- Email: [email protected]
Wound-healing potential of human umbilical cord blood–derived mesenchymal stromal cells in vitro—a pilot study
Cytotherapy, 2015; 0: 1e8
Wound-healing potential of human umbilical cord bloodederived mesenchymal stromal cells in vitro—a pilot study
HI-JIN YOU, SIK NAMGOONG, SEUNG-KYU HAN, SEONG-HO JEONG, EUN-SANG DHONG & WOO-KYUNG KIM Department of Plastic Surgery, Korea University College of Medicine, Seoul, Korea Abstract Background aims. Our previous studies demonstrated that human bone marrowederived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord bloodederived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. Methods. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n ¼ 5) and diabetic (n ¼ 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n ¼ 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-b) production were compared among the three cell groups. Results. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-b production. Conclusions. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Key Words: fibroblast, human umbilical cord blood stem cells, wound healing
Introduction The prevalence of chronic wounds, including diabetic ulcers, is increasing dramatically, as the populations of industrialized countries age and become more sedentary. Chronic wounds respond poorly to conventional treatment, making them very difficult to manage. There has recently been much interest in the use of cell-based therapies for the treatment of diabetic wounds. These therapies have been designed to modulate the levels of biologic molecules, including growth factors and extracellular matrices that may promote wound healing [1,2]. Various types of skin substitutes composed of either allogeneic or autologous fibroblasts and/or keratinocytes have been commercialized for the treatment of diabetic foot ulcers [3e7]. Although allogeneic fibroblasts, derived from healthy donors, are readily accessible, cellular activities are impaired as the result of cryopreservation.
Additionally, they may carry the risk of immunologic reactions or cross-infections. Autologous fibroblasts are advantageous because they may result in permanent engraftment. However, they require a long culture time, and, in the case of diabetic patients, autologous fibroblasts may not exhibit a sufficient capacity to stimulate wound healing. Mesenchymal stromal cells (MSCs) may hold great promise in treating diabetic wounds because they have the advantages of both allogeneic and autologous cells. MSCs demonstrate low levels of immunity-assisted rejection and have the ability to divide without apoptosis. It was demonstrated that even after 20 or 30 cycles of cell doubling in culture, they still retain their initial stem cell properties. Accordingly, MSCs have attracted much attention in the bioengineering field [8]. Bone marrow stroma is one of the main sources of MSCs. Previous studies performed by our group
Correspondence: Seung-Kyu Han, MD, PhD, Department of Plastic Surgery, Korea University, Guro Hospital, 148 Guro-Dong, Guro-Ku Seoul 152e703, Korea. E-mail: [email protected] (Received 31 March 2015; accepted 22 June 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.06.011
2
H.-J. You et al.
demonstrated that bone marrowederived mesenchymal stromal cells (BM-MSCs) synthesize higher amounts of collagen, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in vitro, as compared with dermal fibroblasts. Furthermore, they showed greater activity in granulation formation, epithelialization and angiogenesis in vivo, indicating a potential use in accelerated wound healing [9e11]. However, the number of MSCs in the BM decline with age, and their procurement is an invasive procedure. Furthermore, at present, it is difficult for a clinician to use cultured BM-MSCs for the purpose of wound healing because of issues regarding approval by the Food and Drug Administration (FDA) [12,13]. Meanwhile, a new commercial drug that uses human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) has been developed and approved by the Korean FDA to help regenerate knee cartilage. They can be acquired in massive quantities without significant ethical issues. Furthermore, cord blood stem cells are more immature than adult MSCs and expand easily in vitro [13]. Because the immune system is undifferentiated, successful transplantation without rejection has been reported [14e16]. Therefore, if hUCB-MSCs have greater wound-healing potential than fibroblasts, bioengineered dermal substitutes that are based on allogeneic/autologous fibroblasts, which are the main cell sources in current commercially available cell therapy products to treat diabetic wounds, could be replaced by a superior therapy with hUCB-MSCs. Some researchers have reported that hUCB-MSCs can improve wound healing [16e24]. However, no comparative analysis has been performed to evaluate the wound-healing activity of hUCB-MSCs and fibroblasts. The final purpose of our project is to prove superiority of the hUCB-MSCs over fibroblasts in treating diabetic wounds, and this pilot study was designed to compare the wound-healing activity of hUCB-MSCs with those of healthy and diabetic fibroblasts. Healthy and diabetic fibroblasts were used as representatives of cell sources for allogeneic and autologous fibroblasts in diabetic patients, respectively. This study was especially focused on cell proliferation, collagen synthesis and the production of three growth factors that are essential factors for diabetic wound healing, that is, basic FGF (bFGF), VEGF and transforming growth factor-b (TGF-b). Methods Isolation and culture of healthy fibroblasts, diabetic fibroblasts and hUCB-MSCs Cultured healthy and diabetic fibroblasts used for the experiments were obtained from cryopreserved cells
derived from the dermis of healthy (n ¼ 5) and diabetic (n ¼ 5) adults. The cell donors had provided informed consent for their cells to be used for research purposes. Both types of the fibroblasts were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient (DMEM/F-12; Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 25 mg/mL gentamycin at 37 C in a 5% CO2/95% air atmosphere at 100% humidity. The fibroblasts were dissociated by trypsinization, diluted 2.7-fold in Dulbecco’s phosphate-buffered saline without Mg2þ and Ca2þ (DPBS; Gibco) and collected by centrifugation at 450g for 17 min. The cells were washed twice in 40 mL of DPBS and re-suspended in 5 mL of DPBS. Cell density was determined by use of a hemocytometer, and viability was assessed by use of a trypan blue dye exclusion assay. Three-passage cells were used for all the experiments in this study. Human UCB-MSCs were purchased from Medipost Co Ltd [25]. According to the manufacturer’s protocols, hUCB samples were collected from the umbilical vein of deliveries with informed maternal consent. A 16-gauge needle from an hUCB collection bag containing 23 mL of CPDA-1 anticoagulant (Greencross Co) was inserted into the umbilical vein, and the hUCB was allowed to flow with gravity. UCB harvests were processed in all cases within 24 h of collection, with a viability of >90%. Mononuclear cells were isolated from the hUCB by means of centrifugation through a Ficoll-Hypaque gradient (density, 1.077 g/cm3, Sigma). The separated mononuclear cells were washed, suspended in a-minimum essential medium (a-MEM, Gibco BRL), supplemented with 10% FBS (HyClone) and 25 mg/mL gentamycin and seeded at a concentration of 5 106 cells/cm2. Cultures were maintained at 37 C in a humidified atmosphere containing 5% CO2 with a bi-weekly change of culture medium. When the monolayer of fibroblast-like adherent cells colonies had reached 80% confluence within 1 to 3 weeks, the cells were trypsinized (0.25% trypsin, HyClone), washed, resuspended in culture medium (a-MEM supplemented with 10% FBS) and sub-cultured. Five different cell lines were used in the study. Evaluation of cell proliferation, collagen synthesis and growth factor production For the cell proliferation assay, the cultured fibroblasts and hUCB-MSCs were seeded at 1.5 103 cells/well in 100 mL of a-MEM, supplemented with 5% FBS and 315 mg/dL glucose in 96-well culture plates. The cells were seeded onto a 24-well culture plate at 1.0 104 cells/well in 1.0 mL of the culture medium for the collagen synthesis and growth factor assay. The cells were incubated at 37 C in a 5% CO2/95% air
Umbilical cord bloodederived mesenchymal stromal cells
3
atmosphere at 100% humidity. The post-incubation cell proliferation, collagen synthesis and growth factor production were compared after 3 days.
used as subject data. Statistical comparisons were performed with the Mann-Whitney U test, with values of P < 0.05 considered statistically significant.
