ROCK inhibitor combined with Ca2+ controls the myosin II activation and optimizes human nasal epithelial cell sheets

The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca2+ concentration. However, it was not known whether a combination would influence the behavior of cultured epithelial cells through changes in the phosphorylation of non-muscle myosin light chain II (MLC). Here we show that the combination of ROCK inhibition with Ca2+ elevation regulated the phosphorylation of MLC and improved both cell expansion and cell–cell adhesion during the culture of human nasal mucosal epithelial cell sheets. During explant culture, Ca2+ enhanced the adhesion of nasal mucosal tissue, while ROCK inhibition downregulated MLC phosphorylation and promoted cell proliferation. During cell sheet culture, an elevation of extracellular Ca2+ promoted MLC phosphorylation and formation of cell–cell junctions, allowing the harvesting of cell sheets without collapse. Moreover, an in vitro grafting assay revealed that ROCK inhibition increased the expansion of cell sheets three-fold (an effect maintained when Ca2+ was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca2+ could facilitate the fabrication of many types of cell graft.


Effect of ROCK inhibition on nasal epithelial cell outgrowth patterns.
To better evaluate tissue adhesion during explant culture, we performed our explant culture in temperature-responsive cell culture dishes using KCM-FBS (1.6 mM Ca 2+ ). After cell outgrowth was observed, we carefully detached the tissue as a cell sheet to make paraffin-embedded sections that were subjected to immunohistology ( Fig. 2A,B). In addition, non-cultured nasal mucosal tissues were used as controls (Fig. S2). In native tissue (before explant culture), E-cadherin (an epithelial marker) was expressed in epithelial cells only, cytokeratin-8 (CK8, a marker of differentiated nasal epithelium) was expressed in the upper layer of epithelial cells, vimentin (a marker of fibroblast/epithelial-mesenchymal transition) was expressed only in fibroblasts, Ki67 (a proliferation marker) was expressed sparsely in the epithelial layer, and p63 (a marker of stem/progenitor cells) was expressed on the basal side of epithelial cells. Immunodetection of E-cadherin in the explant culture at day 13 showed that connective tissue was present under the epithelial cells and had adhered before epithelial cell adhesion/migration. CK8, an intermediate filament found in nasal epithelial cells, was expressed in the tissue area but not in migrating cells. Vimentin was expressed not only in fibroblasts in the connective tissue but also in migrating epithelial cells. Interestingly, the proliferation marker Ki67 was observed in leading-edge cells rather than within the tissue area.
These results indicate that cells in the leading edge are important for cell expansion.
In order to observe the leading edge in more detail, we analyzed its behavior using phase-contrast microscopy and time-lapse differential interference microscopy. In normal KCM (1.6 mM Ca 2+ ), the cells migrated collectively (Fig. 2C). Although most cells moved outward, an interesting observation was that part of the cell cluster at the edge moved inward (Movie 1). Since this inward movement was considered actomyosin behavior, we analyzed the distribution of phospho-myosin light chain II (P-MLC) and actin stress fibers (Fig. 2D,E). As expected, P-MLC existed at the edges of each cell in normal KCM, and stress fibers were observed at cell-cell sites along with P-MLC (Fig. 2E). By contrast, when 10 µM Y-27632 was added, epithelial cells only moved toward Scientific RepoRtS | (2020) 10:16853 | https://doi.org/10.1038/s41598-020-73817-3 www.nature.com/scientificreports/ the periphery as an outgrowth, and some of the cells migrated individually (Fig. 2F, Movie 2). In the presence of 10 µM Y-27632, P-MLC existed in the cytoplasm (Fig. 2G), and actin depolymerization was observed (Fig. 2H).
optimization of cell sheet culture conditions. Freeze-thawed cells were able to form a nasal mucosal cell sheet when cultured in KCM-HS (Figs. 3A, S3A), as reported in our previous article 30 . However, defects were observed in the cell sheets when they were cultured in normal KCM-FBS containing 1.6 mM Ca 2+ (Fig. 3B) www.nature.com/scientificreports/ and 10 µM Y-27632 generated cell sheets that were structurally loose (i.e., did not show shrinkage behavior after detachment from the dish) and contained large defects (Fig. 3B). Therefore, we examined whether removal of Y-27632 during cell sheet culture or alteration of its concentration would improve cell sheet morphology. Although excluding Y-27632 from day 0, 3 or 5 produced cell sheets without defects in some cases, defects remained in other cases (Fig. S3B). Reducing the concentration of Y-27632 also led to some improvement, with 1 μM Y-27632 yielding better results than 100 nM or 2.5 μM Y-27632; nevertheless, some defects were still observed (Fig. S3C).
