569913
research-article2015
IJLXXX10.1177/1534734615569913The International Journal of Lower Extremity WoundsChen et al
Basic and Experimental Research
Transdifferentiation of Umbilical Cord– Derived Mesenchymal Stem Cells Into Epidermal-Like Cells by the Mimicking Skin Microenvironment
The International Journal of Lower Extremity Wounds 1–10 © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1534734615569913 ijl.sagepub.com
Deyun Chen, MS1*, Haojie Hao, MD1*, Chuan Tong, BS1, Jiejie Liu, BS1, Liang Dong, BS1, Dongdong Ti, MD1, Qian Hou, MD1, Huiling Liu, BS1, Weidong Han, MD, PhD1, and Xiaobing Fu, MD, PhD1
Abstract Human umbilical cord–derived mesenchymal stem cells (UC-MSCs) are multipotent, primitive, and have been widely used for skin tissue engineering. Their transdifferentiation is determined by the local microenvironment. In this study, we investigated the potential epidermal differentiation of UC-MSCs and the formation of epidermis substitutes in a 3-dimensional (3D) microenvironment, which was fabricated by UC-MSCs embedded into collagen–chitosan scaffolds (CCSs) combined with an air–liquid interface (ALI) culture system. Using fluorescence microscope, we observed that UC-MSCs were spindle-shaped and evenly distributed in the scaffold. Methyl thiazolyl blue tetrazolium bromide assay and Live/Dead assay indicated that the CCSs have good biocompatibility with UC-MSCs. Immunohistochemistry and western blotting assay showed that UC-MSCs on the surface of the CCSs were positive for the epidermal markers cytokeratin 19 and involucrin at 14 days. In addition, hematoxylin–eosin staining indicated that multilayered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and proved to promote wound healing. In conclusion, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering. Keywords human umbilical cord–derived mesenchymal stem cells, epidermal-like cells, collagen–chitosan scaffold, air–liquid interface, wound healing Wound repair is essential for the restoration of the skin barrier function after damage.1 The skin regenerates itself after minor epidermal injury. However, for many pathological conditions, such as diabetes or severe burns, the skin cannot regenerate spontaneously and requires effective coverage to prevent infection and fluid loss.2 Autologous and allogeneic epidermis substitutes are used to treat a variety of these skin injuries.3,4 However, using these approaches, the supporting epidermal cells have limited proliferative abilities, undergo immunological rejection, and are a limited resource, which makes alternative cells a necessity.5 Stem cells have the capacity of multilineage differentiation and can differentiate into epidermal cells for the construction of epidermis substitutes. Embryonic stem cells and induced pluripotent stem cells differentiate into epidermal cells via cytokine induction or co-culture with epidermal cells to construct epidermal substitutes,6,7 but their use is limited because of ethical considerations, teratoma formation, and genetic manipulation.8,9 Mesenchymal stem cells (MSCs), which are derived from adipose tissue,10 bone
marrow,11 and the umbilical cord (UC),12 also possess multipotent capacity.13 In a mouse excisional wound model, bone marrow–derived MSCs (BM-MSCs) labeled with green fluorescent protein (GFP) were injected into the wound, and the coexpression of GFP and a keratinocytespecific protein was observed, suggesting that BM-MSCs may directly differentiate into epidermal cells.14 Furthermore, BM-MSCs could differentiate into epithelial cells induced by epidermal growth factor, keratinocyte 1
Chinese PLA General Hospital, Beijing, China *These authors contributed equally to this work. Corresponding Authors: Xiaobing Fu, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100853, China. Email: [email protected] Weidong Han, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100853, China. Email: [email protected]
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growth factor, and so on or all-trans retinoic acid in vitro.15,16 In addition, when human adipose–derived stem cells are injected subcutaneously into nude mice, they migrate to the skin and differentiate into epidermal cells.17 These findings indicate that the specific microenvironment may trigger MSC differentiation into epidermal cells. In order to study the skin internal structure in vitro, 3-dimensional (3D) culture systems are necessary in which cell–cell and cell–extracellular matrix (ECM) interactions might lead to MSC differentiation into particular lineages. However, to construct the 3D microenvironment requires several key components, including the construction of an ideal scaffold. The collagen–chitosan scaffold (CCS) is popular in skin tissue engineering applications because it promotes wound healing.18 It is fabricated with collage and chitosan, which are ECM materials, and provides a physical support for cell differentiation and proliferation.18 The CCSs also interact with growth factors and ECM components released by the cells to enhance its bioactivity.19 An air–liquid interface (ALI) culture is another critical component for the microenvironment that closely recapitulates the in vivo–like morphologic and biochemical processes of human skin.20 Previous studies reported that the ALI culture system promotes the proliferation and differentiation of keratinocytes21 and the epidermal differentiation of MSCs.22 The constructed 3D microenvironment in vitro is similar to the physiological skin microenvironment in vivo, whereas whether the 3D microenvironment combined with ALI could induce the differentiation of MSCs into skin epidermal cells and the formation of epidermal substitutes in vitro remains undetermined. In this study, we investigated the epidermal differentiation of human umbilical cord–derived MSCs (UC-MSCs) in the 3D microenvironment that mimics human skin. UC-MSCs possess desirable characteristics such as a noninvasive and painless collection procedure23 and are multipotent, primitive,24 and in addition, they have an immunosuppressive effect that made them to avoid host rejection and widely used in cell-based therapy. A previous study showed that UC-MSCs could survive after xenograft transplantation.25 UC-MSCs were grown on the CCS with viable UC-MSCs at an ALI. The results showed that UC-MSCs were induced to express cytokeratin 19 (CK19) and involucrin, and formed an epidermal substitute. The constructed epidermal substitute promoted wound healing in a rat full-thickness wound model. Therefore, the 3D microenvironment for cell differentiation provides a promising surgical tool for skin tissue engineering directed at large area skin injuries and chronic wound applications.
Materials and Methods Ethics Statement Sprague-Dawley (SD) rats were used according to the protocols approved by the Health Sciences Animal Policy and
Welfare Committee of the Chinese People’s Liberation Army (PLA) General Hospital. UC were obtained following signed informed consent with approval from the Ethics Committee of the Chinese PLA General Hospital, Beijing, China.
Isolation of UC-MSCs and Cell Culture The isolation of UC-MSCs was performed according to previously described methods.26 The UC-MSCs isolated from the UCs were cultured in low glucose Dulbecco’s modified Eagle’s medium (L-DMEM; HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Gibco) at 37°C and 5% CO2 in a humidified chamber and passaged when they reached approximately 80% confluence. UC-MSCs of passages 4 to 6 were used for the following experiments.
Flow Cytometry UC-MSCs were trypsinized and prepared as single-cell suspensions. The cells were then incubated with primary antibodies for CD90-FITC, CD105-PE, CD73-PE, CD34-PE, CD45-FITC, anti-HLA-DR-FITC, and CD11a-FITC (BD Biosciences, St Jose, CA). FITC-conjugated antibody and PE-conjugated antibody were used as the isotype control (BD Biosciences) and were detected using flow cytometry (FACS Calibur; BD Biosciences).
Differentiation Assays Induction of adipogenic and osteogenic differentiation was performed as previously described.27 UC-MSCs were cultured in each differentiation medium (Cyagen Biosciences Inc, Santa Clara, CA), and the medium was changed every 2 to 3 days. After 3 weeks, UC-MSCs that differentiated into adipocytes and osteocytes were confirmed by staining with oil-red O and alkaline phosphatase (ALP), respectively.
Fabrication of CCS The collagen I solution (2 mg/mL, BD Biosciences) and chitosan solution (2 mg/mL, Tokyo Chemical Industry Co, Tokyo, Japan) were made by combining at a mass ratio of 1:1 and were neutralized with 0.1 M Na2HPO4 (pH 9.1). These mixtures were frozen at −80°C for 2 hours and were lyophilized for 24 hours in a freeze dryer (LABCONCO Co, Kansas, MO). Next, the samples were immersed in a crosslinking agent containing carbodiimide (EDC, Ruitaibio) and N-hydroxyl succinimide (NHS, Ruitaibio) in 95% ethanol for 12 hours at 4°C. The unreacted chemicals were removed by repeated washing with distilled water at
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Chen et al least 3 times and then freeze-dried. These CCSs were sterilized with ethylene oxide gas.
