The authors have declared that no competing interests exist.
Conceived and designed the experiments: ISP PSC JCA. Performed the experiments: ISP. Analyzed the data: ISP JCA. Contributed reagents/materials/analysis tools: ISP PSC JCA. Wrote the paper: ISP.
We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.
Formation of new blood vessels, either by angiogenesis or by vasculogenesis, is critical for normal wound healing. Angiogenesis aids in the repair of damaged tissue by regenerating blood vessels and thus improves blood flow in chronic, disease-impaired wounds [
Human adipose-derived mesenchymal stem cells (hASCs), which are found in adipose tissue, provide an attractive source of cell therapy for regeneration of damaged skin because they are able to self-renew and are capable of differentiating into various cells [
Low-level light irradiation (LLLI) has been implemented for various purposes for some time, such as to provide pain relief, to reduce inflammation, and to improve local circulation. Moreover, many studies have demonstrated that LLLI has positive biostimulatory effects on stem cells [
Several experimental strategies for endothelial differentiation of stem cells have been developed, including 2D-cell culture in EC growth medium containing VEGF and FGF, 3D spheroid culture on substrates with immobilized polypeptides, and genetic modification of stem cells [
In this study, LLLI was used to promote a hypoxic spheroid of hASCs (which we refer to as a ‘spheroid’) by weakening cell-matrix adhesion. Differentiation and secretion of FGF and VEGF growth factors were also enhanced by LLLI. hASCs can differentiate into ECs without EC growth medium containing VEGF and FGF. The vascularization and potential therapeutic efficacy of ASC spheroids treated with LLLI (L-spheroid) were evaluated by injecting spheroids into a mouse excisional wound splinting model.
The hASCs were supplied by Cell Engineering for Origin, CEFO (Seoul, Korea) under a material transfer agreement. hASCs were isolated from the adipose tissue and were cultured in low-glucose Dulbecco's modified Eagle's medium F-12 (DMEM/F-12; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, Welgene), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator. The hASCs between passage 5 and 8 were used for all experiments.
hASCs were split and seeded on 24 well polystyrene plate (low cell binding surface) at a density of 7.5 × 104 cells/cm2,andallowedtoadhereat37°C. Within 3 days of culture, hASCs formed spheroids by Low-Level light irradiation (L-spheroids). The light source used was LED (light emitting diode; WON Technology Co., Ltd., korea) designed to fit over a standard multi-well plate (12.5 × 8.5 cm) for cell culture. The LED was had an emission wavelength peaked at 660 nm. The irradiance at the surface of the cell monolayer was measured by a power meter (Orion, Ophir Optronics Ltd., UT). To obtain the energy dose of 6 J/cm2,exposure time for LED array was 10 min under power density of 10 mW/cm2 (1 milliwatt × second = 0.001 joules) (
Frequency of irradiation | 10 min during 3days | 10 min daily from day 1 to 20 |
Light irradiated site | 24 well cell culture plate (12.5 × 8.5 cm) | 8-mm (wound site) |
Distance from the LED light | 10 cm | 8 cm |
Power density | 10 mW/cm2 | 50 mW/cm2 |
Light dosage (Fluence) | 6 J/cm2 | 30J/cm2 |
Irradiance (Wavelength) | 660 nm | 660 nm |
After 3 days of culture, the cell viability of the spheroids was analyzed by using a live/dead viability cytotoxicity assay kit (Molecular probes, Carlsbad, CA). Briefly, 1 ml of HEPES-buffered saline solution (HBSS) containing 2 μl of SYTO 10 green fluorescent nucleic acid stain solution and 2 μl of red (ethidium homodimer-2) nucleic acid stain solution were added to plates, and these were then incubated at 37°C in a 5% CO2 incubator for 15 min. The negative control was prepared by freezing cells at -80°C for 30 min. Images were quantified by using the ImageJ software (NIH, Bethesda, MD), and the percentage of live/dead cells was scored by counting pixels in each image.
The cells were washed with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) and were stained in PBS containing 1% BSA, with either isotype controls or antigen specific antibodies, for 60 min. CD34 (BD Biosciences, San Jose, CA), KDR (Beckman Coulter, Brea, CA), CD31 (Beckman Coulter), CD45 (Abcam, Cambridge, MA), CD90 (BD Biosciences), CD105 (Caltac Laboratories, Burlingham, CA), and CD29 (Millipore, Waltham, MA) human antibodies were used. The cells were washed three times with PBS containing 0.5% BSA and were resuspended in PBS for flow cytometry using an Accuri device (BD Biosciences). The isotype IgG was used as a negative control.
