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Online supplementary data
Towards a Clinical Use of Human Embryonic Stem Cell-Derived Cardiac Progenitors: A
Translational Experience
Philippe Menasché1,2,3*, Valérie Vanneaux,4,5,6 Jean-Roch Fabreguettes7, Alain Bel1,3, Lucie
Tosca8, Sylvie Garcia9, Valérie Bellamy3, Yohan Farouz2,3, Julia Pouly1, Odile Damour10,
Marie-Cécile Périer11 Michel Desnos2,3,12, Albert Hagège2,
3, 12
, Onnik Agbulut13, Patrick
Bruneval2,11,14, Gérard Tachdjian8, Jean-Hugues Trouvin15,16, Jérôme Larghero4,5,6.
1
Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of
Cardiovascular Surgery, Paris, France.
2
University Paris Descartes, Sorbonne Paris Cité, F-75475, Paris, France.
3
INSERM U 633, Hôpital Européen Georges Pompidou, Paris, France.
4
Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Louis, Cell Therapy Unit and Clinical
Investigation Center in Biotherapies (CBT501), Paris, France.
5
University Paris Diderot, Sorbonne Paris Cité, F-75475 Paris, France.
6
INSERM UMRS940, Institut Universitaire d’Hématologie, Hôpital Saint-Louis, Paris, France.
7
Assistance Publique-Hôpitaux de Paris, Central Pharmacy, Clinical Trials Department, Paris,
France.
8
Assistance Publique-Hôpitaux de Paris, University Paris Sud, Histology-Embryology-
Cytogenetics, Hôpitaux Universitaires Paris Sud, 92141 Clamart, France.
9
Unité de Biologie des Populations Lymphocytaires, Department of Immunology, Institut
Pasteur, CNRS-URA 1961, Paris, France.
10
Tissues and Cells Bank, Edouard Herriot Hospital, Lyon, France.
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INSERM U 970, Hôpital Européen Georges Pompidou, Paris, France.
12
Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of
Cardiology, Paris, France.
13
UPMC University Paris 6, Sorbonne Universités, Department of Aging, Stress and
Inflammation, Paris, France.
14
Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou,
Department of Pathology, Paris, France.
15
University Paris Descartes, School of Pharmacy, Paris, France.
16
Assistance Publique-Hôpitaux de Paris, Central Pharmacy, Pharmaceutical Innovation
Department, Paris, France.
*To whom correspondence should be addressed : Department of Cardiovascular Surgery,
Hôpital
Européen
Georges
Pompidou,
20,
rue
Leblanc,
75015
Paris,
Phone : 33 1 56 09 36 22; Fax : 33 1 56 09 32 61; philippe.menasche@egp.aphp.fr.
France.
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SUPPLEMENTAL MATERIALS AND METHODS
Procurement, derivation and initial testing of the I6 ESC line.
The I6 cell line was obtained from a leftover zygote after an in vitro fertilization procedure at
the Rambam Medical Center (Israël). This zygote was donated by a couple after it had signed
consent forms. The zygote was cultured to the blastocyst stage using Cook media (Cook,
Queensland, Australia). After zona pellucida digestion by Tyrode’s acidic solution (Sigma, St
Louis, MO, USA), the blastocyst was incubated for 30 minutes with anti-human whole
antiserum antibody. The embryo was washed three times using Gibco® Dulbecco's Modified
Eagle Medium (DMEM, Life Technologies, Grand Island, NY, USA) and then incubated with
0.5% Guinea pig complement (Gibco) for about 20 minutes. At the end of the incubation, the
embryo was washed three times using DMEM and then placed onto mitotically inactivated
mouse embryonic fibroblasts (MEF) derived on-site from Imprinting Control Region (ICR)
mice (Harlan, Israel). Cells were grown in a culture medium consisting of 80% Knock-Out
(KO)-DMEM, supplemented with 20% defined fetal bovine serum (FBS, HyClone, Logan, UT,
USA), 1mM L-glutamine, 0.1 mM β–mercaptoethanol, 1% non-essential amino acid stock (all
from Invitrogen™, Life Technologies, Carlsbad, CA). During the first few passages, the cells
were passaged mechanically using 27 gauge syringes. Later, the cells were passaged every four
to six days using 1mg/ml type IV collagenase (Invitrogen™). A MCB was then prepared after
cells were frozen in liquid nitrogen using a freezing solution consisting of 10% dimethyl
sulfoxide (DMSO, Sigma, St Louis, MO, USA), 20% FBS and 80% KO-DMEM.
