Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice

Journal Pre-proof Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice Yuan Yan, Jie Jiang, Min Zhang, Yinghua Chen, Xueer Wang, Mianbo Huang, Lin Zhang PII:

S0014-4827(19)30489-6

DOI:

https://doi.org/10.1016/j.yexcr.2019.111627

Reference:

YEXCR 111627

To appear in:

Experimental Cell Research

Received Date: 20 July 2019 Revised Date:

29 August 2019

Accepted Date: 17 September 2019

Please cite this article as: Y. Yan, J. Jiang, M. Zhang, Y. Chen, X. Wang, M. Huang, L. Zhang, Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice, Experimental Cell Research (2019), doi: https://doi.org/10.1016/j.yexcr.2019.111627. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Effect of iPSCs-derived Keratinocytes on Healing of Full-Thickness Skin Wounds in mice Yuan Yana,b,1, Jie Jianga,1, Min Zhanga, Yinghua Chena, Xueer Wanga, Mianbo Huanga, Lin Zhanga,b,*

*

Corresponding authors. E-mail address: [email protected]

1

These authors contributed equally to the work

a

Department of Histology and Embryology, College of Basic Medicine, Southern

Medical University, Guangzhou, China b

Key Laboratory of Tissue Construction and Detection of Guangdong Province,

Guangzhou, China Full list of author information is available at the end of the article

Abstract Induced pluripotent stem cells (iPSCs) provide new approaches for the management of severe skin wound healing due to their infinite proliferative capacity, pluripotency into multiple lineages, and important ethical acceptability. In this study, we aimed to differentiate iPSCs into keratinocytes and to observe the therapeutic effects of transplanted iPSCs-derived keratinocytes on wound healing in mice. Here, mouse iPSCs had been successfully differentiated into keratinocytes. Next, iPSCs-derived keratinocytes labeled by CSFE were injected directly into the full-thickness skin wound.

Hematoxylin

&

Eosin,

Masson’s

trichrome,

EdU

staining

and

immunohistochemical staining were performed to assess the effects of iPSCs-derived keratinocytes on wound healing. Our results showed that transplantation of iPSCs-derived keratinocytes into full-thickness skin wound site accelerated re-epithelialization and reduced scar formation. In addition, we found that conditioned medium of iPSCs-derived keratinocytes reduced the expression of α-SMA and COL1 and increased the expression of MMP1 in fibroblasts in vitro. Further mechanism studies show the TNF-α-induced activation of NF-κB is involved in the effect of conditioned medium of iPSCs-derived keratinocytes on fibroblasts. In conclusion, this study has shown that iPSCs-derived keratinocytes decrease the healing time by increasing the epithelization rate and reduce scarring, suggesting a possible new treatment for skin wound healing.

Keywords: iPSCs-derived keratinocytes, Epithelialization, Scar, Collagen, α-SMA, MMP1, TNF-α

Introduction Wound healing is a multifaceted process consisting of four successive and overlapping programming stages, which are hemostasis, inflammation, proliferation and reconstitution [1]. Rapid epithelialization is a mandatory measure to restore skin barrier function and promote healing. Tissue-engineered skin have been used to treat various skin injuries [2-6]. However, these methods have some disadvantages, such as time constraints, limited number of primary cells, and immune response. In the past decade, the role of stem cells in skin wound healing and tissue regeneration has become a hot topic. Many preclinical and clinical trials are testing the ability of stem cells from a variety of sources to promote wound healing and tissue regeneration [7-9]. Traditionally, stem cells can be divided into two categories: embryonic stem cells and adult stem cells. Embryonic stem cells are pluripotent, but their applications are complicated due to the ethical issues in relation to using embryos. Adult stem cells, typically found in mature organs or tissues, might have pluripotency, but most are limited in lineage. In 2006, Shinya Yamanaka transferred a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into differentiated somatic cells to obtain a cell type similar to embryonic stem cells, called induced pluripotency stem cells (iPS cells, iPSCs) [10,11]. This discovery is a major breakthrough in stem cell research and provides new potential for the development of cellular therapies for human diseases. Cells in the skin, blood, or other parts of the body can be reprogrammed into iPS cells, which in turn can differentiate into hepatocytes, nerve cells, and any other cell type that could be used to treatment. This personalized treatment avoids immune rejection and avoids the ethical issues caused by embryonic cell therapy. In the past decade, great progress has been made in how to differentiate iPSCs into keratinocytes [12-16]. In most of these methods, retinoic acid (RA) is used to induce the differentiation of iPSCs into ectoderm [17] and bone morphogenetic protein-4 (BMP4) to block the the commitment to nerve fate [18]. iPSCs-derived keratinocytes have great potential in regenerative medicine and can be used as an infinite cell source

