Molecular and Cell Biology Methods
Stem Cell Spheroid Culturing Conditions
Frozen hMSCs (P2, Lonza Germany) were re-suspended in 5 mL of serum free culture medium (Lonza, Germany) and centrifuged at 500 g for 5 minutes. Cells were then counted, cultured directly onto both tissue culture plate wells (TCP, Nunc, UK) and PhenoDrive (Tissue Click, UK) at a density of 7,000 cells/cm2 and maintained at 37°C in a humidified atmosphere containing 5% CO2 for 7 days.
Spheroid characterisation after culturing on different substrates
After 7 days of culture, the morphology of hMSCs was assessed periodically under a phase contrast microscope (Leica DM2500) with x10 objective lens; whereas their stem marker regulation was investigated by staining cells with primary antibodies against Oct4 and Nanog. These were previously treated with a solution of 1 % w/v bovine serum albumin (BSA, Sigma-Aldrich, UK) to block unspecific sites and immunolabelled with FITC or TRICT-conjugated secondary antibodies (Fisher-Scientific, UK) at a dilution of 1:100 at room temperature for 1 hour. Stained hMSCs were finally imaged with a laser scanning confocal microscopy (Leica TCS SP5, UK).
To calculate the average in number of hMSC spheroids and their diameter, phase contrast images were captured at lower magnification (100 µm) and analysed in NIH ImageJ. The measurements were replicated at least three times and data expressed as mean x standard deviation (n=9).
Image analysis of endothelial cell sprouting and mesenchymal stem cells co-culturing
Phase-contrast images of cells were acquired after 18 and 48 hours of culturing using Leica DM2500 and RM2135 with a 10 and 20x objective lenses. The formation of angiogenic sprouting was evaluated and quantified from these images by measuring the number of (i) tubule-like structures, (ii) their anastomosis-like junctions and (iii) their meshes using an Image J program, Angiosys 1.0 (TCS Cellworks, Buckingham, England). Each tube formation assay was performed as three independent experiments and data were expressed as mean ± standard deviation (SD, n = 9).
For immunofluorescent staining, the cells were fixed with chilled methanol for 10 minutes at -20˚C and washed twice with phosphate buffered saline (PBS, Sigma-Aldrich UK) before being incubated with PBS-Tween (0.05% v/v, Tween-20) and then 1% w/v bovine serum albumin (BSA, Sigma-Aldrich UK) for 1 hour at room temperature. Both HUVECs and HUVECs/MSCs were incubated with antibodies anti-human primary CD31, CD90, RhoA, Rac1, CXCR4 (1:100, Abcam UK) and HIF-1α (1:50, Abcam UK) at 4˚C overnight. Later, samples were incubated with either 488- or 594-conjugated secondary antibodies (1:100, Fisher Scientific UK) for 1 h at room temperature, dark conditions and their nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Fisher Scientific UK). Images were taken by a confocal microscopy (Leica TCS SP5) with 20x objective lens. To facilitate the identification of the different cell phenotypes, HUVEC 594-secondary antibody fluorescence was transformed by the confocal software into blue epifluorescence.
Cell cytoskeleton organisation of endothelial cells when in direct contact with the two different substrates was studied by Rhodamine-Phalloidin staining. Cells were fixed by adding warmed 4% (v/v) paraformaldehyde for 10 minutes and stained using Rhodamine-Phalloidin solution (1:100, Aldrich Sigma UK) in PBS for 1 hour at room temperature. They were then washed twice in PBS and cells at the tip of sprouting structures were analysed by a confocal microscopy as reported above.
Western blotting
After 18 and 48 hours incubation, HUVECs and HUVECs/MSCs were scraped off from PhenoDrive-Y, while those cultured on Matrigel were detachment by treatment with cell recovery solution (BD Biosciences UK) for 1 h at 4˚C. Cells were lysed for 10 min in lysis RIPA buffer supplemented with 50 µg/mL protease inhibitors (Aldrich-Sigma, UK) and then centrifuged at 12,000 x g for 10 minutes at 4°C. The protein concentration in the supernatants was measured using a Bradford reagent (Aldrich Sigma UK). Afterwards, 30 µg from each sample were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred on to a nitrocellulose filter membrane (Amersham UK) at 20 mV overnight. The membranes were treated with 0.01% w/v BSA and incubated with various primary antibodies [β1-integrin (1:500; R&D system UK); pAkt (1:1000, Abcam UK), VEGFR2 (1:1000, Abcam UK), EGFR (1:1000, Abcam UK); PDGFR-β (1:500, Cell Signalling UK) and H3K27me3 (1:1000, Cell Signalling UK) prior to incubation with HRP-conjugated secondary antibodies (1:1000, Fisher Scientific UK) for 1 hour at room temperature. ECL detection kit (Amersham UK) was then applied to detect protein bands which intensity was related to a positive control, GAPDH and quantified using ImageJ.
