produced in this study characterized using 3-8 % gradient SDS-PAGE under reducing

Transcription

1 Supplementary Figure 1 Characterisation of newly produced laminins. Human recombinant LN-521 produced in this study characterized using 3-8 % gradient SDS-PAGE under reducing 1

2 and non-reducing conditions. (a) The protein was visualized using Sypro Ruby protein staining and was shown to contain bands with the expected sizes for the 5 chain and a double band containing the 2 and 1 chains. (b) Western blot analysis of LN-521. SDS polyacrylamide gel electrophoresis was carried out on the purified LN- 521 trimer under reducing (red) and non-reducing (nonred) conditions. The protein was transferred to a PVDF membrane for the immunodetection with anti-human 5 (alpha5), anti-human 2 (beta2), anti-human 1 (beta1), and anti-human 1 (gamma1) antibodies. 2

3 Supplementary Figure 2 Self-renewal of dissociated human ips cells cultured on human recombinant laminin-521 in mtesr1 medium. (a) Immunostaining of CVTB1.2 ips cells after 3 passages on LN-521 in mtesr1 medium with anti-oct4 (depicted as Oct4), anti- Nanog (Nanog), and anti- () antibodies. The right panels are nuclear DAPI staining. Bars: 200 m. (b) Quantitative RT-PCR analysis was used to compare 3

4 numbers of mrna transcripts of pluripotency markers Oct4 (grey bars) and Nanog (black bars) at different time points of the experiments in CVTB1.2 ips cells cultured on LN-521 in mtesr1 medium (the cells were passaged in single cell suspension) or on Matrigel (the cells were passaged in small cluster pieces). Bars represent levels of Oct4 and Nanog expression in control CVTB1.2 cells cultured on Matrigel (Mg) taken as standard values to compare and in the cells cultured on LN-521 after 3 passages (LN-521, p.3). All reactions were done in quadruplicates. Error bars show 95 % confidence intervals. (c) FACS analysis of CVTB1.2 ips cells after three single cell suspension passages on LN-521 for SSEA4. The percentage of positive cells is listed in parentheses. 4

5 Supplementary Figure 3 Karyotyping of HS181 cells after 29 single cell suspension passages onto LN-521 (9 months in culture). No chromosomal abnormalities were observed. 5

6 Supplementary Figure 4 Pluripotency of hes cells cultured on LN-521. Formation of teratomas by pluripotent HS181 hes cells after extensive single cell suspension passaging on LN- 521 (29 passages). (a) + (b) show the formation of cystic structures outlined by a cylindrical epithelium surrounded by a continuous PASD positive basement membrane (b). Some cells have accumulated intracellular PASD positive material and, thus, are compatible with goblet cells (arrows), representing endodermal components. (c) shows a primitive glomerulus (arrow) and associated ducts representing mesoderm. 6

7 (d) + (f) show cartilage (mesoderm) (Car). Ectoderm is represented by a neuroepithelium (depicted as NE) in (e) and ganglion like structures (Gan) in (f). Scale bars: in (a)-(f) 100 m. (g) Immunostaining of embryoid bodies formed from HS181 cells after 32 passages on LN-521 reveals expression of markers of the three embryonic cell layers: smooth muscle actin (depicted as SM Actin), MAP-2 (MAP-2), and -fetoprotein (AFP). Scale bars: 50 m. 7

8 Supplementary Figure 5 Western blot analyses. (a) Western blot analysis of phosphorylated MLC (P-MLC) in dissociated HS181 cells one hour after plating on different substrata. (b) Western blot analysis of Calnexin (normalization control) of the same cells one hour after plating. (c) Western blot analysis of phosphorylated MLC (P-MLC) in dissociated HS181 cells six hours after plating. (d) Western blot analysis of Calnexin (normalization control) of the same cells six hours after plating. 8

9 Supplementary Figure 6 Signalling in hes cells on different substrata. Activation of the PI3K/Akt and Erk pathways, and their role in survival of dissociated hes cells cultured on different matrices. (a) Crystal Violet staining of hes cells 24 hours after plating on LN-521 in O3 medium treated with DMSO, LY , and PD Scale bars: 100 m. (b) Inhibition by different cell-permeable inhibitors on the survival of hes cells grown on LN-521 in the O3 medium 24 hours after plating. Bars represent survival of control HS181 hes cells treated with DMSO taken as standard values for comparison, and survival of cells treated with LY (50 M), PD (40 M), and 9

