Human Umbilical Cord Mesenchymal Stromal Cells Research Articles

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Exosomes Alleviate Hypoxia-Induced Pulmonary Arterial Hypertension in Mice Via Macrophages.

Pulmonary hypertension (PH) is an intractable, severe, and progressive cardiopulmonary disease. Recent findings suggest that human umbilical cord mesenchymal stromal cells (HUCMSCs) and HUCMSC-derived exosomes (HUCMSC-Exos) possess potential therapeutic value for PH. However, whether they have beneficial effects on hypoxic pulmonary hypertension (HPH) is unclear. Exos are released into the extracellular environment by the fusion of intracellular multivesicular bodies with the cell membrane, and they play an important role in cellular communication. Exos ameliorate immune inflammation levels, alter macrophage phenotypes, regulate mitochondrial metabolic function, and inhibit pulmonary vascular remodeling, thereby improving PH. Macrophages are important sources of cytokines and other transmitters and can promote the release of cytokines, vasoactive molecules, and reactive oxygen species, all of which are associated with pulmonary vascular remodeling. Therefore, the aim of this study was to investigate whether HUCMSC-Exos could improve the lung inflammatory microenvironment and inhibit pulmonary vascular remodeling by targeting macrophages and identifying the underlying mechanisms. The results showed that HUCMSC-Exos promoted M2 macrophage polarization, decreased pro-inflammatory factors, increased IL-10 levels, and inhibited IL-33/ST2 axis expression, thereby inhibiting hypoxia-induced proliferation of pulmonary artery smooth muscle cells and ameliorating HPH.

Fecal microbiota transplantation from HUC-MSC-treated mice alleviates acute lung injury in mice through anti-inflammation and gut microbiota modulation.

Acute lung injury (ALI) is a severe respiratory tract disorder facilitated by dysregulated inflammation, oxidative stress and intestinal ecosystem. Fecal microbiota transplantation (FMT) is a rapid method for gut microbiota (GM) reconstruction. Furthermore, our previous studies have confirmed that human umbilical cord mesenchymal stromal cells (HUC-MSCs) can alleviate ALI by improving GM composition. Therefore, we aimed to explore the efficacy and mechanism of FMT from HUC-MSCs-treated mice on ALI. In brief, fresh feces from HUC-MSCs-treated mice were collected for FMT, and the mice were randomly assigned into NC, FMT, LPS, ABX-LPS, and ABX-LPS-FMT groups (n = 12/group). Subsequently, the mice were administrated with antibiotic mixtures to deplete GM, and given lipopolysaccharide and FMT to induce ALI and rebuild GM. Next, the therapeutic effect was evaluated by bronchoalveolar lavage fluid (BALF) and histopathology. Immune cells in peripheral blood and apoptosis in lung tissues were measured. Furthermore, oxidative stress- and inflammation-related parameter levels were tested in BALF, serum, lung and ileal tissues. The expressions of apoptosis-associated, TLR4/NF-κB pathway-associated, Nrf2/HO-1 pathway related and tightly linked proteins in the lung and ileal tissues were assessed. Moreover, 16S rRNA was conducted to assess GM composition and distribution. Our results revealed that FMT obviously improved the pathological damage of lung and ileum, recovered the immune system of peripheral blood, decreased the cell apoptosis of lung, and inhibited inflammation and oxidative stress in BALF, serum, lung and ileum tissues. Moreover, FMT also elevated ZO-1, claudin-1, and occludin protein expressions, activating the Nrf2/HO-1 pathway but hindering the TLR4/NF-κB pathway. Of note, the relative abundances of Bacteroides, Christensenella, Coprococcus, and Roseburia were decreased, while the relative abundances of Xenorhabdus, Sutterella, and Acinetobacter were increased in the ABX-LPS-FMT group. FMT from HUC-MSCs-treated mice may alleviate ALI by inhibiting inflammation and reconstructing GM, additionally, we also found that the TLR4/NF-κB and Nrf2/HO-1 pathways may involve in the improvement of FMT on ALI, which offers novel insights for the functions and mechanisms of FMT from HUC-MSCs-treated mice on ALI.

Exosomes from TNF-α preconditioned human umbilical cord mesenchymal stromal cells inhibit the autophagy of acinar cells of severe acute pancreatitis via shuttling bioactive metabolites.

