Human Adipose-derived Mesenchymal Stem Cells Research Articles

Lineage reprogramming of human adipose mesenchymal stem cells to immune modulatory i-Heps.

Pluripotent stem cell derived-hepatocytes depict fetal -hepatocyte characteristics/maturity and are immunogenic limiting their applications. Attempts have been made to derive hepatocytes from mesenchymal stem cells using developmental cocktails, epigenetic modulators and small molecules. However, achieving a stable terminally differentiated functional state had been a challenge. Inefficient hepatic differentiation could be due to lineage restrictions set during development. Hence a novel lineage reprogramming approach has been utilized to confer competence to adipose-mesenchymal stem cells (ADMSCs) to efficiently respond to hepatogenic cues and achieve a stable functional hepatic state. Lineage reprogramming involved co-transduction of ADMSCs with hepatic endoderm pioneer Transcription factor (TF)-FOXA2, HHEX-a homeobox gene and HNF4α-master TF indispensable for hepatic state maintenance. Lineage priming was evidenced by endogenous HFN4α promoter demethylation and robust responsiveness to minimal hepatic maturation cues. Induced hepatocytes (i-Heps) exhibited mesenchymal-to-epithelial transition and terminal hepatic signatures. Functional characterisation of i-Heps for hepatic drug detoxification systems, xenobiotic uptake/clearance, metabolic status and hepatotropic virus entry validated acquisition of stable hepatic state and junctional maturity Exhaustive analysis of MSC memory in i-Heps indicated loss of MSC-immunophenotype and terminal differentiation to osteogenic/adipogenic lineages. Importantly, i-Heps suppressed phytohemagglutinin-induced T-cell blasts, inhibited allogenic mixed-lymphocyte reactions (MLRs) and secreted immunomodulatory- indoleamine 2,3-dioxygenase in T-cell blast co-cultures akin to native ADMSCs. In a nutshell, the present study identifies a novel cocktail of TFs that reprogram ADMSCs to stable hepatic state. i-Heps exhibit adult hepatocyte functional maturity with robust immune-modulatory abilities rendering suitability for rigorous drug testing, hepatocyte-pathogen interaction studies and transplantation in allogenic settings.

Mesenchymal stem cells exert renoprotection via extracellular vesicle-mediated modulation of M2 macrophages and spleen-kidney network

Adipose-derived mesenchymal stem cells (ASCs) have shown therapeutic potentials against refractory diseases. However, the detailed therapeutic mechanisms remain unclear. Here, we report the therapeutic actions of human ASCs in nephritis, focusing on cellular dynamics and multi-organ networks. Intravenously-administered ASCs accumulated in spleen but not kidneys. Nevertheless, ASCs increased M2 macrophages and Tregs in kidneys and drove strong renoprotection. Splenectomy abolished these therapeutic effects. ASC-derived extracellular vesicles (EVs) were transferred to M2 macrophages, which entered the bloodstream from spleen. EVs induced the transcriptomic signatures of hyperpolarization and PGE2 stimulation in M2 macrophages and ameliorated glomerulonephritis. ASCs, ASC-derived EVs, and EV-transferred M2 macrophages enhanced Treg induction. These findings suggest that EV transfer from spleen-accumulated ASCs to M2 macrophages and subsequent modulation of renal immune-environment underlie the renoprotective effects of ASCs. Our results provide insights into the therapeutic actions of ASCs, focusing on EV-mediated modulation of macrophages and the spleen-kidney immune network.

Electrically controlled mRNA delivery using a polypyrrole-graphene oxide hybrid film to promote osteogenic differentiation of human mesenchymal stem cells.

