The regulation of epithelial cell proliferation and growth by IL-1 receptor antagonist

Biomaterials 34 (2013) 121e129

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The regulation of epithelial cell proliferation and growth by IL-1 receptor antagonist Makoto Kondo a, b, Masayuki Yamato b, *, Ryo Takagi b, Hideo Namiki a, Teruo Okano b a b

Graduate School of Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 July 2012 Accepted 17 September 2012 Available online 8 October 2012

We have performed clinical translation of epithelial cell sheets fabricated on temperature-responsive culture surfaces to treat cornea and esophagus. In the preclinical study using animal models, we found epithelial cell growth potential varied among species. Canine epithelial cell growth was prominent, while rat one was poor under 3T3 feeder layer-free condition. The aim of the present study was to identify growth-promoting factors for epithelial cells. Conditioned medium of canine cell culture harvested at different time points showed different growth promotive activity for rat epithelial cells. Timedependent gene expression was quantitatively evaluated for forty growth factors, and compared with conditioned medium results. Statistically significant promotive activity was observed with IL-1RA, and significant inhibitory activity was observed with IL-1a. Furthermore, neutralizing anti-IL-1a antibody also showed significant promotive activity. Human epidermal keratinocytes were promoted to proliferate by IL-1RA and neutralizing anti-IL-1a antibody, and showed well differentiation to form transplantable, squamous stratified epithelial cell sheets. These findings would be useful to fabricate reproducible, transplantable epithelial cell sheets for regenerative medicine. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Cell proliferation Epithelial cell Stem cell Growth factors Interleukin Gene expression

1. Introduction We have investigated tissue regeneration by transplantation of cell sheets fabricated on temperature-responsive cell culture surfaces, on which a temperature-responsive polymer, poly(N-isopropylacrylamide), is covalently immobilized [1]. Various cell types adhere, spread, and proliferate on the surfaces at 37
C, and confluently cultured cells are harvested as a contiguous single cell sheet by reducing temperature to 20
C without any need for proteolytic enzyme like trypsin or dispase. Therefore, harvested cells intactly retain all the membrane proteins including cadherin, growth factor receptors, and ion channels as well as extracellular matrices (ECM) deposited during culture underneath cell sheets [2]. This non-invasive cell sheet harvest results in quick and ideal integration to transplanted tissue sites. The clinical applications of cell sheet transplantation have been successfully performed to treat skin [3], cornea [4], heart [5], esophagus [6], and periodontal tissue [7] for human patients. Patients’ oral mucosal epithelial cells have been used as a cell source to fabricate transplantable cell sheets in the cases to treat

cornea [4] and esophagus [6]. Usually, murine fibroblastic 3T3 feeder cells derived from mouse embryo and fetal bovine serum (FBS) are used to promote epithelial cell proliferation in vitro [8]. But, we have eliminated these xenogeneic materials from the culture by using patient’s own serum and culture inserts having micro-porous membrane, which supplies culture medium from the cell bottom [9,10] to avoid possibility of xenogeneic infection and contamination. Before, the clinical translation to human patients, we performed the preclinical studies [11e13] using several kinds of experimental animals, and we interestingly found different proliferative capabilities and supplemental requirement of epithelial cells among the species including human (to be submitted). In the present study, we compared canine and rat cell proliferation, and investigated which cytokine has an essential role to control epithelial cell growth in an autocrine manner. The obtained results would be useful to reproducibly fabricate transplantable stratified squamous epithelial cell sheets in the clinical settings for regenerative medicine. 2. Materials and methods 2.1. Fabrication of epithelial cell sheets

* Corresponding author. Tel.: þ81 3 5367 9945x6211; fax: þ81 3 3359 6046. E-mail addresses: [email protected], [email protected] (M. Yamato). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.09.036

All the experimental protocols were approved by the Institutional Animal Care and Use Committee of Tokyo Women’s Medical University. Human epidermal

