Supplementary information Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent stem cells Annabelle Congras 1, Harmonie Barasc 2, Kamila Canale-Tabet 1, lorence Plisson-Petit 1, Chantal Delcros 1, Eva Hadadi 3, Olivier eraud 3, Noufissa Oudrhiri 3, ranck Griscelli 3, Annelise Bennaceur- Griscelli 3, Ali Turhan 3, Marielle Afanassieff 4, Stéphane erchaud 5, Alain Pinton 2, Martine Yerle- Bouissou 1 and Hervé Acloque 1,* Affiliations 1 INA, UM 1388 Génétique, Physiologie et Systèmes d Elevage, -31326 Castanet-Tolosan, rance 2 Université de Toulouse INPT ENVT, UM 1388 Génétique, Physiologie et Systèmes d Elevage, - 31076 Toulouse, rance 3 Inserm, UMS935 ESTeam Malignant and Therapeutic Stem Cell Models, -94800 Villejuif, rance 4 Inserm, U846 Stem Cells and Brain esearch Institute, -69675 Bron, rance 5 INA, UE 1372 GenESI Génétique, Expérimentation et Système Innovants, Surgères, rance 1
SSEA1 A647A SSEA1 A647A A I3 I4 GP GP B LI+2i I3 x5 I4 x5 I20 x5 I3 x20 I4 x20 I20 x10 C NANOG OCT4 SOX2 LIN28B CDH1 I3 LI+2i I4 LI+2i Supplementary igure S1 :ips-like cell lines produced with integrative strategies do not express SSEA- 1 and adapt well to 2i+LI medium. A.low cytometry immunofluorescence analysis indicates the activity of the EOS reporter system through high GP expression together with the absence of SSEA-1 in GP positive cells. B. Cell morphology of I3 and I4 cell lines in LI+2i medium. C. Immunofluorescence analysis reveals the nuclear expression of NANOG, OCT4, SOX2 and the extranuclear expression of LIN28 and CDH1 (A594 and DAPI counterstaining) in LI+2i medium. 2
Supplementary igure S2: Expression levels of exogenous reprogramming factors and endogenous pluripotency markers in NI1 and NI14 lines. eal-time PC were performed on cdnas produced from NI1 and NI14 cell lines at early (p7, blue bars) and late (respectively p34 and p32, red bars) passages. Expression of exogenous factors (A) hsoct4 (SeV OCT4), hssox2 (SeV SOX2), hs KL4 (SeV KL4), hsmyc (SeV MYC) and sendaï virus NA (SeV) was quantified between early and late passages in culture, like for endogenous pluripotency markers (B) ssoct4, sssox2, sskl4, ssmyc, sssall4, ssnanog, ssesb and sslin28. Data are means and SD of two independent experiments (t-test, * P<0.05; ** P<0.01; *** P<0.001, NS: not significant). 3
