector of the indicated genes. Black columns show the copy numbers obtained with primers for the coding sequence, which could detect both endogenous and episomal vector-derived transcripts. As a control, fibroblasts at 4 days after electroporation of Y4 mixture were analyzed (.
44C- 49B- 4F- 44B- 47C- 44E- 44C- KhES KhES H B-6 HDF88 DP74 Supplementary Figure 6. Comparison of the global gene expression profiles. Unsupervised clustering was performed using microarray data from ESC clones (KhES, KhES, H, and, pla-ipsc clones (44C-, 49B-, 44C-, 4F-, 44B-, 44E-, and 47C-, retrovirus-derived ipsc clones (B-6,, and, fibroblasts (HDF88, and DP cells (DP74.
Phase Fluorescence FACS.% week control 67.6% 8.% 4 week week week 4.%.4% count GFP Supplementary Figure 7. Expression of GFP fluorescence from episomal vector. Human fibroblasts were transduced with an episomal vector encoding GFP via electroporation. The Nature cells were Methods: photographed doi..8/nmeth.9 and analyzed by flow cytometry at,,, and 4 weeks after transduction.
Neural tissue Cartilage Gut-like epithelium 44C- 49B- 48C- 4C- 48C- 44E- Supplementary Figure 8. Teratoma derived from pla-ipscs. Nature Hematoxylin Methods: and doi..8/nmeth.9 eosin staining of teratoma derived from the indicated ipsc clones. µm
a no template undif. 49B- 44C- 49B- 44C- neuro-dif. day 4F- 44E- 47C- B-6 OCT/4 SOX MAP PAX6 GAPDH RT(- pla-ipsc retro-ipsc b Nestin/DAPI c Ki67 d Nestin/Ki67/DAPI e Nestin/Ki67/DAPI f PAX6/TH g PAX6/DAPI h TH/Tuj/DAPI i MAPab j MAPab/TH k TH l TH/VMAT Supplementary Figure 9. Differentiation of pla-ipscs into dopamine neurons. (a Expression of neural marker genes (SOX, MAP, and PAX6 detected by RT-PCR analyse. Total RNA was isolated from ESCs, pla-ipscs, and retro-ipscs under undifferentiation condition (undif. or days after neural differentiation (neuro-dif.. (b-l Twenty-nine days after induction using serum-free culture of embryoid body-like aggregates (SFEB combined with double SMAD inhibition by a BMP antagonist and an Activin/Nodal inhibitor, the pla-ipsc clone, 44E-, had differentiated into neurons. (b-d Double immunostaining for Nestin (green and Ki67 (red with DAPI nuclear staining (blue. A high magnification image is shown in (e. (f and g Double immunostaining for PAX6 (red and TH (green with DAPI nuclear staining (blue in a same visual field. (h Double immunostaining for TH (red and TuJ (green with DAPI. (i and j Double immunostaining for TH (red and MAPab (green. (k and l Double immunostaining for TH (Red and the Nature vesicular Methods: monoamine doi..8/nmeth.9 transporter (VMAT, green. Scale bars: μm in (b-e, f-h; μm in (e, i-l.
Removal Supplementary Table. Comparison of integration-free methods of human ipsc generation. Method Efficiency a from adult cells of materials Delivery procedure ipsc generation Preparation of exogenous factors References Adenovirus + Not tested + h ++ ++ l n ( -6 b ( infections Sendai virus +++ ++ + h +++ + m o, p ( - c ( HDF, T cell ( infection Episomal plasmid ++ +++ +++ +++ ++ l (.9-4 d (8 HDF, DP ( transfection Minicircle DNA + ++ + i + ++ l q ( - e ( ASC ( transfections, FACS sorting RNA +++ + + j + +++ r (4.4 - f ( HDF (6 daily transfections Protein + Not tested + k + +++ s (.6 - g (6 cycle treatments a The highest figure reported in each study. b IMR9 embryonic fibroblasts. c BJ neonate fibroblasts. d Adult fibroblasts. e Adult adipose stem cells (ASC, after sorting of transfected cells. f Fibroblasts produced by directed differentiation of H ESC. g Newborn fibroblasts. h Production of virus in packaging cells. i Construction of minicircle vectors. j in vitro transcription with modified nucleotide. k Production of proteins in HEK9 cells. l Rare integration events need to be ruled out. m Sendai virus tend to replicate in cytoplasm. n Zhou, W. & Freed, C.R. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells 7, 667-674 (9. o Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 8, 48-6 (9. p Seki, T., et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7, -4 (. q Jia, F., et al. A nonviral minicircle vector for deriving human ips cells. Nat Methods 7, 97-99 (. r Warren, L., et al. Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mrna. Cell Stem Cell 7, 68- (. s Kim, D.H., et al. Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins. Cell Stem Cell 4, 47-476 (9.
