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Background: Musculoskeletal injuries and chronic degenerative diseases pose significant challenges in equine health, impacting performance and overall well-being. Sex Hormone-Binding Globulin (SHBG) is a glycoprotein determining the bioavailability of sex hormones in the bloodstream, and exerting critical metabolic functions, thus impacting the homeostasis of many tissues including the bone., Methods: In this study, we investigated the potential role of SHBG in promoting osteogenesis and its underlying mechanisms in a model of equine adipose-derived stromal cells (ASCs). An SHBG-knocked down model has been established using predesigned siRNA, and cells subjected to osteogenic induction medium in the presence of exogenous SHBG protein. Changes in differentiation events where then screened using various analytical methods., Results: We demonstrated that SHBG treatment enhances the expression of key osteoconductive regulators in equine ASCs CD34 + cells, suggesting its therapeutic potential for bone regeneration. Specifically, SHBG increased the cellular expression of BMP2/4, osteocalcin (OCL), alkaline phosphatase (ALP), and osteopontin (OPN), crucial factors in early osteogenesis. Furthermore, SHBG treatment maintained adequate apoptosis and enhanced autophagy during osteogenic differentiation, contributing to bone formation and remodeling. SHBG further targeted mitochondrial dynamics, and promoted the reorganization of the mitochondrial network, as well as the expression of dynamics mediators including PINK, PARKIN and MFN1, suggesting its role in adapting cells to the osteogenic milieu, with implications for osteoblast maturation and differentiation., Conclusion: Overall, our findings provide novel insights into SHBG's role in bone formation and suggest its potential therapeutic utility for bone regeneration in equine medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Irwin-Huston, Bourebaba, Bourebaba, Tomal and Marycz.)

In recent years, clinical studies have shown positive results of the application of Mesenchymal Stromal Cells (MSCs) in severe cases of COVID-19. However, the mechanisms of immunomodulation of IFN-γ licensed MSCs in SARS-CoV-2 infection are only partially understood. In this study, we first tested the effect of IFN-γ licensing in the MSC immunomodulatory profile. Then, we established an in vitro model of inflammation by exposing Calu-3 lung cells to SARS-CoV-2 nucleocapsid and spike (NS) antigens, and determined the toxicity of SARS-CoV-2 NS antigen and/or IFN-γ stimulation to Calu-3. The conditioned medium (iCM) generated by Calu-3 cells exposed to IFN-γ and SARS-CoV-2 NS antigens was used to stimulate T-cells, which were then co-cultured with IFN-γ-licensed MSCs. The exposure to IFN-γ and SARS-CoV-2 NS antigens compromised the viability of Calu-3 cells and induced the expression of the inflammatory mediators ICAM-1, CXCL-10, and IFN-β by these cells. Importantly, despite initially stimulating T-cell activation, IFN-γ-licensed MSCs dramatically reduced IL-6 and IL-10 levels secreted by T-cells exposed to NS antigens and iCM. Moreover, IFN-γ-licensed MSCs were able to significantly inhibit T-cell apoptosis induced by SARS-CoV-2 NS antigens. Taken together, our data show that, in addition to reducing the level of critical cytokines in COVID-19, IFN-γ-licensed MSCs protect T-cells from SARS-CoV-2 antigen-induced apoptosis. Such observations suggest that MSCs may contribute to COVID-19 management by preventing the lymphopenia and immunodeficiency observed in critical cases of the disease., (© 2024. The Author(s).)

There is little research concerning the role of stem cells in necrotizing enterocolitis (NEC). Bone marrow-derived mesenchymal stem cells (BMDSC) and amniotic fluid-derived stem cells significantly reduced the amount and severity of NEC in the animal models. ADSCs share similar surface markers and differentiation potential with BMDSCs. Their potential role in the setting of NEC has not been researched before. The hypothesis of the study was that prophylactic intraperitoneal administration of ADSCs before the onset of the disease will result in limiting the inflammatory response, effecting a lower incidence of NEC. On a molecular level, this should result in lowering the levels of inflammatory cytokines IL-1 and IL-6. The local ethical committee for animal experiments approval was acquired (WAW2/093/2021). We utilized a self-modified rat NEC model based on single exposure to hypothermia, hypoxia, and formula feeding. One hundred and twenty-eight rat puppies were divided into two groups-prophylaxis (ADSC-NEC, n = 66) and control group (NEC-PLCB, n = 62)-to measure the influence of ADSCs administration on the inflammatory changes in NEC, the level of cell engraftment, and the histopathology of the disease. The analysis did not show a significant effect on histopathology between groups, H(2) = 2.12; p = 0.347; η²H = 0.00. The intensity of the NEC variable results was similar across the analyzed groups (NEC-PLCB and ADSC-NEC). For IL-1 and IL-6, the difference between the NEC-PLCB group and the ADSC-NEC group was statistically significant, p = 0.002 and p < 0.001, respectively. To conclude, administration of adipose tissue-derived stem cells before the onset of the disease lowers the levels of inflammatory cytokines IL-1 and IL-6 but does not affect the histopathological results in the rat model of NEC.

Intra-articular injection of mesenchymal stem cells (MSCs) is envisioned as a solution for knee osteoarthritis (OA). Although synovial MSCs (SyMSCs) are promising for cartilage regeneration, the clinical choice is usually adipose MSCs (AdMSCs). However, the similarities/differences in the mode of action between SyMSCs and AdMSCs remain unclear. Here, we compared factors secreted by human SyMSCs and AdMSCs after injection into OA knees. Human SyMSCs or AdMSCs were injected into the knees of rat partial meniscectomy models. The next day, the knee joints were collected to analyze the distribution of injected MSCs and transcriptome changes in the human MSCs and rat synovium. Non-injected MSCs were mixed with rat synovium as a control. After injection, no difference was apparent in intra-articular distribution of the SyMSCs or AdMSCs. RNA sequencing demonstrated an enrichment of cytokine-cytokine receptor interaction-related genes in both human SyMSCs and AdMSCs after injection. Differentially expressed genes (DEGs) specific to SyMSCs were associated with cartilage matrix synthesis and homeostasis. PCR analysis of the matrisome-related DEGs showed significantly higher expression of PRG4 in SyMSCs than in AdMSCs after injection. Immunostaining also confirmed a significantly greater expression of lubricin by SyMSCs than by AdMSCs. These findings indicate that SyMSCs will be a more promising treatment for OA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adipose tissue is distributed in diverse locations throughout the human body. Not much is known about the extent to which anatomically distinct adipose depots are functionally distinct, specialized organs, nor whether depot-specific characteristics result from intrinsic developmental programs, as opposed to reversible physiological responses to differences in tissue microenvironment. We used DNA microarrays to compare mRNA expression patterns of isolated human adipocytes and cultured adipose stem cells, before and after ex vivo adipocyte differentiation, from seven anatomically diverse adipose tissue depots. Adipocytes from different depots display distinct gene expression programs, which are most closely shared with anatomically related depots. mRNAs whose expression differs between anatomically diverse groups of depots (e.g., subcutaneous vs. internal) suggest important functional specializations. These depot-specific differences in gene expression were recapitulated when adipocyte progenitor cells from each site were differentiated ex vivo, suggesting that progenitor cells from specific anatomic sites are deterministically programmed to differentiate into depot-specific adipocytes. Many developmental transcription factors show striking depot-specific patterns of expression, suggesting that adipocytes in each anatomic depot are programmed during early development in concert with anatomically related tissues and organs. Our results support the hypothesis that adipocytes from different depots are functionally distinct and that their depot-specific specialization reflects distinct developmental programs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Clemons et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We fabricated three-dimensional (3D)-printed polycaprolactone (PCL) and PCL/graphene oxide (GO) (PGO) scaffolds for bone tissue engineering. An anti-inflammatory and pro-osteogenesis drug dexamethasone (DEX) was adsorbed onto GO and a 3D-printed PGO/DEX (PGOD) scaffold successfully improved drug delivery with a sustained release of DEX from the scaffold up to 1 month. The physicochemical properties of the PCL, PGO, and PGOD scaffolds were characterized by various analytical techniques. The biological response of these scaffolds was studied for adherence, proliferation, and osteogenic differentiation of seeded rabbit adipose-derived stem cells (ASCs) from DNA assays, alkaline phosphatase (ALP) production, calcium quantification, osteogenic gene expression, and immunofluorescence staining of osteogenic marker proteins. The PGOD scaffold was demonstrated to be the best scaffold for maintaining cell viability, cell proliferation, and osteogenic differentiation of ASCs in vitro. In vivo biocompatibility of PGOD was confirmed from subcutaneous implantation in nude mice where ASC-seeded PGOD can form ectopic bones, demonstrated by microcomputed tomography (micro-CT) analysis and immunofluorescence staining. Furthermore, implantation of PGOD/ASCs constructs into critical-sized cranial bone defects in rabbits form tissue-engineered bones at the defect site, observed using micro-CT and histological analysis.