Severe acute pancreatitis (SAP) is a common critical disease of the digestive system, with high mortality and a lack of effective prevention and treatment measures. Despite mesenchymal stromal cell transplantation having the potential to treat SAP, its clinical application prospect is limited, and the mechanism is unclear. Here, we reveal the therapeutic role of exosomes from TNF-α-preconditioned human umbilical cord mesenchymal stromal cells (HUCMSCs) in attenuating SAP and show that it is partly dependent on exosomal metabolites. Bioactive metabolomics analysis showed that 48 metabolites be significantly differentially expressed between the two groups (Exo-Ctrl group versus Exo-TNF-α group). Then, the further functional experiments indicated that 3,4-dihydroxyphenylglycol could be a key molecule mediating the therapeutic effect of TNF-α-preconditioned HUCMSCs. The animal experiments showed that 3,4-dihydroxyphenylglycol reduced inflammation and oxidative stress in the pancreatic tissue and inhibited acinar cell autophagy in a rat model of SAP. Mechanistically, we revealed that 3,4-dihydroxyphenylglycol activated the mTOR pathway to inhibit acinar cell autophagy and alleviate SAP. In summary, our study demonstrated that exosomes from TNF-α-preconditioned HUMSCs inhibit the autophagy of acinar cells of SAP by shuttling 3,4-dihydroxyphenylglycol and inhibiting the mTOR pathway. This study revealed the vital role and therapeutic potential of metabolite-derived exosomes in SAP, providing a new promising method to prevent and therapy SAP.

Efferocytosis of viable versus heat-inactivated MSC induces human monocytes to distinct immunosuppressive phenotypes

BackgroundImmunomodulation by mesenchymal stromal cells (MSCs) can occur through trophic factor mechanisms, however, intravenously infused MSCs are rapidly cleared from the body yet a potent immunotherapeutic response is still observed. Recent work suggests that monocytes contribute to the clearance of MSCs via efferocytosis, the body’s natural mechanism for clearing dead and dying cells in a non-inflammatory manner. This begs the questions of how variations in MSC quality affect monocyte phenotype and if viable MSCs are even needed to elicit an immunosuppressive response.MethodsHerein, we sought to dissect MSC’s trophic mechanism from their efferocytic mechanisms and determine if the viability of MSCs prior to efferocytosis influences the resultant phenotype of monocytes. We cultured viable or heat-inactivated human umbilical cord MSCs with human peripheral blood mononuclear cells for 24 h and observed changes in monocyte surface marker expression and secretion profile. To isolate the effect of efferocytosis from MSC trophic factors, we used cell separation techniques to remove non-efferocytosed MSCs before challenging monocytes to suppress T-cells or respond to inflammatory stimuli. For all experiments, viable and heat-inactivated efferocytic-licensing of monocytes were compared to non-efferocytic-licensing control.ResultsWe found that monocytes efferocytose viable and heat-inactivated MSCs equally, but only viable MSC-licensed monocytes suppress activated T-cells and suppression occurred even after depletion of residual MSCs. This provides direct evidence that monocytes that efferocytose viable MSCs are immunosuppressive. Further characterization of monocytes after efferocytosis showed that uptake of viable-but not heat inactivated-MSC resulted in monocytes secreting IL-10 and producing kynurenine. When monocytes were challenged with LPS, IL-2, and IFN-γ to simulate sepsis, monocytes that had efferocytosed viable MSC had higher levels of IDO while monocytes that efferocytosed heat inactivated-MSCs produced the lowest levels of TNF-α.ConclusionCollectively, these studies show that the quality of MSCs efferocytosed by monocytes polarize monocytes toward distinctive immunosuppressive phenotypes and highlights the need to tailor MSC therapies for specific indications.

