The impact of hypoxia preconditioning on mesenchymal stem cells performance in hypertensive kidney disease.

BACKGROUND: Autologous mesenchymal stem cells (MSCs) have emerged as a therapeutic option for many diseases. Hypertensive kidney disease (HKD) might impair MSCs' reparative ability by altering the biomolecular properties, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, we found hypoxic preconditioning (HPC) enhanced angiogenesis and suppressed senescence gene expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment. METHODS: MSC samples (n = 12 each) were collected from the abdominal fat of healthy kidney donors (HC), hypertensive patients (HTN), and patients with hypertensive kidney disease (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions. MSC functionality was measured by proliferation assays and cytokine released in conditioned media. Senescence was evaluated by senescence-associated beta-galactosidase (SA-beta-gal) activity. Additionally, transcriptome analysis using RNA-sequencing and quantitative PCR (qPCR) were performed. RESULTS: At baseline, normoxic HTN-MSCs had higher proliferation capacity compared to HC. However, HPC augmented proliferation in HC. HPC did not affect the release of pro-angiogenic protein VEGF, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. Under HPC, SA-ß-gal activity tended to decrease, particularly in HC group. HPC upregulated mostly the pro-angiogenic and inflammatory genes in HC and HKD and a few senescence genes in HKD. CONCLUSIONS: HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and modest decrease in senescence, whereas it has little effect on HTN or HKD MSCs.

Exploring the biphasic dose-response effects of photobiomodulation on the viability, migration, and extracellular vesicle secretion of human adipose mesenchymal stem cells.

Photobiomodulation (PBM) is a well-established medical technology that employs diverse light sources like lasers or light-emitting diodes to generate diverse photochemical and photophysical reactions in cells, thereby producing beneficial clinical outcomes. In this study, we introduced an 830 nm near-infrared (NIR) laser irradiation system combined with a microscope objective to precisely and controllably investigate the impact of PBM on the migration and viability of human adipose mesenchymal stem cells (hADSCs). We observed a biphasic dose-response in hADSCs' viability and migration after PBM exposure (0-10 J/cm2), with the 5 J/cm2 group showing significantly higher cell viability and migration ability than other groups. Additionally, at the optimal dose of 5 J/cm2, we used nanoparticle tracking analysis (NTA) and found a 6.25-fold increase in the concentration of extracellular vesicles (EVs) derived from hADSCs (PBM/ADSC-EVs) compared to untreated cells (ADSC-EVs). Both PBM/ADSC-EVs and ADSC-EVs remained the same size, with an average diameter of 56 nm measured by the ExoView R200 system, which falls within the typical size range for exosomes. These findings demonstrate that PBM not only improves the viability and migration of hADSCs but also significantly increases the EV yield.

Adipose mesenchymal stem cells-derived exosomes attenuated hyperoxia-induced lung injury in neonatal rats via inhibiting the NF-κB signaling pathway.

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% O2) 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-1ß, 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.

Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy.

BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. METHODS: In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 µg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10. RESULTS: The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. CONCLUSIONS: Our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.

Engineered hsa-miR-455-3p-Abundant Extracellular Vesicles Derived from 3D-Cultured Adipose Mesenchymal Stem Cells for Tissue-Engineering Hyaline Cartilage Regeneration.

