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Cell Biology International (2010) 34, 1021–1031 (Printed in Great Britain)
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Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines
Ashish Mehta, Sheena Mathew, Chandra Viswanathan and Anish Sen Majumdar1
Regenerative Medicine, Reliance Life Sciences Pvt. Ltd., ThaneBelapur Road, Rabale, Navi Mumbai400 701, India

1To whom correspondence should be addressed (email anish_senmajumdar@relbio.com).

This supplementary data is also available as a PDF

SUPPLEMENTARY ONLINE DATA


Figure S1 Schematic representation of the protocol used during the study

Undifferentiated hESCs were maintained on MEF feeders or in mTeSR1 medium, respectively. Human ES colonies were manually cut into small clumps for embryoid body (EB) formation. Embryoid bodies were kept for 10 days in suspension culture. On day 10, EBs were randomly divided into 2 groups: a) plated, EBs were plated on gelatin-coated dishes until days 20 and 30; and b) suspension, EBs were kept in suspension cultures until days 20 and 30. Cells were collected at each time point (days 0, 10, 20 and 30) for gene expression analysis by real time qRT-PCR and flow cytometric analysis.

Table S1 List of gene expression markers for undifferentiated state, three germ layers and their derivative organs

SNo Gene symbol Gene name Function Cat#
1 18S rRNA 18s rRNA Housekeeping Hs99999901_s1
2 GAPDH Glyceraldehyde-3-phosphate dehydrogenase Housekeeping Hs99999905_m1
3 POU5F1 POU class 5 homeobox 1 Pluripotency Hs01895061_u1
4 NANOG Nanog homeobox Pluripotency Hs02387400_g1
5 hTERT Telomerase reverse transcriptase Pluripotency Hs00972646_m1
6 GDF3 Growth differentiation factor 3 Pluripotency Hs00220998_m1
7 NES Nestin Ectoderm Hs00707120_s1
8 SOX1 SRY (sex determining region Y)-box 1 Ectoderm Hs00534426_s1
9 MSL1 Musashi homolog 1 (Drosophila) Ectodermal Hs00159291_m1
10 NCAM Neuronal cell adhesion molecule Neuronal Hs00170554_m1
11 NEFH Neurofilament, heavy polypeptide 200kDa Neuronal Hs00606024_m1
12 TUBB3 Tubulin, beta 3 Neuronal Hs00801390_s1
13 GFAP Glial fibrillary acidic protein Neuronal Hs00157674_m1
14 KRT14 Keratin 14 Skin Hs00559328_m1
15 T T, brachyury homolog (mouse) Mesendoderm Hs00610080_m1
16 GSC Goosecoid homeobox Mesendoderm Hs00418279_m1
17 GATA4 GATA binding protein 4 Mesendoderm Hs00171403_m1
18 FLK1 (KDR) Kinase insert domain receptor Mesoderm Hs00176676_m1
19 NKX 2.5 NK2 transcription factor related, locus 5 (Drosophila) Cardiac progenitors Hs00231763_m1
20 MYH6 Myosin, heavy chain 6, cardiac muscle, alpha Cardiac myosin heavy chain Hs00411908_m1
21 CD45 (PTPRC) Protein tyrosine phosphatase, receptor type, C Blood cells Hs00236304_m1
22 CD31 (PECAM1) Platelet/endothelial cell adhesion molecule Blood cells Hs00169777_m1
23 CD34 CD34 molecule Blood cells Hs00990732_m1
24 CD105 Endoglin Mesenchymal Hs00164438_m1
25 CD73 (NT5E) 5-Nucleotidase, ecto (CD73) Mesenchymal Hs00159686_m1
26 CD44 CD44 molecule (Indian blood group) Stromal Hs00174139_m1
27 FABP4 Fatty acid binding protein 4, adipocyte Fat Hs00609791_m1
28 PPARγ Peroxisome proliferator-activated receptor gamma Fat Hs01115513_m1
29 CXCR4 Chemokine (C-X-C motif) receptor 4 Meso and endoderm Hs00607978_s1
30 SOX17 SRY (sex determining region Y)-box 17 Definitive endoderm Hs00751752_s1
31 FOXA2 Forkhead box A2 Definitive endoderm Hs00232764_m1
32 SOX7 SRY (sex determining region Y)-box 7 Endoderm Hs00846731_s1
33 AFP Alpha-fetoprotein Liver Hs00173490_m1
34 ALB Albumin Liver Hs00609411_m1
35 PDX1 Pancreatic and duodenal homeobox 1 Pancreas Hs00236830_m1
36 INS Insulin Pancreas Hs00356618_m1
37 CDX2 Caudal type homeobox 2 Intestine Hs00230919_m1
38 FABP2 Fatty acid binding protein 2, intestinal Intestine Hs00164552_m1
39 Cyclin D1 Cyclin D1 Cell cycle Hs00277039_m1
40 CDC2 Cell division cycle 2, G1 to S and G2 to M Cell cycle Hs00364293_m1
41 BAX BCL2-associated X protein Apoptosis Hs00180269_m1
42 BCL2 B-cell CLL/lymphoma 2 Apoptosis Hs00608023_m1
43 CASP3 Caspase 3 Apoptosis Hs00234387_m1
44 BMP4 Bone morphogenetic protein 4 Signal Hs00370078_m1
45 FGF4 Fibroblast growth factor 4 Signal Hs00173564_m1
46 NODAL Nodal homolog (mouse) Signal Hs00250630_s1
47 NOTCH2 Notch homolog 2 (Drosophila) Signal Hs00225747_m1
48 WNT3 Wingless-type MMTV integration site family, member 3 Signal Hs00229135_m1


Table S2 List of antibodies used for flow cytometry and immunofluorescence for undifferentiated and three germinal layers

