Background:Hypertrophic scars (HTSs) are characterized by excessive extracellular matrix deposition and impaired scar remodelling. Fibroblasts are central to HTS pathogenesis, yet clinical strategies remain limited by an incomplete understanding of fibroblast heterogeneity and transcriptional regulation. This study aimed to identify a key fibroblast subpopulation and its regulatory transcription factors to address this translational gap. Methods:Single-cell RNA sequencing was performed on dermal cells from freshly excised human HTS and normal skin (NS) tissues. Fibroblast subsets and transcriptional regulators were identified using Seurat, pseudotime, transcription factor prediction, and cell-cell communication analyses. Functional validation involved lentiviral overexpression of Yin Yang 1 (YY1) in fibroblasts derived from patients with hypertrophic scars, followed by bulk RNA sequencing, western blotting, CUT&Tag, and immunofluorescence assays. Results:Clinical HTS specimens showed characteristic collagen overproduction and vascular hyperplasia. Single-cell analysis of 43 303 cells revealed disease-specific shifts in cellular composition, including pronounced pericyte expansion and reduced fibroblast abundance. Notably, fibroblast subcluster Fib_5 (ADAM12+ COMP+ POSTNhi) increased despite the overall fibroblast decline in HTS and exhibited upregulated fibrotic gene expression. Cross-validation using combined public datasets comprising 21 samples indicated that the Fib_5-like subcluster is conserved across fibrotic conditions. Pseudotime analyses placed Fib_5 within an HTS-dominant branch, State 6; transcription factor prediction from branch-dependent differentially expressed genes identified YY1 as the only predicted transcription factor also differentially expressed in State 6. Functional validation showed that YY1 overexpression in fibroblasts derived from patients with HTS reversed fibrotic gene expression profiles, with Fib_5 identified as a primary responder by Scissor. CUT&Tag analysis validated these findings at the epigenomic level. Cell-cell communication analyses further revealed marked reprogramming of fibroblast-pericyte signalling across multiple fibrosis-related pathways in HTS. Conclusion:This work establishes the Fib_5-YY1 axis as a central hub in HTS pathogenesis, with YY1-mediated fibroblast plasticity as a key transcriptional mechanism underlying skin fibrogenesis.

Alternative splicing (AS) diversifies protein expression and contributes to species-specific differences in organ development. Here, we focused on stage-specific splicing variants and their correlation with disease in humans compared to mice during brain and heart development. Temporal transcriptomic analysis revealed that splicing factors (SFs) can accurately classify organ developmental stages, and 5 SFs were identified specifically upregulated in humans during organogenesis. Additionally, inter-stage splicing variations were identified across analogous human and mouse developmental stages. Developmentally dynamic alternative splicing genes (devASGs) were enriched in various neurodevelopmental disorders in both species, with the most significant changes observed in human newborn brain and 16 weeks post-conception heart. Intriguingly, diseases specifically enriched in humans were primarily associated with neuro-muscular dysfunction, and human-specific neuromuscular devASGs were linked to mannose glycosylation and ciliary motility. These findings highlight the significance of SFs and AS events in organogenesis and inform the selection of appropriate models for translational research.