Supplementary Table. Summary of plasmid mixtures. Mixture name Plasmid name Amount ( g Genes Y pcxle-hoct/4 OCT/4 pcxle-hsk SOX, KLF4 pcxle-hmln c-myc, LIN8, NANOG Y pcxle-hoct/4-shp OCT/4, p shrna pcxle-hsk SOX, KLF4 pcxle-hmln c-myc, LIN8, NANOG Y pcxle-hoct/4 OCT/4 pcxle-hsk SOX, KLF4 pcxle-hul L-MYC, LIN8 Y4 pcxle-hoct/4-shp OCT/4, p shrna pcxle-hsk SOX, KLF4 pcxle-hul L-MYC, LIN8 T pep4eosetk. OCT/4, SOX, SV4LT, KLF4 pep4eosenk. OCT/4, SOX, NANOG, KLF4 pcep4-ml.7 c-myc, LIN8 T pep4eosetk.9 OCT/4, SOX, SV4LT, KLF4 pep4eosckmenl.9 OCT/4, SOX, KLF4, c-myc, NANOG, LIN8 T pep4eosetk.8 OCT/4, SOX, SV4LT, KLF4 pep4eosenl. OCT/4, SOX, NANOG, LIN8 pep4eosemk. OCT/4, SOX, c-myc, KLF4
Supplementary Table. Summary of the ipsc induction experiments. MEF feeder SNL feeder Parental cells Exp. ID Factors non ES-like ES-like non ES-like ES-like HDF49 48 Y Y 4 7 7 Y 8 8 Y4 8 6 66 8 449 Y4 47 4 4 TIG 8 Y Y 4 Y 9 Y4 4 97 TIG 8 Y Y 7 Y 4 6 4 Y4 7 7 7 48 Y Y Y - - Y4 6 448 Y Y 4 Y 4 Y4 9 6 9 T 7 4 T 8 T 4 Y Y 4 8 Y 9 4 9 Y4 7 4 67 T 8 T T 48 6 Y Y 4 4 Y Y4 6 T 4 T T 6 HDF88 44 Y Y Y Y4
49 Y Y Y Y4 T 9 T 4 4 T 6 6 44 Y Y 4 Y Y4 9 T T 6 T 8 4 Y Y Y Y4 T T 8 T 6 47 Y Y Y Y4 4 TIG4 48 Y Y Y Y4 4 HDF77 46 Y Y 78 Y 7 8 Y4 7 6 8 T 6 HDF49 47 Y4 4 4 4 T HDF47 47 a Y Y 9 Y Y4 7 4 46 Y Y 6 Y Y4 4 9 9 T 9
T 8 8 T 9 466 Y Y 4 Y 8 Y4 74 44 8 T 8 T T 8 4 Y Y Y Y4 7 6 T 4 T T 9 HDF4 46 Y Y Y Y4 6 46 Y Y 6 Y 4 Y4 8 468 Y Y 7 Y Y4 TIG7 4 Y - - Y 9 - - Y - - Y4 4 - - control - - 478 Y Y Y Y4 Y4 DP74 4 Y Y 8 Y 7 Y4 7 7 8 T 4 T 44 Y Y 8
Y 4 8 Y4 7 T T 4 T 47 Y Y Y Y4 7 T T T 6 469 Y Y 4 6 4 4 Y 8 4 7 Y4 8 8 97 T T T 4 4 DP94 446 Y Y 7 4 9 Y 4 7 Y4 7 T T T 4 Y Y 8 9 Y 9 8 Y4 4 T T T 4 Y Y 9 Y 9 7 7 Y4 6 4 T T T a In the exp. 47, only.8 cells were plated onto SNL feeder cells. *Exp. ID 448, 4,, 49, 44, 4, 46, 466,, 44, 47, 469, 446, 4, and 4 were used for Fig. d.