Comparison of two hepatocyte differentiation protocols in human umbilical cord mesenchymal stem cells: In vitro study

Human umbilical cord mesenchymal stromal cells (HUCMSCs) are an emerging source of cell therapy due to their self-renew and differentiation ability. They can differentiate into three germ layers, including the potential to generate hepatocytes. This study determined the transplantation efficiency and suitability of HUCMSCs-derived hepatocyte-like cells (HLCs) for their therapeutic application for liver diseases. This study aims to formulate ideal conditions to induce HUCMSCs into the hepatic lineage and investigate the efficiency of the differentiated HLCs based on their expression characteristics and capacity to integrate into the damaged liver of CCl4-challenged mice. Hepatocyte growth factor (HGF) and Activin A, Wnt3a were found to optimally promote the endodermal expansion of HUCMSCs, which showed phenomenal expression of hepatic markers upon differentiation in the presence of oncostatin M and dexamethasone. HUCMSCs expressed MSC-related surface markers and could undergo tri-lineage differentiations. Two hepatogenic differentiation protocols (differentiated hepatocyte protocol 1 [DHC1]: 32 days and DHC2: 15 days) were experimented with. The proliferation rate was faster in DHC2 than in DHC1 on day 7 of differentiation. The migration capability was the same in both DHC1 and DHC2. Hepatic markers like CK18, CK19, ALB, and AFP were upregulated. The mRNA levels of albumin, α1AT, αFP, CK18, TDO2, CYP3A4, CYP7A1, HNF4A, CEBPA, PPARA, and PAH were even higher in the HUCMSCs-derived HCLs than in the primary hepatocytes. Western blot confirmed HNF3B and CK18 protein expression in a step-wise manner differentiated from HUCMSCs. The metabolic function of differentiated hepatocytes was evident by increasing PAS staining and urea production. Pre-treating HUCMSCs with a hepatic differentiation medium containing HGF can drive their differentiation towards endodermal and hepatic lineages, enabling efficient integration into the damaged liver. This approach represents a potential alternative protocol for cell-based therapy that could enhance the integration potential of HUCMSC-derived HLCs.

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells.

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.

Neuroprotective effects of human umbilical cord mesenchymal stromal cells in PD mice via centrally and peripherally suppressing NLRP3 inflammasome-mediated inflammatory responses

Mesenchymal stromal cells (MSCs) exhibit beneficial anti-inflammatory effects against Parkinson’s disease (PD) via immunomodulatory actions. However, the underlying molecular mechanism remains unclear. This study aimed to investigate the potential neuroprotective effects of MSCs and the possible mechanisms involved by infusing human umbilical cord MSCs (hMSCs) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mature male C57BL/6 mice. Subsequently, the striatal content of dopamine (DA) and its metabolites, tyrosine hydroxylase (TH)-positive neurons and activated microglia, circulating inflammatory cytokines, gene expression of cytokines, and NOD-like receptor protein 3 (NLRP3) inflammasome molecules were measured using high-performance liquid chromatography, flow cytometry, immunohistochemistry, immunofluorescent staining, and real-time polymerase chain reaction assays, respectively. Infused hMSCs markedly ameliorated the reduction in striatal DA and loss of TH-positive neurons in the substantia nigra of PD mice. The MPTP-induced activation of microglia and increase in tumor necrosis factor-α and interleukin-1β mRNA expression in the striatum were also attenuated by hMSCs. Furthermore, hMSCs completely reversed the elevated pro-inflammatory cytokine levels in the serum and mRNA expression of cytokines in the peripheral organs of PD mice. Moreover, hMSCs significantly inhibited the expression of caspase-1 and NLRP3 of the NLRP3 inflammasome in both the central and peripheral organs at mRNA level. These data suggest that hMSCs protect dopaminergic neurons in PD mice by suppressing both the central and peripheral inflammatory responses, probably by inhibiting the NLRP3 inflammasome. However, the animals in our study only received several MSC infusions, and the effects of infused MSCs over extended periods on the NLRP3 inflammasome need to be investigated in future studies.