Efforts are made to enhance the inherent potential of extracellular vesicles (EVs) by utilizing 3D culture platforms and engineered strategies for functional cargo-loading. Three distinct types of adipose mesenchymal stem cells-derived EVs (ADSCs-EVs) are successfully isolated utilizing 3D culture platforms consisting of porous gelatin methacryloyl (PG), PG combined with sericin methacryloyl (PG/SerMA), or PG combined with chondroitin sulfate methacryloyl (PG/ChSMA). These correspond to PG-EVs, PG/SerMA-EVs, and PG/ChSMA-EVs, respectively. Unique microRNA (miRNA) profiles are observed in each type of ADSCs-EVs. Notably, PG-EVs encapsulate higher levels of hsa-miR-455-3p and deliver more hsa-miR-455-3p to chondrocytes, which results in the activation of the hsa-miR-455-3p/PAK2/Smad2/3 axis and the subsequent hyaline cartilage regeneration. Furthermore, the functionality of PG-EVs is optimized through engineered strategies, including agomir/lentivirus transfection, electroporation, and Exo-Fect transfection. These strategies, referred to as Agomir-EVs, Lentivirus-EVs, Electroporation-EVs, and Exo-Fect-EVs, respectively, are ranked based on their efficacy in encapsulating hsa-miR-455-3p, delivering hsa-miR-455-3p to chondrocytes, and promoting cartilage formation via the hsa-miR-455-3p/PAK2/Smad2/3 axis. Notably, Exo-Fect-EVs exhibit the highest efficiency. Collectively, the 3D culture conditions and engineered strategies have an impact on the miRNA profiles and cartilage regeneration capabilities of ADSCs-EVs. The findings provide valuable insights into the mechanisms underlying the promotion of cartilage regeneration by ADSCs-EVs.

Therapeutic potential of exosome derived from hepatocyte growth factor-overexpressing adipose mesenchymal stem cells in TGFß1-stimulated hepatic stellate cells.

Cirrhosis is a familiar end-stage of multiple chronic liver diseases. The gene-modified mesenchymal stem cells (MSCs) have become one of the most promising schemes for the treatment of cirrhosis. MSCs exhibit their therapeutic role mainly by secreting hepatocyte growth factor (HGF). The aim of this research was to probe the anti-fibrosis role of exosomes secreted by HGF modified-mouse adipose MSCs (ADMSCs) on activated hepatic stellate cells (HSCs) and to preliminarily explore the possible mechanism. Firstly, mouse ADMSCs were isolated and identified. Quantitative real-time polymerase chain reaction verified the transfection efficiency of ADMSC transfected with HGF lentivirus. Exosomes derived from ADMSC transfecting negative control/HGF (ADMSCNC-Exo/ADMSCHGF-Exo) were extracted by density gradient centrifugation. HSCs were allocated to the control, TGF-ß, TGF-ß + ADMSC-Exo, TGF-ß + ADMSCNC-Exo, and TGF-ß + ADMSCHGF-Exo groups. Moreover, all mice were distributed to the control, CCl4 (40% CCl4 in olive oil), CCl4+ADMSC-Exo, CCl4+ADMSCNC-Exo, and CCl4+ADMSCHGF-Exo groups. Exosomes derived from ADMSCs with or without HGF transfection suppressed HSC activation, as evidenced by attenuating cell viability and cell cycle arrest at S phase but inducing apoptosis. Moreover, ADMSC-Exo, ADMSCNC-Exo, and ADMSCHGF-Exo effectively repressed the gene and protein levels of α-SMA, Col-I, Rho A, Cdc42, and Rac1 in TGF-ß-treated HSCs, and ADMSCHGF-Exo had the best effect. ADMSCHGF-Exo had a stronger regulatory effect on serum liver index than ADMSCNC-Exo in CCl4-induced mice. In conclusion, ADMSCHGF-Exo alleviated liver fibrosis by weakening the Rho pathway, thus reducing collagen production.

