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Hsp90α promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice Ruijun Wu a, 1, Dan Du a, 1, Yunyao Bo a, Min Zhang a, Lin Zhang a, b, **, Yuan Yan a, b, * a b
Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China Key Laboratory of Tissue Construction and Detection of Guangdong Province, Guangzhou, 510515, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2019 Accepted 27 September 2019 Available online xxx
Because of the advantages of induced pluripotent stem cells (iPSCs), iPSCs-derived keratinocyte hold great clinical and research potential in wound repair. Similar to other cell transplantation therapies, the migration ability of iPSCs-derived keratinocyte transplanted into skin is critical to the therapeutic effect. Hsp90a had a positive effect on migration of keratinocytes. Therefore, the aim of this study was to investigate the effects of Hsp90a on transplanted iPSCs-derived keratinocyte in a skin model of deep second degree burns. First, keratinocytes were differentiated from iPSCs by treating with RA and BMP4. Next, we explained the effect Hsp90a on iPSCs-derived keratinocyte in vitro. We found that hsp90a promoted cell migration of iPSCs-derived keratinocyte. Furthermore, activation of AKT was required for Hsp90a-induced iPSCs-derived keratinocyte migration. Then PBS, Hsp90a, iPSCs-derived keratinocyte, and iPSCs-derived keratinocyte plus Hsp90a were applied to the wound bed of deep second degree burns. Wound healing was assessed by gross evaluation and hematoxylin and eosin staining. Our results shown that wound treated with iPSCs-derived keratinocyte plus Hsp90a significantly accelerates the rate of wound healing closure than other groups. In addition, the number of CFSE-labeled iPSCs-derived keratinocyte in regenerated epidermis was increased in iPSCs-derived keratinocyte plus Hsp90a group. In summary, these findings represent that combined administration of iPSCs-derived keratinocyte and Hsp90a may be a promising therapeutic strategy for wound healing. © 2019 Elsevier Inc. All rights reserved.
Keywords: iPSCs Hsp90a Keratinocyte Deep second degree burn Wound healing
1. Introduction Skin wound healing is a complex process involving hemostasis, inflammation, proliferation and remodeling [1]. The goal of normal skin wound healing is to quickly regenerate the natural protective structure of the skin to reduce the risk of infection and provide functional tissue. This led to the discovery of more advanced treatment options, such as gene therapy [2], growth factor therapy [3], platelet-rich plasma therapy [4], stem cell therapy [5], and tissue engineering [6]. Among these methods, stem cell-based therapy is an attractive method for treating acute and chronic wounds. Nowadays, the field of regenerative medicine mainly focuses on stem cells, which can self-renew and differentiate into
* Corresponding author. Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. ** Corresponding author. Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (Y. Yan). 1 These authors contributed equally to the work.
multiple cell types [7e9]. With the great success of Professor Yamanaka's lab, the adult somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) [10,11], which addresses many of the barriers associated with the use of embryonic stem cells, including ethical issues, and allows the generation of patient-specific pluripotent stem cells. Recent studies have shown that iPSCs can produce a variety of differentiated cell types, including keratinocytes [12e14]. Previous studies have delivered cultured keratinocytes directly into the wound as a suspension or combined with skin grafting to accelerate epidermal wound healing in animal models and burn patients [15,16]. However, more research and development should be carried out to address the problems and factors that could affect the outcome of cell therapy. For example, transplanted keratinocytes are usually injected into subcutaneous tissue, so the ability of these cells to migrate to the edge of the wound to promote epithelialization is a critical factor in determining the therapeutic effect. Therefore, this study aimed to identify additional factors that can improve the migration of iPSCs-derived keratinocyte in vitro and in vivo.
https://doi.org/10.1016/j.bbrc.2019.09.120 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Heat shock protein-90a (Hsp90a), secreted by a variety of cell types in response to extracellular stress signals, plays an important role in tissue repair [17]. In previous studies, it has been reported that Hsp90a, secreted by human skin cells at the edge of the wound, had a positive effect on the migration of the three major cell types involved in skin wound healing [18e21]. Therefore, we hypothesized that Hsp90a may improve iPSCs-derived keratinocytemediated wound healing. To test this hypothesis, we evaluated the effect of iPSCs-derived keratinocytes combined with Hsp90a on burn wound healing in mice. In addition, we explored the mechanism of Hsp90a treatment of iPSCs-derived keratinocyte-mediated wound healing in vitro. 2. Methods 2.1. Mouse iPSCs culture and differentiation The mouse iPS cell line OSKM-1 were kindly 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 ESGRO Complete Plus Clonal Grade Medium (Millipore). Replace with fresh ESGRO Complete Plus Clonal Grade Medium every other day. iPSCs were induced to keratinocytes as described previously [14]. Briefly, 1000 iPSCs in 30 mL aliquots of unconditioned medium (UM), which consisted of knockout™ Dulbecco's modified Eagle's medium supplemented with 20% knockout™ serum replacement, 1 mM L-glutamine, 1% non-essential amino acid stock and 1% penicillinestreptomycin (all from Gibco), were cultured in hanging drop for 3 days to form Embryoid body (EB). Then EBs were transferred to Col IV-coated culture plates and cultured with in differentiation medium, which contained UM with 1 mM all-trans RA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D Systems Inc.). On day 5, switch the medium to the defined keratinocyte serum free medium (DKSFM; Life Technologies) with 1 mM all-trans RA and 25 ng/ml of BMP4. On day 9, change the medium to the DKSFM with no RA or BMP4 and replace with fresh media every other day. 2.2. Immunofluorescence assay Cells were fixed in 4% paraformaldehyde for 30 min at room temperature before permeabilizing with 0.5% Triton X-100 for 10 min. After that cells were incubated overnight at 4 C with the primary antibody against K14, loricrin or involucrin (cell signaling technology), and then incubated by Alexa Fluor 647-conjugated donkey anti-rabbit second antibody (Abcam) at room temperature for 2 h. Nuclei were stained with 4,6-diamidino-2phenylindole (DAPI, 500 ng/ml) for 10 min. The positive signals were analyzed by confocal microscope (Zeiss).
