This project was approved by the Institution Review Board of King Saud University Medical College and Hospital (10-2815-IRB).
Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with D-glucose 4500 mg/L, 4 mM L-Glutamine and 110 mg/L Sodium Pyruvate, 10% Fetal Bovine Serum (FBS), 1x penicillin--streptomycin (Pen-strep) and Non essential amino acids (all purchased from Gibco-Invitrogen, USA). hMSC-TERT (bone marrow immortalized cell line) was kindly donated by Dr. Mosthafa Kassem, Department of Endocrinology and Metabolism, University Hospital of Odense, Odense, Denmark . Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza biotech (CC-2517; Walkersville, USA), it was cultured in vascular cell basal medium (ATCC; PCS-100-030) supplemented with microvascular endothelial growth factors (ATCC; PCS-100-041). Adipose tissue and adult dermal skin were received from patients undergoing abdominal bariatric surgery, lipectomy, knee replacement or gastro intestinal operations. Neonatal foreskins were received from voluntary circumcisions of 2-3 days male babies. All donors and/or their parents gave written informed consent for the use of their tissues for scientific purposes. All tissues were washed 3 times with PBS contain 1x Pen-Strep. The epidermis was manually removed from skin, and the dermis was cut into 1-3 mm pieces, placed in 3 cm culture dishes where epidermis layer facing up wards and the dermis layer contacting the culture surface with few drops of culture medium. Tissues were incubated at 37°C and 5% CO2 in a humidified environment. After few hours, and once the tissues were attached, the level of culture medium was raised and culture was maintained for a week or until the outgrowth of fibroblast-like spindle shaped cells was visible. Adipose tissues were minced mechanically then incubated in 1% Collagenase type I (Gibco-Invitrogen, USA) for 45 min with gentle agitation at 37°C. After inactivation of collagenase by culture medium DMEM, debris were separated from pellets of stromal vascular fraction (SVF) by centrifugation at 500 g for 15 min. SVF cells were resuspended in culture medium and plated in 25 cm2 tissue culture flask and maintained in a humidified incubator at 37°C and 5% CO2. The next day, all non adherent cells were removed by washing. In few days, the growth of MSCs was visualized under an inverted microscope. Cells were fed with fresh medium every 3-4 days until cells reached 70-80% confluency. Residual skins were removed from explant cultures and adherent cells were passaged from skin and SVF culture by standard trypsinization method (Trypsin-EDTA (0.05%); Gibco-Invitrogen, USA).
The Formalin-Fixed Paraffin-Embedded human adipose tissue, neonatal and adult skins were stained according to the manufacturer's staining protocol on a Bond-max™ fully automated IHC & ISH staining system (Leica Microsystems GmbH, Germany) and then stained with hematoxylin and eosin (H&E) and Vimentin (clone V9; 1:50), CD31 (clone 1A10; 1:50), CD34 (clone QBend/10; 1:50), Factor VIII (clone 36B11; 1:100), CD146 (abcam, USA; clone P1H12; 1:100) and smooth muscle actin (SMA; clone alpha SM-1; 1:50) using manufacturer's standard protocols (except CD146 all antibodies from Leica Biosystems Newcastle Ltd; UK). Slides were digitalized using High-resolution whole-slide digital ScanScope scanner (Aperio Technologies, Inc.). The digital slide images were then viewed and analyzed using the viewing and image analysis tools of Aperio's ImageScope software (Aperio Technologies, Vista, CA, USA).
Endothelial cell differentiation
To assess the endothelial differentiation potential, hMSC-TERT, ADMSCs, hNFSSCs and hADSSCs were cultured in 25 cm2 tissue culture flasks. When cells reached 70-90% confluency, medium was replaced with endothelial medium DMEM + 2% FBS, 1% Pen-strep, 50 ng/mL VEGF (R&D systems, USA), 10 ng/mL bFGF (Sigma-Aldrich, USA) and 50 μg/mL ECGS (endothelial cell growth supplement, BD Bioscience, USA) for 7 days. Medium was changed every 2 days.
