The use of keratinocytes derived from induced pluripotent stem cells (iPSCs-KCs) may represent a novel cell therapy strategy for burn treatment. There is growing evidence that extracellular vesicles, including exosomes, are primary mediators of the benefits of stem cell therapy. Herein, we thus explored the effects of exosomes produced by iPSCs-derived keratinocytes (iPSCs-KCs-Exos) in a model of deep second-degree burn wound healing and evaluated the mechanistic basis for the observed activity.
iPSCs-KCs-Exos were isolated from conditioned medium of iPSCs-KCs and verified by electron micrograph and size distribution. Next, iPSCs-KCs-Exos were injected subcutaneously around wound sites, and its efficacy was evaluated by measuring wound closure areas, histological examination and immunohistochemistry staining. The effects of iPSCs-KCs-Exos on proliferation and migration of keratinocytes and endothelial cells in vitro were assessed by EdU staining, wound healing assays and transwell assay. Then, high-throughput microRNA sequencing was used to explore the underlying mechanisms. We assessed the roles of miR-762 in iPSCs-KCs-Exos-induced regulation of keratinocytes and endothelial cells migration. Furthermore, the target gene which mediated the biological effects of miR-762 in keratinocytes and endothelial cells was also been detected.
The analysis revealed that iPSCs-KCs-Exos application to the burn wound drove the acceleration of wound closure, with more robust angiogenesis and re-epithelialization being evident. Such iPSCs-KCs-Exos treatment effectively enhanced endothelial cell and keratinocyte migration in vitro. Moreover, the enrichment of miR-762 was detected in iPSCs-KCs-Exos and was found to target promyelocytic leukemia (PML) as a means of regulating cell migration through a mechanism tie to integrin beta1 (ITGB1).
These results thus provide a foundation for the further study of iPSCs-KCs-Exos as novel cell-free treatments for deep second-degree burns.
Burns are a form of serious traumatic injury that cause substantial global morbidity, and there is thus an urgent need to define novel approaches to expediting burn healing [1–3]. Preclinical and clinical evidence suggests that keratinocyte-based therapies offer great promise in the context of burn management [4–6]. However, there are substantial challenges to large-scale keratinocyte preparation efforts owing to the fact that donor keratinocyte yields are often very limited. The ability of these donated cells to proliferate and differentiate in vitro is also highly variable. The Yamananka lab first developed induced pluripotent stem cells (iPSCs) and highlighted their potential as a novel source of many cellular lineages for use in therapeutic contexts [7–9]. The generated iPSCs are extremely similar to embryonic stem cells being capable of generating all cell lineages of different tissues including skin, highlighting a novel method of improving cell-based wound treatment efforts while minimizing ethical concerns and the risk of immune-mediated graft rejection [10, 11].
We have previously shown that iPSCs-derived keratinocytes (iPSCs-KCs) hold great promise for treating wound healing [12, 13]. However, the safety profile of these cells is not known and they are potentially limited by the risk of immunogenicity and genetic instability. Recent work has suggested that small membrane-bound exosomes derived from cells can facilitate a substantial fraction of the paracrine signaling necessary to facilitate intracellular communication [14, 15]. Exosomes of stem cells including bone marrow mesenchymal stem cells, human adipose stem cells, human amniotic epithelial cells and iPSCs can play an anti-inflammatory and anti-fibrosis role, inhibit oxidative stress and enhance angiogenesis, thus promoting skin wound healing [16–20]. As such, we speculate that iPSCs-KCs-derived exosomes (iPSCs-KCs-Eoxs) have the potential to play a direct role in the facilitation of burn wound healing. Up to now, there are no related studies on the iPSCs-KCs-Exos, and there are few studies on the primary keratinocyte’s exosomes in skin wound healing [21–23].
