Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium
- Woojune Hur†1, 2,
- Hoon Young Lee†1,
- Hye Sook Min3,
- Maierdanjiang Wufuer1, 2,
- Chang-won Lee4,
- Ji An Hur5,
- Sang Hyon Kim6,
- Byeung Kyu Kim1, 2 and
- Tae Hyun Choi1Email author
© The Author(s). 2017
Received: 25 June 2016
Accepted: 24 February 2017
Published: 20 April 2017
Fibroblasts are ubiquitous cells in the human body and are absolutely necessary for wound healing such as for injured skin. This role of fibroblasts was the reason why we aimed to differentiate human adipose-derived stem cells (hADSCs) into fibroblasts and to test their wound healing potency. Recent reports on hADSC-derived conditioned medium have indicated stimulation of collagen synthesis as well as migration of dermal fibroblasts in wound sites with these cells. Similarly, human fibroblast-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important for growth of skin. However, it remains unknown whether and how F-CM can stimulate hADSCs to secrete type I collagen.
In this study, we obtained F-CM from the culture of human skin fibroblast HS27 cells in DMEM media. For an in-vivo wound healing assay using cell transplantation, balb/c nude mice with full-thickness skin wound were used.
Our data showed that levels of type I pro-collagen secreted by hADSCs cultured in F-CM increased significantly compared with hADSCs kept in normal medium for 72 h. In addition, from a Sircol collagen assay, the amount of collagen in F-CM-treated hADSC conditioned media (72 h) was markedly higher than both the normal medium-treated hADSC conditioned media (72 h) and the F-CM (24 h). We aimed to confirm that hADSCs in F-CM would differentiate into fibroblast cells in order to stimulate wound healing in a skin defect model. To investigate whether F-CM induced hADSCs into fibroblast-like cells, we performed FACS analysis and verified that both F-CM-treated hADSCs and HS27 cells contained similar expression patterns for CD13, CD54, and CD105, whereas normal medium-treated hADSCs were significantly different. mRNA level analysis for Nanog, Oct4A, and Sox2 as undifferentiation markers and vimentin, HSP47, and desmin as matured fibroblast markers supported the characterization that hADSCs in F-CM were highly differentiated into fibroblast-like cells. To discover the mechanism of type I pro-collagen expression in hADSCs in F-CM, we observed that phospho-smad 2/3 levels were increased in the TGF-β/Smad signaling pathway. For in-vivo analysis, we injected various cell types into balb/c nude mouse skin carrying a 10-mm punch wound, and observed a significantly positive wound healing effect in this full-thickness excision model with F-CM-treated hADSCs rather than with untreated hADSCs or the PBS injected group.
We differentiated F-CM-treated hADSCs into fibroblast-like cells and demonstrated their efficiency in wound healing in a skin wound model.
KeywordsHuman adipose-derived stem cell-derived conditioned medium Type I collagen Wound healing Cell transplantation
Loss of the dermis in extensive full-thickness wounds such as burns, massive avulsion injuries, septic skin necrosis, or extended excision of scars, not completely solved by the application of split-thickness autografts, poses a serious problem [1, 2]. Conventional split-thickness skin grafts, often widely meshed and expanded, are utilized to close large wound deficits .
Recently, it has been recognized that fibroblasts can play an important role in skin regeneration and augmenting healing of wounds through their implantation . However, for autogenic cells, being present in insufficient numbers limits their usage. As an alternative, allogeneic cells pose ethical and safety issues when considered for skin wound repair . After full-thickness dermal injuries it is important to have an effective dermal replacement, because dermal tissue does not regenerate into normal dermis in vivo . For these reasons, although still in the research stage, cells offer a more favorable choice for skin regeneration or wound healing than transplantation of dermal substitutes [4, 7], and stem cell therapy is clinically being investigated as a safe and effective method for repair of several types of tissue damage [8–10].
Wound healing is a well-orchestrated process that can be divided into three overlapping phases, beginning with the inflammatory phase, followed by the proliferative phase, and concluding with the remodeling phase . Several cell types of mesenchymal origin have been implicated in these processes. These include fibroblasts, fibrocytes, and myofibroblasts that play critical roles in both early and late phases, where they contribute to the wound contraction, collagen deposition, and finally fibrosis [12, 13]. Collagens are extracellular matrix (ECM) proteins that are found in nearly all eukaryotic organisms except for plants and protozoa . There are approximately 27 different types of collagens that have been identified, with type I collagen being the most prevalent; type I collagen is found in vertebrate connective tissues such as tendons, ligaments, bone, skin, and the cornea of the eyes .
