Adipose-derived stem cell exosomes loaded with icariin alleviates rheumatoid arthritis by modulating macrophage polarization in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by synovitis and cartilage destruction. The active compound, icariin (ICA), derived from the herb Epimedium, exhibits potent anti-inflammatory properties. However, its clinical utility is limited by its water insolubility, poor permeability, and low bioavailability. To address these challenges, we developed a multifunctional drug delivery system-adipose-derived stem cells-exosomes (ADSCs-EXO)-ICA to target active macrophages in synovial tissue and modulate macrophage polarization from M1 to M2. High-performance liquid chromatography analysis confirmed a 92.4 ± 0.008% loading efficiency for ADSCs-EXO-ICA. In vitro studies utilizing cellular immunofluorescence (IF) and flow cytometry demonstrated significant inhibition of M1 macrophage proliferation by ADSCs-EXO-ICA. Enzyme-linked immunosorbent assay, cellular transcriptomics, and real-time quantitative PCR indicated that ADSCs-EXO-ICA promotes an M1-to-M2 phenotypic transition by reducing glycolysis through the inhibition of the ERK/HIF-1α/GLUT1 pathway. In vivo, ADSCs-EXO-ICA effectively accumulated in the joints. Pharmacodynamic assessments revealed that ADSCs-EXO-ICA decreased cytokine levels and mitigated arthritis symptoms in collagen-induced arthritis (CIA) rats. Histological analysis and micro computed tomography confirmed that ADSCs-EXO-ICA markedly ameliorated synovitis and preserved cartilage. Further in vivo studies indicated that ADSCs-EXO-ICA suppresses arthritis by promoting an M1-to-M2 switch and suppressing glycolysis. Western blotting supported the therapeutic efficacy of ADSCs-EXO-ICA in RA, confirming its role in modulating macrophage function through energy metabolism regulation. Thus, this study not only introduces a drug delivery system that significantly enhances the anti-RA efficacy of ADSCs-EXO-ICA but also elucidates its mechanism of action in macrophage function inhibition.

Recent advances in the use of extracellular vesicles from adipose-derived stem cells for regenerative medical therapeutics.

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) isolated from adipose tissue. They possess remarkable properties, including multipotency, self-renewal, and easy clinical availability. ADSCs are also capable of promoting tissue regeneration through the secretion of various cytokines, factors, and extracellular vesicles (EVs). ADSC-derived EVs (ADSC-EVs) act as intercellular signaling mediators that encapsulate a range of biomolecules. These EVs have been found to mediate the therapeutic activities of donor cells by promoting the proliferation and migration of effector cells, facilitating angiogenesis, modulating immunity, and performing other specific functions in different tissues. Compared to the donor cells themselves, ADSC-EVs offer advantages such as fewer safety concerns and more convenient transportation and storage for clinical application. As a result, these EVs have received significant attention as cell-free therapeutic agents with potential future application in regenerative medicine. In this review, we focus on recent research progress regarding regenerative medical use of ADSC-EVs across various medical conditions, including wound healing, chronic limb ischemia, angiogenesis, myocardial infarction, diabetic nephropathy, fat graft survival, bone regeneration, cartilage regeneration, tendinopathy and tendon healing, peripheral nerve regeneration, and acute lung injury, among others. We also discuss the underlying mechanisms responsible for inducing these therapeutic effects. We believe that deciphering the biological properties, therapeutic effects, and underlying mechanisms associated with ADSC-EVs will provide a foundation for developing a novel therapeutic approach in regenerative medicine.

Role and therapeutic perspectives of extracellular vesicles derived from liver and adipose tissue in metabolic dysfunction-associated steatotic liver disease.

