Tissue Engineering and Regenerative Medicine TISSUE ENGINEERING AND REGENERATIVE MEDICINE Role of Keratinocyte Growth Factor in the Differentiation of Sweat Gland-Like Cells From Human Umbilical Cord-Derived Mesenchymal Stem Cells YONGAN XU,a,b,c,d YUCAI HONG,a,b MENGYAN XU,e KUI MA,c XIAOBING FU,c MAO ZHANG,a,b GUIRONG WANGd Key Words. Burns x Mesenchymal stem cell x Sweat gland x Tissue regeneration x Transdifferentiation x Umbilical cord x Wharton’s jelly Downloaded from aDepartment of Emergency Medicine, Second Affiliated ABSTRACT e ’ Hospital, and Women s Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have higher proliferation potency Hospital, Zhejiang University and lower immune resistance than human bone marrow MSCs and can differentiate into various func- School of Medicine, tional cells. Many regulatory factors, including keratinocyte growth factor (KGF), are involved in the http://stemcellstm.alphamedpress.org/ Hangzhou, Zhejiang, People’s development of skin and cutaneous appendages. Although KGF is important in wound healing, the Republic of China; bInstitute role of KGF in hUC-MSC differentiation remains unknown. In our previous work, we found the mixing of Emergency Medicine, medium (nine parts of basic sweat-gland [SG] medium plus one part of conditioned heat-shock SG Zhejiang University, medium) could induce hUC-MSC differentiation to sweat gland-like cells (SGCs). In this study, we fur- Hangzhou, Zhejiang, People’s ther improved the inducing medium and determined the effects of KGF in hUC-MSC differentiation. Republic of China; cWound We found KGF expression in the SGCs and that recombinant human KGF could induce hUC-MSC dif- ferentiation into SGCs, suggesting KGF plays a pivotal role in promoting hUC-MSC differentiation to Healing and Cell Biology SGCs. Furthermore, the SGCs differentiated from hUC-MSCs were applied to severely burned skin of Laboratory, Burns Institute, the paw of an in vivo severe combined immunodeficiency mouse burn model. Burned paws treated First Affiliated Hospital (304th with SGCs could regenerate functional sparse SGs 21 days after treatment; the untreated control paws Hospital), General Hospital of could not. Collectively, these results demonstrated that KGF is a critical growth factor for SGC differ- People’s Liberation Army, entiation from hUC-MSCs and the differentiated SGCs from hUC-MSCs may have a potential therapeu- Beijing, People’s Republic tic application for regeneration of destroyed SGs and injured skin. STEM CELLS TRANSLATIONAL by guest on October 26, 2016 of China; dDepartment of MEDICINE 2016;5:106–116 Surgery, State University of New York Upstate Medical University, Syracuse, New SIGNIFICANCE York, USA There is growing evidence demonstrating a potential therapeutic application of human umbilical cord- derived mesenchymal stem cells (hUC-MSCs) in injured skin. In the current study, conditioned media Correspondence: Mao Zhang, M.D., Department of Emergency and chemically defined media with recombinant human keratinocyte growth factor (KGF) could induce Medicine, Second Affiliated hUC-MSC differentiation into sweat gland-like cells (SGCs). Moreover, the differentiated SGCs from Hospital, Zhejiang University School hUC-MSCs could regenerate functional sparse sweat glands in a mouse burn model, which provides of Medicine (SAHZU), Hangzhou, further insight into the mechanisms of the role of KGF and a potential therapeutic application of differ- Zhejiang, People’sRepublicof China 310009. Telephone: 86-571- entiated SGCs for regeneration of destroyed sweat glands and injured skin. 87783921; E-Mail: zmhz@hotmail. com; or Guirong Wang, Ph.D., Department of Surgery, UH Room – 8715, SUNY Upstate Medical INTRODUCTION tissue-engineeredskin[5 9].However,theoriginal University, 750 East Adams Street, structure and functions of seriously injured skin, Syracuse, New York 13210, USA. As an essential function of skin, sweating is ma- especially for perspiration (sweating), could not Telephone: 315-464-6283; E-Mail: nipulated by sweat glands (SGs) embedded in be well re-established during rehabilitation. [email protected] the deep dermal layer [1, 2]. When the origi- Mesenchymal stem cells derived from human Received April 24, 2015; accepted nal structure and function of whole skin is bone marrow (BM-MSCs) and human umbilical for publication September 16, destroyed in seriously burned patients, the skin cord Wharton’s jelly (hUC-MSCs) have been used 2015; published Online First on loses its self-regenerative/self-repairing ability as novel and effective sources of stem cell therapy November 16, 2015. [3, 4]. In past decades, the major objective of sur- and regenerative medicine to repair injured cuta- ©AlphaMed Press geons and researchers of burns was to accelerate