for rapid tissue replacement in the treatment of skin diseases. Therefore, further research is required to characterize the role of this iPSCs-derived keratinocytes transplantation in wound care. In this report, initially we induced mouse iPSCs into keratinocytes. Furthermore, we evaluated the therapeutic effects of transplantation of iPSCs-derived keratinocytes on skin wound healing and analyzed the underlying mechanisms.

Methods Mouse iPSCs culture and differentiation The mouse iPS cell line OSKM-1 was provided by Stem Cell Bank, Chinese Academy of Sciences. iPSCs were directly adapted to serum free, feeder-free expansion medium by dissociation cells with Accutase (Millipore) and passaging them into 0.1% gelatin coated plate containing ESCRO Complete Plus Medium (Millipore). Replace with fresh ESGRO Complete Plus Media every other day. iPSCs were induced to keratinocytes as described previously [14]. Briefly, 1000 iPSCs in 30 µl aliquots of unconditioned medium (UM) composed of knockout™ Dulbecco's modified Eagle's medium, 20% knockout™ serum replacement, 1% non-essential amino acid stock, 1 mM L-glutamine, and 1% Penicillin–Streptomycin (all from Gibco), were cultured in hanging drop for 3 days to form EB. On the third day, the EBs were transferred to Col IV-coated culture plates and cultured with differentiation medium, which contained UM with RA (1 mM, Sigma) and BMP4 (25 ng/ml, R&D Systems Inc.). On day 5, switch the medium to the defined keratinocyte serum free medium (DKSFM; Life Technologies) with RA (1 mM, Sigma) and BMP4 (25 ng/ml, R&D Systems Inc.). On day 9, the medium was changed to the DKSFM without RA or BMP4.

Conditioned medium of iPSCs-derived keratinocytes When the third to fifth passages of iPSCs-derived keratinocytes reached 70–80 % confluence, the medium was changed to DKSFM. After 48 hours, the supernatant of

iPSCs-derived keratinocytes was collected, centrifuged at 1000 × g for 5 minutes. and then filtered with a 0.22 µm syringe filter. The supernatant was used as the conditioned medium of iPSCs-derived keratinocytes.

Fibroblasts culture The dermal layers of the skin of C57BL/6 mice were cut into small pieces of less than 0.5 mm. Then the tissue fragments were distributed into plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% Penicillin–Streptomycin (all from Gibco). The medium was changed every other day. Fibroblasts from the second passages were used in the experiment.

Immunofluorescence Assay and confocal microscopy Cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature before permeabilizing with 0.5% Triton X-100 for 10 minutes. After that cells were incubated overnight at 4 ℃ with the primary antibody against NF-κB p65 (cell signaling technology), and then incubated by Alexa Fluor 647-conjugated donkey anti-rabbit second antibody (Abcam) at room temperature for 2 hours. Nucleis were stained with 4,6-diamidino-2-phenylindole (DAPI, 500 ng/ml ) for 10 minutes. The positive signals were analyzed by confocal microscope (Zeiss, Germany).

Labeling of iPSCs-derived keratinocytes The

cells

were

incubated

with

5

µΜ 5(6)-Carboxyfluorescein

diacetate

N-succinimidyl ester (CFSE, Sigma-Aldrich) at 37 ℃ for 30 minutes. After CSFE removal, fresh culture medium was added and the cells were further incubated at 37 ℃ for 30 minutes.

Enzyme-linked immunosorbent assay (ELISA) analysis The concentration of Tumor Necrosis Factor-α (TNF-α) in the in the conditioned medium of iPSCs-derived keratinocytes was measured by ELISA kits (Abcam) according to the manufacturer’s instructions.