Single-cell RNA sequencing reveals the potential mechanism of heterogeneity of immunomodulatory properties of foreskin and umbilical cord mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) are heterogeneous populations. Heterogeneity exists within the same tissue and between different tissues. Some studies have found enormous heterogeneity in immunomodulatory function among MSCs derived from different tissues. Moreover, the underlying mechanism of heterogeneity in immunomodulatory abilities is still unclear.MethodsForeskin mesenchymal stromal cells (FSMSCs) and human umbilical cord mesenchymal stromal cells (HuMSCs) were isolated and cultured until the third passage. According to the International Association for Cell Therapy standard, we confirmed the cell type. Then, FSMSCs and HuMSCs were cocultured with human peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) in vitro. Furthermore, the supernatant was sampled for an enzyme-linked immunosorbent assay to investigate the secretion of IL-1β, IL-6, IL-10, TNF-α, and TGF-β1. Finally, we performed single-cell RNA sequencing (scRNA-seq) of FSMSCs and HuMSCs.ResultsWe successfully identified FSMSCs and HuMSCs as MSCs. When cocultured with LPS pretreated PBMCs, FSMSCs and HuMSCs could effectively reduced the secretion of IL-1β and TNF-α. However, FSMSCs stimulated the PBMCs to secrete more IL-10, TGF-β1, and IL-6. Furthermore, 4 cell subsets were identified from integrated scRNA-seq data, including proliferative MSCs (MKI67+, CD146low+, NG2+, PDGFRB−), pericytes (CD146high+, PDGFRB+, MKI67−, CD31−, CD45−, CD34−), immune MSCs (CXCL12high+, PTGIShigh+, PDGFRB+, CD146−, MKI67−) and progenitor proliferative MSCs (CXCL12low+, PTGISlow+, PDGFRB+, CD146−, MKI67−). Among them, we found that immune MSCs with strengthened transcriptional activity were similar to pericytes with regard to the degree of differentiated. Various of immune-related genes, gene sets, and regulons were also enriched in immune MSCs. Moreover, immune MSCs were determined to be close to other cell subsets in cell–cell communication analysis. Finally, we found that the proportion of immune MSCs in foreskin tissue was highest when comparing the subset compositions of MSCs derived from different tissues.ConclusionsFSMSCs show better immunomodulatory capacity than HuMSCs in vitro. Moreover, immune MSCs may play a vital role in the heterogeneity of immunoregulatory properties. This study provides new insights suggesting that immune MSCs can be isolated to exert stable immunoregulatory functions without being limited by the heterogeneity of MSCs derived from different tissues.

Single-Cell Transcriptome Integration Analysis Reveals the Correlation Between Mesenchymal Stromal Cells and Fibroblasts.

Background: Mesenchymal stromal cells (MSCs) and fibroblasts show similar morphology, surface marker expression, and proliferation, differentiation, and immunomodulatory capacities. These similarities not only blur their cell identities but also limit their application. Methods: We performed single-cell transcriptome sequencing of the human umbilical cord and foreskin MSCs (HuMSCs and FSMSCs) and extracted the single-cell transcriptome data of the bone marrow and adipose MSCs (BMSCs and ADMSCs) from the Gene Expression Omnibus (GEO) database. Then, we performed quality control, batch effect correction, integration, and clustering analysis of the integrated single-cell transcriptome data from the HuMSCs, FMSCs, BMSCs, and ADMSCs. The cell subsets were annotated based on the surface marker phenotypes for the MSCs (CD105 + , CD90 +, CD73 +, CD45 −, CD34 −, CD19 −, HLA-DRA −, and CD11b −), fibroblasts (VIM +, PECAM1 −, CD34 −, CD45 −, EPCAM −, and MYH11 −), and pericytes (CD146 +, PDGFRB +, PECAM1 −, CD34 −, and CD45 −). The expression levels of common fibroblast markers (ACTA2, FAP, PDGFRA, PDGFRB, S100A4, FN1, COL1A1, POSTN, DCN, COL1A2, FBLN2, COL1A2, DES, and CDH11) were also analyzed in all cell subsets. Finally, the gene expression profiles, differentiation status, and the enrichment status of various gene sets and regulons were compared between the cell subsets. Results: We demonstrated 15 distinct cell subsets in the integrated single-cell transcriptome sequencing data. Surface marker annotation demonstrated the MSC phenotype in 12 of the 15 cell subsets. C10 and C14 subsets demonstrated both the MSC and pericyte phenotypes. All 15 cell subsets demonstrated the fibroblast phenotype. C8, C12, and C13 subsets exclusively demonstrated the fibroblast phenotype. We identified 3,275 differentially expressed genes, 305 enriched gene sets, and 34 enriched regulons between the 15 cell subsets. The cell subsets that exclusively demonstrated the fibroblast phenotype represented less primitive and more differentiated cell types. Conclusion: Cell subsets with the MSC phenotype also demonstrated the fibroblast phenotype, but cell subsets with the fibroblast phenotype did not necessarily demonstrate the MSC phenotype, suggesting that MSCs represented a subclass of fibroblasts. We also demonstrated that the MSCs and fibroblasts represented highly heterogeneous populations with distinct cell subsets, which could be identified based on the differentially enriched gene sets and regulons that specify proliferating, differentiating, metabolic, and/or immunomodulatory functions.

