RESEARCH PAPER

The Effects of Microenvironment on Wound Healing by Keratinocytes Derived From Mesenchymal Stem Cells Yi-Han Lin, MS,* Keng-Yen Fu, PhD,Þ Po-Da Hong, PhD,* Hsu Ma, MD, PhD,þ Nien-Hsien Liou, MD, PhD,§ Kuo-Hsing Ma, PhD,§ Jiang-Chuan Liu, PhD,§ Kun-Lun Huang, MD, PhD,|| Lien-Guo Dai, MD, PhD,¶# Shun-Cheng Chang, MD,Þ James Yi-Hsin Chan, MD, PhD,** Shyi-Gen Chen, MD,Þ Tim-Mo Chen, MD,Þ and Niann-Tzyy Dai, MD, PhDÞ Abstract: Embryonic stem cells (ESCs) are pluripotent cells that can differentiate into various cell types, including keratinocyte-like cells, within suitable microniches. In this study, we aimed to investigate the effects of culture media, cell coculture, and a tissue-engineering biocomposite on the differentiation of mouse ESCs (MESCs) into keratinocyte-like cells and applied these cells to a surgical skin wound model. MESCs from BALB/c mice (ESC26GJ), which were transfected using pCX-EGFP expressing green f luorescence, were used to track MESC-derived keratinocytes. Weak expression of the keratinocyte early marker Cytokeratin 14 (CK-14) was observed up to 12 days when MESCs were cultured in a keratinocyte culture medium on tissue culture plastic and on a gelatin/collagen/polycaprolactone (GCP) biocomposite. MESCs cocultured with human keratinocyte cells (HKCs) also expressed CK-14, but did not express CK-14 when cocultured with human fibroblast cells (HFCs). Furthermore, CK-14 expression was observed when MESCs were cocultured by seeding HKCs or HFCs on the same or opposite side of the GCP biocomposite. The highest CK-14 expression was observed by seeding MESCs and HKCs on the same side of the GCP composite and with HFCs on the opposite side. To verify the effectiveness of wound healing in vivo, adipose-derived stem cells were applied to treat surgical wounds in nude mice. An obvious epidermis multilayer and better collagen deposition during wound healing were observed, as assessed by Masson staining. This study demonstrated the potential of keratinocyte-like differentiation from mesenchymal stem cells for use in promoting wound closure and skin regeneration. Key Words: mesenchymal stem cells, mice embryonic stem cells (MESCs), human keratinocyte cells (HKCs), human fibroblast cells (HFCs), skin regeneration, gelatin/collagen/polycaprolactone (GCP) biocomposite (Ann Plast Surg 2013;71: S67YS74)

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tem cells have capabilities of self-renewal and differentiation into different cell types both in vivo and in vitro.1 Stem cells derived from the inner cell mass of early embryos are among these pluripotent

Received October 4, 2013, and accepted for publication, after revision, October 6, 2013. From the *Biomedical Engineering Program, Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology; †Division of Plastic and Reconstructive Surgery, Department of Surgery, TriService General Hospital, National Defense Medical Center; ‡Division of Plastic Surgery, Veteran General Hospital; §Institute of Biology and Anatomy, and ||Graduate Institute of Aerospace and Undersea Medicine, National Defense Medical Center, Taipei; ¶Department of Health-Business Administration, Hungkuang University; #Department of Orthopedics, Kuang-Tien General Hospital, Taichung; and **Graduate Institute of Medical Sciences, National Defense Medical Center, Taiwan, Republic of China. Conflicts of interest and sources of funding: none declared. Reprints: Niann-Tzyy Dai, MD, PhD, Division of Plastic and Reconstructive Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, No. 325, Section 2, Cheng-Kung Rd, Nei-Hu District, Taipei 114, Taiwan, Republic of China. E-mail: [email protected]. Copyright * 2013 by Lippincott Williams & Wilkins ISSN: 0148-7043/13/7101-S067 DOI: 10.1097/SAP.0000000000000045