Supplementary igure S3: Expression levels of exogenous reprogramming factors during differentiation of NI13 and NI20 lines. T-PC amplification fo exogenous reprogramming factors was performed on total NA extracted from NI13 and NI20 cell lines at passage 30, before and after 8 days of differentiation. SeVS: exogenous hssox2; SeVO: exogenous hsoct4 ; SeVK: exogenous hskl4, SeVM: exogenous hsmyc, SeV: sendaï virus; ACTB: endogenous ssactb 4
Supplementary Table S1 : Variation of gene expression level in NI lines compared to I lines Gene Variation p-value POU51 + 2.09E-09 CDC20 + 4.07E-09 c-myc + 3.41E-03 DAX1 + 5.84E-05 DAZL + 6.89E-03 DPPA2 + 7.07E-10 DPPA5 + 2.67E-05 CDH1 + 2.31E-05 EAS + 4.82E-06 EZH2 + 8.88E-10 BX015 + 4.82E-02 GATA6 + 9.10E-05 KL5 + 3.65E-02 LETY2 + 2.34E-02 LIN28B + 7.81E-08 NANOG + 1.54E-05 NODAL + 5.62E-06 N6A1 + 6.23E-05 PDM1 + 2.41E-02 SALL4 + 3.67E-09 SOX2 + 3.52E-06 DPPA3 + 1.70E-10 TET + 3.77E-06 GP - 1.44E-15 OTX2-6.18E-06 TBX3-3.19E-02 5
Supplementary Table S2 : Number of probes differentially expressed for several comparisons. ep = early passage, lp = late passage Number of probes EB13-NI13 EB20-NI20 EB12-NI12 NI13-I3ep NI13-I4ep NI20-I3ep NI20-I4ep Downregulated 1494 1800 1828 2083 2040 2037 2079 Upregulated 1967 1965 2073 2418 2590 2508 2640 I3ep-PE I4ep-PE NI12-PE NI13-PE NI20-PE I20-PE I3lp-I3ep I4lp-I4ep Downregulated 3103 3236 3595 3840 3807 4127 1175 672 Upregulated 2673 2708 3475 4008 4051 3788 904 383 6
Supplementary Table S3 : Primers used for the detection of core pluripotency markers by conventional PC Gene Sequence 5-3 Amplicon size (bp) CCCCGAAGCATCCATTTCC NANOG 214 CGAGGGTCTCAGCAGATGACAT CGGAACTGGCCACTGTAAAT LIN28B 390 TCTGACAGCAATGGCACTTC CTTTCCCTCGGTGTCTGTCA OCT4 125 GTACAAACCCCAGGCTTCTCTC GGTACACTGCCTCTCTCTCACAT SOX2 109 ACTGTTTCTTACTCTCCTCCCATTT CCGTCGGCCGGCCCTTAGAG KL4 426 TGTGGGTTCGCAGGTGTGCC CAACGACAGCAGCTCGCCCA c-myc 758 GGGCCATTCTAGTTCCTCCCTCCA CACGCCATCCTGCGTCTGGA ACTB 380 AGCACCGTGTTGGCGTAGAG PC parameters : 35 cycles, Tm=59 C 7
Supplementary Table S4 : Primers used for the detection of exogenous markers by conventional PC Amplicon size (on Name Sequence 5-3 genomic DNA) (bp) pmxs-as3200 TTATCGTCGACCACTGTGCTGGCG hoct4-s947 CAGGGCCCCATTTTGGTACC 283 hsox2-s807 CCTCCGGGACATGATCAGC 247 hklf4-s1282 TTCGCCCGCTCAGATGAACTG 231 hcmyc-s1199 CAGCATACATCCTGTCCGTCC 221 GGATCACTAGGTGATATCGAGC SeV 181 ACCAGACAAGAGTTTAAGAGATATGTATC CCCGAAAGAGAAAGCGAACCAG Oct4-SeV 483 AATGTATCGAAGGTGCTCAA ATGCACCGCTACGACGTGAGCGC Sox2-SeV 451 