Mauritz – Flk-1 progenitor cells from murine iPSCs
SUPPLEMENTARY MATERIAL
Supplementary methods
Cultivation of iPSCs
The iPSC clone B4 was routinely cultivated and expanded on mitotically inactivated murine
embryonic fibroblasts (MEFs). The culture medium was composed of Dulbecco’s Modified
Eagle’s Medium (Invitrogen, Karlsruhe, Germany) supplemented with 15% FCS (Thermo
Scientific, Bonn, Germany), 0.2mM L-glutamine (Invitrogen), 0.1mM –mercaptoethanol
(Invitrogen), 0.1mM non-essential amino acid stock (Invitrogen), and 0.1% huLIF (human
leukemia inhibitory factor) conditioned medium. The latter had been produced by transient
transfection of a huLIF expression plasmid into human embryonic kidney 293 T cells. Every
three to four days colonies were detached with 0.2% collagenase IV (Invitrogen), dissociated
into single cells with 0.025% trypsin (Sigma-Aldrich, Taufkirchen, Germany) and 0.1%
chicken serum (Invitrogen) in phosphate-buffered saline (PBS) and plated again onto MEFs.
Feeder-free iPSC culture for initiation of differentiation and for colony assays was performed
on 6-well culture dishes (Thermo Scientific) coated with 0.1% gelatin (Figure S1). The
medium was supplemented with 1µM inhibitor of glycogen synthase kinase-3 (Merck,
Darmstadt, Germany)1.
Teratoma assay of iPSCs
2.5x105 viable iPSCs from the B4 clone were injected into lower limb skeletal muscle of
SCID beige mice (n=4). Four weeks later tumours were dissected and paraffin sections were
stained with haematoxylin and eosin (H&E). All sections were analysed by an experienced
pathologist with regard to teratoma formation.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
In vitro differentiation of iPSCs
Before starting differentiation iPSCs were cultivated for several passages without feeder cells
to eliminate contaminating MEFs.
Colonies were detached with 0.2% collagenase IV and dissociated into single cells with
0.025% trypsin and 0.1% chicken serum. To initiate embryoid body (EB) formation in
suspension iPSCs were cultured at 0.5x105 cells/ml in differentiation medium consisting of
Iscove’s Modified Dulbecco’s Medium (Invitrogen) supplemented with 15% FCS, 0.2 mM Lglutamine, 0.1mM –mercaptoethanol and 0.1mM non-essential amino acid stock and kept on
an orbital shaker. For the formation of “hanging drop”-EBs 600 cells were aggregated per
20µl of differentiation medium.
On day 5 of differentiation EBs were either plated onto 0.1% gelatin-coated 6-well culture
dishes or were processed for fluorescent activated cell sorting (FACS). To study the potential
of the iPSC clone B4 to differentiate into cardiovascular lineages, plated EBs were examined
for the appearance of spontaneously beating areas. Cells were immunostained with antibodies
against cardiac troponin T (cTnT), connexin 43 (Cx43), Cd31 and α-smooth muscle actinin
(α-SMA).
Flow cytometry and cell sorting of iPSC-derived Flk-1 cells
On days 3 to 6 of differentiation EBs were dissociated into single cells by incubation for 4min
with 0.025% trypsin and 0.1% chicken serum. Cells were stained with rat anti-mouse Flk-1Allophycocyanin (APC) or with the corresponding rat IgG2a-APC isotype control (BD
Biosciences, Heidelberg, Germany) and the percentage of living Flk-1pos cells excluding
propidium iodide (BD Biosciences) was analysed on a FACSCalibur (BD). On day 5 of
differentiation cells were sorted on a FACSAria IIu cell sorter (BD) into nucVenuspos/Oct4GFPneg/Flk-1-APCneg
(Flk-1neg)
and
nucVenuspos/Oct4-GFPneg/Flk-1-APCpos
(Flk-1pos)
populations. Those populations were either directly used for quantitative real-time RT-PCR
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Mauritz – Flk-1 progenitor cells from murine iPSCs
(qRT-PCR) analyses or for intramyocardial injections or they were further cultivated as
reaggregates. For the formation of reaggregates “hanging drops” composed of 6x10 3 viable
cells in 20µl differentiation medium were generated. Two days later reaggregates were plated
onto gelatin-coated 6-well culture dishes.