Hepatocellular carcinoma patients serum modulates the regenerative capacities of adipose mesenchymal stromal cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers causing the highest mortality rate worldwide. Treatment options of surgery, radiation, cytotoxic drugs and liver transplantation suffer significant side effects and a high frequency of relapse. Stem cell therapy has been proposed as a new effective therapy, however, controversial reports are emerging on the role of mesenchymal stem cells in cancer. In this work, we aimed to assess the regenerative capacities of adipose mesenchymal stem cells when exposed to serum from HCC patients, by assessing the effect of the sera on modulating the regenerative capacities of h-AMSCs and the cancer properties in HCC cells. This will pave the way for maximizing the efficacy of MSCs in cancer therapy. Our data show that HCC serum-treated hA-MSCs suffered oncogene-induced senescence as shown by their altered morphology and ameliorated proliferation and differentiation. The cells were enlarged with small irregular nuclei, swollen rough endoplasmic reticulum cisternae, and aging lysosomes typified by dark residual bodies. HCC serum-treated Huh-7 cancer cells on the other hand displayed higher tumor aggressiveness as depicted by altered morphology, increased cellular proliferation and migration, and decreased percentage of early and late apoptotic cells. Our findings provide evidence that exposure of hA-MSCs to the serum of HCC patients decreases their regenerative capacities and should be considered when employed as a potential therapy in HCC patients.

Engineered three-dimensional bioactive scaffold for enhanced bone regeneration through modulating transplanted adipose derived mesenchymal stem cell and stimulating angiogenesis.

Titanium alloy materials are commonly used in orthopedic clinical treatments. However, conventional titanium implants usually lead to insufficient bone regeneration and integration because of mismatched biomechanics and poor bioactivities. To tackle these challenges, a porous titanium alloy scaffold with suitable mechanical properties was prepared using three-dimensional (3D) printing, and then an adipose-derived mesenchymal stem cell (ADSC) loaded platelet-rich plasma (PRP) gel was placed into the pores of the porous scaffold to construct a bioactive scaffold with dual functions of enhancing angiogenesis and osteogenesis. This bioactive scaffold showed good biocompatibility and supported cell viability proliferation and morphology of encapsulated ADSCs. Osteogenic and angiogenic growth factors in the PRP gel promoted the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and enhanced osteogenic-related gene and protein expression in ADSCs, thus promoting osteogenic differentiation. After implantation into the femoral defects of rabbits, the bioactive scaffold promoted vascular network formation and the expression of osteogenesis-related proteins, thus effectively accelerating bone regeneration. Therefore, the osteogenic and angiogenic bioactive scaffold comprising a 3D printed porous titanium alloy scaffold, PRP, and ADSCs provides a promising design for orthopedic biomaterials with clinical transformation prospects and an effective strategy for bone defect treatment.

Molecular Mechanisms and Therapeutic Implications of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells in an In Vitro Model of Diabetic Retinopathy.

The blood-retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR.

Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis.

BACKGROUND AIMS: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. METHODS: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. RESULTS: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. CONCLUSIONS: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS.

Synergistic Therapeutic Potential of Dual 3D Mesenchymal Stem Cell Therapy in an Ischemic Hind Limb Mouse Model.

Three-dimensional (3D) culture systems have been widely used to promote the viability and metabolic activity of mesenchymal stem cells (MSCs). The aim of this study was to explore the synergistic benefits of using dual 3D MSC culture systems to promote vascular regeneration and enhance therapeutic potential. We used various experimental assays, including dual 3D cultures of human adipose MSCs (hASCs), quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vitro cell migration, Matrigel tube network formation, Matrigel plug assay, therapeutic assays using an ischemic hind limb mouse model, and immunohistochemical analysis. Our qRT-PCR results revealed that fibroblast growth factor 2 (FGF-2), granulocyte chemotactic protein-2 (GCP-2), and vascular endothelial growth factor-A (VEGF-A) were highly upregulated in conventional 3D-cultured hASCs (ASC-3D) than in two-dimensional (2D)-cultured hASCs. Hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and stromal-cell-derived factor-1 (SDF-1) showed higher expression levels in cytokine-cocktail-based, 3D-cultured hASCs (ASC-3Dc). A conditioned medium (CM) mixture of dual 3D ASCs (D-3D; ASC-3D + ASC-3Dc) resulted in higher migration and Matrigel tube formation than the CM of single 3D ASCs (S-3D; ASC-3D). Matrigel plugs containing D-3D contained more red blood cells than those containing S-3D. D-3D transplantation into ischemic mouse hind limbs prevented limb loss and augmented blood perfusion when compared to S-3D transplantation. Transplanted D-3D also revealed a high capillary density and angiogenic cytokine levels and transdifferentiated into endothelial-like cells in the hind limb muscle. These findings highlight the benefits of using the dual 3D culture system to optimize stem-cell-based therapeutic strategies, thereby advancing the therapeutic strategy for ischemic vascular disease and tissue regeneration.