Cell proliferation assay
Results
Cell proliferation was determined by use of a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT; Sigma). Briefly, 10 mL MTT of 5 mg/mL was added to a 100-mL cell monolayer in each well of a 96-well plate, after which an incubation was performed for 3 h at 37 C. Next, 100 mL of 0.04 mol/ L HCL in propan-2-ol was added to each well and mixed thoroughly to dissolve the insoluble blue formazan crystals. The absorbance was measured at a test wavelength of 570 mm and a reference wavelength of 630 mm with the use of an enzyme-linked immunosorbent assay (ELISA) reader.
Monolayer cultures of human dermal fibroblasts consisted of homogeneously spindle-shaped cells (Figure 1). Most hUCB-MSCs were larger and had a more polygonal shape.
Collagen synthesis assay Collagen type I carboxyl-terminal propeptide (CICP) enzyme immunoassay was performed to measure the production of collagen, with the use of a Metra CICP kit (Quidel) according to the manufacturer’s instruction. Briefly, 100 mL of diluted culture supernatants was added to each well of the monoclonal anti-CICP antibody-coated plates, and the plates were then incubated at room temperature for 2 h. One hundred microliters of rabbit anti-CICP antisera was added for 50 min, followed by 100 mL of goat antirabbit alkaline phosphatase conjugates for 50 min. The reaction was stopped and the collagen synthesis was measured at 405 nm.
Cell proliferation After the 3-day incubation, the average cell number of the hUCB-MSCs was 3.8 0.4 103 per well, which was greater than those of the healthy fibroblasts (3.5 0.6 103 per well) and the diabetic fibroblasts (3.3 0.7 103 per well). Cell proliferation in the hUCB-MSC group was 9% and 16% greater than those in the healthy fibroblast and diabetic fibroblast groups, respectively. However, there was no significant statistical difference (P ¼ 0.347 and 0.251, respectively; Figure 2). Collagen synthesis Collagen synthesis of the hUCB-MSC group significantly increased as compared with that of the diabetic fibroblast group (P ¼ 0.009). Healthy fibroblasts also showed significantly increased collagen synthesis compared with that of the diabetic fibroblasts (P ¼ 0.009). However, there was no significant difference between the hUCBMSCs and healthy fibroblasts (P ¼ 0.076, Figure 3).
Growth factor level assay The levels of bFGF, VEGF and TGF-b1 were measured in culture media with an ELISA kit (R&D Systems) according to the manufacturer’s instruction. Briefly, the samples and standards were diluted appropriately in the assay diluent and then incubated at room temperature in wells pre-coated with the specific capturing antibodies. After washing, the respective detection antibody conjugate was added and was then incubated at room temperature before another washing. Addition of a substrate solution resulted in color development. A stop solution was added and the color intensity was measured in an ELISA reader at the appropriate wavelength. The measured TGF-b1 in this study included both the active and latent forms. Statistical analysis All experiments were performed in triplicate on each explant from each individual. The average value was
Growth factor analysis Of the three growth factors studied, the hUCBMSC group showed significantly higher levels of bFGF and VEGF expression. Basic FGF levels in the hUCB-MSC group were 59% (1.6-fold) and 96% (2-fold) greater than those for the healthy fibroblast and diabetic fibroblast groups (P ¼ 0.028 and 0.009, respectively; Figure 4). The most profound difference was observed in the levels of VEGF. The VEFG levels of the hUCB-MSC group were 469% (6-fold) and 586% (7-fold) higher than the VEGF levels of the healthy fibroblast and diabetic fibroblast groups, respectively (P ¼ 0.009, both; Figure 5). However, the TGF-b level of the hUCB-MSC group showed no significant difference when compared with that of the healthy fibroblast and the diabetic fibroblast groups (P ¼ 0.602 and 0.175, respectively; Figure 6).
4
H.-J. You et al.
Figure 1. Human dermal fibroblasts (left), diabetic fibroblasts (center) and hUCB-MSCs (right) in a monolayer culture (original magnification 100).
Discussion Wound-healing strategies with the use of cell transplantation have involved the use of fibroblasts or fibroblast-like cells. A cryopreserved human fibroblastederived dermal substitute (Dermagraft; Shire Regenerative Medicine, Inc) [4] and a bilayered living human skin equivalent (Apligraf; Organogenesis Inc) [7,26] are representative allogeneic skin substitutes that are reportedly effective for treating diabetic foot ulcers. The use of non-cryopreserved fresh human fibroblast allografts was also found to be a safe and effective treatment for diabetic foot ulcers [27,28]. Recently, a new bioengineered dermal substitute composed of cultured autologous fibroblasts seeded on a hyaluronic acid sheet (Hyalograft 3D; ChaBio & Diostech) was developed.
Figure 2. Cell proliferation of the three cell groups (hUCB-MSC, human umbilical cord bloodederived mesenchymal stromal cell group; FB, healthy fibroblast group; DM FB, diabetic fibroblast group). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. The average cell number of the hUCB-MSCs was 3.8 0.4 103 per well, which was greater than those of the healthy fibroblasts (3.5 0.6 103 per well) and the diabetic fibroblasts (3.3 0.7 103 per well).
A randomized, controlled trial showed that the product may provide a safe and effective treatment for diabetic foot ulcers. Human BM-MSCs have been found to promote wound healing [8e11] and have been used in clinical treatment of challenging wounds [12]. Mesenchymal stromal cells isolated from human umbilical cord blood can be easily obtained and refined compared with stem cells from other sources. Because hUCB-MSCs are rich, easy to collect and proliferate up to 4 times the number of cells within 3 to 5 days in appropriate culture conditions in vitro, they could be produced on a larger scale. The most important advantage of hUCB-MSCs is their very low immunogenicity, which provides potential clinical benefits to patients [29]. The acquisition of hUCB-MSCs in contrast to BM-MSCs may not be
Figure 3. Collagen synthesis of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. Collagen synthesis of the hUCB-MSC group (170 28 ng/mL) was significantly increased when compared with that of the diabetic fibroblast group (132 8 ng/mL). The collagen synthesis of the healthy fibroblast group was 210 34 ng/mL, which was also greater than that of the diabetic fibroblasts.
Umbilical cord bloodederived mesenchymal stromal cells
Figure 4. Basic FGF levels of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. The bFGF level of hUCB-MSCs (3.8 0.9 pg/mL) was significantly greater than those of the healthy fibroblasts (2.4 0.6 pg/ mL) and the diabetic fibroblasts (1.9 0.6 pg/mL).
dependent on the overall health status of the recipient [30]. On the basis of these biological advantages, a combination of hUCB-MSCs and sodium hyaluronate (CARTISTEM, Medipost Co Ltd) is intended for use as a single-dose cellular therapeutic agent for cartilage regeneration in human subjects with cartilage defects of the knee caused by aging, trauma or degenerative diseases. With the approval from the Korean FDA, CARTISTEM has become the world’s
Figure 5. VEGF levels of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. The VEFG level of the hUCB-MSC group (638 43 pg/mL) was significantly higher than the VEGF levels of the healthy fibroblast (112 44 pg/mL) and the diabetic fibroblast groups (93 31 pg/mL).
