Supplementary information for:
Drug evaluation in cardiomyocytes derived from human induced pluripotent stem
cells carrying a Long QT Syndrome type 2 mutation
Elena Matsa1, Divya Rajamohan1, Emily Dick1, Lorraine Young1, Ian Mellor2, Andrew Staniforth3,
Chris Denning1.
1
Wolfson Centre for Stem Cells, Tissue Engineering & Modelling (STEM),
Nottingham, University Park, Nottingham, NG7 2RD.
3
2
School of Biology, University of
Department of Cardiovascular Medicine, Queen’s
Medical Centre, Nottingham, NG7 2UH.
1
A
B
C
Supplementary figure 1. Electrocardiogram traces for the LQT2 family. Full 12-lead
electrocardiograms (ECGs) for A) the LQT2 female patient, as well as B) the patient’s mother and
C) father.
2
A
ii.
iii.
v.
vi.
vii.
MAT
HF
i.
B
C
Thr
Cumulative PD
Ala
L
E
L
E
L
MAT-hiPSC
EBs
E
E
E
E
100bp
ladder
L
LQT2-hiPSC
MAT-hiPSC
5
10
15
20
Days in Culture
MAT-hiPSC
iPS
L
HF-iPSC
L
hESC
G
HF-hiPSC
A
G
A
LQT2
G
PCR -
MAT-hiPSC
A
G
A
G
A
LIN28
SOX2
NANOG
OCT4
E
MAT-Fib
HF
HF-iPSC
D
hESCs
0
MAT-hiPSC
HF-ihPSC
EBs
HF-iPSC
20
18
16
14
12
10
8
6
4
2
0
Supplementary figure 2. Characterisation of control pluripotent stem cells. A) Monolayer
cultures of hESC-derived fibroblasts (HF; Ai) and MAT-Fib (Aiv). hiPSC colonies generated from
HF (Aii) and MAT-Fib (Av) following lentiviral transduction with human OCT4, NANOG, SOX2 and
LIN28. Monolayer cultures of HF-hiPSCs (Aiii) and MAT-hiPSCs (Avi) at passage 15, grown in
feeder-free conditions on Matrigel. Scale bars represent 100µm. B) Genomic sequencing in HFhiPSCs and MAT-hiPSCs confirming absence and presence of the KCNH2 G1681A mutation,
respectively. C) Growth kinetics curves for monolayer cultures of hESCs, HF-hiPSC, MAT-hiPSC
and LQT2-hiPSC lines. D) RT-PCR analysis of OCT4, NANOG, SOX2 and LIN28 expression from
the endogenous (E) and lentiviral (L) loci in HF and MAT fibroblasts, hiPSCs and EBs. E) RT-PCR
analysis in EBs derived from hESCs, HF-hiPSCs, MAT-hiPSCs and LQT2-hiPSCs, showing that the
MAT and LQT2 samples express both the wild-type (G) and mutant (A; red arrows) KCNH2 1681
alleles.
3
KLF4
cMYC
REX1
DNMT3B
LIN28
SOX2
NANOG
OCT4
P4HB
A.
Cy3
DAPI MERGE
B.
Cy3
DAPI MERGE
4
Supplementary figure 3. Expression of fibroblast and pluripotency markers.
Immunofluorescence staining shows LQT2-Fib express the fibroblast-specific marker P4HB (A),
while (B) shows LQT2-hiPSCs express the pluripotency markers OCT4, NANOG, SOX2, LIN28,
DNMT3B and REX1. Both cell types express c-MYC and KLF4. Scale bars represent 65µm. The
same profile was seen for HF and MAT fibroblasts and hiPSCs (data not shown).
5
Supplementary Table 1.
Antibodies used for immunofluorescence staining and flow cytometry
Primary
antibody
Supplier
OCT4
SSEA4
SSEA1
TRA-1-81
P4HB
c-MYC
α-actinin
β-III-tubulin
AFP
LIN28
REX1
Troponin-I
SOX2
NANOG
DNMT3B
KLF4
Santa Cruz; SC-5279
Millipore; MAB4304
Millipore; MAB117P
Millipore; MAB4381
Abcam; ab44971
Abcam; ab32
SIGMA; A7811
COVANCE; MMS-435P
Sigma; A8452
Abcam; ab462020
Abcam; ab50828
Spectral Diagnostics; PA-1010
R&D Systems; AF2018
R&D Systems; AF1997
Santa Cruz; SC-10236
R&D Systems; AF3640
Dilution
1:200
1:50
1:50
1:50
1:50
1:250
1:800
1:1000
1:1000
1:1500
1:50
1:1000
1:200
1:50
1:250
1:100
Secondary
antibody
Supplier
Dilution
goat anti-mouse
AlexaFluor® 633
Invitrogen;
A21052
1:400
goat anti-rabbit
AlexaFluor® 633
Invitrogen;
A21071
rabbit anti-goat
AlexaFluor® 568
Invitrogen;
A11079
1:400
1:200
1:400
1:200
6
Supplementary Methods
PATIENT CONSENT
All subjects gave informed consent for blood testing for genetic abnormalities associated
with hereditary LQTS. Isolation and use of patient fibroblasts was approved by the Nottingham
Research Ethics Committee (License 09/H0408/74), and sample collections are registered with the
UK Clinical Research Network under project 8164; “Modelling Long QT Syndrome”.