Human Umbilical Mesenchymal Stromal Cells Mixed with Hyaluronan Transplantation Decreased Cartilage Destruction in a Rabbit Osteoarthritis Model.

Osteoarthritis (OA), the most common type of arthritis, causes pain in joints and disability. Due to the absence of ideal effective medication, stem cell transplantation emerges as a new hope for OA therapy. This study is aimed at evaluating the capability of human umbilical cord mesenchymal stromal cells (HUCMSCs) mixed with hyaluronan (HA) to treat osteoarthritis in a rabbit model. Differentiation capability of HUCMSCs, magnetic resonance image examination, and immunohistochemistry of the cartilage after transplantation of HUCMSCs mixed with HA in a rabbit OA model were explored. HUCMSCs exhibited typical mesenchymal stromal cell (MSC) characteristics, including spindle-shaped morphology, surface marker expressions (positive for human leukocyte antigen- (HLA-) ABC, CD44, CD73, CD90, and CD105; negative for HLA-DR, CD34, and CD45), and trilineage differentiation (chondrogenesis, adipogenesis, and osteogenesis). The gene expression of SOX9, type II collagen, and aggrecan in the HUCMSC-derived chondrocytes mixed with HA was increased after in vitro chondrogenesis compared with HUCMSCs. A gross and histological significant improvement in hyaline cartilage destruction after HUCMSCs mixed with HA was noted in the animal model compared to the OA knees. The International Cartilage Repair Society histological score and Safranin O staining were significantly higher for the treated knees than the control knees (p < 0.05). Moreover, the expression of MMP13 was significantly decreased in the treated knees than in the OA knees. In conclusion, HUCMSCs mixed with HA in vitro and in vivo might attenuate the cartilage destruction in osteoarthritis. Our study provided evidence for future clinical trials.

Individual heterogeneity screened umbilical cord-derived mesenchymal stromal cells with high Treg promotion demonstrate improved recovery of mouse liver fibrosis

BackgroundTo investigate the heterogeneities of human umbilical cord mesenchymal stromal cells (HUCMSCs) derived from different donors and their therapeutic variations when applied to mouse liver fibrosis model.MethodsThe characteristics of HUCMSCs derived from multiple donors were comprehensively analyzed including expressions of surface markers, viability, growth curve, karyotype analysis, tumorigenicity, differentiation potentials, and immune regulation capability. Then, the HUCMSCs with distinct immunomodulatory effects were applied to treat mouse liver fibrosis and their therapeutic effects were observed.ResultsThe HUCMSCs derived from multiple donors kept a high consistency in surface marker expressions, viability, growth curve, and tumorigenicity in nude mice but had robust heterogeneities in differentiation potentials and immune regulations. In addition, three HUCMSC lines applied to mice liver fibrosis model had different therapeutic outcomes, in line with individual immune regulation capability.ConclusionThe HUCMSCs derived from different donors have individual heterogeneity, which potentially lead to distinct therapeutic outcomes in mouse liver fibrosis, indicating we could make use of the donor-variation of MSCs to screen out guaranteed general indicators of MSCs for specific diseases in further stromal cell therapy.

Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells.

AimsPlatelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs).MethodsEVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca2+) content.ResultsOsteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response.ConclusionPL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations.Cite this article: Bone Joint Res 2020;9(10):667–674.