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keratinocytes were purchased from Kohjin Bio (Saitama, Japan). Oral mucosal tissue was excised from the buccal cavities of rats (Lewis, male, 8 weeks old) and canines (beagle, male, 12 months old), disinfected with povidone-iodine (Meiji Seika Pharma, Tokyo, Japan), and washed with Dulbecco’s Modified Eagle Medium (DMEM) (SigmaeAldrich, St Louis, MO) containing 100 IU/mL penicillin and 100 mg/ mL streptomycin (Life Technologies, Carlsbad, CA). Epithelial tissue was peeled off by forceps after incubation with 1000 PU dispase (Godo-shusei, Tokyo, Japan) at 4
C for 15 h. Peeled epithelium was torn with forceps, and dissociated with 1.25% trypsin0.5% ethylenediaminetetraacetate in Dulbecco’s phosphate buffer saline (PBS) (SigmaeAldrich) at 37
C for 15 min. Dissociated cell suspension was filtrated through a 40-mm cell strainer (BD Biosciences, Franklin Lakes, NJ). Keratinocyte culture medium (KCM) was prepared by mixing of DMEM and Ham’s F-12 (Sigmae Aldrich) at the ratio of 3 to 1, supplemented with 5% FBS (Moregate BioTech, Queensland, Australia), 2 nM triiodothyronine (Wako Pure Chemicals, Osaka, Japan), 10 ng/mL recombinant human epidermal growth factor (Protein Express, Chiba, Japan), and pharmaceutical drugs of 5 mg/mL insulin (Humalin: Eli Lilly, Indianapolis, IN), 0.4 mg/mL hydrocortisone (Saxizon: Kowa Pharmaceutical, Tokyo, Japan), 0.25 mg/mL amphotericin B (Fungizone: Bristol-Myers Squibb, Park Avenue, NY) and 40 mg/mL gentamicin (Gentacin: Schering-Plough, Kenilworth, NJ). Cholera toxin (List Biological Labs, Campbell, CA) was eliminated from KCM for canine and rat epithelial cell culture, but added at a concentration of 1 nM for human epidermal keratinocyte culture. Suspension of primary oral mucosal epithelial cells were seeded on temperature-responsive cell culture inserts (23 mm in diameter, UpCell Insert: CellSeed, Tokyo, Japan) at a density of 4.0e8.0  104 cells/cm2, and cultured in an atmosphere of 5% CO2 at 37
C for 11e12 days. Proliferating cells were observed with a phase contrast microscope (ECLIPSE TE2000-U: Nikon, Tokyo, Japan). Stratified squamous epithelial cell sheets were harvested by low temperature treatment at 20
C for 30 min, and subjected to histological analyses. No 3T3 feeder layer was used throughout the experiments.

(Qiagen), and subjected to the synthesis of single stranded cDNA with reverse transcription reaction with PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan) with iCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). For canine gene expression analysis, primer pairs and TaqMan MGB probes were designed for b2 microglobulin (B2M), brain-derived neurotrophic factor (Bdnf), epidermal growth factor (Egf), fibroblast growth factor 1 (Fgf1), Fgf2, Fgf4, Fgf5, Fgf6, Fgf7, Fgf8, Fgf9, Fgf10, Fgf12, Fgf13, Fgf14, Fgf16, Fgf18, Fgf19, glial cell line derived neurotrophic factor (Gdnf), hepatoma-derived growth factor (Hdgf), hepatocyte growth factor (Hgf), c-fos induced growth factor (Figf), growth factor augmenter of liver regeneration (Gfer), heparin-binding EGF-like growth factor (Hbegf), insulin-like growth factor 1 (Igf1), Igf2, interleukin-1a (Il1a), interleukin-1 receptor antagonist (Il1ra), nerve growth factor (Ngf), neuregulin 1 (Nrg1), Nrg2, neurotrophin 3 (Ntf3), Ntf4, platelet-derived growth factor alpha polypeptide (Pdgfa), Pdgfb, placental growth factor (Pgf), pleiotrophin (Ptn), transforming growth factor alpha (Tgfa), Tgfb1, Tgfb2, vascular endothelial growth factor A (Vegfa), and vascular endothelial growth factor C (Vegfc) for TaqMan Gene Expression AssaysÔ (Life Technologies). Quantitative PCR was performed with Premix Ex Taq (Takara Bio) and TaqMan Gene Expression Assays (Life Technologies). For human keratinocyte gene expression assay, primer pairs and TaqMan MGB probes were designed for b2 microglobulin (B2M), Ki67, delta N p63 (dNp63), cytokeratin 15 (CK15), integrin beta 1 (ITGb1), integrin alpha 6 (ITGa6), laminin beta 3 (LAMb3), zonula occludens-1 (ZO1), E-cadherin (CDH1), CK4, CK13, CK1, CK10, filaggrin (FLG), loricrin (LOR), involucrin (IVL), tumor necrosis factora (TNFA), interleukin-1a (IL1A), interleukin-1b (IL1B), interleukin 1 receptor antagonist (IL1RA), interleukin 1 receptor I (IL1RI), and interleukin 1 receptor II (IL1RII) for TaqMan Gene Expression AssaysÔ (Life Technologies). TaqMan Fast Universal PCR Master Mix and StepOnePlusÔ Real-Time PCR Systems (Life Technologies) were used. mRNA expression levels were normalized with the expression level of b2 microglobulin. Human epidermal keratinocytes were seeded at a density of 9.0  104 cells/ cm2, cultured for 6 days, and subjected to RNA extraction. Obtained data from three independent cultures were statistically analyzed by Scheffé’s method.