Background Hair follicle-regenerating drugs are essential for alopecia treatment, yet their discovery remains a key challenge in dermatology. Purpose This study aims to identify small molecule compounds that promote hair growth and investigate their underlying mechanisms. Methods A gene panel was designed based on single-cell and bulk RNA-sequencing data, and chemical genomics was employed to screen potential hair-inducing compounds. The lead candidate was validated using an in vivo mouse model of anagen induction and an ex vivo human hair follicle organ culture system. Transcriptomic, metabolomic, and cellular bioenergetics assays were performed to elucidate the potential mechanism, while molecular docking and surface plasmon resonance confirmed its direct protein targets. Results Rhamnose was identified as a compound that restored the expression of hair-inducing genes in human dermal papilla cells (hDPCs). Rhamnose accelerated hair follicle regeneration by stimulating the hair cycle progression from telogen to anagen in mice and promoting ex vivo cultured human scalp hair follicle growth. Rhamnose promoted glycolysis in both the mouse model and hDPCs at both transcriptional and metabolic levels. Mechanically, rhamnose exerted its metabolic effects not as a hexose energy substrate, but rather by upregulating hexokinase 2 (HK2)—a key rate-limiting glycolytic enzyme—and directly binding to HK2 to stimulate its activity. Conclusion This study identifies the natural monosaccharide rhamnose as a metabolic regulator to enhance glycolysis, thereby promoting hair follicle growth, and highlights its potential therapeutic application in alopecia.

OBJECTIVE:To investigate the biological functions of KHDRBS2 and KHDRBS3 in prostate cancer (PCa) progression and their potential roles in regulating androgen receptor splice variant 7 (AR-V7) expression, a key factor in castration-resistant prostate cancer (CRPC). METHODS:We performed a comprehensive analysis of publicly available datasets to examine the expression patterns of KHDRBS family members in PCa, including CRPC and neuroendocrine subtypes. In vitro experiments were conducted using AR-positive 22RV1 and AR-negative PC3 cell lines to assess the expression and regulatory interactions of KHDRBS2 and KHDRBS3. Functional assays evaluated their effects on cell proliferation and tumor growth in vivo. Additionally, KHDRBS2 protein levels were manipulated in 22RV1 cells to assess their impact on AR-V7 and full-length AR expression. RESULTS:Our dataset analysis revealed distinct expression patterns of KHDRBS2 and KHDRBS3, with higher alteration frequencies in CRPC and neuroendocrine PCa. In vitro, KHDRBS2 and KHDRBS3 exhibited mutually exclusive expression, with KHDRBS2 predominantly found in AR-positive 22RV1 cells and KHDRBS3 in AR-negative PC3 cells. Reciprocal regulation between the two proteins was observed in both cell lines. Functional studies showed that both KHDRBS2 and KHDRBS3 promoted cell proliferation and tumor growth. Notably, silencing KHDRBS2 in 22RV1 cells led to a selective reduction in AR-V7 expression, without affecting full-length AR levels. CONCLUSIONS:These findings uncover novel roles for KHDRBS2 and KHDRBS3 in PCa progression, with KHDRBS2 identified as a potential key regulator of AR-V7 expression. Our results provide new insights into AR splice variant regulation and highlight potential therapeutic targets for PCa treatment.

Pursuing compounds capable of stimulating hair follicle growth or regeneration presents a significant challenge in dermatological research. Dermal papilla (DP) cells, which serve as a dynamic niche for hair follicle stem cells, are often used to identify hair growth-promoting agents as a screening model. Here, we integrated human scalp single-cell RNA sequencing data with human DP cells (hDPCs) gene expression profiles to develop a gene panel for assessing hair follicle inductive potential, then applied a high-throughput sequencing-based high-throughput screening (HTS2) method to quantify the gene expressions of hDPCs perturbed by over 2000 small molecules. Notably, rhamnose was identified as a compound that restored the expression of hair-inducing genes in hDPCs. More importantly, the topical application of rhamnose accelerated hair follicle regeneration by promoting hair cycle into the anagen phase. Transcriptome and central carbon metabolism analyses revealed that rhamnose induced glucose metabolism remodeling favoring glycolysis in mice. A similar phenotype was also observed in rhamnose-treated hDPCs, as demonstrated by glycolysis and mitochondrial stress tests. The present study introduces a novel methodology for evaluating hair-inducing agents and provides valuable insights into the cellular and molecular mechanisms underlying the hair growth-promoting effects of rhamnose. These findings highlight the potential for the therapeutic application of rhamnose in the treatment of alopecia. ### Competing Interest Statement The authors have declared no competing interest.

