Normal skin was obtained from healthy donors undergoing plastic surgery procedures (mostly dermolipectomies), both men and women, excluding pregnant females. Umbilical cords were obtained from pregnant women after planned delivery through caesarian section. Tissues were obtained at the Department of Plastic Surgery and the Department of Obstetrics and Gynecology, respectively, at Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada. Tissue specimens were collected following the Declaration of Helsinki Principles, following Toronto Academic Health Sciences Network (TAHSN) and University of Toronto-affiliated Sunnybrook Research Institute and Sunnybrook Health Sciences Centre Institutional Ethics Review Board approval, and after getting patient signed informed consent.
Primary human normal skin fibroblasts and human WJ-MSCs were obtained from skin tissue samples and umbilical cords, respectively. Skin was dissected to remove any underlying fat, cut into small explant pieces of 2 to 4 mm, and cultured in small dishes. WJ-MSCs were isolated from umbilical cords by gentle dissection of previous sectioned small cord pieces, discarding the outer or epithelial layer, according to earlier described methods . Explants and MSCs were further subcultured in Petri dishes at a density of 3,200 cells/cm2 for 7 days before further passaging. When fibroblasts and/or WJ-MSCs reached 70% confluence, usually within 1 week, they were trypsinized with 0.05% trypsin/0.025% ethylenediamine tetraacetic acid v/v in preparation for subculture. Fibroblasts were subcultured in 75 cm2 tissue culture flasks at a density of 4,500 cells/cm2. Tissue culture plasticware were purchased from BD Falcon™ (Bedford, MA, USA), and all tissue culture media and supplements were obtained from Wisent Inc. (St-Jean-Baptiste, QC, Canada), unless otherwise stated. Fibroblast culture medium consisted of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution. WJ-MSC culture medium consisted of CMRL (Gibco, Carlsbad, CA, USA) with 10% FBS, 2% antibiotic–antimycotic solution and 1% l-glutamine. Media were changed every 48 hours. Collected tissues and cells were cultured at 37°C in a humidified atmosphere with 5% carbon dioxide.
Characterization of human Wharton's jelly-derived MSCs
The cells isolated from the Wharton’s jelly of the umbilical cord were examined to confirm their MSC characteristics. Flow cytometry for MSC cell surface markers (CD90+, CD73+, CD105+, CD45–, CD14–, CD34–, CD19– and HLA-DR–) was performed (Additional file 1). Cells were differentiated into the three main mesenchymal lineages – adipogenic, osteogenic and chondrogenic (Additional file 1). For adipogenic differentiation, cells were seeded at a density of 3,000 cells/cm2 in 24-well plates (BD) with low-glucose DMEM (Wisent Inc., St-Jean-Baptiste, QC, Canada) medium supplemented with 10% FBS, 1% antibiotic–antimycotic solution, 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Saint Louis, MO, USA), 10 μg/ml insulin (SAFC, Saint Louis, MO, USA), 60 μM indomethacin (Sigma-Aldrich), and 1 μM dexamethasone (Sigma-Aldrich). Cultures of cells in low-glucose DMEM medium supplemented with 10% FBS served as a negative control. Lipid accumulation was identified by oil red O staining: 0.3 g oil red O (Sigma-Aldrich) dissolved in 100 ml isopropanol (Sigma-Aldrich), diluted to 60% with distilled water.
For osteogenic differentiation, cells were also seeded at a density of 3,000 cells/cm2 in 24-well plates with low-glucose DMEM supplemented with 10% FBS, 1% antibiotic–antimycotic solution, 0.05 mM ascorbic acid-2-phosphate (Wako Pure Chemicals Industry Ltd, Osaka, Japan), 10 mM β-glycerophosphate (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich). Alizarin red staining (Sigma-Aldrich) was used to identify osteogenic cells (2 g alizarin red dissolved in 100 ml distilled water). For chondrogenic differentiation, cells were seeded in 15 ml polypropylene tubes (2 × 105 cells per tube; BD Falcon, Bedford, MA, USA) with low-glucose DMEM supplemented with 10% FBS, 1% antibiotic–antimycotic solution, 1 mM sodium pyruvate (Sigma-Aldrich), 0.1 mM ascorbic acid-2-phosphate (Wako Pure Chemicals Industry Ltd), 1% insulin–transferrin–selenium (Cellgro, Manassas, VA, USA), 100 nM dexamethasone (Sigma-Aldrich), and 10 ng/ml TGF-β3 (Shenandoah Biotechnology, Inc., Warwick, PA, USA). Chondrocyte pellets were identified with Safranin O staining: 0.1 g Safranin O (Sigma-Aldrich), dissolved in 100 ml distilled water.