neous structures and appendages [10–14]. As a 1066-5099/2015/$20.00/0 closure of burn wounds through the application of prominent cell resource, BM-MSCs have been http://dx.doi.org/ an autograft of a patient’s own skin or allograft widely used for treating various kinds of diseases 10.5966/sctm.2015-0081 transplants of other kinds of skin substitutes, like and injured tissues, like burns and other wounds STEM CELLS TRANSLATIONAL MEDICINE 2016;5:106–116 www.StemCellsTM.com ©AlphaMed Press 2016 Xu, Hong, Xu et al. 107 [11, 15–17]. As shown by previous studies, BM-MSCs can be in- The umbilical cords were kept at 4°C and transported to duced to differentiate into sweat gland-like cells (SGCs) in vitro, the laboratory. They were then repeatedly rinsed by sterile and patients’ own differentiated SGCs from BM-MSCs can be phosphate-buffered saline (PBS). After removing all the vessels, autografted for regenerating injured SGs [10, 13, 18, 19]. Compared the remaining tissues were minced into 1-mm3 cubes and digested with BM-MSCs, hUC-MSCs have higher proliferation potency with type II collagenase (2 mg/ml; Sigma-Aldrich, St. Louis, MO, [20–22], multiple differentiation potentials, and lower immune http://www.sigmaaldrich.com) at 37°Cfor6–8 hours. The samples resistance [22–24]. Additionally, human umbilical cord tissues were centrifuged at 3,000g for 5 minutes at room temperature. The can be stored at 280°C for the preparation of hUC-MSCs [23]. sediments were resuspended and cultured in basic hUC-MSC me- In our previous study, we induced hUC-MSC differentiation to dium (Dulbecco’s modified Eagle’s medium [DMEM] supplemented SGCs in a conditioned induction medium consisting of nine parts with 10% fetal bovine serum [FBS] [Gibco/Thermo Fisher Scientific, basic SG medium and one part sterile supernatants from condi- Waltham, MA, http://www.thermofisher.com]; 100 U/ml penicillin tioned heat-shock SG medium [12]. However, the pivotal induct- (Sigma-Aldrich), 100 mg/ml streptomycin (Sigma-Aldrich), and ing factors in the SGC differentiation have not been identified. In 2mML-glutamine (Sigma-Aldrich) in a cell culture incubator at this study, we further improved the inducing medium by mixing 37°C in a humidified atmosphere containing 5% CO2 (Hera Cell; eight parts basic SG medium and two parts of sterile superna- Thermo Fisher Scientific). hUC-MSCs were routinely examined un- tants from conditioned heat-shock SG medium, referred to as der a phase-contrast inverted microscope (Leica, Wetzlar, Germany, Downloaded from induction medium-mix, and we identified the key regulatory fac- http://www.leica.com). Cells were subcultured when cells reached tors of hUC-MSC differentiation to SGCs. 80% confluence in the plates, and then cells were used for the sub- Several growth factors were found to be involved in the devel- sequent study after 3–5 passages [12]. opment of skin and SGs: keratinocyte growth factor (KGF; one sub- type of fibroblast growth factor [FGFs], also called FGF-7) [25–28], Construction of SGC Differentiation Medium epidermal growth factor (EGF) [28], and anhidrotic ectodermal dys- Normal human skin was collected from five femaleplastic-surgery http://stemcellstm.alphamedpress.org/ plasias (EDAs; one subtype of the tumor necrosis factor family) [27]. patients who had small skin grafts harvested from the inside It has been demonstrated that a deficiency of EDA during embryo of their upper arms. Skin tissue (0.5–1cm2) was minced into development could result in dysfunctional sweating, missing teeth, 1-mm3 skin particles after removal of subcutaneous fat, and and sparse hair [27, 29]. EGF can induce several types of mesenchy- then digested with type II collagenase (2 mg/ml; Sigma-Aldrich) mal stem cell (MSC) differentiation into epidermal lineage cells, at 37°C for 3–4 hours. Mature SGs were cultured in basic SG me- maintain proliferation potencyofepithelial cells,and accelerateskin dium used as a positive control. Proliferated SGs were heat wound healing. Recently, KGFwas found to play an important role in shocked and then recultured with regular culture processes. wound healing and maintenance of cutaneous homeostasis [25, The supernatants of conditioned medium for heat-shock SGs – 30 33]. However, the role of KGF in SGC differentiation is unknown. were collected, filtered through a 0.22-mm diameter filter to In the present study, we hypothesized that KGF is an essential eliminate potential bacteria, and stored at 280°C. The induction factor in hUC-MSC differentiation to SGCs, and could be applied medium-mix consists of 80% basic SG medium and 20% superna- by guest on October 26, 2016 for reconstruction of destroyed sweat glands and skin wounds. tants of conditioned heat-shocked SG medium. Additionally, in- The