Microvascular aging, predominantly driven by endothelial cells (ECs) dysfunction, is a critical early event in cardiovascular diseases. However, the specific effects of aging on ECs across the microvascular network segments and the associated mechanisms are not fully understood. In this study, we detected a microvascular rarefaction and a decreased proportion of venular ECs in the subcutaneous adipose tissue of aged mice using light-sheet immunofluorescence microscopy and single-cell RNA sequencing. Moreover, aged ECs, especially in the venular subtype, exhibited a pseudotemporal transition to a terminal state characterized by diminished oxidative phosphorylation and strengthened cytokine signaling. Metabolic flux balance analysis predicted that among the 13 differentially expressed cytokines identified in aged EC subpopulations, Cxcl9 was strongly correlated with impaired oxidative phosphorylation in aged ECs. It was further validated using microvascular ECs treated with Cxcl9. Notably, the G protein-coupled receptor signaling pathway was subsequently suppressed, in which Aplnr suppression was also observed in aged ECs, contributing to their impaired energy metabolism and reduced angiogenesis. Based on these findings, we propose Cxcl9 as a biomarker for aging-related dysfunction of microvascular ECs, suggesting that targeting Cxcl9 signaling may help combat microvascular aging.

Keloids are pathological scars exhibiting tumour-like aggressiveness and high recurrence rate. Here we find increased proportion of pro-inflammatory and mesenchymal fibroblast subpopulations and senescent fibroblasts, and enhanced expression of senescence-associated secretory phenotype genes using single-cell RNA sequencing analysis, as well as elevated p16 protein and more β-galactosidase-positive cells in keloids. The up-regulated p53-serine15 phosphorylation (p53-pS15) in keloids is identified by phosphospecific protein microarray and western blotting. We further demonstrate that a senolytic FOXO4-D-retro-inverso-isoform peptide (FOXO4-DRI) promotes apoptosis and decreases G0/G1 phase cells in pro-senescence models of keloid organ cultures and fibroblasts, accompanied with p53-pS15 nuclear exclusion. Our study indicates that upregulation of p53-pS15 and p16 maintains a persistent senescent microenvironment to promote cell cycle arrest and apoptosis resistance in keloid fibroblasts. FOXO4-DRI shows potential as a treatment targeting the senescence and apoptosis resistance, and holds promise as an approach to prevent the aggressiveness and relapse of keloids. Senolytic FOXO4-DRI selectively eliminates senescent fibroblasts by inducing nuclear exclusion of p53 phosphorylation, highlighting the role of the senescent microenvironment in keloid overgrowth and relapse.

Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200 g, 300 g and 400 g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. The 300 g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400 g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200 g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. 300 g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

The human auricle has a complex structure, and microtia is a congenital malformation characterized by decreased size and loss of elaborate structure in the affected ear with a high incidence. Our previous studies suggest that inadequate cell migration is the primary cytological basis for the pathogenesis of microtia, however, the underlying mechanism is unclear. Here, we further demonstrate that microtia chondrocytes show a decreased directional persistence during cell migration. Directional persistence can define a leading edge associated with oriented movement, and any mistakes would affect cell function and tissue morphology. By the screening of motility-related genes and subsequent confirmations, active Rac1 (Rac1-GTP) is identified to be critical for the impaired directional persistence of microtia chondrocytes migration. Moreover, Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) are detected, and overexpression of Tiam1 significantly upregulates the level of Rac1-GTP and improves directional migration in microtia chondrocytes. Consistently, decreased expression patterns of Tiam1 and active Rac1 are found in microtia mouse models, Bmp5se/J and Prkralear-3J/GrsrJ. Collectively, our results provide new insights into microtia development and therapeutic strategies of tissue engineering for microtia patients.