AATGTATCGAAGGTGCTCAA TTCCTGCATGCCAGAGGAGCCC Klf4-SeV 410 AATGTATCGAAGGTGCTCAA TAACTGACTAGCAGGCTTGTCG cmyc-sev 532 TCCACATACAGTCCTGGATGATGATG AGAGAAACGAAGGCATGGGA SeV L 418 TGCTAGGTGGATTGCACTCA CAGGTTGGGAGAGGTCAAGT SeV HN 359 GGACTGCGATCGTACTCTCA GCGGTCTCTTCGTGCACCCACTTG hskl4 523 TGTGGGTTCGCAGGTGTGCC TTCCTTCCCCATGGCGGGACA hsoct4 794 GGCTGATCTGCTGCAGTGTGGGTT GGGAAATGGGAGGGGTGCAAAAGAGG hssox2 151 TTGCGTGAGTGTGGATGGGATTGGTG GCCCTGCGTGACCAGATCCC hsmyc 201 TTTTCCTTACGCACAAGAGTTCCGT CACGCCATCCTGCGTCTGGA ssactb 475 AGCACCGTGTTGGCGTAGAG CCGTCGGCCGGCCCTTAGAG sskl4 525 TGTGGGTTCGCAGGTGTGCC AGTGCCCAAAGCCCACTCT ssoct4 200 TTCTGGCGACGGTTGCA GGTACACTGCCTCTCTCTCACAT sssox2 109 ACTGTTTCTTACTCTCCTCCCATTT GCTGGATTTCCTTCGGATAG ssmyc 500 TTGGTGAAGCTGACGTTGAG TCGGAGTGAACGGATTTG ssgapdh 231 CCTGGAAGATGGTGATGG pmxstrangenes PC parameters : 30 cycles, Tm=60 C SeV transgenes PC parameters : 40 cycles, Tm=55 C hskl4 & hsoct4: PC parameters : 40 cycles, Tm=65 C hssox2, hsmyc and porcine sequences: PC parameters : 40 cycles, Tm=60 C 8
9 Supplementary Table S5 : Primers used for eal-time Quantitative PC analysis Gene Sequence 5-3 Gene Sequence 5-3 ACTA2 TGTGCTGGACTCTGGAGATG GTGGTCACGAAGGAGTAGCC LETY2 CAGGGACTATGGAGCTCAGG ACACTCATAGGCCAGGAAGC ACV2B GCTGCTGGCTAGACGACTTC TGGCTCGTACGTGACTTCTG LI TCAATCCTGGTGCTGTGAAC CAGCCCAGCTTCTTCTTCTG CDC20 TTCCTCTGCAGACATTCACG ACCAGAGAATGGAGCACACC LIN28B CGGAACTGGCCACTGTAAAT TCTGACAGCAATGGCACTTC cmyc GCTGGATTTCCTTCGGATAG TTGGTGAAGCTGACGTTGAG MHC1 / SLA1 CCTCTTCCTGCTGCTGTCG AGCGTGTCCTTCCCCATCT DAX1 / N0B1 CTTCCTTGCCAAGTGCTGGA CTGCTGAGTTCCCCACTGAA NANOG AAGAAGCAGAAGATCAGAACTGTGT TGTTTGTAGCTAAGGTTCAGGATGT DAZL GCATTTTCACGACATCATGG TAGCCACCTATTGCCTGTCC NODAL CGTCTCCAGATGGACCTGTT CTGCTCTGGAGAGAGGTTGG DKK1 GAGGGGAAATTGAGGAAACC GTGGCACAGTCTGATGATCG N6A1 TGTCCTCCCTCACCGTTTAC TGCAAGCATACTCCTCGTTG DNTM3B ATGAAGAAGAGGGTGCCAG GCCTCTGATATCTGGGGTGC OCT4 / POU51 AGTGCCCAAAGCCCACTCT TTCTGGCGACGGTTGCA DPPA2 CTTCAAGAGCCGTTCACCCT GGCGAACCCAACCTTCTGTA OTX2 TTGTCCACCTCCTCTTCCTG ACTGAGTGTGGTCCCTGGTC DPPA3 / STELLA TGAACAGAGACCCGCCTTTC TGACATCTTCCCCAAGTCGC PECAM1 TTCACAACGTCTCCTCCACG CACCCTGGGATTGGACACTC DPPA5 GATGCTCCAGTCTATGGCAGAGT GTGAATTCATGGCTTCCTCAAGTC PIWIL2 AGGTCGTGGTATTGGCAGAG TGATCCAGAGAGTGCAGTGG CDH1 CTTCATAACCCGGAGATTAGTTTTC TCTCTTTTCTGTCTTCTGAGACCAT EX1 / ZP42 CTTCAAGGAGAGCGCAAAAC GACAGCCTTCAAAGGGACAC EAS ATGTTTGATCTGGGCCTGGG ACCACCGCTTTGTACTCAGG SALL4 CAGGAGTACCAGAGCCGAAG