Herein, we utilized iPSCs-KCs-Exos to treat murine skin wounds in order to test the therapeutic efficacy of these cell-free vesicles. We found that such iPSCs-KCs-Exos treatment was conducive to more rapid wound closure as well as enhanced endothelial cell and keratinocyte migration. We then evaluated the microRNAs present within these exosomes in an effort to identify important mediators of this wound healing phenotype, and found miR-762 to be highly enriched in iPSCs-KCs-Exos and to serve as an important mediator of cellular migration owing to its ability to target PML. Overall, these data highlight the important roles played by iPSCs-KCs-Exos and miR-762 in the treatment of cutaneous wounds.
The human iPS cell line was provided by Stem Cell Bank, Chinese Academy of Sciences. hiPSCs were cultured on Matrigel (BD Biosciences, CA) in mTeSRTM1 medium (Stemcell Technologies, CA) and passaged every 4-7 days. hiPSCs were induced to keratinocytes as described previously . Briefly, hiPSCs were switched to unconditioned medium (UM) supplemented with retinoic acid (RA): DMEM/F12 containing 20% knockout serum replacer, 1mM L-glutamine, 1× MEM nonessential amino acids, 0.1 mM b-mercaptoethanol (all from Life Technologies, USA) and 1 mM all-trans RA (Sigma-Aldrich, USA). Cells were cultured in UM+RA for 7 days, changing medium daily. On day 8, cells were passaged onto gelatin-coated plates in Keratinocyte Defined Serum-free Medium (K-DSFM, Life Technologies). After 4 weeks, differentiated cells were subcultured using trypsin to obtain high purity keratinocytes populations. Terminal differentiation was induced by adding 1.5 mM CaCl2 in K-DSFM for 10 days. HaCaT cells and HUVECs (COBIOER, China) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), supplemented with 10% fetal bovine serum (Gibico, USA).
Extraction and identification of iPSCs-KCs-Exos
iPSCs-KCs were cultured in K-DSFM for 48 h. After collecting the supernatant, exosomes were isolated and purified by serial ultracentrifugation, using ultracentrifuge (Beckman, Germany). The exosomes was suspended in PBS. Concentration of exosomes was measured by Enhanced BCA Protein Assay Kit (Beyotime, China). Then, transmission electron microscopy (TEM) was used to observe the morphological structure of exosomes. Size distribution and concentration of iPSCs-KCs-Exos were analyzed by nano-particle tracking analysis (NTA) using Nanosight LM 10 (Malvern Panalytical, UK).
Treatment of deep second-degree burns
The 8-week-old C57BL/6 mice were purchased from the Experimental Animal Center of Southern Medical University. After the hair removal from the back of mice, the opening at one end of a cylindrical plastic tube with a diameter of 1.5 cm was affixed to the back skin of mice. Then pour boiling water into the tube at a height of 2 cm and then the tube was removed after 18 seconds. After burn, local subcutaneous injections of PBS or exosomes were given to the wound site.
The skin tissues were fixed with 4% paraformaldehyde for 72 h, embedded in paraffin, and then cut into 5 mm thick sections. The sections were stained with hematoxylin eosin (H&E) staining (Leagene, China) and Masson's tri-chrome staining (MXB Biotechnologies, China). The re-epithelialization was calculated in the accordance with the formula [distance of the re-epithelium]/ [distance of the wound] ×100.
At room temperature, cells were fixed with 4% paraformaldehyde for 30 min and then drilled with 0.5% Triton X-100 for 15 minutes. The cells were incubated overnight at 4°C with primary antibodies against K14, loricrin or involucrin (Proteintech, USA) and then incubated with Alexa Fluor 647-conjugated sheep anti-rabbit second antibody (Abcam, UK) for 1 h at room temperature. DAPI (500 ng/mL) was used to stain nuclei. Fluorescence was observed under an inverted fluorescence microscope (Leica, Germany)
After cells were treated for 48 h, EdU was added to the culture medium and incubated for 4 h. The cells were fixed with 4% paraformaldehyde and then drilled with 0.5% Triton X-100 for 15 minutes. Then, the cells were stained using Beyoclick™ EDU cell proliferation kit Alexa Fluor 488 (Beyotime, China) and imaged using an inverted fluorescence microscope (Leica, Germany).