Wound Healing Assay C57BL/6 mice (8-10 weeks old) were purchased from the Laboratory Animal Centre of Southern Medical University. After shaving the mice, an full-thickness excisional wounds (1 cm × 1 cm) was created on the middle dorsal skin of every mouse. Immediately after the surgery on day 0, 100 µl PBS (vehicle control) or 100 µl iPSCs-derived keratinocytes suspension (1×106 cells) were injected intradermally around wound margins. Macroscopic monitoring of wound healing was performed by digital photography at 0, 3, 5 and 7 days. The wound areas were calculated using the following formula: Relative wound area (%) = [Open area on the indicated time point/Original wound area] × 100. The mice were sacrificed at 3, 5, 7 and 14 days and the skin covering the wounds and surrounding areas were harvested.

Histological assessment The skin samples were fixed in 4 % paraformaldehyde for 48 hours, embedded in paraffin, and then cut into 5 µm thick tissue sections. The sections were used for hematoxylin and eosin (H&E) staining (in accordance with standard procedures) or Masson’s trichrome staining (MaiXin-Bio, China) according to the manufacturer’s manual. The width of wounds and the length of migration tongue were measured in H&E-stained sections at 3, 5 and 7 days. Re-epithelialization was calculated according to the following formula: [distance of the minor axis covered by the epithelium]/ [distance of the minor axis between the edges of the original wound] × 100. The scar tissues from the epidermal-dermal junction to the granuloma and from the edge of the wound were also determined on H&E-stained sections.

5-Ethynyl-2′-deoxyuridine (EdU) incorporation and staining Mice were injected intraperitoneally with 5mg/kg EdU (RiBoBio, China) for 6 hours. Then the mice were sacrificed and paraffin section of skin were prepared. EdU staining was performed using the Cell-LightTM Apollo643 Stain Kit (RiBoBio, China)

according to the manufacturer's instructions.

Immunohistochemical Assay For immunohistochemical staining, the tissue sections were dewaxed, and then the endogenous peroxidase activity of the sections was inactivated by incubation with 3% 3% H2O2 for 20 minutes, and the antigen passage was restored by heating in citrate buffer (pH 6.0). The sections were then blocked with goat serum for 30 min and incubated overnight at 4 ℃ with primary antibodies against COL1 (Abcam) or α-SMA (Abcam). On the second day, the sections were treated with biotinylated secondary antibodies (Zhong Shan Golden Bridge Biotechnology) for 30 minutes at room temperature. Peroxidase activity was detected by diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin. The average optical density values of COL1 and α -SMA expression were analyzed by Image Pro Plus. Five randomly selected fields of view were examined and used to estimate the average optical density value per unit area.

Real time quantitative PCR assay Fibroblasts (1 × 105) were plated in 24℃well plates and treated with vehicle control (DKSFM) and conditioned medium of iPSCs-derived keratinocytes, as well as conditioned medium of iPSCs-derived keratinocytes with mouse TNF-α neutralizing antibody (1µg/ml, Cell Signaling Technology) and Pyrrolidinedithiocarbamate (PDTC, 50 µM, Sigma-Aldrich) for 48 hours. Total RNA extraction from fibroblasts was performed with Trizol™ Reagent (Invitrogen), according to the manufacturer's instructions. Then cDNA was amplified with a HiScript II Q RT SuperMix for qPCR (Vazyme) on an iCycler System (Bio-Rad) and AceQ Universal SYBR qPCR Master Mix (Vazyme) for relative quantification of the indicated genes. The Ct values were normalized for the housekeeping gene GAPDH. The sequences of the primers were as follows:

mouse

downstream,

COL1 5`-

upstream,

5`-GCTCCTCTTAGGGGCCAC-T-3`,

ATTGGGGACCCTTAGGC-CAT-3`;

mouse

and

Matrix

metalloproteinase-1 (MMP-1) upstream, 5`-AACTACATTTAGGGGAGAGGTGT-3`,

and

downstream,

upstream,

5`-GCAGCGTCAAGTTTAACTGGAA-3`;

5`-CCCAGACATCAGGGAGTAATGG-3`,

and

mouse

α-SMA

downstream,

5`-TCTATCGGATAC-TTCAGCGTCA -3`.

Statistical Analysis All data were reported as mean ± SEM of at least three independent experiments (n≥3). The Student’s t test was used to determine the significance of differences in comparisons. Values of p < 0.05 were considered to be statistically significant.