The global epidemic of metabolic diseases has led to the emergence of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), which pose a significant threat to human health. Despite recent advances in research on the pathogenesis and treatment of MASLD/MASH, there is still a lack of more effective and targeted therapies. Extracellular vesicles (EVs) discovered in a wide range of tissues and body fluids encapsulate different activated biomolecules and mediate intercellular communication. Recent studies have shown that EVs derived from the liver and adipose tissue (AT) play vital roles in MASLD/MASH pathogenesis and therapeutics, depending on their sources and intervention types. Besides, adipose-derived stem cell (ADSC)-derived EVs appear to be more effective in mitigating MASLD/MASH. This review presents an overview of the definition, extraction strategies, and characterisation of EVs, with a particular focus on the biogenesis and release of exosomes. It also reviews the effects and potential molecular mechanisms of liver- and AT-derived EVs on MASLD/MASH, and emphasises the contribution and clinical therapeutic potential of ADSC-derived EVs. Furthermore, the future perspective of EV therapy in a clinical setting is discussed.

Histone demethylase KDM3B mediates matrix stiffness-induced osteogenic differentiation of adipose-derived stem cells.

Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that biomechanics, such as the microstructure of nanomaterials, can induce adipose-derived stem cells (ASCs) to differentiate into osteoblasts, mediating gene regulation at the epigenetic level. Therefore, in this study, transcriptome expression levels of histone demethylases in ASCs were screened after treatment with different matrix stiffnesses, and histone lysine demethylase 3B (KDM3B) was found to promote osteogenic differentiation of ASCs in response to matrix stiffness, indicating a positive modulatory effect on this biological process. ASCs exhibited widespread and polygonal shapes with a distinct bundle-like expression of vinculin parallel to the axial cytoskeleton along the cell margins on the stiff matrix rather than round shapes with a smeared and shorter expression on the soft matrix. Comparatively rigid polydimethylsiloxane material directed ASCs into an osteogenic phenotype in inductive culture media via the upregulation of osteocalcin, alkaline phosphatase, and runt-related transcription factor 2. Treatment with KDM3B-siRNA decreased the expression of osteogenic differentiation markers and impaired mitochondrial dynamics and mitochondrial membrane potential. These results illustrate the critical role of KDM3B in the biomechanics-induced osteogenic commitment of ASCs and provide new avenues for the further application of stem cells as potential therapeutics for bone regeneration.

Insights into the role of adipose-derived stem cells and secretome: potential biology and clinical applications in hypertrophic scarring.

Scar tissue is the inevitable result of repairing human skin after it has been subjected to external destructive stimuli. It leads to localized damage to the appearance of the skin, accompanied by symptoms such as itching and pain, which reduces the quality of life of the patient and causes serious medical burdens. With the continuous development of economy and society, there is an increasing demand for beauty. People are looking forward to a safer and more effective method to eliminate pathological scarring. In recent years, adipose-derived stem cells (ADSCs) have received increasing attention from researchers. It can effectively improve pathological scarring by mediating inflammation, regulating fibroblast proliferation and activation, and vascular reconstruction. This review focuses on the pathophysiological mechanisms of hypertrophic scarring, summarizing the therapeutic effects of in vitro, in vivo, and clinical studies on the therapeutic effects of ADSCs in the field of hypertrophic scarring prevention and treatment, the latest application techniques, such as cell-free therapies utilizing ADSCs, and discussing the advantages and limitations of ADSCs. Through this review, we hope to further understand the characterization of ADSC and clarify the effectiveness of its application in hypertrophic scarring treatment, so as to provide clinical guidance.

A novel cell source for therapy of knee osteoarthritis using atelocollagen microsphere-adhered adipose-derived stem cells: Impact of synovial fluid exposure on cell activity.