Background Hypertrophic scarring is a disease of abnormal skin fibrosis caused by excessive fibroblast proliferation. Existing drugs have not achieved satisfactory therapeutic effects. Objectives To explore the molecular pathogenesis of hypertrophic scars and screen effective drugs for their treatment. Methods Existing human hypertrophic scar RNA sequencing data were utilized to search for hypertrophic scar-related gene modules and key genes through weighted gene co-expression network analysis (WGCNA). Candidate compounds were screened in a compound library. Potential drugs were screened by molecular docking and verified in human hypertrophic scar fibroblasts and a mouse mechanical force hypertrophic scar model. Results WGCNA showed that hypertrophic scar-associated gene modules influence focal adhesion, the transforming growth factor (TGF)-β signalling pathway and other biologic pathways. Integrin β1 (ITGB1) is the hub protein. Among the candidate compounds obtained by computer virtual screening and molecular docking, crizotinib, sorafenib and SU11274 can inhibit the proliferation and migration of human hypertrophic scar fibroblasts and profibrotic gene expression. Crizotinib had the best effect on hypertrophic scar attenuation in mouse models. At the same time, mouse ITGB1 small interfering RNA can also inhibit mouse scar hyperplasia. Conclusions ITGB1 and TGF-β signalling pathways are important for hypertrophic scar formation. Crizotinib could be a potential treatment drug for hypertrophic scars.

Background: Mesenchymal stromal cells (MSCs) and Dexamethasone (Dex) are both effective methods to treat inflammatory diseases. However, the interaction between inflammatory factors, Dex, and MSCs in repair is not fully understood. The purpose of this study is to clarify the effects and mechanisms of glucocorticoids on the tissue repair characteristics of MSCs in an inflammatory environment. Methods: This is an experimental study. Human adipose-derived mesenchymal stromal cells (hASCs) were cultured, and Long non-coding RNA (lncRNA) differentiation antagonizing nonprotein coding RNA (DANCR) expression was detected after treatment with Dex and inflammation factors. Additionally, DANCR was knockdown or overexpressed before Dex or tumor necrosis factor-alpha (TNF-α) treatments, respectively. hASC proliferation, cell cycle, and migration ability were analyzed to evaluate the effects of DANCR in hASCs treated with Dex or TNF-α. Nuclear factor-kB (NF-κB) pathway inhibitors were used to clarify the signal pathway that DANCR involved. All data are presented as the mean ± standard deviation. The two-tailed Student's t-test or one-way analysis of variance (ANOVA) was used to determine the statistical differences between groups. Results: Dex decreased the proliferation and migration of hASCs and upregulated DANCR expression in a dosage-dependent relationship. The knockdown of DANCR reversed Dex's repression of hASC proliferation. Moreover, DANCR was decreased by inflammatory cytokines, and overexpressing DANCR alleviated the promotion effects of TNF-α on hASC proliferation and migration. Furthermore, mechanistic investigation validated that DANCR was involved in the NF- κB signaling pathway. Conclusions: We identified a lncRNA, DANCR, that was involved in Dex and inflammation-affected hASC proliferation and migration. Dex reduced the proliferation and migration of hASCs through DANCR while exerting its anti-inflammatory effects. Thus, it is suggested to avoid the simultaneous application of hASCs and steroids in clinical practice. These results enrich our understanding of the versatile function of lncRNAs in the crosstalk of inflammation conditions and MSCs.