Supplementary Table 4. Karyotype of episomal vector-derived ipscs. Clone passage Karyotype origin Age Sex a Race b factors fibroblasts 49B- p9 46,XX [] HDF88 6 F C Y4 44C- p9 46,XX [] HDF88 6 F C Y4 48C- p9 46,XY [] TIG4 6 M J Y4 4C- p9 46,XX [] TIG7 8 F J Y4 46C- p4 46,XX [] HDF4 77 F C Y4 47D-4 p 46,XY, add((q. [] HDF47 6 M C Y4 48C- p 46,XX [] TIG 6 F J Y4 DP cells 4F- p9 46,XX [] DP74 6 F J Y4 4F- p 46,XX [] DP94 6 F J Y4 44B- p 46,XX [] DP74 6 F J Y 44C- p 46,XX [] DP74 6 F J Y 44D- p 46,XX [] DP74 6 F J Y 44E- p 46,XX [] DP74 6 F J Y4 44F- p 46,XX,add(7(q[] / 46,XX[] DP74 6 F J T 47C- p 46,XX [] DP74 6 F J Y4 a M, male; F, female b J, Japanese; C, Caucasian.
SupplementaryTable. STR analyses of pla-ipscs. Locus/clone HDF88 49B- 44C- DP74 44E- 47C- DS8 6 8 6 8 6 8 7 7 7 TH 6 6 6 7 7 7 DS 9 9 9 9 9 9 D8S 9 9 9 Penta_E 7 9 7 9 7 9 DS88 DS7 9 9 9 4 4 4 D7S8 9 9 9 D6S9 9 9 9 CSFPO 4 4 4 Penta_D 9 9 9 AMEL X X X X X X vwa 8 8 8 7 7 7 D8S79 8 8 8 TPOX 8 9 8 9 8 9 8 8 8 FGA 4 4 4
Supplementary Table 6. Pearson correlation coefficient table. Cells 44C- 49B- 44C- 4F- 44B- 44E- 47C- B-6 H KhES KhES HDF88 DP74 CellType 44C-.994.998.9944.99.9998.9888.9944.9764.996.9949.9796.997.989.8664.8 Pla-iPSCs 49B-.994.9969.998.999.997.9946.989.9864.987.998.99.98944.9996.8676.866 Pla-iPSCs 44C-.998.9969.994.99.99.999.99.97867.99466.99.988.9949.9894.847.86 Pla-iPSCs 4F-.9944.998.994.99668.99784.99786.9846.9848.9847.99.989.994.994.899.866 Pla-iPSCs 44B-.99.999.99.99668.9984.9986.9849.989.9847.99.9969.994.9947.88.869 Pla-iPSCs 44E-.9998.997.99.99784.9984.9989.9844.986.98469.9986.9999.9986.9996.84.866 Pla-iPSCs 47C-.9888.9946.999.99786.9986.9989.987.98878.986.99.9986.99.9977.8.868 Pla-iPSCs B-6.9944.989.99.9846.9849.9844.987.976.99.98866.978.994.9796.86.88 Retro-iPSCs.9764.9864.97867.9848.989.986.98878.976.9774.9894.9878.9848.986.879.876 Retro-iPSCs.996.987.99466.9847.9847.98469.986.99.9774.999.976.99.988.86.8 Retro-iPSCs H.9949.998.99.99.99.9986.99.98866.9894.999.98488.99498.999.899.878 ESCs.9796.99.988.989.9969.9999.9986.978.9878.976.98488.98.997.87.8 ESCs KhES.997.98944.9949.994.994.9986.99.994.9848.99.99498.98.999.89.896 ESCs KhES.989.9996.9894.994.9947.9996.9977.9796.986.988.999.997.999.8.87 ESCs HDF88.8664.8676.847.899.88.84.8.86.879.86.899.87.89.8.9668 Fibroblasts DP74.8.866.86.866.869.866.868.88.876.8.878.8.896.87.9668 DP cells