Therapeutic effect of exosomes derived from hepatocyte-growth-factor-overexpressing adipose mesenchymal stem cells on liver injury.

Adipose mesenchymal stem cell-derived exosomes (ADMSC-Exo) are a new strategy for the treatment of liver injury. However, mesenchymal stem cells (MSCs) exert therapeutic effects mainly by secreting hepatocyte growth factor (HGF). Therefore, we investigated the role of exosomes derived from ADMSC that overexpress HGF (ADMSCHGF-Exo) on liver injury. MATERIAL AND METHODS: ADMSCs were isolated from young BALB/c female mice. Then exosomes derived from ADMSC transfecting negative control (ADMSCNC-Exo) and HGF overexpression (ADMSCHGF-Exo) were isolated and identified by quantitative polymerase chain reaction (qPCR), flow cytometry, western blot, transmission electron microscope and Nanosight particle tracking analysis. These exosomes were injected into male mice via tail vein after inducing liver injury by administering 40% carbon tetrachloride (CCl4)-olive oil twice a week (3 mL/kg, subcutaneously) for 6 weeks. Liver injury and liver collagen fiber accumulation were determined by histopathological analysis. Then, the levels of serum liver function indexes (alanine aminotransferase, aspartate aminotransferase, albumin, total bilirubin), hepatocyte-specific markers (albumin, cytokeratin-18 and hepatocyte nuclear factor 4α), hepatic fibrosis-related proteins (α-smooth muscle actin and collagen I) and Rho GTPase (cell division cycle 42 and ras-related C3 botulinum toxin substrate 1) were determined by Enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, Western blot and qPCR. RESULTS: ADMSCs were identified by high expression of CD105 and CD44 molecules and low expression of CD45 and CD34. ADMSCs-Exo, ADMSCNC-Exo and ADMSCHGF-Exo transfected cells had similar expression of exosome-specific membrane proteins (CD63, CD81 and CD9). Mice with CCl4-induced liver injury exhibited abnormal serum liver function indexes, altered expression of hepatocyte-specific markers, hepatic fibrosis-related proteins and Rho GTPase protein as well as histopathological changes and collagen fiber accumulation in the liver. These changes were reversed by ADMSC-Exo, ADMSCNC-Exo and ADMSCHGF-Exo administration with ADMSCHGF-Exo displaying the most significant impact. CONCLUSIONS: ADMSCHGF-Exo exerted a hepatoprotective effect in mice with experimental liver injury by alleviating hepatic fibrosis and restoring liver function.

Hypoxia-treated adipose mesenchymal stem cell-derived exosomes attenuate lumbar facet joint osteoarthritis.