GCTTCGACCTTCCATCTCAG ESB AGGCTCTGACCACTCTCTGT ACACCAGCTTGTCGTCGTAG SOX2 GGTACACTGCCTCTCTCTCACAT ACTGTTTCTTACTCTCCTCCCATTT EZH2 AATGGCGATCACAGGATAGG GTTCCAGAGAGGAGGGGAAG TBX3 GACCATGGAGCCTGAAGAAG TTTATCCAGCCCAGAACACC BXO15 AACAGACCTCGATGGCATTC ATGTGCTCCTCTCCACAAGG TET GAGGAACCCCTCATTTTTCC GAACCAGCTTGAGCAGGAAG G4 3UT GCGATGAGTGCAAGTTCAAA GTCCGAAGAAAGTGCACCAC UT1 CTAAGTTTGGGGTGGGGAAG GTGACATCGTGTCCATCCTG GATA6 CAGGAAACGAAAACCTAAGAGCAT TTCTCGGGATTAGCGCTCTC ACTB CACGCCATCCTGCGTCTGGA AGCACCGTGTTGGCGTAGAG GBX2 AATGCCAACTCCAAGACAGG GGTGGCTGTGAGTTTTGCTT GAPDH TCGGAGTGAACGGATTTG CCTGGAAGATGGTGATGG GD3 TAAGAACCATCCCCAAGCTG CACACATGTGGCTCCTGAAC TBP AACAGTTCAGTAGTTATGAGC AGATGTTCTCAAACGCTTCG KL2 GCCTTCGGTATCTTTGACGA GAGGTGCGAGCTCTTTGTGT GP AGATCCGCCACAACATCGAG TCTCGTTGGGGTCTTTGCTC KL4 CCGTCGGCCGGCCCTTAGAG TGTGGGTTCGCAGGTGTGCC NESTIN TTGAGGACCTAGGGACGGAG TCCTCATCTGCAAACCCGTC KL5 AAGTCCAGATAGACAAGCAGAGATG CTATTGTATCTGACAGGCTGGATGT PDM1 CGCCAAGTTCACCCAGTTTG GAGGCAACTTCAGGAGGGAC C.ACTIN CAGGTATTGCTGATCGCATGCA ATTTGCGGTGGACGATGGA AP TGCTGCAAAGCTGAGAATGC AGACACTCCAGCACGTTTCC PL4 CAAGAGTAACTACAACCTTC GAACTCTACGATGAATCTTC HPT GGACTTGAATCATGTTTGTG CAGATGTTTCCAAACTCAAC
Supplementary Table S7 : Threshold Cycles (Ct) of eal-time Quantitative PC assays in NI lines sspl4 sshpt ssoct4 sssox2 sskl4 ssmyc sssall4 ssnanog ssesb sslin28 NI13 p7 19,90 24,7 27,35 24 26,8 31 25,7 27,7 27,6 28,4 NI13 p29 19,6 24,5 30,1 22,2 28,2 32,3 24,2 26 33 27 NI20 p7 19,8 24,7 27,2 24,4 26,5 31,1 26,2 28,3 28,1 31 NI20 p33 18,6 22,9 25,1 22,5 24,9 31,2 24,5 27,1 25,8 29,8 NI14 p12 19,8 24,2 28,6 24,6 27,8 30,4 27 29,4 28,8 30,8 NI14 p35 20,1 25,4 31,4 23,2 28,3 31,7 25,4 30,6 30,8 26,9 NI1 p12 20,4 24,6 28,6 25 28,9 32 27,6 30,2 27,9 33,3 NI1 p 34 21 25,4 31,3 24,6 29,6 33,7 26,8 30,1 27,9 33,4 sspl4 sshpt SeV SeV hsoct4 SeV hssox2 SeV hskl4 SeV hsmyc NI13 p7 19,90 24,7 19 21,1 27,2 24,9 22,1 NI13 p29 19,6 24,5 19 20 36,7 28,2 22,4 NI20 p7 19,8 24,7 19,9 21,7 26,7 25 22,7 NI20 p33 18,6 22,9 19,1 21,8 34,5 26,7 22,1 NI14 p12 19,8 24,2 20,3 23,1 31,3 26,8 22,8 NI14 p35 20,1 25,4 21 22,5 37,3 28,8 23,8 NI1 p12 20,4 24,6 21,6 24,5 30,6 27,5 25,5 NI1 p 34 21 25,4 21,6 23,4 35 28,7 24,9 10
Supplementary ile S1: Chromosomal formula found in I, NI and I20 lines at early and late passages and clonal analysis Supplementary ile S2: Differentially expressed genes between NI-iPSLCs and their derived Embryoid bodies (EBs) 11