Endothelial cell tube formation (in vitro angiogenesis)
In vitro matrigel angiogenesis assay was utilized to assess the tube-formation capabilities of hMSC-TERT, hADMSCs, hADSSCs, and hNFSSCs under normal and endothelial culture conditions. Matrigel matrix was thawed on ice at 4°C overnight, then 0.3 ml of chilled matrigel solution (10 mg/mL, Basement Membrane Matrix, BD Bioscience) was applied to one well in a 24-well plate using ice-cooled tips and incubated for 1 h at 37°C. After 7 days of endothelial inductions, both control and induced cells were trypsinized and platted on top of the matrigel-coated 24-well plates (2 × 104 cells per well) and were further incubated at 37°C in a 5% CO2 humidified atmosphere for 1-3 days. Tube formation assay was carried out along with HUVEC as a positive control. Tube formation was examined using an inverted phase-contrast microscope Carl Zeiss--Axio observer.1 equipped with a digital camera (Axiocam MRc5).
Immunophenotyping by flow cytometry
HUVEC and cells from induced and non-induced cultures (after 7 days) were harvested using 0.05% trypsin-EDTA and were washed twice in ice-cold PBS supplemented with 0.5% BSA and resuspended at 106 cells per ml. Ten microliter of PE-conjugated mouse anti-human CD146, CD73, CD29 and HLA-DR, FITC-conjugated mouse anti-human CD34, CD90, CD45, CD13, CD184 and CD31, or APC-conjugated mouse anti-human CD105, CD14 and CD44 antibodies (all from BD Biosciences, except the anti-human CD105, which was purchased from R&D systems) was added to 100 μl of cell suspension (105 cells). Negative control staining was performed using a FITC, PE, or APC-conjugated mouse IgG1 isotype control antibodies, respectively. Cells were incubated for 30 min at 4°C in dark, then were washed with PBS to remove excess antibodies, and then were resuspended in 500 μL of PBS and were analyzed using BD FACS Calibour flow cytometer (BD Biosciences). Living cells were gated in a dot plot of forward vs. side scatter signals obtained on linear scale. At least, 5,000 gated events were acquired on a Log fluorescence scale. Data were analyzed using Cell Quest Pro Software Version 3.3 software (BD Biosciences).
Adherent cells were fixed with 4% cold paraformaldehyde (Sigma) for 15 min and permeabilized with 0.1% Triton X-100 (Sigma) for 10 min. After washing with PBS, cells were blocked with 3% bovine serum albumin (BSA, Sigma) for 30 min, followed by incubating with primary antibodies against CD31 (5 μg/mL) VE-Cadherin (BV9; 1/25), eNOS (Endothelial Nitric Oxide Synthase; 5 μl/mL), vWF (von Willebrand Factor; 4 F9; 10 μl/mL) all are from Abcam (USA) and VEGF (VEGF 165; 8 μg/mL; US Biological) at 4°C overnight. After removal of primary antibodies, cells were washed three times with PBS, and the FITC-labeled secondary antibody (goat polyclonal to mouse or rabbit IgG; both 1/4000; Abcam) was added and incubated for 1 h at room temperature. Cells were washed three times with PBS and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear dye and were observed under Leica DM5000 B fluorescence microscope.
Cells were seeded at a density of 0.05 × 106 cell/ml in 6 well plates (for cytochemical staining and RNA isolation) and were grown for 24 h in standard DMEM growth medium. At 70-80% confluence, the medium was replaced in test wells by osteogenic induction medium supplemented with DMEM containing 10% FBS, 1% Pen Strep,50 μg/mL L-ascorbic acid (Wako Chemicals GmbH, Neuss, Germany), 10 mM β-glycerophosphate (Sigma), and 10 nM calcitriol [(1α,25-dihydroxy vitamin D3) (sigma)], 10 nM Dexamethasone (Sigma). The osteogenic medium was changed every 3 days and the experiments were terminated at day 15. Cells cultured in the regular culture medium were considered as experiment control.