SNo Antibody Dilution Company
Pluripotency markers
    1 PE-labelled anti-human Oct-4 1:100 BD Biosciences
    2 PE-labelled anti-human SSEA4 1:100 BD Biosciences
Ectoderm markers
    3 Mouse anti-human Nestin 1:100 Chemicon
    4 Rabbit anti-human NCAM 1:1000 Chemicon
    5 Mouse anti-human NFH 1:1000 Chemicon
Mesoderm markers
    6 Rabbit anti-human Gata4 1:100 Santa Cruz Biotechnology
    7 PE-labelled anti-human CD44 1:100 BD Biosciences
    8 PE-labelled anti-human CD73 1:100 BD Biosciences
    9 Goat anti-human MHC 1:100 Santa Cruz Biotechnology
Endoderm markers
    10 Rabbit anti-human HNF4a 1:100 Santa Cruz Biotechnology
    11 Goat anti-human SOX17 1:1000 R & D Systems
    12 Mouse anti-human albumin 1:5000 Sigma
Secondary antibodies
    13 Donkey anti-goat IgG Alexa Fluor® 488 1:2000 Invitrogen
    14 Goat anti-mouse IgG Alexa Fluor® 488 1:2000 Invitrogen
    15 Goat anti-rabbit IgG Alexa Fluor® 546 1:2000 Invitrogen


Table S3 Differential propensity to differentiate into the three germ layers in different culturing conditions on spontaneous differentiation of hESCs

The data represented are means and S.E.M. of triplicate experiments for each culture condition in both the cell lines. Each marker has been normalized to the lowest expression level in each set of undifferentiated or EBs. The values represent the fold induction of gene expression above the lowest level. To compare the expression during differentiation, the value of markers for undifferentiated state and three germ layers were normalized to the same sample as used at day 0* and day 10* respectively. Undetected samples and those with small values (Ct >35) were defined as 0.00 and were excluded from analysis. Statistical analyses were performed using ANOVA at P<0.05. a−i Same letters within each gene are not statistically significant.

(a) Pluripotency markers
(i)


Marker OCT-4 NANOG
Days D0* D10 D20 D30 D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.000* 0.001a 0.002 0.000c 0.002 0.000c 0.003 0.000c 1.987 0.137a 0.004 0.001c 0.002 0.000c 0.000 0.000
R1FS 1.000* 0.001a 0.002 0.000c 0.002 0.000c 0.002 0.000c 1.987 0.137a 0.004 0.001c 0.001 0.000c 0.001 0.000c
R1mP 3.263 0.638b 0.009 0.003c 0.004 0.000c 0.002 0.000c 7.030 2.282b 0.101 0.058d 0.008 0.002c 0.003 0.000c
R1mS 3.263 0.638b 0.009 0.003c 0.003 0.001c 0.005 0.002c 7.030 2.282b 0.101 0.058d 0.000 0.000 0.007 0.003c
B1FP 1.166 0.120a 0.101 0.012d 0.004 0.001c 0.003 0.000c 1.000* 0.001a 0.157 0.017d 0.014 0.004c 0.013 0.005c
B1FS 1.166 0.120a 0.101 0.012b 0.008 0.000c 0.007 0.001c 1.000* 0.001a 0.157 0.017d 0.010 0.004c 0.008 0.000c
B1mP 3.799 0.634b 0.465 0.128e 0.015 0.001f 0.013 0.001f 7.381 1.855b 3.450 1.901e 0.023 0.004f 0.029 0.004f
B1mS 3.799 0.634b 0.465 0.128e 0.050 0.001g 0.007 0.001c 7.381 1.855b 3.450 1.901e 0.065 0.011g 0.024 0.004f


(ii)


Marker TERT GDF3
Days D0* D10 D20 D30 D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.000* 0.001a 0.151 0.012d 0.000 0.000 0.000 0.000 1.166 0.215a 3.184 0.328c 0.831 0.242a 0.000 0.000
R1FS 1.000* 0.001a 0.151 0.012d 0.012 0.001g 0.002 0.002g 1.166 0.215a 3.184 0.328c 0.440 0.018d 0.000 0.000
R1mP 9.749 2.789b 0.229 0.058e 0.012 0.012g 0.000 0.000 10.117 2.362b 0.000 0.000 0.000 0.000 0.000 0.000
R1mS 9.749 2.789b 0.229 0.058a 0.000 0.000 0.000 0.000 10.117 2.362b 0.000 0.000 0.000 0.000 0.000 0.000
B1FP 3.216 0.755c 0.826 0.191f 0.133 0.015d 0.000 0.000 1.000* 0.001a 0.802 0.094a 0.044 0.022d 0.000 0.000
B1FS 3.216 0.775c 0.826 0.191f 0.223 0.039e 0.170 0.016d 1.000* 0.001a 0.802 0.094a 0.093 0.015f 0.054 0.015f
B1mP 15.492 4.950b 0.695 0.141f 0.166 0.112d 0.193 0.060d 11.941 1.135b 5.597 1.677c 0.358 0.001e 1.115 0.323a
B1mS 15.492 4.950b 0.695 0.141f 0.427 0.045f 0.036 0.024g 11.914 1.135b 5.597 1.677c 0.361 0.030e 0.045 0.018f


(b) Ectodermal markers
(i)


Marker NES NCAM
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.06 0.69a 6.77 0.13b 0.51 0.02a 0.29 0.02a 7.17 4.02a 20.54 1.53b 0.84 0.13a 0.96 0.02a
R1FS 1.06 0.69a 6.77 0.13b 3.54 0.63b 1.29 0.09a 7.17 4.02a 20.54 1.53b 35.15 5.36d 23.87 1.98b
R1mP 1.87 1.01a 5.50 0.69b 2.02 0.10a 1.76 0.02a 1.07 0.49a 7.84 4.48a 0.05 0.01c 0.06 0.01c
R1mS 1.87 1.01a 5.50 0.69b 3.61 0.09b 3.11 0.96b 1.07 0.49a 7.84 4.48a 11.41 0.10a 18.31 4.08b
B1FP 0.60 0.68a 4.90 0.25b 0.61 0.07a 0.68 0.09a 5.91 3.48a 16.97 2.55b 0.30 0.04a 0.77 0.05a
B1FS 0.60 0.68a 4.90 0.25 6.27 0.62b 7.41 0.00b 5.91 3.48a 16.97 2.55b 22.43 0.43b 45.78 20.77d
B1mP 0.45 0.07a 1.00* 0.00a 2.06 0.27a 1.83 0.31a 0.54 1.85a 1.00* 0.00a 0.21 0.01a 0.64 0.25a
B1mS 0.45 0.07 1.00* 0.00a 3.32 0.35b 2.11 0.30a 0.54 1.85a 1.00* 0.00a 4.46 0.50a 19.40 2.93b