RNA extraction from cells using Qiazol
Utilize materials RNAse free. Work on ice with gloves.
Cells in suspension:
1) Centrifuge the cells, discard all the supernatant (keep the supernatant if needed for assays, otherwise discard) and add 800µl-1ml of Qiazol (from now on utilize filter tips) for each sample. Resuspend the pellet with the p1000. If the pellet does not get dissolved, utilize a 1ml syringe. The samples in Qiazol can be stored at -80°C or processed right away. Go to step 5.
Adherent cells:
1) Discard all the supernatant (keep the supernatant if needed for assays, otherwise discard).
2) Rinse the cells once with medium without serum (to wash away the serum-if the cells are cultured without serum skip this) at room temperature (1ml/well for 24 well plates). Carefully aspirate all the medium and discard.
From now on utilize filter RNase free tips
3) Add Qiazol (800µl-1ml for each sample, no more than 1ml/sample. If more wells need to be pooled, add Qiazol to each well so that the pooled sample is no more than 1ml, so that the extraction can be carried out in 1ml Eppendorf tubes. In 24 well plates, 400 µl/well of Qiazol is sufficient).
In a classical experiment in 24 well plates we usually pool 2 wells; we add 400 µl Qiazol/well so that we have 800 µl of Qiazol/sample.
4) The Qiazol lyses the cells completely, anyway it is better to mix several times with the p1000, then transfer to RNAse free Eppendorf tubes. The samples in Qiazol can be stored at -80°C or processed right away.
5) Add 160 µl of cold chloroform for 800 µl of Qiazol (200 µl for 1 ml). Mix manually until the solution is homogeneous (5-10 sec). It is very important to shake energetically to have good RNA extraction. Incubate on ice for 2-3 min.
6) Centrifuge the samples at 12000 x g for 15 min at 4°C. Transfer the upper phase (200 µl) in RNAse-free Eppendorf. Don’t touch the interfase (DNA and proteins).
At this point for primary cells, or when very little material is expected, add 1µl of glycogen/sample.
6) Add the same volume of cold isopropanol and shake gently inverting the Eppendorf tube. Incubate overnight at 4°C.
7) Centrifuge at 12000 x g for 30 min at 4°C. Transfer the tubes on ice. The RNA precipitate should be visible at the bottom of the Eppendorf tube. It is important to keep the tubes on ice so that the pellet does not move.
8) Discard all the supernatant (it is very important in order to have clean RNA to discard all the isopropanol. Aspirate with a p1000 and then with a p200 without touching the pellet).
9) Wash the RNA pellet: add 1ml of cold 75% ethanol, vortex briefly to detach the pellet (it should be seen floating), let the samples on ice for 5 min and centrifuge at 7500 x g for 15 min at 4°C.
10) Discard all the supernatant and let the RNA pellet dry completely (leave the tubes open on the bench at room temperature until the ethanol disappears completely).
11) Resuspend the RNA in RNAse free water (15 µl) pipetting several times and scratching the bottom of the Eppendorf tube. Incubate the samples at 60°C for 10min, then transfer the samples on ice.
12) Calculate the RNA concentration measuring the absorbance at 260 nm (1 OD = 40µg RNA/ml). The RNA quality can be checked measuring the ratio A260/A280 that should be 1.8-2.0.
13) Store the RNA at – 80 °C.
Reagents:
Utilize RNAse-free material
Eppendorf tubes PCR clean (Eppendorf 0030123328; Fisher 10398031)
Sterile serological pipettes individually wrapped
Filter tips (Starlab or AlphaLabs)
Qiazol (QIAGEN code 79306).
Chloroform without isoamyl alcohol (Sigma C2432-500ML).
Isopropanol (Sigma I9516-500ML).
Glycogen ultrapure Invitrogen (Fisher 10757844).
Absolute ethanol (Sigma E7023-500ML; dilute to 75% with RNAse-free water).
RNAse-free water Hyclone (Fisher 10787944)