To analyze the expression profiles of angiogenesis-related proteins, we used a Human Angiogenesis Array Kit (R&D Systems, Ltd., Abingdon, UK). Cell samples (5 × 106 cells) were harvested, and 150 μg of protein were mixed with 15 μl of biotinylated detection antibodies. After pre-treatment, the cocktail was incubated with the array overnight at 4°C on a rocking platform. Following washing to remove unbound material, streptavidin–horseradish and chemiluminescent detection reagents are added sequentially. The signals on the membrane film were detected by scanning on an image reader LAS-3000 (Kodak, Rochester, NY) and were quantified using the MultiGauge 4.0 software (Kodak). The positive signals seen on developed film were identified by placing a transparency overlay on the array image and aligning it with the two pairs of positive control spots in the corners of each array.
Angiogenic growth factor production in the spheroid was assayed with a commercially available ELISA kit (R&D Systems) according to the manufacturer’s protocols. The concentrations are expressed as the amount of angiogenic growth factor per 104 cells at a given time.
Indirect immunofluorescence staining was performed using a standard procedure. In brief, tissues cryosectioned at a 4-μm thickness were fixed with 4% paraformaldehyde, blocked with 5% BSA/PBS (1 h, 24°C), washed twice with PBS, treated with 0.1% Triton X-100/PBS for 1 min, and washed extensively in PBS. The sections were stained with specific primary antibodies and fluorescent-conjugated secondary antibodies (
Antibody | Host | Company | Catalogue number |
---|---|---|---|
anti-human CD29 | mouse | Millipore | MAB2253Z |
anti-human Flk-1 | mouse | Santa Cruz | Sc-6251 |
anti-human CD34 | mouse | Millipore | MAB4211 |
anti-human CD31 | mouse | Dako | M0823 |
anti-human CD31 | rabbit | abcam | ab76533 |
anti-human CD45 | mouse | abcam | ab82595 |
anti-human CD90 | mouse | BD biosciences | 555595 |
anti-human CD105 | mouse | Caltac Laboratories | MHCD10500 |
anti-human αSMA | mouse | Dako | M0851 |
anti-Caspase 3 | rabbit | abcam | ab4051 |
anti-human nuclei | mouse | Millipore | MAB1281 |
HIF-1 alpha | rabbit | Novus | NB100-134 |
anti-human FGF | rabbit | abcam | ab8880 |
anti-human VEGF | rabbit | abcam | ab52917 |
anti-human HGF | rabbit | Santa Cruz | Sc-13087 |
Alexa Fluor 488 anti-mouse IgG | goat | Invitrogen | A11001 |
Alexa Fluor 594 anti-rabbit IgG | goat | Invitrogen | A11012 |
Samples were harvested 14 days after treatment. Specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated in a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4 μm-thick sections and were stained with hematoxylin and eosin (H&E) to examine muscle degeneration and tissue inflammation. Masson’s trichrome collagen staining was performed to assess tissue fibrosisin ischemic regions. The criteria used for the histological scores of wound healing were modified from previous reports [
Scores | Re-epithelializtion | Dermal regeneration | Granulation tissue formation | Angiogenesis |
---|---|---|---|---|
1 | Minimal epidermal regeneration (<50%) | No skin appendage formation | Thin granulation around wound edges only | Little angiogenesis (<10 vessels/HPF) |
2 | Moderate epidermal regeneration (50%) | A few skin appendage formation (<5 appendages/ wound area) | Moderate granulation in the wound bed | Moderate angiogenesis (10–20 vessels/HPF) |
3 | Complete epidermal regeneration (100%) | Considerable skin appendage formation (>5 appendages/ wound area) | Thick granulation in 100% of the wound bed | Marked newly formed and well-structured capillary vessels (>20 vessels/HPF) |
Samples were solubilized in lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, and 2 μg/ml aprotinin] for 1 h at 4°C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4°C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90°C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HIF-1a (Novus), CD31 (Abcam), HGF (Santa cruz), VEGF (Abcam), and FGF2 (Abcam) for 1h at room temperature (
The animal studies were approved by the Dankook University Animal Use and Care Committee. Five-week-old male BALB/c nude mice (20 g body weight; Narabio, Seoul, Korea) were anesthetized with ketamine (100 mg/kg). After aseptically preparing the surgical site, two full-thickness skin wounds were created on the dorsal part using an 8-mm biopsy punch. To inhibit wound contraction, a 0.5-mm thickness silicone splint was applied, as has been previously described [
Light emitting diode (LED; WON Technology, Daejeon, Korea) was applied for 10 min daily from day 1 to 20. The distance from the LED to the skin flap was 8 cm. This LED model exhibited an irradiated wavelength of 660 nm and power density of 50 mW/cm2. The fluence of each flap site was 30 J/cm2 (1 milliwatt × second = 0.001 joules) (
The wounds were photographed using a digital camera at 3, 7, and 14 days after surgery, and the wound area was measured by tracing the wound margin and then performing the calculation using the Image J image analysis program (NIH, MD, USA). The wound area was analyzed by calculating the percentage of the current wound with respect to the original wound area. The wound was considered to be completely closed when the wound area was grossly equal to zero.