Characterization of CCS The morphology and pore size of the CCS were observed using scanning electron microscopy (SEM; JEOL, Tokyo, Japan). The swelling study was performed to assess the water absorption capacity of the CCS. The dry weight of the CCS was measured with an analytic balance, and it was then immersed into PBS. The CCS wet weights were accurately determined at preset time points after removing the PBS from the surface with filter paper. The water absorption of the CCS was calculated with the following formula28: water absorption = (We − W0)/W0, where We is the weight of the wet scaffold and W0 is the initial weight of the dry scaffold. The CCS degradation study was performed in PBS with collagenase I. These reactions were incubated at 37°C and the digestion was terminated with 0.25 M ethylenediamine tetraacetic acid (EDTA) at specific time intervals. At predetermined time points, the samples were subjected to dry weight analysis. The initial weight of the CCS samples was recorded. The residual mass (%) was calculated using the following formula28: residual mass (%) = 100 − ((Wd − WS)/Wd × 100), where Wd represents the sample dry weight before degradation and WS indicates the sample dry weight after degradation.
Green Fluorescence Protein (GFP) Lentiviral Vectors Construction and Labeling of UC-MSCs With GFP UC-MSCs were infected with a lentiviral vector for GFP (provided by our laboratory). GFP lentiviral vectors were obtained by transfection of 293T cells. The GFP lentiviral vectors were mixed with polyethyleneimine (PEI; Polysciences, Eppelheim, Germany; pH 7) at a volume rate of 1:3, transfected into 293T cells, and the supernatant collected. The supernatant was concentrated through PEG6000 (Sigma-Aldrich, St Louis, MO). UC-MSCs were infected with concentrated lentiviral supernatant at a multiplicity of infection (MOI) of 5 to 10.
Chloromethyl-Dialkylcarbocyanine (CM-Dil) Cell Labeling Cell Tracker CM-Dil (Invitrogen Life Technologies, Carlsbad, CA) was prepared using the manufacturer’s instructions. A total of 1 × 106 UC-MSCs in 500 µL PBS were labeled with 2 µL CM-Dil solutions(1 mg/mL). CM-Dil-labeled cells were used for the following experiments.
In Vitro Epidermal-Like Differentiation of UCMSCs The sterilized CCSs were prewetted and placed in 6-well Transwell plates (24 mm Transwell, Corning Incorporated, USA; Figure 4A). A 100-µL UC-MSCs suspension (1 × 106 cells/100 µL) was loaded onto the top surface of each prewetted CCS. The cells/CCS constructs were incubated at 37°C with 5% CO2 for 4 hours for the cells to penetrate into the CCS and adhere. After the cells adhered, 100 µL UC-MSCs suspension was again seeded on the surface of the cells/CCS constructs and incubated at 37°C with 5% CO2 for an additional 4 hours, and cultured in L-DMEM medium. The cells were exposed to air by lowering the medium level to the lower part of the CCS after 2 days in the immersed culture. The medium was changed once each day. At each indicated time interval, the samples were collected for further experimental analysis.
MTT Assay Cell proliferation was determined by methyl thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) absorption as previously described29 and analyzed using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA) at 570 nm.
Live/Dead Assay Cell viability was tested using a Live/Dead assay (Invitrogen, UK). The samples were evaluated following the instruction of the Live/Dead assay kit and were viewed under an Olympus DP72 fluorescence microscope with 528 nm (red, EthD-1) and 494 nm (green, Calcein) excitation filters. For quantitative analysis, a total of 300 cells were counted from each sample over 5 randomly chosen sections, and the live and dead cell counts were recorded.
Collagen Quantification The total amount of collagen secreted by the cells seeded in the scaffold was measured using Sirius Red (SigmaAldrich) stain. The protocol was performed as previously described,30 and the absorbance was measured at 540 nm. By quantifying the amount of collagen in the scaffold without cell seeding and deducting the OD value, the CCS background was excluded.
Western Blot Analysis After 7 and 14 days of UC-MSC culture at the ALI, the total cellular proteins were extracted using cell lysis buffer (Beyotime Biotechnology, Beijing, China), and western blot analysis was performed as previously described.26 The mouse monoclonal antibodies against cytokeratin 19
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(1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and involucrin (1:1000; Abcam, Cambridge, MA) were used as primary antibodies. A mouse monoclonal antibody against β-actin (1:1000; Sigma, St Louis, MO) was used as a loading control. The appropriate horseradish peroxidase–conjugated antibody (1:300; (DAKO, Glostrup, Denmark) was used as a secondary antibody. The immunoreactive proteins were visualized using the SuperECL western blotting detection reagent (Applygen Technologies Inc, Beijing, China).
Wound Healing Assay The in vivo surgery and photographs were performed under 3% pentobarbital sodium anesthesia. Male SD rats (approximately 200 g) were obtained from the Chinese PLA General Hospital. The rats were housed in cages in a temperaturecontrolled room (22-25°C) on a 12-hour/12-hour light/dark schedule with free access to food and water. Forty-five rats were randomly divided into 3 groups, the control group (no treatment), the CCS group (CCS without cells), and the epidermal substitute group (the epidermal substitute which was constituted by the CCS and the cells inner of it and on the surface of it). Following anesthetization, dorsal regions shaved, an open excision-type wound of 1 cm in diameter was created on the middle dorsal skin to the depth of subcutaneous tissue using a dermal punch under aseptic conditions, and a CCS or epidermal substitute was placed on the wound immediately, whereas wounds with no treatment were the control. The animals were sacrificed after 3, 7, 14, and 21 days to assess skin regeneration. The aforementioned experiments were repeated 3 times and showed consistent results.
Histology and Immunohistochemistry The epidermal substitutes and wound skins were fixed in 10% formalin, dehydrated, embedded in paraffin, and cut into 5 µm sections. General histology was visualized by hematoxylin–eosin (H&E) staining. Immunohistochemical analysis of the epidermal substitutes was performed using primary antibodies specific for mouse anti-human CK19 (Santa Cruz) and mouse anti-human involucrin (Abcam), and secondary horseradish peroxidase–conjugated goat anti-mouse antibodies (DAKO) followed by visualization with 3,3-diaminobenzidine tetrahydrochloride (DAB; DAKO). Next, H&E-stained sections and immunostained sections were observed under a light microscope (Olympus DP72, Tokyo, Japan).
Statistical Analysis The data were expressed as the means ± standard deviation (SD). Significant differences were determined using
Student’s 2-sample t test. Differences were considered statistically significant at a P value less than .05.
Results Characterization of UC-MSCs The characteristics of UC-MSCs were analyzed by morphological, multipotent differentiation potential, and flow cytometry analysis. The morphology of UC-MSCs was elongated and spindle-shaped after passage 3 (Figure 1A), and they could differentiate into adipocytes and osteoblasts (Figure 1B and C). Flow cytometry revealed that UC-MSCs were positive for CD90, CD105, and CD73, whereas they were negative for CD34, CD45, HLA-DR, and CD11α (Figure 1D). Therefore, UC-MSCs were successfully isolated for use in the following experiments.
Characterization of CCSs To simulate the 3D microenvironment in vitro, we constructed CCSs using collagen and chitosan through crosslinking and lyophilization.31 The CCS was a sponge-like structure (Figure 2A and B) with a porous structure, and 100 to 200 µm pore size as determined by light microscopy and scanning electron microscopy (Figure 2C and D). Furthermore, the water absorption of the CCS was nearly 13 times their initial weight after 8 hours in phosphate buffer, which demonstrated that the CCS has a good water absorption capacity (Figure 2E). When the CCS was treated with collagenase, the residual mass decreased to approximately 40% after 70 hours of treatment, which demonstrated an appropriate degradation rate (Figure 2F). These results indicated that the CCS with suitable properties was successfully constructed.
