Loxl2 Derived From Amniotic Mesenchymal Stem Cells Accelerates Wound Healing And Its Mechanism

Objective: Skin wound healing is a complex,evolutionarily conserved,dynamic biological process aimed toward to restore the anatomical integrity and physiological function of skin tissue.The process involves the coordinated efforts of multiple cell types(including keratinocytes,fibroblasts,endothelial cells,macrophages,and platelets)and is executed and regulated by numerous growth factors,cytokines and chemokines.Studies have shown that the keratinocyte is the key impact both epidermal homeostasis and the immune defense.And it is crucial in both beginning of traumatic stress and processing of re-epithelialization of wound repair.Amniotic membrane as a new type of biological dressing has been studied for treatment for various wounds.The value of the human amniotic membrane has been thoroughly proven,because of its immunosuppressive properties,simple ethical problems and composition.Amniotic membrane is composed of amniotic mesenchymal stem cells(hAMSCs)and amniotic epithelial cells(hAECs).We have been known that hAECs media conditioned(h AECs-CM)could regulate inflammation and promote neovascularization to promote diabetic wound healing.It also promotes wound healing by facilitating the migration and proliferation of keratinocytes.However,hAECs were found as a less reliable source than hAMSCs and altered morphology during subculture.And cellular therapies would require several billion cells from each cell line for multiple dosing regimens and,importantly,to prevent micro-chimerism and potential immune responses arising from cells that have been pooled from several unrelated donors.Studies showed that expansion led to significant differences in the expression of markers,capacity to differentiate,ability to suppress T cell proliferation and the secretion of immunosuppressive factors by hAEC.It is hard to stably producing conditioned media and clinical product development.Therefore,we are looking for a source of cells that can be effectively replaced.Based on our earlier work,we found that hAMSCs treatment on wound rats could effectively improve the efficiency of wound healing and promote re-epithelialization.To further analyze the mechanism and understand the effect of exocrine proteins in amniotic stem cells on keratinocytes,we compared human amniotic mesenchymal stem cell conditioned medium(hAMSCs-CM)and human amniotic epithelial cell conditioned medium(hAECs-CM)on keratinocytes.Our study objectives are: 1)Preparation of conditioned medium for hAMSCs and hAECs with Epilife to test the effects of the conditioned media on the biological function of keratinocytes.2)Test the effect of hAMSCs-CM on skin wound healing rate and re-epithelialization using mouse skin full-thickness injury model.3)Identify the secreted proteomes of the two cells by Label-free MS analysis and with bioinformatics analysis to find the differences between them.4)Based on the results upon,screening of proteins with positive effects of hAMSCs on wound healing.5)In-depth study of the ability of the target protein(LOXL2)to promote wound healing and its mechanism.Methods: The effect of hAMSCs-CM on keratinocyte biology and wound healing 1.Isolated hAMSCs and hAECs from amniotic membrane tissue,and identify them byflow cytometry,cellular immunofluorescence and multiple differentiation potentials.2.Isolated keratinocytes and identify by immunofluorescence and high calcium culturedifferentiation.3.Use Epilife medium culture amniotic stem cells and collect conditioned medium forsubsequent experiments.hAECs served as a positive control.4.Evaluate the effect of conditioned medium of amniotic stem cells on the migration ofkeratinocytes by cell scratch test.5.Evaluate the changes of keratinocyte proliferation by MTS test and flow cytometry.6.Evaluate the differentiation maker of keratinocytes by real-time PCR and WesternBlot.7.To explore the effect of hAMSCs-CM on the wound healing rate by C57 / BL mouseskin incision wound model.Identification and analysis of hAMSCs and hAECs secreted proteome 1.Using Label-Free mass spectrometry,the proteins of hAMSCs-CM and hAECs-CMwere identified.2.With bioinformatics analysis screen out differentially expressed proteins related towound healing.3.Validate the expression of candidate protein in conditioned medium by ELISA test.The effect of LOXL2 on re-epithelialization and its mechanism 1.Evaluate the migration of keratinocytes with candidate LOXL2,LGALS1 andCTHRC1 by cell scratch test.2.Real-time PCR and Western Blot were used to detect differentiation indicatorschanges in keratinocyte with LOXL2.MTS test to evaluate the effect of LOXL2 onkeratinocyte proliferation.3.The effect of SiLOXL2 hAMSCs-CM on the migration of keratinocytes was testedby cell scratch test.4.To explore the effect of LOXL2 on the wound healing rate through the C57 / BLmouse back skin wound model.5.According to KEGG analysis,screen out the potential pathways.6.Evaluate the effect of JNK inhibitors on the migration and differentiation ofkeratinocytes.7.Detect the changes of JNK,p-JNK and p-YAP1 after treatment with LOXL2 byWestern Blot.Results: hAMSCs-CM promotes keratinocyte migration and accelerates wound re-epithelialization 1.The hAMSCs into a fibrous,fish-like distribution;hAECs were pebbled.Both typesof amniotic stem cells can differentiate into osteoblasts,adipocytes and chondrocytes.Flow cytometric identification of surface markers CD73(+),CD45(-),CD31(-),CD105(+),CD44(+),CD34(-);hAECs: CD90(+),CD29(+),SSEA-3(-),SSEA-4(+),EP-CAM(50% +),HLA-DR(-)and above meet the characteristics of amnioticstem cells.2.Compared with the normal culture system,hAECs-CM obviously promotes themigration of keratinocytes to the scratched area,while hAMSCs-CM is weaker;hAMSCs-CM significantly inhibits the proliferation of keratinocytes thenhAECs-CM;hAMSCs-CM and hAECs-CM can significantly reduce differentiationindicators,there is no significant difference between the two.3.Compared with PBS group,hAMSCs-CM can effectively accelerate the speed ofwound healing In vivo.LOXL2 promotes the migration and differentiation of keratinocytes to accelerates re-epithelializes via JNK-YAP1 pathway 1.LOXL2 can significantly accelerate the migration of keratinocytes to the scratchedarea.2.Loss of LOXL2-hAMSCs-CM significantly reduced the migration ability ofkeratinocytes.3.LOXL2 can significantly increase the expression of keratinocyte differentiationmaker.4.LOXL2 can effectively promote the wound healing rate and restore the integrity ofthe epidermis.5.The pre-treatment of JNK inhibitor obviously affected the ability of LOXL2 topromote the migration of keratinocytes and the increase of differentiation maker.6.LOXL2 can activate keratinocyte JNK and YAP1 phosphorylation.Conclusion: hAMSCs-CM can promote the migration of keratinocytes,accelerate the rate of wound healing in mice with back trauma,and promote re-epithelialization.It is an important research and development resource to identify hAMSCs rich in regulatory factors by MS analysis.The hAMSCs exocrine protein LOXL2 promotes the migration and differentiation of keratinocytes to accelerating wound re-epithelialization via JNK-YAP.