Short-Term Inflammatory Exposure Affects Umbilical Cord-derived Mesenchymal Stem Cells Migration and Differentiation Through Modulation of NLRP3 Inflammasome Expression

Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells.

Human umbilical cord-derived mesenchymal stem cell (UC-MSC) transplantation has shown promise for the treatment of various diseases. For clinical applications, UC-MSCs have been stored in 0.9% saline, 5% dextrose, dextrose and sodium chloride injection, Plasma-Lyte A, 1% human serum albumin (1% HSA), or 5% HSA before administration, but the effect of storage conditions on the viability and biological function of the cells remains unknown. Freshly harvested UC-MSCs were resuspended and incubated in these solutions for 2, 4, or 6 h at 4°C or room temperature (24°C). Cell viability, apoptotic/necrotic fraction, poststorage growth potential, immunophenotype, immunosuppressive capacity, and differentiation capacity were analyzed. When stored in parenteral solutions, UC-MSCs showed progressive deterioration in survival viability and adhesion ability. After 6-h storage, the best viability and attachment rate of UC-MSCs decreased to 83.0 ± 1.6% and 71.8 ± 3.2%, respectively. Our results suggested that UC-MSCs in these conditions lose their viability in a short time. However, it seems that the other biological functions of the surviving UC-MSCs were little affected. Since UC-MSCs suspended in these mediums lose their survival viability in a short time to levels significantly below the permissible limits (70%) by FDA, precautions need to be taken on using these solutions as suspension medium and further studies on the optimal methods for preservation are urgent.

Cell tracking with magnetic resonance imaging (MRI) is important for evaluating the biodistribution of transplanted cells. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have emerged as a promising therapeutic tool in regenerative medicine. We examined the UC-MSCs labeled with superparamagnetic (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) in terms of cell functioning and imaging efficiency in vitro and in vivo. The UC-MSCs were co-incubated with SPIO or USPIO at a concentration of 50 or 100 µg/mL of label. Viability and proliferation were assessed by Trypan blue dye exclusion and MTT assay, respectively. Differentiation (chondrogenesis, osteogenesis, and adipogenesis) was induced to examine the impact of labelling on stemness. For in vitro experiments, we used 7-T MRI to assess the T2 values of phantoms containing various concentrations of cell suspensions. For in vivo experiments, nine neonatal rats were divided into the control, SPIO, and USPIO groups. The UC-MSCs were injected directly into the rat brains. MRI images were obtained immediately and at 7 and 14 days post injection. The UC-MSCs were successfully labeled with SPIO and USPIO after 24 h of incubation. Cell viability was not changed by labelling. Nevertheless, labelling with 100 µg/mL USPIO led to a significant decrease in proliferation. The capacity for differentiation into cartilage was influenced by 100 µg/mL of SPIO. MRI showed that labeled cells exhibited clear hypointense signals, unlike unlabeled control cells. In the USPIO-labeled cells, a significant (P < 0.05) decrease in T2 values (= improved contrast) was observed when compared with the controls and between phantoms containing the fewest and the most cells (0.5 × 106 versus 2.0 × 106 cells/mL). In vivo, the labeled cells were discernible on T2-weighted images at days 0, 7, and 14. The presence of SPIO and USPIO particles at day 14 was confirmed by Prussian blue staining. Microscopy also suggested that the regions occupied by the particles were not as large as the corresponding hypointense areas observed on MRI. Both labels were readily taken up by the UC-MSCs and identified well on MRI. While SPIO and USPIO provide improved results in MRI studies, care must be taken while labelling cells with high concentrations of these agents.

Umbilical cord derived mesenchymal stem cells attenuate rodent Osteoarthritis progression via preserving articular cartilage superficial layer cells and inhibiting synovitis