Cells were seeded at a density of 0.05 × 106 cell/ml in 6 well plates (for cytochemical staining and RNA isolation) and were grown for 24 h in standard DMEM growth medium. At 90-100% confluence, the medium was replaced in all test wells with adipogenic induction medium supplemented with 10% FBS and adipogenic-induction mixture (AIM) containing 10% Horse Serum (Sigma), 1% Pen-strep, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine [(IBMX) (Sigma)], 3 μg/mL insulin (Sigma), and 1 μM Rosiglitazone [(BRL49653) (Novo Nordisk, Bagsvaerd, Denmark)]. The adipogenic medium was replaced every 3 days, and experiments were terminated at day 15. Cells cultured in regular medium were considered as experiment control.
Alkaline phosphatase (ALP) staining for osteoblasts
Cells were induced to osteoblasts differentiation for 15 days as described above, then cells were washed in PBS twice, fixed in acetone/citrate buffer 10 mM (1.5: 1) at pH 4.2 for 5 min at room temperature and incubated with alkaline phosphatase (ALP) substrate solution (naphthol AS-TR phosphate (Sigma) prepared 1:5 in water plus 10 mg Fast red TR (Sigma), in 24 mL of 0.1 M Tris buffer, pH 9.0) for 1 h at room temperature. Cells were rinsed with water, stored in PBS and photographed using Carl Zeiss--Axio observer.1 equipped with a digital camera (Axiocam MRc5).
Oil red-O staining for adipocytes
Oil red-O stain was utilized to assess the accumulation of cytoplasmic lipid droplets. Cells were differentiated into adipocytes for 15 days, washed twice in PBS, fixed in 4% formaldehyde for 10 min at room temperature, rinsed once in 3% isopropanol, and stained for 1 h at room temperature with filtered Oil red-O staining solution (prepared by dissolving 0.5 g Oil red-O powder in 60% isopropanol). Cells were rinsed with water then photographed using Carl Zeiss--Axio observer.1 equipped with a digital camera (Axiocam MRc5).
Quantitative real-time PCR for gene expression
To measure the expression level of endothelial, adipocyte and osteoblast-associated genes, total RNA was isolated using the FastLane cDNA kit (Qiagen) according to manufacturer's instructions. Cell lysate containing RNA from two independent treatments was combined and quantitated. All samples were normalized to the lowest concentration of RNA obtained. Complementary DNA (cDNA) was synthesized from 4 μL of the normalized RNA samples using FastLane cDNA kit according to the manufacturer's instructions. Relative levels of mRNA were determined from cDNA by real time PCR (Applied Biosystem-Real Time PCR Detection System) with QuantiFast SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions.
The sequence for PCR primers (all from Invitrogen limited, UK) were as follows (5'-3'): VEGFR2 (KDR/flk1; Fw: CTTACCCCAGGATATGGAG and Rev: CCGTCAAGGGAAAGACTACG), VE-Cadherin (CD144; Fw: CCCGTCTTTACTCAATCCACA and Rev: GGGTTTGATGATACCCTCGTT), FVIII (AHF; Fw: GTTCGTCCTGGAAGGATCGG and Rev: CACTGACACCTGAGTGAGAC) and VCAM-1 (CD106; Fw: TCTCATTGACTTGCAGCACC and Rev: TTCTTGCAGCTTTGTGGATG), β-actin (Fw: AGCCATGTACGTTGCTA and Rev: AGTCCGCCTAGAAGCA).
Adipocyte and osteoblast-associated genes
The sequence for PCR primers (all from Invitrogen limited, UK) were as follows (5'-3'): PPAR-γ 2 (Fw: CTCCACTTTGATTGCACTTTGG and Rev: TTCTCCTAT TGACCCAGAAAGC), aP2 (Fw: TGGTTGATTTTCCATCCCAT and Rev: GCCAGGAATTTGACGAAGTC), Adiponectin (Fw: ATGTCTCCCTTAGGACCAATAAG and Rev: TGTTGCTGGGAGCTGTTCTACTG), ALP (Fw: ACGTGGCTAAGAATGTCATC and Rev: CTGGTAGGCGATGTCCTTA), Osteocalcin (Fw: AGAGCGACACCCTAGAC and Rev: CATGAGAGCCCTCACA) and Osteopontin (Fw: GGTGATGTCCTCGTCTGTA and Rev: CCAAGTAAGTCCAACGAAAG). The relative abundance of target mRNA was quantified relative to the control β-actin gene expression from the same reaction setup, using the 2-ΔΔCT method .