(ii)


Marker SOX1 MSL1
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.10 0.06a 2.52 0.47c 1.28 0.08b 1.60 0.13b 0.93 0.16a 3.11 0.19c 0.92 0.03a 0.67 0.09a
R1FS 0.10 0.06a 2.52 0.47c 0.58 0.19b 0.71 0.14b 0.93 0.16a 3.11 0.19c 2.49 0.45c 1.14 0.10a
R1mP 0.00 0.00 16.70 5.50d 14.10 4.28d 8.07 1.85d 1.23 0.30a 2.29 0.11c 0.16 0.01b 0.05 0.00d
R1mS 0.00 0.00 16.70 5.50d 13.29 2.25d 4.51 0.44c 1.23 0.30a 2.29 0.11c 3.26 0.66c 3.71 0.18c
B1FP 0.00 0.00 1.00* 0.29b 1.70 0.20b 4.10 0.39c 1.27 0.29a 3.05 0.18c 1.00 0.11a 1.42 0.14a
B1FS 0.00 0.00 1.00* 0.29b 0.37 0.07a 1.20 0.27b 1.27 0.29a 30.5 0.18c 3.23 0.43c 4.59 0.20e
B1mP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.10a 1.00* 0.00a 0.44 0.01a 0.56 0.13a
B1mS 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.10a 1.00* 0.00a 1.45 0.25a 0.76 0.03a


(iii)


Marker NEFH GFAP
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.70 0.46a 1.00* 0.00a 2.14 0.17a 2.13 0.37a 0.06 0.01e 1.00* 0.00a 7.45 1.36a 36.69 2.92c
R1FS 0.70a 0.46a 1.00* 0.00a 1.40 0.56a 10.75 0.80b 0.06 0.01e 1.00* 0.00a 4.83 1.07a 4.52 0.49a
R1mP 1.13 0.78a 3.79 2.35a 0.62 0.10a 5.55 0.77a 4.33 2.65a 2.52 1.48a 12.46 0.85b 78.93 13.57d
R1mS 1.13 0.78a 3.79 23.5a 11.24 1.59b 18.82 1.34c 4.33 2.65a 2.52 1.48a 4.00 2.44a 3.98 0.43a
B1FP 2.65 1.71a 4.30 1.88a 1.71 0.13a 1.57 0.01a 2.25 1.16a 1.58 0.33a 2.00 0.76a 2.29 0.18a
B1FS 2.65 1.71a 4.30 1.88a 5.95 1.06a 8.97 1.95b 2.25 1.16a 1.58 0.33a 4.54 1.56a 5.53 2.54a
B1mP 1.23 0.86a 1.34 0.38a 1.09 0.32a 6.09 0.01a 0.00 0.00 0.00 0.00 2.43 0.45a 13.78 4.44b
B1mS 1.23 0.86a 1.34 0.38a 0.00 0.00 4.13 2.54a 0.00 0.00 0.00 0.00 3.74 0.25a 0.76 0.27a


(iv)


Marker TUBB3 KRT
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 3.10 1.38a 68.58 34.88a 5.46 0.04a 2.32 0.03a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1FS 3.10 1.38a 68.58 34.88a 69.69 9.57a 16.80 0.55a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mP 4.67 0.42a 9.70 5.52a 4.37 0.05a 5.13 0.20a 0.00 0.00 0.00 0.00 50.96 0.45b 26.70 1.73c
R1mS 4.67 0.42 9.70 5.52a 50.27 2.71a 20.11 3.76a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
B1FP 15.41 8.08a 26.88 13.86a 3.57 0.06a 4.87 0.80a 0.00 0.00 1.00* 0.00a 2.20 0.81a 3.35 1.30a
B1FS 15.41 8.08a 26.88 13.86a 62.40 3.43a 79.43 13.03a 0.00 0.00 1.00* 0.00a 2.45 0.98a 4.45 2.18a
B1mP 0.66 7.62a 1.00* 0.00a 5.24 0.32a 5.50 1.02a 0.00 0.00 0.00 0.00 34.01 0.39c 29.21 6.44c
B1mS 0.66 7.62a 1.00* 0.00a 19.67 1.14a 23.80 1.47a 0.00 0.00 0.00 0.00 18.70 9.98c 25.51 0.89c


(c) Mesodermal markers
(i)


Marker T GSC
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.22 0.08b 1.00* 0.00a 0.00 0.00 0.00 0.00 2.99 0.68a 1.00* 0.00a 3.61 1.34a 10.83 0.61c
R1FS 0.22 0.08b 1.00* 0.00a 0.00 0.00 0.00 0.00 2.99 0.68a 1.00* 0.00a 1.58 0.11a 4.45 0.17a
R1mP 1.00 0.55a 3.55 1.97a 0.00 0.00 0.00 0.00 3.51 1.05a 4.31 1.95a 1.04 0.10a 4.35 1.54a
R1mS 1.00 0.55a 3.55 1.97a 0.00 0.00 0.00 0.00 3.51 1.05a 4.31 1.95a 4.78 0.25a 20.50 9.39c
B1FP 0.23 0.15b 14.38 1.17d 0.46 0.16b 0.00 0.00 1.50 0.55a 10.51 3.63c 12.45 2.40c 60.53 2.76e
B1FS 0.23 0.15b 14.38 1.17b 0.37 0.02b 0.92 0.17a 1.50 0.55a 10.51 3.63c 7.95 0.43c 19.06 8.36c
B1mP 0.09 0.05c 66.40 19.97e 0.14 0.00b 0.73 0.04a 18.15 2.62c 41.81 3.51d 14.80 3.05c 39.02 6.61d
B1mS 0.09 0.05c 66.40 19.97e 3.09 1.96a 1.29 0.17a 18.15 2.62c 41.81 3.51d 34.11 5.55d 11.16 4.00c


(ii)