Objective:To explore the cellular heterogeneity and the differences in branched trajectory of pericytes between keloids and localized scleroderma, and to provide new clues for the pathogenesis and therapeutic targets of the two skin fibrotic diseases.Methods:Single cell transcriptome sequencing (scRNA-seq) data of 3 cases of scleroderma, 4 cases of keloid and their corresponding 4 cases of adjacent normal skin samples were selected from GEO and GSA-Human databases, and the expression matrix of the data was drawn. Seurat 4.3.0 of R (4.2.2) was used to process the t-distributed stochastic neighbor embedding ( t-SNE) visualization map. Monocle 2.24.0 was used to analyze the pseudo-temporal trajectory of pericytes. Results:The unsupervised clustering of keloid and scleroderma skin tissues revealed 19 different cell populations, among which C7 and C11 cells were pericytes, marked by high expression levels of PDGFRB and RGS5 genes, accounting for 7.53% of the total cells. Pericytes can be further divided into 8 subgroups. Pseudo-temporal analysis revealed a branched trajectory with two major branches, that is, cell fate 1 and cell fate 2, which could be further divided into 5 cellular states of pericytes (S1-S5). S4 constituted the most of the prebranch, which represented the cellular state of the initial pericyte phenotype. S5 constituted the most of the cell fate 1 branch, which represented the early differentiation state of the pericyte phenotype. S1, S2, S3 constituted the most of the cell fate 2 branch. S3 represented the intermediate differentiation state of the pericyte phenotype, while S1 and S2 represented the terminal differentiation states of the pericyte phenotype. Compared with the uniform distribution of various differentiation states of pericytes in normal skin, the keloid pericytes mainly distributed in the prebranch (S4), cell fate 1 (S5) and the first half of cell fate 2 (S3), representing cellular states of the initial, early and intermediate phases of the pericyte phenotype. Branched expression analysis modeling revealed the overexpression of SOX4, COL4A1, COL6A3, AHR, CXCL3 and IL1R1 genes, et cetera. On the other hand, the localized scleroderma pericytes mainly distributed in the bottom half of cell fate 2 (S1, S2), representing the final differentiated phase of pericyte phenotype, which overexpressed ACTA2 and MYH11 genes.Conclusion:Pericytes in keloid and scleroderma are heterogenous and have different differentiation trajectories. Pericytes in keloid have stem-like characteristics, and play an important role in the pathologic characteristics of invasiveness and recurrence through high expression of genes related to cell stemness, epithelial-mesenchymal transition, invasiveness, and immune microenvironment regulation. However, pericytes in localized scleroderma may mainly transdifferentiate into myofibroblasts, leading to their fibrotic pathological phenotype.

Neural crest-derived cells play essential roles in skin function and homeostasis. However, how they interact with environmental cues and differentiate into functional skin cells remains unclear. Using a combination of single-cell data analysis, neural crest lineage tracing, and flow cytometry, we found that the expression of integrin α6 (ITGA6) in neural crest and its derivatives was developmentally regulated and that ITGA6 could serve as a functional surface marker for distinguishing neural crest derivatives in the skin. Based on the expression of ITGA6, Wnt1-Cre lineage neural crest derivatives in the skin could be categorized into three subpopulations, namely, ITGA6bright, ITGA6dim, and ITGA6neg, which were found to be Schwann cells, melanocytes, and fibroblasts, respectively. We further analyzed the signature genes and transcription factors that specifically enriched in each cell subpopulation, as well as the ligand or receptor molecules, mediating the potential interaction with other cells of the skin. Additionally, we found that Hmx1 and Lhx8 are specifically expressed in neural crest-derived fibroblasts, while Zic1 and homeobox family genes are expressed in mesoderm-derived fibroblasts, indicating the distinct development pathways of fibroblasts of different origins. Our study provides insights into the regulatory landscape of neural crest cell development and identifies potential markers that facilitate the isolation of different neural crest derivatives in the skin.