Introduction: Administration of adipose-derived stem cells (ADSCs) into the joint cavity has been shown to alleviate the symptoms of knee osteoarthritis (OA) by releasing exosomes and anti-inflammatory cytokines. However, the therapeutic effect of these cells is limited by their rapid disappearance after administration. Thus, it is necessary to prolong cell survival in the joint cavity. This study aimed to investigate the potential application of ADSCs adhered to atelocollagen microspheres (AMSs) for cell therapy of knee OA. Methods: ADSCs were cultured for 2, 4, and 7 days in AMS suspension or adherent culture dishes. The supernatants were analyzed for IL-10 and exosome secretion via enzyme-linked immunosorbent assay and Nanosight. The effect of AMS was compared with that of adherent-cultured ADSCs (2D-cultured ADSCs) using transcriptome analysis. Moreover, the solubility of AMS and viability of ADSCs were evaluated using synovial fluid (SF) from patients with knee OA. Results: Compared with 2D-cultured ADSCs, AMS-cultured ADSCs exhibited a significant increase in secretion of exosomes and IL-10, and the expression of several genes involved in extracellular matrix and immune regulation were altered. Furthermore, when AMS-cultured ADSCs were cultured in SF from knee OA patients to mimic the intra-articular environment, the SF dissolved the AMSs and released viable ADSCs. In addition, AMS-cultured ADSCs showed significantly higher long-term cell viability than 2D-cultured ADSCs. Conclusion: Increased survival of AMS-adhered ADSCs was observed in the intra-articular environment, and AMSs were found to gradually dissipate. These results suggest that AMS-adhered ADSCs are promising source for cell therapy of knee OA.

miR-125b-5p delivered by adipose-derived stem cell exosomes alleviates hypertrophic scarring by suppressing Smad2.

Background: Hypertrophic scarring is the most serious and unmet challenge following burn and trauma injury and often leads to pain, itching and even loss of function. However, the demand for ideal scar prevention and treatment is difficult to satisfy. We aimed to discover the effects and mechanisms of adipose-derived stem cell (ADSC) exosomes in hypertrophic scarring. Methods: ADSC exosomes were isolated from the culture supernatant of ADSCs and identified by nanoparticle tracking analysis, transmission electron microscopy and western blotting. The effect of ADSC exosomes on wound healing and scar formation was detected by the wound model of BALB/c mice. We isolated myofibroblasts from hypertrophic scar tissue and detected the cell viability, proliferation and migration of myofibroblasts. In addition, collagen formation and fibrosis-related molecules were also detected. To further disclose the mechanism of ADSC exosomes on fibrosis in myofibroblasts, we detected the expression of Smad2 in hypertrophic scar tissue and normal skin and the regulatory mechanism of ADSC exosomes on Smad2. Injection of bleomycin was performed in male BALB/c mice to establish an in vivo fibrosis model while ADSC exosomes were administered to observe their protective effect. The tissue injury of mice was observed via hematoxylin and eosin and Masson staining and related testing. Results: In this study, we found that ADSC exosomes could not only speed up wound healing and improve healing quality but also prevent scar formation. ADSC exosomes inhibited expression of fibrosis-related molecules such as α-smooth muscle actin, collagen I (COL1) and COL3 and inhibited the transdifferentiation of myofibroblasts. In addition, we verified that Smad2 is highly expressed in both hypertrophic scar tissue and hypertrophic fibroblasts, while ADSC exosomes downregulated the expression of Smad2 in hypertrophic fibroblasts. Further regulatory mechanism analysis revealed that microRNA-125b-5p (miR-125b-5p) is highly expressed in ADSC exosomes and binds to the 3' untranslated region of Smad2, thus inhibiting its expression. In vivo experiments also revealed that ADSC exosomes could alleviate bleomycin-induced skin fibrosis and downregulate the expression of Smad2. Conclusions: We found that ADSC exosomes could alleviate hypertrophic scars via the suppression of Smad2 by the specific delivery of miR-125b-5p.

Identification and verification of AK4 as a protective immune-related biomarker in adipose-derived stem cells and breast cancer.