BACKGROUND: Lumbar facet joint osteoarthritis (LFJ OA) is a common disease, and there is still a lack of effective disease-modifying therapies. Our aim was to determine the therapeutic effect of hypoxia-treated adipose mesenchymal stem cell (ADSC)-derived exosomes (Hypo-ADSC-Exos) on the protective effect against LFJ OA. METHODS: The protective effect of Hypo-ADSC-Exos against LFJ OA was examined in lumbar spinal instability (LSI)-induced LFJ OA models. Spinal pain behavioural assessments and CGRP (Calcitonin Gene-Related Peptide positive) immunofluorescence were evaluated. Cartilage degradation and subchondral bone remodelling were assessed by histological methods, immunohistochemistry, synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR), and 3D X-ray microscope scanning. RESULTS: Hypoxia enhanced the protective effect of ADSC-Exos on LFJ OA. Specifically, tail vein injection of Hypo-ADSC-Exos protected articular cartilage from degradation, as demonstrated by lower FJ OA scores of articular cartilage and less proteoglycan loss in lumbar facet joint (LFJ) cartilage than in the ADSC-Exo group, and these parameters were significantly improved compared to those in the PBS group. In addition, the levels and distribution of collagen and proteoglycan in LFJ cartilage were increased in the Hypo-ADSC-Exo group compared to the ADSC-Exo or PBS group by SR-FTIR. Furthermore, Hypo-ADSC-Exos normalized uncoupled bone remodelling and aberrant H-type vessel formation in subchondral bone and effectively reduced symptomatic spinal pain caused by LFJ OA in mice compared with those in the ADSC-Exo or PBS group. CONCLUSIONS: Our results show that hypoxia is an effective method to improve the therapeutic effect of ADSC-Exos on ameliorating spinal pain and LFJ OA progression.

Extract of Isatidis Radix Inhibits Lipid Accumulation in In Vitro and In Vivo by Regulating Oxidative Stress.

Isatidis Radix (IR), the root of Isatis tinctoria L. belonging to Brassicaceae, has been traditionally used as a fever reducer. Although some pharmacological effects, such as anti-diabetes, anti-virus, and anti-inflammatory, have been reported, there is no study on the anti-obesity effect of IR. This study used 3T3-L1 cells, human mesenchymal adipose stem cells (hAMSCs), and a high-fat diet (HFD)-induced obese mouse model to confirm the anti-adipogenic effect of IR. Intracellular lipid accumulation in 3T3-L1 cells and hAMSCs was decreased by IR treatment.IR extract especially suppressed reactive oxygen species (ROS) production through a cluster of differentiation 36 (CD36)-AMP-activated protein kinase (AMPK) pathway. Consequently, the expressions of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding proteins alpha (C/EBPα), and fatty acid synthesis (FAS) were inhibited by IR extract. In addition, ß-oxidation-related genes were also decreased by treatment of IR extract. IR inhibited weight gain through this cascade in the HFD-induced obese mouse model. IR significantly suppressed lipid accumulation in epididymal white adipose tissue (eWAT). Furthermore, the administration of IR extract decreased serum free fatty acid (FFA), total cholesterol (TC), and LDL cholesterol, suggesting that it could be a potential drug for obesity by inhibiting lipid accumulation.

Hypoxia Treatment of Adipose Mesenchymal Stem Cells Promotes the Growth of Dermal Papilla Cells via HIF-1α and ERK1/2 Signaling Pathways.

Dermal papilla cells (DPCs) cultured in vitro induce hair follicle formation. Using a hypoxic microenvironment to culture adipose mesenchymal stem cells (ADSCs) can promote hair follicle growth. However, the exact molecular mechanisms underlying this process remain unclear. In this study, ADSCs and DPCs from Arbas Cashmere goats were used. A hypoxic microenvironment promoted the proliferation of ADSCs and increased the pluripotency of ADSCs. The growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were upregulated in ADSCs in the hypoxia-conditioned medium (Hypo-cm). Hypo-cm also enhanced the ability of DPCs to induce hair follicle formation. Inhibitors of the ERK1/2 signaling pathway caused the expressions of growth factors that increased in hypoxic microenvironments to decrease; moreover, hypoxia-inducible factor-1α (HIF-1α) increased the expression levels of VEGF, bFGF, and PDGF and inhibited the expression of bone morphogenic protein 7 (BMP7). In conclusion, these findings improve the theoretical basis for the development of gene therapy drugs for the treatment of alopecia areata and hair thinning.

Exosomes Derived from Human Adipose-Derived Stem Cells Cannot Distinctively Promote Graft Survival in Cryopreservation Fat Grafting.