Objective: To investigate the effect and regulation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) on islets function and NOD-like receptor family, pyrin domain containing 3 (NLRP3) and autophagy in type 2 diabetic mellitus (T2DM) mice. Methods: Experimental study. Twenty, 8-week-old, male C57BL/6J mice were selected and divided into a normal control group (n=5) and a high-fat feeding modeling group (n=15). The model of T2DM was established by high-fat feeding combined with intraperitoneal injection of low-dose streptozotocin. After successful modeling, those mice were divided into a diabetes group (n=7) and a UC-MSCs treatment group (n=7). The UC-MSCs treatment group was given UC-MSCs (1×106/0.2 ml phosphate buffer solution) by tail vein infusion once a week for a total of 4 weeks; the diabetes group was injected with the same amount of normal saline, and the normal control group was not treated. One week after the treatment, mice underwent intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests, and then the mice were sacrificed to obtain pancreatic tissue to detect the expressions of interleukin-1β (IL-1β) and pancreatic and duodenal homeobox 1 (PDX-1) by immunofluorescence. The bone marrow-derived macrophages were stimulated with lipopolysaccharide and adenosine triphosphate (experimental group) in vitro, then co-cultured with UC-MSCs for 24 h (treatment group). After the culture, enzyme-linked immunosorbent assay was used to detect the secretion level of IL-1β in the supernatant, and immunofluorescence staining was used to detect the expression of NLRP3 inflammasome, and related autophagy proteins. Statistical analysis was performed using unpaired one-way analysis of variance, repeated measure analysis of variance. Results: In vivo experiments showed that compared with the diabetes group, the UC-MSCs treatment group partially repaired islet structure, improved glucose tolerance and insulin sensitivity (all P<0.05), and the expression of PDX-1 increased and IL-1β decreased in islets under confocal microscopy. In vitro experiments showed that compared with the experimental group, the level of IL-1β secreted by macrophages in the treatment group was decreased [(85.9±74.6) pg/ml vs. (883.4±446.2) pg/ml, P=0.001], the expression of NLRP3 inflammasome and autophagy-related protein P62 was decreased, and the expressions of microtubule-associated protein 1 light chain 3β (LC3) and autophagy effector Beclin-1 were increased under confocal microscopy. Conclusions: UC-MSCs can reduce the level of pancreatic inflammation in T2DM mice, preserving pancreatic function. This might be associated with the ability of UC-MSCs to inhibit the activity of NLRP3 inflammasomes in macrophages and enhance autophagy levels.

PG2, a botanically derived drug extracted from Astragalus membranaceus, promotes proliferation and immunosuppression of umbilical cord-derived mesenchymal stem cells

ObjectivesTransfusion of umbilical cord‐derived mesenchymal stem cells (UC‐MSCs) is a novel strategy for treatment of various liver diseases. However, the therapeutic effect of UC‐MSCs is limited because only a few UC‐MSCs migrate towards the damaged regions. In this study, we observed the effects of autophagy on the migration of UC‐MSCs in vitro and in a model of liver ischaemia/reperfusion (I/R) injury.Materials and MethodsWe investigated the effects of autophagy on the status of the cell, release of anti‐inflammatory factors and migration of UC‐MSCs in vitro. The therapeutic effects and in vivo migration of rapamycin‐preconditioned UC‐MSCs were observed in a C57/B6 mouse model of liver I/R injury.ResultsInduction of autophagy by rapamycin enhanced the ability of UC‐MSCs to migrate and release anti‐inflammatory cytokines as well as increased expression of CXCR4 without affecting cell viability. Inhibition of CXCR4 activation markedly decreased migration of these cells. In a mouse model of liver I/R injury, we found significantly upregulated expression of CXCR12 in the damaged liver. More rapamycin‐preconditioned UC‐MSCs migrated towards the ischaemic regions than 3‐methyladenine‐preconditioned or non‐preconditioned UC‐MSCs, leading to improvement in hepatic performance, pathological changes and levels of inflammatory cytokines. These effects were abolished by AMD3100.ConclusionsPreconditioning of UC‐MSCs by rapamycin afforded increased protection against liver I/R injury by enhancing immunosuppression and strengthening the homing and migratory capacity of these cells via the CXCR4/CXCL12 axis.

Multiple umbilical cord–derived MSCs administrations attenuate rat osteoarthritis progression via preserving articular cartilage superficial layer cells and inhibiting synovitis

Umbilical cord derived - mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical debates or moral constraints, and show excellent pluripotency and proliferation potential. Therefore, the aim of this study was to examine the differentiation potential of UCMSCs into epidermal cells. In this study, UCMSCs were isolated by a tissue attachment culture method and characterized their immunophenotype and their differentiation potency. On the third passage, UCMSCs were induced to differentiate into epidermal cells in induction media. The results showed that UCMSCs exhibited a fibroblast-like morphology, owned strong proliferation and differentiation capacity into adipocytes and osteoblasts. Isolated UCMSCs were strong positive for CD73, CD90, CD105 and CD166, but not CD19, CD34, CD45 and HLA-DR. After differentiation, the induced cells had morphological changes when compared with the non-induced cells, and expressed CK18, CK19, and P63 whereas non-induced cells were not. Interestingly, both induced cells and non-induced cells expressed β1-intergrin. These results demonstrated that UCMSCs might trans-differentiate into epithelial cells in vitro under appropriate culture conditions. These cells may be useful for artificial skin tissue engineering in the future.

