Six-weeks-old Wistar male rats (n = 26, Harlan) were exposed for 3 weeks to chronic hypoxia in a hypobaric chamber (50 kPa) to lead the development of pulmonary hypertension and were compared to control matched rats (n = 26).
The MSCs engraftment and viability control was performed using 4 hypoxic rats and compared to 4 control rats by a direct in-vivo injection of GFP-labeled MSCs into the right lung parenchyma and checked 3 weeks after normoxic or hypoxic condition housing as described below. The early dynamic distribution of infused radiolabeled MSCs was performed using 6 hypoxic rats and compared to 6 control rats. The long-term distribution of infused GFP-labeled MSCs was performed using 6 other hypoxic rats compared to 6 matched control rats. Finally, 5 hypoxic rats and 5 control rats were also sacrificed for DNA extraction and 5 hypoxic rats and 5 control rats were sacrificed for pulmonary enzymatic digestion and culture (see below).
All animal investigations were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publications N°85-23, revised 1996) and European Directives (86/609/CEE).
Cell isolation and culture procedures for MSCs have been established and published previously [11, 12]. Briefly, femurs were aseptically harvested from 6-weeks-old Wistar rats and the adherent soft tissue was removed. The proximal and distal ends of the femur were excised at a level just into the beginning of the marrow cavity. Whole marrow plugs were obtained by flushing the bone marrow cavity with a 18-gauge needle set with a syringe filled with culture medium composed of Modified Eagle Medium Alpha (α-MEM; Invitrogen) supplemented with 20% fetal calf serum (FCS; Hyclone), with antibiotic solution (penicillin/streptomycin: 1%; Invitrogen) and with antimycotic solution (amphotericin B: 0.01%; Bristol-Myers). The marrow plugs were dispersed to obtain a single cell suspension by sequentially passing the dispersion through 18- and 22-gauge needles. The cells were centrifuged and resuspended with culture medium. After counting in Malassez cells following an acetic acid disruption of red blood cells, nucleated cells were plated at a density of 106/cm2 and incubated at 37°C in a humidified atmosphere of 95% air 5% C02. The first medium change was after 2 days and twice a week thereafter. When these primary MSCs reached 80–90% of confluence, they were trypsinized (trypsin-EDTA, Invitrogen), counted and passaged at a density of 104/cm2. For the first study second-passage MSCs were labeled with 111In-oxine as described below and infused intravenously. For the second study MSCs were GFF-labeled after viral gene transduction after the first passage and were used as the second-passage.
Adherent second-passage MSCs were analyzed by flow cytometry with a FACSCalibur flow cytometer (Becton-Dickinson) using a 488 nm argon laser. Cells were incubated for 60 minutes at 4°C with phycoerythrin- or fluorescein isothiocyanate-conjugated monoclonal antibodies against rat CD45 (clone OX-1), rat CD73 (clone 5F/B9), and rat CD90 (Clone OX-7; all from Becton Dickinson). Isotype-identical antibodies served as controls. Samples were analyzed by collecting 10,000 events on a FACSCalibur instrument using Cell-Quest® software (Becton-Dickinson).
Isotopic labeling and Indium-111 labeled MSCs intravenous infusion
The cells were incubated with 111In-oxine (37 MBq/106 cells) and incubated for 60 minutes as previously described . The radiolabeled MSCs were aliquoted at 107 cells/ml and intravenously infused to hypoxic rats within 1 hour and followed by whole body scintigraphic imaging. Preliminary experiments showed that the viability and growth of these labeled MSCs were not adversely affected by this labeling procedure (data not shown); the level of radioisotope was widely sufficient to produce high quality images taken with a gamma camera and to produce high quality autoradiographic images of organs.
Whole body scintigraphic imaging was performed immediately after infusion and within 15 minutes, 30 minutes, 1 hour, 3 hours, 24 hours and 96 hours thereafter. Planar whole body images were acquired with Helix Elscint scanner (GE Healthcare) using a medium energy collimator. Images were acquired on a 256 × 256 matrix using a window centered at 245 keV. The distance between the chest of animals and the detector was fixed at 65 mm. In analysis of the scintigraphic images, regions of interest (ROIs) were placed over lungs, liver and spleen on anterior incidence, and over kidneys on posterior incidence. The whole body count was determined by the mean counts on both incidences. Total counts in the ROIs were corrected with physical decay of 111In and with body count.
After sacrifice lung, liver, heart, spleen, kidneys and bone marrow were harvested. Organs were weighted and assayed for radioactivity using a Muller counter (Ludlum Measurements), after what they were snap-frozen in liquid nitrogen, whereas cytospins of bone marrow were realized. Sample sections (15 μm) and bone marrow cytospins were exposed to a photographic film within 24–96 hours and autoradiographic films were developed.
GFP labeling, in vivo engraftment and viability controls, and GFP-labeled MSCs intravenous infusions
MSCs were labeled by green fluorescent protein (GFP) after stable viral gene transduction with LNCX-GFP vector. GFP fluorescence from first-passage transduced MSCs was checked by flow cytometry. Non-specific fluorescence was determined using MSCs that were not transduced. GFP-labeling stability was assayed by flow cytometry using tenth-passage GFP-labeled MSCs.
