Page1
Ex-vivo Expansion of Limbal Epithelium Optimized by Maintaining Native Niche Cells on Denuded
Amniotic Membrane
Supplementary Table S1: Materials for Cell Isolation and Culture
SHEM
MESCM
Material
Dulbecco’s modified Eagle’s Medium (DMEM)
F-12 nutrient Mixture (F12)
Fetal Bovine Serum (FBS)
Dimethyl Sulfoxide (DMSO)
Cholera Toxin
Hydrocortisone
Insulin-Transferrin-sodium selenite
media supplement (ITS)
Human Epithelial Growth factor (EGF)
Dulbecco’s modified Eagle’s Medium (DMEM)
Knockout serum
Insulin-Transferrin-sodium selenite media supp (ITS)
Supplier
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Sigma-Aldrich, St. Louis, MO
Sigma-Aldrich, St. Louis, MO
Sigma-Aldrich, St. Louis, MO
Roche, Indianapolis, IN
Human Fibroblast Growth Factor-Basic
Leukemia inhibitory factor (LIF)
Mitomycin C
Hank’s Balanced Salt Solutions (HBSS)
Phosphate-Buffered Saline pH7.4 (PBS)
Amphotericin B
Gentamicin
Dispase II
Collagenase A
Trypsin and EDTA (T/E)
Matrigel™ Basement Membrane Matrix
Qdot® nanocrystals (Qtracker)
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Roche, Indianapolis, IN
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Roche, Indianapolis, IN
Roche, Indianapolis, IN
Life Technologies, Carlsbad, CA
BD Biosciences, San Jose, CA
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Life Technologies, Carlsbad, CA
Roche, Indianapolis, IN
Concentration
DMEM/F12 (1:1)
5%
0.50%
1nM
5mg/ml
5mg/ml Insulin, 5mg/ml Transferrin
5ng/ml Sodium selenite
10ng/ml
DMEM/F12 (1:1)
10%
5mg/ml Insulin, 5mg/ml Transferrin
5ng/ml Sodium selenite
4ng/ml
10ng/ml
4mg/ml
1X
1X
1.25mg/ml
50mg/ml
10mg/ml
1mg/ml
0.25% and 1 mM
50%
10nM
Page2
Ex-vivo Expansion of Limbal Epithelium Optimized by Maintaining Native Niche Cells on Denuded
Amniotic Membrane
Supplementary Table S2: Primary and Secondary Antibodies used for Immunofluorescence staining
Primary Antibodies
Antibody
Supplier
Source
Dilution
ABCG2
Bmi-1
CD31
CD34
CXCR4
FLK-1
R&D System
EMD Millipore
Cell Signaling
Abcam
Abcam
Cell Signaling
Mouse
Mouse
Mouse
rat
Rabbit
Rabbit
1 : 100
1 : 100
1 : 100
1:100
1 : 100
1 : 100
Keratin 15
Covance
Chicken
1 : 100
Msi-1
Abcam
Rabbit
1 : 100
Pan-cytokeratins AE1/AE3
Novus
Rabbit
1 : 50
Dako
Mouse
1 : 50
Nanog
Abcam
Rabbit
1:100
p63α
Abcam
Rabbit
1:100
PDGFR-β
Abcam
Rabbit
1:100
Rex1
Abcam
Rabbit
1:100
SDF-1
R&D
Mouse
1 : 100
Vimenin
Abcam
Rabbit
1 : 100
Abcam
Goat
1 : 100
Sigma
-
1 : 500
Hoechst 33342
Antibody
Secondary Antibodies
Supplier
Source
Dilution
Alexa Fluor 488 Anti-Goat
Invitrogen
Donkey
1 : 100
Alexa Fluor 488 Anti-rat
Invitrogen
Donkey
1 : 100
Alexa Fluor 633 Anti-Goat
Invitrogen
Donkey
1 : 100
Alexa Fluor 488 Anti-Mouse
Invitrogen
Donkey
1 : 100
Alexa Fluor 555 Anti-Rabbit
Invitrogen
Donkey
1 : 100
Alexa Fluor® 633 Anti-Chicken
Invitrogen
Goat
1 : 100
Page3
Ex-vivo Expansion of Limbal Epithelium Optimized by Maintaining Native Niche Cells on Denuded
Amniotic Membrane
Supplementary Table S3: Assay ID and Probes Sequence Use for Real-time PCR
Gene
Assay Id (TaqMan® Gene Expression
Uni Gene
Product length
93
from Applied Biosystems)
GAPDH
Hs02758991_g1
Hs.544577
ABCG2
Hs01053796_m1
Hs.480218
96
aSMA
Hs00426835_g1
Hs.500483
105
Bmi-1
Hs00180411_m1
Hs.380403
105
CD31
Hs00169777_m1
Hs.376675
65
CD34
Hs00990734_g1
Hs.374990
101
CEBPd
Hs00270931_s1
Hs.440829
107
CXCR4
Hs00607978_s1
Hs.593413
153
E-cadherin
Hs00170423_m1
Hs.461086
117
FLK-1
Hs00911700_m1
Hs.479756
83
K15
Hs00267035_m1
Hs.654570
81
Musashi-1
Hs00159291_m1
Hs.158311
92
Nanog
Hs02387400_g1
Hs.635882
109
N-cadherin
Hs00354988_m1
Hs.464829
74
Nestin
Hs00707120_s1
Hs.527971
81
NG-2
Hs00361541_g1
Hs.513044
60
OCT 4
Hs00999634_gH
Hs.249184
64
PDGFR-b
Hs01019589_m1
Hs.509067
62
Rex-1
Hs00381890_m1
Hs.192477
109
Sox-2
Hs01053049_s1
Hs.518438
91
Vimentin
Hs00185584_m1
Hs.455493
73
SDF-1
Forward: GGCCTCTGTGGCACTCA
80
Reverse: GCCAATCACTGAGGTTTGAAAGAG
∆Np63α
Forward: GGCTGTTCATCATGTCTGGACTATT
Reverse: CATCCATGGAGTAATGCTCAATCTGA
78