Analysis of mRNA expression by qRT-PCR
Total RNA was isolated using TriZol (Invitrogen), according to the manufacturer’s
instructions. Integrity of total RNA was controlled using an Agilent 2100 Bioanalyzer
(Agilent Technologies, Santa Clara, USA). Potentially contaminating genomic DNA was
digested by DNAse I (Stratagene, La Jolla, CA, USA) for 15min at 37°C followed by
phenol/chloroform-extraction. After precipitation, 300ng RNA was used for Oligo(dT)18primed cDNA synthesis with RevertAidTM H Minus First Strand cDNA Synthesis Kit
(Fermentas, St. Leon-Rot, Germany).
qRT-PCR was performed using a Mastercycler® ep realplex (Eppendorf, Hamburg, Germany)
and the ABsolute™ QPCR SYBR® Green Mix (Thermo Scientific). PCR reaction mixtures
contained cDNA template, primers and 1x ABsolute™ QPCR SYBR® Green Mix in a final
volume of 25µl. Real-time PCR conditions included enzyme activation at 95°C for 15min,
followed by 40 cycles (denaturation at 95°C for 15sec, annealing at TA (for annealing
temperatures of individual primer pairs (see Table S1) for 60sec and extension at 72°C for
60sec, respectively), with a final 10°C step. Size of amplicons and absence of nonspecific
products was controlled by melting curves and gel electrophoresis.
Starting quantities of target cDNAs were calculated by comparing threshold cycle (CT) values
of each sample to CT values of the respective standard curve using Mastercycler® ep realplex
Software Version 2.0 (Eppendorf). Expression levels of target genes were normalized to βactin transcript levels. Primers were designed using Primer3 Software. Controls without
reverse transcriptase excluded false positive results due to contamination with genomic DNA.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Myocardial infarction model
Immunodeficient SCID beige mice (15-19g, 8-10 weeks, Charles River, Germany) were
anaesthetised with isoflurane (2%), orally intubated and mechanically ventilated (MiniVent,
Hugo Sachs Elektronik, March-Hugstetten, Germany). Anaesthesia was maintained using
isoflurane (1.0-1.5%). A left anterior thoracotomy was performed and the left anterior
descending artery (LAD) was permanently ligated using an 8/0 Prolene suture (Ethicon,
Johnson&Johnson Medical, Norderstedt, Germany). Myocardial ischemia was verified by
intraoperative ECG (FMI/MIL MPVS-Ultra ECG cable, FMI, Seeheim-Ober Beerbach,
Germany). Cell or placebo injections were performed using a tuberculine syringe (BD) with a
30G (0.30mm) needle.
Magnetic resonance imaging
We used a 7 Tesla (300 MHz) scanning system (PharmaScan, Bruker, Etlingen, Germany)
with a 160-mm horizontal bore. A 38mm resonator coil (Bruker) was used to transmit and
receive the signals.
Animal body temperature was maintained at 37°C using a water perfused heating mat.
Initially mice were anaesthetised with 3% isoflurane for 3min and placed in a coil with a
pneumatic pillow for respiration monitoring and maintained at 1.5–2% isoflurane. ECG
electrodes were attached to the left fore and right hind limb. ECG- and respiration-triggered
image acquisition as well as retrospective triggered imaging (Intragate®) was performed
using the Bruker Paravision 5.0 software.
On day 2 infarct size was determined by contrast enhanced magnetic resonance imaging
(MRI). The contrast agent gadolinium-DOTA (Dotarem, Guerbet Gorinchem, Netherlands)
was injected via the tail vein to determine infarct size2. To cover the left ventricle (LV), a
multi-slice RARE sequence (14 contiguous, 0.6mm slices, TR 1500, TE 9ms, FA180 deg,
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Mauritz – Flk-1 progenitor cells from murine iPSCs
AVG 3) was used. Late enhancement was quantified as percentage of the left ventricular mass
using the “Mass 4Mice” analysis software (Medis Medical Imaging Systems, Netherlands).
On day 13 the animals were evaluated for cardiac function. We performed single-slice
Intragate FLASH cine imaging using 6-9 contiguous 1-mm thick slices covering the LV to
analyse function. 20 frames were acquired per cardiac cycle. The Intragate cine FLASH
sequences used a 10deg flip angle, 8ms repetition time, 3.6ms echo time, and 300 repetitions.
The field of view was 40x40mm2, projected on a 256x256 matrix resulting in 0.0156cm/pixel.