Direct messenger ribonucleic acid (mRNA) delivery to target cells or tissues has revolutionized the field of biotechnology. However, the applicability of regenerative medicine is limited by the technical difficulties of various mRNA-loaded nanocarriers. Herein, we report a new conductive hybrid film that could guide osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) via electrically controlled mRNA delivery. To find optimal electrical conductivity and mRNA-loading capacity, the polypyrrole-graphene oxide (PPy-GO) hybrid film was electropolymerized on indium tin oxide substrates. We found that the fluorescein sodium salt, a molecule partially mimicking the physical and chemical properties of mRNAs, can be effectively absorbed and released by electrical stimulation (ES). The hADMSCs cultivated on the PPy-GO hybrid film loaded with pre-osteogenic mRNAs showed the highest osteogenic differentiation under electrical stimulation. This platform can load various types of RNAs thus highly promising as a new nucleic acid delivery tool for the development of stem cell-based therapeutics.Electronic Supplementary MaterialSupplementary material (electrochemical and FT-IR analysis on the film, additional SEM, AFM and C-AFM images of the film, optical and fluorescence images of cells, and the primers used for RT-qPCR analysis) is available in the online version of this article at 10.1007/s12274-022-4613-y.

Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells.

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

Effects of human adipose tissue- and bone marrow-derived mesenchymal stem cells on airway inflammation and remodeling in a murine model of chronic asthma

It is challenging to overcome difficult-to-treat asthma, and cell-based therapies are attracting increasing interest. We assessed the effects of mesenchymal stem cell (MSC) treatments using a murine model of chronic ovalbumin (OVA)-challenged asthma. We developed a murine model of chronic allergic asthma using OVA sensitization and challenge. Human adipose-derived MSCs (hADSCs) or human bone marrow-derived MSCs (hBMSCs) were administered. We measured the levels of resistin-like molecule-β (RELM-β). We also measured RELM-β in asthma patients and normal controls. OVA-challenged mice exhibited increased airway hyper-responsiveness, inflammation, and remodeling. hBMSC treatment remarkably decreased airway hyper-responsiveness but hADSC treatment did not. Both MSCs alleviated airway inflammation, but hBMSCs tended to have a more significant effect. hBMSC treatment reduced Th2-cytokine levels but hADSC treatment did not. Both treatments reduced airway remodeling. The RELM-β level decreased in the OVA-challenged control group, but increased in both treatment groups. We found that the serum level of RELM-β was lower in asthma patients than controls. MSC treatments alleviated the airway inflammation, hyper-responsiveness, and remodeling associated with chronic asthma. hBMSCs were more effective than hADSCs. The RELM-β levels increased in both treatment groups; the RELM-β level may serve as a biomarker of MSC treatment efficacy.

Reversion of neuronal differentiation induced in human adipose-derived stem cells

Adipose-derived mesenchymal stem cells are a great alternative to other types of stem cells obtained from other tissues, since they are found in large numbers and are easy to obtain by liposuction and do not have ethical connotations. These cells can differentiate into neuronal lineage using induction media, although the efficacy of these media is determined by their composition. In previous studies, we have demonstrated the differentiation of human adipose-derived mesenchymal stem cells to neuronal lineage by using three induction media, Neu1, Neu2 and Neu3, each one showing a series of neuronal markers in the treated cells. In the present study, a further step is taken, since once a neuronal differentiation is obtained with these three induction media, they are removed from the medium and both morphology and neuronal markers and the ability to maintain neurosphere formation are analyzed. The results obtained show that when these induction media are withdrawn in the three cases, both the neuronal morphological characteristics and the neuronal markers are lost at different times depending on the medium used. In the case of neurospheres, the ability to maintain this shape is also lost when they are not in contact with neuronal induction factors. Therefore, although the neuronal differentiation mechanism is very promising in this type of cells, it is necessary to carry out more studies to elucidate an induction medium that allows cells differentiated into neurons to maintain neuronal characteristics over time without the need to continue applying neuronal differentiation factors.

Stem Cell Fate and Immunomodulation Promote Bone Regeneration via Composite Bio-Oss®/AviteneTM Biomaterial.