Background: Salivary hypofunction leads to debilitating oral symptoms and has major complications for overall quality of life. Two of the most frequent causes of xerostomia are radiotherapy in the head and neck and Sjögren's syndrome. Only symptomatic treatment is available today. An increasing number of both preclinical and clinical studies have suggested that mesenchymal stem cell (MSC) transplantation treatment can increase the salivary flow rate and ameliorate symptoms of xerostomia. However, both adipose-derived and bone marrow-derived MSCs are used, although they differ in important ways. The primary objective of this study is an indirect comparison of the change in the unstimulated salivary flow rate after intervention between patients treated with adipose-derived or bone marrow-derived MSCs., Methods: This systematic review and network meta-analysis will search for eligible studies in the MEDLINE, EMBASE, and Cochrane CENTRAL register of Controlled Trials. Eligible studies are as follows: clinical studies including human patients with salivary hypofunction due to either radiotherapy or Sjogren's syndrome who were subsequently treated with either adipose-derived MSCs or bone marrow-derived MSCs. Studies with no control group will be excluded. The search phrase has been peer-reviewed following the PRESS guidelines. The primary outcome is the change in the unstimulated salivary flow rate after treatment with either adipose-derived or bone marrow-derived MSCs. Secondary outcomes are as follows: change in patient reported outcomes, methods of intervention administration, number of injected MSCs, and safety. Data from included studies will be pooled and compared with a fixed-effects or random effects model dependent on signs of heterogeneity, presented with a forest plot, and indirectly compared with a meta-regression in a network meta-analysis. Risk of bias will be assessed with the tools ROBINS-I or RoB-2 depending on type of study., Discussion: Both adipose-derived and bone marrow-derived MSCs are used today for experimental treatment of salivary hypofunction in humans as no direct or indirect comparisons have been made. Therefore, an evaluation of the effect of adipose-derived vs bone marrow-derived MSC treatment is needed to support future decision-making on the type of MSC used in a clinical trial., Systematic Review Registration: PROSPERO ID CRD42024527183., (© 2024. The Author(s).)

Background: The efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model., Methods: We developed an IAS-targeted in vivo model via rapid freezing (at - 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking., Results: In vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc 20 , Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC 20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups., Conclusions: These findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone., (© 2024. The Author(s).)

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications. First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique. Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

Adipose-derived mesenchymal stem cells (ADMSCs) possess the ability to transform into various cell types, including neurons. It has been proposed that the optimization of this transformation can be achieved by using photobiomodulation (PBM). The objective of this laboratory-based investigation was to induce the transformation of immortalized ADMSCs (iADMSCs) into neurons with chemical triggers and then evaluate the supportive effects of PBM at two different wavelengths, 525 nm and 825 nm, each administered at a dose of 5 J/cm 2 , as well as the combined application of these wavelengths. The results revealed that the treated cells retained their stem cell characteristics, although the cells exposed to the green laser exhibited a reduction in the CD44 marker. Furthermore, early, and late neuronal markers were identified using flow cytometry analysis. The biochemical analysis included the assessment of cell morphology, viability, cell proliferation, potential cytotoxicity, and the generation of reactive oxygen species (ROS). The findings of this study indicate that PBM does not harm the differentiation process and may even enhance it, but it necessitates a longer incubation period in the induction medium. These research findings contribute to the validation of stem cell technology for potential applications in in vivo, pre-clinical, and clinical research environments., (© 2024. The Author(s).)

Human skeletal muscle contains different types of tissues with skeletal muscle fibers (SMFs) and intermuscular adipose tissues (IMATs) as the main components. We maintained human skeletal muscle tissues from 12 study participants under native conditions in vitro for 11 days to investigate the dynamics of macrophages that reside in adjacent IMATs and SMFs simultaneously. The samples were subjected to immunohistochemical analysis for macrophage phenotyping and mitochondrial mass assessment before and after maintenance in vitro. Multiplex protein analysis was used to determine cytokine/chemokine expression in tissue extracts. The results revealed significant correlations between donor age or body mass index (BMI) and distinct phenotypes of resident macrophages in SMFs and IMATs. The dynamics of SMF- and IMAT-resident macrophages differed significantly in vitro and exhibited inverse correlations with chemokine/cytokine expression levels and mitochondrial activity. Moreover, the responses of macrophages to saturated and unsaturated fatty acids (FAs) differed substantially between SMFs and IMATs. These findings showed the functional diversity of phenotypically identical macrophages in adjacent niches. Thus, the currently available macrophage markers cannot capture the functional diversity of human tissue-resident macrophages. The model used in the present study may help elucidate how macrophages affect muscle homeostasis and disease in humans.

Gelatin methacryloyl (GelMA), typically derived from mammalian sources, has recently emerged as an ideal bio-ink for three-dimensional (3D) bioprinting. Herein, we developed a fish skin-based GelMA bio-ink for the fabrication of a 3D GelMA skin substitute with a 3D bioprinter. Several concentrations of methacrylic acid anhydride were used to fabricate GelMA, in which their physical-mechanical properties were assessed. This fish skin-based GelMA bio-ink was loaded with human adipose tissue-derived mesenchymal stromal cells (ASCs) and human platelet lysate (HPL) and then printed to obtain 3D ASCs + HPL-loaded GelMA scaffolds. Cell viability test and a preliminary investigation of its effectiveness in promoting wound closure were evaluated in a critical-sized full thickness skin defect in a rat model. The cell viability results showed that the number of ASCs increased significantly within the 3D GelMA hydrogel scaffold, indicating its biocompatibility property. In vivo results demonstrated that ASCs + HPL-loaded GelMA scaffolds could delay wound contraction, markedly enhanced collagen deposition, and promoted the formation of new blood vessels, especially at the wound edge, compared to the untreated group. Therefore, this newly fish skin-based GelMA bio-ink developed in this study has the potential to be utilized for the printing of 3D GelMA skin substitutes., (© 2024. The Author(s).)