Marker GATA4 KDR
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 12.61 7.22b 1.00* 0.00a 191.44 11.02f 0.38 0.03a 20.94 5.10c 1.00* 0.00a 1.62 0.28a 2.41 0.51a
R1FS 12.61 7.22b 1.00* 0.00a 0.67 0.33a 0.60 0.16a 20.94 5.10c 1.00* 0.00a 2.87 0.13a 2.12 0.25a
R1mP 0.29 0.17a 43.55 3.53c 52.57 2.68c 35.80 1.45c 44.76 8.40d 27.65 8.21c 176.31 26.87e 260.18 19.58f
R1mS 0.29 0.17a 43.55 3.53c 43.38 19.18c 11.83 4.53b 44.76 8.40d 27.65 8.21c 12.39 1.54c 47.35 9.71d
B1FP 9.42 4.98b 344.72 33.14e 59.95 15.70c 49.62 1.11c 6.02 1.73b 71.12 20.00d 72.79 15.97d 47.35 18.63d
B1FS 9.42 4.98b 344.72 33.14e 409.25 17.21d 481.15 14.25g 6.02 1.73b 71.12 20.00d 198.43 12.75e 162.04 21.91e
B1mP 7.68 4.44b 84.70 18.16d 54.50 5.23c 96.41 12.41d 29.96 6.05c 240.34 59.83e 1062.64 102.47h 2282.98 136.58i
B1mS 7.68 4.44b 84.70 18.16d 1410.41 62.78i 837.81 67.99h 29.96 60.5c 240.34 59.83e 582.92 17.98g 304.37 1.91e


(iii)


Marker NKx2.5 MYH
Days D0 D10 D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 2.57 0.99a 2.75 0.64a 2.70 0.55a 3.02 0.33a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1FS 2.57 0.99a 2.75 0.64a 2.05 0.12a 0.79 0.19a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mP 0.67 0.12a 3.56 1.39a 1.45 0.95a 1.22 0.33a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mS 0.67 0.12a 3.56 1.39a 6.14 0.22a 8.82 0.25a 0.00 0.00 0.00 0.00 0.00 0.00 3.82 0.78a
B1FP 0.00 0.00 1.00* 0.00a 1.52 0.08a 0.94 0.06a 0.00 0.00 3.41 2.03a 0.12 0.05b 0.94 0.03a
B1FS 0.00 0.00 1.00* 0.00a 0.40 0.21a 0.71 0.03a 0.00 0.00 3.41 2.03a 0.91 0.12a 7.14 3.81a
B1mP 0.17 0.10a 4.32 1.92a 3.48 2.68a 4.00 0.10a 0.00 0.00 1.00* 0.00a 0.07 0.03b 0.70 0.00a
B1mS 0.17 0.10a 4.32 1.92a 3.29 0.40a 15.09 0.60b 0.00 0.00 1.00* 0.00a 2.59 0.97a 35.45 3.78c


(iv)


Marker ENG CD73
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 2.90 1.86a 1.00* 0.00a 3.18 0.47a 36.47 7.01b 2.29 1.33a 0.00 0.00 1.05 0.25a 2.34 0.83a
R1FS 2.90 1.86a 1.00* 0.00a 0.74 0.32a 1.00 0.26a 2.29 1.33a 0.00 0.00 0.37 0.11a 3.51 0.59a
R1mP 23.24 12.75b 1.11 0.65a 31.17 3.94b 571.66 9.26d 4.76 1.41a 1.00* 0.00a 128.85 34.56c 322.87 13.04e
R1mS 23.24 12.75b 1.11 0.65a 3.00 0.61a 3.83 1.68a 4.76 1.41a 1.00* 0.00a 3.79 0.02a 10.09 4.59a
B1FP 1.00 0.64a 16.30 2.95b 12.37 1.11b 8.82 0.53b 0.00 0.00 6.67 2.45a 3.30 0.20a 10.23 3.47a
B1FS 1.00 0.64a 16.30 2.95b 27.46 0.96b 26.37 5.32b 0.00 0.00 6.67 2.45a 10.36 2.06a 19.17 10.76a
B1mP 15.70 2.05b 10.01 2.19b 69.00 6.19b 227.20 32.05c 12.21 4.15a 8.69 5.75a 223.81 16.96d 352.00 13.42e
B1mS 15.70 2.05b 10.01 2.19b 66.76 22.89b 83.52 27.16b 12.21 4.15a 8.69 5.75a 41.68 1.40b 29.15 0.27a


(v)


Marker CD34 CD45
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.81 0.51a 1.00* 0.00a 0.00 0.00 0.00 0.00 0.00 0.00 1.00* 0.00a 6.88 0.18a 4.50 2.52a
R1FS 0.81 0.51a 1.00* 0.00a 0.42 0.22a 0.00 0.00 0.000 0.00 1.00* 0.00a 12.53 0.28a 8.02 1.15a
R1mP 8.31 4.50b 1.59 0.93a 69.37 14.07c 40.14 3.46c 0.90 0.55a 0.00 0.00 3.34 1.49a 3.97 0.88a
R1mS 8.31 4.50b 1.59 0.93a 0.00 0.00 0.00 0.00 0.90 0.55a 0.00 0.00 279.33 7.76e 637.23 16.81f
B1FP 0.19 0.11a 158.74 29.85c 70.58 8.91c 32.97 8.99c 0.45 0.26a 5.80 1.92a 27.95 1.35b 67.10 7.77c
B1FS 0.19 0.11a 158.74 29.85c 106.45 34.59c 60.95 0.28c 0.45 0.26a 5.80 1.92a 60.44 0.61c 155.61 5.58g
B1mP 19.51 5.78b 302.48 166.7c 405.68 57.18c 353.47 177.4c 0.46 0.27a 8.08 4.74a 43.07 0.20b 85.59 5.55d
B1mS 19.51 5.78b 302.48 166.7c 412.50 96.73c 228.80 70.47c 0.46 0.27a 8.08 4.74a 32.70 3.22b 8.50 1.31a


(vi)