BACKGROUND: Cryopreserved fat has limited clinical applications due to its rapid absorption, high degree of fibrosis, and risk of complications after grafting. Many studies have verified that Adipose-derived mesenchymal stem cell-derived exosomes (ADSC-Exos) can improve fresh fat graft survival. This study assessed whether ADSC-Exos could improve the survival of cryopreserved fat grafts. METHODS: Exosomes were isolated from human ADSCs were subcutaneously engrafted with adipose tissues stored under different conditions (fresh; cryopreserved for 1 month) into the backs of BALB/c nude mice (n = 24), and exosomes or PBS were administered weekly. Grafts were harvested at 1, 2, 4, and 8 weeks, and fat retention rate, histologic, and immunohistochemical analyses were conducted. RESULTS: At 1, 2, and 4 weeks after the transfer, cryopreserved fat grafts in groups of exosome-treated showed better fat integrity, fewer oil cysts, and reduced fibrosis. Further investigations of macrophage infiltration and neovascularization revealed that those exosomes increased the number of M2 macrophages at 2 and 4 weeks (p<0.05), but had limited impact on vascularization (p>0.05). It's important to note that no significant differences (p>0.05) were observed between the two groups in both histological and immunohistochemical evaluations at 8 weeks post-transplantation. CONCLUSIONS: This study suggests that ADSC-Exos could improve the survival of cryopreserved fat grafts in the short term (within 4 weeks), but the overall improvement was poor (after 8 weeks). This suggests that the utility of using ADSC-Exos to treat cryopreserved adipose tissue grafts is limited. 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 Mesenchymal Stem Cell Derived Exosomes Promote Keratinocytes and Fibroblasts Embedded in Collagen/Platelet-Rich Plasma Scaffold and Accelerate Wound Healing.

Engineered skin substitutes derived from human skin significantly reduce inflammatory reactions mediated by foreign/artificial materials and are consequently easier to use for clinical application. Type I collagen is a main component of the extracellular matrix during wound healing and has excellent biocompatibility, and platelet-rich plasma can be used as the initiator of the healing cascade. Adipose mesenchymal stem cell derived exosomes are crucial for tissue repair and play key roles in enhancing cell regeneration, promoting angiogenesis, regulating inflammation, and remodeling extracellular matrix. Herein, Type I collagen and platelet-rich plasma, which provide natural supports for keratinocyte and fibroblast adhesion, migration, and proliferation, are mixed to form a stable 3D scaffold. Adipose mesenchymal stem cell derived exosomes are added to the scaffold to improve the performance of the engineered skin. The physicochemical properties of this cellular scaffold are analyzed, and the repair effect is evaluated in a full-thickness skin defect mouse model. The cellular scaffold reduces the level of inflammation and promotes cell proliferation and angiogenesis to accelerate wound healing. Proteomic analysis shows that exosomes exhibit excellent anti-inflammatory and proangiogenic effects in collagen/platelet-rich plasma scaffolds. The proposed method provides a new therapeutic strategy and theoretical basis for tissue regeneration and wound repair.

Adipose mesenchymal stem cell-derived extracellular vesicles reduce glutamate-induced excitotoxicity in the retina.

Adipose mesenchymal stem cells (ADSCs) have protective effects against glutamate-induced excitotoxicity, but ADSCs are limited in use for treatment of optic nerve injury. Studies have shown that the extracellular vesicles (EVs) secreted by ADSCs (ADSC-EVs) not only have the function of ADSCs, but also have unique advantages including non-immunogenicity, low probability of abnormal growth, and easy access to target cells. In the present study, we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography. In addition, R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium, downregulation of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) subunit GluA2, and phosphorylation of GluA2 and protein kinase C alpha in vitro. A protein kinase C alpha agonist, 12-O-tetradecanoylphorbol 13-acetate, inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells. These findings suggest that ADSC-EVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.

Repairing rat calvarial defects by adipose mesenchymal stem cells and novel freeze-dried three-dimensional nanofibrous scaffolds.