Adipose tissue engineering (ATE) has been gaining increasing interest over the past decades, offering promise for new and innovative breast reconstructive strategies. Animal-derived gelatin-methacryloyl (Gel-MA) has already been applied in a plethora of TE strategies. However, due to clinical concerns, related to the potential occurrence of immunoglobulin E-mediated immune responses and pathogen transmission, a shift towards defined, reproducible recombinant proteins has occurred. In the present study, a recombinant protein based on human collagen type I, enriched with arginine-glycine-aspartic acid was functionalized with photo-crosslinkable methacryloyl moieties (RCPhC1-MA), processed into 3D scaffolds and compared with frequently applied Gel-MA from animal origin using an indirect printing method applying poly-lactic acid as sacrificial mould. For both materials, similar gel fractions (>65%) and biodegradation times were obtained. In addition, a significantly lower mass swelling ratio (17.6 ± 1.5 versus 24.3 ± 1.4) and mechanical strength (Young's modulus: 1.1 ± 0.2 kPa versus 1.9 ± 0.3 kPa) were observed for RCPhC1-MA compared to Gel-MA scaffolds. In vitro seeding assays showed similar cell viabilities (>80%) and a higher initial cell attachment for the RCPhC1-MA scaffolds. Moreover, the seeded adipose-derived stem cells could be differentiated into the adipogenic lineage for both Gel-MA and RCPhC1-MA scaffolds, showing a trend towards superior differentiation for the RCPhC1-MA scaffolds based on the triglyceride and Bodipy assay. RCPhC1-MA scaffolds could result in a transition towards the exploitation of non-animal-derived biomaterials for ATE, omitting any regulatory concerns related to the use of animal derived products., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Purpose: This study explores the application of adipose-derived mesenchymal stromal cells (adMSCs) as a therapy for ocular inflammatory diseases utilizing a chronic GVHD model., Methods: Human adMSCs were administered via subconjunctival injection into mice with chronic ocular GVHD. Clinical scores and changes in T cell populations were analyzed., Results: The study showed significant improvement in corneal integrity, including epithelial damage, opacity, thickness, and structure, after subconjunctival adMSC transplantation. Additionally, adMSC transplantation increased CD45 + and Foxp3 + Tregs while decreasing CD4 + T cells, 1IL17A + Th17 cells, and IFNγ + Th1 cells in local cervical lymph nodes. Moreover, adMSC-conditioned media enhanced wound closure and cell migration toward the wound bed in vitro. The cells disappeared within a week suggesting that trophic factors were involved., Conclusion: The dual benefit of adMSCs in immune-related ocular disorders underscores their potential for clinical application. This study focuses on subconjunctival delivery, effects of adMSCs and migration post-injection, with implications for optimizing cellular therapy application. The observed dual action, combining immunomodulation and tissue repair enhancement, underscores holistic approach of adMSC therapy in regenerative medicine, making it a potent treatment for diseases involving inflammation and tissue damage in the ocular surface., Competing Interests: Disclosure of potential conflicts of interest R. T. and H. N are affiliated with Rohto Pharmaceuticals, a company with a vested interest in the clinical application of the cells utilized in this study. The study was supported by a grant from the Japan Society for the Promotion of Science (JSPS) to S. S. The study was funded in part by Rohto Pharmaceuticals., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sad and UNC84 domain 1 (SUN1) is a kind of nuclear envelope protein with established involvement in cellular processes, including nuclear motility and meiosis. SUN1 plays an intriguing role in human adipose-derived stem cells (hASCs) differentiation; however, this role remains largely undefined. This study was undertaken to investigate the role of SUN1 in hASCs differentiation, as well as its underlying mechanisms. Employing siRNAs, we selectively downregulated SUN1 and CD36 expression. Microtubules were depolymerized using nocodazole, and PPARγ was activated using rosiglitazone. Western blotting was performed to quantify SUN1, PPARγ, α-tubulin, CD36, OPN, and adiponectin protein expression levels. Alkaline phosphatase and Oil red O staining were used to assess osteogenesis and adipogenesis, respectively. Downregulated SUN1 expression increased osteogenesis and decreased adipogenesis in hASCs, concomitant with upregulated α-tubulin expression and downregulated CD36 expression, alongside reduced nuclear localization of PPARγ. Microtubule depolymerization increased CD36 expression. Rescue experiments indicated that microtubule depolymerization counteracted the downregulated SUN1-induced phenotypic changes. This study demonstrates that SUN1 influences the differentiation of hASCs towards osteogenic and adipogenic lineages, indicating its essential role in cell fate., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Osteoarthritis (OA) is a prevalent and disabling disease that affects a significant proportion of the global population. Urine-derived stem cells (USCs) have shown great prospects in the treatment of OA, but there is no study that has compared them with traditional stem cells., Purpose: This study aimed to compare the therapeutic efficacy and mechanisms of USCs and adipose-derived stem cells (ADSCs) for OA treatment., Study Design: Controlled laboratory study., Methods: We compared the biological properties of USCs and ADSCs using CCK-8, colony formation, EdU, adhesion, and apoptosis assays. We evaluated the protective effects of USCs and ADSCs on IL-1β-treated OA chondrocytes by chemical staining, immunofluorescence, and Western blotting. We assessed the effects of USCs and ADSCs on chondrocyte autophagy by transmission electron microscopy, immunofluorescence, and Western blotting. We also compared the therapeutic efficacy of intra-articular injections of USCs and ADSCs by gross, histological, micro-computed tomography, and immunohistochemical analyses in an OA rat model induced by anterior cruciate ligament transection., Results: USCs showed higher proliferation, colony formation, DNA synthesis, adhesion, and anti-apoptotic abilities than ADSCs. Both USCs and ADSCs increased the expression of cartilage-specific proteins and decreased the expression of matrix degradation-related proteins and inflammatory factors in OA chondrocytes. USCs had a greater advantage in suppressing MMP-13 and inflammatory factors than ADSCs. Both USCs and ADSCs enhanced autophagy in OA chondrocytes, with USCs being more effective than ADSCs. The autophagy inhibitor 3-MA reduced the enhanced autophagy and protective effects of USCs and ADSCs on OA chondrocytes., Conclusion: To our knowledge, this is the first study to explore the efficacy of USCs in the treatment of knee OA and to compare them with ADSCs. Considering the superior properties of USCs in terms of noninvasive acquisition, a high cost-benefit ratio, and low ethical concerns, our study suggests that they may be a more promising therapeutic option than ADSCs for OA treatment under rigorous regulatory pathways., Clinical Relevance: USCs may be a superior cell source for stem cells to treat knee OA, and this study strengthens the evidence for the application of USCs., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by the National Natural Science Foundation of China (82172508, 82372490), the Sichuan Province Science and Technology Program (2024NSFJQ0041), and the 1.3.5 Project for Disciplines of Excellence from West China Hospital of Sichuan University (ZYJC21030). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Human adipose-derived stem cells (hASCs) are commonly used in bone tissue regeneration. The N6-methyladenosine (m 6 A) modification has emerged as a novel regulatory mechanism for gene expression, playing a critical role in osteogenic differentiation of stem cells. However, the precise role and mechanism of alkylation repair homolog 5 (ALKBH5) in hASC osteogenesis remain incompletely elucidated and warrant further investigation. Herein, we employed methylated RNA immunoprecipitation sequencing, RNA sequencing, and weighted gene coexpression network analysis to identify a key long noncoding RNA (lncRNA) in hASCs: lncRNA AK311120. Functional experiments demonstrated that lnc-AK311120 promoted the osteogenic differentiation of hASCs, while a mutation at the m 6 A central site A of lnc-AK311120 was found to decrease the level of m 6 A modification. The osteogenic effect of ALKBH5 was confirmed both in vitro and in vivo using a mandibular defect model in nude mice. Subsequent investigations revealed that knockdown of ALKBH5 resulted in a significant increase in the m 6 A modification level of lnc-AK311120, accompanied by a downregulation in the expression level of lnc-AK311120. Additional rescue experiments demonstrated that overexpression of lnc-AK311120 could restore the phenotype after ALKBH5 knockdown. We observed that AK311120 interacted with the RNA-binding proteins DExH-Box helicase 9 (DHX9) and YTH domain containing 2 (YTHDC2) to form a ternary complex, while mitogen-activated protein kinase kinase 7 (MAP2K7) served as the shared downstream target gene of DHX9 and YTHDC2. Knockdown of AK311120 led to a reduction in the binding affinity between DHX9/YTHDC2 and the target gene MAP2K7. Furthermore, ALKBH5 facilitated the translation of MAP2K7 and activated the downstream JNK signaling pathway through the AK311120-DHX9-YTHDC2 complex, without affecting its messenger RNA level. Collectively, we have investigated the regulatory effect and mechanism of ALKBH5-mediated demethylation of lncRNA in hASC osteogenesis for the first time, offering a promising approach for bone tissue engineering., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cultured fat is an important part of cultured meat, and the ability of adipose-derived mesenchymal stem cells (ADSCs) to differentiate into mature adipose tissue affects the quality of cultured fat. Thus, the primary aim of this study was to screen for combinations of differentiation-inducing factors (DIF) using single-factor experiment and orthogonal experimental design under two-dimensional culture conditions for ADSCs. The results showed that a combination of DIF consisting of 1 μmol/L dexamethasone, 0.1 mmol/L 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, 0.1 mmol/L indomethacin, and 2 μmol/L rosiglitazone was a good choice for the differentiation of ADSCs. An combination of DIF was applied to the preparation of cultured fat with collagen as scaffolds. Forty-eight fatty acids were detected in cultured fat by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Among them, the content of twenty-one fatty acids in cultured fat was significantly higher than that of conventional porcine subcutaneous adipose tissue (P < 0.05), and the content of 14 fatty acids was not significantly different (P > 0.05). The ratio of ω-6 polyunsaturated fatty acids content to ω-3 polyunsaturated fatty acids content was 1.23:1, which meant cultured fat was beneficial for human health. This study provides a method to improve the differentiation ability of ADSCs while also providing a reference for indicating the nutritional value of cultured fat., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Shi-Jie Ding is the co-founder of Joes Future Food company, which is committed to the commercialization of cultured meat. Other authors have no conflict of interest.], (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: In facial plastic surgery, patients with nasal deformity are often treated by rib cartilage transplantation. In recent years, cartilage tissue engineering has developed as an alternative to complex surgery for patients with minor nasal defects via injection of nasal filler material. In this study, we prepared an injectable nasal filler material containing poly-L-lactic acid (PLLA) porous microspheres (PMs), hyaluronic acid (HA) and adipose-derived mesenchymal stem cells (ADMSCs)., Methods: We seeded ADMSCs into as-prepared PLLA PMs using our newly invented centrifugation perfusion technique. Then, HA was mixed with ADMSC-incorporated PLLA PMs to form a hydrophilic and injectable cell delivery system (ADMSC-incorporated PMH)., Results: We evaluated the biocompatibility of PMH in vitro and in vivo. PMH has good injectability and provides a favorable environment for the proliferation and chondrogenic differentiation of ADMSCs. In vivo experiments, we observed that PMH has good biocompatibility and cartilage regeneration ability., Conclusion: In this study, a injectable cell delivery system was successfully constructed. We believe that PMH has potential application in cartilage tissue engineering, especially in nasal cartilage regeneration., (© 2024 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Objective: Bronchopulmonary dysplasia (BPD) is the most common chronic morbidity in extremely preterm infants. Mesenchymal stem cells-derived exosomes (MSC-Exos) therapies have shown prospects in animal models of BPD. Our study aimed to evaluate the effect of adipose mesenchymal stem cells-derived exosomes (AMSC-Exos) on BPD and the role of the NF-κB signaling pathway in this process., Methods: The AMSCs were extracted and AMSC-Exos were isolated by ultracentrifugation method. Newborn rats were exposed to hyperoxia (90% O 2 ) continuously for 7 days to establish a BPD model. The rats were treated with AMSC-Exos by intratracheal administration on postnatal day 4 (P4). Pulmonary morphology, pulmonary vasculature, inflammatory factors, and NF-κB were assessed. Hyperoxia-induced primary type II alveolar epithelial cells (AECIIs) and AMSC-Exos treatment with or without a pan-NF-κB inhibitor (PDTC) were established to explore the potential mechanism., Results: Hyperoxia-exposed rats showed alveolar simplification with decreased radial alveolar count and increased mean linear intercept, low CD31, and vascular endothelial growth factor expression, reduced microvessel density, increased the expression of TNF-α, IL-1β, and IL-6 and decreased the expression of IL-10, and induced NF-κB phosphorylation. AMSC-Exos protected the neonatal lung from the hyperoxia-induced arrest of alveolar and vascular development, alleviated inflammation, and inhibited NF-κB phosphorylation. Hyperoxia decreased viability, increased apoptosis, enhanced inflammation, and induced NF-κB phosphorylation of AECIIs but improved by AMSC-Exos, PDTC, or AMSC-Exos+PDTC. The effect of AMSC-Exos+PDTC in AECIIs was the same as AMSC-Exos, but more notable than PDTC alone., Conclusion: AMSC-Exos attenuated the hyperoxia-induced lung injury in neonatal rats by inhibiting the NF-κB signaling pathway partly., (© 2024 Wiley Periodicals LLC.)