Results Identification of keratinocytes derived from iPSCs Embryoid bodies, which developed using the hanging drop method, have round shape (Figure 1Aa). On day 3, embryoid bodies were transferred onto the collagen IV-coated plates and cultured with RA and BMP4. By day 7, some of the cells shape appeared epithelioid (Figure 1Ab), and many cells exhibited an epithelial-like phenotype by day14 (Figure 1Ac). Differentiated cells were passaged on new collagen IV-coated plates to provide further proliferation (Figure 1Ad). To identify the characteristics of differentiated cells, we observed cytokeratin 14 (K14), an early keratinocyte differentiation marker, in differentiated cells by immunohistochemical analysis (Figure 1B). In addition, this K14-expressing keratinocyte precursor was capable of terminal differentiation markers such as loricrin and involucrin in DKSFM, supplemented with 1.5 mmol/L calcium chloride for one week (Figure 1C). Taken together, these results demonstrate that we successfully differentiated iPSCs cells into keratinocytes.

iPSCs-derived keratinocytes accelerate wound healing due to increased re-epithelialization To investigate the effect of iPSCs-derived keratinocytes on skin wound healing process, we performed full-thickness excisional wounds on the back of C57BL/6 mice

and observed the healing process. The wound areas of mice were measured at 0, 3, 5 and 7 d post-wounding. Our data showed that the iPSCs-derived keratinocytes group exhibited significantly smaller wound areas than did the PBS group at 3, 5 and 7 days post-wounding, indicating that the iPSCs-derived keratinocytes group accelerated wound closure (Figure 2A). To investigate the mechanism by which iPSCs-derived keratinocyte accelerated wound healing, we performed skin biopsies on these wound sites on days 3, 5, and 7 after injury (Figure 2B). Re-epithelialization is widely accepted as one of the main processes of wound healing to ensure successful repair [19]. As shown in Figure 2C, H&E staining of the wound site indicated a significant increase in the degree of re-epithelialization of the wound after 3, 5 and 7 days in the iPSCs-derived keratinocyte group compared to the PBS group. The lengths of migrating epithelial tongues were also correspondingly increased on days 3, 5 and 7 after injury in iPSCs-derived keratinocytes group (Figure 2D). Compared with mice in the PBS group, EdU-positive proliferating keratinocytes were increased in iPSCs-derived keratinocytes group per area of migrating epidermis and epithelial tongues in wound sites on days 5 after the injury (Figure 3A). To determine the presence of CFSE-labeled iPSCs-derived keratinocytes, tissue sections from day 14 were examined by fluorescent microscope. In PBS group, fluorescence could not be detected in any section. In iPSCs-derived keratinocytes groups, CFSE-labeled iPSCs-derived keratinocytes appeared as green fluorescent cells in regenerated epidermis after two weeks (Figure 3B). Together these data suggest that iPSCs-derived keratinocytes accelerates wound healing by enhanced re-epithelialization.

iPSCs-derived keratinocytes suppress scar formation Fibrosis is the formation of scar tissue formed by nascent collagen and other extracellular matrix (ECM) components during the proliferative phase of wound healing [20]. Smaller fibrotic areas indicate histological evidence of less scar formation. Densely packed collagen, which indicated fibrosis, was identified by H&E

and Masson’s Trichrome staining. In Figure 4A, the fibrotic area is divided by a black frame and calculated. Compared with the PBS group, scar areas were significant decrease in iPSCs-derived keratinocytes group on day 14 (Figure 4A). Collagen deposition and increased amounts of α-SMA, a mark of myofibroblast differentiation, are pathological features of fibrosis tissues [21-24]. By immunohistochemistry staining, we also detected expressions of COL1 and α-SMA were significant lower in wounds from iPSCs-derived keratinocytes group compared with PBS group (Figure 4B, C). Collectively, the above results indicate that iPSCs-derived keratinocytes suppressed scar formation