2.2. Gene expression analyses 2.3. Conditioned medium Expression of mRNA was quantified by realtime quantitative RT-PCR with 7300 Real Time PCR System (Life Technologies). Cultured cells were lysed with QIAshredder (Qiagen, Hilden, Germany). Total RNA was isolated with RNeasy Plus Mini Kit

The scheme of conditioned medium experiment is shown in Fig. 2. Primary canine oral mucosal epithelial cells were cultured with KCM at a density of

Day5

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Fig. 1. Culture of canine and rat oral mucosal epithelial cells. The cells were seeded at a density of 4.0  104 cells/cm2. Scale bar indicates 200 mm. Phase contrast microscope images in the upper and lower rows represent canine and rat oral mucosal epithelial cells observed at 5, 7, 9, and 11 days after cell seeding, respectively. The graph shows the number of the cultured oral mucosal epithelial cells of canine and rat (n ¼ 3). Canine oral mucosal epithelial cell sheet was successfully fabricated as shown in the left photograph. White scale bar indicates 10 mm. The section of the cell sheet was fixed and stained with hematoxylin and eosin shows a stratified cell sheet structure with approximately 50 mm in thick. Black scale bar indicates 50 mm.

M. Kondo et al. / Biomaterials 34 (2013) 121e129

Medium change:

Day 3

Canine cell culture

Day 5

Day 7

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Day 9

Day 11

48-h 48-h 48-h 48-h 48-h Incubation Incubation Incubation Incubation Incubation

Supernatant collection:

Control medium

Day 5

Day 7

Day 9

Day 11

Day 13

Rat cell culture Fig. 2. Scheme of rat oral mucosal epithelial cell culture with conditioned medium. Conditioned media were collected from canine oral mucosal epithelial cell culture at 5, 7, 9, 11, and 13 days after 48-h culture. The volumes of collected supernatant samples were adjusted with fresh KCM for obtaining a certain ratio of the number of the cultured cells and the volume.

40

30

20

10

Day 13

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0 Day 9

Canine and human epidermal cell sheets were harvested by temperature reduction to 20
C after 11 days culture. For histological analyses, harvested cell sheets were fixed with 10% neutral buffered formalin, and routinely processed into 3-mm thick paraffin-embedded sections. Hematoxylin and eosin staining was performed by conventional methods. For immunohistochemistry, de-paraffinized sections were washed with PBS, and subjected to proteinase K treatment (DakoCytomation, Glostrup, Denmark) or heat treatment with Target Retrieval Solution, Citrate pH6 (DakoCytomation). Sections were then treated with each of the following primary antibodies; mouse monoclonal anti-E-cadherin (1:50 dilution) (NCH-38: DakoCytomation), mouse monoclonal anti-filaggrin (1:100 dilution) (FLG01: Thermo Fisher Scientific, Waltham, MA), mouse monoclonal anti-p63 (1:100 dilution) (4A4: Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-Ki67 (1:100 dilution) (MIB-1: Lab Vision, Fremont, CA), and mouse monoclonal anti-pancytokeratin (1:200 dilution) (AE1/AE3: Progen Biotechnik, Heidelberg, Germany) at 4
C overnight. All sections were peroxidase-stained using LSAB2 kit (DakoCytomation), according to the manufacturer’s suggested protocol.