Genetic stability : The human ESC line I6 was tested for karyotypic stability 7 months after its
derivation (at passage 29). This analysis was conducted at the Cytogenetic Unit of Rambam
Health Care Campus. G-band standard staining (Giemsa, Merck, Darmstadt, Germany) was
used for chromosome visualization. The karyotypes were analyzed and reported according to
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the "International System for Human Cytogenetic Nomenclature" (ISCN). One-hundred
metaphases were examined and the cell line was found as normal 46, XY.
Sterility: Different batches were tested for mycoplasma, using a commercial kit (Biological
Industries, Kibbutz Beit-Haemek, Israel) or Polymerase Chain Reaction (PCR) according to the
protocol provided by the British National Cell Bank and were found negative.
Viral testing : No evidence for murine leukemia viruses (MuLVs) transmission from MEFs to
the I-6 line was found1. The HSV-1, HSV-2, CMV and HBV viruses, tested by nested PCR at
the Rambam Medical Center Virology Laboratory were also found negative. Such was also the
case for the HIV virus tested by PCR at the Sheba Medical Center Virology Laboratory.
hESCs features: The pluripotency of the cell line was confirmed by its positive immunostaining
for undifferentiated stem cell-associated genes (SSEA-3, SSEA-4, Oct 4, tumor rejection
antibody [TRA]-1-60, TRA-1-81), the formation of embryoid bodies and the generation of
teratomas following cell injection into the rear leg muscle of 4-week-old male SCID-beige
mice.2
Cell expansion.
Cell expansion was performed in a similar way in the two laboratories (Hôpital Saint-Louis and
MAbgène). Briefly, thawed cells (viability > 50%) were washed in10 mL of Nutristem™ hESC
XF (Biological Industries, Kibbutz Beit-Haemek, Israel) and centrifuged at 800 rpm for 4
minutes. The pellet was then collected and suspended in Nutristem™. Cells were seeded onto
15cm dishes (B15) previously seeded with clinical-grade irradiated human foreskin fibroblasts
(Tissue Bank, Hôpital Edouard Herriot, Lyon) at 37°C with 5% CO2 and the medium was
changed daily. When I6 ES cells reached 80-90% confluence, cells were dissociated from the
feeder fibroblasts by treatment with collagenase (1mg/mL, Collagenase NB6, GMP grade,
batch #22540105, Nordmark, Uetersen, Germany) during 40±10 minutes. The I6 ESC colonies
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were removed from the surface of dishes by collecting the supernatant. After the washing step
of dishes, the pooling of the supernatant and the washing medium and centrifugation of the
pool contents at 800 rpm for 4 minutes, the I6 cells were replated onto fibroblast-pre-seeded
dishes at the usual 1:5 split ratio. Preliminary experiments have estimated that, at this stage, the
number of cells present in a B15 dish is approximately 10 million. All calculations have thus
been based on this metric. Care was taken to passage the cells in small clumps to enhance their
viability. At the time of banking, cells were cryopreserved in FBS (HyClone, Thermo-Fisher,
Illkirch, France) and 10% DMSO (B.Braun Medical, Boulogne, France). Back-and forth
transfers of the cells between the two sites (Hôpital Saint-Louis and MAbgène) were performed
under well controlled transport conditions in vapour nitrogen dry shippers. As mentioned in the
main manuscript, for the pilot phase I clinical trial, it has been decided to pool the master and
the working cell banks together. A two-tiered banking system will be established later on
depending on the outcome of the trial.