captured randomly for each transwell membrane using an inverted microscope (Leica). 2.4. Labeling of iPSCs-derived keratinocytes The cells were incubated with 5 mМ 5 (6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich) at 37 C for 30 min. After CSFE removal, fresh culture medium was added and the cells were further incubated at 37 C for 30 min. 2.5. Western blot analysis The cells were harvested and lysed in ice-cold Radio-Immunoprecipitation Assay (RIPA, Sigma) lysis buffer. The amount of protein were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by electrophoresis on SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking 5% fat-free milk, the proteins were incubated with antibodies for b-actin, AKT, p-AKT (Abcam) overnight at 4 C. Subsequently, the menbranes were incubated with horseradish peroxidase conjugated secondary antibodies (Thermo Fisher Scientific). The bands were visualized using the ECL detection system (Abcam). Quantity One software (BioRad) was used to detect the band intensity. 2.6. Deep second-degree burns in mice and treatments C57BL/6 mice aged 8e10 weeks were purchased from the Laboratory Animal Centre of Southern Medical University. After hair removal from the bilateral sites of the abdomens of mice, the opening at one end of a cylindrical plastic tube having a diameter of 1 cm was attached to the skin of the abdomen of mice. The hot water was then poured into the tube at a height of 2 cm and taken out after 15 s. Four animal groups were set in present study. Hsp90a Group received intradermal injection of Hsp90a (1 mg/ml) daily in wound sites for 5 days following burn injury, while PBS Group received PBS with the same manner. In addition to injection of Hsp90a, iPSCs-derived keratinocyte plus Hsp90a Group were also injected with CFSE-labeled iPSCs-derived keratinocyte (1 106 cells) immediately after burn, while iPSCs-derived keratinocyte Group were injected only with cells. The wounds were recorded with a digital camera on days 0, 5 and 9 after burn. 2.7. Histological observations The skin samples were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and then cut into 5 mm thick tissue sections. The sections were used for hematoxylin and eosin (H&E) staining (in accordance with standard procedures). 2.8. Statistical analysis
2.3. Transwell assay The miPSCs-KCs were harvested and resuspended in DKSFM to a density of 1 105 cell per mL after washing with DPBS. For each transwell of a 24-well plate, 500 mL of DKSFM supplemented with Hsp90a was added to the lower chamber, and 100 mL of the cell suspension was added to the transwell upper chamber. After 24h incubation at 5% CO2 and 37 C, the transwell upper chamber was taken out and washed with PBS before being fixed using 4% paraformaldehyde for 30 min. The cells that adhered to the lower side of the transwell membrane were stained with 0.5% crystal violet (Tianjin Zhiyuan Chemical Reagent Co., Ltd.) for 30 min after the non-migrated cells were carefully removed from the upper side of the transwell membrane using cotton swabs. Five images were
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. 3. Results 3.1. Identification of keratinocytes derived from iPSCs In order to characterize the differentiated cells, we performed immunohistochemical analysis and the early stage keratinocyte differentiation marker, cytokeratin-14 (K14), was observed in differentiated cells (Fig. 1A). In addition, this K14-expressing
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 1. Characterization of keratinocytes differentiated from mouse iPSCs. (A) Representative photomicrographs of immunohistochemical staining for K14 (red) in iPSCs-derived keratinocytes. Scale bars ¼ 100 mm. (B) 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 ¼ 100 mm. Blue staining is DAPI, which labels the nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