CCS Biocompatibility The biocompatibility of CCS was analyzed by morphological analysis, MTT assay, Live/Dead staining, and collagen production assay. UC-MSCs were infected with GFPexpressing lentivirus and transplanted into the CCS. Based on the fluorescence micrographs, we observed that UC-MSCs were spindle-shaped and distributed evenly in the scaffold (Figure 3A). SEM micrographs showed that UC-MSCs attached well to the CCS in the form of cell layers (Figure 3B). MTT assay (Figure 3C) showed that UC-MSCs in the CCS were not proliferating. Live/Dead staining (Figure 3E and F) revealed that the live percentage of the cells was 98% at 3 days, 95% at 7 days, and 86% at 14 days. Sirius Red staining (Figure 3D) showed the total amount of collagen in the CCS was significantly increased at 3 days. The results confirmed that the scaffold has good biocompatibility with UC-MSCs.
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Figure 1. Characterization of UC-MSCs.
(A) Morphology of UC-MSCs under light microscopy showing the spindle-shaped appearance of cell colonies after passage 3 (scale bar = 200 µm). (B, C) Multilineage differentiation potential of UC-MSCs showing UC-MSCs differentiated into adipocytes, which are indicated by the accumulation of lipid vesicles in the cells (scale bar = 50 µm) and osteoblasts, which express alkaline phosphatase, as indicated in blue (scale bar = 100 µm). (D) UC-MSC surface marker expression by flow cytometry showing the percentages of UC-MSCs for mesenchymal antigens, CD90, CD73, and CD105, which were 99.94%, 97.11%, and 97.34%, respectively, whereas the percentages of CD34, CD45, CD11a, and HLA-DR were 2.01%, 0.07%, 0.01%, and 0.35%, respectively.
UC-MSCs Transdifferentiate Into Epidermal-Like Cells for Engineering Epidermal Substitutes To induce UC-MSC transdifferentiation into epidermal-like cells in vitro, UC-MSCs were seeded onto the surface of the CCSs populated with UC-MSCs. Following 2 days of immersion in medium, UC-MSCs were grown at an ALI, which mimics the microenvironment of human skin in vivo.32 Immunohistochemistry results showed that several of the cell layers on the surface of the CCS were positive for CK19 and involucrin after culturing at the ALI for 7 days and 14 days (Figure 4D). This observation was corroborated by western blot analysis. The results also showed that both CK19 and involucrin were more highly expressed at 14 days than 7 days (Figure 4C). H&E staining (Figure 4B) showed cells distribution throughout the porous inside of the CCS and revealed that a multilayered epidermal substitute had formed on the superficial area of the CCS with an organization similar to the skin epidermis at both 7 days and
14 days. These results demonstrated that UC-MSCs are differentiated into epidermal-like cells in the 3D microenvironment and formed a stratified epidermal substitute.
In Vivo Wound Healing The role of the constructed epidermal substitute in wound healing was evaluated in a rat full-thickness wound model of 1 cm in diameter. The results revealed that the stratified epidermal substitutes accelerated wound closure (Figure 5A and B). At 3, 7, and 14 days after surgery, the epidermal substitute groups showed significantly better wound closure compared with the control group. At 21 days, all wounds in the epidermal substitutes and CCS groups had achieved complete wound closure, but not all the wounds of the control groups were completely closed. Although no significant difference was observed between the epidermal substitutes and CCS group at any time, H&E (Figure 5C) staining showed that the epidermal substitutes reduced the inflammation response at
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Figure 2. Characterization of CCS.
(A, B) Macroscopic view of the CCS structure. (C) The optical micrographs indicate the presence of porosity in the scaffold structure (scale bar = 200 µm). (D) Scanning electron microscopy shows that the scaffold has a porous structure and the pore size is 100 to 200 µm (scale bar = 200 µm). (E) Swelling kinetics of the CCS in PBS (pH 7.4). (F) Degradation kinetics of the CCS in PBS (pH 7.4).
3 days and enhanced reepithelialization and granulation at 7 and 14 days compared with the CCS group and control group. After 21 days, all wounds showed a regenerated epithelium with several viable layers. Interestingly, in epidermal substitutes group, bud-like structures were observed, appearing as epidermal ingrowth and hair follicle differentiation. In addition, the tracer experiment (Figure 5D) demonstrated the CM-Dil-positive cells remained at the subcutaneous tissue of the rats at 21 days and they did not participate in the neo-epidermis formation. These results demonstrate that epidermal substitutes promote wound healing in a rat full-thickness wound model.
Discussion In this study, we investigated whether the 3D microenvironment in which the CCS harbors viable UC-MSCs could induce UC-MSC differentiation into epidermal-like cells in ALI culture and construct epidermal substitutes. These results demonstrated that UC-MSCs successfully differentiated into epidermal-like cells and formed epidermal substitutes. Moreover, when the constructed epidermal substitutes were used to treat the full-thickness wounds in rats, successful wound healing with a well-differentiated epidermis was observed.
One crucial factor of the 3D microenvironment in vitro is the construction of a scaffold. A 3D scaffold provides an ECM analog for cell penetration and a physical support for cell proliferation and differentiation.18 Previous studies demonstrated that the scaffold widely used in tissue engineering possesses a microstructure with 100 to 200 µm pore size and porosity greater than 90%, high swelling efficiency, suitable biodegradability, and excellent biocompatibility.33 The CCS with the above-mentioned characteristics embedded with dermal fibroblasts showed that the scaffold accelerated cell infiltration and growth in vitro. Moreover, animal experiments demonstrated that the scaffold promoted fibroblasts infiltration from the adjacent tissue in vivo.18 In a rat full-thickness wound model, a similar collagen/HA/gelatin scaffold harboring cells had been shown to promote wound healing.30 In the present study, we used EDC and NHS to cross-link the collagen and chitosan to construct a CCS. Consistent with previous studies,33 CCS also has excellent properties, including a pore size of 100 to 200 µm and porosity greater 90%, biodegradability, and excellent biocompatibility. These results indicated that CCS can be used as a scaffold for constructing the 3D microenvironment. The seed cell is another important factor of the 3D microenvironment in vitro. In a conventional culture model, the fibroblasts are usually used as the seed cells that are incorporated into the dermal substitutes to facilitate the formation of stratified epidermis in vitro.34 However, when MSCs replaced the fibroblasts, a stratified epidermis was also observed.35 In the keratinocyte-MSC skin model, MSCs were used as seed cells and were seeded into a deepidermalized dermis, and the keratinocytes were cultured on the de-epidermalized dermis at an ALI to induce epidermal differentiation.36 These results demonstrated that MSCs contribute to epidermal morphology formation and ECM production similar to fibroblasts. Numerous studies have reported that MSCs play a role in wound repair through paracrine effects by releasing bioactive molecules that affect cell migration and proliferation, anti-inflammation, and anti-scar.37,38 Therefore, these attributes make them useful for cell-based bioengineered products in skin repair therapy. UC-derived cells are a group of impure cell population and still have not a definite marker so far. In our study, they were isolated and purified by adherent culture method,26 and the majority of isolated UC-derived cells come to meet the minimal criteria proposed by the International Society for Cellular Therapy, including growing adherent to plastic, exhibiting a multilineage differentiation potential, and expressing CD105, CD73, and CD90 and lack the expression of CD45, CD34, CD11b, and HLA-DR, for defining as multipotent MSCs.39 So we termed UC-derived cells as UC-MSCs. UC-MSCs were used as seed cells and cultured in the inner of CCS, in which they not only survived well but also expressed collagen. These results indicate that the 3D microenvironment
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Figure 3. The CCS biocompatibility with UC-MSCs.
(A) Morphology of UC-MSCs within the CCS observed by fluorescence microscope (scaffold preloaded with GFP-expressing UC-MSCs) (scale bar = 100 µm). (B) Scanning electron micrographs of UC-MSCs seeded on the CCS (scale bar = 200 µm). (C) Proliferation patterns (MTT assay) of UC-MSC culture on tissue culture plates (as a control) and CCSs. UC-MSCs were cultured for 3, 7, and 14 days and evaluated for proliferation using the MTT assay. (D) The total collagen amounts in the scaffold are shown. (E) Live/Dead staining results after 3, 7, and 14 days culture. Green spots indicate living cells, and red spots indicate dead cells (scale bar = 100 µm). (F) Quantitative analysis of Live/Dead staining results.
