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Hypoxia rejuvenates ASC properties from old donors
SUPPLEMENTARY MATERIALS AND METHODS
Adipose tissue cell isolation and culture of hASC
Subcutaneous adipose tissue was obtained from donors aged 20 to 35 years (mean ± standard
deviation: 29 ± 5 years old) and over 50 years (61 ± 7 years old) undergoing elective abdominal
dermolipectomy in no obese patients (BMI < 26). No objection certificates were obtained
according to the bioethic law n° 2004-800 of august 6, 2004. Cells were isolated as previously
described [44]. Briefly, adipose tissue (AT) was digested in DMEM-F12 medium (Invitrogen,
Carlsbad, USA) supplemented with 2 mg/ml collagenase A (Roche Diagnostic, Indianapolis, IN)
and bovine serum albumin (BSA) (2%) for 45 min at 37°C, filtered through 100 μm and 25 μM
nylon membrane and centrifuged at 600 g for 10 min to separate floating mature adipocytes from
the stromal-vascular fraction (SVF). SVF was incubated in erythrocyte lysis buffer (ammonium
chloride solution, StemCell Technologies, Vancouver, Canada) for 5 min at 4°C and washed in
PBS (phosphate-buffered saline). SVF cells were counted and either used for flow cytometry
analysis or seeded in vitro at 4 000 cell/cm2 in hASC expansion medium in 75 cm2 flasks The
hASC culture medium was Good Manufacturing Practice (GMP): -MEM medium (Invitrogen)
supplemented with 2% human plasma (EFS Midi-Pyrénées, France; enriched with human
platelet factors), 100 µg/ml streptomycin, 100 U/ml penicillin, 25 µg/mL amphotericin
(Invitrogen) and 10 U/mL heparin Choay (Sigma, St Louis, USA). Adherent cells were cultured
for 10-14 days at 37°C in a humidified atmosphere of 5% CO2 until they reached confluence.
The medium was renewed every 2 to 3 days.
Colony Forming Unit-Fibroblasts Assay
Freshly prepared SVF cells were seeded in 25 cm2 flasks at a final concentration of 8 cells/cm2
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Hypoxia rejuvenates ASC properties from old donors
in ASC expansion medium. The medium was renewed every 2 or 3 days. After 12 days, cells
were washed with PBS and fixed with methanol for 15 min. The colony forming unit-fibroblasts
(CFU-f) were stained with Giemsa (6%) for 30 min and scored under an optical microscope.
Colonies were considered as clusters of more than 50 cells.
In vivo vascular function analysis
Vascular function was assessed 14 days after the onset of ischemia for control (0,9% NaCl) and
treated (hASC) animal. Ischemic hindlimb conservation was first evaluated as percent of control.
To provide evidence for ischemia-induced changes in vascularisation of the rescued limb, laser
Doppler perfusion imaging experiments were performed in mice limbs, as previously described
[13]. Mice were placed on a heating plate at 37°C to minimize temperature variation for imaging
and limb perfusion was expressed as a ratio of right ischemic (I) to left non-ischemic (NI) leg.
For capillary density mice were anesthetized by an intra peritoneal injection of ketaminexylazine before retro-orbital injection of Lectin GSL TRITC (Eurobio-Abcys) at 150 µl per
mouse. After 30 min, mice received an intracardiac infusion of 3.6% formaldehyde before
muscles (gastrocnemius, gracilis, and quadriceps) were removed, post-fixed at 4°C overnight,
sunk in PBS, and frozen at -50°C in isopentane. Muscles were cut into 20 µm serial sections on a
cryostat (Leica). Capillary density was evaluated using a Leica DM-RB microscope and NIS-AR
image analysis software (Nikon).