Wound healing assay
Cells were seeded into 24-well plates and cultured overnight. After each well was filled with 95% monolayer of cells, the tip of the 200 μL pipette was used to form linear scratch wounds. At 0 h and 24 h, the wound healing was recorded by a microscope (Leica, Germany). Image J software was used to quantitatively evaluate the wound healing rate.
200 μL cell suspension (serum-free DMEM) was added to the upper chamber of Transwell, and 600 μL DMEM containing 2% FBS was added to the lower chamber. After 24 h incubation at 5% CO2 and 37℃, the cells were fixed with 4% paraformaldehyde. The cells at the lower side of the membrane were stained with Crystal Violet Staining Solution (Beyotime China) for 15 min. An inverted microscope (Leica, Germany) randomly collected 10 images from each sample.
miRNA microarray analysis
miRNA microarray analysis was performed by Epibiotek (Guangzhou, China) as previously described .
Western blot analysis
The Western blot assay was performed as previously described . Antibodies used were described as follow: anti-PML, anti-ITGB1or anti b-actin (all from Abcam, UK).
Reverse transcription quantitative polymerase chain (qRT-PCR)
Trizol™ reagent (Invitrogen, USA) was used to extract the total RNA from cells, according to the manufacturer's instructions. RNA was reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). qRT-PCR analysis was using ACEQ Universal SYBR qPCR Master Mix (Vazyme, China). Midtect A TrackTM miRNA qRT-PCR Starter Kit (RiboBio, China) was used for synthesis of miRNA cDNA. Then, Midtect A TrackTM miRNA qRT-PCR Starter Kit (RiboBio, China) were used for qRT-PCR.
Luciferase report assay
The wild type and mutant miR-762 binding sites of PML 3′UTR sequence were subcloned into psiCHECK-2 vector. MiR-762 mimics or negative control mimics were co-transfected with recombinant plasmid into HEK-293 T cells using Lipofectamine 2000 (Invitrogen, USA). According to the manufacturer’s instructions, the Firefly and Renilla luciferase activities were detected by Dual Luciferase Reporter Gene Assay Kit (Beyotime, China).
Data were presented as mean ± standard deviation (SD) of at least three independent experiments (n ≥ 3). Statistical analysis was performed by independent samples t-test for comparison between two groups or one-way ANOVA among the groups. p < 0.05 was considered statistically significant.
Characterization of iPSCs-KCs and iPSCs-KCs-Exos.
To characterize prepared iPSCs-KCs, we conducted immunohistochemical (IHC) staining for cytokeratin-14 (K14), which is a keratin marker confirming commitment of the ectoderm to a keratinocyte fate. As shown as in Fig. 1A, K14 was detectable in iPSCs-KCs. In addition, these K14+ cells expressed involucrin and loricrin (Fig. 1B), markers of terminal epidermal differentiation, after induced differentiation in high calcium medium (1.5 mM CaCl2), indicating that these iPSCs-KCs have the ability of epidermal differentiation.
Next, iPSCs-KCs-Exos preparations were obtained by ultracentrifuging iPSCs-KCs culture supernatants (Fig. 1C) and analyzing them via transmission electron microscopy (TEM), revealing the presence of exosomes with the expected cup-shaped morphology (Fig. 1D). Nanoparticle tracking analysis (NTA) revealed these particles to have an average diameter of 75 nm (Fig. 1E). All these data suggested that these nanoparticles were actually exosomes.