Background: Autologous adipose tissue is an ideal material for soft tissue filling and transplantation; however, high volumes of fat absorption over time lead to a relatively low overall survival percentage. The survival and differentiation of adipose-derived stem cells (ADSCs) in the transplanted microenvironment might improve adipose graft survival. Adipocytes have been reported to affect ADSC activation. However, its underlying mechanisms remain unclear., Methods: Human ADSCs were incubated in a culture medium supplemented with hypoxic or normoxic conditioned culture medium (CM) derived from human adipocytes. Neuronal Pentraxin 1 (NPTX1) was overexpressed or knocked down in human adipocytes using an overexpression vector (NPTX1 OE) or small interfering RNA (siRNA) transfection, respectively. ADSC differentiation and paracrine secretion were assessed. Nude mice were implanted with human adipocytes and ADSCs. The adipose tissue was subsequently evaluated by histological analysis., Results: CM from hypoxic-stimulated human adipocytes significantly facilitated the differentiation ability and paracrine levels of ADSCs. NPTX1 was significantly up-regulated in human adipocytes exposed to hypoxic conditions. In vitro, CM derived from hypoxia-stimulated human adipocytes or NPTX1-overexpressing human adipocytes exposed to normoxia promoted ADSC differentiation and paracrine; after silencing NPTX1, the facilitating effects of hypoxia-treated human adipocytes on ADSC activation were eliminated. Similarly, in vivo, the NPTX1 OE + normoxia-CM group saw improved histological morphology and fat integrity, less fibrosis and inflammation, and increased vessel numbers compared with the OE NC + normoxia-CM group; the adipocyte grafts of the si-NC + hypoxia-CM group yielded the most improved histological morphology, fat integrity, and the most vessel numbers. However, these enhancements of ADSC activation and adipose graft survival were partially abolished by NPTX1 knockdown in human adipocytes., Conclusion: NPTX1 might mediate the facilitating effects of hypoxia-stimulated human adipocytes on ADSC activation, thereby improving adipose tissue survival rate after autologous fat transplantation and the effectiveness of autologous fat transplantation through promoting ADSC activation., Level of Evidence Iii: This journal requires that authors assign a level of evidence to each article. 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 ., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature and International Society of Aesthetic Plastic Surgery.)

In the context of improving the efficacy of autologous fat grafts (AFGs) in reconstructive surgery, this study delineates the novel use of adipose-derived mesenchymal stem cells (ADSCs) and their extracellular vesicles (EVs) as vehicles for delivering delta-like ligand 4 (DLL4) siRNA. The aim was to inhibit DLL4, a gene identified through transcriptome analysis as a critical player in the vascular endothelial cells of AFG tissues, thereby negatively affecting endothelial cell functions and graft survival through the Notch signaling pathway. By engineering ADSC EVs to carry DLL4 siRNA (ADSC EVs-siDLL4), the research demonstrated a marked improvement in endothelial cell proliferation, migration, and lumen formation, and enhanced angiogenesis in vivo, leading to a significant increase in the survival rate of AFGs. This approach presents a significant advancement in the field of tissue engineering and regenerative medicine, offering a potential method to overcome the limitations of current fat grafting techniques. NEW & NOTEWORTHY This study introduces a groundbreaking method for enhancing autologous fat graft survival using adipose-derived stem cell extracellular vesicles (ADSC EVs) to deliver DLL4 siRNA. By targeting the delta-like ligand 4 (DLL4) gene, crucial in endothelial cell dynamics, this innovative approach significantly improves endothelial cell functions and angiogenesis, marking a substantial advancement in tissue engineering and regenerative medicine.

Understanding the impact of various culturing strategies on the secretome composition of adipose-derived stromal cells (ASC) enhances their therapeutic potential. This study investigated changes in the secretome of perirenal ASC (prASC) under different conditions: normoxia, cytokine exposure, high glucose, hypoxia, and hypoxia with high glucose. Using mass spectrometry and enrichment clustering analysis, we found that normoxia enriched pathways related to extracellular matrix (ECM) organization, platelet degranulation, and insulin-like growth factor (IGF) transport and uptake. Cytokine exposure influenced metabolism, vascular development, and protein processing pathways. High glucose affected the immune system, metabolic processes, and IGF transport and uptake. Hypoxia impacted immune and metabolic processes and protein processing. Combined hypoxia and high glucose influenced the immune system, IGF transport and uptake, and ECM organization. Our findings highlight the potential of manipulating culturing conditions to produce secretomes with distinct protein and functional profiles, tailoring therapeutic strategies accordingly., (© 2024. The Author(s).)

Object: Adipose-derived mesenchymal stem cells (ADSCs) have received significant attention in the field of cartilage tissue repair. Angelica sinensis polysaccharide (ASP) can enhance both the proliferation and differentiation of mesenchymal stem cells. Therefore, we intend to explore the effect of ASP on chondrogenic differentiation of ADSCs in vitro, and elucidate the underlying mechanisms., Method: ADSCs were treated with different concentrations of ASP to determine the optimal concentration. The chondrogenic differentiation of ADSCs was evaluated using Alcian blue staining, qRT-PCR, western blot, and IF staining. Transcriptome sequencing was performed to identify the expression profiles of ADSCs before and after ASP treatment, followed by bioinformatic analyses including differential expression analysis, enrichment analysis, and construction of PPI networks to identify differentially expressed genes (DEGs) associated with ASP and chondrogenic differentiation., Result: Surface markers of isolated rat-derived ADSCs were identified by CD44 + CD90 + CD45 - CD106 - , and exhibited the capacity for lipogenic, osteogenic, and chondrogenic differentiation. With increasing concentration of ASP treatment, there was an upregulation in the activity and acidic mucosubstance of ADSCs. The levels of Aggrecan, COL2A1, and Sox9 showed an increase in ADSCs after 28 days of 80 µg/ml ASP treatment. Transcriptome sequencing revealed that ASP-associated DEGs regulate extracellular matrix synthesis, immune response, inflammatory response, and cell cycle, and are involved in the NF-κB, AGE-RAGE, and calcium pathways. Moreover, Edn1, Frzb, Mdk, Nog, and Sulf1 are hub genes in DEGs. Notably, ASP upregulated MDK levels in ADSCs, while knockdown of MDK mitigated ASP-induced elevations in acidic mucosubstance, chondrogenic differentiation-related markers (Aggrecan, COL2A1, and Sox9), and the activity of the PI3K/AKT pathway., Conclusion: ASP enhances the proliferation and chondrogenic differentiation of ADSCs by activating the MDK-mediated PI3K/AKT pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background and Aims: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions., Methods: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 10 6 AD-MSCs (G2), MSC-EVs produced by 2.5 × 10 6 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 10 6 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4)., Results: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions., Conclusions: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo., Competing Interests: Declaration of competing interest Authors MP and PM declare competing financial interests as owners of the company Bio. All other authors declare that they have no conflicting interests., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The influence of surface topography on stem cell behavior and differentiation has garnered significant attention in regenerative medicine and tissue engineering. The cell-imprinting method has been introduced as a promising approach to mimic the geometry and topography of cells. The cell-imprinted substrates are designed to replicate the topographies and dimensions of target cells, enabling tailored interactions that promote the differentiation of stem cells towards desired specialized cell types. In fact, by replicating the size and shape of cells, biomimetic substrates provide physical cues that profoundly impact stem cell differentiation. These cues play a pivotal role in directing cell morphology, cytoskeletal organization, and gene expression, ultimately influencing lineage commitment. The biomimetic substrates' ability to emulate the native cellular microenvironment supports the creation of platforms capable of steering stem cell fate with high precision. This review discusses the role of mechanical factors that impact stem cell fate. It also provides an overview of the design and fabrication principles of cell-imprinted substrates. Furthermore, the paper delves into the use of cell-imprinted polydimethylsiloxane (PDMS) substrates to direct adipose-derived stem cells (ADSCs) differentiation into a variety of specialized cells for tissue engineering and regenerative medicine applications. Additionally, the review discusses the limitations of cell-imprinted PDMS substrates and highlights the efforts made to overcome these limitations., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nowadays, regenerative medicine techniques are usually based on the application of mesenchymal stromal cells (MSCs) for the repair or restoration of injured damaged tissues. However, the effectiveness of autologous therapy is limited as therapeutic potential of MSCs declines due to patient's age, health condition and prolonged in vitro cultivation as a result of decreased growth rate. For that reason, there is an urgent need to develop strategies enabling the in vitro rejuvenation of MSCs prior transplantation in order to enhance their in vivo therapeutic efficiency. In presented study, we attempted to mimic the naturally occurring mitochondrial transfer (MT) between neighbouring cells and verify whether artificial MT (AMT) could reverse MSCs aging and improve their biological properties. For that reason, mitochondria were isolated from healthy donor equine adipose-derived stromal cells (ASCs) and transferred into metabolically impaired recipient ASCs derived from equine metabolic syndrome (EMS) affected horses, which were subsequently subjected to various analytical methods in order to verify the cellular and molecular outcomes of the applied AMT. Mitochondria recipient cells were characterized by decreased apoptosis, senescence and endoplasmic reticulum stress while insulin sensitivity was enhanced. Furthermore, we observed increased mitochondrial fragmentation and associated PARKIN protein accumulation, which indicates on the elimination of dysfunctional organelles via mitophagy. AMT further promoted physioxia and regulated autophagy fluxes. Additionally, rejuvenated ASCs displayed an improved anti-inflammatory activity toward LPS-stimulated synoviocytes. The presented findings highlight AMT as a promising alternative and effective method for MSCs rejuvenation, for potential application in autologous therapies in which MSCs properties are being strongly deteriorated due to patients' condition., Competing Interests: Declaration of competing interest The authors confirm that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Atherosclerosis (AS) is a common disease seriously detrimental to human health. AS is a chronic progressive disease related to inflammatory reactions. The present study aimed to characterize and evaluate the effects of adipose tissue stem cells (ADSCs) in high-fat diet-induced atherosclerosis in a rat model. The present study comprises thirty-six rats and they were divided into three groups: the control group, the high-fat diet (HFD) group; which received a high-fat diet, and the high-fat diet + stem cells (HFD+SC) group; which was fed with a high-fat diet along with the administration of intravenous ADSCs. Food was given to the animals for 20 weeks to establish dyslipidemia models. After 20 weeks, animals were sacrificed by cervical dislocation; blood was collected to measure total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL); aortae were collected to detect morphologic changes. Rats of the HFD group showed a significant increase in body weight (B.Wt), altered lipid profile increased expression of inducible nitric oxide synthase (iNOS), and decreased expression of endothelial nitric oxide synthase (eNOS). However, in HFD+SC there was a significant decrease in body weight gain and an improvement in lipid profile. Histopathological and ultrastructural variations observed in the aorta of the HFD group when treated with ADSCs showed preserved normal histological architecture and reduced atherosclerosis compared with the HFD group. This was evidenced by laboratory, histological, immunohistochemical, and morphometric studies. Thus, ADSCs reduced TC, TG, and LDL, reduced the expression of iNOS, and increased the expression of eNOS. The high-fat diet was likely to cause damage to the wall of blood vessels. Systemically transplanted ADSCs could home to the aorta, and further protect the aorta from HFD-induced damage., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: In plastic surgery tissue transplantation, tissue ischemia limits transplanted tissue survival. Adipose-derived stem cells (ASCs) and stromal vascular fraction (SVF) show potential for promoting angiogenesis and rescuing ischemic conditions. However, when SVF and ASC suspensions are utilized without the protection of extracellular matrix, the retention rate of transplanted cells tends to be diminished, leading to an unsatisfactory therapeutic outcome. To overcome this, adipose tissue-derived microvascular fragments (ad-MVFs) have emerged as a promising solution., Methods: We conducted enzymatic digestion on human adipose tissue to generate ad-MVFs. These fragments underwent a thorough characterization process, utilizing electron microscopy to assess their structural attributes and enabling a detailed analysis of their intricate morphology. Furthermore, our team investigated the cellular composition of these microvascular fragments, subsequently confirming their ability to enhance the viability of ischemic skin flaps., Results: The resulting product primarily comprised fragments with sizes ranging from 20 to 50 µm, and some exhibited a sophisticated network-like structure. Electron microscopy examination revealed the presence of collagen components in the product. Additionally, flow cytometry analysis indicated a substantial abundance of adipose-derived stem cells and endothelial cells within these microvascular fragments. Significantly, when tested in treating an ischemic skin flap in a nude mouse model, the product exhibited superior therapeutic efficacy compared to SVF cell suspension., Conclusion: We have successfully generated human ad-MVFs and established standardized procedures. Compared with SVF, Ad-MVFs have a better effect in the treatment of ischemic diseases., Level of Evidence Ii: This journal requires that authors assign a level of evidence to each article. 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 ., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature and International Society of Aesthetic Plastic Surgery.)