keratinocyte precursors were able to undergo terminal differentiation and expressed suprabasal keratinocyte markers, such as loricrin and involucrin in DKSFM, supplemented with 1.5 mmol/L of calcium chloride for one week (Fig. 1B). Taken together, these results demonstrate that we successfully differentiated iPSCs into keratinocytes. 3.2. Hsp90a enhanced iPSCs-derived keratinocyte migration via activation of Akt Pathway Migration of iPSCs-derived keratinocyte are key biological processes during transplantation to wound healing. The effect of Hsp90a on the migration of iPSCs-derived keratinocyte was analyzed by transwell assay. Results showed treated with Hsp90a significantly promoted migration of iPSCs-derived keratinocyte compared with the PBS control group (Fig. 2A). It has been reported that Akt pathway is essential for Hsp90a -stimulated migration of primary human dermal fibroblasts [22]. To further determine whether Hsp90a can enhance iPSCs-derived keratinocyte migration via activation of the Akt pathway, we determined the phosphorylation status of Akt in iPSCs-derived keratinocyte treated with 10 ng/mL Hsp90a by Western blotting analysis. Compared with the control group, the relative expression of p-Akt/Akt was significantly increased after Hsp90a treatment; however, the total Akt protein level was not affected by Hsp90a (Fig. 2B). To further determine the role of Akt signaling in the Hsp90a for promotion of iPSCs-derived keratinocyte migration, the activation of Akt was blocked using perifosine, a selective inhibitor of Akt. Through transwell assay,
perifosine reversed the increased number of iPSCs-derived keratinocyte migrated in the lower chamber, which was mediated by Hsp90a (Fig. 2C). In conclusion, these data suggest that Hsp90a promotes the migration of iPSCs-derived keratinocyte by enhancing the activation of Akt signaling pathway. 3.3. HSP90a promotes deep second-degree burn wound healing mediated by iPSCs-derived keratinocyte Until now, we have shown the effects of Hsp90a on migration of iPSCs-derived keratinocyte in vitro. To evaluated whether Hsp90a can promote iPSCs-derived keratinocyte mediated wound healing, we created 1 cm round deep second-degree burn on the abdominal skin. Hematoxylin and eosin (H&E) staining of burn sites on day 1 showed that the structures of the whole epidermis and a deeper part of the dermis were damaged (Fig. 3A), suggesting that we have successfully established a skin model for deep second degree burns. Then, we applied PBS, Hsp90a, iPSCs-derived keratinocyte, or iPSCs-derived keratinocyte plus Hsp90a to wound sites. The healing process was observed through days 0e9 after burn (Fig. 3B). Macroscopic evaluation revealed that iPSCs-derived keratinocyte plus Hsp90a significantly promoted the rates of wound closure compared with PBS, Hsp90a, or iPSCs-derived keratinocyte on days 5 and 9 after treatment (Fig. 3C). Then, we performed skin biopsies at these wounds on days 5 and 9 after burn and evaluated the skin wound healing process microscopically (Fig. 3C). Hematoxylin and eosin (H&E) staining showed that re-epithelialization was significantly enhanced by iPSCs-derived keratinocyte plus Hsp90a
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 2. Hsp90a Enhanced iPSCs-derived keratinocyte Migration via activation of Akt Pathway. (A) Representative images of migrated iPSCs-derived keratinocyte treated with PBS or Hsp90a by transwell assay (left panel). Quantitative analysis of the migrated cells (right panel). *P < 0.05. Scale bar ¼ 100 mm. (B) Western blot analysis of protein levels of phosphorylated AKT (p-AKT), total AKT and b-actin of iPSCs-derived keratinocyte treated with PBS or Hsp90a (left panel). Quantification presented as the relative densitometry intensity of the p-AKT to total AKT in the corresponding same lane (right panel). (C) Representative images of migrated iPSCs-derived keratinocyte treated with PBS or Hsp90a by transwell migration assay (left panel). Quantitative analysis of the migrated cells (right panel). *P < 0.05. Scale bar ¼ 100 mm.
(Fig. 3D). To determine the presence of CFSE-labeled iPSCs-derived keratinocytes, tissue sections from day 14 of all groups were examined by fluorescent microscope. In PBS and Hsp90a group, fluorescence could not be detected in any section. In iPSCs-derived keratinocyte and iPSCs-derived keratinocyte plus Hsp90a groups, CFSE-labeled iPSCs-derived keratinocyte appeared as green fluorescent cells in regenerated epidermis after two weeks (Fig. 4A). In addition, the number of CFSE-labeled iPSCs-derived keratinocyte in regenerated epidermis was increased in iPSCs-derived keratinocyte plus Hsp90a group compared with that in iPSCs-derived keratinocyte group (Fig. 4B). These data are consistent with previous in vitro results, demonstrating that a Hsp90a can promote iPSCs-derived keratinocyte migration to the wound margin, resulting in faster healing of the wound.