Figure 4. UC-MSCs transdifferentiate into epidermal-like cells for engineering epidermal substitutes.
(A) Schematic diagram showing the protocol for inducing UC-MSC differentiation. The CCS was first seeded within UC-MSCs until the cells adhered, and then seeded with UC-MSCs on the top of the CCS cultured for 2 days immersed in culture medium, and 7 to 14 additional days at the air–liquid interface to promote epidermal differentiation. (B) H&E staining for UC-MSCs cultured on CCS (scale bar = 100 µm). (C) Immunohistochemistry staining: UC-MSCs were cultured for 7 and 14 days. Cells on the top of the CCS were positive for CK19 and involucrin (scale bar = 100 µm). (D) Western blotting analysis for CK19 and involucrin expression.
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Figure 5. In vivo wound healing.
(A) Representative macroscopic images of full-thickness dorsal skin wounds in the control group, the scaffold group, and the epidermis substitutes at 0, 3, 7, 14, and 21 days after injury. (B) Quantitation of the wound area of full-thickness dorsal skin wounds at 0, 3, 7, 14, and 21 days after injury. Each value is the mean ± SD, n = 3, *P < .05, **P < .01. (C) Representative H&E-stained sections of the full-thickness dorsal skin wounds from rats with scaffold or epidermis substitutes at 3, 7, 14, and 21 days after wounding (scale bar = 100 µm). (D) CM-Dil-labeled cells in the rat skin were observed under fluorescent microscope at 21 days after operation. The red fluorescence that indicated the CM-Dil-labeled cells were obviously visible in subcutaneous tissue at 21 days after operation. Up: scale bar = 100 µm; down: scale bar = 50 µm.
fabricated by viable MSCs, CCS, and ECM, cytokines selected by MSCs, can create an artificial niche. Skin epidermal cells grow on the basement membrane and are exposed to air in vivo.40 Therefore, we cultured these cells at the ALI in vitro in similar physiological conditions. A previous study showed that the ALI culture stimulated the early differentiation of human amnion epithelial cells seeded on a fibrin layer embedded with human amnion mesenchymal cells to epidermal cells, which indicate that the ALI culture system is crucial for enhancing epidermal differentiation.20 Consistent with the above-mentioned finding, BM-MSCs cultured in an induction medium on a contractible fibroblast-embedded collagen gel with an ALI also committed MSCs to an epidermal lineage.22 In our study, when MSCs were placed on the surface of MSCembedded CCSs and grown at the ALI, they were positive for K19 and involucrin at 7 and 14 days. H&E staining showed that a multilayered epidermal substitute with an organization similar to the skin epidermis was formed. In
the previous researches, the successful differentiation of hUC-MSCs into epidermal-like cells was also confirmed by indirect co-culture with epidermal stem cells,41 which is mainly due to the role of the factors selected by epidermal stem cells. However, another study has demonstrated that UC-MSCs failed to differentiate into epidermal cells after they were cultured at an ALI on dermal equivalents that consist of collagen I/III and human dermal fibroblasts and were further treated with 5-azacytidine or all-trans retinoic acid.24 The failure of differentiation may be attributed to the culture system, which is not enough to stimulate UC-MSC differentiation into an epithelial phenotype. Compared with the previous studies, we speculated that the suitable 3D microenvironment including the suitable scaffold, cytokines, and ECM secreted by UC-MSCs might be the prominent inducers for epidermal differentiation. The constructed epidermis substitutes have been proven to promote wound healing in a rat full-thickness model. To explore the role of UC-MSCs, the cells on the surface of the
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Chen et al CCSs were labeled with CM-Dil, and the cells inner of the CCSs were labeled with GFP. The results showed that there are no GFP-positive cells in the rats probably because the cells inner of the CCSs were dead. However, CM-Dilpositive cells on the surface of the CCSs remained in the rats until 21 days after surgery but they did not participate in the neo-epidermis formation. Therefore, we conclude that UC-MSCs in promoting wound healing are probably due to their paracrine effect. This is supported by numerous previous studies, which have demonstrated that MSCs could secrete lots of factors that regulate cellular responses to skin damage and their paracrine effect is also supported by MSC-conditioned medium enhanced wound closure.42 In conclusion, our study demonstrates that UC-MSCs differentiate into epidermal-like cells in the 3D microenvironment in vitro, which mimics the environment of native skin, and can be used to construct epidermal substitutes. In vivo animal tests showed that the constructed epidermis substitutes have the ability to promote wound healing. These findings suggested that mimicking the 3D microenvironment can be used for epidermal-like cells differentiation to construct epidermal substitutes, which provide an alternative strategy for skin tissue engineering. Authors’ Note Deyun Chen and Haojie Hao contributed equally to this work.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported in part by the National Basic Science and Development Program (2012CB518103, 2012CB518105), the 863 Projects of Ministry of Science and Technology of China (2013AA020105, 2012AA020502), Military Medical Foundation (AWS11J008), and National Natural Science Foundation of China (81121004, 81230041).
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6. Guenou H, Nissan X, Larcher F, et al. Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study. Lancet. 2009;374: 1745-1753. 7. Bilousova G, Chen J, Roop DR. Differentiation of mouse induced pluripotent stem cells into a multipotent keratinocyte lineage. J Invest Dermatol. 2011;131:857-864. 8. Fu X, Li H. Mesenchymal stem cells and skin wound repair and regeneration: possibilities and questions. Cell Tissue Res. 2009;335:317-321. 9. Hassan WU, Greiser U, Wang W. Role of adipose-derived stem cells in wound healing. Wound Repair Regen. 2014;22:313-325. 10. Gruber HE, Deepe R, Hoelscher GL, et al. Human adiposederived mesenchymal stem cells: direction to a phenotype sharing similarities with the disc, gene expression profiling, and coculture with human annulus cells. Tissue Eng Part A. 2010;16:2843-2860. 11. Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells: isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol. 2009;482:281-294. 12. Li W, Ye B, Cai XY, Li JH, Gao WQ. Differentiation of human umbilical cord mesenchymal stem cells into prostatelike epithelial cells in vivo. PLoS One. 2014;9:e102657. 13. Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells. 2007;25:2896-2902. 14. Wu Y, Chen L, Scott PG, Tredgett EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 2007;25:2648-2659. 15. Paunescu V, Deak E, Herman D, et al. In vitro differentiation of human mesenchymal stem cells to epithelial lineage. J Cell Mol Med. 2007;11:502-508. 16. Takebayashi T, Horii T, Denno H, et al. Human mesenchymal stem cells differentiate to epithelial cells when cultured on thick collagen gel. Biomed Mater Eng. 2013;23:143-153. 17. Lin YH, Fu KY, Hong PD, et al. The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells. Ann Plast Surg. 2013;71(suppl 1): S67-S74. 18. Ma L, Gao C, Mao Z, et al. Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials. 2003;24:4833-4841. 19. Han S, Zhao Y, Xiao Z, et al. The three-dimensional collagen scaffold improves the stemness of rat bone marrow mesenchymal stem cells. J Genet Genomics. 2012;39:633-641. 20. Fatimah SS, Chua K, Tan GC, Azmi TI, Tan AE, Abdul Rahman H. Organotypic culture of human amnion cells in airliquid interface as a potential substitute for skin regeneration. Cytotherapy. 2013;15:1030-1041. 21. Sugihara H, Toda S, Miyabara S, Kusaba Y, Minami Y. Reconstruction of the skin in three-dimensional collagen gel matrix culture. In Vitro Cell Dev Biol. 1991;27A:142-146. 22. Ma K, Laco F, Ramakrishna S, Liao S, Chan CK. Differentiation of bone marrow-derived mesenchymal stem cells into multi-layered epidermis-like cells in 3D organotypic coculture. Biomaterials. 2009;30:3251-3258.