Recently, articles concerning human adipose-derived stem cell-derived conditioned medium (hADSC-CM) have reported on its ability to stimulate collagen synthesis as well as migration of dermal fibroblasts. hADSCs are known to promote wound healing mainly through a paracrine mechanism, and it is plausible that ADSCs may exert their effect by secreting cytokines and growth factors that act on neighboring cells to repair the damaged tissues [16, 17]; thus the enriched media from these cells may be a good source of tissue repair factors. As such, hADSC-CM stimulated both collagen synthesis and migration of dermal fibroblasts, which improved the appearance of wrinkles and accelerated wound healing in animal models [18–20].
Similarly, human fibroblast (HS27)-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important in the growth of skin [21–23], and from research on hADSCs it is still not clear how F-CM stimulates hADSCs to secrete type I collagen. Our study goals were to investigate whether F-CM-treated hADSCs could be efficient in wound healing in an in-vitro model, and whether regeneration of full-thickness skin defects could be achieved by having the hADSCs transdifferentiate into fibroblast-like cells by F-CM in vivo.
hADSC isolation and culture
Discarded tissue from the abdomen was collected as skin and fat samples from Seoul National University Hospital (SNUH) with the written consent from the patients prior to surgery. The study was approved by the SNUH Ethics Committee (approval IRB No. 1108-098-374).
Human subcutaneous adipose tissue samples were separated from blood vessels, hair, and excess fat, and cut into small pieces prior to digestion with collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) under gentle agitation for 1 h at 37 °C. The samples were then filtered with 70-μm mesh filters, and mixed with low-glucose DMEM supplemented with 10% fetal bovine serum (FBS; Gibco/Thermo Fisher, Waltham, MA, USA) and 1% penicillin/streptomycin solution (P/S; Gibco), and then centrifuged at 300 × g for 20 min. The ADSC fraction was washed with Hank’s balanced salt solution (HBSS) and centrifuged at 300 × g for 10 min; the supernatant was discarded. The cell pellet was resuspended in DMEM supplemented with 10% FBS and cultured in a humidified 5% CO2, 37 °C incubator. The culture medium was changed every 2 days.
Preparation of F-CM
To obtain F-CM, human skin fibroblast HS27 cells (CRL-1634, 5 × 105 cells; ATCC, Manassas, VA, USA) were cultured in high-glucose DMEM (Invitrogen-Gibco/Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS and 1% P/S. After reaching 80% confluency, the normal grown medium was discarded and the cells were washed twice with phosphate-buffered saline (PBS; 3 M, USA). Serum-free high-glucose DMEM supplemented with 1% P/S was added to HS27 cells and the cells were continued for culture at 37 °C and in a humidified atmosphere containing 5% CO2. After incubation for 2 days, the culture medium was collected and centrifuged at 300 × g for 5 min, and then filtered through a 0.2-μm syringe filter (Millipore, Billerica, MA, USA) for later use. The cell experiments were carried out with early passage (passage 1–5) cells.
Western blot analysis
Antibody information for western blot analysis
Antibody size (kDa)
Santa Cruz, USA
Cell Signaling Technology, USA
Santa Cruz, USA
Cell Signaling Technology, USA
Cell Signaling Technology, USA
Cell Signaling Technology, USA
Cell Signaling Technology, USA
Pro-collagen type I
Santa Cruz, USA
Cell Signaling Technology, USA
Sircol collagen assay
The manufacturer’s protocol was followed for the Sircol collagen assay (SCA) (Biocolor Life Science Assays, UK). In brief, 200 μl of conditioned medium was added to 1 ml of the colorimetric reagent and centrifuged for 30 min at 5000 × g for 20 min. The sample was released from the pellet with an alkali reagent (1.5 N NaOH) and was measured at 500 nm on a high-performance monochromator multimode microplate reader (BMG Labtech, Offenburg, Germany). Absolute values were obtained with a standard graph composed from collagen type I standard, which was supplied with the kit in the range of 5–100 μg/ml.
FACS analysis was performed for characterization of hADSCs, HS27 cells, and F-CM-treated ADSCs after differentiation for 72 h. The cells in each group were incubated with FITC-conjugated antibodies for CD14, CD19, CD34, CD45, and CD105 and PE-conjugated antibodies for CD13, CD54, and CD73 (all from BD Pharmingen/BD, Franklin Lakes, NJ, USA) for 30 min at room temperature. As control, the cells were stained with FITC-isotype control IgG and PE-isotype control IgG (BD Pharmingen). Cells were subsequently washed twice with FACS buffer and analyzed on a FACScan flow cytometer (Becton Dickson, Franklin Lakes, NJ, USA) using the CellQuest Pro software (Becton Dickson).