5
Figure 6. TGF-b levels of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. TGF-b levels of the hUCB-MSCs, healthy fibroblasts and diabetic fibroblasts were 7685 1263 pg/mL, 8393 906 pg/mL and 6283 1121 pg/ mL, respectively. Statistical significance was observed between the healthy fibroblasts and the diabetic fibroblasts (P ¼ 0.028).
first allogeneic, off-the-shelf stem cell drug and is now available in Korea. The therapeutic potential of hUCB-MSCs has been widely explored as a promising tool in the treatment of various diseases; however, its effect on the healing of chronic wounds has not been studied to the same degree [15] There are few reports on the application of MSCs from hUCB to improve wound healing. Zhao et al. [29] demonstrated that hUCBMSCs specifically localize to the target ulcerated tissue and may promote the epithelialization of diabetic foot ulcers in rats. Luo et al. [23] demonstrated that the local application of hUCB-MSCs may not only be able to accelerate the speed of the wound healing but also may improve the quality of wound healing in mice. Tark et al. [16] used the diabetic mouse as a model of delayed wound healing. They concluded that hUCB-MSCs had a positive effect on wound healing, based on TGF-b levels. However, the majority of studies on hUCB-MSCs are limited and the underlying mechanisms have not yet been determined. Furthermore, the majority are animal studies. We have attempted to assess cell characteristics and phenotypes in vitro in comparison to healthy and diabetic fibroblasts. This is the first quantitative comparison of MSCs from human UCB and somatic cells. Human UCB-MSCs produced slightly lower amounts of TGF-b when compared with healthy fibroblasts, though there was no statistical difference. This finding may be related to the lower degree of collagen synthesis seen in hUCB-MSC
6
H.-J. You et al.
group because TGF-b is a potent stimulator of fibroblast proliferation and collagen synthesis. Our results may conceptually reflect a previous report that wound contraction is inhibited by the local administration of MSCs [23]. The fibroblast is central to the normal wound-healing process [31]. Diabetes influences this process at the molecular level, resulting in fibroblast senescence and slower growth factor production [32]. Our results also demonstrated the impaired functions of diabetic fibroblasts. The diabetic fibroblast group showed significantly decreased levels of TGF-b and a lower degree of collagen synthesis compared with the healthy fibroblast group. Human UCB-MSCs, on the other hand, produced significantly greater amounts of VEGF and bFGF, as compared with dermal fibroblasts and diabetic fibroblasts. The observed VEGF levels were especially striking. Basic FGF, an angiogenic growth factor that stimulates endothelial cell proliferation and migration, is an efficient mediator of VEGF activity. VEGF, a family of platelet-derived growth factors, induces angiogenesis and endothelial cell proliferation while stimulating vascular permeability. Additionally, VEGF can cause vasodilatation. It can also stimulate cell migration and inhibit apoptosis. VEGF and bFGF are potent angiogenic factors. Angiogenesis is a critical step in the wound healing process, particularly in chronic wound healing. Neovascularization, the formation of new blood vessels, is necessary to sustain the newly formed granulation tissues and the survival of keratinocytes. This study showed that there are justifiably high expectations for the use of hUCB-MSCs in wound healing because they have the potential to induce neovascularization and release the various necessary growth factors and cytokines. Clinical applicability of hUCB-MSCs might be hindered by potential graft rejection caused by the immunological barrier imposed by human leukocyte antigen mismatching. However, the immunomodulatory properties of hUCB-MSCs allow for an allogeneic cell source for stem cell-based therapy. Oh et al. [13] demonstrated that hUCB-MSCs can suppress the allogeneic responsiveness of human lymphocytes, as they suppress their proliferation and reduce their production of immunostimulatory cytokines. Lee et al. [33] used a humanized mouse model to provide evidence that the low immunogenicity of allogeneic hUCB-MSCs can be maintained in vivo. Their data suggested that hUCB-MSCs are “immunologically safe” for use in allogeneic cell therapies. Possible drawbacks of autologous cells can be exaggerated in patients with chronic wounds, owing to the attenuated cell activity. For example, even BM-MSCs show reduced growth in culture in
patients with chronic wounds. The therapeutic benefits of using allogeneic MSCs from healthy donors might surpass those of autologous MSCs derived from patients with chronic wounds. Human UCBMSCs rather than BM-MSCs or other somatic cells may therefore be the preferred choice in stem cellebased bioengineered products for wound healing. The route of cell administration, the number of cells transplanted, the number of treatments and the interval between individual treatments may be key points in cell therapy for wound healing. Allogeneic MSCs have been usually delivered in a single dose by various routes in animal wound-healing models. However, the most optimal condition to accelerate healing of diabetic wounds has not yet been determined. The effective delivery method must be determined to develop commercial bioengineered products with hUCB-MSCs. One limitation of the present study is that the cells were not grown in the same medium. The optimal medium for fibroblasts is DMEM/F-12, based on our previous studies, and that for hUCBMSCs is a-MEM, according to recommendation by the manufacturer. A preliminary study was done to select the optimal culture medium for both cells. Attempts to culture hUCB-MSCs in DMEM/F-12 under the same conditions used with fibroblasts were unsuccessful, as were attempts to culture fibroblasts in a-MEM. Consequently, both cell types were cultured in their own optimal media. The results of this study suggested that hUCBMSCs transplanted into a wound might actively secrete growth factors that stimulate angiogenesis, such as VEGF and bFGF, to a much greater extent than fibroblasts. However, further studies are required to determine the effect of the hUCBMSCs allograft on the activity of diabetic fibroblasts. We would have liked to see additional in vitro characterization that further explores the cells’ ability to promote angiogenesis in particular, for example, through co-culture with endothelial cells/vascular tubule formation assays or similar. When we focus on cell therapies, we should consider interaction between resident cells and grafted cells. In this study, we evaluated only the capacities of grafted cells. The evaluations regarding the effects of hUCB-MSCs on resident cells should be performed in the future. In addition, it is difficult to assess the effect of hUCB-MSC transplantation on the overall wound-healing process because in vivo wound healing is a complex process requiring multiple factors. Future in vivo studies will be needed on the mechanisms of transplanted hUCB-MSCs in healing a diabetic wound.