CULTURE OF PATIENT-SPECIFIC FIBROBLASTS
Dermal fibroblasts from the LQT2 patient and her mother were prepared from 4mm skin
punch biopsies, after informed consent was given by the patients. Skin samples were cleaned from
adipose and epidermal tissue and were minced to 1mm pieces. They were then digested with 2.5%
trypsin (Invitrogen) for 15min, and 1mg/ml Collegenase IV (Invitrogen) for 90min, both at 37 oC.
Digested cells were plated in Chang’s-D medium (Metachem) supplemented with 2mM L-glutamine
(Invitrogen) and 100mM HEPES (SIGMA), and were grown for 2-3 weeks until fibroblast populations
emerged. Fibroblasts were grown in DMEM medium (Invitrogen), supplemented with 10% FBS,
0.1mM non-essential amino acids (Invitrogen) and 2mM L-glutamine.
GENERATION OF PATIENT-SPECIFIC hiPSCS
Fibroblast cells were seeded onto tissue culture-treated 6 well plates (NUNC) at a density of
100,000 cells/well and left to adhere to the plastic for 4h. Cells were infected with lentiviral particles,
prepared as described in [1]. Briefly, lentiviral particles encoding human OCT4, SOX2, LIN28 and
NANOG [2] were produced using the HEK293T cell line, BL15, modified to express a biotin acceptor
peptide domain on its cell surface [3, 4]. Viral supernatants were precipitated via incubation in
60mM CaCl2 at 37 °C for 30-40min and then purified by conjugation to streptavidin paramagnetic
beads (Promega), for 3-5h at room temperature on a rolling platform. Viral particles were finally
resuspended in DMEM with 10% FBS, and were used to transduce fibroblasts at an MOI of 5 for
each virus. A medium change with fibroblast culture medium was performed 24h post transduction
7
(PTD), and 48h PTD the cells were transferred to 90mm dishes where they were cultured onto
mitomycin C inactivated MEFs (3.6x106 cells/plate) in hiPSC culture medium (DMEM-F12
supplemented with 15% KSR, 100µM β-mercaptoethanol, 10ng/ml bFGF (Sigma-Aldrich), 2mM
GlutaMAX™-1 (Invitrogen), and 0.1mM non-essential amino acids). The medium was changed daily
with hiPSC culture medium for 1 week, and with MEF conditioned medium [5] supplemented with 10
ng/ml bFGF thereafter. At 18-23 days PTD, reprogrammed hiPSCs were isolated by TRA-1-81
based positive selection over a magnetic column (Miltenyi Biotec), as described previously [1].
hiPSCs were then transferred to feeder-free conditions on Matrigel-coated flasks, as described
previously [6] and cultured in MEF conditioned medium supplemented with 10ng/ml bFGF. Two
independent hiPSC lines were derived from each type of fibroblast sample.
GROWTH AND MAINTENANCE OF hESCS
The hESC line HUES7 was maintained under feeder-free conditions on Matrigel-coated
flasks, and cultured in MEF conditioned medium supplemented with 4ng/ml bFGF, as previously
described [6].
DETERMINATION OF POPULATION DOUBLING TIME
Population doublings (PDs) at each passage were calculated using the formula [log10 (total
cell counts/cells seeded)/ log10 (2)], where the cell number seeded was 1.2x106 cells/t25 flask.
Proliferation rates were calculated by total hours in test culture/ cumulative PDs.
DETERMINATION OF KARYOTYPE STABILITY
Metaphase spreads were prepared as previously described [5, 7] from hiPSCs at passage 15, and
karyotype analysis was performed by G-banding of 30 metaphase spreads in each sample,
according to guidelines from the International System for Human Cytogenetic Nomenclature (ISCN,
2005).
DETECTION OF PLURIPOTENCY MARKER EXPRESSION
8
For flow cytometry analysis, cells were harvested and fixed in 4% paraformaldehyde (PFA,
Sigma-Aldrich), then resuspended in 2% FBS in PBS. Cells were incubated in the dark with PEconjugated antibodies, or relevant isotype controls, for 20min at room temperature, washed in 2%
FBS and analysed in a flow cytometer (FC500, BD Biociences).
For immunofluorescence, cells were grown to confluence in 8-chamber glass slides (NUNC)
and then fixed in 4% PFA. Cells were permeabilised with 0.1% Triton-X-100 (Sigma-Aldrich), and
blocked with 8% goat or rabbit serum (Sigma-Aldrich) in PBS, as appropriate. Primary antibody
incubations were performed overnight at 4°C and secondary antibodies at room temperature for 1h
(antibody dilutions shown in supplementary table 1). Cells were imaged using a Nikon Eclipse 90i
fluorescence microscope and images were analysed using Volovity software (Improvision).