50

Day 7

2.5. Histology

60

Day 5

Each of 13 recombinant cytokines was added to KCM, and the proliferation of primary rat oral mucosal epithelial cells and human epidermal keratinocyte were examined. Culture condition for rat cells was the same as the conditioned medium experiment (n ¼ 3), but the initial cell number was 9.0  104 and 1.5  104 cells/cm2 for human epidermal cell growth and cell sheet formation experiments, respectively. Numbers of cultured human epidermal cells were counted at day 6 (n ¼ 3). Obtained data from three independent cultures were statistically analyzed by Student’s t-test. Recombinant human heparin-binding EGF-like growth factor (HBEGF), recombinant human interleukin-1a (IL-1a), recombinant human IL-1 receptor antagonist (IL-1RA), recombinant human brain-derived neurotrophic factor (BDNF), recombinant human neuregulin, recombinant human hepatoma-derived growth factor Isoform 1 (HDGF), recombinant Rat Platelet derived growth factor-BB (PDGFBB), recombinant human vascular endothelial growth factor-C (VEGF-C), recombinant rat b-NGF, recombinant human pleiotrophin were purchased from R&D Systems (Minneapolis, MN). Recombinant human fibroblast growth factor 18 (FGF18) and recombinant human transforming growth factor-a (TGF-a) were purchased from Biovision (Milpitas, CA). Recombinant growth factor, augmenter of liver regeneration (GFER) and recombinant placental growth factor (PGF) were purchased from Abnova (Taipei, Taiwan). Each cytokine was added at a final concentration of 10 ng/mL. IL-1a and IL-1RA were examined at concentrations of 0.001e10 ng/mL and 0.01e100 ng/mL, respectively. Anti-lL-1a neutralizing antibody was obtained from R&D Systems. Normal goat IgG (R&D Systems) was used as a control. Antibodies were added at a final concentration of 10 mg/mL. KCM with PBS or normal goat IgG was used for a control culture condition.

Primary canine and rat oral mucosal epithelial cells were cultured with KCM under a 3T3 feeder layer-free condition. Primary canine oral mucosal epithelial cells exhibited a small, cobblestonelike cell shape, and a higher proliferation than primary rat oral mucosal epithelial cells (n ¼ 3) (Fig. 1). At 11 days of culture, canine cells reached confluency, and kept their confluence more than 13 days of culture. Cultured canine oral mucosal epithelial cells were successfully harvested as a contiguous cell sheet from a temperature-responsive cell culture insert at 12 days after seeding (Fig. 1). On the other hand, rat cells were flat and large in cell shape. Their cell proliferation was significantly poor, and failed to reach confluency under the culture condition (Fig. 1), while these cells showed high proliferative capacity in the presence of cholera toxin and 3T3 feeder layer (data not shown). This observation

Control

2.4. Cytokines and neutralizing antibodies

3. Results

Cell number (104 cells/cm2)

4  104 cells/cm2. Conditioned media were collected at the time points of 5, 7, 9, 11, and 13 days culture 48 h after medium change. Collected conditioned media were diluted with fresh KCM to normalize corresponding cell numbers in culture. Then, primary rat oral mucosal epithelial cells were seeded at an initial cell density of 4  104 cells/cm2 with conditioned media for seven days, and the cell numbers were counted (n ¼ 3). KCM pre-incubated at 37
C for 48 h was used for a control culture condition.

Fig. 3. Culture of rat oral mucosal epithelial cells with conditioned medium. The number of rat oral mucosal epithelial cells cultured with conditioned medium was counted at 7 days after the cell seeding (n ¼ 3).

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convincingly revealed that supplemental requirement for cell proliferation is different between the two species, and implies canine epithelial cells would secrete autocrine factors to promote cell proliferation, that lack in rat epithelial cell culture. Then, we examined whether conditioned media collected from canine epithelial cell culture can promote primary rat epithelial cell proliferation (Fig. 2). Interestingly, potent promotive activity of cell proliferation was observed with conditioned media collected during 5e9 day of culture (Fig. 3). Therefore, we examined which cytokine plays a crucial role in the cell proliferation promotive activity. First, gene expression of canine epithelial cells was quantified for 40 cytokine genes by quantitative RT-PCR with genespecific TaqMan probes. Referring to the time-course change of the cell proliferation promotive activity, cytokine genes were categorized into four groups; descending, ascending, non-descript, and undetectable (Table 1). Obtained candidate genes to promote epithelial cell growth were Bdnf, Fgf1, Fgf18, Gfer, Hbegf, Hdgf, IL1a, Il1ra, Ngf, Nrg1, Pdgfb, Ptn, Tgfa, and Vegfc which showed descending pattern (Fig. 4). Next, thirteen recombinant cytokine proteins among these fourteen genes were added to culture of primary rat epithelial cells. As a result, only IL-1RA enhanced the epithelial cell proliferation dose-dependently (Fig. 5A, B). By contrast, IL-1a significantly inhibited rat epithelial cell growth (Fig. 5A, B). Other cytokines except GFER didn’t show significant cell proliferation promotive or inhibitory activity. The promotive