In vivo GFP-hASC tracking
Replication defective, self-inactivating lentiviral vectors were provided by BiviC core vector
production (IFR 150, Toulouse, France). Vectors were generated in a BSL-3 facility
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Hypoxia rejuvenates ASC properties from old donors
concentrated by ultracentrifugation for viral titration, expressed in transduction unit per ml
(TU/ml). hASC 3,300 cell/cm2 were transduced overnight in 4 ml of transduction medium
containing serum-free medium, 15% BIT9500 (Stem cells Technologies, Grenoble, France) and
4 μg/ml Protamine Choay (Sanofi Aventis France, Paris, France) in the presence of purified
lentiviral vectors at MOI 8. Cells were collected at least 72 h after transduction for EGFPpositive cells quantification by flow cytometry analysis on a fluorescent-activated cell sorter
(FACS Canto II, Becton Dickinson, Mountain View, CA). For GFP-hASC injected mice, 200
µm muscle sections were done with vibroslice on fixed tissue and fluorescence analysis was
performed utilizing a Zeiss LSM510 NLO confocal microscope.
Quantification of hASC in mice muscle tissue
14 days after hASC injection, DNA from muscle was extracted using DNA kit extraction
(Qiagen, Courtaboeuf, France) according to the manufacturer’s recommendations for tissue, and
was used for PCR. To determine how many human cells were present in mouse muscle, DNA
samples were amplified with the human alu-specific primers and mouse actineB primers and
were compared to a standard curve performed with a mix of increasing amount of hASC (106105-104-103-102) to a muscle sample before DNA extraction. 20 ng of gDNA was analyzed by
real time PCR in a final volume of 10 µl using Sso Advanced SYBR Green supermix (BIORAD)
with the primers for alu, alu for: CAT GGT GAA ACC CCG TCT CTA, alu rev: GCC TCA
GCC TCC CGA GTA G and moACTB, moACTB for: GAT GCA CAG TAG GTC TAA GTG
GAG, moACTB rev: CAC TCA GGG CAG GTG AAA CT (0.3 µM final).
qPCR conditions were as follows: 2 minutes at 95°C, followed by 40 cycles of 5 seconds at
95°C, 30 seconds at 60°C. This was followed by a dissociation stage for 5 seconds at 95 °C to
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Hypoxia rejuvenates ASC properties from old donors
ensure the presence of a single amplicon. Real-time PCR assays were run on StepOne detection
system instrument (Life Technologies/ Applied Biosystem).
Real Time PCR
Total RNAs were isolated from hASC using RNeasy Mini Kit (QIAGEN).About 1µg of
total RNAs was reverse transcribed using random hexamers and Multiscribe reverse transcriptase
(kit High Capacity cDNA Reverse Transcription kit, Life Technologies/ Applied Biosystem). 20
ng of cDNA were analyzed by real time PCR in a final volume of 20 µl using Power SYBRgreen
master mix (Life Technologies/Applied Biosystem) with the primers for VEGF, HGF, IL6TNF and PUM (0.3 µM final) or using Taqman Universal PCR Master Mix (Life
Technologies/Applied Biosystem) with primers and probe Taqman Gene Expression Assay for
IGF1, TGF-2, IL8, CXCL1, p16INK4A and PUM (Life Technologies/Applied Biosystem). All
primers sequences are detailed in Table S1. qPCR conditions were as follows: 10 minutes at
95°C, followed by 40 cycles of 15 seconds at 95°C, 1 minute at 60°C. This was followed by a
dissociation stage for 15 seconds at 95 °C to ensure the presence of a single amplicon. Real-time
PCR assays were run on StepOne detection system instrument (Life Technologies/ Applied
Biosystem). Relative gene expression was calculated by the dCT method and normalized to
PUM. Primers used for qPCR are detailed in Table S1. Primers were designed using Primer
Express software (Applied Biosystems,) and validated by testing PCR efficiency using standard
curves (85%≤ efficiency ≤115%), allowing us to use the ΔΔCt method for absolute
quantification. PCR product specificity was evaluated by generating a dissociation curve
following the manufacturer's recommendation.