Supplementary Table 7. HLA Type of 7 Individuals. No. Cell line HLA-A HLA-B HLA-DRB Match DP *4 * *4 *44 *4 * DP4 * *4 *7 * * *7 DP6 * * * *4 *4 *9 4 DP7 * *4 *44 * *9 * DP74 DP *4 *6 * * * * DP74 6 DP *4 * *7 *6 * *4 7 DP * - *4 * *8 *9 8 DP4 *4 * * * *9 * DP74 9 DP * * * *46 *8 *4 DP7 *4 *6 * *4 *9 * DP74 DP *6 * *44 *46 *4 *8 DP * *4 * * *4 *8 DP6 * * * * *4 * 4 DP8 * * * *46 *4 *8 DP *4 * * * *9 * DP74 6 DP * * *48 * *9 * 7 DP *4 *6 *4 *4 *4 *8 8 DP *4 *6 * *4 *4 * DP74 9 DP * - *44 - *8 * DP8 * * *4 * *4 *4 DP9 * *4 * *4 *4 * DP94 DP4 *4 *6 * * *4 * DP74 DP4 *4 *6 *4 *4 * *4 4 DP4 *4 *4 *7 * * *8 DP44 *4 *4 *4 * * * DP74 6 DP46 * *4 * * *4 *4 DP94 7 DP48 *4 *6 * * *4 * DP74 8 DP49 *4 * *7 *44 * *8 9 DP * *6 * *67 *8 *6 DP *4 * *7 *4 * *4 DP4 * *6 *4 *46 *8 *9 DP6 *4 *4 *4 * *9 * DP74 DP7 *4 * *44 *4 *4 * 4 DP8 *4 *6 *4 *4 * *9 DP9 *4 *4 *7 * * *4 6 DP6 * *4 *46 * *8 * DP74 7 DP6 *4 *4 *48 *9 *4 *9 8 DP64 * * *4 *46 *8 *4 9 DP6 *4 *4 *4 *4 *9 *4 4 DP66 *4 *4 *4 * *8 * 4 DP68 *4 * * * *4 *4 4 DP69 * *4 *46 * *9 * DP74 4 DP7 * * *4 *46 *8 * 44 DP7 * * * *4 *8 *9 4 DP74 a *4 - * - * - DP74 46 DP7 * *4 * * *8-47 DP8 * * *44 * *9 * 48 DP8 *4 *4 *7 * * * DP74 49 DP8 * *6 * *4 *8 * DP86 *4 * * * *9 * DP74 DP87 *4 * * * *4 * DP74 DP9 * *4 * * *4 * DP94 a * - * - *4 - DP94 4 DP9 *4 *4 * *4 *4 *9 DP96 * *4 *4 *8 *8 * 6 DP97 *4 *4 * *4 *4 * DP74 7 DP98 * *4 *48 *4 *4-8 DP99 *4 * * * *8 * DP74
9 DP * * * * *4 *4 6 DP * - *44 - *4 * 6 DP * *4 * *4 *4 *4 6 DP6 *4 *6 * *4 *4 * 6 DP9 *4 *4 *46 *4 *4 * 64 DP * - *44 - *9 * 6 DP *6 * *44 * * * 66 DP * *4 * *4 *4 *8 67 DP * *4 * *4 *9 *4 68 DP8 *4 *4 * *7 *9 * 69 DP9 * * *9 *67 *4 * 7 DP4 * * *44 * *4 * 7 DP * - * *44 * *4 7 DP6 * *4 * *4 *8 * DP74 7 DP8 * *4 *4 *46 *8 * 74 DP4 * * * *9 *9 * 7 DP4 * *4 *4 * * * DP74 76 DP4 * * * *44 *9 * 77 DP4 *4 *4 *4 * *4 * DP74 78 DP44 * * *44 - * - 79 DP47 *4 *4 * * *4 * DP74 8 DP * * * * *4-8 DP4 * *4 * * * * DP74 8 DP7 * * *7 *44 * *4 8 DP8 *4 * * * *9 * DP74 84 DP9 * *4 * *46 *8 * 8 DP6 * *4 *4 *4 *4 * 86 DP6 *4 *6 *4 *46 *4 *9 87 DP64 * *4 *9 *4 * - 88 DP6 *4 *4 *7 * * *9 89 DP66 *4 * *4 *44 *4 *6 9 DP67 *4 *4 *4 * *4 * DP74 9 