Table 1 Forty cytokine gene expressions in cultured canine oral mucosal epithelial cells. Expression patterns

Gene symbol

Descending

Bdnf, Fgf1, Fgf18, Gfer, Hbegf, Hdgf, Il1a, Il1ra, Ngf, Nrg1, Pdgfb, Ptn, Tgfa, Vegfc Egf, Igf2, Ntf3, Pgf Fgf2, Fgf7, Fgf9, Fgf13, Gdnf, Hgf, Nrg2, Ntf4, Pdgfa, Tgfb2, Vegfa Fgf4, Fgf5, Fgf6, Fgf8, Fgf10, Fgf12, Fgf14, Fgf16, Fgf19, Figf, Igf1, Tgfb1

Ascending Non-descript Undetectable

Relative gene expressions were quantified at 5, 7, 9, 11, and 13 days after cell seeding. The abbreviation of gene of brain-derived neurotrophic factor is Bdnf; hepatoma-derived growth factor, Hdgf; fibroblast growth factor 1, Fgf1; fibroblast growth factor 18, Fgf18; growth factor, augmenter of liver regeneration, Gfer; heparin-binding EGF-like growth factor, Hbegf; interleukin 1 alpha, Il1a; interleukin 1 receptor antagonist, Il1ra; nerve growth factor, Ngf; neuregulin 1, Nrg1; plateletderived growth factor beta polypeptide, Pdgfb; pleiotrophin, Ptn; transforming growth factor alpha, Tgfa; vascular endothelial growth factor C, Vegfc; epidermal growth factor, Egf; insulin-like growth factor 2, insulin-like growth factor 2, Igf2; neurotrophin 3, Ntf3; placental growth factor, Pgf; fibroblast growth factor 2, Fgf2; fibroblast growth factor 7, Fgf7; fibroblast growth factor 9, Fgf9; fibroblast growth factor 13, Fgf13; glial cell line derived neurotrophic factor, Gdnf: hepatocyte growth factor, Hgf; neuregulin 2, Nrg2; neurotrophin 4, Ntf4; platelet-derived growth factor alpha polypeptide, Pdgfa; transforming growth factor, beta 2, Tgfb2; vascular endothelial growth factor A, Vegfa; fibroblast growth factor 4, Fgf4; fibroblast growth factor 5, Fgf5; fibroblast growth factor 6, Fgf6; fibroblast growth factor 8, Fgf8; fibroblast growth factor 10, Fgf10; fibroblast growth factor 12, Fgf12; fibroblast growth factor 14, Fgf14; fibroblast growth factor 16, Fgf16; fibroblast growth factor 19, Fgf19; c-fos induced growth factor, Figf; insulin-like growth factor 1, Igf1; transforming growth factor, beta 1, Tgfb1.

Bdnf

Fgf1

Fgf18

Gfer

Hbegf

Hdgf

Il1a

Il1ra

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Pdgfb

Ptn

Tgfa

Vegfc

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Relative gene expression (%)

0

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Time (day) Fig. 4. Gene expression of selected cytokines showing a descending pattern (n ¼ 3). The cytokine genes were brain-derived neurotrophic factor (Bdnf), fibroblast growth factor 1 (Fgf1), fibroblast growth factor 18 (Fgf18), growth factor, augmenter of liver regeneration (Gfer), heparin-binding EGF-like growth factor (Hbegf), hepatoma-derived growth factor (Hdgf), interleukin-1 alpha (Il1a), interleukin-1 receptor antagonist (Il1ra), nerve growth factor (Ngf), neuregulin 1 (Nrg1), platelet-derived growth factor subunit B (Pdgfb), pleiotrophin (Ptn), transforming growth factor, alpha (Tgfa), vascular endothelial growth factor C (Vegfc). Relative gene expressions at 7, 9, 11, and 13 days after cell seeding were calculated by allowing the expression at day 5 to be 100%.

