Juliana Girón Bastidas, Natasha Maurmann, Juliete Nathali Scholl, Augusto Ferreira Weber, Raíssa Padilha Silveira, Fabricio Figueiró, Marco Augusto Stimamiglio, Bruna Marcon, Alejandro Correa, Patricia Pranke
{"title":"人脱落乳牙(SHED)干细胞及其细胞外囊泡的分泌组改善了角质形成细胞的迁移、活力和H2诱导的细胞毒性的衰减。","authors":"Juliana Girón Bastidas, Natasha Maurmann, Juliete Nathali Scholl, Augusto Ferreira Weber, Raíssa Padilha Silveira, Fabricio Figueiró, Marco Augusto Stimamiglio, Bruna Marcon, Alejandro Correa, Patricia Pranke","doi":"10.1111/wrr.13131","DOIUrl":null,"url":null,"abstract":"<p><p>Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H<sub>2</sub> O<sub>2</sub> in vitro was performed with H<sub>2</sub> O<sub>2</sub> followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 μg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H<sub>2</sub> O<sub>2</sub> -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.</p>","PeriodicalId":23864,"journal":{"name":"Wound Repair and Regeneration","volume":" ","pages":"827-841"},"PeriodicalIF":3.8000,"publicationDate":"2023-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H<sub>2</sub> O<sub>2</sub> -induced cytotoxicity.\",\"authors\":\"Juliana Girón Bastidas, Natasha Maurmann, Juliete Nathali Scholl, Augusto Ferreira Weber, Raíssa Padilha Silveira, Fabricio Figueiró, Marco Augusto Stimamiglio, Bruna Marcon, Alejandro Correa, Patricia Pranke\",\"doi\":\"10.1111/wrr.13131\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H<sub>2</sub> O<sub>2</sub> in vitro was performed with H<sub>2</sub> O<sub>2</sub> followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 μg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H<sub>2</sub> O<sub>2</sub> -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.</p>\",\"PeriodicalId\":23864,\"journal\":{\"name\":\"Wound Repair and Regeneration\",\"volume\":\" \",\"pages\":\"827-841\"},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2023-11-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Wound Repair and Regeneration\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1111/wrr.13131\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2023/12/10 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Wound Repair and Regeneration","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1111/wrr.13131","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/12/10 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Secretome of stem cells from human exfoliated deciduous teeth (SHED) and its extracellular vesicles improves keratinocytes migration, viability, and attenuation of H2 O2 -induced cytotoxicity.
Therapies for wound healing using the secretome and extracellular vesicles (EVs) of mesenchymal stem/stromal cells have been shown to be successful in preclinical studies. This study aimed to characterise the protein content of the secretome from stem cells from human exfoliated deciduous teeth (SHED) and analyse the in vitro effects of SHED-conditioned medium (SHED-CM) and SHED extracellular vesicles (SHED-EVs) on keratinocytes. EVs were isolated and characterised. The keratinocyte viability and migration of cells treated with SHED-EVs and conditioned medium (CM) were evaluated. An HaCaT apoptosis model induced by H2 O2 in vitro was performed with H2 O2 followed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live/dead assays. Finally, the expression of vascular endothelial growth factor (VEGF) in keratinocytes treated with secretome and EVs was evaluated by immunofluorescence staining and confirmed with RT-qPCR. SHED-EVs revealed a cup-shaped morphology with expression of the classical markers for exosomes CD9 and CD63, and a diameter of 181 ± 87 nm. The internalisation of EVs by HaCaT cells was confirmed by fluorescence microscopy. Proteomic analysis identified that SHED-CM is enriched with proteins related to stress response and development, including cytokines (CXCL8, IL-6, CSF1, CCL2) and growth factors (IGF2, MYDGF, PDGF). The results also indicated that 50% CM and 0.4-0.6 μg/mL EVs were similarly efficient for improving keratinocyte viability, migration, and attenuation of H2 O2 -induced cytotoxicity. Additionally, expression of VEGF on keratinocytes increased when treated with SHED secretome and EVs. Furthermore, VEGF gene expression in keratinocytes increased significantly when treated with SHED secretome and EVs. Both SHED-CM and SHED-EVs may therefore be promising therapeutic tools for accelerating re-epithelialization in wound healing.
期刊介绍：
Wound Repair and Regeneration provides extensive international coverage of cellular and molecular biology, connective tissue, and biological mediator studies in the field of tissue repair and regeneration and serves a diverse audience of surgeons, plastic surgeons, dermatologists, biochemists, cell biologists, and others.
Wound Repair and Regeneration is the official journal of The Wound Healing Society, The European Tissue Repair Society, The Japanese Society for Wound Healing, and The Australian Wound Management Association.
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