Adipose-derived stem cells (ASCs) from distinct age groups possess different characteristics; however, the age-associated changes in ASCs heterogenicity remain largely unknown. In this study, several publicly available single-cell RNA sequencing (RNA-seq) data cohorts of inguinal adipose tissues, including young (2 weeks), adult (8 weeks), and old (18 months) C57BL/6 mice, were analyzed. Transcriptomic clustering of integrated single-cell RNA-seq data from different age groups revealed the existence of five ASCs subtypes. Interestingly, ASCs showed a loss of heterogeneity with aging, and ASCs subtype 4 (ASC-4) was the dominant subpopulation accounting for more than 98% of aged ASCs converging to the terminal differentiation state. The multidirectional differentiation potentials of different ASCs subtypes were largely distinct while the adipogenic ability of ASC-4 increased with age persistently. Regulon analysis of ASC subtypes further identified Cebpb as the ASC-4-specific transcription factor, which was known as one of the major adipogenic regulators. Analysis of ligand-receptor pairs between ASCs and other cell types in adipose tissue identified age-associated upregulation of inflammatory responses-associated factors including CCL2 and CCL7. Treatment with 100 ng/mL CCL2 in vitro could significantly promote the adipogenesis of ASCs through enhanced phosphorylation of AKT and decreased expression of beta-catenin. In addition, supplementation of 100 ng/mL CCL7 could significantly increase the expression of inflammatory genes and ASC-4-specific transcriptional factors in 2-week-old ASCs, potentially acting as a driver of ASCs convergence. Our findings help to delineate the complex biological processes of ASCs aging and shed light on better regenerative and therapeutic applications of ASCs.

BACKGROUND:Hypertrophic scar is a fibrotic disease following wound healing and is characterized by excessive extracellular matrix deposition. Autologous microfat grafting proves an effective strategy for the treatment thereof as it could improve the texture of scars and relieve relevant symptoms. This study aims to explore the potential mechanisms underlying the anti-fibrotic effect of microfat on hypertrophic scars.METHODS:In this study, we injected microfat into transplanted hypertrophic scars in mouse models and investigated the subsequent histological changes and differential expression of mRNAs therein. As for in vitro studies, we co-cultured microfat and hypertrophic scar fibroblasts (HSFs) and analyzed molecular profile changes in HSFs co-cultured with microfat by RNA sequencing. Moreover, to identify the key transcription factors (TFs) which might be responsible for the anti-fibrotic function of microfat, we screened the differentially expressed TFs and transfected HSFs with lentivirus to overexpress or knockdown certain differentially expressed TFs. Furthermore, comparative secretome analyses were conducted to investigate the proteins secreted by co-cultured microfat; changes in gene expression of HSFs were examined after the administration of the potential anti-fibrotic protein. Finally, the relationship between the key TF in HSFs and the microfat-secreted anti-fibrotic adipokine was analyzed.RESULTS:The anti-fibrotic effect of microfat was confirmed by in vivo transplanted hypertrophic scar models, as the number of α-SMA-positive myofibroblasts was decreased and the expression of fibrosis-related genes downregulated. Co-cultured microfat suppressed the extracellular matrix production of HSFs in in vitro experiment, and the transcription factor ETV4 was primarily differentially expressed in HSFs when compared with normal skin fibroblasts. Overexpression of ETV4 significantly decreased the expression of fibrosis-related genes in HSFs at both mRNA and protein levels. Fetuin-A secreted by microfat could also downregulate the expression of fibrosis-related genes in HSFs, partially through upregulating ETV4 expression.CONCLUSIONS:Our results demonstrated that transcription factor ETV4 is essential for the anti-fibrotic effect of microfat on hypertrophic scars, and that fetuin-A secreted by microfat could suppress the fibrotic characteristic of HSFs through upregulating ETV4 expression. Microfat wields an alleviative influence over hypertrophic scars via fetuin-A/ETV4 axis.