Background: Breast cancer (BC) remains the most common cancer among women, and novel post-surgical reconstruction techniques, including autologous fat transplantation, have emerged. While Adipose-derived stem cells (ADSCs) are known to impact the viability of fat grafts, their influence on breast cancer progression remains unclear. This study aims to elucidate the genetic interplay between ADSCs and breast cancer, focusing on potential therapeutic targets. Methods: Using the GEO and TCGA databases, we pinpointed differentially expressed (DE) mRNAs, miRNAs, lncRNAs, and pseudogenes of ADSCs and BC. We performed functional enrichment analysis and constructed protein-protein interaction (PPI), RNA binding protein (RBP)-pseudogene-mRNA, and lncRNA-miRNA-transcription factor (TF)-gene networks. Our study delved into the correlation of AK4 expression with 33 different malignancies and examined its impact on prognostic outcomes across a pan-cancer cohort. Additionally, we scrutinized immune infiltration, microsatellite instability, and tumor mutational burden, and conducted single-cell analysis to further understand the implications of AK4 expression. We identified novel sample subtypes based on hub genes using the ConsensusClusterPlus package and examined their association with immune infiltration. The random forest algorithm was used to screen DE mRNAs between subtypes to validate the powerful prognostic prediction ability of the artificial neural network. Results: Our analysis identified 395 DE mRNAs, 3 DE miRNAs, 84 DE lncRNAs, and 26 DE pseudogenes associated with ADSCs and BC. Of these, 173 mRNAs were commonly regulated in both ADSCs and breast cancer, and 222 exhibited differential regulation. The PPI, RBP-pseudogene-mRNA, and lncRNA-miRNA-TF-gene networks suggested AK4 as a key regulator. Our findings support AK4 as a promising immune-related therapeutic target for a wide range of malignancies. We identified 14 characteristic genes based on the AK4-related cluster using the random forest algorithm. Our artificial neural network yielded excellent diagnostic performance in the testing cohort with AUC values of 0.994, 0.973, and 0.995, indicating its ability to distinguish between breast cancer and non-breast cancer cases. Conclusions: Our research sheds light on the dual role of ADSCs in BC at the genetic level and identifies AK4 as a key protective mRNA in breast cancer. We found that AK4 significantly predicts cancer prognosis and immunotherapy, indicating its potential as a therapeutic target.

Adipose-derived stem cells derived decellularized extracellular matrix enabled skin regeneration and remodeling.

The tissues or organs derived decellularized extracellular matrix carry immunogenicity and the risk of pathogen transmission, resulting in limited therapeutic effects. The cell derived dECM cultured in vitro can address these potential risks, but its impact on wound remodeling is still unclear. This study aimed to explore the role of decellularized extracellular matrix (dECM) extracted from adipose derived stem cells (ADSCs) in skin regeneration. Methods: ADSCs were extracted from human adipose tissue. Then we cultivated adipose-derived stem cell cells and decellularized ADSC-dECM for freeze-drying. Western blot (WB), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) were conducted to analyzed the main protein components in ADSC-dECM. The cell counting assay (CCK-8) and scratch assay were used to explore the effects of different concentrations of ADSC-dECM on the proliferation and migration of human keratinocytes cells (HaCaT), human umbilical vein endothelia cells (HUVEC) and human fibroblasts (HFB), respectively. Moreover, we designed a novel ADSC-dECM-CMC patch which used carboxymethylcellulose (CMC) to load with ADSC-dECM; and we further investigated its effect on a mouse full thickness skin wound model. Results: ADSC-dECM was obtained after decellularization of in vitro cultured human ADSCs. Western blot, ELISA and mass spectrometry results showed that ADSC-dECM contained various bioactive molecules, including collagen, elastin, laminin, and various growth factors. CCK-8 and scratch assay showed that ADSC-dECM treatment could significantly promote the proliferation and migration of HaCaT, human umbilical vein endothelia cells, and human fibroblasts, respectively. To evaluate the therapeutic effect on wound healing in vivo, we developed a novel ADSC-dECM-CMC patch and transplanted it into a mouse full-thickness skin wound model. And we found that ADSC-dECM-CMC patch treatment significantly accelerated the wound closure with time. Further histology and immunohistochemistry indicated that ADSC-dECM-CMC patch could promote tissue regeneration, as confirmed via enhanced angiogenesis and high cell proliferative activity. Conclusion: In this study, we developed a novel ADSC-dECM-CMC patch containing multiple bioactive molecules and exhibiting good biocompatibility for skin reconstruction and regeneration. This patch provides a new approach for the use of adipose stem cells in skin tissue engineering.