M. Kondo et al. / Biomaterials 34 (2013) 121e129

Gene expressions of normal human epidermal keratinocyte cultured for 6 days in the presence of each of PBS, IL-1RA, IL-1a, normal IgG, and anti-IL-1a neutralizing antibody were analyzed in terms of epithelial cell differentiation, stem/progenitor cell marker, cell proliferation, ECM, IL-1 and its receptors (Fig. 7). The gene expression of proliferation marker; Ki-67, was significantly higher in the presence of IL-1RA. A putative epithelial stem/progenitor cell marker, dNp63 was expressed in a higher degree in the presence of IL-1RA or anti-IL-1a antibody, although stem progenitor marker CK15 was expressed in a higher degree only in the presence of IL1RA. To the contrary, IL-1a down-regulated the expressions of dNp63 and CK15. The gene expression of epithelial extracellular matrix component; laminin beta 3 was slightly higher in IL-1RA

activity of GFER was statistically significant, but much smaller than that of IL-1RA (Fig. 5A). Then, the activities of interleukins and receptor antagonist were examined with normal human epidermal keratinocytes under a 3T3 feeder layer-free condition. Certainly, the epithelial cell proliferation was significantly inhibited by IL-1a, but enhanced by IL-1RA. In addition, the cell size was kept smaller, and the cell shape was kept more cuboidal in the presence of IL-1RA than a control added with PBS (Fig. 6). IL-1b showed a small but significant negative effect on the cell growth (Fig. 6), and no difference of cell shape was observed (data not shown). Furthermore, anti-IL-1a neutralizing antibody also significantly promoted human epithelial cell proliferation (Fig. 6).

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Fig. 5. Growth inhibitory and promotive effects of exogenous cytokines on epithelial cells. Rat oral mucosal epithelial cells were cultured with supplement of each of 13 recombinant cytokines for seven days, then cell number was counted (n ¼ 3) (A). **shows p < 0.01 IL-1a and IL-1RA showed dose-dependent effects on rat oral mucosal epithelial cell proliferation (n ¼ 3) (B).

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Fig. 6. Effect of IL-1 signaling on normal human epidermal keratinocyte growth. Normal human epidermal keratinocytes cultured with KCM supplemented with each of PBS (control), 100 ng/mL IL-1RA, 10 ng/mL IL-1a, 10 ng/mL IL-1b, 10 mg/mL normal goat immunoglobulin G (normal IgG) as the control, or 10 mg/mL neutralizing anti-IL-1a antibody (anti-IL-1a). Phase contrast microscope images show the cells cultured with KCM containing PBS, IL-1RA, normal IgG, and anti-IL1a at 6 days after cell seeding. The relative cell number at day 6 after cell seeding was calculated as the ratio to the control (PBS or normal IgG) (n ¼ 3). Scale bar indicates 200 mm. ** and * show p < 0.01 and 0.05, respectively.

condition than its control, and slightly lower in IL-1a, whereas antiIL-1a showed no difference. Gene expressions of epithelial cell differentiation markers of CK4, CK13, CK1, and CK10 were downregulated in the presence of IL-1a, but up-regulated in the presence of IL-1RA, whereas anti-IL-1a antibody showed no effect. The gene expression of filaggrin was significantly lower in the presence of IL-1a, but higher in IL-1RA and anti-IL-1a. For TNFA, not significant but slight down-regulation in IL-1RA and significant downregulation in anti-IL-1a condition, and not significant upregulation was observed in the presence of IL-1a. Loricrin, IL-1a, IL-1b, IL-1 receptor antagonist, IL-1 receptor I, and IL-1 receptor II showed no differences among five conditions (Fig. 7). Histological evaluation after H-E staining and immunohistochemistry revealed that the cell sheets cultured in the presence of IL-1RA or anti-IL-1a

antibody showed cell stratification and normal epidermal cell differentiation (Fig. 8). 4. Discussion For establishing epithelial regenerative medicine as a widespread and standard treatment, a stable epithelial cell culture condition should be established. The present study aimed to sophisticate epithelial cell culture by investigating applicable materials having a growth promotional effect on stratified squamous epithelial cells. Primary canine oral mucosal epithelial cells showed a significantly higher growth potential than primary rat oral mucosal epithelial cells under 3T3 feeder-free condition (Fig. 1), and the supernatant of canine cell culture conditioned medium

