Supplementary Information Escobedo-Lucea et al.
Development of a Human Extracellular Matrix for Applications
Related with Stem Cells and Tissue Engineering
Escobedo-Lucea C, Ayuso-Sacido A, Xiong C, Prado-López S, Sanchez del
Pino M, Melguizo D, Bellver-Estellés C, Gonzalez –Granero S, Valero L,
Moreno R, Burks DJ, and Stojkovic M.
Supplementary Figure 1. Bioinformatics classification of the hffECM protein
components: Babelomics analysis of gene ontology cellular compartment and KEGG.
Diagram represents the cell components enriched in the eluted hffECM. Using the list of
proteins identified by proteomic analysis, functional categories were assigned using
bioinformatic tools (Babelomics) and enriched components were identified using GO. Note that
the three groups of enriched proteins reflect components of the extracellular matrix, basal part
of the cell, or the cell surface.1
Supplementary Figure 2. hESC cultured on hffECM retain markers of the undifferentiated
state.
Analysis of pluripotent markers (Tra1-81 and SSEA-4) in H9 colonies maintained during 21
passages on either human foreskin fibroblasts (a2 and a4) or after 21 passages on hffECM
(see fluorescence for Tra1-81 (b2) and SSEA-4 (b4). Nuclei were stained with DAPI (blue) and
Alexa 488 secondary antibodies (green) were used to detect Tra 1-81 or SSEA-4. Cultures
maintained on hffECM were also stained for fibronectin and revealed using Alexa 647 (far red).
A1, a3, b1 and b3 are brightfield images. All images were captured at 10X magnification with a
Zeiss Axiovert 200M fluorescence microscope.
Supplementary Figure 3. hffECM supports differentiation of hESC cells.
(a and b) Fluorescent images of hESC which were allowed to spontaneously differentiate on
hffECM. Neuronal differentiation was suggested by detection of Tuj-1 positive cells in a.
Differentiation to cardiac cells was suggested by detection of cardiac actin in b. Scale bars
100µm. (c) Directed differentiation of H9 plated on hffECM to endoderm precursors. Phase
contrast image of hESC glandular structures cultured over hffECM, surrounded by fibroblast like
cells, formed 3 weeks after the triggering of endoderm differentiation. (d) Fluorescence
immunocytochemistry of differentiated induced cells co-expressing SOX-17(in green) and
CXCR4 (in red). (e) Electron microscopy of endoderm differentiated cells, e-1.Immature vesicles
with undefined content (white arrows) and hemidesmosomes (black arrows).e-2 Detail of
secretory canaliculi structures (white asterisk). (f) RT-PCR of endoderm markers comparing
hffECM
differentiated
versus
hffECM
control
cells
along
the
differentiation
stages:
mesendoderm, definitive endoderm and secreting cells.β-2 microglobulin expression was used
as a positive control for cDNA templates. The samples were assessed using bioanalizer Agilent.
Scale bars, 100 μm (a, b and c), 47.62 μm (d), 1μm (e).
Supplementary videos 1, 2 and 3. Migration pattern of U87 cells maintained over plastic,
matrigel and hECM.
Video images of the movement of U87 cells plated on hECM, Matrigel or plastic.
Supplementary Methods
Escobedo-Lucea et al. Nature Methods.
Assesment of spontaneous differentiation of hESC over hECM by immunocytochemistry.
Spontaneous in vitro differentiation was achieved by allowing hESC to overgrow on extracellular
matrix in chamber slides in the corresponding growth media which lacked basic fibroblast
growth factor bFGF (Invitrogen). After two weeks in culture, cells were fixed with 4%
paraformaldehyde and permeabilized with Triton X-100. Cultures were stained two hours hour
at room temperature for human -tubulin isotype III (SDL.3D10) (Sigma) or muscle actin
(HHF35) (Dako), respectively. Detection with secondary antibodies was with Alexa fluor 488
goat anti-mouse IgG2b (A-21141) and Alexa fluor 555 goat anti mouse IgG (A-21424, Molecular
Probes). Chamber slides were mounted and counterstained using Prolong antifade reagent
containing DAPI (Molecular Probes).