Since the best results were obtained using 1 μM Y-27632, we next examined whether increasing the Ca 2+ concentration from 1.6 mM to either 2.6 mM, 3.6 mM or 5.6 mM would improve cell-cell adhesion and yield cell sheets that did not develop defects or shrink after detachment from the dish following culture in medium containing 1 μM Y-27632. Medium containing 1 μM Y-27632 and either 2.6 mM Ca 2+ or 3.6 mM Ca 2+ produced cell sheets that were easily detached and shrank spontaneously without the development of defects (Fig. 3B, S3D), and the effects of increasing the Ca 2+ concentration to 3.6 mM were reproducible irrespective of the condition of the original tissue (Fig. S4). Detachable cell sheets were also fabricated in medium containing 1 μM Y-27632 and 5.6 mM Ca 2+ (Fig. S3D), but numerous crystals and floating dead cells were observed (data not shown). Structural loosening of the cell sheet was observed when it was cultured in medium containing 10 μM Y-27632 and 3.6 mM Ca 2+ , and exclusion of Y-27632 from KCM containing 3.6 mM Ca 2+ resulted in a cell sheet with many defects (Fig. S3E,F). Based on the above findings, it was concluded that optimal culture of nasal cell sheets was achieved using KCM-FBS containing 1 μM Y-27632 and 3.6 mM Ca 2+ .
Phosphorylation of MLC during cell sheet culture. To examine whether the effects of Y-27632 and Ca 2+ on the shrinkage behavior of the cell sheet was related to MLC phosphorylation, we performed immunofluorescence and Western blotting to detect MLC, P-MLC (Ser19) and double-phospho-myosin light chain II (Thr18, Ser19; PP-MLC). Immunofluorescence experiments showed that PP-MLC and P-MLC were present at the periphery of each cell in medium containing 1.6 mM Ca 2+ (normal KCM-FBS) but were barely detectable at the cell-cell interfaces in medium containing 1.6 mM Ca 2+ and 10 µM Y-27632 (Fig. 4A). Interestingly, increasing the Ca 2+ concentration from 1.6 mM to 3.6 mM recovered the phosphorylation of MLC at the cell periphery in the presence of 1 μM Y-27632 ( Fig. 4A) but not in the presence of 10 μM Y-27632 (Fig. S5). The results of Western blotting experiments showed that phosphorylation of MLC was downregulated by the addition of Y-27632 to medium containing 1.6 mM Ca 2+ but was recovered at both day 3 and day 7 when the Ca 2+ concentration was increased from 1.6 mM to 3.6 mM ( Fig. 4B; full-length Western blot in Fig. S7). Thus, the Western blot findings support the immunohistology data. In additional experiments, P-MLC was found to be weakly expressed in 0.1 mM Ca 2+ (KGM), and the expression of P-MLC was downregulated when Y-27632 was added, www.nature.com/scientificreports/ indicating that ROCK signaling might be active under these conditions (Fig. S5). Furthermore, increasing the level of Ca 2+ to ~ 2.2 mM (by adding extra Ca 2+ to KGM) led to P-MLC being expressed at the periphery of each cell, suggesting that it might be promoting cell-cell adhesion (Fig. S5). These results show that an increase in extracellular Ca 2+ can recover the downregulation of MLC phosphorylation that is induced by ROCK inhibition.

Immunohistological analyses of nasal mucosal cell sheets.