Cell purification.
To maximize the safety of the anti-SSEA-1 antibody with regard to viral contaminations, we
were initially committed to use an IgG-type of antibody and consequently ordered the
development of such a customized clinical-grade product. Unfortunately, the antibody that was
developed, although found to be ultimately free from any contamination, turned out to be
unsuitable because of its poor affinity at the end of the purification process. We then had to
redirect our attention towards a commercially available research-grade microbead-coupled IgM
anti-SSEA-1 antibody (Miltenyi Biotec, Teterow, Germany) but were then requested by the
regulatory agency to submit it to extensive viral testing (Supplemental Table 1) before it could
be approved for use in our phase 1 clinical trial. Multiple preclinical runs were performed to
optimize the sorting procedure which ended in a protocol that entailed the following steps : (1)
withdrawal of the specification medium ; (2) wash-out of cells with phosphate-buffered saline
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([PBS] 10 mL/one B15 dish), detachment with trypsin (5 mL trypsin-EDTA [Hyclone] 0.25%
per one B15; 4 minutes at 37°C) and blockade of trypsin by a buffer (PBS + Human Serum
Albumin ([HSA, Albunorm, Octapharma, Boulogne-Billancourt, France] + 1% DNAse
[Dornase alpha, Pulmozyme®, Roche, Boulogne-Billancourt, France]); (3) centrifugation of the
cells (800 rpm, 4 minutes) which were then resuspended in the sorting buffer (PBS + HSA +
1% EDTA 2mM); (4) filtration of the cell suspension (100 µm) and cell counting in an
hemocytometer; (5) additional cell washing in the sorting buffer before suspension in 100 µL
of buffer for 10 million cells; (6) addition of the anti-SSEA-1 beads at a concentration of 20 µL
per 10 million cells in 1 mL of the sorting buffer. Cells and beads were then incubated for 15
minutes at 4°C. They were then washed and resuspended in the same sorting buffer. The
suspension was transferred into a LS column, up to 20 million cells per column, held within a
magnet (Miltenyi Biotec). One column was used for 20 million cells. The magnetically labeled
SSEA-1+ cells were eluted from the column after it had been removed from the magnetic field
of the separator whereas the SSEA-1- cells were collected in the flow-through fraction. The
positive fraction was again sorted on a second column, according to a similar procedure, to
minimize contamination by still undifferentiated I6 cells and improve the purity of the cell
population (≥95%). Specifications for raw and ancillary materials were sourced and
documented as extensively as possible.
Global gene expression analysis.
RNA Extraction from ESC and ESC-derived CD15+ and CD15- cells: Total RNA was
extracted from cells retrieved from three different batches using Trizol reagent (Roche). RNA
integrity was evaluated by microfluidic analysis using the Agilent 2100 Bioanalyzer with an
RNA LabChip® KIT (Agilent Technologie). RNA concentration was determined using a
Nanovue (GE Healthcare).
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Microarray Analysis : An aliquot of 200 ng of total RNA was used to synthesize doublestranded cDNA, and then produce biotin-tagged cRNA using the 3’IVT Express kit
(Affymetrix®). The resulting bio-tagged cRNA were fragmented into strands of 35–200 bases
in length according to the protocols from Affymetrix® (Santa Clara, CA, USA). The
fragmented cRNA was hybridized to a Human Genome U219 array strip (Affymetrix®),
containing more than 36,000 transcripts and variants. Hybridization was performed at 45°C in
the Affymetrix® GeneAtlas™ Hybridization station for 20h. The GeneChip arrays strips were
washed and then stained (streptavidin–phycoerythrin) on an Affymetrix® Fluidics Station 450,
followed by scanning on the GeneAtlas™ Imaging Station (Affymetrix®).
Normalization and Data Analysis : The hybridization data were analyzed using Expression
Console (EC version 1.3.0.187, Affymetrix®). The scanned images were first assessed by
visual inspection, then analyzed to generate raw data files saved as CEL files using the default
setting of Expression Consol. Data were normalized using RMA algorithm. In a comparison
analysis, genes were determined to be significantly differentially expressed with a selection
threshold of ratio >2.0 or ratio <0.5 in the output result.