1.2

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CK10 2.5

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IL1B

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PBS IL-1RA IL-1α IgG anti-IL-1α 0.03 0.02 0.01

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3

TNFA

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120 40

PBS IL-1RA IL-1α IgG anti-IL-1α

IVL

60

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15

30

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30

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IL1RII

M. Kondo et al. / Biomaterials 34 (2013) 121e129

1.5

90

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CK13

100

60

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6

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PBS IL-1RA IL-1α IgG anti-IL-1α

CK4

Relative Gene Expressions (% of B2M)

CDH1 120

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*

PBS IL-1RA IL-1α IgG anti-IL-1α

0

120

90

ZO1

PBS IL-1RA IL-1α IgG anti-IL-1α

20

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40

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PBS IL-1RA IL-1α IgG anti-IL-1α

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60

ITGa6 120

PBS IL-1RA IL-1α IgG anti-IL-1α

4

40

PBS IL-1RA IL-1α IgG anti-IL-1α

0.2 PBS IL-1RA IL-1α IgG anti-IL-1α

0.4

8

**

PBS IL-1RA IL-1α IgG anti-IL-1α

12

0.6

ITGb1

**

PBS IL-1RA IL-1α IgG anti-IL-1α

**

CK15

PBS IL-1RA IL-1α IgG anti-IL-1α

16

0.8

60

**

PBS IL-1RA IL-1α IgG anti-IL-1α

dNp63

PBS IL-1RA IL-1α IgG anti-IL-1α

**

Relative Gene Expressions (% of B2M)

Relative Gene Expressions (% of B2M)

Ki67 1

Fig. 7. Gene expression of human normal epidermal keratinocyte. The cells were cultured with KCM containing PBS, 100 ng/mL IL-1RA, 10 ng/mL IL-1a, 10 mg/mL normal IgG, or 10 mg/mL neutralizing anti-IL-1a antibody (anti-IL-1a). Gene expressions were determined by quantitative RT-PCR (n ¼ 3). The analyzed genes were Ki67, delta N p63 (dNp63), cytokeratin 15 (CK15), integrin beta 1 (ITGb1), integrin alpha 6 (ITGa6), laminin beta 3 (LAMb3), zonula occludens1 (ZO1), E-cadherin (CDH1), CK4, CK13, CK1, CK10, filaggrin (FLG), loricrin (LOR), involucrin (IVL), tumor necrosis factor-a (TNFA), interleukin-1a (IL1A), interleukin-1b (IL1B), interleukin 1 receptor antagonist (IL1RA), interleukin 1 receptor I (IL1RI), and interleukin 1 receptor II (IL1RII). Relative gene expression against b2 microglobulin was shown.

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IL-1RA

Anti-IL-1α

HE

p63

Ki67

Filaggrin

E-cadherin

Pancytokeratin Fig. 8. Histology of harvested human normal epidermal cell sheets. The photographs on the upper row are cell sheets cultured in keratinocyte culture medium containing 100 ng/ mL IL-1RA and 20 mg/mL neutralizing anti-IL-1a antibody (anti-IL-1a), respectively. White scale bars of the upper row photographs indicate 10 mm. HE staining and immunohistochemistry of p63, Ki67, filaggrin, E-cadherin, and pan-cytokeratin (panCK) were also performed. Black scale bars indicate 50 mm.