4. Discussion In this study, we found that Hsp90a promotes migration by activating Akt signaling in iPSCs-derived keratinocyte. In addition, administration of iPSCs-derived keratinocyte plus Hsp90a could accelerate wound healing of deep second-degree burn in mice. Therefore, our research provides a new strategy for the application of iPSCs technology in the treatment of skin wound healing. The re-epithelialization process begins within a few hours after injury and is absolutely necessary for optimal wound closure in view of its role in restoring the intact epidermal barrier [23,24]. Keratinocytes play a key role in epidermal restoration through proliferation and re-epithelialization after injury [25,26]. To date, cultured keratinocytes and non-cultured epidermal isolates are the devices most commonly used to treat burns. iPSCs are a promising source of keratinocytes. In our study, iPSCs-derived keratinocytes were positive for K14, a keratin marker confirming commitment of
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 3. Hsp90a promotes iPSCs-derived keratinocyte -mediated Burn Wound Healing. (A) Representative photomicrographs of H&E-stained burned skin from mice 24 h after burns. Scale bar ¼ 100 mm. (B) Representative macroscopic images of wounds treated with PBS, iPSCs-derived keratinocytes, Hsp90a or iPSCs-derived keratinocytes plus Hsp90a on days 0, 5 and 9 after burn (left panel). Quantitative analysis of wound area per group on 0, 5 and 9 after burn, expressed as the percentage of the initial wound size at day 0 (right panel). *P < 0.05. (C) Representative photomicrographs of H&E-stained wounds treated with PBS and iPSCs-derived keratinocytes on days 5 and 9 after burn. (D) Time-course of changes of the re-epithelialization ration of wounds treated with PBS and iPSCs-derived keratinocytes on days 5 and 9 after burn. 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. *P < 0.05. miPSCs-KCs: iPSCs-derived keratinocytes.
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 4. Hsp90 promotes the migration of keratinocytes to regenerated epidermis. (A) Representative photomicrographs of immunohistochemical staining for CFSE (green) of wounds treated with PBS and iPSCs-derived keratinocytes on day 14 (left panel). Scale bars ¼ 100 mm. (B) Quantitative analysis of the percentage of CFSE-positive cells in regenerated epidermis. *P < 0.05. miPSCs-KCs: iPSCs-derived keratinocytes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
the ectoderm to a keratinocyte fate [27] and were able to express terminal differentiation markers under high Ca2þ cultures. Hsp90, including Hsp90a and hsp90b two isoforms, is a highly conserved family of intracellular molecular chaperones, accounting for 1e2% of all cellular proteins in most cells. They regulate the folding, transport, maturation and degradation of a variety of client proteins, many of which are signaling proteins. Interestingly, Li and his colleagues found that hypoxia promotes migration of human dermal fibroblast (HDF) by inducing the secretion of Hsp90a into the extracellular environment through hypoxia-inducible factor-1 alpha (HIF-1a) [28]. Their subsequent research work further demonstrated that topical application of human recombinant Hsp90a protein could promote wound closure in excision, burn, and diabetic wounds [17,29]. A therapeutic entity of Hsp90a, a 115 amino acid fragment called F-5, promotes human skin cell migration in vitro and wound healing in vivo [30]. Therefore, we hypothesized that Hsp90a can also enhance the migratory ability of iPSCs-derived keratinocyte. In this study, the transwell assay, was utilized to investigate iPSCs-derived keratinocyte migration. Similarly, our work revealed that in the Hsp90a group, more cells passed through the transwell membrane, which suggests that Hsp90a facilitated iPSCs-derived keratinocyte migration. The Akt signaling pathway is involved in many cellular processes, including proliferation, apoptosis, cell migration, angiogenesis, invasion, and metastasis [31,32]. Previous studies have demonstrated that Hsp90a promotes migration of fibroblasts through the Akt pathway. Therefore, we assumed that Hsp90a can enhance the migration of iPSCs-derived keratinocyte via Akt activation. Indeed, the western blotting results indicate that Hsp90a activates the Akt in iPSCs-derived keratinocyte. Furthermore, we used the AKT inhibitor perifosine in the transwell assay. The results showed that perifosine blocks the migratory character of iPSCsderived keratinocyte, which is promoted by Hsp90a. Therefore, these findings suggest that Hsp90a promotes iPSCs-derived keratinocyte migration by activating the Akt. We further investigated how Hsp90a improves the efficacy of iPSCs-derived keratinocyte for the treatment of skin burns. In accordance with the in vitro results, Hsp90a enhanced the migration of transplanted iPSCs-derived keratinocyte into the wound
sites. Moreover, combination treatment with Hsp90a and iPSCsderived keratinocyte improves wound healing compared with single treatment, suggesting that this combination could be an effective therapeutic approach. In summary, we demonstrated that Hsp90a exert beneficial effects on treatment of iPSCs-derived keratinocyte on skin wound healing. Therefore, iPSCs-derived keratinocyte plus Hsp90a may be used as novel therapeutic approach in cutaneous wound healing.
Declaration of competing interest The authors declare that they have no competing interests.
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).
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.120.
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 analyzed during this study are included in this published article.
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Consent for publication Not applicable.
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Authors’ contributions
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RJ W and D D performed the experimental work, Y Y wrote the paper, M Z performed the data collection, L Z designed the study. All authors read and approved the final manuscript.