Marker CD44 CD31
Days D0 D10* D20 D30 D0 D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 25.15 14.43a 2.37 1.33a 0.38 0.01a 0.83 0.02a 2.51 1.44a 0.00 0.00 0.12 0.12a 0.12 0.12a
R1FS 25.15 14.43a 2.37 1.33a 7.82 3.26a 10.22 1.98a 2.51 1.44a 0.00 0.00 0.12 0.12a 0.01 0.01a
R1mP 11.99 5.93a 1.00* 0.00a 11.15 0.31a 31.40 0.64a 0.00 0.00 1.00* 0.00a 47.56 11.26b 33.64 5.91b
R1mS 11.99 5.93a 1.00* 0.00a 3.73 0.36a 4.42 2.88a 0.00 0.00 1.00* 0.00a 0.00 0.00 0.00 0.00
B1FP 3.71 2.44a 3.48 1.90a 0.54 0.08a 0.43 0.13a 3.04 2.11a 168.38 87.7c 13.19 0.87a 6.57 2.39a
B1FS 3.71 2.44a 3.48 1.90a 19.29 3.25a 21.28 8.32a 3.04 2.11a 168.38 87.7c 448.41 8.22c 340.17 160.1c
B1mP 35.03 21.84a 1.15 0.07a 20.69 0.86a 64.51 11.18a 1.16 0.58a 77.14 40.68c 167.72 42.18c 171.54 63.48c
B1mS 35.03 21.84a 1.15 0.07a 83.43 32.9a 48.08 13.27a 1.16 0.58a 77.14 40.68c 175.80 43.02c 85.55 9.92c


(vii)


Marker PPARG FABP4
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.69 0.42a 1.00* 0.00a 0.89 0.08a 2.39 0.23a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1FS 0.69 0.42a 1.00* 0.00a 0.66 0.02a 1.30 0.08a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mP 1.58 0.50a 20.19 11.23a 26.02 1.39a 9.63 0.31a 0.00 0.00 0.00 0.00 3.67 1.28a 14.71 1.77b
R1mS 1.58 0.50a 20.19 11.23a 3.82 0.15a 1.15 0.21a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
B1FP 0.37 0.22a 7.72 0.77a 4.32 0.13a 5.88 1.16a 0.00 0.00 1.00* 0.00a 0.00 0.00 0.00 0.00
B1FS 0.37 0.22a 7.72 0.77a 7.65 1.39a 9.14 2.70a 0.00 0.00 1.00* 0.00a 8.08 0.51b 11.68 3.51b
B1mP 0.47 0.28a 20.70 10.54a 24.39 1.21a 80.09 8.90b 0.00 0.00 0.00 0.00 22.69 0.09c 52.51 5.39d
B1mS 0.47 0.28a 20.70 10.54a 17.86 1.04a 5.97 1.52a 0.00 0.00 0.00 0.00 2.99 0.29a 3.17 1.03a


(d) Endodermal markers
(i)


Marker AFP FOXA2
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.00 0.00 1.00* 0.00a 0.00 0.00 0.00 0.00 7.09 2.38b 1.00* 0.00a 0.00 0.00 0.00 0.00
R1FS 0.00 0.00 1.00* 0.00a 0.00 0.00 0.00 0.00 7.09 2.38b 1.00* 0.00a 1.46 0.56a 0.88 0.08a
R1mP 0.00 0.00 1.56 0.31a 0.54 0.03a 1.25 0.14a 1.71 1.04a 25.35 16.18b 0.00 0.00 0.00 0.00
R1mS 0.00 0.00 1.56 0.31a 1.53 0.53a 31.86 3.91b 1.71 1.04a 25.35 16.18b 154.20 65.13c 20.78 1.08b
B1FP 0.38 0.07a 152.69 7.31c 283.71 16.41d 210.53 0.00d 4.22 0.65b 202.34 18.11c 25.29 5.59b 33.31 0.64b
B1FS 0.38 0.07a 152.69 7.31c 334.08 15.51d 347.29 43.92d 4.22 0.65b 202.34 18.11c 270.39 18.93d 349.18 44.66d
B1mP 0.00 0.00 593.79 94.05d 2100.61 29.26e 2024.71 312.1e 1.26 0.86a 135.78 54.28c 12.67 3.15b 21.97 1.18b
B1mS 0.00 0.00 593.79 94.05d 3855.46 716.6f 1895.47 200.9e 1.26 0.86a 135.78 54.28c 1269.7 44.35f 554.47 24.12e


(ii)


Marker CXCR4 SOX17
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.26 0.13a 22.85 11.57b 2.10 0.14a 0.65 0.01a 9.59 4.11a 1.00* 0.00a 0.00 0.00 0.00 0.00
R1FS 0.26 0.13a 22.85 11.57b 18.71 3.17b 4.47 0.04b 9.59 4.11a 1.00* 0.00a 0.00 0.00 0.00 0.00
R1mP 0.34 0.16a 9.26 5.29b 0.27 0.01a 0.13 0.03a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mS 0.34 0.16a 9.26 5.29b 28.51 0.71b 15.80 6.44b 0.00 0.00 0.00 0.00 0.00 0.00 9.89 3.67a
B1FP 0.28 0.14a 13.34 6.31b 1.24 0.01a 1.33 0.15a 36.31 28.89a 783.34 45.02d 244.95 17.5c 827.28 92.07d
B1FS 0.28 0.14a 13.34 6.31b 15.45 0.87b 21.17 1.73b 36.31 28.89a 783.34 45.02d 253.68 24.20c 329.61 58.84c
B1mP 0.55 0.19a 1.00* 0.00a 1.07 0.01a 1.38 0.27a 1.00 0.88a 485.70 86.21c 80.18 10.21b 99.27 10.24b
B1mS 0.55 0.19a 1.00* 0.00a 11.65 2.48b 8.55 1.74b 1.00 0.88a 485.70 86.21c 292.38 36.80c 320.47 79.48c


(iii)