Osteoarthritis (OA) is a clinical state which is identified by the degeneration of articular cartilage. OA is a common condition (>500 millions of people affected worldwide), whose frequency is anticipated to continue to rise (> 110 % increase worldwide since 2019). The treatment for early-stage OA is based on a combination of therapeutic approaches, which can include regenerative medicine based on Adipose Derived Stem Cells (ADSCs). Germanium embedded Incrediwear® functional Cred40 fabric has been shown to have positive effects on OA clinically and is envisaged to give encouraging effects also on tissue regeneration. Still, the biological mechanisms underlying this therapeutic modality have not yet been fully defined. We tested the hypothesis that Germanium-embedded Incrediwear® functional Cred40 fabric could enhance chondrogenic differentiation. To this purpose, we applied Incrediwear® to human adipose-derived stem cells (hADSCs) induced to chondrogenic differentiation in vitro. Chondrogenic markers (ACAN, SOX9, RUNX2, COL2A1, COL10A1) were quantified following 21 days of treatment. We also assessed extracellular matrix (ECM) deposition (specifically Collagen and glycosaminoglycans (GAGs)) using Alcian Blue and Sirius Red staining. Here, we provide pilot data to demonstrate that Germanium-embedded Incrediwear® functional Cred40 fabric can enhance hADSCs chondrogenic differentiation and maturity and potentially induce events of cartilage regeneration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

When delivering cells on a scaffold to treat a bone defect, the cell seeding technique determines the number and distribution of cells within a scaffold, however the optimal technique has not been established. This study investigated if human adipose-derived stem cells (ASCs) transduced with a lentiviral vector to overexpress bone morphogenetic protein 2 (BMP-2) and loaded on a scaffold using dynamic orbital shaker could reduce the total cell dose required to heal a critical sized bone defect when compared with static seeding. Human ASCs were loaded onto a collagen/biphasic ceramic scaffold using static loading and dynamic orbital shaker techniques, compared with our labs standard loading technique, and implanted into femoral defects of nude rats. Both a low dose and standard dose of transduced cells were evaluated. Outcomes investigated included BMP-2 production, radiographic healing, micro-computerized tomography, histologic assessment, and biomechanical torsional testing. BMP-2 production was higher in the orbital shaker cohort compared with the static seeding cohort. No statistically significant differences were noted in radiographic, histomorphometric, and biomechanical outcomes between the low-dose static and dynamic seeding groups, however the standard-dose static seeding cohort had superior biomechanical properties. The standard-dose 5 million cell dose standard loading cohort had superior maximum torque and torsional stiffness on biomechanical testing. The use of orbital shaker technique was labor intensive and did not provide equivalent biomechanical results with the use of fewer cells., (© 2024 Wiley Periodicals LLC.)

Stem cell-based therapies offer promising avenues for treating inflammatory diseases owing to their immunomodulatory properties. However, challenges persist regarding their survival and efficacy in inflamed tissues. Our study introduces a novel approach by engineering adipose-derived stem cells (ADSCs) to enhance their viability in inflammatory environments and boost the secretion of paracrine factors for treating inflammatory bowel disease (IBD). An arginine-glycine-aspartate peptide-poly (ethylene glycol)-chlorin e6 conjugate (RPC) was synthesized and coupled with ADSCs, resulting in RPC-labeled ADSCs (ARPC). This conjugation strategy employed RGD-integrin interaction to shield stem cells and allowed visualization and tracking using chlorin e6. The engineered ARPC demonstrated enhanced viability and secretion of paracrine factors upon light irradiation, regulating the inflammatory microenvironment. RNA-sequencing analysis unveiled pathways favoring angiogenesis, DNA repair, and exosome secretion in ARPC(+) while downregulating inflammatory pathways. In in vivo models of acute and chronic IBD, ARPC(+) treatment led to reduced inflammation, preserved colon structure, and increased populations of regulatory T cells, highlighting its therapeutic potential. ARPC(+) selectively homed to inflammatory sites, demonstrating its targeted effect. Overall, ARPC(+) exhibits promise as an effective and safe therapeutic strategy for managing inflammatory diseases like IBD by modulating immune responses and creating an anti-inflammatory microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tissue engineering (TE) of adipose tissue (AT) is a promising strategy that can provide 3D constructs to be used for in vitro modelling, overcoming the limitations of 2D cell cultures by closely replicating the complex breast tissue extracellular matrix (ECM), cell-cell, and cell-ECM interactions. However, the challenge in developing 3D constructs of AT resides in designing artificial matrices that can mimic the structural properties of native AT and support adipocytes biological functions. Herein, we developed photocrosslinkable hydrogels by employing gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) to mimic the collagenous and glycosaminoglycan components of AT microenvironment, respectively. The physico-mechanical properties of the hydrogels were tuned to target AT biomimetic properties by varying the hydrogel formulation (with or without hyaluronic acid), and the amount of photoinitiator (ruthenium/sodium persulfate) used to crosslink the hydrogels via visible light. The physical and mechanical properties of the developed hydrogels were tuned by varying the material formulation and the photoinitiator concentration. Preadipocytes were encapsulated inside the hydrogels and differentiated into mature adipocytes. Findings enlightened that HAMA addition in hybrid hydrogels boosted an increased lipid accumulation. The engineered biomimetic adipocyte-based constructs resulted promising as scaffolds or 3D in vitro models of AT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mesenchymal stem cells (MSCs) have demonstrated considerable potential in tissue repair and the treatment of immune-related diseases, but there are problems with homing efficiency during MSCs transplantation. Exercise, as an intervention, has been shown to have an important impact on the properties of MSCs. This review summarizes the effects of exercise on the properties (including proliferation, apoptosis, differentiation, and homing) of bone marrow-derived MSCs and adipose-derived MSCs. Studies indicated that exercise enhances bone marrow-derived MSCs proliferation, osteogenic differentiation, and homing while reducing adipogenic differentiation. For adipose-derived MSCs, exercise enhances proliferation and reduces adipogenic differentiation. In addition, studies have investigated the therapeutic effects of combined therapy of MSCs transplantation with exercise on diseases of the bone, cardiac, and nervous systems. The combined therapy improves tissue repair by increasing the homing of transplanted MSCs and cytokine secretion (such as neurotrophin 4). Furthermore, MSCs transplantation also has potential for the treatment of obesity. Although the effect is not significant in weight loss, MSCs transplantation shows effects in controlling blood glucose, improving dyslipidemia, reducing inflammation, and improving liver disease. Finally, the potential role of combined MSCs transplantation and exercise therapy in addressing obesity is discussed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Diabetic retinopathy, a leading cause of vision impairment, is marked by microvascular complications in the retina, including pericyte loss, a key indicator of early-stage disease. This study explores the therapeutic potential of exosomes derived from immortalized adipose-mesenchymal stem cells differentiated into pericyte-like cells in restoring the function of mouse retinal microvascular endothelial cells damaged by high glucose conditions, thereby contributing to the understanding of early diabetic retinopathy intervention strategies. To induce immortalized adipose-mesenchymal stem cells differentiation into pericyte-like cells, the study employed pericyte growth supplement. And confirmed the success of cell differentiation through the detection of α-smooth muscle actin and neural/glial antigen 2 expression by Western blot and immunofluorescence. Exosomes were isolated from the culture supernatant of immortalized adipose-mesenchymal stem cells using ultracentrifugation and characterized through Western blot for exosomal markers (CD9, CD81, and TSG101), transmission electron microscopy, and nanoparticle tracking analysis. Their influence on mouse retinal microvascular endothelial cells under high glucose stress was assessed through various functional assays. Findings revealed that exosomes, especially those from pericyte-like immortalized adipose-mesenchymal stem cells, were efficiently internalized by retinal microvascular endothelial cells and effectively counteracted high glucose-induced apoptosis. These exosomes also mitigated the rise in reactive oxygen species levels and suppressed the migratory and angiogenic properties of retinal microvascular endothelial cells, as demonstrated by Transwell and tube formation assays, respectively. Furthermore, they preserved endothelial barrier function, reducing hyperglycemia-induced permeability. At the molecular level, qRT-PCR analysis showed that exosome treatment modulated the expression of critical genes involved in angiogenesis (VEGF-A, ANG2, MMP9), inflammation (IL-1β, TNF-α), gap junction communication (CX43), and cytoskeletal regulation (ROCK1), with the most prominent effects seen with exosomes from pericyte-like immortalized adipose-mesenchymal stem cells. High glucose increased the expression of pro-angiogenic and pro-inflammatory markers, which were effectively normalized post-exosome treatment. In conclusion, this research highlights the reparative capacity of exosomes secreted by pericyte-like differentiated immortalized adipose-mesenchymal stem cells in reversing the detrimental effects of high glucose on retinal microvascular endothelial cells. By reducing apoptosis, oxidative stress, inflammation, and abnormal angiogenic behavior, these exosomes present a promising avenue for therapeutic intervention in early diabetic retinopathy. Future studies can focus on elucidating the precise molecular mechanisms and exploring their translational potential in vivo., Competing Interests: Declarations of interest All authors disclosed no relevant relationships., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization. Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue-derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein-expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system. In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence-positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule. Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