Bone defects in maxillofacial regions lead to noticeable deformity and dysfunctions. Therefore, the use of biomaterials/scaffolds for maxillofacial bone regrowth has been attracting great interest from many surgical specialties and experts. Many approaches have been devised in order to create an optimal bone scaffold capable of achieving desirable degrees of bone integration and osteogenesis. Osteogenesis represents a complex physiological process involving multiple cooperating systems. A tight relationship between the immune and skeletal systems has lately been established using the concept of “osteoimmunology,” since various molecules, particularly those regulating immunological and inflammatory processes, are shared. Inflammatory mediators are now being implicated in bone remodeling, according to new scientific data. In this study, a profiler PCR array was employed to evaluate the expression of cytokines and chemokines in human adipose derived-mesenchymal stem cells (hASCs) cultured on porous hydroxylapatite (HA)/Collagen derived Bio-Oss®/Avitene scaffolds, up to day 21. In hASCs grown on the Bio-Oss®/Avitene biomaterial, 12 differentially expressed genes (DEGs) were found to be up-regulated, together with 12 DEG down-regulated. Chemokine CCL2, which affects bone metabolism, tested down-regulated. Interestingly, the Bio-Oss®/Avitene induced the down-regulation of pro-inflammatory inter-leukin IL-6. In conclusion, our investigation carried out on the Bio-Oss®/Avitene scaffold indicates that it could be successfully employed in maxillofacial surgery. Indeed, this composite material has the advantage of being customized on the basis of the individual patients favoring a novel personalized medicine approach.

Nitrogen-doped titanium dioxide films fabricated via magnetron sputtering for vascular stent biocompatibility improvement

Nowadays, vascular stents are commonly used to treat cardiovascular diseases. This article focuses on the influence of nitrogen doping of titanium dioxide thin films, utilized for coating metallic stents to improve their biological properties and biocompatibility. The hereby-investigated titanium oxide thin films are fabricated by magnetron sputtering in a reactive gas atmosphere consisting of argon and oxygen in the first case and argon, nitrogen and oxygen in the second case. Control of the nitrogen and oxygen gas flow rates, and hence their mixing ratios, allows adjustment of the nitrogen-doping level within the titanium dioxide thin films. A correlation of the thin film internal structure on the in vitro behavior of human mesenchymal stem cells derived from adipose tissue is hereby demonstrated. Different nitrogen doping levels affect the surface energy, the wettability, the cell adhesion and thus the cellular proliferation on top of the thin films. The surface colonization of cells on titanium dioxide thin films decreases up to a nitrogen-doping level of ∼ 3.75 at.%, which is associated with a decreasing polar component of the surface energy. For non-doped titanium dioxide thin films, a weak chondrogenesis of adult human adipose-derived mesenchymal stem cells with lower chondrogenic differentiation compared to glass is observed. An increasing nitrogen-doping level leads to linear increase in the chondrogenic differentiation rate, which is comparable to the control value of uncoated glass. Other investigated differentiated cell types do not display this behavior.

Effects of Triiodothyronine on Human Osteoblast-Like Cells: Novel Insights From a Global Transcriptome Analysis.

Background: Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. Methods: To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Results: Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Conclusions: Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.

Poly-caprolactone nanofibrous coated with sol-gel alginate/ mesenchymal stem cells for cartilage tissue engineering

Human adipose-derived mesenchymal stem cells (AMSCs) were encapsulated in alginate sol and coated on PCL nanofibers to produce a scaffold. The alginate solution was generated in three different concentrations (0.5%, 2%, and 3.5%) in the specified scaffold, and the resulting solutions were ionic cross-linked to prepare coated networks on the PCL nanofibers. We analyzed the samples using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) tests. The electrospinning method and coating alginate on PCL with a calcium chloride cross-linker were both successful, according to the SEM and FTIR data. The behaviors of AMSCs in in-vitro conditions were evaluated by viability and chondrogenic potential of alginate gel through MTT, DAPI, alcian staining, and immunofluorescence (ICC) assay, respectively. The results showed that PCL/Alg scaffolds not only support the adhesion and proliferation of AMSCs but also can enhance their chondrogenic differentiation without applying any external differential agent. The cells adequately exhibited the COLLAGEN type Ⅱ expression as a chondrogenic differentiation marker. As a result, alginate gels with cells coated on PCL nanofibers have a lot of potential for building bioactive networks as a substrate in cartilage tissue engineering.