Marker SOX7 ALB
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 3.75 0.51b 1.00* 0.00a 1.48 0.49a 0.00 0.00 0.20 0.12a 3.17 0.75a 2.46 0.23a 1.21 0.15a
R1FS 3.75 0.51b 1.00* 0.00a 1.19 0.11a 0.46 0.00a 0.20 0.12a 3.17 0.75a 0.48 0.03a 0.28 0.04a
R1mP 7.11 3.34b 57.12 5.51d 215.77 49.5d 27.15 6.51c 0.66 0.39a 1.35 0.74a 0.14 0.01a 0.83 0.02a
R1mS 7.11 3.34b 57.12 5.51d 1.61 0.40a 0.00 0.00 0.66 0.39a 1.35 0.74a 4.68 1.42a 3.16 1.21a
B1FP 3.88 0.51b 22.18 4.35c 8.70 1.19b 7.31 3.66b 0.02 0.01a 11.77 3.13a 283.23 24.48d 203.94 14.02d
B1FS 3.88 0.51b 22.18 4.35c 11.22 1.09b 10.16 3.88b 0.02 0.01a 11.77 3.13a 99.69 13.48b 89.11 3.01b
B1mP 8.41 2.36b 141.86 83.89d 99.06 26.63d 99.78 43.53d 0.00 0.00 1.00* 0.00a 218.68 2.12d 341.95 18.34d
B1mS 8.41 2.36b 141.86 83.89d 38.63 7.27c 12.89 2.48b 0.00 0.00 1.00* 0.00a 456.54 17.35e 152.87 5.41c


(iv)


Marker PDX1 INS
Days D0 D10* D20 D30 D0 D10 D20* D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1FS 0.00 0.00 0.00 0.00 0.00 0.00 0.50 0.22a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mS 0.00 0.00 0.00 0.00 0.00 0.00 9.89 3.67a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
B1FP 0.00 0.00 1.00* 0.00a 7.60 2.27a 14.86 8.39a 0.00 0.00 0.00 0.00 0.00 0.00 6.26 1.95b
B1FS 0.00 0.00 1.00* 0.00a 1.61 0.50a 4.45 3.18a 0.00 0.00 0.00 0.00 1.00* 0.00a 2.64 0.40a
B1mP 0.00 0.00 0.00 0.00 51.00 32.42b 255.60 13.66d 0.00 0.00 0.00 0.00 0.00 0.00 212.09 60.20d
B1mS 0.00 0.00 0.00 0.00 176.85 22.46c 145.05 54.95c 0.00 0.00 0.00 0.00 0.00 0.00 94.45 43.69c


(v)


Marker CDX2 IFABP
Days D0 D10* D20 D30 D0 D10* D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.94 1.20a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1FS 1.94 1.20a 0.00 0.00 0.00 0.00 0.09 0.07a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mP 0.00 0.00 1.00* 0.00a 0.88 0.34a 0.27 0.07a 0.00 0.00 1.00* 0.00a 2.26 0.14a 0.73 0.16a
R1mS 0.00 0.00 1.00* 0.00a 0.00 0.00 2.75 0.88a 0.00 0.00 1.00* 0.00a 0.00 0.00 0.00 0.00
B1FP 0.00 0.00 19.58 7.55b 5.51 1.81b 7.97 2.69b 0.00 0.00 0.00 0.00 1.81 0.72a 20.92 5.29b
B1FS 0.00 0.00 19.58 7.55b 15.38 5.85b 22.35 12.27b 0.00 0.00 0.00 0.00 3.86 1.78a 47.96 0.66c
B1mP 0.00 0.00 12.70 7.86b 5.76 1.48b 8.39 0.01b 0.00 0.00 0.00 0.00 6.89 3.57a 35.70 3.49c
B1mS 0.00 0.00 12.70 7.86b 26.74 5.06b 22.43 7.89b 0.00 0.00 0.00 0.00 49.38 1.10c 98.79 4.32d


(e) Apoptotic markers
(i)


Marker BAX BCL2
Days D0* D10 D20 D30 D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.00* 0.00 0.59 0.09 0.37 0.01 0.45 0.07 2.35 0.39 4.83 1.04 4.46 0.60 5.83 1.25
R1FS 1.00* 0.00 0.59 0.09 0.40 0.01 0.35 0.05 2.35 0.39 4.83 1.04 6.66 0.31 5.41 1.91
R1mP 3.21 0.86 0.62 0.18 1.80 0.16 1.77 0.15 1.48 0.46 5.31 0.89 2.25 0.38 2.99 0.47
R1mS 3.21 0.86 0.62 0.18 0.30 0.01 0.31 0.04 1.48 0.46 5.31 0.89 5.33 0.41 5.32 0.18
B1FP 1.00 0.02 0.90 0.20 0.54 0.04 0.60 0.05 1.00* 0.00 7.07 1.89 6.05 1.14 15.20 0.95
B1FS 1.00 0.02 0.90 0.20 1.11 0.31 0.99 0.19 1.00* 0.00 7.07 1.89 11.28 2.81 26.73 16.95
B1mP 6.81 0.36 1.15 0.49 3.17 0.09 5.38 0.24 5.13 1.41 4.98 2.41 10.61 1.00 22.59 1.59
B1mS 6.81 0.36 1.15 0.49 1.44 0.11 0.71 0.09 5.13 1.41 4.98 2.41 15.37 2.16 8.80 2.88


(ii)


Marker CASP3
Days D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.00* 0.00 0.93 0.08 1.01 0.05 0.86 0.11
R1FS 1.00* 0.00 0.93 0.08 0.76 0.07 0.49 0.03
R1mP 9.35 3.58 0.77 0.18 1.34 0.10 2.73 0.11
R1mS 9.35 3.58 0.77 0.18 1.10 0.07 0.98 0.19
B1FP 1.27 0.14 1.21 0.12 1.14 0.07 1.20 0.12
B1FS 1.27 0.14 1.21 0.12 1.38 0.07 1.57 0.20
B1mP 15.42 4.81 1.78 0.96 4.12 0.15 7.90 0.98
B1mS 15.42 4.81 1.78 0.96 1.53 0.01 0.74 0.06


(f) Signalling molecules
(i)