Human normal fibroblasts and Wharton's jelly-derived MSC one-way paracrine signaling indirect co-culture
First, primary human fibroblasts were seeded into six-well plates (Grenier-Bio-One Cellstar, Frieckenhausen, Germany) at a density of 22,000 cells/cm2 with DMEM. To design a one-way indirect or paracrine co-culture system between human normal skin fibroblasts and WJ-MSCs, the WJ-MSCs were separately seeded at the same cell density in the upper three wells of a six-well plate with CMRL media; the lower three wells of the same six-well plate were filled with CMRL medium alone. When refreshing the media (once every other day), the six-well plate containing the fibroblasts was filled with the medium from the WJ-MSC six-well plate: the three upper wells (treatment wells) with the WJ-MSC-CM, and the three lower wells (control wells) with the CMRL media alone. On day 5 of culture, the amount of FBS in the medium was reduced from 10 to 2%, to avoid TGF-β1 false measurements. On day 7, total RNA extraction was started for further gene expression studies. Experiments were performed with low-passage cells (less than passage 5) and in triplicate (unless otherwise stated), on one set of cells from different patients.
RNA isolation and real-time quantitative polymerase chain reaction
For RNA isolation, cells were lysed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the RNeasy MicroKit was used (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s instructions. The total RNA yield was determined using a NanoDrop-2000 spectrophotometer (ThermoScientific, Waltham, MA, USA). cDNA was synthesized in a thermocycler (AB Applied Biosystems, Foster City, CA, USA), after mixing 10 μg RNA and a master mix prepared with the high-capacity cDNA synthesis reverse transcription kit (AB Applied Biosystems). Real-time polymerase chain reaction (PCR) was conducted using SYBR® Green PCR Master Mix (Applied Biosystems) to relatively quantify the mRNA transcript products of the following genes of interest: TGF-β1, TGF-β2, TGF-β3, plasminogen activator inhibitor-1, connective tissue growth factor, fibroblast growth factor-2, hypoxia-inducible factor-1α, VEGF, collagen I, collagen III and decorin. 18S was used as housekeeping gene. The primer sequences of the above genes used are listed in Additional file 2. Amplification and analysis of cDNA fragments were carried out using the StepOnePlus RT-PCR System (AB Applied Biosystems).
Relative gene expression was measured as the cycle threshold and was normalized with individual housekeeping gene control cycle threshold values. Quantitative PCR was loaded in duplicate, and cycle threshold values from triplicates of the same treatment group sample were averaged. The ΔΔCt method was used to report quantitative PCR results.
Proliferation assay: Ki67 antigen staining
Normal skin fibroblasts and WJ-MSCs were seeded in different eight-chamber culture slides at a cell density of 715 cells/cm2 and cultured for 7 days, following methods described above. Briefly, the media from the wells containing WJ-MSC (WJ-MSC-CM) were used to fill the wells from the normal skin fibroblasts from the treated group every other day, whereas wells of the control group were filled with WJ-MSC nonconditioned media.