10 Wortmannin (0.3 M). Error bars show s.e.m. (n = 4). P-value was calculated by the Student s t-test. (c) Inhibition of survival of hes cells grown in O3 medium without bfgf for 24 hours after plating on LN-521. Names of inhibitors are denoted as in b. Error bars show s.e.m. (n = 4). (d) Western blot analysis carried out for Akt activity with phospho-akt (P-Akt) and total Akt (T-Akt) antibodies on untreated hes cells, DMSO treated, and LY treated hes cells one hour after plating on LN-521 in O3. (e) Western blot analysis of Erk activity in DMSO and PD treated hes cells one hour after plating on LN-521 in O3 medium with and without bfgf using phospho-erk (P-Erk) and total Erk (T-Erk) antibodies. (f) Western blot analysis of phosphorylated Akt (P-Akt) and total Akt (T-Akt) in dissociated HS181 cells one hour after plating on LN-521 and Matrigel. (g)elisa of human ES cell lysates collected one hour after plating on Matrigel (Mg), LN-111, LN-511, and LN-521 in O3 medium. Bars represent endogenous level of phospho-akt2 in control HS181 cells plated on Matrigel taken as a standard value to compare and levels of phospho-akt2 in cells plated on LN-111, LN-511, and LN-521. Error bars show s.e.m. (n = 4). P- value was calculated by the Student s t-test: ***, p<.002; **, p<.02. (h) ELISA of human ES cell lysates collected one hour after plating on Matrigel (Mg), LN-111, LN-511, or LN-521 in O3 medium. Bars represent endogenous levels of phospho- Akt1 on different coatings. The coatings are denoted as in g. Error bars show s.e.m. (n = 4). 10

11 Supplementary Figure 7 Pluripotency of clonal hes cells and new HS983a cells. Immunostaining of embryoid bodies formed (a) from HS181 cells after cloning of one cell in a well of a 96-well plate on LN-521/E-cadherin matrix, and (b) from HS983a cells after 19 passages in culture. Expression of markers of the three embryonic cell layers was revealed: smooth muscle actin (depicted as SM Actin), MAP-2 (MAP-2), and - fetoprotein (AFP). Scale bars: 100 m. 11

12 12

13 Supplementary Figure 8 Characterisation of HS181 cells after 20 passages on LN-521/E-cadherin coating. (a) Immunostaining of the cells. Bars: 100 m. (b) FACS analysis of hes cells after 20 single cell suspension passages on the new matrix for Oct4 and SSEA4. The percentage of positive cells is listed in parentheses. (c) Karyotyping of HS181 cells after 20 single cell suspension passages onto a LN-521/E-cadherin coating. No chromosomal abnormalities were observed. (d) Immunostaining of embryoid bodies formed from HS181 cells after 21 passages on LN-521/E-cadherin revealed expression of markers of the three embryonic cell layers: smooth muscle actin (depicted as SM Actin), MAP-2 (MAP-2), and -fetoprotein (AFP). Scale bars: 200 m. 13

14 Supplementary Figure 9 14

15 Karyotyping of HS1001, HS999, HS980, HS983a, and HS975 cell lines derived on a LN-521/E-cadherin matrix in a chemically defined and xeno-free environment. 15

16 16

17 17

18 18

19 Supplementary Figure 10 Maps of CNVs. Figures showing maps of CNV segments in new hes lines established and grown under chemically defined and xeno-free conditons for various number of passages (p) on LN-521, and their comparison with previously described HS401 (p.25) and H1 (p.52) cell lines established on feeders, as well as with samples 19

20 from blood and saliva of healthy individuals (female control 1-4, male controls 1-4). Blue triangles represent multiplications, red deletions. The Y-chromosomal CNV segment seen in female samples probably reflected X-Y homology rather than a stemcell-related abnormality, since a similar segment is observed in all female reference samples (four shown, e-f). 20