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23. Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica. 2006;91:1017-1026. 24. Schneider RK, Pullen A, Kramann R, et al. Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents. Differentiation. 2010;79:182-193. 25. Arufe MC, De la Fuente A, Fuentes I, Toro FJ, Blanco FJ. Umbilical cord as a mesenchymal stem cell source for treating joint pathologies. World J Orthop. 2011;2(6):43-50. 26. Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells. 2004;22:1330-1337. 27. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143-147. 28. Adekogbe I, Ghanem A. Fabrication and characterization of DTBP-crosslinked chitosan scaffolds for skin tissue engineering. Biomaterials. 2005;26:7241-7250. 29. Liu J, Xu HH, Zhou H, Weir MD, Chen Q, Trotman CA. Human umbilical cord stem cell encapsulation in novel macroporous and injectable fibrin for muscle tissue engineering. Acta Biomater. 2013;9:4688-4697. 30. Wang HM, Chou YT, Wen ZH, Wang CZ, Chen CH, Ho ML. Novel biodegradable porous scaffold applied to skin regeneration. PLoS One. 2013;8:e56330. 31. Zhu Y, Liu T, Song K, Jiang B, Ma X, Cui Z. Collagenchitosan polymer as a scaffold for the proliferation of human adipose tissue-derived stem cells. J Mater Sci Mater Med. 2009;20:799-808. 32. Yee CH, Aoki S, Uchihashi K, et al. The air liquid-interface, a skin microenvironment, promotes growth of melanoma cells, but not their apoptosis and invasion, through activation of mitogen-activated protein kinase. Acta Histochem Cytochem. 2010;43:1-7.
33. Han CM, Zhang LP, Sun JZ, Shi HF, Zhou J, Gao CY. Application of collagen-chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering. J Zhejiang Univ Sci B. 2010;11:524-530. 34. Gangatirkar P, Paquet-Fifield S, Li A, Rossi R, Kaur P. Establishment of 3D organotypic cultures using human neonatal epidermal cells. Nat Protoc. 2007;2:178-186. 35. Fioretti F, Lebreton-DeCoster C, Gueniche F, et al. Human bone marrow-derived cells: an attractive source to populate dermal substitutes. Wound Repair Regen. 2008;16:87-94. 36. Ojeh NO, Navsaria HA. An in vitro skin model to study the effect of mesenchymal stem cells in wound healing and epidermal regeneration. J Biomed Mater Res A. 2014;102: 2785-2792. 37. Jackson WM, Nesti LJ, Tuan RS. Concise review: clinical translation of wound healing therapies based on mesenchymal stem cells. Stem Cells Transl Med. 2012;1:44-50. 38. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000;18:307-316. 39. Lindenmair A, Hatlapatka T, Kollwig G, et al. Mesenchymal stem or stromal cells from amnion and umbilical cord tissue and their potential for clinical applications. Cells. 2012;1:1061-1088. 40. Prunieras M, Regnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J Invest Dermatol. 1983;81(1 suppl):28s-33s. 41. Li D, Chai J, Shen C, Han Y, Sun T. Human umbilical cordderived mesenchymal stem cells differentiate into epidermallike cells using a novel co-culture technique. Cytotechnology. 2014;66:699-708. 42. Liu L, Yu Y, Hou Y, et al. Human umbilical cord mesenchymal stem cells transplantation promotes cutaneous wound healing of severe burned rats. PLoS One. 2014;9:e88348.
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research-article2015
IJLXXX10.1177/1534734615569913The International Journal of Lower Extremity WoundsChen et al
Basic and Experimental Research
Transdifferentiation of Umbilical Cord– Derived Mesenchymal Stem Cells Into Epidermal-Like Cells by the Mimicking Skin Microenvironment
The International Journal of Lower Extremity Wounds 1–10 © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1534734615569913 ijl.sagepub.com
Deyun Chen, MS1*, Haojie Hao, MD1*, Chuan Tong, BS1, Jiejie Liu, BS1, Liang Dong, BS1, Dongdong Ti, MD1, Qian Hou, MD1, Huiling Liu, BS1, Weidong Han, MD, PhD1, and Xiaobing Fu, MD, PhD1
Abstract Human umbilical cord–derived mesenchymal stem cells (UC-MSCs) are multipotent, primitive, and have been widely used for skin tissue engineering. Their transdifferentiation is determined by the local microenvironment. In this study, we investigated the potential epidermal differentiation of UC-MSCs and the formation of epidermis substitutes in a 3-dimensional (3D) microenvironment, which was fabricated by UC-MSCs embedded into collagen–chitosan scaffolds (CCSs) combined with an air–liquid interface (ALI) culture system. Using fluorescence microscope, we observed that UC-MSCs were spindle-shaped and evenly distributed in the scaffold. Methyl thiazolyl blue tetrazolium bromide assay and Live/Dead assay indicated that the CCSs have good biocompatibility with UC-MSCs. Immunohistochemistry and western blotting assay showed that UC-MSCs on the surface of the CCSs were positive for the epidermal markers cytokeratin 19 and involucrin at 14 days. In addition, hematoxylin–eosin staining indicated that multilayered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and proved to promote wound healing. In conclusion, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering. Keywords human umbilical cord–derived mesenchymal stem cells, epidermal-like cells, collagen–chitosan scaffold, air–liquid interface, wound healing Wound repair is essential for the restoration of the skin barrier function after damage.1 The skin regenerates itself after minor epidermal injury. However, for many pathological conditions, such as diabetes or severe burns, the skin cannot regenerate spontaneously and requires effective coverage to prevent infection and fluid loss.2 Autologous and allogeneic epidermis substitutes are used to treat a variety of these skin injuries.3,4 However, using these approaches, the supporting epidermal cells have limited proliferative abilities, undergo immunological rejection, and are a limited resource, which makes alternative cells a necessity.5 Stem cells have the capacity of multilineage differentiation and can differentiate into epidermal cells for the construction of epidermis substitutes. Embryonic stem cells and induced pluripotent stem cells differentiate into epidermal cells via cytokine induction or co-culture with epidermal cells to construct epidermal substitutes,6,7 but their use is limited because of ethical considerations, teratoma formation, and genetic manipulation.8,9 Mesenchymal stem cells (MSCs), which are derived from adipose tissue,10 bone
marrow,11 and the umbilical cord (UC),12 also possess multipotent capacity.13 In a mouse excisional wound model, bone marrow–derived MSCs (BM-MSCs) labeled with green fluorescent protein (GFP) were injected into the wound, and the coexpression of GFP and a keratinocytespecific protein was observed, suggesting that BM-MSCs may directly differentiate into epidermal cells.14 Furthermore, BM-MSCs could differentiate into epithelial cells induced by epidermal growth factor, keratinocyte 1
Chinese PLA General Hospital, Beijing, China *These authors contributed equally to this work. Corresponding Authors: Xiaobing Fu, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100853, China. Email: [email protected] Weidong Han, Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100853, China. Email: [email protected]
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growth factor, and so on or all-trans retinoic acid in vitro.15,16 In addition, when human adipose–derived stem cells are injected subcutaneously into nude mice, they migrate to the skin and differentiate into epidermal cells.17 These findings indicate that the specific microenvironment may trigger MSC differentiation into epidermal cells. In order to study the skin internal structure in vitro, 3-dimensional (3D) culture systems are necessary in which cell–cell and cell–extracellular matrix (ECM) interactions might lead to MSC differentiation into particular lineages. However, to construct the 3D microenvironment requires several key components, including the construction of an ideal scaffold. The collagen–chitosan scaffold (CCS) is popular in skin tissue engineering applications because it promotes wound healing.18 It is fabricated with collage and chitosan, which are ECM materials, and provides a physical support for cell differentiation and proliferation.18 The CCSs also interact with growth factors and ECM components released by the cells to enhance its bioactivity.19 An air–liquid interface (ALI) culture is another critical component for the microenvironment that closely recapitulates the in vivo–like morphologic and biochemical processes of human skin.20 Previous studies reported that the ALI culture system promotes the proliferation and differentiation of keratinocytes21 and the epidermal differentiation of MSCs.22 The constructed 3D microenvironment in vitro is similar to the physiological skin microenvironment in vivo, whereas whether the 3D microenvironment combined with ALI could induce the differentiation of MSCs into skin epidermal cells and the formation of epidermal substitutes in vitro remains undetermined. In this study, we investigated the epidermal differentiation of human umbilical cord–derived MSCs (UC-MSCs) in the 3D microenvironment that mimics human skin. UC-MSCs possess desirable characteristics such as a noninvasive and painless collection procedure23 and are multipotent, primitive,24 and in addition, they have an immunosuppressive effect that made them to avoid host rejection and widely used in cell-based therapy. A previous study showed that UC-MSCs could survive after xenograft transplantation.25 UC-MSCs were grown on the CCS with viable UC-MSCs at an ALI. The results showed that UC-MSCs were induced to express cytokeratin 19 (CK19) and involucrin, and formed an epidermal substitute. The constructed epidermal substitute promoted wound healing in a rat full-thickness wound model. Therefore, the 3D microenvironment for cell differentiation provides a promising surgical tool for skin tissue engineering directed at large area skin injuries and chronic wound applications.
