Mechanisms Of Adipose-derived Stem Cells Ameliorating Localized Scleroderma

Objective:This study aims to collect the clinical data of the fat retention of localized scleroderma(LoS)patients,analyze the differences in the composition of adipose tissues in LoS patients,and explore the potential mechanism of adipose-derived stem cells(ASCs)in the treatment of LoS.Methods:1)The imaging data of LoS patients undergoing autologous fat grafting were evaluated,whose fat retention was measured by magnetic resonance imaging(MRI).2)The stromal vascular fraction(SVF)derived from the adipose tissues of LoS patients and healthy people were analyzed by single-cell sequencing.3)The LoS mice models were established by bleomycin.Mice were divided into 6 groups:healthy blank control,LoS disease model group,fat grafting group,low-dose ASCs+fat grafting group,medium-dose ASCs+fat grafting group,and high-dose ASCs+fat grafting group.The backs of nude mice were injected with phosphate buffer,fat,or fat mixed with different doses of ASCs,respectively.The skin fibrosis and fat retention were measured after one or three months.Results:1)Six LoS patients,including four female and two male,were assessed by MRI scan,whose fat retention was 21.86%±1.68%six months post-operation.2)The SVF isolated from the subcutaneous adipose tissue specimens from the thighs of four linear scleroderma patients and five healthy people were subjected to single-cell sequencing,and a transcriptome of 58,425 cells was obtained(Scleroderma group:18,421 cells;Control group:40,004 cells),which was divided into 21 clusters and 10 cell lineages.The proportion of ASCs in SVF was 49.17%(58.73%in the scleroderma group and 44.76%in the control group),and the proportion of macrophages was 14.62%(7.42%in the scleroderma group and 17.94%in the control group).The visualization of cell density showed that the relative proportions of multiple lineages including ASCs,macrophages and T cells significantly changed.Among the five subpopulations of ASCs characterized,c5 subpopulation expressed a series of fibrosis-related markers,indicating it may be the subpopulation closely related to the fibrosis and pathological changes of scleroderma.Among the three subpopulations of macrophage characterized,c4 subpopulation,confirmed as a subpopulation of tissue-resident macrophages with the M2 phenotype,was found to be significantly reduced in the scleroderma group.The dysregulated pathways and genes of ASCs in LoS patients were identified:the expression of genes related to collagen biosynthesis was up-regulated,and the expression of genes involved in the regulation of transforming growth factor?-regulated "SMAD2-SMAD3-SMAD4 heterotrimers regulated transcription" was down-regulated.3)The histological analysis showed that compared with the LoS disease model group,the skin fibrosis and fat retention in ASCs-fat grafting groups significantly improved.The immunohistochemical relative density of type ?collagen and TGF-?1 in ASCs-fat grafting groups decreased significantly with the increase of ASC concentration.The fat retention was 0.022±0.015g in the fat grafting group,0.026±0.012g in the low-dose ASCs-fat grafting group,0.041±0.004g in the medium-dose ASCs-fat grafting group,and 0.068±0.013g in the high-dose ASCs-fat grafting group.The expression levels of angiogenesis-related cytokines,platelet endothelial cell adhesion molecules,as well as the expression of upstream and downstream signaling molecules in the AKT/ERK signaling pathway increased significantly with the increase of ASC concentration.Conclusion:1)The fat retention of LoS patients is significantly lower than that of healthy people reported in the literature.2)This study conducted the first comprehensive analysis of the heterogeneity and cell lineage of SVF cells derived from the normal adipose tissue of LoS patients,showing that an immune dysfunction characterized by significant consumption of anti-inflammatory macrophages occurred in the adipose tissue of scleroderma patients as well as a change of the cell density of ASCs and the cell subpopulations and gene expression related to fibrosis regulation of ASCs.3)ASCs promote angiogenesis and adipogenesis in a dose-dependent fashion,attenuating the skin fibrosis as well as improving the fat retention in the LoS models.