Synthesis and characterizations of sugar-glass nanoparticles mediated protein delivery system for tissue engineering application

The present work focuses on the synthesis and characterization of a sugar-glass nanoparticle (SGnP) based reservoir type protein delivery system pertinent to tissue engineering applications. The SGnP nanocarriers were prepared via inverse micelle of sodium bis(2-ethylhexyl) sulfosuccinate based on an anionic surfactant and subsequent flash-freezing technique. Initially, a total of five different grades of protein-free SGnPs have been prepared to examine the effects of systematic changes in starting concentrations of the aqueous phase, organic solvent, the molar ratio of water, and surfactant in controlling the size, shape, and uniformity of micelles. Evidently, the Fourier transform infrared (FTIR) and scanning electron microscope (SEM) results confirmed that the SGnP can be successfully prepared. Subsequently, SGnP based protein depot has been validated using bovine serum albumin (BSA), horseradish peroxidase (HRP) and growth and differentiation factor-5 (GDF-5). The particle size, morphology, protein encapsulation efficiency and in vitro release kinetics were assessed using SEM, FTIR, UV–visible spectroscopy and Bradford protein assays. Excellent encapsulation efficiency (93%–94%) and sustained release behaviour of BSA (∼22% protein release after 14 d) and GDF-5 proteins (∼29% protein release after 30 d) were exhibited by the optimal grades of SGnP constructs with an average particle size of 266 nm and 93 nm, respectively. Furthermore, FTIR, differential scanning calorimeter (DSC), polyacrylamide gel electrophoresis (PAGE) and NATIVE-PAGE studies results confirm successful encapsulation, stability and preserving the structural integrity of proteins placed into the core of the SGnP constructs. Evidently, a very high (93%) residual HRP enzyme activity signifies the capability of our SGnP system to protect the encapsulated proteins from process-related stresses. In vitro cytotoxicity and fluorescence cell morphology analyses using human adipose-derived mesenchymal stem cells affirmed good cytocompatibility of protein encapsulated SGnP. Overall, the study findings indicate SGnP nanocarrier-mediated protein delivery systems as a promising approach complementary to conventional techniques in tissue engineering and therapeutic applications.

Proteomic analysis of MSC-derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions.

Apoptotic vesicles (apoVs) are apoptotic cell-derived nanosized vesicles that play a crucial role in multiple pathophysiological settings. However, their detailed characteristics, specific surface markers, and biological properties are not fully elucidated. In this study, we compared mesenchymal stem cell (MSC)-derived apoVs and exosomes from three different types of MSCs including human bone marrow MSCs (hBMSCs), human adipose MSCs (hASCs), and mouse bone marrow MSCs (mBMSCs). We established a unique protein map of MSC-derived apoVs and identified the differences between apoVs and exosomes in terms of functional protein cargo and surface markers. Furthermore, we identified 13 proteins specifically enriched in apoVs compared to exosomes, which can be used as apoV-specific biomarkers. In addition, we showed that apoVs inherited apoptotic imprints such as Fas to ameliorate haemophilia A in factor VIII knockout mice via binding to the platelets' FasL to activate platelet functions, and therefore rescuing the blood clotting disorder. In summary, we systemically characterized MSC-derived apoVs and identified their therapeutic role in haemophilia A treatment through a previously unknown Fas/FasL linkage mechanism.

Treatment of Abdominal Aortic Aneurysm Utilizing Adipose-Derived Mesenchymal Stem Cells in a Porcine Model