Directed differentiation towards endoderm over hECM sustract.
For directed pancreatic differentiation, we used a modification of the D´Amour protocol, whereby
different growth factors were added to low serum culture media
2,3.
For immunocytochemistry,
cells were stained prior to fixation with CXCR4-PE conjugated (551510) (BD-Pharmingen)
during 1h at room temperature. Subsequently, cells were fixed in 4% paraformaldehyde and
permeabilized with Triton X-100 before incubation o.n. with human Sox-17(Millipore).
Secondary detection was performed with goat anti-rabbit IgG, FITC conjugate (AP187F)
(Millipore).
Before fixation for Transmission Electron Microscopy (TEM), differentiated cultures as well as
the corresponding controls, were repeatedly washed in a 0,1M phosphate buffer (PB; pH 7.4)
solution. Fixation was performed in 3% glutaraldehyde solution in PB for 30 minutes at 37ºC
and postfixed in 2% OsO4 in PBS. Samples were stained with 2% uranyl acetate (Electron
Mycroscopy Science, Washington). Dehydration was achieved by a graded series of ethanol
and samples were embedded in propylene oxide (Lab Baker, Deventry, Holland). Finally, plates
were infiltrated in araldite (Durkupan, Fluka) overnight. Following polymerization, embedded
cells were detached from the chamber slide and glued to Araldite blocks. Serial semi-thin (1.5
µm) sections were cut with an Ultracut UC-6 (Leica, Heidelberg, Germany) and mounted onto
slides and stained with 1% toluidine blue. Selected semi-thin sections were glued (Super Glue,
Loctite) to araldite blocks and detached from the glass slide by repeated freezing (in liquid
nitrogen) and thawing. Ultrathin (0.07 µm) sections were prepared with the Ultracut and stained
with lead citrate. Finally, photomicrographs were obtained under a transmission electron
microscope (FEI Tecnai Spirit G2) using a digital camera (Morada, Soft Imaging System,
Olympus).
For reverse transcription and PCR, total RNA was extracted using RNeasy kit (Quiagen) and
digested with deoxyribonuclease (DNase) I (Quiagen). cDNA was generated with 1µg of total
RNA using high capacity cDNA reverse transcription kit (Applied Biosistems). RT–PCR and
primer sequences are described in the Supplementary Table S1. Results were visualized using
Agilent 2100 Bioanalyzer (Agilent Technologies).
SUPPLEMENTARY TABLES
Supplementary Table 1. Proteins of the enriched cellular component terms of gene ontology.
FAMILY (name)
ID
PROCESS (UniprotKB)
SCORE PEPTIDES %Cov
1.ECM (Go:0031012) p-value 2.66E-11
P02751
Fibronectin
Basement membrane-specific HS proteoglycan P98160
P24821
Tenascin
P35555
Fibrillin-1
Collagen alpha-3(VI) chain
Fibrillin-2
P12111
P35556
Involved in cell adhesion, cell motility, opsonization,
w ound healing, and maintenance of cell shape
Integral component of basement membranes
Extracellular matrix protein implicated in guidance of
migrating neurons as w ell as axons during
development, synaptic plasticity as w ell as
neuronal regeneration
Provide force bearing structural support in
connective tissue
Bind ECM proteins, organizing matrix components
Elastic fiber assembly
Annexin A2
P07355
TGF-beta-induced protein ig-h3
Q15582
Collagen alpha-2(I) chain
Fibulin-2
P08123
Blood vessel development ,collagen biosynthetic
process ,collagen fibril organization, embryonic
development ,skin morphogenesis
Positive regulation of vesicle fusion, skeletal system
development
This adhesion protein may play an important role in
cell-collagen interactions
Type I collagen is a member of group I collagen
(fibrillar forming collagen).