Introduction: Treatment of critical-sized bone defects is challenging. Tissue engineering as a state-of-the-art method has been concerned with treating these non-self-healing bone defects. Here, we studied the potentials of new three-dimensional nanofibrous scaffolds (3DNS) with and without human adipose mesenchymal stem cells (ADSCs) for reconstructing rat critical-sized calvarial defects (CSCD). Methods: Scaffolds were made from 1- polytetrafluoroethylene (PTFE), and polyvinyl alcohol (PVA) (PTFE/ PVA group), and 2- PTFE, PVA, and graphene oxide (GO) nanoparticle (PTFE/ PVA/GO group) and seeded by ADSCs and incubated in osteogenic media (OM). The expression of key osteogenic proteins including Runt-related transcription factor 2 (Runx2), collagen type Iα (COL Iα), osteocalcin (OCN), and osteonectin (ON) at days 14 and 21 of culture were evaluated by western blot and immunocytochemistry methods. Next, 40 selected rats were assigned to five groups (n=8) to create CSCD which will be filled by scaffolds or cell-containing scaffolds. The groups were denominated as the following order: Control (empty defects), PTFE/PVA (PTFE/PVA scaffolds implant), PTFE/PVA/GO (PTFE/PVA/GO scaffolds implant), PTFE/PVA/Cell group (PTFE/PVA scaffolds containing ADSCs implant), and PTFE/PVA/GO/Cell group (PTFE/PVA/GO scaffolds containing ADSCs implant). Six and 12 weeks after implantation, the animals were sacrificed and bone regeneration was evaluated using computerized tomography (CT), and hematoxylin-eosin (H&E) staining. Results: Based on the in-vitro study, expression of bone-related proteins in ADSCs seeded on PTFE/PVA/GO scaffolds were significantly higher than PTFE/PVA scaffolds and TCPS (P<0.05). Based on the in-vivo study, bone regeneration in CSCD were filled with PTFE/PVA/GO scaffolds containing ADSCs were significantly higher than PTFE/PVA scaffolds containing ADSCs (P<0.05). CSCD filled with cell-seeded scaffolds showed higher bone regeneration in comparison with CSCD filled with scaffolds only (P<0.05). Conclusion: The data provided evidence showing new freeze-dried nanofibrous scaffolds formed from hydrophobic (PTFE) and hydrophilic (PVA) polymers with and without GO provide a suitable environment for ADSCs due to the expression of bone-related proteins. ADSCs and GO in the implanted scaffolds had a distinct effect on the bone regeneration process in this in-vivo study.

Extracellular vesicle-loaded hydrogels for tissue repair and regeneration.

Extracellular vesicles (EVs) are a collective term for nanoscale or microscale vesicles secreted by cells that play important biological roles. Mesenchymal stem cells are a class of cells with the potential for self-healing and multidirectional differentiation. In recent years, numerous studies have shown that EVs, especially those secreted by mesenchymal stem cells, can promote the repair and regeneration of various tissues and, thus, have significant potential in regenerative medicine. However, due to the rapid clearance capacity of the circulatory system, EVs are barely able to act persistently at specific sites for repair of target tissues. Hydrogels have good biocompatibility and loose and porous structural properties that allow them to serve as EV carriers, thereby prolonging the retention in certain specific areas and slowing the release of EVs. When EVs are needed to function at specific sites, the EV-loaded hydrogels can stand as an excellent approach. In this review, we first introduce the sources, roles, and extraction and characterization methods of EVs and describe their current application status. We then review the different types of hydrogels and discuss factors influencing their abilities to carry and release EVs. We summarize several strategies for loading EVs into hydrogels and characterizing EV-loaded hydrogels. Furthermore, we discuss application strategies for EV-loaded hydrogels and review their specific applications in tissue regeneration and repair. This article concludes with a summary of the current state of research on EV-loaded hydrogels and an outlook on future research directions, which we hope will provide promising ideas for researchers.