Annals of Plastic Surgery

cells. They can maintain a normal karyotype when cultured in a basal medium and differentiate into various cell types under suitable conditions.2,3 The skin, which is the largest human organ, is a barrier against harmful environmental factors and unregulated water loss. Keratinocytes primarily constitute this barrier and originate from the basal epidermal layer to form the epidermis. During keratinocyte maturation, type I keratin Cytokeratin 14 (CK-14) is constitutively expressed during the immature, proliferative state before differentiation of the stratified epithelium. Precursor proteins of the cornified envelope, such as involucrin and loricrin, are also produced during keratinocyte stratification.4 On epidermal injury, a sequence of events occur to repair the wound, including keratinocyte migration to the injured dermis.5,6 During the later stage of wound repair, fibroblasts and myofibroblasts interact and produce extracellular matrix (ECM) components, primarily collagens, which form the bulk of a mature scar.6 Therefore, keratinocytes play a critical role in early skin regeneration during wound repair. To overcome insufficient sources of autologous epidermis, embryonic stem cells (ESCs) have been used as a substitute source of keratinocytes. Cell fate decisions, including maintaining self-renewal capability or forming any somatic cell type, largely depend on a specific microenvironmental stimulus applied within the cellular milieu or niche. Several components, including soluble factors, appear in the surrounding tissue or culture media, ECM, or cell substrate. The biophysical environment, including mechanical forces, shear, spatial organization, and nearby cells affecting cell-to-cell signaling, are also critical for establishing an appropriate stem cell niche.7 Different methods have been explored to generate keratinocyte precursors from ESCs. In addition, seeding cells into 3-dimensional biocomposites can induce the generation of organ-like structures because of the incorporation of in vivo counterparts. Previous reports have indicated that the integrated effects of appropriate microenvironmental factors, including 3-dimensional biocomposites and growth factors, can maintain cell lineages and differentiation of embryonic cells.8Y10 In this study, we aimed to understand the mechanism by which different microniches induce the differentiation of MESCs, transfected with enhanced green fluorescent protein (EGFP) vectors, into keratinocytes during wound repair. We investigated the effects on keratinocyte differentiation by culture media and coculturing keratinocytes and fibroblasts in addition to investigating the spatial effects using a multiporous gelatin/collagen/polycaprolactone (GCP) biocomposite developed in our laboratory and the effects of the cell seeding procedure. The expression of autologous green fluorescent protein (GFP) confirmed the fates of MESCs.

MATERIALS AND METHODS Culture of GFP-Transfected MESCs In this experiment, MESCs were obtained from Taipei Veterans General Hospital using transgenic technology to clone the

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GFP gene (pCX-EGFP) into MESCs (ESC26GJ9012-8-2).11 The cells were incubated in T25 culture f lask using Dulbecco modified Eagle medium/Ham F-12 medium (DMEM/F12) (Gibco/Invitrogen) for cell expansion and passage and then seeded on the 24-well tissue culture plastic (TCP) using basal DMEM/F12 medium and EpiLife HKM (Cat No. M-EPIcf-500 with the addition of 0.06 mM calcium chloride and human keratinocyte growth supplement (HKGS) kit: Cat No. S-001-5; Cascade Biologics, Inc, USA) for testing keratinocyte differentiation.

in Figure 1. To investigate the inf luence on the differentiation of MESCs to keratinocytes, each keratinocyte or fibroblast was cocultured with MESCs in vitro test. In brief, MESC and HKCs were both seeded on TCP at a cell density of 2  104 cells per cm2 and cultured in keratinocyte culture medium. Meanwhile, the coculture of MESCs and HFCs were also carried out according to the previously mentioned procedure, which also grew in keratinocyte culture medium. In addition, MESCs were cultured individually on TCP after the same procedure described previously.