Cells were washed with phosphate-buffered saline (PBS) and fixed for 15 minutes in 4% paraformaldehyde (Alfa Aesar, Karlsruhe, Germany). Fixed cells were washed in PBS and permeabilized for 10 minutes with PBS/0.5% Triton X-100 solution. After another washing step, cells were blocked for 30 minutes with 1% bovine serum albumin in PBS/0.5% Triton X-100. A monoclonal mouse anti-human Ki67 (1:100, clone MIB-1; Dako, Markham, ON, Canada) primary antibody was added and incubated overnight at 4°C. After washing with PBS, the secondary antibody was added in 1% bovine serum albumin in PBS/0.5% Triton X-100 and incubated for 1 hour at room temperature in the dark (Alexa Fluor 488 donkey anti-mouse, 1:500; Life Technologies, Eugene, OR, USA). After three final washes with PBS, slides were mounted with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Cells were examined and photographed using an Apotome Axiovert fluorescent imaging system at 10× magnification (Zeiss, Oberkochen, Germany). Three images were taken per well and two wells were imaged per treatment. Quantification was performed by counting the number of Ki67-positive cells in the high-power field as well as the total number of DAPI-positive nuclei. Data are presented as means with 95% confidence intervals of duplicate measurements for three different normal skin samples.
Terminal transferase TdT-mediated dUTP biotin end-labeling apoptosis assay
Normal fibroblasts cultured with WJ-MSC-CM and with nonconditioned medium were seeded into eight-chamber culture slides at a cell density of 3,000 cells/cm2 for half a week. The terminal transferase TdT-mediated dUTP biotin end-labeling apoptosis kit (Promega, Fitchburg, WI, USA) was used as per the manufacturer’s instructions.
Briefly, cells were fixed in 4% paraformaldehyde for 25 minutes at 4°C and washed with PBS. They were permeabilized with 0.25% Triton X-100 in PBS for 5 minutes, and then washed with PBS. Cells were incubated with equilibration buffer for 10 minutes, followed by labeling with terminal deoxynucleotidyl transferase reaction mix (10% nucleotide mix, 0.02% rTdT enzyme in equilibration buffer) for 1 hour at 37°C in humidified chamber. Cells were immersed in 2× SSC for 15 minutes to stop the reaction, followed by washes with PBS. Slides were mounted with Vectashield mounting medium with DAPI (H-1200; Vector Laboratories). Images were taken on an Apotome Axiovert fluorescent imaging system at 10× magnification (Zeiss); three images were taken per well. Quantification was performed by counting the number of terminal transferase TdT-mediated dUTP biotin end-labeling-positive and DAPI-positive nuclei, or apoptotic and alive cells, respectively.
Cell migration study: scratch wound assay
Normal skin fibroblasts were seeded in four-chamber culture slides at a cell density of 1,000 cells/cm2, with WJ-MSC-CM (or non-conditioned medium as control) for 48 hours. Two scratches were performed with a 200 μl pipette tip. After 24 hours, cells were fixed with 4% paraformaldehyde. The staining protocol followed the same procedure as the aforementioned proliferation assay. Briefly, cells were incubated with phalloidin antibody conjugated to fluorescein isothiocyanate (1:30; Invitrogen, Eugene, OR, USA) in blocking solution for 1 hour. Cells were washed three times with PBS and mounted with Vectashield mounting medium with DAPI. Images were taken on laser scanning META 510 confocal microscope (Zeiss) with 5× magnification. Three images were taken per scratch. Quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). A set area with a height of 0.5 mm and a width spanning the high-power field was placed in the center of the scratch, and the cells within this area were counted as the cells in scratch zone.
In vivo wound healing model
Eight BALB/c mice (13 weeks old, male, body weight 28 to 34 g) were obtained from Jackson Laboratory under the guidelines of the Sunnybrook Research Institute and Sunnybrook Health Sciences Animal Policy and Welfare Committee of the University of Toronto. Animal procedures were reviewed and approved by Sunnybrook Research Institute and Sunnybrook Health Sciences Centre at University of Toronto animal care and use committee. Animals were anesthetized and back cutaneous hair was removed by electrical shaving under anesthesia as stated in the Animal Protocol. Two pairs of 4 mm diameter full-thickness skin excisional wounds were created on each side of the midline. The animals were randomly divided into two groups: treatment (WJ-MSC-CM and Matrigel; BD Biosciences, San Jose, CA, USA) and sham (nonconditioned medium and Matrigel). Matrigel was of high concentration and was applied dropwise in liquid form and then allowed to gel. Each wound topically received 100 μl treatment or sham mix.