In Vitro Study of Thymosin Beta 4 Promoting Transplanted Fat Survival by Regulating Adipose-Derived Stem Cells.

BACKGROUND: Autologous fat grafting (AFG) has emerged as a highly sought-after plastic surgery procedure, although its success has been hampered by the uncertain fat survival rate. Current evidence suggests that adipose-derived stem cells (ADSCs) may contribute to fat retention in AFG. In previous studies, it was confirmed that thymosin beta 4 (Tß4) could enhance fat survival in vivo, although the precise mechanism remains unclear. METHODS: ADSCs were isolated from patients undergoing liposuction and their proliferation, apoptosis, anti-apoptosis, and migration were analyzed under Tß4 stimulation using cell counting kit-8, flow cytometry, wound healing assay, and real-time quantitative PCR. The mRNA levels of genes relating to angiogenesis and Hippo signaling were also determined. RESULTS: Tß4 at 100 ng/mL (p-value = 0.0171) and 1000 ng/mL (p-value = 0.0054) significantly increased ADSC proliferation from day 1 compared to the control group (0 ng/mL). In addition, the mRNA levels of proliferation-associated genes were elevated in the Tß4 group. Furthermore, Tß4 enhanced the anti-apoptotic ability of ADSCs when stimulated with Tß4 and an apoptotic induction reagent (0 ng/mL vs. 1000 ng/mL, p-value = 0.011). Crucially, the mRNA expression levels of angiogenesis-related genes and critical genes in the Hippo pathway were affected by Tß4 in ADSCs. CONCLUSIONS: Tß4 enhances adipose viability in AFG via facilitating ADSC proliferation and reducing apoptosis, and acts as a crucial positive regulator of ADSC-associated angiogenesis. Additionally, Tß4 could be accountable for the phenotypic adjustment of ADSCs by regulating the Hippo pathway. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

[Adipose-derived stem cell-derived exosomes regulate Th2/Treg balance in peripheral blood of AR patients through the mTOR pathway].

Objective:To investigate the mechanism of adipose derived stem cell exosomes（ADSC-exos） regulating Th2/Treg balance in peripheral blood of patients with allergic rhinitis（AR）. Methods:Thirty patients with AR who were treated in Department of Otolaryngology Head and Neck Surgery, the First Affiliated Hospital of Zhengzhou University from March 2022 to October 2022 were selected, and 30 patients with simple deviation of nasal septum who were treated in our department during the same period were selected as the control group. 10 mL peripheral venous blood was collected from all patients. The levels of IL-4 and TGF-ß in plasma were analyzed by ELISA. PBMCs were isolated by density gradient centrifugation. Then, protein and RNA were further extracted, and the expression levels of IL-4, TGF-ß, GATA3 and Foxp3 genes were detected by qRT-PCR. Western Blotting detected p-PI3K（P85）, p-AKT（Ser473） in PBMCs of AR patients and healthy controls. Protein expression levels of p-mTOR（Ser2448）, p-p70S6K（Thr389）, and the proportion of Th2 and Treg cells were analyzed by flow cytometry. PBMCs of AR patients were stimulated to differentiate and co-cultured with exosomes of adipose stem cells. p-PI3K（P85）, p-AKT（Ser473）, p-mTOR（Ser2448） were detected in exosome treated group and untreated group by Western Blotting. The expression level of p-p70S6K（Thr389） protein, the proportion of Th2 and Treg cells were analyzed by flow cytometry, and the levels of IL-4 and TGF-ß in the supernatant of cell culture were detected by ELISA. Results:Compared with the control group, the mTOR pathway in peripheral blood of AR group was significantly activated, the level of IL-4 in plasma was increased, and the level of TGF-ß was decreased（P<0.05）. Compared with the control group, the proportion of Th2 cells in peripheral blood was increased, and the proportion of Treg cells was decreased（P<0.01）. Compared with the untreated group, the expression level of mTOR pathway protein decreased, the level of IL-4 decreased, and the level of TGF-ß increased. The proportion of Th2 cells decreased, and the proportion of Treg cells increased（P<0.01）. Conclusion:There is an imbalance of Th2 and Treg cells in peripheral blood mononuclear cells of AR patients; the PI3K/AKT/mTOR/p70S6K pathway is activated in peripheral blood mononuclear cells of AR patients Exosomes derived from adipose mesenchymal stem cells may regulate Th2/Treg balance in AR patients through the PI3K/AKT/mTOR/p70S6K pathway.