Umbilical cord bloodederived mesenchymal stromal cells Conclusions Cell proliferation, collagen synthesis and growth factor production of hUCB-MSCs were compared with those of healthy dermal fibroblasts and diabetic fibroblasts in vitro. The most significant differences were shown in VEGF and bFGF secretion. Human UCB-MSCs produced significantly higher amounts of VEGF and bFGF when compared with both healthy and diabetic fibroblasts. Human UCB-MSCs were superior to diabetic fibroblasts but not superior to healthy fibroblasts in collagen synthesis. Cell proliferation and TGF-b production were not significantly different. These results suggested that cell therapy with the use of hUCB-MSCs may be superior to that using allogeneic/autologous fibroblasts for diabetic wound healing, especially in angiogenesis. Acknowledgments This study was supported by grant no. K1430391 from Korea University College of Medicine. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
References [1] Lu G, Huang S. Bioengineered skin substitutes: key elements and novel design for biomedical applications. Int Wound J 2013;10:365e71. [2] Papanas N, Eleftheriadou I, Tentolouris N, Maltezos E. Advances in the topical treatment of diabetic foot ulcers. Curr Diabetes Rev 2012;8:209e18. [3] You HJ, Han SK, Lee JW, Chang H. Treatment of diabetic foot ulcers using cultured allogeneic keratinocytesea pilot study. Wound Repair Regen 2012;20:491e9. [4] Marston WA, Hanft J, Norwood P, Pollak R. Dermagraft Diabetic Foot Ulcer Study G. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care 2003;26:1701e5. [5] Warriner RA 3rd, Cardinal M, TIDE Investigators. Human fibroblast-derived dermal substitute: results from a treatment investigational device exemption (TIDE) study in diabetic foot ulcers. Adv Skin Wound Care 2011;24:306e11. [6] Uccioli L, Giurato L, Ruotolo V, Ciavarella A, Grimaldi MS, Piaggesi A, et al. Two-step autologous grafting using HYAFF scaffolds in treating difficult diabetic foot ulcers: results of a multicenter, randomized controlled clinical trial with longterm follow-up. Int J Low Extrem Wounds 2011;10:80e5. [7] Steinberg JS, Edmonds M, Hurley DP Jr, King WN. Confirmatory data from EU study supports Apligraf for the treatment of neuropathic diabetic foot ulcers. J Am Podiatr Med Assoc 2010;100:73e7. [8] Kim JB, Chun KW, Han SK, Kim WK. Effect of human bone marrow stromal cell allograft on proliferation and collagen synthesis of diabetic fibroblasts in vitro. J Plast Reconstr Aesthet Surg 2010;63:1030e5.
7
[9] Lee CH, Han SK, Choi WI, Kim WK. Effect of human bone marrow stromal cells and dermal fibroblasts on collagen synthesis and epithelization. Ann Plast Surg 2007;59:713e9. [10] Han SK, Chun KW, Gye MS, Kim WK. The effect of human bone marrow stromal cells and dermal fibroblasts on angiogenesis. Plast Reconstr Surg 2006;117:829e35. [11] Han SK, Yoon TH, Lee DG, Lee MA, Kim WK. Potential of human bone marrow stromal cells to accelerate wound healing in vitro. Ann Plast Surg 2005;55:414e9. [12] Zebardast N, Lickorish D, Davies JE. Human umbilical cord perivascular cells (HUCPVC): A mesenchymal cell source for dermal wound healing. Organogenesis 2010;6:197e203. [13] Oh W, Kim DS, Yang YS, Lee JK. Immunological properties of umbilical cord blood-derived mesenchymal stromal cells. Cell Immunol 2008;251:116e23. [14] Malgieri A, Kantzari E, Patrizi MP, Gambardella S. Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. Int J Clin Exp Med 2010;3:248e69. [15] Kim JY, Jeon HB, Yang YS, Oh W, Chang JW. Application of human umbilical cord blood-derived mesenchymal stem cells in disease models. World J Stem Cells 2010;2:34e8. [16] Tark KC, Hong JW, Kim YS, Hahn SB, Lee WJ, Lew DH. Effects of human cord blood mesenchymal stem cells on cutaneous wound healing in leprdb mice. Ann Plast Surg 2010;65:565e72. [17] Liao Y, Itoh M, Yang A, Zhu H, Roberts S, Highet AM, et al. Human Cord Blood Derived Unrestricted Somatic Stem Cells Promote Wound Healing and Have Therapeutic Potential for Patients With Recessive Dystrophic Epidermolysis Bullosa. Cell Transplant 2014;23:303e17. [18] Chotinantakul K, Dechsukhum C, Dejjuy D, Leeanansaksiri W. Enhancement of wound closure in diabetic mice by ex vivo expanded cord blood CD34þ cells. Cell Mol Biol Lett 2013;18:263e83. [19] Shohara R, Yamamoto A, Takikawa S, Iwase A, Hibi H, Kikkawa F, et al. Mesenchymal stromal cells of human umbilical cord Wharton’s jelly accelerate wound healing by paracrine mechanisms. Cytotherapy 2012;14:1171e81. [20] Elsharawy MA, Naim M, Greish S. Human CD34þ stem cells promote healing of diabetic foot ulcers in rats. Interact Cardiovasc Thorac Surg 2012;14:288e93. [21] Cabrera C, Carriquiry G, Pierinelli C, Reinoso N, AriasStella J, Paino J. The role of biologically active peptides in tissue repair using umbilical cord mesenchymal stem cells. Ann N Y Acad Sci 2012;1270:93e7. [22] Reddi AS, Ende N. The use of human umbilical cord blood for wound healing, burns, and brain injury in combat zones. Mil Med 2011;176:361e3. [23] Luo G, Cheng W, He W, Wang X, Tan J, Fitzgerald M, et al. Promotion of cutaneous wound healing by local application of mesenchymal stem cells derived from human umbilical cord blood. Wound Repair Regen 2010;18:506e13. [24] Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns 2009;35:171e80. [25] Yang SE, Ha CW, Jung M, Jin HJ, Lee M, Song H, et al. Mesenchymal stem/progenitor cells developed in cultures from UC blood. Cytotherapy 2004;6:476e86. [26] Edmonds M, European and Australian Apligraf Diabetic Foot Ulcer Study Group. Apligraf in the treatment of neuropathic diabetic foot ulcers. Int J Low Extrem Wounds 2009;8:11e8. [27] Han SK, Choi KJ, Kim WK. Clinical application of fresh fibroblast allografts for the treatment of diabetic foot ulcers: a pilot study. Plast Reconstr Surg 2004;114:1783e9. [28] Han SK, Kim HS, Kim WK. Efficacy and safety of fresh fibroblast allografts in the treatment of diabetic foot ulcers. Dermatol Surg 2009;35:1342e8.
8
H.-J. You et al.
[29] Zhao QS, Xia N, Zhao N, Li M, Bi CL, Zhu Q, et al. Localization of human mesenchymal stem cells from umbilical cord blood and their role in repair of diabetic foot ulcers in rats. Int J Biol Sci 2013;10:80e9. [30] Li XY, Zheng ZH, Li XY, Guo J, Zhang Y, Li H, et al. Treatment of foot disease in patients with type 2 diabetes mellitus using human umbilical cord blood mesenchymal stem cells: response and correction of immunological anomalies. Curr Pharm Des 2013;19:4893e9. [31] Lerman OZ, Galiano RD, Armour M, Levine JP, Gurtner GC. Cellular dysfunction in the diabetic fibroblast:
impairment in migration, vascular endothelial growth factor production, and response to hypoxia. Am J Pathol 2003;162: 303e12. [32] You HJ, Han SK, Rhie JW. Randomised controlled clinical trial for autologous fibroblast-hyaluronic acid complex in treating diabetic foot ulcers. J Wound Care 2014;23. 521e2, 4, 6e30. [33] Lee M, Jeong SY, Ha J, Kim M, Jin HJ, Kwon SJ, et al. Low immunogenicity of allogeneic human umbilical cord bloodderived mesenchymal stem cells in vitro and in vivo. Biochem Biophys Res Commun 2014;446:983e9.