Primers for RT-PCR analysis
Forward: CCT CCT CCC ATC CCT CAT
Reverse: GGA TGG GCA TCA TGG AAA
Forward:CCC TCC AGG TGG TGG
Reverse:GGC CTT GGAAGC TTA
Forward: CTC CGG GAC ATG ATC AGC
Reverse: AGT GCT GGG ACA TGT GAA
Forward: AGG ATG TTG ACAATG CGT
Reverse: ATC GAT TTG GAC ATG CTG
Forward: AGC AGC AAG CAG CAC TAC
Reverse: AAT TTC TCG TCC CAG TGT
Forward: CAG ACC TAC TCT GCC CTC
Reverse: GAT CAT CAC CGT CTT CTT
Forward: TCC ACA TGC TTT ATT CCA
Reverse: TGG CAC CCA GCA CAA TGA
For staining collagen, tissue sections were cut and mounted on slides. Using the Discovery XT Automated Immunohistochemistry Stainer (Ventana Medical Systems Inc., Tucson, AZ, USA), the slides were incubated in DAB + H2O2 substrate for 8 min at 37 °C, followed by hematoxylin/Bluing Reagent counterstain at 37 °C. Reaction buffer (pH 7.6 Tris buffer) was also used for washing. The 12-μm thick sections were mounted on slides precoated with 10% poly-l-lysine (Sigma-Aldrich), and were blocked with goat serum for 1 h, followed by incubation with primary rabbit antibodies to anti-collagen type I (rabbit polyclonal, 1:40; Sigma-Aldrich) at 4 °C overnight. After washing with PBS, the sections were allowed to incubate with secondary antibodies conjugated with fluorochrome TRITC (goat anti-rabbit, 1:250; Sigma-Aldrich) at room temperature in the dark for 2 h. Following further PBS washes, the sections were mounted in fluorescent mounting medium and observed under a confocal microscope.
Full-thickness skin wound model and cell transplantation
In the in-vivo wound healing assay using cell transplantation, balb/c nude mouse (male; 11 weeks old; weighing 20 ± 3 g) were fed and maintained under a 12-h light/dark cycle at 22–25 °C with 55–60% humidity. All of the conditions including diet, drinking, and defecation were normal. All experimental protocols were approved in strict accordance with the guidelines for the SNUH Institutional Animal Care and Use Committee (IACUC No. 13-0292-C0A3).
Wound healing analysis
To evaluate whether injected F-CM-treated cells enhanced wound healing in a full-thickness skin wound by repair involving a dense cell structure and secreted collagen, we sacrificed the animals 12 and 24 weeks after cell injection. The specimens were fixed in 10% buffered neutral formalin (v/v) for 1 day. After washing with tap water, the specimens were deparaffined in xylene and dehydrated in graded ethanol series (80–100%), and embedded in paraffin. The sample tissues were sectioned at 6–7 μm thickness and were stained with hematoxylin and eosin (H&E). The sections were examined under light microscopy (DMLA; Leica, Wetzlar, Germany).
All data are presented as mean ± SE, and the statistical difference between each group was assessed by Kruskal–Wallis analysis with pairwise comparisons using a Tukey’s post-hoc test (GraphPad Prism, version 5.01; GraphPad). P < 0.05, P < 0.01, and P < 0.001 were considered statistically significant as specified for the measurement.
Increased expression of type I collagen in hADSC conditioned media by F-CM in vitro
Moreover, the amount of total collagen measured by the Sircol assay indicated a similar pattern as western blot analysis occurring at about baseline concentration density (Fig. 3b).
Characterization of hADSCs, HS27 cells, and F-CM hADSCs
hADSCs have been well characterized with cluster of differentiation (CD) markers and along with mesenchymal stem cells they express CD105, CD73, and CD90 and lack the expression of hematopoietic lineage markers c-kit, CD14, CD11b, CD34, CD45, CD79, CD19, and HLA-DR [24–27].
Differentiation of hADSCs into fibroblast-like cells by F-CM in vitro
Mechanism of pro-collagen type I increase in F-CM-treated hADSCs in vitro
Wound healing on animals in vivo
At 2 weeks post implantation of cells, the F-CM-treated hADSC injection group wound samples showed an increase in dermal thickness with little fibrosis. In addition, the follicle densities were no different between the wounded skin and the normal skin (Fig. 10). However, for the wound samples in the untreated hADSC and PBS injection groups, there was a slight reduction in the follicle density and a slight shrinkage in the structure of dermis. In general, when inflammation is very severe and wound healing is lengthy, there is abundant fibrosis. A paucity of fibrosis means rapid wound healing, leading to a normal skin structure. Abundant fibrosis causes dense scarring, and the dermis structure will disappear following a reduction in follicle densities. An increase in levels of collagen type I in the F-CM-treated hADSC injection group (82.12 ± 2.31) was significantly evident with intense staining in brown rather than the untreated hADSC (38.29 ± 1.26) and PBS (36.82 ± 1.39) injection groups (Fig. 11a, b; P < 0.05). The other two groups only showed blue staining, indicating minimal or no increase in collagen type I levels in the dermis.