Wound measurements were taken and wound closure was examined in a timely manner on days 1, 3 and 7. Wounds with a complete re-epithelialization were considered healed wounds.
Mice were sacrificed at day 7, when skin biopsies including the wound/scar and 2 mm of satellite skin were harvested for further histologic analysis. Twenty-four hours before sacking, animals received an intraperitoneal injection of bromodeoxyuridine (BrdU) (Calbiochem, San Diego, CA, USA).
Tissue specimens were fixed in 10% buffered formalin overnight at room temperature, preserved in 70% ethanol and embedded in paraffin. Specimens were cut into 5 μm sections. Tissue specimens were cut simultaneously at different sites, the center or midline and both sides, eliciting a cross-section through the whole wound and satellite area. A serial section of the scar or healing wound was performed. The largest wound diameter or central wound section was stained for trichrome staining. Trichrome reagents were from EMS (Hatfield, PA, USA) unless otherwise stated. Briefly, paraffin-embedded slides were deparaffinized with citrosol, followed by rehydration through 100%, 95%, 70% and 50% ethanol to water. Slides were placed in Bouin’s solution (26367–01; EMS, Hatfield, PA, USA) overnight at room temperature and washed next. Hematoxylin stain (HHS16; Sigma, Saint Louis, MO, USA) and Biebrich scarlet-acid fuchsin solution were applied sequentially for 10 minutes. Washes were performed after each stain addition. Slides were differentiated in phosphomolybdic–tungstic acid for 15 minutes, and were transferred to aniline blue for 5 minutes. They were next rinsed and differentiated in 1% acetic acid for 2 minutes. Slides were dehydrated through 95% ethanol and absolute ethanol followed by clearing in citrosol. Slides were mounted with SHUR/Mount xylene-based liquid mounting media (Triangle Biomedical Sciences, Durham, NC, USA). Images were acquired using a Zeiss Axiovert 200 light microscope at 10× and 40× magnification. Quantification was carried out using merged 10× images to measure the wound bed and satellite area.
For immunohistochemistry staining, paraffin-embedded skin tissue slides were deparaffinized with xylene followed by rehydration. Antigen decloaker (1×; Biocare Medical, Concord, CA, USA) was added to the slides in a preheated decloaking chamber for 4 minutes at 110°C. For BrdU staining, samples were denatured with 1.5 N HCl for 30 minutes at 37°C and neutralized with 0.1 M borate buffered twice for 5 minutes. Samples were blocked with 3% H2O2 for 10 minutes, and then washed with washing buffer (0.05 M Tris–HCl, 0.15 M NaCl, 0.05% Tween 20 in deionized water). The primary antibody (mouse monoclonal anti-BrdU, 1:200; Cell Signaling, Beverly, MA, USA) was diluted in PBS and incubated at room temperature for 1 hour. Slides were then incubated for 15 minutes first with MACH3 mouse probe (Biocare Medical), and secondly with MACH3 rabbit or mouse horseradish peroxidase polymer, with before and after washes. The betazoid diaminobenzidine chromogen kit (Biocare Medical) was mixed and added for 5 minutes or until brown stain was noticeable. The reaction was terminated with running water. Nuclear staining was carried out with hematoxylin for 30 seconds, followed by differentiation with three dips in 1.5% acid alcohol and bluing in 0.1% sodium bicarbonate for 10 seconds. Sections were dehydrated through 95% and absolute ethanol to citrosol and mounted with SHUR/Mount as described previously. Images were acquired using a Zeiss Axiovert 200 light microscope at 10× magnification to image the whole section followed by 40× magnification to further focus on the wound margins and the wound center. The higher magnification images of BrdU staining were quantified by counting using ImageJ software, and normalized to the number of cells in the high-power field.
The statistical comparisons between the groups were performed using an unpaired Student’s t test with GraphPad Prism software (GraphPad, LaJolla, CA, USA). Two-tailed P ≤ 0.05 was considered significant. Data were graphically expressed as the mean of the target group ± the standard error of the mean or 95% confidence interval.