Wound-healing potential of human umbilical cord bloodederived mesenchymal stromal cells in vitro—a pilot study
HI-JIN YOU, SIK NAMGOONG, SEUNG-KYU HAN, SEONG-HO JEONG, EUN-SANG DHONG & WOO-KYUNG KIM Department of Plastic Surgery, Korea University College of Medicine, Seoul, Korea Abstract Background aims. Our previous studies demonstrated that human bone marrowederived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord bloodederived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. Methods. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n ¼ 5) and diabetic (n ¼ 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n ¼ 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-b) production were compared among the three cell groups. Results. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-b production. Conclusions. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Key Words: fibroblast, human umbilical cord blood stem cells, wound healing
Introduction The prevalence of chronic wounds, including diabetic ulcers, is increasing dramatically, as the populations of industrialized countries age and become more sedentary. Chronic wounds respond poorly to conventional treatment, making them very difficult to manage. There has recently been much interest in the use of cell-based therapies for the treatment of diabetic wounds. These therapies have been designed to modulate the levels of biologic molecules, including growth factors and extracellular matrices that may promote wound healing [1,2]. Various types of skin substitutes composed of either allogeneic or autologous fibroblasts and/or keratinocytes have been commercialized for the treatment of diabetic foot ulcers [3e7]. Although allogeneic fibroblasts, derived from healthy donors, are readily accessible, cellular activities are impaired as the result of cryopreservation.
Additionally, they may carry the risk of immunologic reactions or cross-infections. Autologous fibroblasts are advantageous because they may result in permanent engraftment. However, they require a long culture time, and, in the case of diabetic patients, autologous fibroblasts may not exhibit a sufficient capacity to stimulate wound healing. Mesenchymal stromal cells (MSCs) may hold great promise in treating diabetic wounds because they have the advantages of both allogeneic and autologous cells. MSCs demonstrate low levels of immunity-assisted rejection and have the ability to divide without apoptosis. It was demonstrated that even after 20 or 30 cycles of cell doubling in culture, they still retain their initial stem cell properties. Accordingly, MSCs have attracted much attention in the bioengineering field [8]. Bone marrow stroma is one of the main sources of MSCs. Previous studies performed by our group
Correspondence: Seung-Kyu Han, MD, PhD, Department of Plastic Surgery, Korea University, Guro Hospital, 148 Guro-Dong, Guro-Ku Seoul 152e703, Korea. E-mail: [email protected] (Received 31 March 2015; accepted 22 June 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.06.011
2
H.-J. You et al.
demonstrated that bone marrowederived mesenchymal stromal cells (BM-MSCs) synthesize higher amounts of collagen, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in vitro, as compared with dermal fibroblasts. Furthermore, they showed greater activity in granulation formation, epithelialization and angiogenesis in vivo, indicating a potential use in accelerated wound healing [9e11]. However, the number of MSCs in the BM decline with age, and their procurement is an invasive procedure. Furthermore, at present, it is difficult for a clinician to use cultured BM-MSCs for the purpose of wound healing because of issues regarding approval by the Food and Drug Administration (FDA) [12,13]. Meanwhile, a new commercial drug that uses human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) has been developed and approved by the Korean FDA to help regenerate knee cartilage. They can be acquired in massive quantities without significant ethical issues. Furthermore, cord blood stem cells are more immature than adult MSCs and expand easily in vitro [13]. Because the immune system is undifferentiated, successful transplantation without rejection has been reported [14e16]. Therefore, if hUCB-MSCs have greater wound-healing potential than fibroblasts, bioengineered dermal substitutes that are based on allogeneic/autologous fibroblasts, which are the main cell sources in current commercially available cell therapy products to treat diabetic wounds, could be replaced by a superior therapy with hUCB-MSCs. Some researchers have reported that hUCB-MSCs can improve wound healing [16e24]. However, no comparative analysis has been performed to evaluate the wound-healing activity of hUCB-MSCs and fibroblasts. The final purpose of our project is to prove superiority of the hUCB-MSCs over fibroblasts in treating diabetic wounds, and this pilot study was designed to compare the wound-healing activity of hUCB-MSCs with those of healthy and diabetic fibroblasts. Healthy and diabetic fibroblasts were used as representatives of cell sources for allogeneic and autologous fibroblasts in diabetic patients, respectively. This study was especially focused on cell proliferation, collagen synthesis and the production of three growth factors that are essential factors for diabetic wound healing, that is, basic FGF (bFGF), VEGF and transforming growth factor-b (TGF-b). Methods Isolation and culture of healthy fibroblasts, diabetic fibroblasts and hUCB-MSCs Cultured healthy and diabetic fibroblasts used for the experiments were obtained from cryopreserved cells
derived from the dermis of healthy (n ¼ 5) and diabetic (n ¼ 5) adults. The cell donors had provided informed consent for their cells to be used for research purposes. Both types of the fibroblasts were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient (DMEM/F-12; Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 25 mg/mL gentamycin at 37 C in a 5% CO2/95% air atmosphere at 100% humidity. The fibroblasts were dissociated by trypsinization, diluted 2.7-fold in Dulbecco’s phosphate-buffered saline without Mg2þ and Ca2þ (DPBS; Gibco) and collected by centrifugation at 450g for 17 min. The cells were washed twice in 40 mL of DPBS and re-suspended in 5 mL of DPBS. Cell density was determined by use of a hemocytometer, and viability was assessed by use of a trypan blue dye exclusion assay. Three-passage cells were used for all the experiments in this study. Human UCB-MSCs were purchased from Medipost Co Ltd [25]. According to the manufacturer’s protocols, hUCB samples were collected from the umbilical vein of deliveries with informed maternal consent. A 16-gauge needle from an hUCB collection bag containing 23 mL of CPDA-1 anticoagulant (Greencross Co) was inserted into the umbilical vein, and the hUCB was allowed to flow with gravity. UCB harvests were processed in all cases within 24 h of collection, with a viability of >90%. Mononuclear cells were isolated from the hUCB by means of centrifugation through a Ficoll-Hypaque gradient (density, 1.077 g/cm3, Sigma). The separated mononuclear cells were washed, suspended in a-minimum essential medium (a-MEM, Gibco BRL), supplemented with 10% FBS (HyClone) and 25 mg/mL gentamycin and seeded at a concentration of 5 106 cells/cm2. Cultures were maintained at 37 C in a humidified atmosphere containing 5% CO2 with a bi-weekly change of culture medium. When the monolayer of fibroblast-like adherent cells colonies had reached 80% confluence within 1 to 3 weeks, the cells were trypsinized (0.25% trypsin, HyClone), washed, resuspended in culture medium (a-MEM supplemented with 10% FBS) and sub-cultured. Five different cell lines were used in the study. Evaluation of cell proliferation, collagen synthesis and growth factor production For the cell proliferation assay, the cultured fibroblasts and hUCB-MSCs were seeded at 1.5 103 cells/well in 100 mL of a-MEM, supplemented with 5% FBS and 315 mg/dL glucose in 96-well culture plates. The cells were seeded onto a 24-well culture plate at 1.0 104 cells/well in 1.0 mL of the culture medium for the collagen synthesis and growth factor assay. The cells were incubated at 37 C in a 5% CO2/95% air
Umbilical cord bloodederived mesenchymal stromal cells
3
atmosphere at 100% humidity. The post-incubation cell proliferation, collagen synthesis and growth factor production were compared after 3 days.
used as subject data. Statistical comparisons were performed with the Mann-Whitney U test, with values of P < 0.05 considered statistically significant.