Left ventricular volumetric parameters and ejection fraction (LV-EF) were measured and
calculated with the “Mass 4Mice” analysis software (Medis Medical Imaging Systems).
Conductance catheter analysis
On day 14 the animals were anaesthetised with ketamine (25-50mg/kg) and xylazine (510mg/kg) and placed supine on a heating plate. A longitudinal cervical incision was
performed to expose the trachea and cervical vessels. The trachea was incised and an
endotracheal tube was placed. Animals were mechanically ventilated and anaesthesia was
maintained using isoflurane (1.0-1.5%). A 1.0 F conductance catheter (MIL-PVR-1030,
Millar Instruments, FMI, Seeheim, Germany) was positioned into the left ventricle via the
right carotid artery3. The left external jugular vein was cannulated for hypertonic saline
injection to obtain parallel conductance of the left ventricular myocardium. Volumetric
calibration for slope factor α was performed via a calibration cuvette (Mo.910-1049, Millar
Instruments). Data were obtained using a Millar transducer system (MPVS-Ultra, Millar
Instruments) and PowerLab data acquisition platform (ADInstruments, FMI). Pressurevolume signals were recorded in steady state to acquire volumetric and functional
parameters3. Preload independent functional parameters were determined from pressure
volume loop relations during load reduction via temporary occlusion of the inferior vena cava.
Following the operation animals were sacrificed for histological evaluation.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Cryosectioning and immunostaining
Hearts were perfused with PBS, explanted and fixed in 4% paraformaldehyde (PFA) followed
by overnight cryopreservation in 30% sucrose (Sigma-Aldrich). Hearts were embedded in
OCT (Sakura Finetek, Heppenheim, Germany) and stored at -80°C. Subsequently, starting at
the atrio-ventricular transition a series of twenty 8μm cryosections was produced (160µm).
The following 144µm were discarded. This procedure was repeated until the entire heart was
cut. Masson’s Trichrome staining was performed on the first section of each series, the other
slices were used for immunostaining.
Cultivated cells as well as cryosections were fixed with 4% PFA for 5min at 4°C and
subsequently, blocked with 5% donkey serum and 0.25% Triton X-100 (Sigma-Aldrich)
diluted in Tris-buffered saline for 20min at room temperature (RT). The following primary
antibodies were used: rat monoclonal IgG2a anti-Cd31 antibody (working concentration:
0.078µg/ml; clone MEC 13.3, BD Biosciences), rabbit polyclonal IgG anti-Cx43 antibody
(10µg/ml; Millipore, Schwalbach, Germany), mouse monoclonal IgG2b anti-Oct4 antibody
(2µg/ml; clone sc-5279, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit polyclonal
IgG anti-α-SMA (2µg/ml; Abcam, Cambridge, UK), mouse monoclonal IgM anti-SSEA-1
antibody (3.1µg/ml; clone MC-480, Developmental Studies Hybridoma Bank, Iowa City,
USA) and mouse monoclonal IgG1 anti-cTnT antibody (2µg/ml; clone 13-11, Thermo
Scientific). Cells and cryosections were incubated for 1h at RT with the primary antibody or
the corresponding isotype control diluted in phosphate buffered saline (PBS, without Ca2+ and
Mg2+) with 1% bovine serum albumin, then rinsed three times for 5min with PBS. Further
incubation was performed with the appropriate secondary antibody for 30min at RT:
DyLight549TM-labeled donkey anti-mouse IgG or IgM, DyLight549TM-labeled donkey antirabbit IgG or DyLight549TM-labeled donkey anti-rat IgG (1:300; Dianova, Hamburg,
Germany). For microscopy we used an Axio Observer A1 fluorescence microscope (Zeiss,
Oberkochen, Germany).
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Morphometry
Infarct size was determined on Masson’s Trichrome stained sections at 304µm intervals by
calculating the ratio of the circumferential infarct length to the circumferential length of
healthy LV myocardium as described by others4 and depicted in Figure S5. In addition,
minimal left ventricular wall thickness (LV-WT) and the relative area of viable myocardium
(VM) were measured within the infarct region (Figure S5)5. Data were obtained by computerassisted morphometry (Image J 1.40g, NIH, USA).
Quantification of in vivo expression of cardiovascular markers
Cardiovascular marker expression following immunostaining was determined based on a
semiquantitative scoring system. The amount of immunopositive cells within the graft was
determined by two different observers. Score values: 0: no positive cells found; 1: 1-10% of
grafted cells positive; 2: 11-25% of grafted cells positive 3: 26-50% of grafted cells positive;
4: >50% of grafted cells positive (Figure S8).