iPSCs-derived keratinocytes exert anti-fibrotic effects by affecting the expression of fibrosis-associated factors It is known that keratinocytes can interact with dermis and affect the activity of fibroblasts in dermis during wound healing through secretion of several growth factors [25-27]. Therefore, in order to detect whether iPSCs-derived keratinocytes affect the expression of fibrosis-associated factors in fibroblasts through the paracrine pathway, we applied conditioned medium of iPSCs-derived keratinocytes to fibroblasts. In vehicle control group, the cell bodies were large and the cells were spindle-shaped or star-shaped, while the cell bodies became smaller and the cells were spindle-shaped after treatment with conditioned medium of iPSCs-derived keratinocytes (Figure 5A). High levels of COL1 and α-SMA expression are thought to be fibrotic features of fibroblasts. Notably, our in vitro studies showed that conditioned medium of iPSCs-derived keratinocytes reduced the expression of COL1 and α-SMA (Fig. 5b), suggesting that iPSCs-derived keratinocytes could reduce the deposition of COL1 and α-SMA by paracrine. One of the key factors regulating the total amount of collagen deposited by fibroblasts during scar formation is MMP-1, which is capable of promoting collagen degradation in ECM. Our further findings showed a significant increase in mRNA expression of MMP1 in fibroblasts after treatment with conditioned medium of iPSCs-derived keratinocytes compared to control cells (Figure 5B). The results indicate that iPSCs-derived keratinocytes

exhibite anti-fibrotic effect by decreasing the expression of COL1 and α-SMA and increasing the expression of MMP1 in mouse fibroblast.

TNF-α-induced activation of NF-κB is involved in the anti-fibrotic effects of iPSCs-derived keratinocytes TNF-α secreted by keratinocytes and inflammatory cell, has been shown to be a strong anti-fibrotic cytokine during wound healing [28-30]. ELISA showed that TNF-α existed in conditioned medium of iPSCs-derived keratinocytes and reached the highest level of 301 pg/ml in 48 hours (Figure 6A). Therefore, we speculated that TNF-α may be involved in the anti-fibrotic function of iPSCs-derived keratinocytes. To further explore the role of TNF-α in the conditioned medium of iPSCs-derived keratinocytes for inhibition of fibroblast, the active TNF-α was blocked using TNF-α neutralizing antibody, prior to its being given to fibroblast. As show as Figure 6B, fibroblasts cultured with TNF-α neutralizing antibody treated conditioned medium of iPSCs-derived keratinocytes expressed more COL1 and α-SMA and less MMP1 than those cultured with only conditioned medium of iPSCs-derived keratinocytes. Nuclear factor-kappa B (NF-kappa B) is a ubiquitous transcription factor that can be rapidly activated by TNF-α. It controls the expression of various genes, including MMP1 [31,32]. We investigated the NF-κB activity in fibroblasts treated with conditioned medium of iPSCs-derived keratinocytes. The results showed that nuclear protein level of p65, which is subunit of NF-κB [29], was increased by stimulation of conditioned medium of iPSCs-derived keratinocytes and the increased p65 protein in nucleus was largely decreased by TNF-α neutralizing antibody treated conditioned medium of iPSCs-derived keratinocytes (Figure 6C). Furthermore, the NF℃кB specific inhibitor PDTC reversed the reduced expression of α-SMA and COL1 and enhanced expression of MMP1 (Figure 6D), which were mediated by conditioned medium of iPSCs-derived keratinocytes. These results indicate that TNF-α-induced activation of NF-κB participates in the effect of conditioned medium of iPSCs-derived keratinocytes on fibroblasts. Taken together, these data suggest that TNF-α secreted by iPSCs-derived

keratinocytes participates in the anti-fibrotic effect of iPSCs-derived keratinocytes.

Discussion In this study, we investigated whether iPSCs-derived keratinocytes can play a therapeutic role in a cutaneous wound and explored the underlying mechanisms involved. Our findings demonstrated that iPSCs-derived keratinocytes accelerated wound healing with increased re-epithelialization. In addition, we identified that iPSCs-derived keratinocytes could exert an anti-fibrotic ability by secreting TNF-α. These observations suggest that the iPSCs-derived keratinocytes can be served as a new therapeutic strategy. Epithelialization is an essential component of wound healing and serves as a decisive parameter of its success. It is important in the treatment of a skin injury to focus on rapid re-epithelialization. Treatment of wound healing with cultured epithelial keratinocytes has been well established in clinical practice [33-36]. Therefore, a large number of highly proliferative keratinocytes are required to support tissue regeneration. iPSCs will be an important source of keratinocytes. In our study, we used the previously published protocol for the differentiation of mouse iPSCs into keratinocytes. First, the embryoid bodies developed from iPSCs using hanging drop method. Embryoid bodies were then treated with RA to induce iPSCs to differentiate into ectodermal fate, BMP4 was used to block the commitment to neural fate. The resulting iPSCs-derived keratinocyte-like cells were positive for K14, which was a keratin marker confirming commitment of the ectoderm to a keratinocyte fate, and were able to express terminal differentiation markers under high Ca2+ medium. In this study, iPSCs-derived keratinocytes were directly injected into the wounds. The present work demonstrated better and faster healing of iPSCs-derived keratinocytes -treated wound compared with the control group. We observed significantly accelerated wound re-epithelialization in iPSCs-derived keratinocytes groups. These findings could be attributed to the injected iPSCs-derived keratinocytes. This explanation was further supported by detection of red fluorescent CFSE-labeled iPSCs-derived keratinocytes within the regenerated epidermal of the treated groups.