Primary Culture of Human Keratinocyte Cells and Human Fibroblast Cells

Preparation of GCP Biocomposite

The primary human epidermal keratinocytes and dermal fibroblasts were obtained from human child foreskin after the surgery of circumcision. Approval was obtained from the institutional review board in the Tri-Service General Hospital, Republic of China. EpiLife HKM was used for keratinocyte culture. DMEM (400 mL) supplemented with 10% fetal calf serum and 5 mL penicillin/streptomycin (100 U/mL and 100 mg/mL) was applied for fibroblast culture. The isolation procedure, cell expansion, and cell counting were performed according previous literature.12 In brief, the foreskin was firstly transferred to 10 mL of 0.2% Dispase II solution and kept at room temperature overnight. Next, epidermis was isolated and incubated in 0.025% trypsin-ethylenediaminetetraacetic acid solution for 10 minutes. After centrifugation, the keratinocyte cells were seeded in a fibronectin/collagen-coated f lask by adding keratinocyte culture medium. For the dermis sample, it was first rinsed in 0.05% collagenase solution and incubated for 24 hours. After centrifugation, the fibroblast cells were resuspended in fibroblast culture medium.

Aliquots of gelatin/10% collagen solution was prepared by dissolving 0.2% wt/vol gelatin 0.2 mL in double-distilled water followed by mixing 0.25% wt/vol collagen 0.025 mL (10% of the amount of collagen used for preparation of collagen: polycaprolactone biocomposites). The dissolution of gelatin/collagen was facilitated by stirring using a heating magnetic stirrer in a 25-mL glass shell vial at the temperature of 80-C. After complete dissolution, aliquots (0.2 mL) of the gelatin/collagen solution were added to 7-mL glass vials followed by elimination of air bubbles and frozen at j20-C for approximately 45 to 50 minutes. In the second stage, samples were transferred to a freezer at j72-C for 35 minutes. Finally, the frozen samples were placed in a freeze dryer (Edwards Modulyo) at j44-C under 42 mbar vacuum for 24 hours. Aliquots (0.5 mL) of 0.74% and 1.85% wt/vol polycaprolactone/dichloromethane solution were added carefully to the freeze-dried gelatin/collagen mats to prepare GCP biocomposites. The vials were kept stopped for 30 minutes before removing the lids to allow solvent evaporation overnight.

The Coculture of MESCs With HKCs or HFCs and Individual Culture on TCP

The Coculture of MESCs With HKCs or HFCs and Individual Culture on GCP Biocomposite

The f low chart of in vitro experiment and associated schematic diagram of combination for cells and materials were present

To mimic the skin environment, coculture of MESCs with other 2 cells (HKCs or HFCs) was prepared by seeding MESCs and

FIGURE 1. The f low chart of in vitro experiment (A) and schematic diagram of combination for MESCs and other cells seeded on different materials (B) in this study. S68

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HKCs (or HFCs) on the same side or on the opposite side of GCP biocomposite in a coculture system at a cell density of 2  104 cells per cm2 incubated on keratinocyte culture medium up to 12 days. Furthermore, MESCs and HKCs were seeded on the same side of biocomposite and fibroblast was seeded on opposite side at a cell density of 2  104 cells per cm2 supplemented with keratinocyte culture medium for 12 days to observe keratinocyte differentiation. Meanwhile, MESCs were cultured individually on GCP biocomposite following the same procedure described previously.

Immunofluorescence Staining Samples from in vitro experiments were obtained and fixed by acetic acid/alcohol solution. Next, the immunof luorescence staining of CK-14 marker was performed according to previous literature.13 Samples were treated with primary antibodies, rabbit anti-CK14 (1:100; Bioworld Technology) at 4-C overnight. After rinsing with PBS, samples were incubated with goat anti-rabbit secondary rhodamineconjugated antibodies (1:200; Chemicon, Billerica, Mass) for 45 minutes at 37-C. The nucleus was stained with 4¶,6-diamidino-2-phenylindole (DAPI). Samples were imaged on an inverted microscope system (Leica DMIL/5.0MC).