Secreted responses of umbilical cord mesenchymal cells to full thickness swine burns are refined by the zone of injury

Background & Aim Primary treatment of battlefield burns is often inadequate due to delayed field evacuation or limited care in mass casualty incidents. Untreated burns expand in depth and surface area and elicit a hyper-immune response until the damaged tissue is removed. Third degree (full-thickness) burns penetrate the dermis and comprise 3 concentric zones of injury. The central zone of coagulation is irrevocably damaged while the surrounding zone of stasis is at risk. Tissue in the outermost zone of hyperemia can be saved despite an acute inflammatory response and increased perfusion. Mesenchymal stromal cells (MSCs) reportedly improve outcomes as post-surgical burn treatments. We assessed in vitro whether MSCs may also benefit burn sequelae if applied prior to surgical intervention. Methods, Results & Conclusion Nine anaesthetized swine received 3rddegree burns on the inner flank by a heated brass bar. After 30 minutes, the burn was excised ∼1 cm outside the zone of hyperemia. Control (unburned) samples were also excised. The burn center (zones of coagulation and stasis) and margin (zone of hyperemia) were isolated by dissection. Human umbilical cord MSCs (UC-MSCs), were co-incubated with 0.4 +/- 0.005g of tissue using a 6-well Transwell system for 24, 48 or 72 hours (h) in xeno-free media. Secreted proteins were assessed by multiplex ELISA of 115 analytes. Within 24h of exposure to any burn tissue, UC-MSCs increased output of inflammation mediators including fractalkine, GCP-2, GRO-β, I-309, IL-19, MCP-3, MIP3-α, MMP-2 and TSLP. IL-2Ra and MIG were specifically elevated in response to the necrotic center tissue, followed by β-NGF and IL-3 at 48h and SCF and SCGF-β at 72h. Intriguingly, the burn margin stimulated production of wound healing molecules in addition to inflammation mediators. G-CSF, HGF, IL-1β, IL-1RA, LIF and TECK increased significantly at 24 hours, followed by IL-20, IL-35, MDC, sCD163 and sTNF-R1 at 72h. Thus, UC-MSCs exhibit refined soluble responses that may improve outcomes for severe burn patients that initially receive sub-optimal treatment and delayed definitive care. The observed zone-specific responses suggest that the molecular mechanisms of MSC treatments may differ if the cells are applied before or after surgical debridement and depending on their localization within the wound. UC-MSCs delivered to unsalvageable tissue can produce a response focused on managing inflammation, while UC-MSCs delivered to the viable peripheral zone additionally promote wound healing.

Investigation of bioactive molecules released by MSCs seeded on decellularized tracheal scaffolds for airway tissue engineering

Background & Aim The use of Mesenchymal stromal cells (MSCs) in tissue engineering applications has expanded exponentially. Their relative ease of isolation, expansion and differentiation potential in addition to the ever-expanding knowledge of their paracrine mediated effects such as anti-inflammatory, immunomodulatory and anti-scarring and supportive effects. This has made them an attractive source that could improve tissue healing, repair and regeneration. We and others have used MSCs for recellularization of decellularized, cadaveric donor tracheal scaffolds in several clinical compassionate cases of airway tissue engineering for stenosis. However, the role of these cells in the engineered airway remains not fully explored. The aim of this study was to investigate viability, proliferation and differential gene expression of selected angiogenic and growth factors of human MSCs seeded onto decellularized tracheal scaffolds. Methods, Results & Conclusion Methods Human umbilical cord MSCs were isolated by expanded in GMP-compliant, serum-free culture conditions to a maximum of 10 population doublings. Cells were recovered, suspended in culture media and seeded by passive adhesion onto human decellularized tracheal scaffolds at a density of 500 × 103/cm2. The seeded constructs were cultured in 5% CO2 at 37°C and assessed at days 3, 7 and 14 for viability and proliferation using live/ dead staining and KI67 staining. RNA extraction was performed on day 3 and reverse transcriptase polymerase chain reaction (RT-PCR) was used to test gene expression changes of VEGF, PTGES (PGE2), CSF2 (GM-CSF), TGF-B and BMP4 as opposed to two-dimensional culture flasks. Results & conclusion The study demonstrated the cytocompatibility of the scaffolds. Cells remained viable and continued to proliferate during 14 days of culture. RT-PCR analysis revealed an upregulation of VEGFA, PTGES (PGE2), CSF2 (GM-CSF), TGF-B and downregulation of BMP4. The results of this preliminary study indicate that seeded MSCs are able to engraft into the scaffold and proliferate whilst producing angiogenic and supportive factors that could enhance constructive remodelling of tissue engineered airway upon implantation.