Cell-cell junctions were evaluated using immunofluorescence experiments and Western blotting to detect E-cadherin, claudin-1 and ZO-2 (Figs. 4C, S6A,B; full-length western blot in Fig. S7). Since cells were unable to form cell sheets under low Ca 2+ conditions, immunohistochemistry experiments could not be performed for cell sheets cultured in 0.1 mM Ca 2+ . Each cell sheet was composed of several layers, so immunohistology was performed to evaluate the cell-cell junctions in each layer. Immunohistology detected E-cadherin in almost all cells under all cell culture conditions. However, poor cell-cell adhesions were observed in the basal layer of cell sheets cultivated in 1.6 mM Ca 2+ (KCM-FBS) or 1.6 mM Ca 2+ with 10 µM Y-27632. By contrast, tight adhesions between cells were observed when the medium contained 3.6 mM Ca 2+ and 1 µM Y-27632, and E-cadherin was observed in the basal cell layer, suggesting that elevation of the Ca 2+ level improved cell-cell adhesions in the basal layer directly or indirectly. We also confirmed the reproducibility of the P-MLC and E-cadherin expression regardless of the condition of the original tissue (n = 4, Fig. S4). TEM images also demonstrated gaps between cells in the basal layer when medium containing 1.6 mM Ca 2+ or medium containing 1.6 mM Ca 2+ and 10 µM Y-27632 was used but little or no gaps when medium containing 3.6 mM Ca 2+ and 1 µM Y-27632 was used ( Fig. S6C-E). Immunohistology showed that clau- www.nature.com/scientificreports/ din-1 and ZO-2 were present at the periphery of each cell when medium containing 1.6 mM Ca 2+ or medium containing 3.6 mM Ca 2+ and 1 µM Y-27632 was used, but these proteins were rarely observed when medium containing 1.6 mM Ca 2+ and 10 µM Y-27632 was utilized (Fig. 4C). Western blotting revealed no notable differences in the expressions of E-cadherin, claudin-1 and ZO-2 between the various culture conditions (Fig. S6B), and tight junctions were observed in TEM images ( Fig. S6C-E). P-MLC was detected almost exclusively in the upper layer of the cell sheet when medium containing 1.6 mM Ca 2+ or medium containing 1.6 mM Ca 2+ and 10 µM Y-27632 was used. When the culture medium contained 3.6 mM Ca 2+ and 1 µM Y-27632, P-MLC was present in all layers, suggesting that this combination of Ca 2+ and Y-27632 promoted strong cell-cell adhesion within the basal layer. Expression of the proliferation marker Ki67 was similar between the various experimental conditions.
Potential wound healing effects of cultured nasal epithelial cell sheets. To analyze the wound healing effect, we performed a cell sheet grafting assay in vitro (Fig. 5A), in which a cell sheet adhered to and migrated on a collagen gel (Fig. 5B). The expansion rate at 6 days after grafting was significantly higher for sheets fabricated in 1.6 mM Ca 2+ and 10 µM Y-27632 (240 ± 26%, n = 6) or 3.6 mM Ca 2+ and 1 µM Y-27632 (301 ± 62%, n = 6) than for sheets fabricated in 1.6 mM Ca 2+ (81 ± 29%, n = 8, P < 0.0001; Fig. 5C). These findings imply that cell sheet culture in the presence of a ROCKi would increase the potential wound healing effects of nasal epithelial cell sheets. Colony forming assays (CFAs) using 2000 cells from each cell sheet were also performed to assess proliferation potential. Colonies were formed by cells obtained from sheets fabricated under all three culture conditions (Fig. S8A), but the colony forming efficiency was significantly higher for culture medium containing 1.6 mM Ca 2+ and 10 µM Y-27632 (3.6 ± 1.6%, n = 6, P < 0.01) or 3.6 mM Ca 2+ and 1 µM Y-27632 (1.9 ± 0.9%, n = 6, P < 0.05) than for culture medium containing 1.6 mM Ca 2+ (0.5 ± 0.4%, n = 6; Fig. S8B).

Immunohistological analyses of cell sheets after grafting onto a collagen gel. Although cell
sheets fabricated in 1.6 mM Ca 2+ (normal KCM) showed maintenance of adhesion in macroscopic images (Fig. 5B), the detachment of some cells from the collagen gel was observed in microscopic images (Fig. 5D). Furthermore, P-MLC was rarely detected in cell sheets fabricated in 1.6 mM Ca 2+ , and although CK8 was evident, Ki67 was poorly expressed (Fig. 5D). These results suggest that cell sheets produced using KCM containing 1.6 mM Ca 2+ underwent differentiation rather than proliferation after grafting onto collagen. By contrast, grafted cell sheets that had been fabricated in the presence of Y-27632 exhibited extensive expression of E-cadherin and P-MLC, slight expression of CK8 and notable expression of Ki67. These findings imply that cell sheets fabricated in the presence of a ROCKi underwent proliferation after grafting, which would be favorable for wound healing.