Furthermore, Our data revealed an increased number of EdU-positive proliferating keratinocytes in the iPSCs-derived keratinocytes group compared with the control group. Studies have shown that activated keratinocytes are the main source of cytokines in the epidermis. A number of growth factors secreted by keratinocytes, including transforming growth factor alpha (TGF-α), keratinocyte growth factor, epidermal growth factor (EGF), and heparin-binding EGF, are known to stimulate the motility and proliferation of keratinocytes in injured epidermis [37,38]. Therefore, the accelerated re-epithelialization may be partly due to the growth stimulator secreted by the transplanted keratinocyte. Taken together, iPSCs-derived keratinocytes may hold great promise for treating wound healing through autocrine or paracrine e℃ects and direct involvement in the formation of new epidermis. Collagen deposition and increased α-SMA expression in the dermis are thought to be the most common features of scar tissue. By means of immunostaining, the weaker protein expression of α-SMA and COL1 in wounds treated by iPSCs-derived keratinocytes suggested a potential of iPSCs-derived keratinocytes for anti-scar. Increasing evidences indicate that the epidermal-dermal interaction during wound healing play a key role in controlling the expression of ECM, which often leads to fibrotic disorders [25-27]. With little direct cell-to-cell, fibroblast-keratinocyte communication

is

mainly

regulated

by

releasable

factors

acting

in

an

autocrine/paracrine loop. In our study, conditioned medium of iPSCs-derived keratinocytes has showed the effect of inhibiting the expression of α-SMA and COL1. The expression of MMP-1, well known as collagenase, was increased following treatment with conditioned medium of iPSCs-derived keratinocytes. Collectively, our data reveal that iPSCs-derived keratinocytes could prevent scar formation in mouse skin through anti-fibrotic secretory factors. TNF-α is a cytokine that mediates multi-directional inflammatory response and immune regulation and has a wide range of biological activities. TNF-α is produced by mononuclear macrophages and activated T lymphocytes, and can also be produced by keratinocytes. TNF-α has been shown to inhibit several cell responses induced by TGF-β1, including the differentiation of myofibroblasts and the production of key

molecules involved in tissue repair, such as fibronectin, type I collagen, and periostin [39-41]. TNF-α also induces MMP1 expression in human lung fibroblasts and rat skin fibroblasts [42, 43]. In our study, we found that TNF-α was secreted by iPSCs-derived keratinocytes. This prompts us to conclude that TNF-α participate in anti-fibrotic effect of iPSCs-derived keratinocytes. Results of our study showed that treatment with TNF-α neutralizing antibody in conditioned medium of iPSCs-derived keratinocytes partially reversed the inhibition of COL1 and α-SMA expressions and enhancement of MMP1 expression in fibroblast. Therefore, these results provide evidences for our hypothesis that anti-fibrotic effects of iPSCs-derived keratinocytes may be related to the secretory of TNF-α. TNF-α rapidly activates many transcription factors, including NF-κB, a gene regulator involved in inflammation and immune responses [44, 45]. Expression of TNF-a and active NF-κB is elevated in proliferative inflammatory conditions and correlate with increased levels of matrix metalloproteinases [46, 47]. NF-κB is also involved in reduction of extracellular matrix deposition to exhibit an anti-fibrosis effect in many tissues [48-50]. In our study, NF-κB has also been activated by conditioned medium of iPSCs-derived keratinocytes, and this activation could be inhibited by treatment with TNF-α neutralizing antibody. In addition, PDTC (NF℃кB specific inhibitor) reversed the downregulation COL1 and α-SMA and upregulation of MMP1, as did TNF-α neutralizing antibody. Our results imply that anti-fibrotic effects of iPSCs-derived keratinocytes is mediated by TNF-α-mediated NF-κB activation. Specific mechanisms involved in the factors and the pathways should be further explored.