enhanced the proliferation of rat cells (Fig. 3). These observations strongly implied that cultured canine oral mucosal epithelial cells secreted growth promotive factors in an autocrine manner. By focusing on the time-course change of cytokine gene expression (Table 1 and Fig. 4) and growth promotive activity (Fig. 5A), IL-1RA was identified to promote epithelial cell growth, while IL-1a inhibited the growth (Fig. 5A, B). Previous reports [14e17] show that IL-1a have a growth promotional effect on epidermal keratinocytes under a 3T3 feeder co-cultured condition under the rationale that IL1a allows 3T3 feeder layer to release KGF into culture medium. In the present study, we utilized the culture condition which eliminated 3T3 feeder layer [9,10], and discovered direct negative effect of IL-1a on epithelial cell growth (Fig. 5A, B, Fig. 6). Cultured normal keratinocyte store a large amount of IL-1a in the cytoplasm [18e21], and secrete IL-1a and IL-1RA consistently [22]. Although many reports have been published on IL-1 signaling, and IL-1 pathway is drawn as a huge correlation diagram [23], the mechanism of IL-1a-mediating epithelial cell growth inhibition is unproven directly. IL-1a agonize IL-1 receptor, and the signal is transduced through MyD88, IRAK1, IRAK4, and TRAF6 [24]. NFkB activation by IL-1a through TRAF6 and its effector proteins including NEMO, IKKa, and IKKb has been reported [25], and overexpressed NFkB has a growth inhibitory role for epidermal cells and inhibition of NFkB causes hyperplasia in vivo [26]. Also, NFkB profoundly inhibits cell cycle progression in vitro [27], and activation of NFkB is required for PMA-induced keratinocyte growth arrest [28]. Presumably, in this study, exogenous IL-1RA inhibited the signal transduction described above. The balance of IL1a and IL-1RA in vitro and in vivo would be of importance to maintain

normal epithelial cell growth. Gene expression of p63 was much higher in the presence of IL-1RA or anti-IL-1a antibody, while IL-1a (10 ng/mL) down-regulated p63 expression (Fig. 7). p63 is reported to bind directly to the transcription regulating region of IL-1a, and IL1a gene expression is lower in p63 knockout mouse [29]. Negative feedback system of p63 expression by IL-1a signaling might underlie. As well as p63 expression, Ki67, a proliferation marker, various types of keratin, cellecell junction, and cell-substrate junction were up-regulated by hampering IL-1a signaling (Fig. 7). These results indicated that the control of IL-1a signaling should be important to fabricate well-differentiated, robust, stratified squamous epithelial cell sheets for regenerative medicine. 5. Conclusion Under 3T3 feeder-free cell culture condition, IL-1a was found to inhibit the proliferation of stratified squamous epithelial cells, whereas IL-1RA to enhance the proliferation. Moreover, significant growth promotion was confirmed for human normal epidermal keratinocytes by adding exogenous IL-1RA or anti-IL-1a antibody to culture medium. Acknowledgments The author thanks Mr. H. Sugiyama of Tokyo Women’s Medical University for the useful comments and technical criticism. This work was supported by JSPS research fellowship, Formation of Innovation Center for Fusion of Advanced Technologies in the

M. Kondo et al. / Biomaterials 34 (2013) 121e129

Special Coordination Funds for Promoting Science and Technology ‘Cell Sheet Tissue Engineering Center (CSTEC)’ and the Global COE program, the Multidisciplinary Education and Research Center for the Establishment of Regenerative Medicine (MERCREM), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. References [1] Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermoresponsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol Chem Rapid Commun 1990;11:571e6. [2] Kushida A, Yamato M, Konno C, Kikuchi A, Sakurai Y, Okano T. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. J Biomed Mater Res 1999;45:355e62. [3] Yamato M, Utsumi M, Kushida A, Konno C, Kikuchi A, Okano T. Thermoresponsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng 2001;7:473e80. [4] Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 2004;77:379e85. [5] Sawa Y, Miyagawa S, Sakaguchi T, Fujita T, Matsuyama A, Saito A, et al. Tissue engineered myoblast sheets improved cardiac function sufficiently to discontinue LVAS in a patient with DCM: report of a case. Surg Today 2012;42:181e4. [6] Ohki T, Yamato M, Ota M, Takagi R, Murakami D, Kondo M, et al. Prevention of esophageal stricture after endoscopic submucosal dissection using tissueengineered cell sheets. Gastroenterology 2012;143(3):582e8. [7] Iwata T, Yamato M, Tsuchioka H, Takagi R, Mukobata S, Washio K, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model. Biomaterials 2009;30:2716e23. [8] Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331e43. [9] Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, et al. The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets. Biomaterials 2006;27:5518e23. [10] Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, et al. Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells. J Artif Organs 2006;9(3):185e91. [11] Takagi R, Yamato M, Murakami D, Kondo M, Yang J, Ohki T, et al. Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J Tissue Eng Regen Med 2011;5:e63e73. [12] Takagi R, Yamato M, Murakami D, Kondo M, Ohki T, Sasaki R, et al. Fabrication and validation of autologous human oral mucosal epithelial cell sheets to

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