During the aging process, elastin is degraded and the level of elastin-derived peptides (EDPs) successively increases. The main peptide released from elastin during its degradation is a peptide with the VGVAPG sequence. To date, several papers have described that EDPs or elastin-like peptides (ELPs) affect human mesenchymal stem cells (hMSCs) derived from different tissues. Unfortunately, despite the described effect of EDPs or ELPs on the hMSC differentiation process, the mechanism of action of these peptides has not been elucidated. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG and VVGPGA peptides on the hMSC stemness marker and elucidation of the mechanism of action of these peptides. Our data show that both studied peptides (VGVAPG and VVGPGA) act with the involvement of ERK1/2 and c-SRC kinases. However, their mechanism of activation is probably different in hMSCs derived from adipose tissue. Both studied peptides increase the KI67 protein level in hMSCs, but this is not accompanied with cell proliferation. Moreover, the changes in the NANOG and c-MYC protein expression and in the SOX2 and POU5F1 mRNA expression suggest that EDPs reduced the hMSC stemness properties and could initiate cell differentiation. The initiation of differentiation was evidenced by changes in the expression of AhR and PPARγ protein as well as specific genes (ACTB, TUBB3) and proteins (β-actin, RhoA) involved in cytoskeleton remodeling. Our data suggest that the presence of EDPs in tissue can initiate hMSC differentiation into more tissue-specific cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Stem cell therapy is currently the most promising strategy for the treatment of myocardial infarction. However, the development of injectable cell carriers that can scavenge reactive oxygen species (ROS) in the infarct zone to improve transplanted cell survival remains a challenge. Here, we developed a ROS responsive conductive microsphere based on chitosan (CS) and dextran (DEX) with 4-formylphenylboronic acid (4-FPBA) as a cross-linking agent and the addition of graphite oxide (GO) and the anti-inflammatory agent salvianolic acid B (SalB), as a cell delivery carrier for myocardial infarction. These microspheres were crosslinked by dual dynamic networks of Schiff base and phenylborate bonds. The relationship between CS concentration and microsphere particle size, as well as the biocompatibility, ROS responsiveness, anti-inflammatory properties, and effects on myogenic differentiation of H9C2 cells were fully investigated. The microspheres exhibit good biocompatibility, proliferation promoting, differentiation promoting, antioxidant, and anti-inflammatory properties. When applied to mice myocardial infarction models, the ROS responsive conductive microspheres loaded with SalB and adipose derived stem cells (ADSC) exhibited excellent in vivo repair ability. In addition, they reduced myocardial fibrosis and promoted ventricular wall regeneration by promoting the expression of Connexin 43 (Cx43) and CD31, ultimately reshaping the infarcted myocardium, suggesting their great potential as cell delivery carriers for myocardial infarction treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Prunella vulgaris L.(PV) and Tussilago farfara (TF) are perennial herbs rich in flavonoids and phenolic compounds with immense medicinal value. PV extract (PV-E) possesses potent antipyretic, anti-inflammtory, antioxidant, antiseptic, anti-cancer and immune stimulatory properties and have been traditionally known for the treatment of wounds, ulcers and sores. TF extract (TF-E) has been known for antibacterial, antioxidant, anti-inflammatory, anti-viral, anti-diabetic, anti-cancer, anti-obesity and wound healing effects. Additionally, TF-E infusions have been used for asthma, cough, and bronchopneumonia treatments., Aim of the Study: The therapeutic efficacy of transplanted human adipose stem cells (hASCs) is abrogated under the deteriorating effects of heat stress offered by burn wounds. Earlier researches has documented antioxidant priming as an effective strategy to enhance stem cell performance. As both PV-E and TF-E are known for their potent antioxidant effects. The present study aims to examine the cryoprotective effects of PV-E and TF-E priming on hASCs against in-vitro heat-induced thermal stress. Moreover, we determined the anti-inflammatory potential of both PV-E and TF-E on rabbits., Methods: Antioxidant capacity of both PV-E and TF-E is examined via DPPH assay and anti-inflammatory activity is assessed in rabbits using carrageen-induced paw edema model of inflammation. Next, we investigate the efficacy of different doses (1.25-100 μg/ml) of PV-E and TF-E on hASCs; MTT, LDH, calcein AM staining, and wound scratch assay were used to assess cell viability, cytotoxicity, proliferation ability and cell migration potential in the cells. Then, hASCs were pretreated for 24 h with optimum doses of PV-E and TF-E determined from MTT assay results and were subsequently exposed to in-vitro thermal injury (51 °C,10 min). The cytoprotective effects of both PV-E and TF-E priming under thermal stress were investigated via MTT, LDH, annexin-V staining and gene expression analysis., Results: Both PV-E and TF-E extracts demonstrated potent antioxidant and effective anti-inflammatory activities, with a clear reduction in inflammation. Study on hASCs exhibited improved cell viabilities, enhanced cell proliferation and migration abilities of both extracts. While heat stress data revealed that PV-E (2.5 μg/ml) and TF-E (5 μg/ml) pretreatment significantly ameliorated effects of thermal-injuries in hASCs as depicted by significantly enhanced cell viabilities, low LDH release profile, and lower annexin-V expression and regulated gene expression of the pretreated cells., Conclusion: PV-E and TF-E priming effectively enabled hASCs to combat thermal injury by significantly promoting cell survival than untreated cells. Hence, these findings suggest that PV-E and TF-E priming could be used to attain improved cellular responses and enhanced therapeutic efficacy in burnt tissue., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Osteoarthritis (OA) is a progressive degenerative disease in older cats, and often leads to decreased quality of life (QOL). Mesenchymal stem cells (MSCs) have been used in novel therapies for inflammatory diseases. We aimed to evaluate quantitatively allogeneic adipose-derived MSC (ADSC) therapy in cats with naturally occurring OA, based on QOL assessment resources. To characterize the in vitro properties of ADSCs, we estimated ADSCs from four healthy cats with respect to morphology, differentiation potential, and immunomodulatory potential. Six cats with OA were administered a single intravenous injection of allogeneic ADSCs. Based on the feline musculoskeletal pain index (FMPI), the outcome measure was QOL. The cultured cells were adherent, exhibited a spindle shape without becoming flattened or large, and maintained doubling time until passage 5. After induction, the cells had osteogenic, adipogenic, and chondrogenic phenotypes. These cells expressed CD44 and CD90 and lacked expression of CD14 and CD45, had significantly suppressed the production of interferon -ɤ released from mitogen-stimulated lymphocytes (P < 0.05). The FMPI of all cats with OA significantly increased one month after ADSC therapy (P < 0.05). No adverse effects associated with ADSC administration were observed during follow-up in any of the cats. In conclusion, ADSC therapy with immunomodulatory potential could have beneficial effects on the QOL in cats with OA. Further research is necessary to carry out larger studies of the effectiveness of ADSC therapy., Competing Interests: Declaration of competing interest Y. Takene is a Chief Executive Officer and stockholder of J-ARM Co., Ltd., the company that markets the cell culture kit used in the present study. K. Mitani and Y. Ito are employees of J-ARM Co., Ltd. T. Inaba declares no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is an intractable aseptic disease. Modulating the transition of macrophages from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype offers an attractive therapeutic approach. Recently, small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) reportedly have potent modulatory abilities, however, their applications are limited by suboptimal targeting. Our group hypothesized that surface modification of sEVs derived from ADSCs are useful for the management of CP/CPPS by promoting M1/M2 macrophage phenotypic transformation. In this study, a novel nanomaterial (CD86-sEVs) is designed for CP/CPPS treatment using click chemistry, a bioconjugation technique enabling robust covalent linkages. The results of immunofluorescence staining, western blot and ELISA confirmed that azide-modified CD86 antibody was successfully conjugated onto the sEVs surface. In vitro, CD86-sEVs significantly accelerated M1 macrophage polarization to M2 and upregulated anti-inflammatory factors. In vivo, CD86-sEVs targeted the prostatic lesion region, alleviated chronic pelvic pain, and inhibited inflammation by promoting M1/M2 phenotype shift. Furthermore, miRNA array analysis identified specific miRNAs (miR-26a, miR-147, miR-17, miR-21, miR-182, miR-451a) within CD86-sEVs that likely contributed to these observed effects. In sum, this study presents a novel paradigm for the treatment of CP/CPPS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The regenerative capacity of mesenchymal stem cells (MSCs) is now attributed to their ability to release paracrine factors into the extracellular matrix that boost tissue regeneration, reduce inflammation and encourage healing. Understanding the MSC secretome is crucial for shifting the prototypic conventional cell-based therapies to cell-free regenerative treatments. This systematic review aimed to analyse the functional annotations of the secretome of human adult adipose tissue and bone marrow MSCs and unveil the gene clusters responsible for bone formation. Bioinformatics tools were used to identify the biological processes, molecular functions, hallmarks and KEGG pathways of adipose and bone marrow MSC secretome proteins. We found a substantial overlap in the functional annotations and protein compositions of both adipose and bone marrow MSC secretome indicating that MSC source may be noninfluencial with regards to tissue regeneration. Additionally, a novel network pharmacology-based analysis of the secreted proteins revealed that the commonly secreted proteins within a single source interact with multiple drugable targets of bone diseases and regulate various KEGG pathway. This study unravels the secretome profile of human adult adipose and bone marrow MSCs based on the current literature and provides valuable insights into the therapeutic use of the MSC secretome for cell-free therapies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Although mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are as effective as MSCs in the suppression of allergic airway inflammation, few studies have evaluated the immunomodulatory capacity of MSC-derived EVs in patients with asthma. Thus, we assessed the effects of adipose stem cell (ASC)-derived EVs on cytokine expression and regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) of asthmatic patients. PBMCs (1 × 10 6 cells/mL) were isolated from asthmatic patient and healthy controls and co-cultured with 1 μg/mL of ASC-derived EVs. Th (T helper) 1-, Th2-, and Treg-related cytokine expression, fluorescence-activated cell sorting analysis of CD4 + CD25 + FOXP3 + T cells, and co-stimulatory molecules were analyzed before and after ASC-derived EV treatment. The expression levels of IL-4 and costimulatory molecules such as CD83 and CD86 were significantly higher in PBMCs of asthmatic patients than in control PBMCs. However, ASC-derived EV treatment significantly decreased the levels of interleukin (IL)-4 and co-stimulatory molecules such as CD83 and CD86 in the phytohemagglutinin (PHA)-stimulated PBMC of asthmatic patients. Furthermore, ASC-derived EVs remarkably increased the transforming growth factor-β (TGF-β) levels and expression of Tregs in the PBMC of asthmatic patients. ASC-derived EVs induce Treg expansion and have immunomodulatory effects by downregulating IL-4 and upregulating TGF-β in PBMCs of asthmatic patients.