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Supplementary tables
Table S1
Gene
Primer sequence (5’-3’)
Annealing
temperature (°C)
60
Product size (bp)
β-actin
F-CAAGAGATGGCCACTGCC
R-CTTGATCTTCATGGTGCTAGGA
Cd31
F-CACCAAGCGGTCGTGAATG
R-CAATGACAACCACCGCAATG
60
193
Flk-1
F-GGTCCTTGCATCAGAAGAGCTG
R-CCTCGTCGCTGGAGTACACG
60
185
Mesp1
F-GCTCTTCAGCAGCGACATGC
R-GCCGCCTCTTCCAGGATCTAC
F-GGACAGGTTTCAGAAGCAGAAG
R-ACCATTGCTAGTCTTCAACCAC
60
176
60
160
Nkx2.5
F-TGCAGAAGGCAGTGGAGCTG
R-CGCTGTCGCTTGCACTTGTAG
60
244
Oct4
F-GCGTTCTCTTTGGAAAGGTG
R-AGCCTCATACTCTTCTCGTTGG
60
386
α-SMA
F-CATCTTTCATTGGGATGGA
R-GCGTTCTGGAGGGGCAAT
60
215
cTnT
F-GCGGAAGAGTGGGAAGAGACA
R-CCACAGCTCCTTGGCCTTCT
60
127
Nanog
Table S1. Sequences of primers used for qRT-PCR.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Table S2
Control groups
Cell treated groups
PBS
Flk-1neg
Flk-1pos
ESV (µl)
47±15
39±13
29±9 *
EDV (µl)
59±17
52±14
42±13 *
SV (µl)
13±3
13±3
13±6
LV-EF (%)
23±3
28±6
32±10 *
449±17
423±26
451±14
416±14
ESV (µl)
7±2
24±7 †
21±3 #,††
15±4 *,†
EDV (µl)
13±5
27±8 †
23±5
19±6 *,††
ESP (mmHg)
80±15
90±41
79±14
94±34
EDP (mmHg)
5±2
7±3
7±2
7±3
SV (µl)
8±3
4±2 †††
6±2
7±3
LV-EF (%)
59±9
16±3 †
23±6 *, †
27±8 **, †
CO (µl/min)
3670±1392
1917±874
2694±930
2764±1127
Stroke work
(mmHg/µl)
479±217
211±159
235±111
323±297
dP/dtMAX
(mmHg/sec)
5591±1399
4707±1991
5919±1889
5478±2204
Tau (msec)
8±2
10±2
9±1
11±3
Maximal power
(mWatts)
4±2
2±2
4±2
3±2
Preload adjusted
maximal power
(mWatts/µl²)
268±64
28±10 †
73±21 *,†
87±60 **,†
Sham
MRI
CC
HF (beats/min)
Table S2. Hemodynamic evaluation by magnetic resonance imaging (MRI) and
conductance catheter analysis (CC). Values are given as mean±SD. MRI was not performed
on non-infarcted control animals (sham). HF = heart frequency; ESV = end systolic volume;
EDV = end diastolic volume; ESP = end systolic pressure; EDP = end diastolic pressure; SV
= stroke volume; LV-EF = left ventricular ejection fraction; CO = cardiac output; dP/dtMAX
= maximum pressure increase; Tau = relaxation time constant. Sham = (non infarcted) sham
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Mauritz – Flk-1 progenitor cells from murine iPSCs
operated group; PBS = placebo treated infarct group; * P<0.05 versus PBS; ** P<0.01 vs.
PBS; # P<0.05 vs. Flk-1neg; † P<0.001 vs. sham; †† P<0.01 vs. sham; ††† P<0.05 vs. sham.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Supplementary figure legends
Figure S1. iPSC clone B4 expresses pluripotency markers. A: Phase contrast of iPSC
colonies grown on feeder cells. B: iPSC colonies on gelatin without feeder cells. C: Oct4eGFPpos cells (green, cytoplasmic expression) were immunopositive for endogenous Oct4
(red, D). E: Oct4-eGFPpos cells (green, cytoplasmic expression) were immunopositive for
stage specific antigen-1 (SSEA-1) (F). nucVenus expression (bright green, nuclear
expression). Scale bars: 100µm.