21 Supplementary Table 1 Summary of long-term pluripotent stem cell culturing experiments on LN-521 and LN-521/E-cadherin substrata. Line Coating Medium Number of passages HS181 LN521 mtesr1 37 HS401 LN521 mtesr1 25 H1 LN521 mtesr1 35 CVTB1.2 LN521 mtesr1 15 ChiPSW LN521 mtesr1 10 H1 LN521 TeSR2 14 HS401 LN521 TeSR2 12 HS181 LN521/E-Cad mtesr1 24 H1 LN521/E-Cad mtesr1 20 HS916 HS975 HS983a derived on LN-521/E- Cad; cultured on LN521 derived on LN-521/E- Cad; cultured on LN521 derived on LN-521/E- Cad; cultured on LN521 mtesr2 for 12 passages; after that NutriStemhESC XF mtesr2 for 7 passages; after that NutriStemhESC XF mtesr2 for 1 passage; after that NutriStemhESC XF Immunostaing, positive FACS, positive Charachterization Karyotyping in vitro pluripotency test, passed Oct4, SSEA4 normal, p.29 p.32 p.29 Oct4, SSEA4 normal, p.20 nd nd nd normal, p.31 p.20 p.12 Oct4, SSEA4 nd nd nd nd nd nd nd Oct4, SSEA4 normal, p.7 p12 p.7 Oct4, SSEA4 nd nd nd Oct4, SSEA4 normal, p.20 p.21 p.20 nd normal, p.17 p.19 nd Oct4, SSEA4 normal, p.10 p.10 p10 Oct4, SSEA4 Oct4, SSEA4 trisomy of chr. 8, p.35 normal,p.15 abnormal, p.20 p.38 p40 p.19 nd in vivo pluripitency test,passed 21

22 HS980 derived on LN-521/E- Cad; cultured on LN521 NutriStemhESC XF 45 Oct4, SSEA4 normal, p.20; normal, p.36 p.37 p.35 HS999 derived on LN-521/E- Cad; cultured on LN521 NutriStemhESC XF 15 nd normal, p.10 p.14 p.9 HS1001 derived on LN-521/E- Cad; cultured on LN521 NutriStemhESC XF 20 nd normal, p.12 p.19 ongoing 22

23 Bounds Contrast QC Contrast QC (Random) Contrast QC (Nsp) Contrast QC (Nsp/Sty Overlap) Contrast QC (Sty) Computed Gender # CHP/CEL Supplementary Table 2 QC-contrast values for the samples used in SNP 6.0 analysis. File IF2_13_980 P31_(GenomeWideSNP_6).CEL In female 0 LA01_HS181 p0_b_(genomewidesnp_6).cel In female 0 LA02_HS181 p19_(genomewidesnp_6).cel In female 0 LA03_HS181 p0_ecad_(genomewidesnp_6).cel In female 0 LA04_HS181 p20_ecad_(genomewidesnp_6).cel In female 0 LA09c_1001 p5_(genomewidesnp_6).cel In male 0 LA10c_1001 p15_(genomewidesnp_6).cel In male 0 LA11_975 p6_(genomewidesnp_6).cel In female 0 LA12b_980 p6_(genomewidesnp_6).cel In female 0 LA2_999_p10_(GenomeWideSNP_6).CEL In female 0 LA3_975_p35_(GenomeWideSNP_6).CEL In female 0 LA05_983a p15_(genomewidesnp_6).cel In male 0 LA01_916 p17_(genomewidesnp_6).cel In male 0 LA02_CVTB1.2 p10_(genomewidesnp_6).cel In male 0 LA03_CVTB p22_(genomewidesnp_6).cel In male 0 LA04_983a p5_(genomewidesnp_6).cel In male 0 23

24 Supplementary Table 3 Number of loss-of-heterozygosity (LOH) regions in different cell lines characterized in this study. The columns represent total number of LOHs and total number of LOHs without X-chromosome. sample total number of LOHs total w/o X-chr. 975_p _p _p _p _p female control N female control N female control N female control N ES401_p H1_p HS181feeders_p HS181feeders_p HS181feeders_p75 + LN521_p HS181feeders_p52 +LN521/E-Cad_p _p _p _p a_p a_p CVBT1.2_p CVBT1.2_p