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Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice Ruijun Wu a, 1, Dan Du a, 1, Yunyao Bo a, Min Zhang a, Lin Zhang a, b, **, Yuan Yan a, b, * a b
Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China Key Laboratory of Tissue Construction and Detection of Guangdong Province, Guangzhou, 510515, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2019 Accepted 27 September 2019 Available online xxx
Because of the advantages of induced pluripotent stem cells (iPSCs), iPSCs-derived keratinocyte hold great clinical and research potential in wound repair. Similar to other cell transplantation therapies, the migration ability of iPSCs-derived keratinocyte transplanted into skin is critical to the therapeutic effect. Hsp90a had a positive effect on migration of keratinocytes. Therefore, the aim of this study was to investigate the effects of Hsp90a on transplanted iPSCs-derived keratinocyte in a skin model of deep second degree burns. First, keratinocytes were differentiated from iPSCs by treating with RA and BMP4. Next, we explained the effect Hsp90a on iPSCs-derived keratinocyte in vitro. We found that hsp90a promoted cell migration of iPSCs-derived keratinocyte. Furthermore, activation of AKT was required for Hsp90a-induced iPSCs-derived keratinocyte migration. Then PBS, Hsp90a, iPSCs-derived keratinocyte, and iPSCs-derived keratinocyte plus Hsp90a were applied to the wound bed of deep second degree burns. Wound healing was assessed by gross evaluation and hematoxylin and eosin staining. Our results shown that wound treated with iPSCs-derived keratinocyte plus Hsp90a significantly accelerates the rate of wound healing closure than other groups. In addition, the number of CFSE-labeled iPSCs-derived keratinocyte in regenerated epidermis was increased in iPSCs-derived keratinocyte plus Hsp90a group. In summary, these findings represent that combined administration of iPSCs-derived keratinocyte and Hsp90a may be a promising therapeutic strategy for wound healing. © 2019 Elsevier Inc. All rights reserved.
Keywords: iPSCs Hsp90a Keratinocyte Deep second degree burn Wound healing
1. Introduction Skin wound healing is a complex process involving hemostasis, inflammation, proliferation and remodeling [1]. The goal of normal skin wound healing is to quickly regenerate the natural protective structure of the skin to reduce the risk of infection and provide functional tissue. This led to the discovery of more advanced treatment options, such as gene therapy [2], growth factor therapy [3], platelet-rich plasma therapy [4], stem cell therapy [5], and tissue engineering [6]. Among these methods, stem cell-based therapy is an attractive method for treating acute and chronic wounds. Nowadays, the field of regenerative medicine mainly focuses on stem cells, which can self-renew and differentiate into
* Corresponding author. Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. ** Corresponding author. Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (Y. Yan). 1 These authors contributed equally to the work.
multiple cell types [7e9]. With the great success of Professor Yamanaka's lab, the adult somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) [10,11], which addresses many of the barriers associated with the use of embryonic stem cells, including ethical issues, and allows the generation of patient-specific pluripotent stem cells. Recent studies have shown that iPSCs can produce a variety of differentiated cell types, including keratinocytes [12e14]. Previous studies have delivered cultured keratinocytes directly into the wound as a suspension or combined with skin grafting to accelerate epidermal wound healing in animal models and burn patients [15,16]. However, more research and development should be carried out to address the problems and factors that could affect the outcome of cell therapy. For example, transplanted keratinocytes are usually injected into subcutaneous tissue, so the ability of these cells to migrate to the edge of the wound to promote epithelialization is a critical factor in determining the therapeutic effect. Therefore, this study aimed to identify additional factors that can improve the migration of iPSCs-derived keratinocyte in vitro and in vivo.
https://doi.org/10.1016/j.bbrc.2019.09.120 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Heat shock protein-90a (Hsp90a), secreted by a variety of cell types in response to extracellular stress signals, plays an important role in tissue repair [17]. In previous studies, it has been reported that Hsp90a, secreted by human skin cells at the edge of the wound, had a positive effect on the migration of the three major cell types involved in skin wound healing [18e21]. Therefore, we hypothesized that Hsp90a may improve iPSCs-derived keratinocytemediated wound healing. To test this hypothesis, we evaluated the effect of iPSCs-derived keratinocytes combined with Hsp90a on burn wound healing in mice. In addition, we explored the mechanism of Hsp90a treatment of iPSCs-derived keratinocyte-mediated wound healing in vitro. 2. Methods 2.1. Mouse iPSCs culture and differentiation The mouse iPS cell line OSKM-1 were kindly 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 ESGRO Complete Plus Clonal Grade Medium (Millipore). Replace with fresh ESGRO Complete Plus Clonal Grade Medium every other day. iPSCs were induced to keratinocytes as described previously [14]. Briefly, 1000 iPSCs in 30 mL aliquots of unconditioned medium (UM), which consisted of knockout™ Dulbecco's modified Eagle's medium supplemented with 20% knockout™ serum replacement, 1 mM L-glutamine, 1% non-essential amino acid stock and 1% penicillinestreptomycin (all from Gibco), were cultured in hanging drop for 3 days to form Embryoid body (EB). Then EBs were transferred to Col IV-coated culture plates and cultured with in differentiation medium, which contained UM with 1 mM all-trans RA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D Systems Inc.). On day 5, switch the medium to the defined keratinocyte serum free medium (DKSFM; Life Technologies) with 1 mM all-trans RA and 25 ng/ml of BMP4. On day 9, change the medium to the DKSFM with no RA or BMP4 and replace with fresh media every other day. 2.2. Immunofluorescence assay Cells were fixed in 4% paraformaldehyde for 30 min at room temperature before permeabilizing with 0.5% Triton X-100 for 10 min. After that cells were incubated overnight at 4 C with the primary antibody against K14, loricrin or involucrin (cell signaling technology), and then incubated by Alexa Fluor 647-conjugated donkey anti-rabbit second antibody (Abcam) at room temperature for 2 h. Nuclei were stained with 4,6-diamidino-2phenylindole (DAPI, 500 ng/ml) for 10 min. The positive signals were analyzed by confocal microscope (Zeiss).