Figure S2. iPSC clone B4 forms teratomas and shows robust cardiovascular
differentiation. A-C: H&E staining of iPSC-derived teratomas. A: Ectodermal (keratinizing
epithelium), B: mesodermal (cartilage) and C: endodermal (palisade-like epithelium)
structures. D: iPSC-derived embryoid bodies (EBs) with contracting areas in % from day 7 to
day 19 of differentiation. E-H: Immunostaining of iPSC-derived EBs. Scale bars: 100µm.
Figure S3. Colony-forming Oct4pos cells are still present after FACS-sorting. A: Oct4eGFPpos cells in % before and after FACS-sorting. *** P<0.001 vs. unsorted B: Day 5
unsorted, Flk-1neg and Flk-1pos cells were further cultivated in pluripotency-supporting
medium. Developed colonies of Oct4 immunopositive (Oct4pos) cells per originally plated
cells (in %) were evaluated on day 8. * P<0.05 vs. unsorted C-J: Anti-Oct4 immunostaining
of Oct4-eGFPpos colonies on day 8 (3 days after FACS-sorting) and corresponding phase
contrasts. C+D: Undifferentiated iPSC colonies on gelatin as positive control. E+F: Unsorted
cells. G+H: Flk-1neg cells. I+J: Flk-1pos cells. Scale bars: 100µm.
Figure S4. Flk-1pos cells form larger reaggregates than Flk-1neg cells: Following FACSsorting on day 5 reaggregates were generated for in vitro differentiation. Depicted are day 7
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Mauritz – Flk-1 progenitor cells from murine iPSCs
reaggregates in suspension of A: unsorted, B: Flk-1pos and C: Flk-1neg cells. Scale bars:
100µm.
Figure S5. Morphometry, Masson’s Trichrome staining. A: Myocardial infarction after
LAD-ligation. B: The mean infarct size per section was calculated after determining the inner
and outer length of the infarcted and non-infarcted left ventricle (orange versus green lines).
Yellow bars divide infarcted from non-infarcted myocardium. Within the infarct zone the
minimal LV wall thickness (LV-WT; white arrow) as well as the relative area of viable
myocardium (VM; red coloured pixels) were calculated.
Figure S6. Left ventricular function and volume load of infarcted hearts in comparison
to healthy controls. Conductance catheter analysis (CC) A: Left ventricular ejection fraction
(LV-EF) is significantly reduced after LAD-ligation in all infarct groups compared to sham
operated controls. *** P<0.001 vs. sham.
B/C: Enddiastolic and endsystolic volume
(EDV/ESV) is increased after LAD-ligation in infarcted animals compared to sham operated
controls. *** P<0.001, ** P<0.01 vs. sham. D: Differences in function and volume load result
in a marked reduction of preload adjusted maximum power in infarcted animals. *** P<0.001
vs. sham. Differences between infarct groups are presented in the main manuscript. Detailed
values are given in Table S2.
Figure S7. Transplantation of Flk-1pos cells improves cardiac function and reduces left
ventricular volume load after myocardial infarction. Magnetic resonance imaging (MRI).
Additional volumetric data. A: Left ventricular ejection fraction (LV-EF) improves
significantly after intramyocardial transplantation of Flk-1pos cells. * P<0.05 vs. PBS. B/C:
Enddiastolic and endsystolic volume (EDV/ESV) is significantly decreased after treatment
with Flk-1pos progenitor cells. * P<0.05 vs. PBS. Detailed values are given in Table S2.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
Figure S8. Flk-1pos and Flk-1neg cells form cardiovascular grafts in vivo. Flk-1pos grafts
contain more Flk-1/Cd31 immunopositive vascular cells. Expression of representative
differentiation markers as calculated from semiquantitative scoring of immunostained
sections. A: cTnT immunopositive cells (score): Flk-1neg: 1.1±0.3; Flk-1pos: 1.3±0.2; P=ns. B:
Cd31 immunopositive cells (score): Flk-1neg: 1.2±0.4; Flk-1pos: 1.8±0.2; * P<0.05. C: Flk-1
immunopositive cells (score): Flk-1neg: 0.0±0.0; Flk-1pos: 1.9±0.2; *** P<0.001.
Supplementary movies
Movie S1. Flk-1pos cell-derived reaggregates with spontaneously contracting areas.
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Mauritz – Flk-1 progenitor cells from murine iPSCs
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