Investigation of Expanded Human Adipose-derived Stem Cell Dosage and Timing for Improved Defecation Function.

BACKGROUND/AIM: Although certain treatment options exist for intestinal incontinence, none are curative. Adipose-derived stem cells (ADSCs) have emerged as promising therapeutic agents, but most preclinical studies of their effectiveness for anal function have used autologous or allogeneic ADSCs. In this study, the effectiveness, timing of administration, and required dosage of human ADSCs were investigated for clinical application. MATERIALS AND METHODS: A 10-mm balloon catheter was used to induce anal sphincter injury in immunodeficient mice in the following experimental groups (n=4 per group): ADSC (injected ADSCs after injury), PBS (injected phosphate-buffered saline after injury), and control (uninjured). The effects of different timing (immediately after injection and 30 days following injury) and number of human ADSCs administered was compared among groups based on defecation status and pathological evaluation. RESULTS: In terms of defecation status, groups receiving ≥1×104 human ADSCs after injection showed improvement. Pathological images showed that compared to the PBS group, the thinnest part of the sphincter was thicker for animals that received ≥1×104 human ADSCs, and fibrosis of the sphincter was notable in those treated with 1×103 human ADSCs or PBS. Furthermore, defecation status was improved by administration of human ADSCs, not only immediately after injury, but also at 30 days following injury. CONCLUSION: Human ADSC administration in a mouse model of anal sphincter injury was effective. Injection of ≥1×104 human ADSCs was the amount necessary to improve defecation status, an effect detected in both the acute and chronic phases.

Bee Venom Stimulates Growth Factor Release from Adipose-Derived Stem Cells to Promote Hair Growth.

Limited evidence suggests that stimulating adipose-derived stem cells (ASCs) indirectly promotes hair growth. We examined whether bee venom (BV) activated ASCs and whether BV-induced hair growth was facilitated by enhanced growth factor release by ASCs. The induction of the telogen-to-anagen phase was studied in mice. The underlying mechanism was investigated using organ cultures of mouse vibrissa hair follicles. When BV-treated ASCs were injected subcutaneously into mice, the telogen-to-anagen transition was accelerated and, by day 14, the hair weight increased. Quantitative polymerase chain reaction (qPCR) revealed that BV influenced the expression of several molecules, including growth factors, chemokines, channels, transcription factors, and enzymes. Western blot analysis was employed to verify the protein expression levels of extracellular-signal-regulated kinase (ERK) and phospho-ERK. Both the Boyden chamber experiment and scratch assay confirmed the upregulation of cell migration by BV. Additionally, ASCs secreted higher levels of growth factors after exposure to BV. Following BV therapy, the gene expression levels of alkaline phosphatase (ALP), fibroblast growth factor (FGF)-1 and 6, endothelial cell growth factor, and platelet-derived growth factor (PDGF)-C were upregulated. The findings of this study suggest that bee venom can potentially be utilized as an ASC-preconditioning agent for hair regeneration.

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion.

BACKGROUND: Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. METHOD: To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. RESULTS: ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. CONCLUSIONS: HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells.