IntroductionThe current treatment paradigm of abdominal aortic aneurysms (AAA) focuses on observing patients until their disease reaches certain thresholds for intervention, with no preceding treatment available. There is an opportunity to develop novel therapies to prevent further aneurysmal growth and decrease the risk of a highly morbid rupture. We used a porcine model of aortic dilation to assess the ability of human adipose-derived mesenchymal stem cells (MSCs) to attenuate aortic dilation. Materials and methodsTwelve Yorkshire pigs received periadventitial injections (collagenase and elastase) into a 4-cm segment of infrarenal aorta. Animals were treated with either 1 × 106 MSCs placed onto Gelfoam or treated with media as a control. Aortic diameters were measured at the time of surgery and monitored at postoperative day (POD) 7 and 14 with ultrasound. Animals were sacrificed on POD 21. Aortic tissue was harvested for histopathological analyses and immunohistochemistry. Groups were compared with paired t-tests or Mann–Whitney U-tests. ResultsAll animals survived until POD 21. The mean aortic diameter was reduced in the aortic dilation + MSC treatment group compared to aortic dilation control animals (1.10 ± 0.126 versus 1.48 cm ± 0.151, P < 0.001). Aortic media thickness was reduced in the aortic dilation group compared to the aortic dilation + MSC group (609.14 IQR 445.21-692.93 μm versus 643.55 IQR 560.91-733.88 μm, P = 0.0048). There was a significant decrease in the content of collagen and alpha-smooth muscle actin and elastin perturbation in the aortic dilation group as compared to the aortic dilation + MSC group. Immunohistochemistry demonstrated an increased level of vascular endothelial growth factor, tissue inhibitor of matrix metalloproteinase 1, and tissue inhibitor of matrix metalloproteinase 3 expression in the aorta of aortic dilation + MSC animals. ConclusionsStem cell therapy suppressed the aortic dilation in a porcine model. Animals from the aortic dilation group showed more diseased gross features, histologic changes, and biochemical properties of the aorta compared to that of the aortic dilation + MSC treated animals. This novel finding should prompt further investigation into translatable drug and cell therapies for aneurysmal disease.

Nebulized exosomes derived from allogenic adipose tissue mesenchymal stromal cells in patients with severe COVID-19: a pilot study

BackgroundExisting clinical studies supported the potential efficacy of mesenchymal stromal cells as well as derived exosomes in the treatment of COVID-19. We aimed to explore the safety and efficiency of aerosol inhalation of the exosomes derived from human adipose-derived MSCs (haMSC-Exos) in patients with COVID-19.MethodsThe MEXCOVID trial is a phase 2a single-arm, open-labelled, interventional trial and patients were enrolled in Jinyintan Hospital, Wuhan, China. Eligible 7 patients were assigned to receive the daily dose of haMSCs-Exos (2.0 × 108 nano vesicles) for consecutively 5 days. The primary outcomes included the incidence of prespecified inhalation-associated events and serious adverse events. We also observed the demographic data, clinical characteristics, laboratory results including lymphocyte count, levels of D-dimer and IL-6 as well as chest imaging.ResultsSeven severe COVID-19 related pneumonia patients (4 males and 3 females) were enrolled and received nebulized haMSC-Exos. The median age was 57 year (interquartile range (IQR), 43 year to 70 year). The median time from onset of symptoms to hospital admission and administration of nebulized haMSC-Exos was 30 days (IQR, 15 days to 40 days) and 54 d (IQR, 34 d to 69 d), respectively. All COVID-19 patients tolerated the haMSC-Exos nebulization well, with no evidence of prespecified adverse events or clinical instability during the nebulization or during the immediate post-nebulization period. All patients presented a slight increase of serum lymphocyte counts (median as 1.61 × 109/L vs. 1.78 × 109/L). Different degrees of resolution of pulmonary lesions after aerosol inhalation of haMSC-Exos were observed among all patients, more obviously in 4 of 7 patients.ConclusionsOur trial shows that a consecutive 5 days inhalation dose of clinical grade haMSC-Exos up to a total amount of 2.0 × 109 nano vesicles was feasible and well tolerated in seven COVID-19 patients, with no evidence of prespecified adverse events, immediate clinical instability, or dose-relevant toxicity at any of the doses tested. This safety profile is seemingly followed by CT imaging improvement within 7 days. Further trials will have to confirm the long-term safety or efficacy in larger population.Trial Registration: MEXCOVID, NCT04276987.

Assessing the biocompatibility of bovine tendon scaffold, a step forward in tendon tissue engineering.