To test the effects of iPSCs-KCs-Exos treatment on the healing of burn wounds, deep second-degree burns were generated in the abdominal skin of model mice, after which iPSCs-KCs-Exos or an equivalent volume of PBS was locally injected into these areas. Beginning on day 7 post-wounding, significant decreases in wound area in the iPSCs-KCs-Exos group were evident relative to the PBS group, and these improvements grew through day 11 (Fig. 2A). This suggests that iPSCs-KCs-Exos can promote the acceleration of cutaneous wound repair in vivo.
We additionally conducted histological analyses of these healing wounds. Improved re-epithelialization of the wound site was observed in the iPSCs-KCs-Exos treatment group on days 7 and 11 after wounding as compared to the control group when H&E staining was conducted (Fig. 2B, C). Furthermore, there was a significant increase in the length of epithelial tongues on days 7 and 11 in the iPSCs-KCs-Exos group compared to the PBS group (Fig. 2B, D). Immunohistochemical staining for CD31 further revealed that significant improvements in capillary density were evident in the iPSCs-KCs-Exos-treated wounds. (Fig. 2E). However, no differences in collagen deposition were observed when comparing iPSCs-KCs-Exos and PBS group (Fig. 2F). Together, these data revealed a role for iPSCs-KCs-Exos as drivers of re-epithelialization and angiogenesis, thereby enabling them to accelerate the wound healing process.
iPSCs-KCs-Exos promote the migration of keratinocytes and endothelial cells in vitro
To better elucidate the mechanisms where by iPSCs-KCs-Exos treatment can accelerate the burn wound healing, the impact of these exosomes on healing-related cell types (endothelial cells and keratinocytes) was next assessed. EdU assays were applied to determine the effect of iPSCs-KCs-Exos on the proliferation of these two kinds of cells. As shown as in Fig. 3A and B, these iPSCs-KCs-Exos did not alter keratinocytes or HUVECs proliferation at all doses in vitro. In a wound healing assay, iPSCs-KCs-Exos treatment was associated with a significantly increased wound closure rate compared to PBS at doses of 1, 2, and 4 µg/ml (Fig. 2C). When evaluated using Transwell assays, HUVECs migratory activity was also found to be significantly increased following iPSCs-KCs-Exos treatment at doses of 1, 2, and 4 µg/ml (Fig. 2D). As such, iPSCs-KCs-Exos can enhance the migration of endothelial cells and keratinocytes migration without altering the proliferative activity of these cells.
miR-762 is enriched within iPSCs-KCs-Eoxs and serves as a key mediator of iPSCs-KCs-Exos-induced migration of keratinocytes and endothelial cells
While microRNAs (miRNAs) account for a relatively small fraction of the total content within extracellular vesicles, they have nonetheless been shown to be important modulators of cellular function . To test the potential relevance of such miRNAs in the context of iPSCs-KCs-Exos-induced migratory activity and wound healing, miRNA microarray analysis was conducted using these exosomes. The result showed that miR-762 was the most abundant miRNA among these detected miRNAs (Table 1). Thus, we focused on exosomal miR-762 for next investigation. In a wound healing assay with HaCaT cells, the overexpression of miR-762 resulted in enhanced wound closure rates at 24 h post-wounding (Fig. 4A). To further confirm the functional importance of this miRNA, iPSCs-KCs-Exos-treated HaCaT cells were treated with a specific inhibitor of miR-762, which attenuated this enhanced migratory activity (Fig. 4A). The same effect was also observed in HUVECs (Fig. 4B). In addition, the expression of miR-762 in HaCaT cells and HUVECs was indeed significantly elevated at 48 h post-iPSCs-KCs-Exos treatment, confirming the ability of these exosomes to deliver miR-762 to recipient cells (Fig. 4C). These data thus suggest that miR-762 is an important mediator of iPSCs-KCs-Exos-induced migration of keratinocyte and endothelial cells in vitro.