Conclusion In conclusion, our findings suggest that keratinocytes derived from iPSCs are a promising therapeutic agent for skin wound. Due to the tumorigenicity of stem cells, further researches is needed to confirm the safety and efficacy of this therapeutic strategy.

Abbreviations iPSCs: induced pluripotent stem cells; RA: retinoic acid; BMP4: bone morphogenetic protein-4; UM: unconditioned medium; DKSFM: defined keratinocyte serum free medium; DMEM: Dulbecco’s modified Eagle’s medium; PBS: phosphate-buffered saline;

DAPI:

4,6-diamidino-2-phenylindole;

CFSE:

5(6)-Carboxyfluorescein

diacetate N-succinimidyl ester; ELISA: enzyme-linked immunosorbent assay (ELISA)TNF-α: tumor necrosis factor-α; H&E: hematoxylin and eosin; EdU: 5-Ethynyl-2′-deoxyuridine; COL1: collagen type I; α-SMA: alpha smooth muscle actin; DAB: diaminobenzidine; PDTC: pyrrolidinedithiocarbamate; MMP-1: matrix metalloproteinase-1; K14: cytokeratin-14; ECM: extracellular matrix; NF-κB: nuclear factor-κB.

Declarations Ethics approval and consent to participate The Bioethics Committee of Southern Medical University approved all animal procedures, which were in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests The authors declare that they have no competing interests.

Consent for publication Not applicable.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (No: 81501677, No: 81772095, No: 81872514) and Natural Science Foundation of Guangdong Province, China (No: 2016A030313575, No: 2014A030312013).

Authors’ contributions Y Y and J J performed the experimental work, Y Y wrote the paper, M Z, YH C, XE W MB H and D D performed the data collection, L Z designed the study. All authors read and approved the final manuscript.

Funding This work was supported by National Natural Science Foundation of China (No: 81501677, No: 81772095, No: 81872514) and Natural Science Foundation of Guangdong Province, China (No: 2016A030313575, No: 2014A030312013).

Author details 1

Department of Histology and Embryology, College of Basic Medicine, Southern

Medical University, Guangzhou, China. 2Key Laboratory of Tissue Construction and Detection of Guangdong Province, Guangzhou, China.

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Figures Figure 1 Characterization of keratinocytes differentiated from mouse iPSCs. (A) Light microscopy images of morphological changes occurring during iPSCs differentiation into keratinocytes. (a) EBs in suspension culture on day 2. (b) EB outgrown on a Col IV-coated plate on day 7. (c) EB outgrown on a Col IV-coated plate on day 14. (d) iPSCs-derived keratinocytes on day 25 of differentiation. Scale bars = 100 µm. (B) Representative photomicrographs of immunohistochemical staining for K14 (red) in iPSCs-derived keratinocytes. Scale bars = 10 µm. (C) Representative photomicrographs of immunohistochemical staining for involucrin (red) and loricrin (red) in iPSCs-derived keratinocytes culture in DKSFM supplemented with 1.5 mmol/L of calcium chloride for one week. Scale bars = 100 µm. Blue staining is DAPI, which labels the nuclei.

Figure 2 iPSCs-derived keratinocytes accelerated wound healing in mice. (A) Representative macroscopic images of wounds treated with PBS and iPSCs-derived keratinocytes (miPSCs-KCs) on days 0, 3, 5 and 7 after wounding (left panel). Quantitative analysis of wound area per group on days 3, 5 and 7 after injury, expressed as the percentage of the initial wound size at day 0 (right panel). Values are

mean ± SEM (n = 3), *P

0.05. (B) Representative photomicrographs of

H&E-stained wounds treated with PBS and iPSCs-derived keratinocytes on days 3, 5 and 7 after injury. Black arrows represent the dermal border; green arrows represent the epidermal margin. Scale bar = 500 µm. (C) Time-course of changes of the re-epithelialization ration of wounds treated with PBS and iPSCs-derived keratinocytes on days 3, 5 and 7 after injury. The re-epithelialization was calculated as described in Materials and Methods. (D) Time-course of changes of the length of migration tongues of wounds treated with PBS and iPSCs-derived keratinocytes on days 3, 5 and 7 after injury. s. Values are mean ± SEM (n = 3), *P