Stem cells are a subject of intense research in the cell replacement and tissue engineering fields. There have been many reports of stem cells for regeneration of wounded tissue using specific differentiated cells. However, we did not come across a study where stem cells were transdifferentiated into fibroblasts in order to accelerate healing. Fibroblasts are very important in the promotion of wound healing, and we recognized that fibroblasts are ubiquitous cells in wound areas undergoing healing, but their numbers may not be adequate to promote optimal healing in normal circumstances. Therefore, we tried to differentiate hADSCs into fibroblast-like cells and tested their potency in mediating wound healing.
In the present study, we demonstrated that hADSCs could be differentiated into fibroblast-like cells by culturing them in F-CM. By FACS analysis, we demonstrated that the stem cell properties of hADSCs allowed them to differentiate into fibroblast-like cells. Compared with other MSCs, hADSCs can be harvested easily from patients and can be cultured and expanded rapidly. In addition, long-term cultured ADSCs retain their mesenchymal pluripotency . In this study, we confirmed that untreated hADSCs not only expressed characteristic surface markers (positive for CD105, CD13, CD54, and CD73; negative for CD149, CD19, CD45, and CD34), but were also significantly different from HS27 cells and F-CM-treated hADSCs. We aim to validate that hADSCs would differentiate into fibroblast-like cells with F-CM in order to stimulate wound healing in a skin defect model. With hADSCs harvested from human adipose tissue, we induced differentiation of hADSCs into fibroblast-like cells by F-CM treatment to meet the requirement of massive fibroblast numbers for thick skin wound healing. To confirm that hADSCs induced by F-CM for 72 h had differentiated into fibroblast-like cells, FACS analysis was performed; this showed that F-CM-treated hADSCs and HS27 cells had similar surface markers, and those were significantly different from untreated hADSCs (Fig. 4a, b). In addition, RT-PCR and immunohistochemistry also indicated increased expression of matured fibroblast-like markers in F-CM-treated hADSCs, an obvious result of differentiation (Fig. 5a, b).
Human dermal skin fibroblasts play key roles in wound healing by secretion of type I collagen and cytokines [28, 29]. We confirmed the expression of type I pro-collagen protein in F-CM-treated hADSC conditioned media with western blot analysis as well as the expression of total collagen protein level with the Sircol assay. Figures 2 and 3a indicated that the band size was significantly increased for type I pro-collagen, indicating a sizable increased expression in F-CM-treated hADSC conditioned media (baseline concentration, double concentration) at 72 h after treatment. In addition, the amount of total collagen also showed a similar result, with a significantly increased amount in F-CM-treated hADSC conditioned media (baseline concentration, double concentration) at 72 h post treatment (Fig. 3b).
In recent studies, transplantation of BMSCs has been reported to promote the healing process due to the cells’ capacity to differentiate into the skin epidermis and appendages, thus mediating dermal regeneration . Also, several studies have recently demonstrated accelerated rates of wound closure after transplantation of BMSCs, mesenchymal stem cells, and ADSCs [31, 32]. Moreover, in cutaneous wounds, partial or whole epidermis is destroyed, but the vascular structure is not damaged. Unaltered ADSCs exert only a moderate therapeutic potential compared with fibroblast-like differentiated ADSCs. These unaltered ADSCs can also differentiate into endothelial cells and smooth muscle cells, which contribute to about 9% of the ADSC-mediated angiogenesis, and as such unaltered ADSCs induce a beneficial effect that is predominantly mediated by a paracrine mechanism, with direct in-vivo differentiation playing a minor role .
To study the effect of F-CM-treated hADSCs in wounded skin in vivo, we intradermally injected F-CM-treated hADSCs and untreated hADSCs into the backs of balb/c nude mice in a full-thickness excision wound model (Fig. 9). The F-CM-treated hADSC injection group showed accelerated wound contraction and reepithelialization reduced the wound size compared with the unaltered hADSC and PBS injection groups at 10 days. To evaluate the histology of wound healing in vivo, we sacrificed mice sequentially at 1 and 2 weeks post implantation. With H&E staining, samples from the F-CM-treated hADSC injection group indicated an increased wound healing pattern at 2 weeks (Fig. 10). Immunohistochemical analysis indicated a sizable increase in staining of collagen type I with intense brown staining clearly observed in the F-CM-treated hADSC injection group compared with the other two groups (Fig. 11).
