Patient biopsies were performed in accordance with relevant government guidelines and approved by the institutional review board of the Southern Medical University. Each patient provided informed consent.
All experimental procedures using animals were performed in accordance with relevant guidelines and approved by the animal experimental committee of the Southern Medical University.
Isolation and culture of human mesenchymal stem cells (MSCs) and ECFCs
Primary human ASCs were isolated from white adipose tissue obtained during surgical liposuction procedures as previously described . ASCs were maintained in an adherent monolayer culture on untreated tissue culture plates for up to nine passages in MSC-medium (MSCGM (Lonza) supplemented with 10% FBS (Hyclone)). Human ECFCs were isolated from peripheral blood as described previously . ECFCs were cultured on 1% gelatin-coated plates using ECFC-medium (EGM-2 (Lonza) supplemented with hydrocortisone and 20% FBS. ECFCs were used for experiments after secondary colony formation (passage number 3-5). All cell cultures were maintained in a humidified incubator at 37°C with 5% CO2, unless otherwise stated.
3D cultures with HA gel
Prior to experimentation, HA gel was prepared by mixing HA powder and culture medium overnight in a plastic dish (Fig. 1A). The HA powder was a pharmaceutical grade product and non-cross-linked with an average molecular weight of 1,000 kDa (Kikkoman, Tokyo, Japan). Monolayer-cultures of ASCs at nine-passages (p0, p2, p4, p6, p8, p9) were trypsinized, and the cells were counted and resuspended in 1 ml DMEM culture medium containing 3% (w/v) HA gel at a density of 1-6x106/35mm dish, or 0.11-0.68x105/cm2. Suspended ASCs were incubated for 24-72 hours. In the presence of HA gel, 3D ASC aggregates formed in suspension.
To produce 3D co-cultured ASC/ECFC spheroids, 4 × 105 ASCs (passage 8) were co-cultured in suspension with 4× 104 ECFCs in the presence of 3% HA gel for 24 hours.
For flow cytometry analysis, suspended ECFCs and ASCs were filtered, washed and stained with the following antibodies and corresponding isotype controls: Anti-CD45-Cy5 (Miltenyi Biotec, Bergisch Gladbach, Germany), Anti-CD133-PE (eBioscience, Inc., CA, USA), Anti-CD31-FITC (BD Biosciences), Anti-CD105-FITC (BD Biosciences), Rat IgG2a Kappa cy5(BD Biosciences), and Rat IgG2a Kappa Control PE (BD Biosciences). Samples and controls were analyzed with a MACSQuant Analyzer 10 (Miltenyi Biotec). Each channel was gated respective to the control samples.
3D cultured ASCs, ECFC or ASC+ECFC (10% of ASCs) co-cultures were extracted from HA gels. Monolayer-cultured ASCs, ECFCs or ASC+ECFC (10% of ASCs) were seeded at a density of 1.0 × 105 cells in 6 cm2 plates and cultured in full growth medium for 24 hours. Subsequently, the medium was exchanged for serum-free DMEM, and each dish was incubated under normoxic (6% O2) conditions. After 48 hours, the culture media was collected and filtered through a 0.22-μm syringe filter prior to analysis. Commercially available Enzyme-linked immunosorbent assay (ELISA) kits (Ray Biotech, Inc., Norcross, US) for hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) were used. The absorbance was measured at 450 nm using an infinite microplate reader (Tecan Group, Männedorf, Switzerland).
Nude mice radiation-ulcer model and treatments
12-week-old male nude mice (BALB/cAJcl-FOXN1nu/nu) were purchased from Southern Medical University Experimental Animal Center (Guangzhou, China). Prior to experimentation, mice were anaesthetized by isoflurane inhalation. Anaesthetized mice were then placed in a lateral position and the dorsal skin pulled back to create a skin fold. The remaining body and tail was covered with a 3mm lead shield, leaving the dorsal skin fold unprotected, which was treated with 15 Gy ionizing radiation using an MX-160Labo X-ray radiation machine (Medixtec, Japan). Experimentally treated mice were housed separately for 4 weeks. To induce a chronic injury, a 8mm full-thickness cutaneous ulcer was created on the back of each mouse as described previously .
There were four groups: a. ASC+ECFC group: irradiated mice with cutaneous ulcer treated by 3D cultured hASC (product from 4 × 105 passage 8 hASCs+4× 104 hECFC, cultured in 3% HA gel for 24 hours;); b. ECFC group: monolayer cultured 4× 105 ECFCs suspended in 3% HA gel in DMEM; c. ASC group: monolayer cultured 4× 105 hASC (passage 8) suspended in 3% HA gel in DMEM; d. vehicle group:3% HA gel in DMEM. n = 3 mice/group and we photographed the wounds on days 0, 4, 7, 14 and 18.
DiI labeling of ASCs
ASCs (passage 8) were cultured in suspension in a petri dish with DMEM supplemented with 10% FBS. To label, ASCs were collected and suspended with 2 μM CM-DiI (Thermo Fisher Scientific) in Hank’s balanced salt solution (HBSS) at 37 °C for 5 minutes, followed by incubation at 4 °C for 15 minutes. Labelled ASCs were then re-plated back onto the petri dish for maintenance. The staining procedure was repeated daily for 2 subsequent days until the optimal cellular fluorescence was obtained.
3D spheroids were fixed and set in paraffin blocks as previously described. To prepare for immunolabelling, the paraffin blocks were sectioned with a cryostat, deparaffinized and rehydrated. To retrieve antigens, sections were incubated in target-retrieval solution followed by boiling water for 20 minutes. Sections were permeabilized and then blocked with 1% BSA buffer. All primary and secondary antibody solutions were prepared in TBST (0.1% Tween 20, 1% BSA). Sections were incubated with Guinea pig anti-Perilipin/PLIN1 (1:1000, Progen, Heidelberg, Germany) and goat anti- α smooth muscle actin (α-SMA, N-term, GeneTex, TX, USA) antibodies for 16 hours at 4 °C. After a series of washes, the sections were incubated with Isolectin IB4 Alexa Fluor dye conjugates (1:1000, Invitrogen) or Hoechst 33324 (1:1000 Thermo Fisher Scientific) for 1 hour. Stained sections were washed three times and then mounted. Stained sections were imaged with a fluorescent microscope. Vessel density/viable fat tissue quantification was based on α-SMA or perilipin labelling. A representative field of 500×500μm was imaged for each sample (n=5) in each treatment group. Fluorescent intensity was measured with ImageJ (imagej.nih.gov/ij/).
Wound size in the irradiated mouse model was compared by the Kruskal-Wallis test. The viability assay and fluorescent intensity experiments were analyzed by one-way ANOVA and Tukey’s HSD post-hoc. All statistical analyses were performed with the statistical program available at https://astatsa.com/. All statistical results were represented by the mean ± standard error. p-values < 0.05 were considered as statistically significant.