Materials and Methods Ethics Statement Sprague-Dawley (SD) rats were used according to the protocols approved by the Health Sciences Animal Policy and
Welfare Committee of the Chinese People’s Liberation Army (PLA) General Hospital. UC were obtained following signed informed consent with approval from the Ethics Committee of the Chinese PLA General Hospital, Beijing, China.
Isolation of UC-MSCs and Cell Culture The isolation of UC-MSCs was performed according to previously described methods.26 The UC-MSCs isolated from the UCs were cultured in low glucose Dulbecco’s modified Eagle’s medium (L-DMEM; HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Gibco) at 37°C and 5% CO2 in a humidified chamber and passaged when they reached approximately 80% confluence. UC-MSCs of passages 4 to 6 were used for the following experiments.
Flow Cytometry UC-MSCs were trypsinized and prepared as single-cell suspensions. The cells were then incubated with primary antibodies for CD90-FITC, CD105-PE, CD73-PE, CD34-PE, CD45-FITC, anti-HLA-DR-FITC, and CD11a-FITC (BD Biosciences, St Jose, CA). FITC-conjugated antibody and PE-conjugated antibody were used as the isotype control (BD Biosciences) and were detected using flow cytometry (FACS Calibur; BD Biosciences).
Differentiation Assays Induction of adipogenic and osteogenic differentiation was performed as previously described.27 UC-MSCs were cultured in each differentiation medium (Cyagen Biosciences Inc, Santa Clara, CA), and the medium was changed every 2 to 3 days. After 3 weeks, UC-MSCs that differentiated into adipocytes and osteocytes were confirmed by staining with oil-red O and alkaline phosphatase (ALP), respectively.
Fabrication of CCS The collagen I solution (2 mg/mL, BD Biosciences) and chitosan solution (2 mg/mL, Tokyo Chemical Industry Co, Tokyo, Japan) were made by combining at a mass ratio of 1:1 and were neutralized with 0.1 M Na2HPO4 (pH 9.1). These mixtures were frozen at −80°C for 2 hours and were lyophilized for 24 hours in a freeze dryer (LABCONCO Co, Kansas, MO). Next, the samples were immersed in a crosslinking agent containing carbodiimide (EDC, Ruitaibio) and N-hydroxyl succinimide (NHS, Ruitaibio) in 95% ethanol for 12 hours at 4°C. The unreacted chemicals were removed by repeated washing with distilled water at
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Characterization of CCS The morphology and pore size of the CCS were observed using scanning electron microscopy (SEM; JEOL, Tokyo, Japan). The swelling study was performed to assess the water absorption capacity of the CCS. The dry weight of the CCS was measured with an analytic balance, and it was then immersed into PBS. The CCS wet weights were accurately determined at preset time points after removing the PBS from the surface with filter paper. The water absorption of the CCS was calculated with the following formula28: water absorption = (We − W0)/W0, where We is the weight of the wet scaffold and W0 is the initial weight of the dry scaffold. The CCS degradation study was performed in PBS with collagenase I. These reactions were incubated at 37°C and the digestion was terminated with 0.25 M ethylenediamine tetraacetic acid (EDTA) at specific time intervals. At predetermined time points, the samples were subjected to dry weight analysis. The initial weight of the CCS samples was recorded. The residual mass (%) was calculated using the following formula28: residual mass (%) = 100 − ((Wd − WS)/Wd × 100), where Wd represents the sample dry weight before degradation and WS indicates the sample dry weight after degradation.
Green Fluorescence Protein (GFP) Lentiviral Vectors Construction and Labeling of UC-MSCs With GFP UC-MSCs were infected with a lentiviral vector for GFP (provided by our laboratory). GFP lentiviral vectors were obtained by transfection of 293T cells. The GFP lentiviral vectors were mixed with polyethyleneimine (PEI; Polysciences, Eppelheim, Germany; pH 7) at a volume rate of 1:3, transfected into 293T cells, and the supernatant collected. The supernatant was concentrated through PEG6000 (Sigma-Aldrich, St Louis, MO). UC-MSCs were infected with concentrated lentiviral supernatant at a multiplicity of infection (MOI) of 5 to 10.
Chloromethyl-Dialkylcarbocyanine (CM-Dil) Cell Labeling Cell Tracker CM-Dil (Invitrogen Life Technologies, Carlsbad, CA) was prepared using the manufacturer’s instructions. A total of 1 × 106 UC-MSCs in 500 µL PBS were labeled with 2 µL CM-Dil solutions(1 mg/mL). CM-Dil-labeled cells were used for the following experiments.
In Vitro Epidermal-Like Differentiation of UCMSCs The sterilized CCSs were prewetted and placed in 6-well Transwell plates (24 mm Transwell, Corning Incorporated, USA; Figure 4A). A 100-µL UC-MSCs suspension (1 × 106 cells/100 µL) was loaded onto the top surface of each prewetted CCS. The cells/CCS constructs were incubated at 37°C with 5% CO2 for 4 hours for the cells to penetrate into the CCS and adhere. After the cells adhered, 100 µL UC-MSCs suspension was again seeded on the surface of the cells/CCS constructs and incubated at 37°C with 5% CO2 for an additional 4 hours, and cultured in L-DMEM medium. The cells were exposed to air by lowering the medium level to the lower part of the CCS after 2 days in the immersed culture. The medium was changed once each day. At each indicated time interval, the samples were collected for further experimental analysis.
MTT Assay Cell proliferation was determined by methyl thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) absorption as previously described29 and analyzed using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA) at 570 nm.
Live/Dead Assay Cell viability was tested using a Live/Dead assay (Invitrogen, UK). The samples were evaluated following the instruction of the Live/Dead assay kit and were viewed under an Olympus DP72 fluorescence microscope with 528 nm (red, EthD-1) and 494 nm (green, Calcein) excitation filters. For quantitative analysis, a total of 300 cells were counted from each sample over 5 randomly chosen sections, and the live and dead cell counts were recorded.
Collagen Quantification The total amount of collagen secreted by the cells seeded in the scaffold was measured using Sirius Red (SigmaAldrich) stain. The protocol was performed as previously described,30 and the absorbance was measured at 540 nm. By quantifying the amount of collagen in the scaffold without cell seeding and deducting the OD value, the CCS background was excluded.
Western Blot Analysis After 7 and 14 days of UC-MSC culture at the ALI, the total cellular proteins were extracted using cell lysis buffer (Beyotime Biotechnology, Beijing, China), and western blot analysis was performed as previously described.26 The mouse monoclonal antibodies against cytokeratin 19
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(1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and involucrin (1:1000; Abcam, Cambridge, MA) were used as primary antibodies. A mouse monoclonal antibody against β-actin (1:1000; Sigma, St Louis, MO) was used as a loading control. The appropriate horseradish peroxidase–conjugated antibody (1:300; (DAKO, Glostrup, Denmark) was used as a secondary antibody. The immunoreactive proteins were visualized using the SuperECL western blotting detection reagent (Applygen Technologies Inc, Beijing, China).