Microbiological testing.
The detailed list of the tests is indicated in the Supplemental Table 1. These tests were done by a
subcontractor (Texcell, Evry, France) on pluripotent cells from both the MCB and late-production cell
bank and were repeated on the SSEA-1-positive progenitor cells derived from these two banks. Viral
testing was also performed on the anti-SSEA-1 antibody (hybridoma and final microbead-conjugated
antibody used for cell sorting).
Cytogenetic studies.
Conventional cytogenetic analysis: Chromosome analyses by standard karyotype were
performed from cultured cells using standard procedures (RHG and GTG bandings). For
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mitotic preparations, cells were supplemented with 0.02 mg/ml colchicin (Eurobio,
Courtaboeuf, France) for up to 1 h and 45 min. The cells were harvested and warm hypotonic
solution of 0.075 M KCl was added in the preparation for up to 15-20 min. Finally, cells were
fixed several times in cold Carnoy’s fixative (methanol/acetic acid, 3:1).
Fluorescent in situ hybridization (FISH): FISH analyses were performed on interphasic nuclei
and metaphase spreads. The centromeric probes specific for chromosome 12 and 17 were used
according to the manufacturer’s recommendations (Vysis, Abbott France, Rungis, France).
BAC clone RP5-857M17 specific for the 20q11.21 chromosomal region were used.
Oligonucleotide based-array comparative genomic hybridization (array-CGH): The genomic
imbalances were analysed by array-CGH using 105K oligonucleotide arrays (Hu-105A, Agilent
Technologies, Massy, France). All array hybridizations were performed according to the
manufacturer’s recommended protocols. In brief, 3 µg of genomic DNA was digested with
AluI (5 units) and RsaI (5 units) for 2 h at 37°C and fluorescently labelled with the Agilent
Genomic DNA labelling kit PLUS (Agilent Technologies). A male human genomic DNA
(Promega, Charbonnière, France) was used as reference. Experiments were conducted in dyeswap. Cy5-dUTP patient DNA and its gender-matched reference labelled with Cy3-dUTP were
denatured and preannealed with Cot-1 DNA and Agilent blocking reagent before hybridization
for 40 h at 20 rpm in a 65°C rotating hybridization oven (Agilent Technologies). After
washing, the slides were scanned on an Agilent Microarray Scanner. Captured images were
processed with Feature Extraction 9.1 (Agilent Technologies) software and data analysis was
performed with CGH Analytics 3.5 (Agilent Technologies). Copy number variations (CNVs)
were considered significant if they were defined by three or more oligonucleotides spanning at
least 50 Kb and contained at least one gene and were not identified in the Database of Genomic
Variants.
Fabrication of the fibrin patch.
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Both fibrinogen and thrombin were components of the Evicel® kit (Ethicon Biosurgery, Omrix
Biopharmaceuticals-Ethicon Biosurgery, Rhode Saint Genèse, Belgium) which is approved for
clinical use and widely used as an hemostatic sealant in surgery. However, the
fibrinogen/thrombin ratio had to be revisited as compared to what is indicated in the
instructions for use of Evicel® to generate a scaffold meeting three major objectives : (1) an
elasticity best suited for mesodermal gene expression (we ended up with an elasticity modulus
of the patch, as measured by shear wave elastography, in the range of 6 kPa), (2) a fiber
network dense enough to retain cells while remaining loose enough to prevent their excessively
tight packing and to facilitate in vivo scaffold vascularization from the host vessels, and (3) a
robustness compatible with easy intraoperative manipulations. At the end of the screening
phase, concentrations of 20 mg/mL and 4 U/mL of fibrinogen and thrombin, respectively, were
found to represent an appropriate combination, which is consistent with the finding that, at least
with MSC, fibrinogen concentrations in the low range are best suited for promoting cell
proliferation.3
SUPPLEMENTAL RESULTS
Safety studies
Among mice subcutaneously injected with cells mixed with matrigel, only those receiving
undifferentiated hESC, SSEA-1-positive progenitor cells intentionally “contaminated” with 5%
undifferentiated I6 cells and SSEA-1-negative cells prepared early in our first lab-scale
experiments developed, although not consistently, a teratoma. Conversely, no teratoma
developed in any of the 12 mice injected with 500,000 to 900,000 SSEA-1-positive progenitor
cells (follow-up: 3 to 7 months), nor in those (n=8) exclusively injected with SSEA-1-negative
cells generated at the completion of a selection step entailing the same protocol as the one
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planned for the clinical trial (follow-up: 3 months). No teratoma was either seen in the group of
mice (n=6) injected with SSEA-1-positive progenitor cells spiked with 2% undifferentiated I6
hESC. The 5 mice injected with a 10% fraction of undifferentiated cells mixed with the
progenitors all died during follow-up, without apparent tumor but no necropsy could be made.