Discussion
The present study examined the use of Y-27632 (a ROCKi) and different concentrations of extracellular Ca 2+ to optimize both explant culture and cell sheet culture. Furthermore, evaluation of the expressions of P-MLC and PP-MLC under different experimental conditions provided insights into the role of phosphorylated MLC in cell proliferation and differentiation during culture (Fig. 6). Our findings have enabled us to improve the fabrication of human nasal epithelial cell sheets and produce constructs that do not collapse after harvesting and have high wound healing potential in vitro.
Nasal mucosal tissue is a thin pseudostratified columnar epithelium 31 . Although the dispase dissociation technique is often used in the primary culture of skin grafts or oral mucosal cell sheets 32,33 , it is not considered suitable for the cultivation of nasal mucosal epithelial cells 34 . As we agree with the findings of this latter report, we used primary explant culture as a cell expansion stage (Fig. 1A). Since low-Ca 2+ medium is widely used to expand epithelial cells 26 , we examined the use of KGM (0.1 mM Ca 2+ ) as a low-Ca 2+ medium for explant culture. However, the tissue adhesion rate was notably lower in medium containing 0.1 mM Ca 2+ than in medium containing 1.1 mM Ca 2+ (Fig. 1B). As the adhesion of fibroblasts requires an extracellular Ca 2+ concentration of at least 0.5 mM 26,35 , we suggest that increasing the level of Ca 2+ in the medium facilitated the initial adhesion of the explant culture. This proposal is supported by our histological analysis of the explant culture, which demonstrated a connective tissue layer beneath the epithelial layer as well as the occurrence of fibroblast adhesion before epithelial cell migration (Fig. 2B). Therefore, we believe that connective tissue is key to adhesion in explant culture and that Ca 2+ is important for the development of adhesion. Bivalent metal ions such as Mg 2+ and Mn 2+ are known promoters of cell adhesion via integrin 36 . Since our optimization of explant culture based on ROCK inhibition and Ca 2+ level allowed us to collect a sufficient number of cells for cell sheet fabrication, we did not examine the roles of other bivalent ions; thus, it is likely that there is further room for improvement of the explant culture technique. Epithelial mesenchymal transition (EMT) is understood to involve multiple steps of transition between epithelial and mesenchymal tissue, and these are now referred to as partial EMT 37,38 . In our experiments, CK8, a marker of differentiated epithelium, was not expressed in proliferating/migrating cells, whereas the epithelial marker E-cadherin and the mesenchymal marker vimentin were expressed in migrating cells (Fig. 2B). The above results suggest that partial EMT occurred in the explant culture.