Wound Healing Assay The in vivo surgery and photographs were performed under 3% pentobarbital sodium anesthesia. Male SD rats (approximately 200 g) were obtained from the Chinese PLA General Hospital. The rats were housed in cages in a temperaturecontrolled room (22-25°C) on a 12-hour/12-hour light/dark schedule with free access to food and water. Forty-five rats were randomly divided into 3 groups, the control group (no treatment), the CCS group (CCS without cells), and the epidermal substitute group (the epidermal substitute which was constituted by the CCS and the cells inner of it and on the surface of it). Following anesthetization, dorsal regions shaved, an open excision-type wound of 1 cm in diameter was created on the middle dorsal skin to the depth of subcutaneous tissue using a dermal punch under aseptic conditions, and a CCS or epidermal substitute was placed on the wound immediately, whereas wounds with no treatment were the control. The animals were sacrificed after 3, 7, 14, and 21 days to assess skin regeneration. The aforementioned experiments were repeated 3 times and showed consistent results.
Histology and Immunohistochemistry The epidermal substitutes and wound skins were fixed in 10% formalin, dehydrated, embedded in paraffin, and cut into 5 µm sections. General histology was visualized by hematoxylin–eosin (H&E) staining. Immunohistochemical analysis of the epidermal substitutes was performed using primary antibodies specific for mouse anti-human CK19 (Santa Cruz) and mouse anti-human involucrin (Abcam), and secondary horseradish peroxidase–conjugated goat anti-mouse antibodies (DAKO) followed by visualization with 3,3-diaminobenzidine tetrahydrochloride (DAB; DAKO). Next, H&E-stained sections and immunostained sections were observed under a light microscope (Olympus DP72, Tokyo, Japan).
Statistical Analysis The data were expressed as the means ± standard deviation (SD). Significant differences were determined using
Student’s 2-sample t test. Differences were considered statistically significant at a P value less than .05.
Results Characterization of UC-MSCs The characteristics of UC-MSCs were analyzed by morphological, multipotent differentiation potential, and flow cytometry analysis. The morphology of UC-MSCs was elongated and spindle-shaped after passage 3 (Figure 1A), and they could differentiate into adipocytes and osteoblasts (Figure 1B and C). Flow cytometry revealed that UC-MSCs were positive for CD90, CD105, and CD73, whereas they were negative for CD34, CD45, HLA-DR, and CD11α (Figure 1D). Therefore, UC-MSCs were successfully isolated for use in the following experiments.
Characterization of CCSs To simulate the 3D microenvironment in vitro, we constructed CCSs using collagen and chitosan through crosslinking and lyophilization.31 The CCS was a sponge-like structure (Figure 2A and B) with a porous structure, and 100 to 200 µm pore size as determined by light microscopy and scanning electron microscopy (Figure 2C and D). Furthermore, the water absorption of the CCS was nearly 13 times their initial weight after 8 hours in phosphate buffer, which demonstrated that the CCS has a good water absorption capacity (Figure 2E). When the CCS was treated with collagenase, the residual mass decreased to approximately 40% after 70 hours of treatment, which demonstrated an appropriate degradation rate (Figure 2F). These results indicated that the CCS with suitable properties was successfully constructed.
CCS Biocompatibility The biocompatibility of CCS was analyzed by morphological analysis, MTT assay, Live/Dead staining, and collagen production assay. UC-MSCs were infected with GFPexpressing lentivirus and transplanted into the CCS. Based on the fluorescence micrographs, we observed that UC-MSCs were spindle-shaped and distributed evenly in the scaffold (Figure 3A). SEM micrographs showed that UC-MSCs attached well to the CCS in the form of cell layers (Figure 3B). MTT assay (Figure 3C) showed that UC-MSCs in the CCS were not proliferating. Live/Dead staining (Figure 3E and F) revealed that the live percentage of the cells was 98% at 3 days, 95% at 7 days, and 86% at 14 days. Sirius Red staining (Figure 3D) showed the total amount of collagen in the CCS was significantly increased at 3 days. The results confirmed that the scaffold has good biocompatibility with UC-MSCs.
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Figure 1. Characterization of UC-MSCs.
(A) Morphology of UC-MSCs under light microscopy showing the spindle-shaped appearance of cell colonies after passage 3 (scale bar = 200 µm). (B, C) Multilineage differentiation potential of UC-MSCs showing UC-MSCs differentiated into adipocytes, which are indicated by the accumulation of lipid vesicles in the cells (scale bar = 50 µm) and osteoblasts, which express alkaline phosphatase, as indicated in blue (scale bar = 100 µm). (D) UC-MSC surface marker expression by flow cytometry showing the percentages of UC-MSCs for mesenchymal antigens, CD90, CD73, and CD105, which were 99.94%, 97.11%, and 97.34%, respectively, whereas the percentages of CD34, CD45, CD11a, and HLA-DR were 2.01%, 0.07%, 0.01%, and 0.35%, respectively.
UC-MSCs Transdifferentiate Into Epidermal-Like Cells for Engineering Epidermal Substitutes To induce UC-MSC transdifferentiation into epidermal-like cells in vitro, UC-MSCs were seeded onto the surface of the CCSs populated with UC-MSCs. Following 2 days of immersion in medium, UC-MSCs were grown at an ALI, which mimics the microenvironment of human skin in vivo.32 Immunohistochemistry results showed that several of the cell layers on the surface of the CCS were positive for CK19 and involucrin after culturing at the ALI for 7 days and 14 days (Figure 4D). This observation was corroborated by western blot analysis. The results also showed that both CK19 and involucrin were more highly expressed at 14 days than 7 days (Figure 4C). H&E staining (Figure 4B) showed cells distribution throughout the porous inside of the CCS and revealed that a multilayered epidermal substitute had formed on the superficial area of the CCS with an organization similar to the skin epidermis at both 7 days and
14 days. These results demonstrated that UC-MSCs are differentiated into epidermal-like cells in the 3D microenvironment and formed a stratified epidermal substitute.
In Vivo Wound Healing The role of the constructed epidermal substitute in wound healing was evaluated in a rat full-thickness wound model of 1 cm in diameter. The results revealed that the stratified epidermal substitutes accelerated wound closure (Figure 5A and B). At 3, 7, and 14 days after surgery, the epidermal substitute groups showed significantly better wound closure compared with the control group. At 21 days, all wounds in the epidermal substitutes and CCS groups had achieved complete wound closure, but not all the wounds of the control groups were completely closed. Although no significant difference was observed between the epidermal substitutes and CCS group at any time, H&E (Figure 5C) staining showed that the epidermal substitutes reduced the inflammation response at
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Figure 2. Characterization of CCS.
(A, B) Macroscopic view of the CCS structure. (C) The optical micrographs indicate the presence of porosity in the scaffold structure (scale bar = 200 µm). (D) Scanning electron microscopy shows that the scaffold has a porous structure and the pore size is 100 to 200 µm (scale bar = 200 µm). (E) Swelling kinetics of the CCS in PBS (pH 7.4). (F) Degradation kinetics of the CCS in PBS (pH 7.4).
3 days and enhanced reepithelialization and granulation at 7 and 14 days compared with the CCS group and control group. After 21 days, all wounds showed a regenerated epithelium with several viable layers. Interestingly, in epidermal substitutes group, bud-like structures were observed, appearing as epidermal ingrowth and hair follicle differentiation. In addition, the tracer experiment (Figure 5D) demonstrated the CM-Dil-positive cells remained at the subcutaneous tissue of the rats at 21 days and they did not participate in the neo-epidermis formation. These results demonstrate that epidermal substitutes promote wound healing in a rat full-thickness wound model.
Discussion In this study, we investigated whether the 3D microenvironment in which the CCS harbors viable UC-MSCs could induce UC-MSC differentiation into epidermal-like cells in ALI culture and construct epidermal substitutes. These results demonstrated that UC-MSCs successfully differentiated into epidermal-like cells and formed epidermal substitutes. Moreover, when the constructed epidermal substitutes were used to treat the full-thickness wounds in rats, successful wound healing with a well-differentiated epidermis was observed.