the optimum temperatures and mediums storage for stability of umbilical cord derived mesenchymal stem cell (UC-MSC) and stromal vascular fraction (svf) viability

Background:Acute respiratory distress syndrome (ARDS) is a severe disorder that is related to a high mortality. Mesenchymal stem cells (MSCs) have shown strong effects in relieving lung injury.Aims:To determine the role of umbilical cord-derived MSCs (UC-MSCs) together with surfactant protein B (SP-B) in ARDS.Study Design:Animal experimentation.Methods:Immunophenotypic characteristics of UC-MSCs were identified. BALB/c mice were intratracheally administrated with lipopolysaccharide (LPS) and received UC-MSCs or UC-MSCs transfected with SP-B (UC-MSCs-SP-B). Pathological changes and lung injury degrees after transplantation were assessed by histological and biochemical analyses. Inflammatory chemokine and cytokine production in the bronchoalveolar lavage fluid (BALF) was measured using enzyme-linked immunoassay. Flow cytometry was used to examine macrophage phenotypes and differentiation of T-helper 17 (Th17) and T-regulatory (Treg) in the BALF.Results:Our results showed that isolated UC-MSCs possessed multilineage differentiation potential. SP-B transfection into UC-MSCs strengthened the effects of UC-MSCs on lung function repair in LPS-induced ARDS. UC-MSCs and UC-MSCs-SP-B attenuated cellular infiltration. Additionally, UC-MSCs and UC-MSCs-SP-B inhibited the inflammatory response by promoting M2-like polarization, as well as reduced Th17 differentiation and promoted Treg differentiation.Conclusion:UC-MSCs in combination with SP-B, alleviates inflammatory reaction in ARDS by regulating macrophage polarization.

Adequate hypothermic storage of human mesenchymal stem cells (hMSCs) is of fundamental importance since they have been explored in several regenerative medicine initiatives. However, the actual clinical application of hMSCs necessitates hypothermic storage for long periods, a process that requires the use of non-toxic and efficient cryo-reagents capable of maintaining high viability and differentiating properties after thawing. Current cryopreservation methods are based on cryoprotectant agents (CPAs) containing dimethylsulphoxide (DMSO), which have been shown to be toxic for clinical applications. In this study, we describe a simple and effective trehalose (TRE)-based solution to cryo-store human umbilical cord-derived MSCs (UC-MSCs) in liquid nitrogen. Cells viability, identity, chromosomal stability, proliferative and migration capacity, and stress response were assessed after cryopreservation in TRE as CPA, testing different concentrations by itself or in combination with ethylene glycol (EG). Here we show that TRE-stored UC-MSCs provided lower cell recovery rates compared with DMSO-based solution, but maintained good functional properties, stability, and differentiating potential. The best cell recovery was obtained using 0.5 M TRE with 10% EG showing no differences in the osteogenic, adipogenic, and chondrogenic differentiation capacity. A second cycle of cryopreservation in this TRE-based solution had no additional impact on the viability and morphology, although slightly affected cell migration. Furthermore, the expression of the stress-related genes, HSPA1A, SOD2, TP53, BCL-2, and BAX, did not show a higher response in UC-MSCs cryopreserved in 0.5 M TRE + 10% EG compared with DMSO. Together these results, in addition to ascertained therapeutic properties of TRE, provide sufficient evidence to consider TRE-based medium as a low-cost and efficient solution for the storage of human UC-MSCs cells and potentially substitute DMSO-based cryo-reagents.

Transplantation of stem cells is a promising, emerging treatment for cardiovascular diseases in the modern era. Mesenchymal stem cells (MSCs) derived from the umbilical cord are one of the most promising cell sources because of their capacity for differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells in vitro/in vivo. In addition, umbilical cord‐derived MSCs (UC‐MSCs) secrete many effective molecules regulating apoptosis, fibrosis and neovascularization. Another important and specific characteristic of UC‐MSCs is their low immunogenicity and immunomodulatory properties. However, the application of UC‐MSCs still faces some challenges, such as low survivability and tissue retention in a harmful disease environment. Gene engineering and pharmacological studies have been implemented to overcome these difficulties. In this review, we summarize the differentiation ability, secretion function, immunoregulatory properties and preclinical/clinical studies of UC‐MSCs, highlighting the advantages of UC‐MSCs for the treatment of cardiovascular diseases.