BISULPHITE SEQUENCING OF GENOMIC DNA
Cell pellets from fibroblasts (passage 6), reprogrammed hiPSC lines (passage 15-17) and
their differentiated progeny (day 25) were analysed to determine the methylation status of key
regulatory regions in the endogenous OCT4 and NANOG promoters. Genomic DNA was extracted
from frozen cell pellets (QIAGEN DNeasy Blood and Tissue kit) and bisulfite conversion of genomic
DNA, PCR and DNA methylation analysis were performed as described previously [1].
ENDOGENOUS AND LENTIVIRAL EXPRESSION OF OCT4, NANOG, SOX2 AND LIN28
Reactivation of endogenous OCT4, NANOG, SOX2 and LIN28, and silencing of lentiviral
transgenes in hiPSC lines and their differentiated progeny was performed by RT-PCR, as described
previously [1].
IN VITRO DIFFERENTIATION TO THE THREE EMBRYONIC GERM LAYERS
To determine whether hiPSC lines could generate derivatives of the three embryonic germ
layers in vitro, cells were differentiated via embryoid body (EB) formation as described above. On
day 25 of differentiation, EBs were seeded onto 16-chamber glass slides and fixed on day 30, in 4%
9
PFA. Analysis for the expression of proteins representing the three germ layers; β-III-tubulin for
ectoderm, α-fetoprotein for endoderm, and α-actinin or troponin-I for mesoderm, was performed by
immunofluorescence staining as described above (antibody dilutions in Supplementary Table 1).
IN VITRO DIFFERENTIATION TO CARDIAC MYOCYTES
hiPSC lines were differentiated via embryoid body (EB) formation, using forced aggregation
of defined numbers of cells, as described previously [8]. Briefly, on day 0 of differentiation, EB
formation was initiated by seeding V-96 plates with 3,000 or 6,000 cells/well in MEF conditioned
medium and centrifuging at 950g for 5min, at room temperature. EBs were maintained in V-96
plates for 4 days in conditioned medium, and were then transferred to untreated 90mm dishes in DFBS (DMEM supplemented with 20% Foetal Bovine Serum, 100mM b-ME, 1% NEAA, and 2mM
GlutaMAX). EBs were maintained in suspension for 6 days to allow further differentiation and then
transferred to untreated U-96 well plates at a density of one EB/well, in D-FBS. Spontaneously
beating clusters were observed from day 11 of differentiation onwards. Between days 25-30,
beating clusters were either mounted onto multi-electrode arrays (MEAs) for electrophysiology
analysis or were dissociated into single cells for patch clamp analysis.
ELECTROPHYSIOLOGICAL ANALYSIS
For the measurement of extracellular field potentials by MEA analysis, beating clusters were
plated directly onto Matrigel-coated MEAs (Scientifica) as described previously [9]. Recordings were
performed at 37oC in Normal Tyrode’s Buffer (140mM NaCl, 10mM Glucose, 10mM HEPES, 4mM
KCl, 1mM MgCl2, 1.8mM CaCl2 pH 7.45) using 2 kHz sampling rate. Data were captured and
processed using MC_Rack and MC_DataTool software (Multi Channel Systems).
For whole-cell patch recordings of action potentials, beating clusters were disaggregated as
described previously [10, 11]. Briefly, beating areas were manually dissected, washed in PBS, and
then incubated for 30min at room temperature in buffer 1 (120mM NaCl, 5.4mM KCl, 5mM MgSO 4 ,
5mM sodium pyruvate, 20mM taurine, 10mM HEPES, 20mM glucose, pH 6.9), for 45min at 37°C in
buffer 2 (120mM NaCl, 5.4mM KCl, 5mM MgSO, 5mM sodium pyruvate, 20mM taurine, 10mM
10
HEPES, 0.3mM CaCl2, 20mM glucose, 1mg/ml collagenase B, pH 6.9) and for 1 hour at room
temperature in buffer 3 (85mM KCl, 5mM MgSO4, 5mM sodium pyruvate, 20mM taurine, 1mM
EGTA, 5mM creatine, 30mM K2HPO4, 20mM glucose, 1mg/ml Na2ATP, pH 7.2). Finally, cell clusters
were dissociated by repeated pipetting through a P1000 tip, and the liberated cells were seeded
onto 35mm dishes with a glass coverslip (MatTek).
Whole-cell patch-clamp recordings of spontaneously beating single cells were obtained
using an Axopatch 200A patch-clamp amplifier (Axon Instruments/Molecular Devices) in normal
current-clamp mode [12]. Cells were maintained at 37oC, in Normal Tyrode’s Buffer during
recordings and pipettes, pulled on a Sutter P-97 programmable micropipette puller, had resistances
of 5–10 M when filled with ionic solution (145mM KCl, 5mM NaCl, 2mM CaCl2, 2mM MgCl2, 4mM
ethylene glycol tetra-acetic acid, 10mM HEPES, pH 7.3). Data were transferred to a PC via an A/D
interface (Model PCI-6014, National Instruments) and were recorded and analyzed using WinEDR
v3.1.4 software (Strathclyde Electrophysiology Software).
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