Although some cell types have been successfully cultured in the absence of serum 39 , serums are still considered essential supplements for cell cultivation. Unexpectedly, nasal cells reached only 15% confluence in a 60-mm dish at day 13 when cultured in FBS-containing medium (normal KCM, Fig. 1C), whereas substantially higher confluence was achieved when human serum was used. Since improving the expansion of epithelial cells in medium containing γ-irradiated FBS would facilitate future preclinical and clinical research, we examined the effects of 10 μM Y-27632, a ROCKi that is known to enhance cell proliferation 17 . As expected, Y-27632 significantly promoted cell proliferation (Fig. 1C,D). Interestingly, proliferating epithelial cells mainly existed at the Scientific RepoRtS | (2020) 10:16853 | https://doi.org/10.1038/s41598-020-73817-3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ edge of the explant culture (Fig. 2B), indicating that cell behavior at the migrating edge plays an important role in cell expansion. When cultured in normal KCM (1.6 mM Ca 2+ ), cells in the migrating edge exhibited partial backward movements (Movie 1) similar to those reported in our previous study 40 , and P-MLC and stress fibers were detected at the cell-cell interfaces (Fig. 2D,E), implying that P-MLC and actin may operate to connect migrating cells to each other, as suggested by a prior investigation using keratinocytes 41 . By contrast, P-MLC was not strongly detected, and actin depolymerization occurred at the cell-cell interfaces when culture was carried out in medium containing 1.6 mM Ca 2+ and 10 µM Y-27632, implying weaker connections between migrating cells; this may have contributed to the enhancement of cell proliferation and some of the cells migrating individually (Fig. 2F). Based on these results, we suggest that P-MLC at the cell periphery prevents cell proliferation (Fig. 2D,G), consistent with a previous report that cell proliferation is induced by the mosaic loss of myosin and 50-75% of MLC 11 . The purpose of cell sheet culture is to connect cells to each other so that all the cells can be detached as an intact construct. Claudin-1 and ZO-2 were expressed in the upper layer of the cell sheet under all experimental conditions (Fig. S6), indicating the presence of tight junctions 42,43 . Although adherens junctions were observed in the basal layer by TEM when normal KCM (1.6 mM Ca 2+ ) was used (Fig. S6), the basal layer was found to contain many intercellular gaps (Fig. 4C). Therefore, we speculated that the addition of a ROCKi to promote continuous proliferation would prevent the formation of basal layer gaps. However, although we confirmed that Y-27632 downregulated P-MLC (Fig. 4A,B), the resulting cell sheets were structurally loose and still contained some defects (Figs. 3B, S4). Based on these results, we concluded that the prevention of intercellular defects in the basal layer required both cell proliferation and MLC activation, which would necessitate the recovery of MLC phosphorylation in the presence of the ROCKi. In cardiovascular smooth muscle, phosphorylation of MLC is also regulated by Ca 2+ through a different pathway to that recruited by ROCK 44 . In epithelia, non-muscle P-MLC enhances the accumulation of E-cadherin-related proteins 45 . Therefore, we examined whether increasing the level of Ca 2+ in Y-27632-containing medium (3.6 mM Ca 2+ and 1 µM Y-27632) would recover MLC phosphorylation (and thus activation) and promote cell-cell adhesion. When medium containing 3.6 mM Ca 2+ and 1 µM Y-27632 was used, P-MLC and E-cadherin were expressed at the cell-cell interfaces of all layers (Fig. 4A-C), and the cell sheet could be harvested without collapse and shrinkage (Fig. 3B). We propose that P-MLC participates in the shrinkage behavior of nasal mucosal cell sheets, which has also been observed in keratinocyte sheets 40,46 . These results suggest that increasing the Ca 2+ concentration in the culture medium recovered MLC phosphorylation and thereby promoted the maturation of adherens junctions (Figs. 3B, 4A-C). Overall, our findings demonstrate www.nature.com/scientificreports/ that the combination of a ROCKi with Ca 2+ induces MLC phosphorylation/activation in all layers of the cell sheet, thereby promoting cell-cell adhesion and facilitating the harvesting of an intact cell sheet that does not collapse. Finally, we used an in vitro cell sheet grafting assay and CFA to analyze the wound healing ability of the optimized cell sheet (Figs. 5, S8). We previously established an experimental model to evaluate grafted cell sheets in vitro and used this model to confirm the behavior of normal human epidermal keratinocyte cell sheets 40 . Therefore, we used this model system to analyze the behavior of cell sheets fabricated under three culture conditions (Fig. 5A). Cell sheets fabricated in KCM containing 1.6 mM Ca 2+ and 10 µM Y-27632 or KCM containing 3.6 mM Ca 2+ and 1 µM Y-27632 expanded nearly three-fold relative to their original size and approximately three-fold relative to the expansion of cell sheets made using medium containing 1.6 mM Ca 2+ (Fig. 5B,C). Ki67 was rarely detected in cell sheets before grafting, suggesting that proliferation had ceased because of confluence (Fig. 4C). However, cell sheets expressed Ki67 after grafting, indicating that they had proliferated on the collagen gel (Fig. 5D). The CFA results supported the findings of the cell sheet grafting assays (Fig. S8A, B). Although the wound healing potential in vitro was improved by the combination of a ROCKi with Ca 2+ , it should be emphasized that the environment in which the cell sheet was grafted was different to that in vivo. Nevertheless, since maintaining proliferative potential is important for effective therapy 47-49 , we consider our graft to have potential for use in the clinical setting.