Cell proliferation assay
Results
Cell proliferation was determined by use of a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT; Sigma). Briefly, 10 mL MTT of 5 mg/mL was added to a 100-mL cell monolayer in each well of a 96-well plate, after which an incubation was performed for 3 h at 37 C. Next, 100 mL of 0.04 mol/ L HCL in propan-2-ol was added to each well and mixed thoroughly to dissolve the insoluble blue formazan crystals. The absorbance was measured at a test wavelength of 570 mm and a reference wavelength of 630 mm with the use of an enzyme-linked immunosorbent assay (ELISA) reader.
Monolayer cultures of human dermal fibroblasts consisted of homogeneously spindle-shaped cells (Figure 1). Most hUCB-MSCs were larger and had a more polygonal shape.
Collagen synthesis assay Collagen type I carboxyl-terminal propeptide (CICP) enzyme immunoassay was performed to measure the production of collagen, with the use of a Metra CICP kit (Quidel) according to the manufacturer’s instruction. Briefly, 100 mL of diluted culture supernatants was added to each well of the monoclonal anti-CICP antibody-coated plates, and the plates were then incubated at room temperature for 2 h. One hundred microliters of rabbit anti-CICP antisera was added for 50 min, followed by 100 mL of goat antirabbit alkaline phosphatase conjugates for 50 min. The reaction was stopped and the collagen synthesis was measured at 405 nm.
Cell proliferation After the 3-day incubation, the average cell number of the hUCB-MSCs was 3.8 0.4 103 per well, which was greater than those of the healthy fibroblasts (3.5 0.6 103 per well) and the diabetic fibroblasts (3.3 0.7 103 per well). Cell proliferation in the hUCB-MSC group was 9% and 16% greater than those in the healthy fibroblast and diabetic fibroblast groups, respectively. However, there was no significant statistical difference (P ¼ 0.347 and 0.251, respectively; Figure 2). Collagen synthesis Collagen synthesis of the hUCB-MSC group significantly increased as compared with that of the diabetic fibroblast group (P ¼ 0.009). Healthy fibroblasts also showed significantly increased collagen synthesis compared with that of the diabetic fibroblasts (P ¼ 0.009). However, there was no significant difference between the hUCBMSCs and healthy fibroblasts (P ¼ 0.076, Figure 3).
Growth factor level assay The levels of bFGF, VEGF and TGF-b1 were measured in culture media with an ELISA kit (R&D Systems) according to the manufacturer’s instruction. Briefly, the samples and standards were diluted appropriately in the assay diluent and then incubated at room temperature in wells pre-coated with the specific capturing antibodies. After washing, the respective detection antibody conjugate was added and was then incubated at room temperature before another washing. Addition of a substrate solution resulted in color development. A stop solution was added and the color intensity was measured in an ELISA reader at the appropriate wavelength. The measured TGF-b1 in this study included both the active and latent forms. Statistical analysis All experiments were performed in triplicate on each explant from each individual. The average value was
Growth factor analysis Of the three growth factors studied, the hUCBMSC group showed significantly higher levels of bFGF and VEGF expression. Basic FGF levels in the hUCB-MSC group were 59% (1.6-fold) and 96% (2-fold) greater than those for the healthy fibroblast and diabetic fibroblast groups (P ¼ 0.028 and 0.009, respectively; Figure 4). The most profound difference was observed in the levels of VEGF. The VEFG levels of the hUCB-MSC group were 469% (6-fold) and 586% (7-fold) higher than the VEGF levels of the healthy fibroblast and diabetic fibroblast groups, respectively (P ¼ 0.009, both; Figure 5). However, the TGF-b level of the hUCB-MSC group showed no significant difference when compared with that of the healthy fibroblast and the diabetic fibroblast groups (P ¼ 0.602 and 0.175, respectively; Figure 6).
4
H.-J. You et al.
Figure 1. Human dermal fibroblasts (left), diabetic fibroblasts (center) and hUCB-MSCs (right) in a monolayer culture (original magnification 100).
Discussion Wound-healing strategies with the use of cell transplantation have involved the use of fibroblasts or fibroblast-like cells. A cryopreserved human fibroblastederived dermal substitute (Dermagraft; Shire Regenerative Medicine, Inc) [4] and a bilayered living human skin equivalent (Apligraf; Organogenesis Inc) [7,26] are representative allogeneic skin substitutes that are reportedly effective for treating diabetic foot ulcers. The use of non-cryopreserved fresh human fibroblast allografts was also found to be a safe and effective treatment for diabetic foot ulcers [27,28]. Recently, a new bioengineered dermal substitute composed of cultured autologous fibroblasts seeded on a hyaluronic acid sheet (Hyalograft 3D; ChaBio & Diostech) was developed.
Figure 2. Cell proliferation of the three cell groups (hUCB-MSC, human umbilical cord bloodederived mesenchymal stromal cell group; FB, healthy fibroblast group; DM FB, diabetic fibroblast group). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. The average cell number of the hUCB-MSCs was 3.8 0.4 103 per well, which was greater than those of the healthy fibroblasts (3.5 0.6 103 per well) and the diabetic fibroblasts (3.3 0.7 103 per well).
A randomized, controlled trial showed that the product may provide a safe and effective treatment for diabetic foot ulcers. Human BM-MSCs have been found to promote wound healing [8e11] and have been used in clinical treatment of challenging wounds [12]. Mesenchymal stromal cells isolated from human umbilical cord blood can be easily obtained and refined compared with stem cells from other sources. Because hUCB-MSCs are rich, easy to collect and proliferate up to 4 times the number of cells within 3 to 5 days in appropriate culture conditions in vitro, they could be produced on a larger scale. The most important advantage of hUCB-MSCs is their very low immunogenicity, which provides potential clinical benefits to patients [29]. The acquisition of hUCB-MSCs in contrast to BM-MSCs may not be
Figure 3. Collagen synthesis of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. Collagen synthesis of the hUCB-MSC group (170 28 ng/mL) was significantly increased when compared with that of the diabetic fibroblast group (132 8 ng/mL). The collagen synthesis of the healthy fibroblast group was 210 34 ng/mL, which was also greater than that of the diabetic fibroblasts.
Umbilical cord bloodederived mesenchymal stromal cells
Figure 4. Basic FGF levels of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. The bFGF level of hUCB-MSCs (3.8 0.9 pg/mL) was significantly greater than those of the healthy fibroblasts (2.4 0.6 pg/ mL) and the diabetic fibroblasts (1.9 0.6 pg/mL).
dependent on the overall health status of the recipient [30]. On the basis of these biological advantages, a combination of hUCB-MSCs and sodium hyaluronate (CARTISTEM, Medipost Co Ltd) is intended for use as a single-dose cellular therapeutic agent for cartilage regeneration in human subjects with cartilage defects of the knee caused by aging, trauma or degenerative diseases. With the approval from the Korean FDA, CARTISTEM has become the world’s
Figure 5. VEGF levels of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. The VEFG level of the hUCB-MSC group (638 43 pg/mL) was significantly higher than the VEGF levels of the healthy fibroblast (112 44 pg/mL) and the diabetic fibroblast groups (93 31 pg/mL).