Marker BMP4 FGF4
Days D0* D10 D20 D30 D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 3.46 0.26 0.31 0.06 0.25 0.05 1.09 0.10 1.00* 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1FS 3.46 0.26 0.31 0.06 0.40 0.01 1.19 0.27 1.00* 0.00 0.00 0.00 0.00 0.00 0.00 0.00
R1mP 3.95 0.87 6.55 3.62 29.62 2.22 35.57 2.50 6.49 2.77 0.00 0.00 0.00 0.00 0.00 0.00
R1mS 3.95 0.87 6.55 3.62 1.21 0.08 1.30 0.31 6.49 2.77 0.00 0.00 0.00 0.00 0.00 0.00
B1FP 1.00* 0.00 46.60 9.94 22.49 3.26 19.92 3.31 5.99 1.87 8.42 3.69 0.00 0.00 0.00 0.00
B1FS 1.00* 0.00 46.60 9.94 51.36 6.81 45.74 1.91 5.99 1.87 8.42 3.69 0.00 0.00 0.00 0.00
B1mP 12.64 1.34 65.47 29.05 171.59 3.62 396.86 48.75 18.46 13.49 8.10 4.81 0.00 0.00 0.00 0.00
B1mS 12.64 1.34 65.47 29.05 89.47 8.39 25.16 1.83 18.46 13.49 8.10 4.81 0.00 0.00 0.00 0.00


(ii)


Marker NODAL NOTCH
Days D0* D10 D20 D30 D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 1.00* 0.00 0.52 0.07 0.14 0.14 0.44 0.13 1.16 0.11 0.55 0.10 0.47 0.05 0.99 0.00
R1FS 1.00* 0.00 0.52 0.07 0.18 0.01 0.14 0.07 1.16 0.11 0.55 0.10 0.44 0.09 0.94 0.40
R1mP 43.87 24.50 0.59 0.22 1.00 0.35 1.48 0.27 1.53 0.27 1.02 0.30 3.66 0.52 2.19 0.05
R1mS 43.87 24.50 0.59 0.22 1.06 0.04 0.66 0.15 1.53 0.27 1.02 0.30 0.72 0.18 0.78 0.03
B1FP 7.80 1.64 7.12 0.93 1.35 0.12 1.23 0.05 1.00* 0.00 1.44 0.27 1.14 0.11 1.19 0.08
B1FS 7.80 1.64 7.12 0.93 1.11 0.30 1.21 0.08 1.00* 0.00 1.44 0.27 1.76 0.64 2.14 0.83
B1mP 101.62 44.58 26.46 14.56 8.27 1.06 8.91 1.48 2.70 0.73 2.43 1.08 6.45 0.26 8.40 1.71
B1mS 101.62 44.58 26.46 14.56 5.19 0.32 0.99 0.21 2.70 0.73 2.43 1.08 2.00 0.33 1.24 0.19


(iii)


Marker WNT
Days D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 5.86 2.74 72.28 37.27 3.51 0.86 1.92 0.56
R1FS 5.86 2.74 72.28 37.27 41.06 3.40 17.88 2.33
R1mP 2.31 0.58 23.18 13.31 0.03 0.02 0.18 0.02
R1mS 2.31 0.58 23.18 13.31 58.17 0.16 32.60 8.90
B1FP 1.00* 0.00 27.90 14.60 2.38 0.60 2.95 0.06
B1FS 1.00* 0.00 27.90 14.60 40.12 4.45 58.41 15.60
B1mP 6.86 3.06 29.21 5.33 12.85 0.22 13.73 0.07
B1mS 6.86 3.06 29.21 5.33 119.65 15.84 221.21 41.05


(g) Cell-cycle regulators
(i)


Marker CCND1 CDC2
Days D0* D10 D20 D30 D0* D10 D20 D30
Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M. Mean S.E.M.
R1FP 2.22 0.20 1.81 0.22 0.83 0.07 0.57 0.04 1.00* 0.00 1.20 0.22 0.26 0.00 0.06 0.01
R1FS 2.22 0.20 1.81 0.22 0.86 0.09 0.38 0.07 1.00* 0.00 1.20 0.22 0.48 0.03 0.08 0.02
R1mP 1.85 0.32 0.30 0.14 5.28 0.15 11.13 0.11 3.94 1.28 1.08 0.28 1.15 0.09 1.07 0.17
R1mS 1.85 0.32 0.30 0.14 0.76 0.06 0.56 0.08 3.94 1.28 1.08 0.28 0.46 0.01 0.26 0.03
B1FP 1.00* 0.00 2.08 0.35 1.10 0.12 1.67 0.32 1.21 0.13 2.17 0.41 0.82 0.12 1.18 0.12
B1FS 1.00* 0.00 2.08 0.35 1.87 0.24 2.45 0.75 1.21 0.13 2.17 0.41 1.84 0.22 2.28 0.50
B1mP 3.00 0.29 0.89 0.36 6.25 0.79 9.03 1.54 11.76 2.36 2.51 1.15 5.75 0.25 9.07 0.62
B1mS 3.00 0.29 0.89 0.36 1.70 0.06 0.65 0.02 11.76 2.36 2.51 1.15 3.74 0.03 1.91 0.13


Table S4 Comparison of gene expression in ReliCellhES1 and BG01 of various markers at day 30 time point

Gene expression of markers of three germinal layers; apoptosis and signalling are represented in terms of fold change at day 30. Note the clustering of high expression (++, +++ or ++++) of mesodermal and endodermal markers in BG01 as compared with ReliCellhES1. Results also suggest that ectodermal differentiation is quite similar in both the cell lines, indicating it to be the default pathway of hESC differentiation.

ReliCellhES1 BG01
Feeders mTeSR1 Feeders mTeSR1
Gene Plated Suspension Plated Suspension Plated Suspension Plated Suspension
NES ± + + + ± + + +
NCAM ± ++ ± ++ ± ++ ± ++
SOX1 + ± + + + + nd nd
MSL1 ± + ± + + + ± ±
NEFH + + + ++ + + + +
GFAP ++ ++ ++ + + + ++ ±
TUBB3 + ++ + ++ + ++ + ++
KRT nd nd ++ nd + + ++ ++
T nd nd nd nd nd ± ± ±
GSC ++ + + ++ ++ ++ ++ ++
KDR + + +++ ++ ++ +++ ++++ +++
GATA4 ± ± ++ ++ ++ +++ ++ +++
NKX2.5 + ± + + ± ± nd +
MYH nd nd nd + ± + ± ++
FABP4 nd nd ++ nd nd ++ ++ +
PPARG + + + + + + ++ +
CD44 ± ++ ++ + ± ++ ++ ++
CD34 nd nd ++ nd ++ ++ +++ +++
ENG ++ +++ + + ++ +++ ++
NT5E + + +++ ++ ++ ++ +++ ++
PTPRC + + + +++ ++ +++ ++ +
PECAM ± ± ++ nd + +++ +++ ++
AFP nd nd + ++ +++ +++ ++++ ++++
FOXA2 nd ± nd ++ ++ +++ ++ +++
CXCR4 ± + ± ++ + ++ + +
SOX17 nd nd nd ++ +++ +++ ++ +++
SOX7 nd ± ++ nd + ++ ++ ++
ALB + ± ± + +++ ++ +++ +++
PDX1 nd ± nd + ++ + +++ +++
INS nd nd nd nd + + +++ ++
CDX2 nd ± ± + + ++ + ++
FABP2 nd nd ± nd ++ ++ ++ ++
BMP4 + + ++ + ++ ++ +++ ++
FGF4 nd nd nd nd nd nd nd nd


