Supplementary Figure 1 Characterisation of newly produced ... · 1 Supplementary Figure 1...

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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

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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.

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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-Sox2 (Sox2) antibodies. The right panels are nuclear DAPI

staining. Bars: 200 m. (b) Quantitative RT-PCR analysis was used to compare

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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.

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Karyotyping of HS181 cells after 29 single cell suspension passages onto LN-521

(9 months in culture). No chromosomal abnormalities were observed.

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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.

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(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.

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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.

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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 294002, and PD 98059. 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 294002 (50 M), PD 98059 (40 M), and

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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 294002 treated hES cells one hour after plating on LN-521 in

O3. (e) Western blot analysis of Erk activity in DMSO and PD 98059 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).

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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.

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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.

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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.

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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

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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 stem-

cell-related abnormality, since a similar segment is observed in all female reference

samples (four shown, e-f).

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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

Charachterization

Immunostaing,

positive

FACS,

positive Karyotyping

in vitro

pluripotency

test, passed

in vivo

pluripitency

test,passed

HS181 LN521 mTeSR1 37 Oct4, Nanog,

Sox2 Oct4, SSEA4 normal, p.29 p.32 p.29

HS401 LN521 mTeSR1 25 Oct4, Nanog,

Sox2 Oct4, SSEA4 normal, p.20 nd nd

H1 LN521 mTeSR1 35 Oct4, Nanog,

Sox2 nd normal, p.31 p.20 p.12

CVTB1.2 LN521 mTeSR1 15 Oct4, Nanog,

Sox2 Oct4, SSEA4 nd nd nd

ChiPSW LN521 mTeSR1 10 Oct4, Nanog,

Sox2 nd nd nd nd

H1 LN521 TeSR2 14 Oct4, Nanog,

Sox2 Oct4, SSEA4 normal, p.7 p12 p.7

HS401 LN521 TeSR2 12 Oct4, Nanog,

Sox2 Oct4, SSEA4 nd nd nd

HS181 LN521/E-Cad mTeSR1 24 Oct4, Nanog,

Sox2 Oct4, SSEA4 normal, p.20 p.21 p.20

H1 LN521/E-Cad mTeSR1 20 Oct4, Nanog,

Sox2 nd normal, p.17 p.19 nd

HS916 derived on LN-521/E-

Cad; cultured on LN521

mTeSR2 for 12 passages;

after that NutriStemhESC

XF

20 Oct4, Nanog,

Sox2 Oct4, SSEA4 normal, p.10 p.10 p10



mTeSR2 for 7 passages;

after that NutriStemhESC

XF

40 Oct4, Nanog,

Sox2 Oct4, SSEA4

trisomy of chr.

8, p.35 p.38 p40

HS983a derived on LN-521/E-


mTeSR2 for 1 passage; after

that NutriStemhESC XF 27

Oct4, Nanog,

Sox2 Oct4, SSEA4

normal,p.15

abnormal, p.20 p.19 nd

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Cad; cultured on LN521 NutriStemhESC XF 45

Oct4, Nanog,

Sox2 Oct4, SSEA4

normal, p.20;

normal, p.36 p.37 p.35



Oct4, Nanog,

Sox2 nd normal, p.10 p.14 p.9



Oct4, Nanog,

Sox2 nd normal, p.12 p.19 ongoing

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Supplementary Table 2 QC-contrast values for the samples used in SNP 6.0 analysis.

File

Bo

un

ds

Co

ntr

ast

QC

Co

ntr

ast

QC

(R

and

om

)

Co

ntr

ast

QC

(N

sp)

Co

ntr

ast

QC

(N

sp/S

ty

Ov

erla

p)

Co

ntr

ast

QC

(S

ty)

Co

mp

ute

d G

end

er

# C

HP

/CE

L

IF2_13_980 P31_(GenomeWideSNP_6).CEL In 1.21 1.21 0.68 1.28 1.53 female 0

LA01_HS181 p0_B_(GenomeWideSNP_6).CEL In 1.33 1.33 1.41 1.91 1.17 female 0

LA02_HS181 p19_(GenomeWideSNP_6).CEL In 0.59 0.59 0.91 1.3 0.94 female 0

LA03_HS181 p0_ECad_(GenomeWideSNP_6).CEL In 0.45 0.45 0.85 1.02 1.07 female 0

LA04_HS181 p20_ECad_(GenomeWideSNP_6).CEL In 0.79 0.79 1.43 1.37 1.35 female 0

LA09c_1001 p5_(GenomeWideSNP_6).CEL In 1.2 1.2 1.6 1.76 1.79 male 0

LA10c_1001 p15_(GenomeWideSNP_6).CEL In 1.08 1.08 1.38 1.53 1.55 male 0

LA11_975 p6_(GenomeWideSNP_6).CEL In 0.81 0.81 0.76 0.88 0.58 female 0

LA12b_980 p6_(GenomeWideSNP_6).CEL In 1.01 1.01 1.55 1.77 1.38 female 0

LA2_999_p10_(GenomeWideSNP_6).CEL In 1.83 1.83 1.36 1.97 1.75 female 0

LA3_975_p35_(GenomeWideSNP_6).CEL In 1.41 1.41 1.05 1.55 1.01 female 0

LA05_983a p15_(GenomeWideSNP_6).CEL In 0.84 0.84 0.34 1.37 1.11 male 0

LA01_916 p17_(GenomeWideSNP_6).CEL In 0.91 0.91 0.51 0.88 0.85 male 0

LA02_CVTB1.2 p10_(GenomeWideSNP_6).CEL In 1 1 0.59 0.93 0.65 male 0

LA03_CVTB p22_(GenomeWideSNP_6).CEL In 1.01 1.01 0.68 1.08 0.94 male 0

LA04_983a p5_(GenomeWideSNP_6).CEL In 0.95 0.95 0.36 1.1 0.49 male 0

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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_p35 104 89

975_p6 121 102

980_p31 97 82

980_p6 100 83

999_p10 105 85

female control N1 85 62




ES401_p25 100 100

H1_p56 86 86

HS181feeders_p75 136 120

HS181feeders_p52 148 129

HS181feeders_p75 +

LN521_p19 140 122

HS181feeders_p52

+LN521/E-Cad_p20 136 118

1001_p15 97 97

1001_p5 81 81

916_p17 104 104

983a_p15 103 103

983a_p5 109 109

CVBT1.2_p10 93 93

CVBT1.2_p22 98 98

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