In Vivo Animal Study To investigate the skin regeneration promoted by adiposederived stem cells (ASCs) in vivo, a skin wound healing model of nude mice was used following previous report.14 Two experiment groups were designed: ASCs seeding group and no-ASCs seeding group as the control. All animals were acquired, housed, and studied according the protocol approved by the Institutional Animal Care and Use Committee of National Defense Medical Center, Republic of China. Two 8-week-old nude mice (BALB/c-nu; BioLASCO) were used in this experiment. The surgical instruments were sterilized and all the wound surgery was performed under laminar f low. The surgical sites were sterilized using Easy Antiseptic Liquid 2% (Panion & BF, Taipei, Taiwan) before surgical process. After anesthesia, a rounded,

Keratinocyte Differentiation From Stem Cells

full-thickness, 8-mm cutaneous wound was produced by impressing a punch biopsy instrument on each mouse dorsum of the hind thighs, followed by grabbing, pulling the circular region with a forceps, and excising the full-thickness tissue with scissors. ASCs (1  105) were seeded onto the wound surface and covered with Tegaderm films (3M Health Care, Minn) to prevent catching, biting, or wound infections. The wounds were observed within a period of 2 weeks. After 2 weeks, the wound tissues were removed and frozen sections were made following standard procedure. Masson trichrome staining was performed based on previous study15 and observed under an optical microscope (Olympus BX50, Hamburg, Germany). The collagen component of the ECM deposited in skin substitutes was stained green.

RESULTS To investigate differentiated cells, we used immunof luorescent staining for CK-14, a marker in the early stage of keratinocyte differentiation. Positive staining indicated that MESCs had begun differentiation into keratinocytes. The constitutive expression of eGFP by mouse embryonic stem ESC26GJ clones allowed us to confirm differentiation. DAPI staining was used to identify cell nuclei positions. Staining for different markers, including EGFP, DAPI, and CK14, was used to determine the cell fates of MESCs when these cells were seeded on TCP for 1 day using DMEM/F12 or keratinocyte culture medium (Fig. 2). These results indicated that MESCs did not express CK-14 when incubated in the DMEM/F12 medium, suggesting that MESCs could not directly differentiate into keratinocyte under these conditions. However, a weak CK-14 expression was observed when MESCs were transferred into the keratinocyte culture medium. Thus, the culture medium played a critical role in MESCsderived keratinocyte differentiation. The induction of CK14 expression by MESCs occurred when these cells were cocultured with HKCs or HFCs (Fig. 3). MESCs that interacted with keratinocytes could undergo continuous keratinocyte differentiation. In contrast, MESCs coculture with fibroblasts inhibited

FIGURE 2. MESCs seeded on TCP with either DMEM/F12 or keratinocyte culture medium. After incubaton for 1 day, immunostaining for DAPI, EGFP, and CK-14 was done and MESCs immunf luorescent images were acquired using an inverted microscope system. * 2013 Lippincott Williams & Wilkins

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FIGURE 3. MESCs cocultured with other stem cells (HKCs or HFCs) on TCP in keratinocyte culture medium. Immunostaining for DAPI, EGFP, and CK-14 was performed and MESCs imunof luorescent images were acquired using an inverted microscope system until 12 days of incubation.

keratinocyte differentiation at 8 days, although weak CK-14 expression was observed when cocultured for 12 days. We also attempted to mimic the microniche of MESCs where differentiation would occur in the skin microenvironment. MESCs were seeded on a 3-dimensional GCP biocomposite supplemented with the DMEM/F12 or keratinocyte culture medium to assess these effects on differentiation (Fig. 4). A weak CK-14 expression was observed only when MESCs were immersed in the DMEM/F12 culture medium. In addition, increased CK-14 induction was observed when MESCs were incubated in the keratinocyte culture medium compared with that in the DMEM/F12 culture medium. The combined effects of the appropriate microenvironmental factors, including a 3-dimensional GCP biocomposite and nearby cells (HKCs or HFCs), was explored by seeding 2 kinds of cells on the same side of the GCP biocomposite (Fig. 5). CK-14 expression was induced regardless of whether MESCs interacted with HKCs or HFCs on the same side of the GCP biocomposite in this coculture system. However, MESCs cocultured with HFCs had higher CK-14 expression than that of MESCs cocultured with HKCs after 12 days. S70