5
Figure 6. TGF-b levels of the three cell groups (*P < 0.05). All experiments were performed in triplicate on each explant from each individual. The average value was used for the subject’s data. TGF-b levels of the hUCB-MSCs, healthy fibroblasts and diabetic fibroblasts were 7685 1263 pg/mL, 8393 906 pg/mL and 6283 1121 pg/ mL, respectively. Statistical significance was observed between the healthy fibroblasts and the diabetic fibroblasts (P ¼ 0.028).
first allogeneic, off-the-shelf stem cell drug and is now available in Korea. The therapeutic potential of hUCB-MSCs has been widely explored as a promising tool in the treatment of various diseases; however, its effect on the healing of chronic wounds has not been studied to the same degree [15] There are few reports on the application of MSCs from hUCB to improve wound healing. Zhao et al. [29] demonstrated that hUCBMSCs specifically localize to the target ulcerated tissue and may promote the epithelialization of diabetic foot ulcers in rats. Luo et al. [23] demonstrated that the local application of hUCB-MSCs may not only be able to accelerate the speed of the wound healing but also may improve the quality of wound healing in mice. Tark et al. [16] used the diabetic mouse as a model of delayed wound healing. They concluded that hUCB-MSCs had a positive effect on wound healing, based on TGF-b levels. However, the majority of studies on hUCB-MSCs are limited and the underlying mechanisms have not yet been determined. Furthermore, the majority are animal studies. We have attempted to assess cell characteristics and phenotypes in vitro in comparison to healthy and diabetic fibroblasts. This is the first quantitative comparison of MSCs from human UCB and somatic cells. Human UCB-MSCs produced slightly lower amounts of TGF-b when compared with healthy fibroblasts, though there was no statistical difference. This finding may be related to the lower degree of collagen synthesis seen in hUCB-MSC
6
H.-J. You et al.
group because TGF-b is a potent stimulator of fibroblast proliferation and collagen synthesis. Our results may conceptually reflect a previous report that wound contraction is inhibited by the local administration of MSCs [23]. The fibroblast is central to the normal wound-healing process [31]. Diabetes influences this process at the molecular level, resulting in fibroblast senescence and slower growth factor production [32]. Our results also demonstrated the impaired functions of diabetic fibroblasts. The diabetic fibroblast group showed significantly decreased levels of TGF-b and a lower degree of collagen synthesis compared with the healthy fibroblast group. Human UCB-MSCs, on the other hand, produced significantly greater amounts of VEGF and bFGF, as compared with dermal fibroblasts and diabetic fibroblasts. The observed VEGF levels were especially striking. Basic FGF, an angiogenic growth factor that stimulates endothelial cell proliferation and migration, is an efficient mediator of VEGF activity. VEGF, a family of platelet-derived growth factors, induces angiogenesis and endothelial cell proliferation while stimulating vascular permeability. Additionally, VEGF can cause vasodilatation. It can also stimulate cell migration and inhibit apoptosis. VEGF and bFGF are potent angiogenic factors. Angiogenesis is a critical step in the wound healing process, particularly in chronic wound healing. Neovascularization, the formation of new blood vessels, is necessary to sustain the newly formed granulation tissues and the survival of keratinocytes. This study showed that there are justifiably high expectations for the use of hUCB-MSCs in wound healing because they have the potential to induce neovascularization and release the various necessary growth factors and cytokines. Clinical applicability of hUCB-MSCs might be hindered by potential graft rejection caused by the immunological barrier imposed by human leukocyte antigen mismatching. However, the immunomodulatory properties of hUCB-MSCs allow for an allogeneic cell source for stem cell-based therapy. Oh et al. [13] demonstrated that hUCB-MSCs can suppress the allogeneic responsiveness of human lymphocytes, as they suppress their proliferation and reduce their production of immunostimulatory cytokines. Lee et al. [33] used a humanized mouse model to provide evidence that the low immunogenicity of allogeneic hUCB-MSCs can be maintained in vivo. Their data suggested that hUCB-MSCs are “immunologically safe” for use in allogeneic cell therapies. Possible drawbacks of autologous cells can be exaggerated in patients with chronic wounds, owing to the attenuated cell activity. For example, even BM-MSCs show reduced growth in culture in
patients with chronic wounds. The therapeutic benefits of using allogeneic MSCs from healthy donors might surpass those of autologous MSCs derived from patients with chronic wounds. Human UCBMSCs rather than BM-MSCs or other somatic cells may therefore be the preferred choice in stem cellebased bioengineered products for wound healing. The route of cell administration, the number of cells transplanted, the number of treatments and the interval between individual treatments may be key points in cell therapy for wound healing. Allogeneic MSCs have been usually delivered in a single dose by various routes in animal wound-healing models. However, the most optimal condition to accelerate healing of diabetic wounds has not yet been determined. The effective delivery method must be determined to develop commercial bioengineered products with hUCB-MSCs. One limitation of the present study is that the cells were not grown in the same medium. The optimal medium for fibroblasts is DMEM/F-12, based on our previous studies, and that for hUCBMSCs is a-MEM, according to recommendation by the manufacturer. A preliminary study was done to select the optimal culture medium for both cells. Attempts to culture hUCB-MSCs in DMEM/F-12 under the same conditions used with fibroblasts were unsuccessful, as were attempts to culture fibroblasts in a-MEM. Consequently, both cell types were cultured in their own optimal media. The results of this study suggested that hUCBMSCs transplanted into a wound might actively secrete growth factors that stimulate angiogenesis, such as VEGF and bFGF, to a much greater extent than fibroblasts. However, further studies are required to determine the effect of the hUCBMSCs allograft on the activity of diabetic fibroblasts. We would have liked to see additional in vitro characterization that further explores the cells’ ability to promote angiogenesis in particular, for example, through co-culture with endothelial cells/vascular tubule formation assays or similar. When we focus on cell therapies, we should consider interaction between resident cells and grafted cells. In this study, we evaluated only the capacities of grafted cells. The evaluations regarding the effects of hUCB-MSCs on resident cells should be performed in the future. In addition, it is difficult to assess the effect of hUCB-MSC transplantation on the overall wound-healing process because in vivo wound healing is a complex process requiring multiple factors. Future in vivo studies will be needed on the mechanisms of transplanted hUCB-MSCs in healing a diabetic wound.