All quantitative results were obtained from triplicate samples. Data were expressed as a mean ± SD, and the statistical analyses were carried out using two-sample t tests to compared two groups of samples and a One-way Analysis of Variance (ANOVA) for the three groups. A value of p < 0.05 was considered to be statistically significant.
hASCs obtained from human adipose tissue were expanded
A wound scratch test showed that the migration of LLLI-treated hASCs markedly improved relative to that of control cells cultured without LLLI after 24 hours (
hASCs were cultured on non-tissue culture-treated 24-well plates in the presence of FBS and formed a floating spheroid after irradiation with low-level light (
(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis and quantification of HIF1-α in hASCs cultured as spheroids, L-spheroids and monolayers (*
The endothelial phenotype of the L-spheroid cells was also evaluated via immunofluorescent staining for a variety of endothelial cell surface markers. First, the surface markers of hASCs expanded in DMEM-F12/FBS were examined. The cells expressed CD29 (β1 integrin), CD90 (Thy-1), and CD105 (endoglin), as well as MSC surface antigens, but not CD34, CD31, or KDR (
(A) Monitoring cell surface markers via immunofluorescence staining. L-spheroids cultured for 3 days were cryosectioned and stained with anti human CD34, CD31, and KDR antibodies. Scale bar = 500 μm. (B) Flow cytometry analysis.
After 14 days, fluorescence microscopy was used to identify caspase 3-positive cells and HNA-positive cells throughout the wound bed to determine whether locally transplanted ASCs were incorporated into the healing wound. In the L-spheroid and L-spheroid + LLLT groups, ASCs were observed in the regenerated skin tissue (
(A) For the ASCs, L-spheroid and L-spheroid + LLLT groups, DAPI (blue) and caspase 3 (apoptotic marker; red)-positive cells were detected after immunostaining at 14 days. The hASCs were stained with HNA (green). Apoptosis of transplanted hASCs (arrows) was reduced in the L-spheroid + LLLT group. (B) The ratio of HNA-positive cells (transplanted hASCs) to DAPI-positive cells (total cells) in the wound bed (
Transplantation of hASCs into the wound bed enhanced the paracrine secretion of angiogenic growth factors. Double immunofluorescent staining of HNA and human angiogenic growth factors bFGF, VEGF, and HGF indicated the presence of secretion from transplanted hASCs in the ASC or spheroid group (
(A) Immunostaining was performed with anti-bFGF and anti-VEGF or anti-HGF antibody (red) at 14 days. The scale bar indicates 100 μm. (B) Western blot indicated the expression of bFGF, VEGF, and HGF at 14 days. The results of the Western blot were analyzed as relative density (
Many of the CD31+ cells in the L-spheroid + LLLT group were double stained for smooth muscle actin (SMA). ECs and perivascular cells differentiated from injected human cells were detected via αSMA and human CD31 antibodies, respectively (
(A) The implants were removed on day 14 after transplantation and were stained with anti human CD31 and human αSMA antibody. The scale bars indicate 200 μm. (B) Vessel density in the wound bed (
To determine whether the L-spheroid ASCs could contribute to the epidermal structure, immunohistochemistry for pan-cytokeratin was performed at 14 days (
Immunofluorescence images show cytokeratin-positive epithelial cells (red) at 14 days. The scale bar indicates 20 μm.
An excisional wound splinting model was prepared, and the silicon splints remained tightly adherent to the skin and restricted wound contraction during the experimental period (
(A) The prepared excisional wound splinting model. (B) Photographs of the wounds. (C) The percentage of the wound area was calculated using photographs of the wounds at 1, 7, and 14 days. *
The formation of spheroids is affected by the cell-matrix adhesion strength [
Clinical studies of stem cell transplantation have raised several questions concerning cell therapy. The density and complexity of vascular networks formed by the synergistic dual cell system were reported to be was many times greater than those observed with ASC- containing and EC- or SMC-containing implants [
hASC L-spheroids transplantation accelerates tissue regeneration through the differentiation of ECs and through growth factor secretion. We emphasize the significance of the application of a 3D spheroid culture of stem cells with LLLI to achieve a high-ratio of EC differentiation of hASCs and to enhance treatment efficiency of L-spheroid transplantation relative to single-cell transplantation in the wound bed. These results may provide more effective therapeutic methods to treat delayed skin regeneration.
hASCs (passage 4) were stained with CD29, CD90 and CD105 for mesenchymal stem cell identification, with KDR, CD31 and CD34 for endothelial lineage cell identification, and SMA for smooth muscle cell identification. Scale bar: 200 μm (B) Flow cytometry analysis; hASCs cultured for 1 days were stained for CD29, CD90, CD105, CD45, CD31, CD34 and KDR expression and analyzed by flow cytometry.
(TIF)
LLLI treated hASCs scratch wound at 24 h (*,
(TIF)
The middle-section was 500 μm from the L-spheroid surface. Live cells were stained by calcein AM (green), and dead ones were stained with ethidium homodimer (red). Scale bar: 500 μm.
(TIF)