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MESCs were also investigated for interactions with other cells (HKCs or HFCs) when incubated on the opposite side of the GCP biocomposite (Fig. 6A). In addition, a bilayered skin model that seeded keratinocytes on the same side and fibroblasts on the opposite side of the GCP biocomposite was used (Fig. 6B). These conditions were assessed for keratinocyte differentiation. Similar trends were observed for induced CK-14 expression when seeding MESCs and HKCs or HFCs on the opposite side of the GCP biocomposite in a coculture system. However, MESCs that interacted with HFCs seemed to have higher CK-14 expression compared with those that interacted with HKCs. The greatest CK-14 expression was observed when seeding MESCs and HKCs on the same side and HFCs on the opposite side in the model, which mimicked a bilayered skin microenvironment. From the observation, we can summarize the productive grade of CK-14 positive cells induced by varieties of factors such as culture medium, cell effects, and growth environment derived from different experimental groups (Table 1). Wound closure and skin regeneration were assessed with and without (control) ASCs seeding on the wound surface. After 2 weeks, * 2013 Lippincott Williams & Wilkins

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Keratinocyte Differentiation From Stem Cells

FIGURE 4. MESCs seeded on a GCP biocomposite with either DMEM/F12 or keratinocyte culture medium. After incubation for 12 days, immunostaining for DAPI, EGFP, and CK-14 was performed and MESCs immunof luorescent images were acquired using an inverted microscope system.

these conditions were assessed using Masson staining (Fig. 7). The nonseeding control showed loose collagen deposition even after 14 days in the open wound model (Fig. 7A). With ASC seeding,

complete wound closure with a differentiated epidermis and abundant dermal parallel-arranged fibrous collagen deposition was observed that was nearly similar to the collagen content of proximal normal

FIGURE 5. MESCs cocultured with other cells (HKCs or HFCs) seeded on the same side of a GCP biocomposite and with keratinocyte culture medium. Immunostaining for DAPI, EGFP, and CK-14 was performed and MESCs immunof luorescent images were aquired using an inverted microscope system until 12 days of incubation. * 2013 Lippincott Williams & Wilkins

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FIGURE 6. MESCs cocultured with other cells (HKCs or HFCs) seeded on the opposite side of a GCP biocomposite (A) and MESCs cocultured with HKCs seeded on the same side, but with HFCs seeded on the opposite side of a GCP biocomposite (B) and with keratinocyte culture medium. Immunostaining for DAPI, EGFP, and CK-14 was performed and MESCs immunof luorescent images were acquired using an inverted microscope system until 12 days of incubation.

tissue after 14 days (Fig. 7BYD). At higher magnification, a thick epidermal layer was confirmed in the outside portion of the wound.

DISCUSSION In this study, we investigated the inf luence of microniche in guiding stem cell fate. Mouse GFP-transfected ESC was a useful tool for the purpose of tracking the keratinocyte differentiation from MESCs and therefore was chosen for the in vitro study. We used the developed GCP biocomposite as a tissue-engineering platform to mimic the skin microniche in vitro. Actually, the seeding of MESCs was proven to differentiate to keratinocytes in suitable microniche. However, the use of ESCs was problematic due to the oncogenic potential and ethical concerns. These reasons currently forbade the clinical application in patients. In contrast with ESC, adipose stem cells are now considered as an accessible, abundant, and reliable S72

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source for the isolation of adult stem cells suitable for tissue engineering and regenerative medicine applications. Therefore, mesenchymal stem cell instead of ESC was made in skin wound healing model of nude mice for the investigation whether suitable microniche could promote keratinocytes differentiation in vivo. From our result, the successful wound closure with a well-differentiated epidermis was observed. On the other hand, the ASCs were confirmed to differentiate into keratinocytes by the stain using RNA-binding motif on Y chromosome (RBMY), a human novel male-specific protein (data are shown in another submitting manuscript). Moreover, we investigated several factors that could inf luence the cell fate decisions of ESCs for their differentiation into keratinocytes, including the culture media, spatial organization, and nearby cell effects in coculture systems. When ESCs were transferred from the basal DMEM/F12 to keratinocyte culture medium, a weak CK-14 expression was observed during the incubation period, which * 2013 Lippincott Williams & Wilkins

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TABLE 1. The Rank of CK-14 Positive Cells Among Different Groups Group No. 1 2 3 4 4 5 6 7 8 9 10 11