Secreted factors FGF, TGF-β1, and PDGF promote synthesis, deposition, and organization of new ECM collagen, fibronectin, and additional FGF, and TGF-β, which all contribute to wound healing, cell signaling, and tissue remodeling [34, 35]. The TGF-β signaling pathway plays an important role in each of these processes. The TGF-β1 signaling mechanism functions through the TGF-β type I (TbRI) and TGF-β type II (TbRII) transmembrane serine/threonine protein kinase receptors. Upon TGF-β1 binding to its type II receptor, TbRI is recruited to TbRII where it forms a ligand–receptor heterotetrameric complex [36, 37]. Under physiological conditions, TLP binds the type II receptor even when the pathway has been previously activated by TGF-β1, and the type II receptor is constitutively active. It transphosphorylates and activates the type I receptor, whose direct substrates are Smad2 and Smad3. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are then imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes [38, 39]. In the process of tissue fibrosis, TGF-β1 is likely to facilitate the expression of the ECM genes for an increase in the synthesis and deposition of collagen, fibronectin, and proteoglycan .
We demonstrated a mechanism for upregulation of pro-collagen type I in F-CM-treated hADSCs with F-CM containing TGF-β, FGF2, and VEGF cytokines (Fig. 7). F-CM-treated hADSCs exhibited increased pro-collagen type I expression via activated phospho-smad2, phospho-smad3, and smad4, not seen with the other groups of cells. hADSCs treated by F-CM, untreated hADSCs, and HS27 cells for 72 h were also analyzed for human collagen type I levels with immunocytochemistry in vitro (Fig. 8). In future studies, we plan to compare the levels of F-CM-treated hADSC fibroblasts and endogenous fibroblasts without ADSC stimulation in the different wound healing scenarios.
In conclusion, our group aimed to differentiate F-CM-treated hADSCs into fibroblast-like cells and confirmed the efficiency of differentiated cells in promoting collagen type I synthesis in a wound healing model. With potential applications in the clinic, our study is the first research of its kind with differentiated hADSCs being applied for treating full-thickness skin wounds.
F-CM contains a variety of factors known to be important in healing skin wounds. Our study revealed that F-CM had factors promoting collagen synthesis in hADSCs as well as transdifferentiation of hADSCs into fibroblast-like cells. These cells could stimulate wound healing in a skin defect model. After hADSC transdifferentiation by F-CM, the changes in expression of target molecules involved the use of western blot analysis, Sircol collagen assay for collagen increase, as well as FACS and RT-PCR. We confirmed an increased expression of type I pro-collagen through smad 2/3 protein upregulation in F-CM-treated hADSCs. In vivo, we verified a significant increase in the level of wound healing in balb/c nude mice skin, implanted with F-CM-treated hADSCs. Our findings may contribute to applications in stem cell therapy and regenerative medicine. Furthermore, these findings may argue for using cell therapy without gene or protein modification.
Fibroblast-derived conditioned medium
Human adipose-derived stem cell
Human adipose-derived stem cell-derived conditioned medium
This study was supported by grants from the Ministry of Health & Welfare (HI14C2310), KRIBB (KGM4891511), the Disaster and Safety Management Institute funded by the Ministry of Public Safety and Security of Korean government (MPSS-CG-2016-02), and Projects for Research and Development of Police Science and Technology under the Center for Research and Development of Police Science and Technology and the Korean National Police Agency (PA-H000001), Republic of Korea.
Availability of data and materials
The supporting data for this publication are available upon request.
WH was responsible for the in-vitro and in-vivo experiments as well as data interpretation & manuscript writing. HYL was responsible for the in-vivo experiment and support. HSM performed the histological data analysis. MW was responsible for animal model establishment. CL carried out the cell cultures. JAH and SHK were responsible for conception and design of the experiments. BKK was also responsible for cell cultures and also the F-CM analysis. THC was responsible for the conception and design of the study, edited the manuscript, and provided the funding. All authors read and approved the manuscript for publication.
The authors declare that they have no competing interests.
Consent for publication
All authors provide consent for publication of this manuscript.
Ethics approval and consent to participate
All experimental protocols were approved in strict accordance with the guidelines for Seoul National University Hospital’s Institutional Animal Care and Use Committee (IACUC No. 13-0292-C0A3).
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