Skin healing is a complex and dynamic physiological process that follows mechanical alteration of the skin barrier. Under normal conditions, this complex process can be divided into at least three continuous and overlapping phases: an inflammatory reaction, a proliferative phase that leads to tissue reconstruction and a phase of tissue remodeling. Macrophages critically contribute to the physiological cascade for tissue repair. In fact, as the inflammatory phase progresses, macrophage gene expression gradually shifts from pro-inflammatory M1-like to pro-resolutive M2-like characteristics, which is critical for entry into the repair phase. A dysregulation in this macrophage' shift phenotype leads to the persistence of the inflammatory phase. Mesenchymal stromal cells and specifically the MSC-derived from adipose tissue (ADSCs) are more and more use to treat inflammatory diseases and several studies have demonstrated that ADSCs promote the wound healing thanks to their neoangiogenic, immunomodulant and regenerative properties. In several studies, ADSCs and macrophages have been injected directly into the wound bed, but the delivery of exogenous cells directly to the wound raise the problem of cell engraftment and preservation of pro-resolutive phenotype and viability of the cells. Complementary approaches have therefore been explored, such as the use of biomaterials enriched with therapeutic cell to improve cell survival and function. This review will present a background of the current scaffold models, using adipose derived stromal-cells and macrophage as therapeutic cells for wound healing, through a discussion on the potential impact for future applications in skin regeneration. According to the PRISMA statement, we resumed data from investigations reporting the use ADSCs and bioscaffolds and data from macrophages behavior with functional biomaterials in wound healing models. In the era of tissue engineering, functional biomaterials, that can maintain cell delivery and cellular viability, have had a profound impact on the development of dressings for the treatment of chronic wounds. Promising results have been showed in pre-clinical reports using ADSCs- and macrophages-based scaffolds to accelerate and to improve the quality of the cutaneous healing., (© 2024. The Author(s).)

Background: Human adipose-derived stromal/stem cells (hASCs) play important roles in regenerative medicine and numerous inflammatory diseases. However, their cellular heterogeneity limits the effectiveness of treatment. Understanding the distinct subtypes of hASCs and their phenotypic implications will enable the selection of appropriate subpopulations for targeted approaches in regenerative medicine or inflammatory diseases., Methods: hASC subtypes expressing dipeptidyl peptidase-4 (DPP4) were identified via fluorescence-activated cell sorting (FACS) analysis. DPP4 expression was knocked down in DPP4 + hASCs via DPP4 siRNA. The capacity for proliferation, hepatocyte differentiation, inflammatory factor secretion and T-cell functionality regulation of hASCs from DPP4 - , DPP4 + , and control siRNA-treated DPP4 + hASCs and DPP4 siRNA-treated DPP4 + hASCs were assessed., Results: DPP4 + hASCs and control siRNA-treated DPP4 + hASCs presented a lower proliferative capacity but greater hepatocyte differentiation capacity than DPP4 - hASCs and DPP4 siRNA-treated DPP4 + hASCs. Both DPP4 + hASCs and DPP4 - hASCs secreted high levels of vascular endothelial growth factor-A (VEGF-A), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6), whereas the levels of other factors, including matrix metalloproteinase (MMP)-1, eotaxin-3, fractalkine (FKN, CX3CL1), growth-related oncogene-alpha (GRO-alpha, CXCL1), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP)-1beta, and macrophage colony-stimulating factor (M-CSF), were significantly greater in the supernatants of DPP4 + hASCs than in those of DPP4 - hASCs. Exposure to hASC subtypes and their conditioned media triggered changes in the secreted cytokine profiles of T cells from healthy donors. The percentage of functional T cells that secreted factors such as MIP-1beta and IL-8 increased when these cells were cocultured with DPP4 + hASCs. The percentage of polyfunctional CD8 + T cells that secreted multiple factors, such as IL-17A, tumour necrosis factor alpha (TNF-α) and TNF-β, decreased when these cells were cocultured with supernatants derived from DPP4 + hASCs., Conclusions: DPP4 may regulate proliferation, hepatocyte differentiation, inflammatory cytokine secretion and T-cell functionality of hASCs. These data provide a key foundation for understanding the important role of hASC subpopulations in the regulation of T cells, which may be helpful for future immune activation studies and allow them to be customized for clinical application., (© 2024. The Author(s).)

Single-cell omics technologies that profile genes (genomic and epigenomic) and determine the abundance of mRNA (transcriptomic), protein (proteomic and secretomic), lipids (lipidomic), and extracellular matrix (matrisomic) support the dissection of adipose tissue heterogeneity at unprecedented resolution in a temporally and spatially defined manner. In particular, cell omics technologies may provide innovative biomarkers for the identification of rare specific progenitor cell subpopulations, assess transcriptional and proteomic changes affecting cell proliferation and immunomodulatory potential, and accurately define the lineage hierarchy and differentiation status of progenitor cells. Unraveling adipose tissue complexity may also provide for the precise assessment of a dysfunctional state, which has been associated with cancer, as cancer-associated adipocytes play an important role in shaping the tumor microenvironment supporting tumor progression and metastasis, obesity, metabolic syndrome, and type 2 diabetes mellitus. The information collected by single-cell omics has relevant implications for regenerative medicine because adipose tissue is an accessible source of multipotent cells; alternative cell-free approaches, including the use of adipose tissue stromal cell-conditioned medium, extracellular vesicles, or decellularized extracellular matrix, are clinically valid options. Subcutaneous white adipose tissue, which is generally harvested via liposuction, is highly heterogeneous because of intrinsic biological variability and extrinsic inconsistencies in the harvesting and processing procedures. The current limited understanding of adipose tissue heterogeneity impinges on the definition of quality standards appropriate for clinical translation, which requires consistency and uniformity of the administered product. We review the methods used for dissecting adipose tissue heterogeneity and provide an overview of advances in omics technology that may contribute to the exploration of heterogeneity and dynamics of adipose tissue at the single-cell level., (© 2024. The Author(s).)