Characterization of Acellular Cartilage Matrix-Sodium Alginate Scaffolds in Various Proportions.

The development of three-dimensional (3D) bioprinting technology has provided a new solution to address the shortage of donors, multiple surgeries, and aesthetic concerns in microtia reconstruction surgery. The production of bioinks is the most critical aspect of 3D bioprinting. Acellular cartilage matrix (ACM) and sodium alginate (SA) are commonly used 3D bioprinting materials, and there have been reports of their combined use. However, there is a lack of comprehensive evaluations on ACM-SA scaffolds with different proportions. In this study, bioinks were prepared by mixing different proportions of decellularized rabbit ear cartilage powder and SA and then printed using 3D bioprinting technology and crosslinked with calcium ions to fabricate scaffolds. The physical properties, biocompatibility, and toxicity of ACM-SA scaffolds with different proportions were compared. The adhesion and proliferation of rabbit adipose-derived stem cells on ACM-SA scaffolds of different proportions, as well as the secretion of Collagen Type II, were evaluated under an adipose-derived stem cell chondrogenic induction medium. The following conclusions were drawn: when the proportion of SA in the ACM-SA scaffolds was <30%, the printed structure failed to form. The ACM-SA scaffolds in proportions from 1:9 to 6:4 showed no significant cytotoxicity, among which the 5:5 proportion of ACM-SA scaffold was superior in terms of adhesiveness and promoting cell proliferation and differentiation. Although a higher proportion of SA can provide greater mechanical strength, it also significantly increases the swelling ratio and reduces cell proliferation capabilities. Overall, the 5:5 proportion of ACM-SA scaffold demonstrated a more desirable biological and physical performance.

GDNF facilitates the differentiation of ADSCs to Schwann cells and enhances nerve regeneration through GDNF/MTA1/Hes1 axis.

Adipose tissue-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential, which mainly restore tissue repair function and promote cell regeneration. It can be directionally differentiated into Schwann-like cells to promote the repair of peripheral nerve injury. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in the repair of nerve injury, but the underlying mechanism remains unclear, which seriously limits its further application.The study aimed to identify the molecular mechanism by which overexpression of glial cell line-derived neurotrophic factor (GDNF) facilitates the differentiation of ADSCs into Schwann cells, enhancing nerve regeneration after injury. In vitro, ADSCs overexpressing GDNF for 48 h exhibited changes in their morphology, with 80% of the cells having two or more prominences. Compared with that of ADSCs, GDNF-ADSCs exhibited increased expression of the Schwann cell marker S100, nerve damage repair-related factors.ADSC cells in normal culture and ADSC cells were overexpressing GDNF(GDNF-ADSCs) were analysed using TMT-Based Proteomic Analysis and revealed a significantly higher expression of MTA1 in GDNF-ADSCs than in control ADSCs. Hes1 expression was significantly higher in GDNF-ADSCs than in ADSCs and decreased by MTA1 silencing, along with a simultaneous decrease in the expression of S100 and nerve damage repair factors. These findings indicate that GDNF promotes the differentiation of ADSCs into Schwann cells and induces factors that accelerate peripheral nerve damage repair.

Glutathione supplementation improves fat graft survival by inhibiting ferroptosis via the SLC7A11/GPX4 axis.