Seeding Cells

Culture Medium

Seeded Materials

Incubating Time, d

CK-14 Expression

MESCs MESCs MESCs + HKCs MESCs + HFCs MESCs + HFCs MESCs MESCs MESCs + HKCs MESCs + HFCs MESCs + HKCs MESCs + HFCs MESCs + HKCs + HFCs

DMEM + F12 Keratinocyte medium Keratinocyte medium Keratinocyte medium Keratinocyte medium DMEM + F12 Keratinocyte medium Keratinocyte medium Keratinocyte medium Keratinocyte medium Keratinocyte medium Keratinocyte medium

TCP TCP TCP TCP TCP GCP GCP GCP same side GCP same side GCP opposite side GCP opposite side GCP both sides

1 1 12 8 12 12 12 12 12 12 12 12

j + + j + + ++ ++ ++ ++ ++ +++

j, Negative; +, weak positive; ++, medium positive; +++, strong positive.

FIGURE 7. Collagen deposition in a wound bed assessed using Masson staining. Histology of the cultured skin model after (A) no ASCs seeding as a control (A, 100; scale bar, 30 Hm) and (B) ASCs seeding (B, 100; scale bar, 30 Hm) on the surfaces of full-thickness skin defects at 14 days after wounds were inf licted in nude mice. Panels (C) and (D) are magnified images of the highlighted areas (yellow rectangles) in (B). * 2013 Lippincott Williams & Wilkins

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indicated that the culture medium contained factors that contributed to keratinocyte differentiation. A previous report indicated that a culture medium supplemented with insulin, epidermal growth factor, and cholera toxin could support the growth of mature, epidermal keratinocytes.10 In addition, a weak CK-14 expression was detected when MESCs were seeded on a GCP biocomposite, even when cultured using the basal DMEM/F12 medium. Thus, MESC differentiation also resulted from the effects of this GCP biocomposite. Replacing the DMEM/F12 medium with keratinocyte culture medium further enhanced CK-14 induction on a GCP biocomposite. The major components of our GCP biocomposite were type I collagen and gelatin, whereas the keratinocyte culture was serum free and supplemented with calcium. Other studies also used culture dishes coated with type I collagen and keratinocyte culture medium supplemented with calcium to culture human ESCs, which resulted in 25% stem cells expressing CK-14 after 28 days.16 In our analysis of proximate cell effects, we investigated the effects of the interactions between HKCs and ESCs. CK-14 expression, either on TCP or GCP biocomposite, was observed when HKCs were included in the coculture system. Comparable CK-14 expression was detected when HKCs were seeded on the same and opposite side of a biocomposite, which suggested that the capability to differentiate did not change by introducing the biocomposite. We assumed that the mass transfer of differentiation factors released from HKCs was not retarded through the pores of this biocomposite. In contrast, CK-14 expression was not induced by coculturing HFCs and ESCs on TCP, which suggested that HFCs may have inhibited keratinocyte differentiation in a 2-dimensional environment. Moreover, when coculturing HFCs and ESCs on a 3-dimensional biocomposite, the inhibitory effect was weakened and CK-14 expression was detectable after 12 days. In a previous study, numerous stem cells expressed CK-14 after 15 days when a culture dish was treated with the ECM components produced by fibroblasts and used to culture MESCs, which were also induced by bone morphogenetic protein-4.17 In addition, fibroblasts could promote the growth of keratinocytes by releasing numerous growth factors and, thus, also promoted wound healing.15 Therefore, we speculate that MESCs can differentiate into keratinocytes during the late phase because of the ECM components released from fibroblasts. Stem cell fate decisions are guided during early development by various signals that are mediated by paracrine factors, growth regulatory factors, and structural proteins that provide dynamic crosstalk within tissues.18 A previous study found that cells that were seeded on a collagen matrix and applied to an air-liquid interface resulted in stratification and terminal differentiation of epidermal keratinocytes.19 Successful stratification and cornification from human embryonic stem cells in 3-dimensional culture has also been shown.20 Another study indicated that incorporating these cells into the stromal and epithelial compartments of 3-dimensional tissues could result in the formation of multilayer epithelia.10 However, the mature of keratinocytes was regulated by various signals such as components of medium, paracrine factors, and the structure of biomaterial, it is difficult to determine the main factors regulating the cell fate and their interactive mechanisms from this study. The critical factors regulating keratinocyte differentiation need to be investigated by further research. In this study, a novel bilayer skin substrate, the GCP biocomposite, was used as the support matrix for the growth and differentiation of fibroblasts and keratinocytes. As we hypothesized, the highest keratinocyte differentiation was observed when seeding keratinocytes and fibroblasts in this biocomposite, which mimicked actual skin tissue. Thus, we have established a microenvironmental