Expression level 1 or less: ±; expression level >1-<10: +; expression level >10-<100: ++; expression level >100-<1000: +++; expression level >1000: ++++; not detected: nd

Table S5 Flow cytometric analysis of undifferentiated and three germ layers markers under different culture conditions showing percentage positive cells

The data is a representation of three independent experiments, depicted as means±S.E.M.

(a)


ReliCellhES1 BG01
Day 0 Day 10 Day 20 Day 30 Day 20 Day 30
Cells on feeders Plated Susp Plated Susp Day 0 Day 10 Plated Susp Plated Susp
Oct4 97.3±0.2 9.1±0.5 9.3±6.0 5.5±2.3 5.9±0.7 5.0±1.4 95.0±0.6 89.0±1.5 47.3±8.1 46.8±7.6 1.4±0.9 5.4±0.6
SSEA4 88.0±0.8 86.5±4.5 77.5±1.5 82.0±3 27.0±5 20.5±1.5 96.7±1.2 87.4±9.0 59.3±17 49.1±7.8 23.4±11 20.0±9.8
NCAM 16.7±2.0 94.4±1.9 94.9±1.5 83.3±4 51.9±1.6 71.6±12 10.2±1.8 90.9±5.5 83.9±5.5 68.8±10 41.9±2.4 69.3±3.5
NFH 20.3±0.8 92.9±2.4 63.4±14 30.4±7 82.6±0.5 26.8±1.4 18.3±2.4 89.7±2.5 88.7±3.4 82.3±15 82.8±4.4 25.1±11
CD44 16.7±3.7 11.3±1.4 28.8±5.5 7.3±5 82.3±3 7.1±0.2 15.2±1.7 5.4±2.5 16.7±2.5 13.5±0.8 16.9±1.4 1.9±1.3
CD73 1.0±0.2 15.0±5.2 25.2±6.0 17.7±3.5 33.8±10 25.3±6.1 0.8±0.3 15.8±3.5 33.6±4.3 15.8±3.4 35.6±1.8 8.7±5.5
MHC 0.9±0.2 2.2±0.5 3.3±0.3 0.8±0.4 7.0±3.5 5.0±0.4 1.8±0.3 7.9±0.8 17.3±2.0 27.2±6.0 21.0±1.4 32.7±1.5
SOX17 6.7±0.6 1.3±0.4 5.4±4.1 7.4±2.1 0.8±0.5 8.2±0.3 6.8±1.3 80.1±1.1 87.5±11 67.8±15 28.9±0.5 33.3±2.9
ALB 0.5±0.3 1.0±0.3 3.0±2.3 4.5±2.4 1.6±1.2 4.3±0.6 1.7±0.3 6.4±1.6 22.8±3.5 20.5±10 38.0±4.8 37.9±4.3


(b)


ReliCellhES1 BG01
Day 0 Day 10 Day 20 Day 30 Day 20 Day 30
Cells in mTeSR1 Plated Susp Plated Susp Day 0 Day 10 Plated Susp Plated Susp
Oct4 88.8±2.0 2.1±0.9 3.1±0.6 4.3±1.6 0.6±0.2 3.0±0.9 89.6±2.7 39.5±5.0 33.8±12 21.8±9.5 0.5±0.3 5.3±1.0
SSEA4 93.3±4.1 88.7±9.5 63.9±15 82.4±3.4 31.3±3.5 20.8±1.0 91.3±1.6 88.4±10 78.4±11 60.8±11 27.7±6.5 23.8±11
NCAM 5.2±2.5 71.9±4.7 65.8±7.5 73.3±14 38.4±6.1 64.9±4.9 2.3±0.1 36.3±9.9 17.8±7.6 53.2±20 15.0±2.5 60.0±2.1
NFH 1.7±0.4 17.7±1.5 20.8±8.5 25.2±6.0 76.7±9.4 11.7±0.8 2.8±1.3 2.0±1.2 34.8±21 40.2±4.0 83.7±7.4 13.8±3.6
CD44 1.9±0.4 16.5±1.3 33.7±5.7 8.6±4.2 40.8±1.4 11.0±1.2 1.8±0.4 6.4±1.9 17.8±5.4 11.7±7.5 40.2±1.0 0.7±0.2
CD73 1.0±0.2 9.8±2.1 21.6±6.8 13.3±0.9 31.2±1.4 9.4±1.5 2.7±1.3 1.5±0.8 10.8±8.5 3.0±1.3 29.3±8.1 27.3±1.9
MHC 0.8±0.5 2.6±1.1 1.0±0.3 2.0±0.3 2.8±0.4 1.2±0.3 0.3±0.1 1.6±0.9 15.8±3.5 27.7±3.5 20.4±1.0 15.0±2.4
SOX17 5.4±3.4 0.5±0.1 2.7±1.2 15.4±2.6 5.6±1.8 30.9±1.0 3.3±0.8 34.2±3.0 60.7±12 89.2±2.0 27.1±0.8 71.1±2.1
ALB 1.8±0.4 0.9±0.5 8.3±6.5 11.2±1.5 2.0±0.1 7.2±1.5 1.7±0.4 0.4±0.1 9.4±3.8 15.8±3.4 14.1±0.6 12.0±0.5


Published as Cell Biology International Immediate Publication 30 June 2010, doi:10.1042/CBI20100283

© The Author(s) Journal compilation © 2010 Portland Press Limited
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