To further clarify the regulatory role of iPSCs-KCs-Exos-derived miR-762 in the context of wound healing, potential target genes for this miRNA were next identified using the Targetscan, miRDB, and microRNA.org databases. This approach identified PML as a putative target of this miRNA. To confirm this prediction, luciferase reporter constructs containing wild-type (WT) or mutated (MUT) versions of the putative miR-762 binding site located within the PML 3’-untranslated region (UTR) were prepared (Fig. 5A). When 293T cells were transfected with these reporters and miR-762 mimics, this miRNA was confirmed to suppress the activity of the WT but not the MUT reporter, confirming the predicted regulatory relationship (Fig. 5B). Moreover, mRNA and protein levels of PML were significantly lower in HaCaT cells and HUVECs following miR-762 mimic or iPSCs-KCs-Exos treatment (Fig. 5C, D). PML is thus a direct miR-762 target gene. To further examine the link between PML downregulation and the effects of miR-762 and iPSCs-KCs-Exos on the migration of keratinocytes and endothelial cells, the PML was overexpressed with pcDNA3.1 vector. As shown as in Fig. 5E, overexpression of PML inhibited the migration of HaCaT cells and could restraine the cell migration that was promoted by miR-762 mimics or iPSCs-KCs-Exos. The similar results were also observed in HUVECs (Fig. 5F). Taken together, above results suggest that miR-762 and iPSCs-KCs-Exos induced acceleration of keratinocytes and endothelial cells is mediated at least partly by inhibiting PML expression.
PML regulated ITGB1 is involved in the promotion of keratinocytes and endothelial cells migration by miR-762 and iPSCs-KCs-Exos.
Reineke et al. previously demonstrated that ITGB1 was responsible for mediating PML-induced changes in migratory activity . In this study, we examined the effects of PML knockdown on ITGB1 expression by siRNA. When PML expression decreased, we observed an increase in ITGB1 mRNA and protein expression in keratinocytes and endothelial cells (Fig. 6A, B). We further found that the expression of ITGB1 also increased after treatment with miR-762 mimic or iPSCs-KCs-Eoxs (Fig. 6C, D). Moreover, siRNA-mediated ITGB1 knockdown ablated the miR-762 mimic and iPSCs-KCs-Eoxs induced migration of HaCaT cells and HUVECs (Fig. 6E, F). Together, these data suggest that miR-762 can target PML to enhance the migration of keratinocyte and endothelial cells through a mechanism involving ITGB1.
Herein, we found that iPSCs-KCs-Exos treatment was sufficient to promote keratinocyte migration and to thereby accelerate the healing of deep second-degree burn wounds in mice. We additionally determined that the most abundant miRNA within these exosomes, miR-762, was a key mediator of this enhanced migratory effect owing to its ability to target PML.
In the process of wound healing, keratinocytes, fibroblasts and endothelial cells are the main cell populations involved in re-epithelialization and granulation tissue construction . Therefore, communication between these cells plays a key role in skin repair. This network is likely regulated by cell-derived exosomes, which carry a range of macromolecular cargos that can be delivered to target cells, enabling them to regulate recipient cell responses in a variety of complex manners [14, 15]. Both HaCaT cells and primary keratinocytes can release exosomes and other extracellular vesicles [27, 28]. In their deep sequencing analysis of microRNAs within keratinocyte-derived extracellular vesicles, Than et al. found that the most enriched miRNAs found therein were linked to key physiological processes such as fibroblast migration and dermal-epithermal wound healing, suggesting a role for these vesicles in the context of cutaneous wound healing .
This study was designed to explore the ability of iPSC-KC-derived exosomes to regulate the process of burn wound healing. We successfully generated and characterized iPSCs-KCs-Exos, which were of the expected size and exhibited characteristic morphological features. Cutaneous wound healing processes are complicated and necessitate interplay between inflammatory, angiogenic, remodeling, proliferative, epithelialization, and fibrotic processes . Histological analyses revealed that increases in re-epithelialization and capillary density were evident in wound sites following iPSCs-KCs-Exos treatment, but collagen formation was not affected. Thus, we then further examined the effects of these exosomes on endothelial cells and keratinocytes, which are the key mediators of angiogenesis and epithelialization, respectively. These analyses revealed that iPSCs-KCs-Exos significantly enhanced the migratory activity of these cells without impacting their proliferative activity. As such, the beneficial impact of these exosomes in the context of cutaneous wound healing may be primarily attributable to their ability to promote keratinocyte and endothelial cell migration.