Background: The uncultured adipose-derived stromal vascular fraction (SVF), consisting of adipose-derived stromal cells (ADSCs), M2 macrophages (M2Φ) and others, has shown therapeutic potential against osteoarthritis (OA), however, the mechanisms underlying its therapeutic effects remain unclear. Therefore, this study investigated the effects of the SVF on OA in a human-immunodeficient rat xenotransplantation model., Methods: OA model was induced in the knees of female immunodeficient rats by destabilization of the medial meniscus. Immediately after the surgery, human SVF (1 × 10 5 ), ADSCs (1 × 10 4 ), or phosphate buffered saline as a control group were transplanted into the knees. At 4 and 8 weeks postoperatively, OA progression and synovitis were analyzed by macroscopic and histological analyses, and the expression of collagen II, SOX9, MMP-13, ADAMTS-5, F4/80, CD86 (M1), CD163 (M2), and human nuclear antigen (hNA) were evaluated immunohistochemically. In vitro, flow cytometry was performed to collect CD163-positive cells as M2Φ from the SVF. Chondrocyte pellets (1 × 10 5 ) were co-cultured with SVF (1 × 10 5 ), M2Φ (1 × 10 4 ), and ADSCs (1 × 10 4 ) or alone as a control group, and the pellet size was compared. TGF-β, IL-10 and MMP-13 concentrations in the medium were evaluated using enzyme-linked immunosorbent assay., Results: In comparison with the control and ADSC groups, the SVF group showed significantly slower OA progression and less synovitis with higher expression of collagen II and SOX9, lower expression of MMP-13 and ADAMTS-5, and lower F4/80 and M1/M2 ratio in the synovium. Only the SVF group showed partial expression of hNA-, CD163-, and F4/80-positive cells in the rat synovium. In vitro, the SVF, M2Φ, ADSC and control groups, in that order, showed larger pellet sizes, higher TGF-β and IL-10, and lower MMP-13 concentrations., Conclusions: The M2Φ in the transplanted SVF directly affected recipient tissue, enhancing the secretion of growth factors and chondrocyte-protecting cytokines, and partially improving chondrocytes and joint homeostasis. These findings indicate that the SVF is as an effective option for regenerative therapy for OA, with mechanisms different from those of ADSCs., (© 2024. The Author(s).)

Background: At present, fat transplantation is widely used in the plastic surgery industry, but the long-term preservation rate of transplanted fat decreases because of complications such as oil cysts due to the inability in macrophages to metabolize absorption. In cell-assisted lipotransfer technology, adipose-derived stem cells (ASCs) can influence the inflammatory response of grafts through the immunoregulation in macrophages, and the lipid metabolism in macrophages plays an important role in this process. Therefore, we hypothesized ASCs could improve the retention rate of fat grafts by regulating the progress of lipid metabolism in macrophages., Methods: We established fat transplantation and ASC-assisted fat transplantation model in C57BL/6 mice in vivo, and bone marrow-derived macrophages cocultured with apoptotic adipocytes were treated with or without ASCs in vitro. Graft retention, tissue structure, fibrosis, macrophage phenotype transformation, lipid deposition, mitochondrial morphology, oxygen consumption rate (OCR), fatty acid β-oxidation (FAO) level, and ATP production were assessed. Additionally, fat transplantation and ASC-assisted fat transplantation model was treated with etomoxir which inhibits mitochondrial FAO. Macrophages pretreated with etomoxir were co-cultured with apoptotic adipocytes and treated with or without ASCs. The method aboved was used for detection and verification., Results: In vivo, ASC-assisted fat transplantation improved macrophage mitochondrial expression and FAO level, promoted the early transformation of M2 macrophages, reduced the long-term lipid deposition of macrophages, and improved the retention rate of fat grafts. In vitro, ASCs up-regulated the level of mitochondrial FAO, OCR and ATP production in macrophages, reduced lipid deposition of macrophages and promoted M2 macrophages polarization by paracine function. The ability of ASCs in group pretreated with etomoxir to reduce the foaming of macrophages, promote the transformation to M2 macrophages, and improve the retention rate of fat transplantation was weakened., Conclusions: ASCs increased the retention rate of transplanted fat by upregulating mitochondrial FAO to promote M2 polaration in macrophages. In addition, ASCs up-regulate mitochondrial FAO by paracrine effect to reduce foam cells formation and promote M2 transformation in macrophages in vitro., (© 2024. The Author(s).)

Background: Chronic radiation dermatitis (CRD) is a late consequence of radiation with high incidence in patients receiving radiotherapy. Conventional therapies often yield unsatisfactory results. Therefore, this study aimed to explore the therapeutic potential and mechanism of adipose-derived stem cells (ADSCs) for CRD, paving the way for novel regenerative therapies in clinical practice., Methods: Clinical CRD skin biopsies were analyzed to character the pathological changes of CRD skin and guided the animal modeling scheme. Subsequently, an in vivo analysisusing mouse CRD models was conducted to explore their effects of ADSCs on CRD, monitoring therapeutic impact for up to 8 weeks. Transcriptome sequencing and histologic sections analysis were performed to explore the potential therapeutic mechanism of ADSCs. Following observing extensive apoptosis of transplanted ADSCs, the therapeutic effect of ADSCs were compared with those of apoptosis-inhibited ADSCs. Multiphoton imaging and analysis of collagen morphologic features were employed to explain how translated ADSCs promote collagen remodeling at the microscopic level based on the contrast of morphology of collagen fibers., Results: Following injection into CRD-afflicted skin, ADSCs therapy effectively mitigated symptoms of CRD, including acanthosis of the epidermis, fibrosis, and irregular collagen deposition, consistent with the possible therapeutic mechanism suggested by transcriptome sequencing. Notably, in vivo tracking revealed a significant reduction in ADSCs number due to extensive apoptosis. Inhibiting apoptosis in ADSCs partially tempered their therapeutic effects. Mechanically, analysis of collagen morphologic features indicated that translated ADSCs might promote dermal extracellular matrix remodeling through enlarging, lengthening, crimping, and evening collagen, counteracting the atrophy and rupture caused by irradiation., Conclusions: This study demonstrated that ADSCs underwent substantial apoptosis upon local skin transplantation, and paradoxically, this apoptosis is essential for their efficacy in promoting the regeneration of late radiation-impaired skin. Mechanically, transplanted ADSCs could promote the remodeling and rearrangement of radiation-damaged dermal collagen matrix., (© 2024. The Author(s).)

Background: Fibrosis is a pathological scarring process characterized by persistent myofibroblast activation with excessive accumulation of extracellular matrix (ECM). Fibrotic disorders represent an increasing burden of disease-associated morbidity and mortality worldwide for which there are limited therapeutic options. Reversing fibrosis requires the elimination of myofibroblasts, remodeling of the ECM, and regeneration of functional tissue. Multipotent mesenchymal stromal cells (MSC) have antifibrotic properties mediated by secreted factors present in their conditioned medium (MSC-CM). However, there are no standardized in vitro assays to predict the antifibrotic effects of human MSC. As a result, we lack evidence on the effect of cytokine priming on MSC's antifibrotic effects. We hypothesize that the MSC-CM promotes fibrosis resolution in vitro and that this effect is enhanced following MSC cytokine priming., Methods: We compared the antifibrotic effects of resting versus interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) primed MSC-CM in four in vitro assays: prevention of fibroblast activation, myofibroblasts deactivation, ECM degradation and fibrosis resolution in lung explant cultures. Furthermore, we performed transcriptomic analysis of myofibroblasts treated or not with resting or primed MSC-CM and proteomic characterization of resting and primed MSC-CM., Results: We isolated MSC from adipose tissue of 8 donors, generated MSC-CM and tested each MSC-CM independently. We report that MSC-CM treatment prevented TGF-β induced fibroblast activation to a similar extent as nintedanib but, in contrast to nintedanib, MSC-CM reduced fibrogenic myofibroblasts (i.e. transcriptomic upregulation of apoptosis, senescence, and inflammatory pathways). These effects were larger when primed rather than resting MSC-CM were used. Priming increased the ability of MSC-CM to remodel the ECM, reducing its content of collagen I and fibronectin, and reduced the fibrotic load in TGF-β treated lung explant cultures. Priming increased the following antifibrotic proteins in MSC-CM: DKK1, MMP-1, MMP-3, follistatin and cathepsin S. Inhibition of DKK1 reduced the antifibrotic effects of MSC-CM., Conclusions: In vitro, MSC-CM promote fibrosis resolution, an effect enhanced following MSC cytokine priming. Specifically, MSC-CM reduces fibrogenic myofibroblasts through apoptosis, senescence, and by enhancing ECM degradation. Future studies will establish the in vivo relevance of MSC priming to fibrosis resolution., (© 2024. The Author(s).)