Objective:To establish mesenchymal cell bicaudal-C (Bicc1) gene conditional knockout mice models and analyze their phenotypes.Methods:Bicc1 f/+ mice were crossed with Pdgfra promoter-driven Cre mice to obtain the offspring mice. Genomic DNA was extracted from the toe and tail tissues from 1-2 weeks old mice, amplified by PCR and detected at the DNA level by agarose gel electrophoresis. Three Bicc1 gene conditional knockout mice (experimental group) and three wild-type mice (control group) were selected after identification and grew to 3 weeks of age for follow-up experiments. The Bicc1 gene was knocked out by the induction of tamoxifen intraperitoneal injection. After 1 week, the kidney, skeletal muscle, skin and adipose tissue samples were collected. Real-time quantitative PCR (RT-qPCR) was performed to determine the expression levels of Bicc1 mRNA in the collected tissue samples. HE and Masson staining were performed with tissue samples fixed in 10% paraformaldehyde, and observed with a light microscope. The SPSS 28.0 software was used to analyze the data, t-test was used for comparison between groups, and P<0.05 was considered statistically significant. Results:Mesenchymal cell Bicc1 gene conditional knockout mice models were obtained by breeding, and the genotype was Bicc1 f/fCre +/-. The genotype of the wild-type mice was Bicc1 f/fCre -/-. RT-qPCR showed that the expression levels of Bicc1 mRNA in kidney, skeletal muscle, skin and adipose tissue of the experimental mice were significantly lower than those of the control group (all P<0.01). HE staining and Masson staining showed that compared with the control group, glomerular atrophy could be observed in the experimental group, renal capsules were irregular in shape, and some renal capsules disappeared. The arrangement of skeletal muscle fibers were loose and scattered, and the accumulation of muscle fibers was not dense. There were no significant differences between the skin and adipose tissue. Conclusion:Mesenchymal cell Bicc1 gene conditional knockout mice models were successfully established, which could provide models for studying the mechanisms of action of Bicc1 gene in different tissues and organs. Mesenchymal cell conditional Bicc1 gene knockout affected the phenotypes of kidney and skeletal muscle in mice.

The molecular mechanism underlying white adipogenesis in humans has not been fully elucidated beyond the transcriptional level. Here, we found that the RNA-binding protein NOVA1 is required for the adipogenic differentiation of human mesenchymal stem cells. By thoroughly exploring the interactions between NOVA1 and its binding RNA, we proved that NOVA1 deficiency resulted in the aberrant splicing of DNAJC10 with an in-frame premature stop codon, reduced DNAJC10 expression at the protein level and hyperactivation of the unfolded protein response (UPR). Moreover, NOVA1 knockdown abrogated the down-regulation of NCOR2 during adipogenesis and up-regulated the 47b+ splicing isoform, which led to decreased chromatin accessibility at the loci of lipid metabolism genes. Interestingly, these effects on human adipogenesis could not be recapitulated in mice. Further analysis of multispecies genomes and transcriptomes indicated that NOVA1-targeted RNA splicing is evolutionarily regulated. Our findings provide evidence for human-specific roles of NOVA1 in coordinating splicing and cell organelle functions during white adipogenesis.

目的 比较两种消化试剂传代对人脂肪干细胞生物学特性的影响,探寻可替代胰蛋白酶的细胞消化试剂.方法 采用类胰蛋白酶(TrypLE Express)与胰蛋白酶(Trypsin)两种细胞消化试剂,对原代提取的人脂肪干细胞生物学特性及成脂分化能力进行比较.结果 第三代脂肪干细胞在TrypLETM Express与Trypsin试剂消化条件下活细胞百分比无显著性差异(P>0.05).流式细胞仪检测结果表明,CD90、CD73、CD105等细胞表面抗原标志物呈现阳性表达,CD11b、CD34、CD19、CD45、HLA-DR等呈现阴性表达,两组消化试剂作用下阳性细胞的百分比无显著性差异(P>0.05).成脂诱导细胞脂滴染色结果用Imagepro plus进行半定量分析,两组消化试剂作用下成脂分化率无显著性差异(P>0.05),接种第1～7天细胞增殖能力无明显差异(P>0.05).结论 两种消化试剂作用对人脂肪干细胞生物学特性的影响无显著性差异,TrypLE Express可完全代替Trypsin用于脂肪干细胞体外传代培养.