Tendon is a collagen-enriched, tough, and intricately arranged connective tissue that connects muscle to the bone and transmits forces, resulting in joint movement. High mechanical demands can affect normal tissues and may lead to severe disorders, which usually require replacement of the damaged tendon. In recent decades, various decellularization methods have been studied for tissue engineering applications. One of the major challenges in tendon decellularization is preservation of the tendon extracellular matrix (ECM) architecture to maintain natural tissue characteristics. The aim of the present study was to create a decellularized bovine Achilles tendon scaffold to investigate its cytocompatibility with seeded hAd-MSCs (human adipose derived-mesenchymal stem cells) and blastema tissue in vitro. Here, we describe a reliable procedure to decellularize bovine Achilles tendon using a combination of physical and chemical treatments including repetitive freeze-thaw cycles and the ionic detergent SDS, respectively. The decellularization effectiveness and cytocompatibility of the tendon scaffolds were verified by histological studies and scanning electron microscopy for up to 30days after culture. Histological studies revealed hAd-MSC attachment and penetration into the scaffolds at 5, 10, 15 and 20days of culture. However, a decrease in cell number was observed on days 25 and 30 after culture in vitro. Moreover, migration of the blastema tissue cells into the scaffold were shown at 10 to 25days post culture, however, destruction of the scaffolds and reduction in cell number were observed on 30th day after culture. Our results suggest that this decellularization protocol is an effective and biocompatible procedure which supports the maintenance and growth of both hAd-MSCs and blastema cells, and thus might be promising for tendon tissue engineering.

Human Adipose-Derived Stem Cells Combined with Nano-Hydrogel Promote Functional Recovery after Spinal Cord Injury in Rats.

Simple SummaryNerve regeneration and functional recovery after spinal cord injury (SCI) are worldwide problems. Scientists have achieved encouraging results in the repair of spinal cord injuries using natural or synthetic materials. In this paper, we report that nano-hydrogel combined with human adipose-derived stem cells regulate the inflammatory microenvironment, protect neurons and axons, and promote motor function recovery. In addition, three proteins related to neuronal and axonal growth were screened by Liquid chromatography-mass spectrometry. These results provide evidence for clinical treatment of spinal cord injury.The treatment of spinal cord injury aims to reconstruct the fiber connection and restore the interrupted neural pathways. Adipose mesenchymal stem cells (ADSCs) can promote the recovery of motor functions in spinal cord injury. However, poor survival of ADSCs and leakage outside of the injury site after local transplantation reduce the number of cells, which seriously attenuates the cumulative effect. We performed heterotopic transplantation on rats with severe spinal cord injury using human ADSCs loaded within self-assembly hydrogel RADA16-RGD (R: arginine; A: alanine; D: aspartic acid; G: glycine). Our results indicate that the combined transplantation of human ADSCs with RADA16-RGD improved the survival of ADSCs at the injured site. The inflammatory reaction was inhibited, with improved survival of the neurons and increased residual area of nerve fibers and myelin protein. The functional behaviors were promoted, as determined by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale score and electrophysiological measurements. ADSCs can promote the repair of spinal cord injury. This study provides new ideas for the treatment of spinal cord injury.

Human adipose-derived mesenchymal stem cells prevent type 1 diabetes induced by immune checkpoint blockade