Purity Determines the Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) have been recently identified as vital components of cell-based therapies based on the observation that conditioned media from cultured stromal cells reproduce some of the beneficial effects of intact cells. In order to obtain clinically active EVs derived from Mesenchymal Stromal Cells (MSCs) different procedures have been reported in the literature. Usually, non-confluent cells are incubated with culture medium for 48 h either with EV-depleted Fetal Bovine Serum (FBS) or without FBS. Our aim was to compare the effects of EVs isolated by ultracentrifugation from human umbilical cord MSC conditioned media obtained using these two conditions: with EV-depleted FBS (UC) or without FBS (UCw/o) on the mRNA expression levels of extracellular matrix related genes using the mouse chondrogenic cell line ATDC-5. We observed a deleterious effect on chondrogenic cells treated with UCw/o, showing higher mRNA expression levels of different metalloproteinases and decorin (Dcn) and lower collagen (Col1a1 and Col2a1) and aggrecan (Acan) mRNA levels. To elucidate whether this deleterious effect was induced by the EVs or by any proteins co-purified in the EV pellet, we used size exclusion chromatography (SEC) to further purify the EV pellet, obtaining an EV enriched fraction (EV or EVw/o) and a protein enriched fraction (Prot or Protw/o). Our results pointed that the negative effect on the chondrogenic cell line was due to the contaminant proteins coisolated with the EVs by ultracentrifugation and not from the EVs themselves. Thus, these results highlight the importance of working with well purified EV preparations to specifically achieve their therapeutic effect.

Overexpression of IL-10 Enhances the Efficacy of Human Umbilical-Cord-Derived Mesenchymal Stromal Cells in E. coli Pneumosepsis.

Enhancing the immunomodulatory effects of mesenchymal stromal cells (MSCs) may increase their effects in sepsis. We tested the potential for overexpression of Interleukin-10 (IL-10) in human umbilical cord (UC) MSCs to increase MSC efficacy in Escherichia coli (E. coli) pneumosepsis and to enhance human macrophage function. Pneumonia was induced in rats by intratracheal instillation of E. coli ((2.0–3.0) × 109 Colony forming units (CFU)/kg). One hour later, animals were randomized to receive (a) vehicle; (b) naïve UC-MSCs; or (c) IL-10 overexpressing UC-MSCs (1 × 107 cells/kg). Lung injury severity, cellular infiltration, and E. coli colony counts were assessed after 48 h. The effects and mechanisms of action of IL-10 UC-MSCs on macrophage function in septic rodents and in humans were subsequently assessed. Survival increased with IL-10 (9/11 (82%)) and naïve (11/12 (91%)) UC-MSCs compared to vehicle (9/15 (60%, p = 0.03). IL-10 UC-MSCs—but not naïve UC-MSCs—significantly decreased the alveolar arterial gradient (455 ± 93 and 520 ± 81, mmHg, respectively) compared to that of vehicle animals (544 ± 52, p = 0.02). Lung tissue bacterial counts were significantly increased in vehicle- and naïve-UC-MSC-treated animals but were not different from sham animals in those treated with IL-10 overexpressing UC-MSCs. IL-10 (but not naïve) UC-MSCs decreased alveolar neutrophils and increased alveolar macrophage percentages compared to vehicle. IL-10 UC-MSCs decreased structural lung injury compared to naïve UC-MSC or vehicle therapy. Alveolar macrophages from IL-10-UC-MSC-treated rats and from human volunteers demonstrated enhanced phagocytic capacity. This was mediated via increased macrophage hemeoxygenase-1, an effect blocked by prostaglandin E2 and lipoxygenase A4 blockade. IL-10 overexpression in UC-MSCs enhanced their effects in E. coli pneumosepsis and increased macrophage function. IL-10 UC-MSCs similarly enhanced human macrophage function, illustrating their therapeutic potential for infection-induced acute respiratory distress syndrome (ARDS).

Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice.

We speculate that exosomes derived from human umbilical cord mesenchymal stromal cells (HUC-MSCs) will accumulate within tumors and have the potential for both tumor location or drug delivery.Methods: To determine proof of concept, HUC-MSC exosomes were labeled with an MRI contrast agent, gadolinium, or a near infrared dye. Exosome accumulation within ectopic osteosarcoma tumor-bearing mice was determined by 14.1 T MRI or bioimaging over 24-48 h after injection. In vitro studies examine the accumulation and physiological effect of exosomes on human and mouse osteosarcoma cell lines by MTT assay, confocal microscopy, and flow cytometry.Results: Systemic HUC-MSC exosomes accumulated continuously in tumor over a 24-48 h post-injection period. In contrast, synthetic lipid nanoparticles accumulate in tumor only for the first 3 h post-injection.Conclusion: These results suggest that HUC-MSCs exosomes accumulate within human or mouse osteosarcoma cells in vitro and in vivo over a 24 to 48 h after infusion.