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model using a 3-dimensional environment, culture media, and cells to optimize their crosstalk during keratinocytes differentiation from both stem cells. ACKNOWLEDGMENTS Dr. Niann-Tzyy Dai acknowledges the financial support for this work provided by the Ministry of National Defense, ROC (MAB101-45, MAB102-5), National Defense Medical Center and Tri-Service General Hospital, ROC (TSGH-C101-008-013-S02, TSGH-C102-006-008013-S02). We are also grateful to the financial support by Teh-Tzer Study Group for Human Medical Research Foundation. REFERENCES 1. Friel R, van der Sar S, Mee PJ. Embryonic stem cells: understanding their history, cell biology and signalling. Adv Drug Deliv Rev. 2005;57:1894Y1903. 2. Evan MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154Y156. 3. Robertson EJ. Embryo derived stem cell lines. In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford, London: IRL Press; 1987:71Y112. 4. Fuchs E. Epidermal differentiation: the bare essentials. J Cell Biol. 1990;111: 2807Y2814. 5. Rennekampff HO, Hansbrough JF, Kiessig V, et al. Bioactive interleukin-8 is expressed in wounds and enhances wound healing. J Surg Res. 2000;93: 41Y54. 6. Gurtner GC, Werner S, Barrandon Y, et al. Wound repair and regeneration. Nature. 2008;453:314Y321. 7. Metallo CM, Mohr JC, Detzel CJ, et al. Engineering the stem cell microenvironment. Biotechnol Prog. 2007;23:18Y23. 8. Levenberg S, Huang NF, Lavik E, et al. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc Natl Acad Sci U S A. 2003;100:12741Y12746. 9. Inanc B, Elcin AE, Elcin YM, et al. Human embryonic stem cell differentiation on tissue engineering scaffolds: effects of NGF and retinoic acid induction. Tissue Eng Part A. 2008;14:955Y964. 10. Hewitt KJ, Shamis Y, Carlson MW, et al. Three-dimensional epithelial tissues generated from human embryonic stem cells. Tissue Eng Part A. 2009;15: 3417Y3426. 11. Lin HT, Kao CL, Lee KH, et al. Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method. World J Gastroenterol. 2007;13:1672Y1679. 12. Dai NT, Yeh MK, Chiang CH, et al. Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer. Biochem Biophys Res Commun. 2009;386:21Y25. 13. Ning F, Guo Y, Tang J, et al. Differentiation of mouse embryonic stem cells into dental epithelial-like cells induced by ameloblasts serum-free conditioned medium. Biochem Biophys Res Commun. 2010;394:342Y347. 14. Huang SP, Hsu CC, Chang SC, et al. Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect. Ann Plast Surg. 2012;69:656Y662. 15. Roeder HA, Cramer SF, Leppert PC, et al. A look at uterine wound healing through a histopathological study of uterine scars. Reprod Sci. 2012;19: 463Y473. 16. Ji L, Allen-Hoffmann BL, de Pablo JJ, et al. Generation and differentiation of human embryonic stem cell-derived keratinocyte precursors. Tissue Eng. 2006;12:665Y679. 17. Coraux C, Hilmi C, Rouleau M, et al. Reconstituted skin from murine embryonic stem cells. Curr Biol. 2003;13:849Y853. 18. Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132: 661Y680. 19. Itoh M, Kiuru M, Cairo MS, et al. Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2011;108:8797Y8802. 20. Dabelsteen S, Hercule P, Barron P, et al. Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types. Stem Cells. 2009;27:1388Y1399.

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The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells.

Embryonic stem cells (ESCs) are pluripotent cells that can differentiate into various cell types, including keratinocyte-like cells, within suitable m...
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