Aims/hypothesisImmunomodulators blocking cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) have improved the treatment of a broad spectrum of cancers. These immune checkpoint inhibitors (ICIs) reactivate the immune system against tumour cells but can also trigger autoimmune side effects, including type 1 diabetes. Mesenchymal stem cell (MSC) therapy is the most prevalent cell therapy, with tissue-regenerating, anti-fibrosis and immunomodulatory functions provided by the secretome of the cells. Here, we examined whether systemic MSC treatment could prevent the development of type 1 diabetes in a NOD mouse model.MethodsThe purified PD-L1 monoclonal antibody was administered to induce diabetes in male NOD mice which normally do not develop diabetes. Human adipose-derived MSCs were administered by tail vein injections. T cells, macrophages and monocyte-derived macrophages expressing C-X-C motif chemokine ligand 9 (CXCL9) in pancreatic sections of NOD mice and a cancer patient who developed diabetes following the ICI treatments were analysed by immunofluorescence. Tissue localisation of the injected MSCs, plasma exosome levels and plasma cytokine profiles were also investigated.ResultsPD-1/PD-L1 blockade induced diabetes in 16 of 25 (64%) NOD mice which received anti-PD-L1 mAb without hMSCs [MSC(−)], whereas MSC administration decreased the incidence to four of 21 (19%) NOD mice which received anti-PD-L1 mAb and hMSCs [MSC(+)]. The PD-1/PD-L1 blockade significantly increased the area of CD3-positive T cells (6.2-fold) and macrophage-2 (Mac-2) antigen (2.5-fold)- and CXCL9 (40.3-fold)-positive macrophages in the islets. MSCs significantly reduced T cell (45%) and CXCL9-positive macrophage (67%) accumulation in the islets and the occurrence of diabetes. The insulin content (1.9-fold) and islet beta cell area (2.7-fold) were also improved by MSCs. T cells and CXCL9-positive macrophages infiltrated into the intricate gaps between the beta cells in the islets by PD-1/PD-L1 blockade. Such immune cell infiltration was largely prevented by MSCs. The most striking difference was observed in the CXCL9-positive macrophages, which normally did not reside in the beta cell region in the islets but abundantly accumulated in this area after PD-1/PD-L1 blockade and were prevented by MSCs. The CXCL9-positive macrophages were also observed in the islets of a cancer patient who developed diabetes following the administration of ICIs but few CXCL9-positive macrophages were observed in a control patient. Mechanistically, the injected MSCs accumulated in the lung but not in the pancreas and strongly increased plasma exosome levels and changed plasma cytokine profiles.Conclusions/interpretationOur results suggest that MSCs can prevent the incidence of diabetes associated with immune checkpoint cancer therapy and may be worth further consideration for new adjuvant cell therapy.Graphical abstract

Gentamicin and fluconazole loaded electrospun polymethylmethacrylate (PMMA) fibers as a novel platform for the treatment of corneal keratitis

In this study, 10 mg fluconazole (10 FCZ) and 10 mg gentamicin (10 GEN) loaded with 40% polymethylmethacrylate (40% PMMA) fibers were fabricated by electrospinning and the performance of the formulations (40% PMMA, 40% PMMA/10 FCZ, 40% PMMA/10 GEN, and 40% PMMA/10 FCZ/10 GEN) was tested. 10 mg Gentamicin-loaded 40% PMMA fiber mat showed a more significant zone of inhibition against the S. aureus compared to the zone of 40% PMMA/10 GEN/10 FCZ fiber. The biocompatibility test using a human adipose-derived mesenchymal stem cell (MSCs) as a cell model proved that the amount of loaded and released GEN has no toxic effects on the MSCs.

PD43-10 CANCER-ASSOCIATED ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROMOTE MIGRATION AND INVASION IN KIDNEY CANCER