There are two main limitations to this study. First, inhibitors of ROCK affect not only myosin regulation but also gene expression 50,51 , but we did not perform gene expression assays. It will be important to investigate the expressions of genes at each step of the cell culture process (original tissue, explant culture, cell sheet and grafted cell sheet) to better understand the changes that occur and how these are influenced by inhibition of ROCK or variations in Ca 2+ level, since this knowledge may facilitate further optimization of cell sheets suitable for grafting. Second, we did not examine the populations of various cell types in the cell sheet. Native nasal mucosal tissue contains different cell types, including fibroblasts, basal epithelial cells, goblet cells and ciliated cells, and these various cell types potentially influence the wound healing potential. Further work is needed to address these limitations.
Regenerative medicine using cultured cell grafts is an important research field in cell biology. Recent studies fabricating similar cell grafts have utilized multiple cell culture steps including cell expansion and cell differentiation 52,53 . The fabrication of a cell graft that is fully compatible with the transplant site will require an optimal balance of several agents in the culture medium. Here, we demonstrate that a balanced combination of a ROCKi and Ca 2+ can maintain MLC phosphorylation and optimize both explant culture and cell sheet culture using human nasal mucosal cells. Our study provides important new insights into the role of phosphorylated MLC in the behavior of cell clusters, and we anticipate that our findings will facilitate the fabrication of stable cell grafts for use in regenerative medicine.

Materials and methods
Surgical collection of human nasal mucosal tissue. All methods were performed in accordance with the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan. In addition, this study was approved by the Institutional Review Board of Jikei University, Tokyo, Japan. All volunteers, who were scheduled to undergo endoscopic sinus surgery, provided informed consent and were confirmed not to be infected with human immunodeficiency virus, syphilis, hepatitis B virus or hepatitis C virus. Nasal mucosal tissue was collected from the inferior turbinate. In some experiments, KCM was supplemented with 5% autologous human serum (KCM-HS) instead of FBS. Human blood was collected from volunteers scheduled to undergo endoscopic sinus surgery (each supplied 70 mL), and serum was extracted according to our previous paper 30 . explant culture. Nasal mucosal tissues underwent explant culture using a modification of our previously described method 30 . In short, sterilized mucosal tissues were cut into 1.5-mm cubes, placed in cell culture dishes (Primaria Dish, Corning, Corning, NY, USA) and incubated in 750 μL of medium at 37 °C with 5% CO 2 (Fig. 1A). A further 1 mL of medium was added to each dish 1 h later, and another 2 mL of medium was added the following day. The medium was changed on days 6 and 10. Following 13 days of cultivation, cells were collected by trypsin-EDTA treatment and preserved with freezing medium (STEM-CELLBANKER, Nippon Zenyaku Kogyo, Asakamachi, Fukushima, Japan). In order to track migration behavior, cell outgrowth was monitored for 8 h on day 7 of the explant culture process using differential interference contrast microscopy (CytoWatcher, Atto, Motoasakusa, Tokyo, Japan). www.nature.com/scientificreports/ cell sheet culture. Cells were thawed, seeded on temperature-responsive cell culture dishes (CellSeed, Ōme, Tokyo, Japan) at a density of 1.0 × 10 5 cells/cm 2 and cultured for 7 days at 37 °C (Fig. 3A). The medium was changed on days 3 and 5. After 7 days of culture, the cells were detached as a cell sheet by reducing the temperature to 20 °C for 40 min.
Immunofluorescence analysis of cultured cells. Nasal mucosal tissues or nasal mucosal cells were cultured on a slide chamber (Thermo Fisher Scientific) and fixed in 100% ethanol or 4% paraformaldehyde (Wako Pure Chemical Industries). Sections were exposed to blocking buffer (Nacalai Tesque, Nijo Karasuma, Nakagyo-ku, Kyoto, Japan) and then incubated with phosphate-buffered saline (PBS) containing primary antibody (Table S1) at 4 °C overnight. After washing with PBS, the samples were incubated with Alexa Fluor Plus 488-conjugated secondary antibody (Table S1) for 1 h at room temperature. The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific), and F-actin was stained by phalloidin (Thermo Fisher Scientific) at room temperature for 1 h.