One crucial factor of the 3D microenvironment in vitro is the construction of a scaffold. A 3D scaffold provides an ECM analog for cell penetration and a physical support for cell proliferation and differentiation.18 Previous studies demonstrated that the scaffold widely used in tissue engineering possesses a microstructure with 100 to 200 µm pore size and porosity greater than 90%, high swelling efficiency, suitable biodegradability, and excellent biocompatibility.33 The CCS with the above-mentioned characteristics embedded with dermal fibroblasts showed that the scaffold accelerated cell infiltration and growth in vitro. Moreover, animal experiments demonstrated that the scaffold promoted fibroblasts infiltration from the adjacent tissue in vivo.18 In a rat full-thickness wound model, a similar collagen/HA/gelatin scaffold harboring cells had been shown to promote wound healing.30 In the present study, we used EDC and NHS to cross-link the collagen and chitosan to construct a CCS. Consistent with previous studies,33 CCS also has excellent properties, including a pore size of 100 to 200 µm and porosity greater 90%, biodegradability, and excellent biocompatibility. These results indicated that CCS can be used as a scaffold for constructing the 3D microenvironment. The seed cell is another important factor of the 3D microenvironment in vitro. In a conventional culture model, the fibroblasts are usually used as the seed cells that are incorporated into the dermal substitutes to facilitate the formation of stratified epidermis in vitro.34 However, when MSCs replaced the fibroblasts, a stratified epidermis was also observed.35 In the keratinocyte-MSC skin model, MSCs were used as seed cells and were seeded into a deepidermalized dermis, and the keratinocytes were cultured on the de-epidermalized dermis at an ALI to induce epidermal differentiation.36 These results demonstrated that MSCs contribute to epidermal morphology formation and ECM production similar to fibroblasts. Numerous studies have reported that MSCs play a role in wound repair through paracrine effects by releasing bioactive molecules that affect cell migration and proliferation, anti-inflammation, and anti-scar.37,38 Therefore, these attributes make them useful for cell-based bioengineered products in skin repair therapy. UC-derived cells are a group of impure cell population and still have not a definite marker so far. In our study, they were isolated and purified by adherent culture method,26 and the majority of isolated UC-derived cells come to meet the minimal criteria proposed by the International Society for Cellular Therapy, including growing adherent to plastic, exhibiting a multilineage differentiation potential, and expressing CD105, CD73, and CD90 and lack the expression of CD45, CD34, CD11b, and HLA-DR, for defining as multipotent MSCs.39 So we termed UC-derived cells as UC-MSCs. UC-MSCs were used as seed cells and cultured in the inner of CCS, in which they not only survived well but also expressed collagen. These results indicate that the 3D microenvironment
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Figure 3. The CCS biocompatibility with UC-MSCs.
(A) Morphology of UC-MSCs within the CCS observed by fluorescence microscope (scaffold preloaded with GFP-expressing UC-MSCs) (scale bar = 100 µm). (B) Scanning electron micrographs of UC-MSCs seeded on the CCS (scale bar = 200 µm). (C) Proliferation patterns (MTT assay) of UC-MSC culture on tissue culture plates (as a control) and CCSs. UC-MSCs were cultured for 3, 7, and 14 days and evaluated for proliferation using the MTT assay. (D) The total collagen amounts in the scaffold are shown. (E) Live/Dead staining results after 3, 7, and 14 days culture. Green spots indicate living cells, and red spots indicate dead cells (scale bar = 100 µm). (F) Quantitative analysis of Live/Dead staining results.
Figure 4. UC-MSCs transdifferentiate into epidermal-like cells for engineering epidermal substitutes.
(A) Schematic diagram showing the protocol for inducing UC-MSC differentiation. The CCS was first seeded within UC-MSCs until the cells adhered, and then seeded with UC-MSCs on the top of the CCS cultured for 2 days immersed in culture medium, and 7 to 14 additional days at the air–liquid interface to promote epidermal differentiation. (B) H&E staining for UC-MSCs cultured on CCS (scale bar = 100 µm). (C) Immunohistochemistry staining: UC-MSCs were cultured for 7 and 14 days. Cells on the top of the CCS were positive for CK19 and involucrin (scale bar = 100 µm). (D) Western blotting analysis for CK19 and involucrin expression.
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Figure 5. In vivo wound healing.
(A) Representative macroscopic images of full-thickness dorsal skin wounds in the control group, the scaffold group, and the epidermis substitutes at 0, 3, 7, 14, and 21 days after injury. (B) Quantitation of the wound area of full-thickness dorsal skin wounds at 0, 3, 7, 14, and 21 days after injury. Each value is the mean ± SD, n = 3, *P < .05, **P < .01. (C) Representative H&E-stained sections of the full-thickness dorsal skin wounds from rats with scaffold or epidermis substitutes at 3, 7, 14, and 21 days after wounding (scale bar = 100 µm). (D) CM-Dil-labeled cells in the rat skin were observed under fluorescent microscope at 21 days after operation. The red fluorescence that indicated the CM-Dil-labeled cells were obviously visible in subcutaneous tissue at 21 days after operation. Up: scale bar = 100 µm; down: scale bar = 50 µm.
fabricated by viable MSCs, CCS, and ECM, cytokines selected by MSCs, can create an artificial niche. Skin epidermal cells grow on the basement membrane and are exposed to air in vivo.40 Therefore, we cultured these cells at the ALI in vitro in similar physiological conditions. A previous study showed that the ALI culture stimulated the early differentiation of human amnion epithelial cells seeded on a fibrin layer embedded with human amnion mesenchymal cells to epidermal cells, which indicate that the ALI culture system is crucial for enhancing epidermal differentiation.20 Consistent with the above-mentioned finding, BM-MSCs cultured in an induction medium on a contractible fibroblast-embedded collagen gel with an ALI also committed MSCs to an epidermal lineage.22 In our study, when MSCs were placed on the surface of MSCembedded CCSs and grown at the ALI, they were positive for K19 and involucrin at 7 and 14 days. H&E staining showed that a multilayered epidermal substitute with an organization similar to the skin epidermis was formed. In
the previous researches, the successful differentiation of hUC-MSCs into epidermal-like cells was also confirmed by indirect co-culture with epidermal stem cells,41 which is mainly due to the role of the factors selected by epidermal stem cells. However, another study has demonstrated that UC-MSCs failed to differentiate into epidermal cells after they were cultured at an ALI on dermal equivalents that consist of collagen I/III and human dermal fibroblasts and were further treated with 5-azacytidine or all-trans retinoic acid.24 The failure of differentiation may be attributed to the culture system, which is not enough to stimulate UC-MSC differentiation into an epithelial phenotype. Compared with the previous studies, we speculated that the suitable 3D microenvironment including the suitable scaffold, cytokines, and ECM secreted by UC-MSCs might be the prominent inducers for epidermal differentiation. The constructed epidermis substitutes have been proven to promote wound healing in a rat full-thickness model. To explore the role of UC-MSCs, the cells on the surface of the
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Chen et al CCSs were labeled with CM-Dil, and the cells inner of the CCSs were labeled with GFP. The results showed that there are no GFP-positive cells in the rats probably because the cells inner of the CCSs were dead. However, CM-Dilpositive cells on the surface of the CCSs remained in the rats until 21 days after surgery but they did not participate in the neo-epidermis formation. Therefore, we conclude that UC-MSCs in promoting wound healing are probably due to their paracrine effect. This is supported by numerous previous studies, which have demonstrated that MSCs could secrete lots of factors that regulate cellular responses to skin damage and their paracrine effect is also supported by MSC-conditioned medium enhanced wound closure.42 In conclusion, our study demonstrates that UC-MSCs differentiate into epidermal-like cells in the 3D microenvironment in vitro, which mimics the environment of native skin, and can be used to construct epidermal substitutes. In vivo animal tests showed that the constructed epidermis substitutes have the ability to promote wound healing. These findings suggested that mimicking the 3D microenvironment can be used for epidermal-like cells differentiation to construct epidermal substitutes, which provide an alternative strategy for skin tissue engineering. Authors’ Note Deyun Chen and Haojie Hao contributed equally to this work.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported in part by the National Basic Science and Development Program (2012CB518103, 2012CB518105), the 863 Projects of Ministry of Science and Technology of China (2013AA020105, 2012AA020502), Military Medical Foundation (AWS11J008), and National Natural Science Foundation of China (81121004, 81230041).
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