You have accessJournal of UrologyCME1 May 2022PD43-10 CANCER-ASSOCIATED ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROMOTE MIGRATION AND INVASION IN KIDNEY CANCER Yumina Muto, Kazuyuki Numakura, Yuya Sekine, Mizuki Kobayashi, Soki Kashima, Ryohei Yamamoto, Atsushi Koizumi, Taketoshi Nara, Mitsuru Saito, Shintaro Narita, Yasufumi Omori, and Tomonori Habuchi Yumina MutoYumina Muto More articles by this author , Kazuyuki NumakuraKazuyuki Numakura More articles by this author , Yuya SekineYuya Sekine More articles by this author , Mizuki KobayashiMizuki Kobayashi More articles by this author , Soki KashimaSoki Kashima More articles by this author , Ryohei YamamotoRyohei Yamamoto More articles by this author , Atsushi KoizumiAtsushi Koizumi More articles by this author , Taketoshi NaraTaketoshi Nara More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Yasufumi OmoriYasufumi Omori More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002604.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Adipose-derived mesenchymal stem cells (A-MSCs) are known to be a preeminent contributor to tumor microenvironments. Their role in renal cell carcinoma (RCC) has not been entirely investigated. This study aimed to clarify the significance of A-MSCs in RCC. METHODS: Human A-MSCs were isolated from the perirenal fat in RCC patients (RCC-MSC) and kidney transplantation donors (Donor-MSC) for use as controls. Several RCC cell lines were co-cultured with the A-MSCs, then analyzed for their migration and invasive ability. Molecular pathways activated in the RCC-MSCs and isolated RCC cultures were assessed. Beta-catenin in perirenal fat from surgical specimens from RCC patients was stained by immunohistochemistry (IHC). RESULTS: RCC-MSCs expressed higher levels of mRNAs encoding Hes1, Hey1, and Twist, higher protein levels of beta-catenin, and reduced protein levels of phospho-Akt (Ser473) and phospho-p38 MAPK relative to Donor-MSCs (Figure 1A and B). These results suggest that RCC-MSC stemness is reduced compared with Donor-MSCs in terms of stemness associated molecular pathways. This finding was confirmed by the aldehyde dehydrogenase enzymatic activity (ALDH) assays, in which fewer RCC-MSCs were ALDH+ than Donor-MSCs (Figure 1C). We next analyzed cell function by co-culturing with RCC cell lines. Tumor cell migration was significantly elevated in RCC-MSC cultures compared with RCC cell lines cultured alone (Figure 2A and B). Pathway screening using the RCC cell line revealed elevated urokinase-type plasminogen activator (uPA) mRNA expression in RCC-MSCs than cultured alone (Figure 2C). In RCC surgical specimens, IHC of perirenal stromal tissue for beta-catenin showed a high positive correlation with tumor aggressiveness (Figure 2D). CONCLUSIONS: Co-culturing of RCC-MSCs with RCC cell lines exacerbated tumor invasiveness. Interaction between A-MSCs and tumors may promote A-MSC differentiation from pluripotent stem cells, and facilitate RCC migration and invasion by increasing expression of uPA in tumor cells. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e706 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yumina Muto More articles by this author Kazuyuki Numakura More articles by this author Yuya Sekine More articles by this author Mizuki Kobayashi More articles by this author Soki Kashima More articles by this author Ryohei Yamamoto More articles by this author Atsushi Koizumi More articles by this author Taketoshi Nara More articles by this author Mitsuru Saito More articles by this author Shintaro Narita More articles by this author Yasufumi Omori More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement PDF DownloadLoading ...

Secretome of adipose-derived mesenchymal stem cells establish anti-inflammatory milieu by developing M2b/c macrophage

Abstract Mesenchymal stem cells (MSCs) express immunomodulatory and anti-inflammatory effect toward immune system with several mechanisms such as secreting bioactive molecules. Distinct mechanisms of their immune actions, however, are not fully understood. As one of the important cells in immune system, macrophages play a vital role in innate immunity with two functional phenotypes, classically activated (M1) and alternatively activated (M2) macrophages. In this study, immunomodulatory ability of adipose-derived MSCs (AD-MSCs) secretome toward peritoneal macrophage was studied. Secretome was concentrated from human AD-MSCs using 3K TFF membrane. Murine peritoneal macrophages were cultured for 24 hours with secretome of AD-MSCs and 4 hours with LPS. Macrophages were analyzed by flow cytometry, ELISA, and real-time PCR. LC-MS/MS proteomics analysis of secretome was performed and acquired protein sets were analyzed by DAVID Bioinformatics. Secretome of AD-MSCs increased cell viability of peritoneal macrophages. Secretome treated peritoneal macrophages resulted in reduced M1 markers such as CD86, iNOS, and TNFα and increased M2 markers such as CD206, Arginase-1, and IL-10. Also, peritoneal macrophage expressed upregulation of mRNA level of LIGHT and MerTK, M2b and M2c markers, respectively. 83 proteins were identified in secretome according to the proteomics analysis. Several proteins among them were selected as candidates after confirmation of inhibiting the effect of secretome with neutralizing antibodies. In conclusion, secretome of AD-MSCs induced polarization of peritoneal macrophages from M1 macrophages to M2 macrophages and made them create anti-inflammatory milieu by several key secreted proteins. Supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2021R1F1A1049630) and the Ministry of Health and Welfare, Republic of Korea (HI14C1324)