Efficacy studies
In keeping with data showing that hearts receiving the progenitor cell-loaded fibrin patch
yielded the best recovery of LVEF, the cell/patch treatment was also associated with the
greatest limitation of adverse LV remodeling, as demonstrated by a striking stability of LV
enddiastolic volumes (mm3) : 361,74 [322.55;400.93] at 2 months vs 357.63 [323.89;391.37] at
baseline. Conversely, these volumes markedly increased in hearts receiving a cell-free patch
(380.79 [343.92;417.66] vs 365.12 [328.88;401.35] at baseline) and to a greater extent in the
sham-operated rats (387.02 [341.59;432.45] vs 344.81 [319.38;370.23] at baseline; p = 0.034
vs the treated group). Changes in LV endsystolic volumes featured similar patterns as they
increased (absolute difference between 2-month and baseline values, mean ± SEM) by 22.71 ±
12.56mm3 and 11.25 ± 13.85 mm3 in the sham and control groups, respectively, whereas they
decreased by 14.92 ± 11.05 mm3 in hearts receiving the cell-loaded fibrin patch.
SUPPLEMENTAL FIGURES LEGENDS
Supplemental Table 1. Listing of the tests used for the viral testing.
Supplemental Table 2. Summary of safety experiments in immunodeficient mice
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Supplemental Figure 1. Morphological aspect of the undifferentiated I6 cell colonies
throughout the scale-up process (P: Passage).
Supplemental Figure 2. Expression of the cardiac transcription factors Isl-1 (panel A) and
Mef2c (panel B) by the SSEA-1+ committed progenitor cell population embedded into the
fibrin patch. For each panel, the top rows correspond to a patch loaded with undifferentiated I6
cells (negative control). Bar: 20 µm.
Supplemental Figure 3. Expression of residual pluripotency (Panel A), mesodermal (Panel B)
and endodermal (Panel C) genes in the CD15-negative fraction. These data were determined by
a transcriptomic analysis based on 3 different sorting runs, using the AffymetrixGeneAtlasTM
system coupled to the new Human Genome U219 array. Data are given as fold changes relative
to undifferentiated I6 hESC. Blue bar graphs refer to the I6 line and red bar graphs refer to the
CD15-negative fraction of the sorted cells.
SUPPLEMENTAL REFERENCES
1. Amit M, Winkler ME, Menke S, Brüning E, Büscher K, Denner J, Haverich A, ItskovitzEldor J, Martin U. No evidence for infection of human embryonic stem cells by feeder cellderived murine leukemia viruses. Stem Cells. 2005;23:761–771.
2. Amit M, Itskovitz-Eldor J. Derivation and spontaneous differentiation of human embryonic
stem cells. J Anat. 2002;200:225–232.
3. Ho W, Tawil B, Dunn JCY, Wu BM. The behavior of human mesenchymal stem cells in 3D
fibrin clots: dependence on fibrinogen concentration and clot structure. Tissue Eng.
2006;12:1587–1595.