Umbilical cord bloodederived mesenchymal stromal cells Conclusions Cell proliferation, collagen synthesis and growth factor production of hUCB-MSCs were compared with those of healthy dermal fibroblasts and diabetic fibroblasts in vitro. The most significant differences were shown in VEGF and bFGF secretion. Human UCB-MSCs produced significantly higher amounts of VEGF and bFGF when compared with both healthy and diabetic fibroblasts. Human UCB-MSCs were superior to diabetic fibroblasts but not superior to healthy fibroblasts in collagen synthesis. Cell proliferation and TGF-b production were not significantly different. These results suggested that cell therapy with the use of hUCB-MSCs may be superior to that using allogeneic/autologous fibroblasts for diabetic wound healing, especially in angiogenesis. Acknowledgments This study was supported by grant no. K1430391 from Korea University College of Medicine. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
References [1] Lu G, Huang S. Bioengineered skin substitutes: key elements and novel design for biomedical applications. Int Wound J 2013;10:365e71. [2] Papanas N, Eleftheriadou I, Tentolouris N, Maltezos E. Advances in the topical treatment of diabetic foot ulcers. Curr Diabetes Rev 2012;8:209e18. [3] You HJ, Han SK, Lee JW, Chang H. Treatment of diabetic foot ulcers using cultured allogeneic keratinocytesea pilot study. Wound Repair Regen 2012;20:491e9. [4] Marston WA, Hanft J, Norwood P, Pollak R. Dermagraft Diabetic Foot Ulcer Study G. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care 2003;26:1701e5. [5] Warriner RA 3rd, Cardinal M, TIDE Investigators. Human fibroblast-derived dermal substitute: results from a treatment investigational device exemption (TIDE) study in diabetic foot ulcers. Adv Skin Wound Care 2011;24:306e11. [6] Uccioli L, Giurato L, Ruotolo V, Ciavarella A, Grimaldi MS, Piaggesi A, et al. Two-step autologous grafting using HYAFF scaffolds in treating difficult diabetic foot ulcers: results of a multicenter, randomized controlled clinical trial with longterm follow-up. Int J Low Extrem Wounds 2011;10:80e5. [7] Steinberg JS, Edmonds M, Hurley DP Jr, King WN. Confirmatory data from EU study supports Apligraf for the treatment of neuropathic diabetic foot ulcers. J Am Podiatr Med Assoc 2010;100:73e7. [8] Kim JB, Chun KW, Han SK, Kim WK. Effect of human bone marrow stromal cell allograft on proliferation and collagen synthesis of diabetic fibroblasts in vitro. J Plast Reconstr Aesthet Surg 2010;63:1030e5.
7
[9] Lee CH, Han SK, Choi WI, Kim WK. Effect of human bone marrow stromal cells and dermal fibroblasts on collagen synthesis and epithelization. Ann Plast Surg 2007;59:713e9. [10] Han SK, Chun KW, Gye MS, Kim WK. The effect of human bone marrow stromal cells and dermal fibroblasts on angiogenesis. Plast Reconstr Surg 2006;117:829e35. [11] Han SK, Yoon TH, Lee DG, Lee MA, Kim WK. Potential of human bone marrow stromal cells to accelerate wound healing in vitro. Ann Plast Surg 2005;55:414e9. [12] Zebardast N, Lickorish D, Davies JE. Human umbilical cord perivascular cells (HUCPVC): A mesenchymal cell source for dermal wound healing. Organogenesis 2010;6:197e203. [13] Oh W, Kim DS, Yang YS, Lee JK. Immunological properties of umbilical cord blood-derived mesenchymal stromal cells. Cell Immunol 2008;251:116e23. [14] Malgieri A, Kantzari E, Patrizi MP, Gambardella S. Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. Int J Clin Exp Med 2010;3:248e69. [15] Kim JY, Jeon HB, Yang YS, Oh W, Chang JW. Application of human umbilical cord blood-derived mesenchymal stem cells in disease models. World J Stem Cells 2010;2:34e8. [16] Tark KC, Hong JW, Kim YS, Hahn SB, Lee WJ, Lew DH. Effects of human cord blood mesenchymal stem cells on cutaneous wound healing in leprdb mice. Ann Plast Surg 2010;65:565e72. [17] Liao Y, Itoh M, Yang A, Zhu H, Roberts S, Highet AM, et al. Human Cord Blood Derived Unrestricted Somatic Stem Cells Promote Wound Healing and Have Therapeutic Potential for Patients With Recessive Dystrophic Epidermolysis Bullosa. Cell Transplant 2014;23:303e17. [18] Chotinantakul K, Dechsukhum C, Dejjuy D, Leeanansaksiri W. Enhancement of wound closure in diabetic mice by ex vivo expanded cord blood CD34þ cells. Cell Mol Biol Lett 2013;18:263e83. [19] Shohara R, Yamamoto A, Takikawa S, Iwase A, Hibi H, Kikkawa F, et al. Mesenchymal stromal cells of human umbilical cord Wharton’s jelly accelerate wound healing by paracrine mechanisms. Cytotherapy 2012;14:1171e81. [20] Elsharawy MA, Naim M, Greish S. Human CD34þ stem cells promote healing of diabetic foot ulcers in rats. Interact Cardiovasc Thorac Surg 2012;14:288e93. [21] Cabrera C, Carriquiry G, Pierinelli C, Reinoso N, AriasStella J, Paino J. The role of biologically active peptides in tissue repair using umbilical cord mesenchymal stem cells. Ann N Y Acad Sci 2012;1270:93e7. [22] Reddi AS, Ende N. The use of human umbilical cord blood for wound healing, burns, and brain injury in combat zones. Mil Med 2011;176:361e3. [23] Luo G, Cheng W, He W, Wang X, Tan J, Fitzgerald M, et al. Promotion of cutaneous wound healing by local application of mesenchymal stem cells derived from human umbilical cord blood. Wound Repair Regen 2010;18:506e13. [24] Branski LK, Gauglitz GG, Herndon DN, Jeschke MG. A review of gene and stem cell therapy in cutaneous wound healing. Burns 2009;35:171e80. [25] Yang SE, Ha CW, Jung M, Jin HJ, Lee M, Song H, et al. Mesenchymal stem/progenitor cells developed in cultures from UC blood. Cytotherapy 2004;6:476e86. [26] Edmonds M, European and Australian Apligraf Diabetic Foot Ulcer Study Group. Apligraf in the treatment of neuropathic diabetic foot ulcers. Int J Low Extrem Wounds 2009;8:11e8. [27] Han SK, Choi KJ, Kim WK. Clinical application of fresh fibroblast allografts for the treatment of diabetic foot ulcers: a pilot study. Plast Reconstr Surg 2004;114:1783e9. [28] Han SK, Kim HS, Kim WK. Efficacy and safety of fresh fibroblast allografts in the treatment of diabetic foot ulcers. Dermatol Surg 2009;35:1342e8.
8
H.-J. You et al.
[29] Zhao QS, Xia N, Zhao N, Li M, Bi CL, Zhu Q, et al. Localization of human mesenchymal stem cells from umbilical cord blood and their role in repair of diabetic foot ulcers in rats. Int J Biol Sci 2013;10:80e9. [30] Li XY, Zheng ZH, Li XY, Guo J, Zhang Y, Li H, et al. Treatment of foot disease in patients with type 2 diabetes mellitus using human umbilical cord blood mesenchymal stem cells: response and correction of immunological anomalies. Curr Pharm Des 2013;19:4893e9. [31] Lerman OZ, Galiano RD, Armour M, Levine JP, Gurtner GC. Cellular dysfunction in the diabetic fibroblast:
impairment in migration, vascular endothelial growth factor production, and response to hypoxia. Am J Pathol 2003;162: 303e12. [32] You HJ, Han SK, Rhie JW. Randomised controlled clinical trial for autologous fibroblast-hyaluronic acid complex in treating diabetic foot ulcers. J Wound Care 2014;23. 521e2, 4, 6e30. [33] Lee M, Jeong SY, Ha J, Kim M, Jin HJ, Kwon SJ, et al. Low immunogenicity of allogeneic human umbilical cord bloodderived mesenchymal stem cells in vitro and in vivo. Biochem Biophys Res Commun 2014;446:983e9.