0.05

Figure 3 iPSCs-derived keratinocytes promoted the proliferation of keratinocytes in the wound site and integrated into the regenerated epidermis. (A) Representative photomicrographs of immunohistochemical staining for EdU (red) of wounds treated with PBS and iPSCs-derived keratinocytes (miPSCs-KCs) on day 5 after wounding (left panel). Scale bars = 100 µm. EdU-positive cells were counted in the transitional epidermis and the epithelial tongues of wound sites treated with PBS and iPSCs-derived keratinocytes and were related to the area of the same part of the epidermis (right panel). Statistics regarding the number of EdU-positive cells were obtained using five randomly selected fields of view for each group. Values are mean ± SEM (n = 3), *P

0.05. (B) CFSE-labeled cells (green) in the wound wounds

treated with PBS and iPSCs-derived keratinocytes were observed under fluorescent microscope after wounding. Scale bar = 100 µm. Blue staining is DAPI, which labels the nuclei.

Figure 4 Anti-fibrotic effect of iPSCs-derived keratinocytes on wound healing. (A) Representative photomicrographs of H&E-stained and Masson’s trichrome-stained wounds treated with PBS and iPSCs-derived keratinocytes on day 14 after wounding. In the H&E images, the black boxes approximate fibrotic area (left panel). Scale bar = 100 µm. Fibrosis areas of wounds treated with PBS or iPSCs-derived keratinocyte

(right panel). Values are mean ± SEM (n = 3), *P

0.05. (B) Representative

photomicrographs of immunohistochemical staining for COL1 of wounds treated with PBS and iPSCs-derived keratinocytes on day 14 after wounding (left panel). Scale bar = 100 µm. The areas stained with COL1 were determined by planimetric image analysis using ImageJ software (right panel). Values are mean ± SEM (n = 3), *P 0.05. (C) Representative photomicrographs of immunohistochemical staining for α-SMA of wounds treated with PBS and iPSCs-derived keratinocytes on days 5 and 7 after injury (left panel). The areas stained with α-SMA were determined by planimetric image analysis using ImageJ software (right panel). Scale bar = 50 µm. Values are mean ± SEM (n = 3), *P

0.05.

Figure 5 iPSCs-derived keratinocytes decreased expression of COL1 and α-SMA and increased expression of MMP1 in fibroblasts through paracrine effect. (A) Representative light microscopy images of fibroblasts cultured with vehicle control, conditioned medium of iPSCs-derived keratinocytes (miPSCs-KCs-CM). Scale bar = 100 µm. (B) Relative COL1, α-SMA and MMP1 mRNA expressions of fibroblasts cultured with vehicle control or conditioned medium of iPSCs-derived keratinocytes. Values are mean ± SEM (n = 3), *P

0.05.

Figure 6 iPSCs-derived keratinocytes secreted TNF-α to decrease expression of COL1 and α-SMA and increase expression of MMP1 in fibroblasts. (A) The secretion of TNF-α in the conditioned medium of iPSCs-derived keratinocytes (miPSCs-KCs-CM) measured by ELISA. Values are mean ± SEM (n = 3), *P

0.05.

(B) Relative COL1, α-SMA and MMP1 mRNA expressions of fibroblasts cultured with control, conditioned medium of iPSCs-derived keratinocytes or conditioned medium of iPSCs-derived keratinocytes with TNF-α neutralizing antibody. Values are mean ± SEM (n = 3), *P

0.05. (C) Representative confocal IF microscopy images of

the NF-κB p65 (red), subunit of the NF-κB complex, subcellular distribution in

fibroblasts cultured with control, conditioned medium of iPSCs-derived keratinocytes or conditioned medium of iPSCs-derived keratinocytes with TNF-α neutralizing antibody (left panel). Blue staining is DAPI, which labels the nuclei. Scale bar = 10 µm. Mean nuclear vs. cytoplasm p65 fluorescence intensity ratio in more than 200 cells scored in random fields (right panel). Values are mean ± SEM (n = 3), *P

0.05.

(D) Relative COL1, α-SMA and MMP1 mRNA expressions of fibroblasts cultured with control, conditioned medium of iPSCs-derived keratinocytes or conditioned medium of iPSCs-derived keratinocytes with PDTC. Values are mean ± SEM (n = 3), *P

0.05.