BackgroundPeriventricular leukomalacia (PVL) is a type of multifactorial brain injury that causes cerebral palsy in premature infants. To date, effective therapies for PVL have not been available. In this study, we examined whether mesenchymal stem cells (MSCs) possess neuroprotective property in a lipopolysaccharide (LPS)-induced neonatal rat PVL-like brain injury.MethodsHuman umbilical cord-derived MSCs (UCMSCs) were used in this study. Four-day-old rats were intraperitoneally injected with LPS (15 mg/kg) to cause the PVL-like brain injury and were treated immediately after the LPS-injection with UCMSCs, conditioned medium prepared from MSCs (UCMSC-CM) or interferon-gamma (IFN-γ)-pretreated MSC (IFN-γ-UCMSC-CM). To assess systemic reaction to LPS-infusion, IFN-γ in sera was measured by ELISA. The brain injury was evaluated by immunostaining of myelin basic protein (MBP) and caspase-3. RT-PCR was used to quantitate pro-inflammatory cytokine levels in the brain injury, and the expression of tumor necrosis factor-stimulated gene-6 (TSG-6) or indoleamine 2,3-dioxygenase (IDO) to evaluate anti-inflammatory or immunomodulatory molecules in UCMSCs, respectively. A cytokine and growth factor array was employed to investigate the cytokine secretion profiles of UCMSCs.ResultsElevated serum IFN-γ was observed in LPS-infused rats. The expression of IL-6, tumor necrosis factor-alpha (TNF-α), IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) were increased in the brain by LPS-infusion in comparison to saline-infused control. LPS-infusion increased caspase-3-positive cells and decreased MBP-positive area in neonatal rat brains. A cytokine and growth factor array demonstrated that UCMSCs secreted various cytokines and growth factors. UCMSCs significantly suppressed IL-1ß expression in the brains and reversed LPS-caused decrease in MBP-positive area. UCMSC-CM did not reverse MBP-positive area in the injured brain, while IFN-γ-UCMSC-CM significantly increased MBP-positive area compared to control (no treatment). IFN-γ-pretreatment increased TSG-6 and IDO expression in UCMSCs.ConclusionWe demonstrated that bolus intraperitoneal infusion of LPS caused PVL-like brain injury in neonatal rats and UCMSCs infusion ameliorated dysmyelination in LPS-induced neonatal rat brain injury. Conditioned medium prepared from IFN-γ-pretreated UCMSCs significantly reversed the brain damage in comparison with UCMSC-CM, suggesting that the preconditioning of UCMSCs would improve their neuroprotective effects. The mechanisms underline the therapeutic effects of MSCs on PVL need continued investigation to develop a more effective treatment.

BackgroundInsulin resistance is one of the most common and important pathological features of type 2 diabetes (T2D). Recently, insulin resistance is increasingly considered to be associated with systemic chronic inflammation. Elevated levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in blood are predictive indicators of the development of T2D. Mesenchymal stem cell (MSC)-based therapies have been proven to have potential immunomodulation and anti-inflammatory properties through their paracrine effects; however, the mechanism for the anti-inflammatory effect of MSCs in enhancing insulin sensitivity is still uncertain.MethodsIn the present experiment, we used HepG2 cells, a human hepatoma cell line, and a MSC-HepG2 transwell culturing system to investigate the anti-inflammatory mechanism of human umbilical cord-derived MSCs (UC-MSCs) under palmitic acid (PA) and lipopolysaccharide (LPS)-induced insulin resistance in vitro. Insulin resistance was confirmed by glycogen assay kit and glucose assay kit. Inflammatory factor release was detected by ELISA, gene expression was tested by quantitative real-time PCR, and insulin signaling activation was determined by western blotting analysis. The changes of inflammatory factors and insulin signaling protein were also tested in T2D rats injected with UC-MSCs.ResultsTreating HepG2 cells with PA–LPS caused NLRP3 inflammation activation, including overexpression of NLRP3 and caspase-1, and overproduction of IL-1β and IL-18 as well as TNF-α from HepG2 cells. The elevated levels of these inflammatory cytokines impaired insulin receptor action and thereby prevented downstream signaling pathways, exacerbating insulin resistance in HepG2 cells. Importantly, UC-MSCs cocultured with HepG2 could effectively alleviate PA and LPS-induced insulin resistance by blocking the NLRP3 inflammasome activation and inflammatory agents. Furthermore, knockdown of NLRP3 or IL-1β partially improved PA and LPS-induced insulin signaling impairments in the presence of UC-MSCs. Similarly, UC-MSC infusion significantly ameliorated hyperglycemia in T2D rats and decreased inflammatory activity, which resulted in improved insulin sensitivity in insulin target tissues.ConclusionsOur results indicated that UC-MSCs could attenuate insulin resistance and this regulation was correlated with their anti-inflammatory activity. Thus, MSCs might become a novel therapeutic strategy for insulin resistance and T2D in the near future.