P98095
Calcium binding.Interacts w ith laminin2 (LAMA2).
P02452
Collagen alpha-1(I)
P27797
Calreticulin
P09382
Galectin-1
Collagen alpha-1(VI) chain
Elastin
Alpha-2-HS-glycoprotein
Collagen alpha-2(VI) chain
EMILIN-1
Fibulin-1
P12109
P15502
P02765
P12110
Q9Y6C2
P23142
Thrombospondin-1
Serotransferrin
Galectin-3
Alpha-1-antitrypsin
Vitronectin
Coiled-coil domain-containing protein 80
Metalloproteinase inhibitor 3
Lumican
P07996
Molecular calcium-binding chaperone promoting
folding, oligomeric assembly and quality control in
the ER via the calreticulin/calnexin cycle
May regulate apoptosis, cell proliferation and cell
differentiation.Interacts w ith laminin (via poly-Nacetyllactosamine).
Cell adhesion
Component of elastic fibers
Tissue development
Cell adhesion
Development of elastic tissues
Binding to laminin and nidogen
Mediates cell-to-cell and cell-to-matrix interactions
(fibronectin, laminin)
P02787 Iron transport
P17931 Cell adhesion apoptosis
P01009 Serin protease inhibitor
P04004 Cell adhesion and spreading promoter
Q76M96 Interacts w ith protein NS5A.
P35625 Inhibitor of ECM degradation
P51884 Control migration, grow th and tissue repair
214,33
114,26
107
57
66,3
34,4
69,78
35
42,1
59,94
53,36
47,86
30
27
24
25,5
36,6
24,42
32,74
16
57,7
27,47
14
87,6
23,16
12
34,5
19,47
18
9
9
49,2
18
15,37
7
43,1
14
11,39
10
8,98
6,61
6,56
5,44
7
6
5
5
4
4
3
74,8
24,6
57,1
29,9
28,4
16,3
16,3
4,92
7,18
4,62
3,81
2,56
2,17
2
2
3
4
3
2
2
2
1
1
12,9
20,9
22,8
17,4
17,6
17,8
11,37
5,32
24,45
13
47,11
21,07
20,87
11
11
42,12
40,25
19,73
10
36,8
15,37
10,8
7
6
43,1
37,9
9,06
6,05
5
3
64,6
30,03
4,93
3
26,8
4,92
3
12,9
2,16
2,05
2
2
29,35
22,66
2.CELL SURFACE (Go: 0009986) p-value 1.09634E-4
60 kDa heat shock protein, mitochondrial
P10809
Protein disulfide-isomerase
ATP synthase subunit beta, mitochondrial
P07237
Mitochondrial protein import and macromolecular
assembly
Multifunctional protein.catalyzes the formation,
breakage and rearrangement of disulfide bonds
P06576
Mitochondrial membrane ATP synthase
78 kDa glucose-regulated protein
P11021
Calreticulin
Heat shock cognate 71 kDa protein
P27797
Probably plays a role in facilitating the assembly of
multimeric protein complexes inside the ER
Molecular calcium-binding chaperone promoting
folding, oligomeric assembly and quality control in
the ER via the calreticulin/calnexin cycle
P11142
Chaperone
Caveolin-1
Stress-70 protein, mitochondrial
Q03135
May act as a scaffolding protein w ithin caveolar
membranes
P38646
Stress response
Heat shock protein beta-1
P04792
Involved in stress resistance and actin organization
Thrombospondin-1
P07996
Gremlin-1
Heterogeneous nuclear ribonucleoprotein U
O60565
Mediates cell-to-cell and cell-to-matrix interactions
(fibronectin, laminin)
Cytokine that may play an important role during
carcinogenesis and organogenesis
Q00839
Promotes MYC mRNA stabilization
Supplementary Table 2. PCR primers and conditions.