DP69 * *6 *4 * *8 * 9 DP7 * *4 * *4 *4 *9 9 DP7 * *4 *4 * *4 * DP74 94 DP7 * * *7 *44 * *4 9 DP74 * *4 *44 *48 *4 * 96 DP7 * * *7 *44 * *4 97 DP76 *4 *4 * *4 *4 *9 98 DP77 *4 * *4 *44 * *4 99 DP78 *4 *4 *4 * * * DP74 DP79 * *4 *9 * *8 * DP8 * *6 *46 *48 *8 *9 DP8 *4 * *7 *9 * * DP84 *4 * *44 * * * DP74 4 DP8 * *4 *9 * * - DP86 * * *4 - *4 *4 6 DP87 * * * *46 *8 * 7 DP9 *4 *6 *9 * *8 * DP74 a DP lines used for induction of pla-ips cells in this study. This data was based on the results of 4-digit PCR-rSSOP. DP74 covers 9, and DP94 covers lines. Total coverage is 9.9% (/7.
Supplementary Table 9. Pla-iPSC clone list. Source Gene expression Differentiation Clone Origin Copy Age Sex Race Factors RT-PCR Microarray Bisulfite number Karyotyping HLA STR Teratoma Dopa RPE analysis 44C- HDF88 6 F C Y4 49B- HDF88 6 F C Y4 44C- HDF88 6 F C Y4 48C- TIG4 6 M J Y4 4C- TIG7 8 F J Y4 46C- HDF4 77 F C Y4 47D-4 HDF47 6 M C Y4 48C- TIG 6 F J Y4 4F- DP74 6 F J Y4 4F- DP94 6 F J Y4 44B- DP74 6 F J Y 44C- DP74 6 F J Y 44D- DP74 6 F J Y 44E- DP74 6 F J Y4 44F- DP74 6 F J T 47C- DP74 6 F J Y4 ND ND, we could not detect RPE differentiation in our differentiation protocol (n =.
Supplementary Table. Primer list for quantitative PCR. OCT/4 (CDS CCC CAG GGC CCC ATT TTG GTA CC ACC TCA GTT TGA ATG CAT GGG AGA GC OCT/4 (pla CAT TCA AAC TGA GGT AAG GG TAG CGT AAA AGG AGC AAC ATA G KLF4 (CDS ACC CAT CCT TCC TGC CCG ATC AGA TTG GTA ATG GAG CGG CGG GAC TTG KLF4 (pla CCA CCT CGC CTT ACA CAT GAA GA TAG CGT AAA AGG AGC AAC ATA G SOX (CDS TTC ACA TGT CCC AGC ACT ACC AGA TCA CAT GTG TGA GAG GGG CAG TGT GC SOX (pla TTC ACA TGT CCC AGC ACT ACC AGA TTT GTT TGA CAG GAG CGA CAA T L-MYC (CDS GCG AAC CCA AGA CCC AGG CCT GCT CC CAG GGG GTC TGC TCG CAC CGT GAT G L-MYC (pla GGC TGA GAA GAG GAT GGC TAC TTT GTT TGA CAG GAG CGA CAA T LIN8 (CDS AGC CAT ATG GTA GCC TCA TGT CCG C TCA ATT CTG TGC CTC CGG GAG CAG GGT AGG LIN8 (pla AGC CAT ATG GTA GCC TCA TGT CCG C TAG CGT AAA AGG AGC AAC ATA G GAPDH ACC ACA GTC CAT GCC ATC AC TCC ACC ACC CTG TTG CTG TA EBNA- ATC AGG GCC AAG ACA TAG AGA TG FBXO GCC AAT GCA ACT TGG ACG TT GCC AGG AGG TCT TCG CTG TA AAT GCA CGG CTA GGG TCA AA CDS; for detection of cording sequence. pla; for detection of plasmid vector-derived expression.