In-vivo engraftment and viability controls
Animals were lightly anesthetized and GFP-labeled MSCs were injected, at a dose of 2.106 cells, through the rib cage, into the right lung lower lobe. After recovering, animals were housed 3 weeks either in normoxic condition, or hypoxic condition. Animals were sacrificed after the 3 weeks and the lung was harvested, snap-frozen in liquid nitrogen. The frozen sample sections (15 μm) were analyzed by tree-dimensional confocal laser microscopy.
GFP-labeled MSCs intravenous infusions
Second-passage GFP-labeled MSCs were sequentially infused intravenously at the dose of 106 MSCs. The first infusion indicated the first day of the 3 weeks chronic hypoxia. Both hypoxic and control rats were infused twice a week during 3 weeks.
After sacrifice lung, liver, heart, spleen, kidneys and bone marrow were harvested. Organs were weighed and snap-frozen in liquid nitrogen. The frozen sample sections (15 μm) of the different organs were analyzed by tree-dimensional confocal laser microscopy. Data was collected with sequential laser excitation to eliminate bleed through and acquired on a 1024 × 1024 matrix using a 110 μm pinhole and an optical section thickness of 0.31 μm. The system was made up of a FV500 confocal microscope (Olympus) using FluoView500 software and a 488 nm argon laser.
The GFP protein was also researched on frozen sections by immunohistochemistry. Sections of harvested organs were incubated with a rabbit polyclonal antibody against GFP (1/200, Santa Cruz Biotechnology) and were revealed either by a conjugated goat anti-rabbit alexa-594 (1/400, Molecular Probes) or by a conjugated goat anti-rabbit horseradish peroxydase (1/400, Biosource).
Bone marrow homing detection
Cytospins of bone marrow aspirates from control and hypoxic rats were realized 3 days after a unique GFP-labeled MSCs infusion and 3 days after the end of GFP-labeled MSCs infusion during the 3-week hypoxia exposure. The percentage of fluorescent cells was estimated for each rat in five random fields by microscopy using Optimas software (Imasys). Thin slices (12 μm) of frozen bone sections were cut in the metaphysis of tibia from five injected rats. Fluorescence (GFP) was directly observed by confocal microscopy and adipocytes were detected after counterstaining with DAPI (4,6-diamidino-2-phenylindole, AbCys) .
Detection of GFP transgene and protein by PCR and western blotting
After sequential infusions, organs were harvested. From each animal, GFP transgene and protein were assayed by PCR and Western blotting.
Total DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. It was analyzed by PCR for GFP transgene presence using a set of primer generating a 249 bp amplicon: forward, GCGACGTAAACGGCCACAAGTTC and reverse, CGTCCTTGAAGAAGATGGTGCGC. DNA was subjected to PCR for 35 cycles of 94°C for 30 seconds, 58°C for 60 seconds, 72°C for 30 seconds, with a final elongation step of 10 minutes at 72°C.
Organs were crushed by Turrax and homogenized with lysis buffer [1% sodium deoxycholate, 0.1% SDS, 1% triton X-100, 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and an inhibitor protease cocktail (chymotrypsin-, thermolysin-, papain-, pronase-, pancreatic extract- and trypsin-inhibitor; Roche)] and centrifuged at 20,000 g for 1 h. After purifying and concentrating small proteins from each sample (Centriprep Centrigugal Devices YM-30MW, Millipore) with a nominal molecular weight limit of 30 kDa, proteins were separated on a SDS/12% polyacrylamide gel and then transferred to a nitrocellulose membrane (Amersham). Blots were blocks for 2 h at room temperature with 5% (w/v) non-ft dried milk in Tris-buffered saline [10 mM Tris-HCl (pH 8.0) and 150 mM NaCl] containing 0.05% Tween 20. The membrane was incubated overnight at 4°C with rabbit polyclonal antibody against GFP (1/400, Santa Cruz Biotechnology). The blot was then incubated with the conjugated goat anti-rabbit horseradish peroxydase (1/1000, Biosource) 2 h at room temperature. Immunoreactive proteins were detected with the ECL Western blotting detection system (Amersham).
Pulmonary enzymatic digestion
Lung from 5 non-hypoxic and 5 hypoxic MSCs-injected rats were cultured after enzymatic digestion. Briefly, rat lungs were harvested, mechanically dissected and the thin pieces were digested with collagenase (0.5 mg/ml, 1 hour at 37°C, Sigma). After wash, the suspension was passed through a cell strainer to remove undigested block and wash in PBS with FCS (20%, Hyclone). Then, the suspension was incubated in trypsin (30 minutes at 37°C, Invitrogen), wash twice in PBS-FCS, counted, plated and incubated at 37°C in a humidified atmosphere of 95% air 5% C02. The first medium change was after 2 days and twice a week thereafter. The GFP fluorescence was checked after 1 and 2 weeks.
Data are presented as mean +/-SEM with statistical significance tested using the two tailed paired t-test or the Mann-Whitney test.