Immunohistological analysis of nasal mucosal cell sheets. Immunohistology was performed according to methods described in our previous article 30 . Fabricated cell sheets were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries), dehydrated, embedded in paraffin, sectioned (4 μm) and mounted on glass slides. The slides were de-paraffinized, and antigen retrieval was performed by autoclaving with citrate buffer (Histo-VT One, Nacalai Tesque). Peroxidase blocking (Dako) and protein blocking (Nacalai Tesque) were carried out to prevent non-specific reactions. The slides were then incubated overnight at 4 °C in PBS containing primary antibody (Table S2) Western blot analysis. Western blotting was performed using a modification of the method described in our previous study 54 . An entire cell sheet was solubilized in 855 μL Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 45 μL 2-mercaptoethanol and 9 μL proteinase/phosphatase inhibitor (Cell Signaling Technology). Samples (5 μL) were loaded onto a polyacrylamide gel (Thermo Fisher Scientific), and electrophoresis was performed. After transfer to a nitrocellulose membrane using a gel transfer device (Thermo Fisher Scientific), the membrane was blocked with 5% skim milk (Cell Signaling Technology) in Tris-buffered saline/Tween 20 (TBS-T) for 1 h at room temperature. The membrane was incubated with primary antibodies (Table S3) diluted in 2.5% blocking buffer at 4 °C overnight. After being washed with TBS-T, the membrane was incubated with secondary antibody (Table S3) for 1 h at room temperature. The membrane was washed with TBS-T and treated with luminol (ECL Prime, GE Healthcare, Chicago, IL, USA) for 5 min to detect the HRP. Photographs were taken using a chemiluminescence imager (LAS 4000; GE Healthcare).
In vitro wound healing assay. Wound healing assays (Fig. 4C) were performed in vitro using a modification of a method described previously 40 . Briefly, a nasal cell sheet was grafted onto type I collagen gel in a 60-mm dish, and silicone that was covered by nylon mesh (Sansyo, Kandasakumacho, Tokyo, Japan) was used as a weight to promote adhesion of the cell sheet to the collagen gel. The weight was removed after incubation at 37 °C for 2-4 h. On the following day, 2 mL of KCM plus 1 μM Y-27632 was added to the culture dish. Adherent cell sheets were cultured for 6 days, with the medium changed on days 3 and 5.
Colony-forming assay (CFA). Colony-forming efficiency was calculated according to a previously reported method 55 . A CFA was used to measure the proliferative potential of cell sheets cultured under various experimental conditions. Harvested cell sheets were treated with trypsin-EDTA at 37 °C to obtain a suspension of dissociated cells. Two thousand cells were seeded in a 60-mm dish containing KCM and 1 μM Y-27632. The medium was changed on days 5 and 10. After 14 days, the colonies were fixed by 4% paraformaldehyde and stained with crystal violet solution (Merck, Darmstadt, Germany).
transmission electron microscopy (teM). TEM was carried out using a modification of a method reported previously 56 . Briefly, harvested cell sheets were fixed in 2.0% glutaraldehyde in 0.1 M phosphate buffer, treated with 1% osmium tetroxide in 0.1 M phosphate buffer, dehydrated in ethanol, immersed in absolute propylene oxide and embedded in Epok 812 (Okensyoji, Ginza, Tokyo, Japan). We confirmed the trimming regions using toluidine blue-stained sections. Ultrathin sections (approximately 60 nm) were prepared, mounted on grids, stained with uranyl acetate and lead citrate, and observed using TEM (H-7500, Hitachi, Marunouchi, Tokyo, Japan) at 80 kV.
Statistical analysis. Prism 7.0 (GraphPad, San Diego, CA, USA) was used for the statistical analysis. Data are presented as the mean ± standard error of the mean (SEM). Comparisons between two groups were made using Student's t-test. Comparisons between three groups were made using one-way analysis of variance and the Bonferroni or Tukey-Kramer multiple comparisons test as the post-hoc test. P-values < 0.05 were considered statistically significant.