Combinatory effect of mesenchymal stromal cells transplantation and quercetin after spinal cord injury in rat.

To investigate the synergistic effects of quercetin (Qu) administration and transplantation of human umbilical cord mesenchymal stromal cells (HUMSCs) following spinal cord injury (SCI). HUMSCs were isolated, cultured and certificated via flow cytometry. Sixty Sprague-Dawley (SD) female rats were used and SCI models were made. All rats were divided into five experimental groups: culture medium treated group (n=28); HUSMCs + quercetin-treated group (n = 28); HUMSCs treated group (n=28); quercetin-treated group (n = 28); sham group (n = 20). Basso, Beattie, and Bresnahan (BBB) were used to assess neurological function recovery. Axons at the injury epicenter of the injury were checked by immunohistochemical analysis. Cystic cavity was measured and rat cytokine Luminex custom 8-plex kits (for interleukin (IL)-4, IL-1β, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-β1) were checked. The combination treatment with Qu and delayed transplantation of HUMSCs after rat SCI improved neurological functional recovery, increased axonal preservation, promoted macrophage polarization, decreased the size of the cystic cavity, reduced the proinflammatory cytokines, including IL-1β and IL-6. Also, it increased anti-inflammatory cytokines, including IL-4, IL-10, and transforming growth factor (TGF)-β1. We showed that HUMSCs transplantation in combination with Qu was a potential strategy for reducing secondary damage and promoting functional recovery following SCI.

Sevoflurane suppresses microglial M2 polarization.

Microglia can be polarized into the classical (M1) or alternative (M2) activation states in response to various stimuli. The M2 phenotype has its own set of receptor profiles, cytokine production, and chemokine secretion, of which arginase 1 (Arg1), chitinase 3-like 3 (Chi3l3, Ym1), and IL-10 have neuroprotective properties. Sevoflurane is one of the most commonly used volatile anesthetics in clinics. Previous studies have shown that sevoflurane promotes microglial M1 activation. However, it remains unclear whether sevoflurane regulates microglial M2 activation. In this study, we found that sevoflurane treatment abolished interleukin 4 (IL-4)-induced M2 microglial activation. Our results indicate that IL-4-mediated induction of the characteristic M2 marker genes and proteins Arg1, Ym1, and IL-10 was significantly attenuated by sevoflurane pretreatment in primary microglia. Microglial M2 polarization induced by incubation with culture supernatant from human umbilical cord mesenchymal stromal cells (HUC-MSCs) was abolished by treatment with 2% or 4% sevoflurane. Upregulation of SOCS1 and suppression of SOCS3 were shown to play a crucial role in the process of M2 microglial polarization. Our results also indicate that SOCS1 expression was induced by IL-4, but it was inhibited by pretreatment with sevoflurane. In contrast, IL-4 suppressed SOCS3 expression, which was restored by pretreatment with sevoflurane. Mechanistically, it was shown that sevoflurane suppresses STAT6 phosphorylation in primary microglia.

Anti-inflammatory Effect of Mesenchymal Stromal Cell Transplantation and Quercetin Treatment in a Rat Model of Experimental Cerebral Ischemia.

Here, we have investigated the synergistic effect of quercetin administration and transplantation of human umbilical cord mesenchymal stromal cells (HUMSCs) following middle cerebral artery occlusion in rat. Combining quercetin treatment with delayed transplantation of HUMSCs after local cerebral ischemia significantly (i) improved neurological functional recovery; (ii) reduced proinflammatory cytokines (interleukin(IL)-1β and IL-6), increased anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor-β1), and reduced ED-1 positive areas; (iii) inhibited cell apoptosis (caspase-3 expression); and (iv) improved the survival rate of HUMSCs in the injury site. Altogether, our results demonstrate that combined HUMSC transplantation and quercetin treatment is a potential strategy for reducing secondary damage and promoting functional recovery following cerebral ischemia.