captured randomly for each transwell membrane using an inverted microscope (Leica). 2.4. Labeling of iPSCs-derived keratinocytes The cells were incubated with 5 mМ 5 (6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich) at 37 C for 30 min. After CSFE removal, fresh culture medium was added and the cells were further incubated at 37 C for 30 min. 2.5. Western blot analysis The cells were harvested and lysed in ice-cold Radio-Immunoprecipitation Assay (RIPA, Sigma) lysis buffer. The amount of protein were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by electrophoresis on SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking 5% fat-free milk, the proteins were incubated with antibodies for b-actin, AKT, p-AKT (Abcam) overnight at 4 C. Subsequently, the menbranes were incubated with horseradish peroxidase conjugated secondary antibodies (Thermo Fisher Scientific). The bands were visualized using the ECL detection system (Abcam). Quantity One software (BioRad) was used to detect the band intensity. 2.6. Deep second-degree burns in mice and treatments C57BL/6 mice aged 8e10 weeks were purchased from the Laboratory Animal Centre of Southern Medical University. After hair removal from the bilateral sites of the abdomens of mice, the opening at one end of a cylindrical plastic tube having a diameter of 1 cm was attached to the skin of the abdomen of mice. The hot water was then poured into the tube at a height of 2 cm and taken out after 15 s. Four animal groups were set in present study. Hsp90a Group received intradermal injection of Hsp90a (1 mg/ml) daily in wound sites for 5 days following burn injury, while PBS Group received PBS with the same manner. In addition to injection of Hsp90a, iPSCs-derived keratinocyte plus Hsp90a Group were also injected with CFSE-labeled iPSCs-derived keratinocyte (1 106 cells) immediately after burn, while iPSCs-derived keratinocyte Group were injected only with cells. The wounds were recorded with a digital camera on days 0, 5 and 9 after burn. 2.7. Histological observations The skin samples were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and then cut into 5 mm thick tissue sections. The sections were used for hematoxylin and eosin (H&E) staining (in accordance with standard procedures). 2.8. Statistical analysis
2.3. Transwell assay The miPSCs-KCs were harvested and resuspended in DKSFM to a density of 1 105 cell per mL after washing with DPBS. For each transwell of a 24-well plate, 500 mL of DKSFM supplemented with Hsp90a was added to the lower chamber, and 100 mL of the cell suspension was added to the transwell upper chamber. After 24h incubation at 5% CO2 and 37 C, the transwell upper chamber was taken out and washed with PBS before being fixed using 4% paraformaldehyde for 30 min. The cells that adhered to the lower side of the transwell membrane were stained with 0.5% crystal violet (Tianjin Zhiyuan Chemical Reagent Co., Ltd.) for 30 min after the non-migrated cells were carefully removed from the upper side of the transwell membrane using cotton swabs. Five images were
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. 3. Results 3.1. Identification of keratinocytes derived from iPSCs In order to characterize the differentiated cells, we performed immunohistochemical analysis and the early stage keratinocyte differentiation marker, cytokeratin-14 (K14), was observed in differentiated cells (Fig. 1A). In addition, this K14-expressing
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 1. Characterization of keratinocytes differentiated from mouse iPSCs. (A) Representative photomicrographs of immunohistochemical staining for K14 (red) in iPSCs-derived keratinocytes. Scale bars ¼ 100 mm. (B) 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 ¼ 100 mm. Blue staining is DAPI, which labels the nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