Exosomes contain a variety of miRNAs, mRNAs, lipids, and proteins that are compiled within the ExoCarta database [14, 15]. Of these, miRNAs are the best-studied exosomal cargos owing to their ability to regulate target mRNA expression. Herein, we detected many miRNAs that were highly abundant within iPSCs-KCs-Exos, of which miR-762 was detected at the highest levels. Moreover, we found that miR-762 was able to promote endothelial cell and keratinocyte migration in a manner similar to iPSCs-KCs-Exos treatment, suggesting that it is likely a primary mediator of the phenotypic effects-KCs-Exos in the context of wound healing. In prior research, miR-762 has been found to regulate a range of target genes in different contexts. The upregulation of miR-762 in non-small cell lung cancer, bladder cancer, breast cancer and nasopharyngeal carcinoma has been linked to enhanced tumor cell invasivity, migration, and proliferation [30–33]. However, there have been no prior studies regarding the role of this specific miRNA in keratinocytes or endothelial cells. In line with these past findings, we observed a pro-migratory role for miR-762 in both of these cell types.
Herein, we identified promyelocytic leukemia (PML) as a novel miR-762 target gene associated with the ability of this miRNA to regulate endothelial cell and keratinocyte migration. PML plays a role in a number of important cellular processes, including tumor suppression, apoptosis, transcriptional regulation, DNA damage response, senescence and viral defense mechanisms [34–36]. Herein, we confirmed that PML was a direct miR-762 target, and we found that overexpressing PML was sufficient to reverse the beneficial impact of miR-762 on cellular migration. In prior reports, the ability of PML to suppress the migration of MDA-MB-231 cells was attributed to its ability to promote ITGB1 downregulation . ITGB1 are important for attachment to and integrity of cell-extracellular matrix (BME) interaction by mediating binding to BME components, and they are generally required for migration of keratinocytes and endothelial cells [37, 38]. We thus explored the link between ITGB1 and this miR-762/PML regulatory axis, leading us to conclude that PML overexpression suppressed ITGB1 expression while miR-762 was able to reverse this effect. We further confirmed a role for ITGB1 in the miR-762/PML-mediated migratory phenotypes observed in this experimental system. However, as miRNAs target multiple genes, these results do not offer any insight into the ability of miR-762 to regulate other signaling pathways within keratinocytes. Moreover, while PML and ITGB1 clearly play a role in this process, further work is required to elucidate the underlying mechanisms.
In conclusion, these results indicate that iPSCs-KCs-Exos treatments can significantly improve burn wound healing outcomes, highlighting novel approaches to expediting cutaneous wound healing and providing a foundation for future research efforts.
induced pluripotent stem cells
keratinocyte defined serum-free Medium
Dulbecco’s modified eagle's medium
human umbilical vein endothelial cells
transmission electron microscopy
nanoparticle tracking analysis
hematoxylin and eosin
quantitative real-time polymerase chain reaction
L.Z. and Y.Y. participated in all aspects of the study, including design, financial support, analyses, final approval of manuscript and revised the manuscript. YY. B. and LJ. Y. performed the experimental work and analyzed the data. BT. L., GP. T. and CX. L. performed the collection and/or assembly of data and revised the manuscript; All authors agreed to be accountable for all aspects of the work.
This work was funded by the National Natural Science Foundation of China (No: 82072186, No: 81872514).
Availability of data and materials
The authors agree to share data and materials related to this manuscript.
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.
Consent for publication
The authors declared no potential conflicts of interest.