BACKGROUND: Autologous fat grafting is hampered by unpredictable graft survival, which is potentially regulated by ferroptosis. Glutathione (GSH), a powerful antioxidant used in tissue preservation, has ferroptosis-regulating activity; however, its effects on fat grafts are unclear. This study investigated the effects and mechanisms of GSH in fat graft survival. METHODS: Human lipoaspirates were transplanted subcutaneously into the backs of normal saline-treated (control) or GSH-treated nude mice. Graft survival was evaluated by magnetic resonance imaging and histology. RNA sequencing was performed to identify differentially expressed genes and enriched pathways. GSH activity was evaluated in vitro using an oxygen and glucose deprivation (OGD) model of adipose-derived stem cells. RESULTS: Compared with control group, GSH induced better outcomes, including superior graft retention, appearance, and histological structures. RNA sequencing suggested enhanced negative regulation of ferroptosis in the GSH-treated grafts, which showed reduced lipid peroxides, better mitochondrial ultrastructure, and SLC7A11/GPX4 axis activation. In vitro, OGD-induced ferroptosis was ameliorated by GSH, which restored cell proliferation, reduced oxidative stress, and upregulated ferroptosis defense factors. CONCLUSIONS: Our study confirms that ferroptosis participates in regulating fat graft survival and that GSH exerts a protective effect by inhibiting ferroptosis. GSH-assisted lipotransfer is a promising therapeutic strategy for future clinical application.

Sulfated Chitosan Nanofibrous Scaffolds Seeded With Adipose Stem Cells Promote Ischemic Wound Healing in a Proangiogenic Strategy.

Ischemic wounds are chronic wounds with poor blood supply that delays wound reconstruction. To accelerate wound healing and promote angiogenesis, adipose-derived stem cells (ADSCs) are ideal seed cells for stem cell-based therapies. Nevertheless, providing a favorable environment for cell proliferation and metabolism poses a substantial challenge. A highly sulfated heparin-like polysaccharide 2-N, 6-O-sulfated chitosan (26SCS)-doped poly(lactic-co-glycolic acid) scaffold (S-PLGA) can be used due to their biocompatibility, mechanical properties, and coagent 26SCS high affinity for growth factors. In this study, a nano-scaffold system, constructed from ADSCs seeded on electrospun fibers of modified PLGA, was designed to promote ischemic wound healing. The S-PLGA nanofiber membrane loaded with adipose stem cells ADSCs@S-PLGA was prepared by a co-culture in vitro, and the adhesion and compatibility of cells on the nano-scaffolds were explored. Scanning electron microscopy was used to observe the growth state and morphological changes of ADSCs after co-culture with PLGA electrospun fibers. The proliferation and apoptosis after co-culture were detected using a Cell Counting Kit-8 kit and flow cytometry, respectively. An ischemic wound model was then established, and we further studied the ability of ADSCs@S-PLGA to promote wound healing and angiogenesis. We successfully established ischemic wounds on the backs of rats and demonstrated that electrospun fibers combined with the biological effects of adipose stem cells effectively promoted wound healing and the growth of microvessels around the ischemic wounds. Phased research results can provide a theoretical and experimental basis for a new method for promoting clinical ischemic wound healing.

Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Background: Stem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells. Methods: Human ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes. Results: The transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified. Conclusions: This study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Trends in Adipose-Derived Stem Cell-Conditioned Medium: A Bibliometric and Visualized Review.

Adipose-derived stem cell-conditioned medium (ADSC-CM) has been widely studied and used as a stem cell-based cell-free therapy. Due to the explosion of scientific publications in this field, it is difficult to review all relevant publications systematically, not mention quantitively. In this study, we combined bibliometrics with the conventional review method to summarize, analyze, and visualize the characteristics of nearly all published articles related to ADSC-CM using CiteSpace-a bibliometrics software. We applied this software to quantitively and vividly show (a) annual publications and citations; (b) distributions and co-occurrence networks of countries/regions, authors, journals, and institutions; (c) keyword co-occurrence networks and clusters in different time periods; (d) cocitation networks of references; and (e) ongoing challenges and new topics in ADSC-CM. Altogether, we found that ADSC-CM is at a hot stage with an increasing number of publications and citations, extensive and close scientific collaborations, and dense cocited networks. Impact statement To our best knowledge, it is the first bibliometric and visualized review in the field of adipose-derived stem cell-conditioned medium (ADSC-CM). This review systematically and quantitatively revealed the developments, challenges, and emerging hot spots of ADSC-CM, providing a panoramic view to assist researchers to decide the direction of their future study in the fields of ADSCs and CM derived from stem cells.