keratinocyte precursors were able to undergo terminal differentiation and expressed suprabasal keratinocyte markers, such as loricrin and involucrin in DKSFM, supplemented with 1.5 mmol/L of calcium chloride for one week (Fig. 1B). Taken together, these results demonstrate that we successfully differentiated iPSCs into keratinocytes. 3.2. Hsp90a enhanced iPSCs-derived keratinocyte migration via activation of Akt Pathway Migration of iPSCs-derived keratinocyte are key biological processes during transplantation to wound healing. The effect of Hsp90a on the migration of iPSCs-derived keratinocyte was analyzed by transwell assay. Results showed treated with Hsp90a significantly promoted migration of iPSCs-derived keratinocyte compared with the PBS control group (Fig. 2A). It has been reported that Akt pathway is essential for Hsp90a -stimulated migration of primary human dermal fibroblasts [22]. To further determine whether Hsp90a can enhance iPSCs-derived keratinocyte migration via activation of the Akt pathway, we determined the phosphorylation status of Akt in iPSCs-derived keratinocyte treated with 10 ng/mL Hsp90a by Western blotting analysis. Compared with the control group, the relative expression of p-Akt/Akt was significantly increased after Hsp90a treatment; however, the total Akt protein level was not affected by Hsp90a (Fig. 2B). To further determine the role of Akt signaling in the Hsp90a for promotion of iPSCs-derived keratinocyte migration, the activation of Akt was blocked using perifosine, a selective inhibitor of Akt. Through transwell assay,
perifosine reversed the increased number of iPSCs-derived keratinocyte migrated in the lower chamber, which was mediated by Hsp90a (Fig. 2C). In conclusion, these data suggest that Hsp90a promotes the migration of iPSCs-derived keratinocyte by enhancing the activation of Akt signaling pathway. 3.3. HSP90a promotes deep second-degree burn wound healing mediated by iPSCs-derived keratinocyte Until now, we have shown the effects of Hsp90a on migration of iPSCs-derived keratinocyte in vitro. To evaluated whether Hsp90a can promote iPSCs-derived keratinocyte mediated wound healing, we created 1 cm round deep second-degree burn on the abdominal skin. Hematoxylin and eosin (H&E) staining of burn sites on day 1 showed that the structures of the whole epidermis and a deeper part of the dermis were damaged (Fig. 3A), suggesting that we have successfully established a skin model for deep second degree burns. Then, we applied PBS, Hsp90a, iPSCs-derived keratinocyte, or iPSCs-derived keratinocyte plus Hsp90a to wound sites. The healing process was observed through days 0e9 after burn (Fig. 3B). Macroscopic evaluation revealed that iPSCs-derived keratinocyte plus Hsp90a significantly promoted the rates of wound closure compared with PBS, Hsp90a, or iPSCs-derived keratinocyte on days 5 and 9 after treatment (Fig. 3C). Then, we performed skin biopsies at these wounds on days 5 and 9 after burn and evaluated the skin wound healing process microscopically (Fig. 3C). Hematoxylin and eosin (H&E) staining showed that re-epithelialization was significantly enhanced by iPSCs-derived keratinocyte plus Hsp90a
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 2. Hsp90a Enhanced iPSCs-derived keratinocyte Migration via activation of Akt Pathway. (A) Representative images of migrated iPSCs-derived keratinocyte treated with PBS or Hsp90a by transwell assay (left panel). Quantitative analysis of the migrated cells (right panel). *P < 0.05. Scale bar ¼ 100 mm. (B) Western blot analysis of protein levels of phosphorylated AKT (p-AKT), total AKT and b-actin of iPSCs-derived keratinocyte treated with PBS or Hsp90a (left panel). Quantification presented as the relative densitometry intensity of the p-AKT to total AKT in the corresponding same lane (right panel). (C) Representative images of migrated iPSCs-derived keratinocyte treated with PBS or Hsp90a by transwell migration assay (left panel). Quantitative analysis of the migrated cells (right panel). *P < 0.05. Scale bar ¼ 100 mm.
(Fig. 3D). To determine the presence of CFSE-labeled iPSCs-derived keratinocytes, tissue sections from day 14 of all groups were examined by fluorescent microscope. In PBS and Hsp90a group, fluorescence could not be detected in any section. In iPSCs-derived keratinocyte and iPSCs-derived keratinocyte plus Hsp90a groups, CFSE-labeled iPSCs-derived keratinocyte appeared as green fluorescent cells in regenerated epidermis after two weeks (Fig. 4A). In addition, the number of CFSE-labeled iPSCs-derived keratinocyte in regenerated epidermis was increased in iPSCs-derived keratinocyte plus Hsp90a group compared with that in iPSCs-derived keratinocyte group (Fig. 4B). These data are consistent with previous in vitro results, demonstrating that a Hsp90a can promote iPSCs-derived keratinocyte migration to the wound margin, resulting in faster healing of the wound.