Gene
B-2Microglobulin
GAPDH
18s rRNA
NANOG
OCT-3/4
FGF-4
TERT
AFP
DBH
CAC
LPL
ADIPOQ
FABP4
PPARgamma
PLIN
SREBF1
BRACHYURY
WNT3
FGF4
FOXA2
Primer sequencies
HOUSEKEEPING GENES
F:CTCGCGCTACTCTCTCTTTCTG
R:GCTTACATGTCTCGATCCCACT
F: GAGTCAACGGATTTGGTCGT
R: TTGATTTTGGAGGGATCTCG
F: GGAAGGGCACCACCAGGAGT
R: TGCAGCCCCGGACATCTAAG
UNDIFFERENTIATION ASSAY GENES
F: AATGGTGTGACGCAGAAGG
R: ACTGGATGTTCTGGGTCTGG
F:CCTGTCTCCGTCACCACTCT
R:CAAAAACCCTGGCACAAACT
F:GGCGTGGTGAGCATCTTC
R:CGTAGGCGTTGTAGTTGTTGG
F: GCGTTTGGTGGATGATTTCT
R: AGCTGGAGTAGTCGCTCTGC
F:ACACAAAAAGCCCACTCCAG
R:GGTGCATACAGGAAGGGATG
F:TGACTGGGAGAAAGGTGGTC
R:TACGTGCAGGAGGTGATGAG
F:TGCTGATCGTATGCAGAAGG
R:GCTGGAAGGTGGACAGAGAG
DIFFERENTIATION ASSAY GENES
F:CAAAGCCCTGCTCGTGCTGA
R:CAGCCAGTCCACCACAATGA
F:TCCTTACAGAACACGCTTTCA
R:AGGGCCACAGAACGAGAG
F: ATTGGGATGGAAAATCAACCA
R:GTGGAAGTGACGCCTTTCAT
F:TCAGCGGGAAGGACTTTATGTATG
R:TCAGGTTTGGGCGGATGC
F:GCAGTCAACAAAGGCCTCAC
R:AAGCTACTGGCGCTCTGCAC
F:GGAGCCATGGATTGCACTTTC
R:ATCCTTCAATGGAGTGGGTGCAG
F:ACCCAGTTCATAGCGGTGAC
R:ATGAGGATTTGCAGGTGGAC
F:TGTGAGGTGAAGACCTGCTG
R: AAAGTTGGGGGAGTTCTCGT
F:GAAGACCAAGAAGGGGAACC
R:AAAAACACACCGCAGAACT
F:CTACGCCAACATGAACTCCA
R:CGGTAGAAGGGGAAGAGGTC
Annealing
Temperature
55ºC
55ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
60ºC
Size
Detection
335 pb
RT-PCR
QRT-PCR
RT-PCR
QRT-PCR
238 pb
157pb
QRT-PCR
128 pb
139 pb
QRT-PCR
QRT-PCR
254 pb
QRT-PCR
147pb
QRT-PCR
160 pb
QRT-PCR
135 pb
QRT-PCR
334 pb
RT-PCR
501 pb
RT-PCR
88 pb
RT-PCR
147 pb
RT-PCR
200 pb
RT-PCR
261 pb
RT-PCR
216 pb
RT-PCR
207 pb
RT-PCR
250 pb
RT-PCR
213 pb
RT-PCR
60ºC
60ºC
INS
PROINS
F:AGCCTTTGTGAACCAACACC
R:GCTGGTAGAGGGAGCAGATG
F:CTGCATCAGAAGAGGCCATCAAGC
R:GGCTTTATTCCATCTCTCTCGGTGC
60ºC
60ºC
157 pb
RT-PCR
464 pb
RT-PCR
Supplementary references
1.
2.
3.
Al-Shahrour, F. et al. BABELOMICS: a systems biology perspective in the functional annotation of genome-scale experiments. Nucleic
Acids Res 34, W472-476 (2006).
D'Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23, 1534-1541
(2005).
D'Amour, K. A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol
24, 1392-1401 (2006).