4. Discussion In this study, we found that Hsp90a promotes migration by activating Akt signaling in iPSCs-derived keratinocyte. In addition, administration of iPSCs-derived keratinocyte plus Hsp90a could accelerate wound healing of deep second-degree burn in mice. Therefore, our research provides a new strategy for the application of iPSCs technology in the treatment of skin wound healing. The re-epithelialization process begins within a few hours after injury and is absolutely necessary for optimal wound closure in view of its role in restoring the intact epidermal barrier [23,24]. Keratinocytes play a key role in epidermal restoration through proliferation and re-epithelialization after injury [25,26]. To date, cultured keratinocytes and non-cultured epidermal isolates are the devices most commonly used to treat burns. iPSCs are a promising source of keratinocytes. In our study, iPSCs-derived keratinocytes were positive for K14, a keratin marker confirming commitment of
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 3. Hsp90a promotes iPSCs-derived keratinocyte -mediated Burn Wound Healing. (A) Representative photomicrographs of H&E-stained burned skin from mice 24 h after burns. Scale bar ¼ 100 mm. (B) Representative macroscopic images of wounds treated with PBS, iPSCs-derived keratinocytes, Hsp90a or iPSCs-derived keratinocytes plus Hsp90a on days 0, 5 and 9 after burn (left panel). Quantitative analysis of wound area per group on 0, 5 and 9 after burn, expressed as the percentage of the initial wound size at day 0 (right panel). *P < 0.05. (C) Representative photomicrographs of H&E-stained wounds treated with PBS and iPSCs-derived keratinocytes on days 5 and 9 after burn. (D) Time-course of changes of the re-epithelialization ration of wounds treated with PBS and iPSCs-derived keratinocytes on days 5 and 9 after burn. 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. *P < 0.05. miPSCs-KCs: iPSCs-derived keratinocytes.
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Fig. 4. Hsp90 promotes the migration of keratinocytes to regenerated epidermis. (A) Representative photomicrographs of immunohistochemical staining for CFSE (green) of wounds treated with PBS and iPSCs-derived keratinocytes on day 14 (left panel). Scale bars ¼ 100 mm. (B) Quantitative analysis of the percentage of CFSE-positive cells in regenerated epidermis. *P < 0.05. miPSCs-KCs: iPSCs-derived keratinocytes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
the ectoderm to a keratinocyte fate [27] and were able to express terminal differentiation markers under high Ca2þ cultures. Hsp90, including Hsp90a and hsp90b two isoforms, is a highly conserved family of intracellular molecular chaperones, accounting for 1e2% of all cellular proteins in most cells. They regulate the folding, transport, maturation and degradation of a variety of client proteins, many of which are signaling proteins. Interestingly, Li and his colleagues found that hypoxia promotes migration of human dermal fibroblast (HDF) by inducing the secretion of Hsp90a into the extracellular environment through hypoxia-inducible factor-1 alpha (HIF-1a) [28]. Their subsequent research work further demonstrated that topical application of human recombinant Hsp90a protein could promote wound closure in excision, burn, and diabetic wounds [17,29]. A therapeutic entity of Hsp90a, a 115 amino acid fragment called F-5, promotes human skin cell migration in vitro and wound healing in vivo [30]. Therefore, we hypothesized that Hsp90a can also enhance the migratory ability of iPSCs-derived keratinocyte. In this study, the transwell assay, was utilized to investigate iPSCs-derived keratinocyte migration. Similarly, our work revealed that in the Hsp90a group, more cells passed through the transwell membrane, which suggests that Hsp90a facilitated iPSCs-derived keratinocyte migration. The Akt signaling pathway is involved in many cellular processes, including proliferation, apoptosis, cell migration, angiogenesis, invasion, and metastasis [31,32]. Previous studies have demonstrated that Hsp90a promotes migration of fibroblasts through the Akt pathway. Therefore, we assumed that Hsp90a can enhance the migration of iPSCs-derived keratinocyte via Akt activation. Indeed, the western blotting results indicate that Hsp90a activates the Akt in iPSCs-derived keratinocyte. Furthermore, we used the AKT inhibitor perifosine in the transwell assay. The results showed that perifosine blocks the migratory character of iPSCsderived keratinocyte, which is promoted by Hsp90a. Therefore, these findings suggest that Hsp90a promotes iPSCs-derived keratinocyte migration by activating the Akt. We further investigated how Hsp90a improves the efficacy of iPSCs-derived keratinocyte for the treatment of skin burns. In accordance with the in vitro results, Hsp90a enhanced the migration of transplanted iPSCs-derived keratinocyte into the wound
sites. Moreover, combination treatment with Hsp90a and iPSCsderived keratinocyte improves wound healing compared with single treatment, suggesting that this combination could be an effective therapeutic approach. In summary, we demonstrated that Hsp90a exert beneficial effects on treatment of iPSCs-derived keratinocyte on skin wound healing. Therefore, iPSCs-derived keratinocyte plus Hsp90a may be used as novel therapeutic approach in cutaneous wound healing.
Declaration of competing interest The authors declare that they have no competing interests.
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).
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.120.
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 analyzed during this study are included in this published article.
Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120
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Authors’ contributions
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RJ W and D D performed the experimental work, Y Y wrote the paper, M Z performed the data collection, L Z designed the study. All authors read and approved the final manuscript.
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Please cite this article as: R. Wu et al., Hsp90a promotes the migration of iPSCs-derived keratinocyte to accelerate deep second-degree burn wound healing in mice, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.120