Compliant substrates mitigate the senescence associated phenotype of stress induced mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are a promising cell population for musculoskeletal cell-based therapies due to their multipotent differentiation capacity and complex secretome. Cells from younger donors are mechanosensitive, evidenced by changes in cell morphology, adhesivity, and differentiation as a function of substrate stiffness in both two- and three-dimensional culture. However, MSCs from older individuals exhibit reduced differentiation potential and increased senescence, limiting their potential for autologous use. While substrate stiffness is known to modulate cell phenotype, the influence of the mechanical environment on senescent MSCs is poorly described. To address this question, we cultured irradiation induced premature senescent MSCs on polyacrylamide hydrogels and assessed expression of senescent markers, cell morphology, and secretion of inflammatory cytokines. Compared to cells on tissue culture plastic, senescent MSCs exhibited decreased markers of the senescence associated secretory phenotype (SASP) when cultured on 50 kPa gels, yet common markers of senescence (e.g., p21, CDKN2A, CDKN1A) were unaffected. These effects were muted in a physiologically relevant heterotypic mix of healthy and senescent MSCs. Conditioned media from senescent MSCs on compliant substrates increased osteoblast mineralization compared to conditioned media from cells on TCP. Mixed populations of senescent and healthy cells induced similar levels of osteoblast mineralization compared to healthy MSCs, further indicating an attenuation of the senescent phenotype in heterotypic populations. These data indicate that senescent MSCs exhibit a decrease in senescent phenotype when cultured on compliant substrates, which may be leveraged to improve autologous cell therapies for older donors.

Standard of Care Versus Octreotide in Angiodysplasia-Related Bleeding (the OCEAN Study): A Multicenter Randomized Controlled Trial.

BACKGROUND & AIMS: Gastrointestinal angiodysplasias are vascular anomalies that may result in transfusion-dependent anemia despite endoscopic therapy. An individual patient data meta-analysis of cohort studies suggests that octreotide decreases rebleeding rates, but component studies possessed a high risk of bias. We investigated the efficacy of octreotide in reducing the transfusion requirements of patients with angiodysplasia-related anemia in a clinical trial setting. METHODS: The study was designed as a multicenter, open-label, randomized controlled trial. Patients with angiodysplasia bleeding were required to have had at least 4 red blood cell (RBC) units or parental iron infusions, or both, in the year preceding randomization. Patients were allocated (1:1) to 40-mg octreotide long-acting release intramuscular every 28 days or standard of care, including endoscopic therapy. The treatment duration was 1 year. The primary outcome was the mean difference in the number of transfusion units (RBC + parental iron) between the octreotide and standard of care groups. Patients who received at least 1 octreotide injection or followed standard of care for at least 1 month were included in the intention-to-treat analyses. Analyses of covariance were used to adjust for baseline transfusion requirements and incomplete follow-up. RESULTS: We enrolled 62 patients (mean age, 72 years; 32 men) from 17 Dutch hospitals in the octreotide (n = 31) and standard of care (n = 31) groups. Patients required a mean number of 20.3 (standard deviation, 15.6) transfusion units and 2.4 (standard deviation, 2.0) endoscopic procedures in the year before enrollment. The total number of transfusions was lower with octreotide (11.0; 95% confidence interval [CI], 5.5-16.5) compared with standard of care (21.2; 95% CI, 15.7-26.7). Octreotide reduced the mean number of transfusion units by 10.2 (95% CI, 2.4-18.1; P = .012). Octreotide reduced the annual volume of endoscopic procedures by 0.9 (95% CI, 0.3-1.5). CONCLUSIONS: Octreotide effectively reduces transfusion requirements and the need for endoscopic therapy in patients with angiodysplasia-related anemia. CLINICALTRIALS: gov, NCT02384122.

Macromolecular crowding and decellularization method increase the growth factor binding potential of cell-secreted extracellular matrices.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

Barrett Esophagus: Quality of life and factors associated with illness perception.

BACKGROUND: Health-related Quality of life (HRQoL) in patients with Barrett's esophagus (BE), a premalignant condition, may be influenced by gastroesophageal reflux disease (GERD) symptoms and the risk of developing esophageal adenocarcinoma. METHODS: We aim to investigate HRQoL in non-dysplastic Barrett Esophagus (NDBE) patients, identify factors associated with a negative illness perception of the diagnosis BE and compare outcomes between patients treated in a specialized BE center with non-expert centers. In this multi-center cross-sectional study, HRQoL of NDBE patients were assessed using the Short Form 36, Hospital Anxiety and Depression Scale, Cancer worry Scale, and Reflux Disease Questionnaire. A multivariable, linear regression analysis was conducted to assess factors associated with illness perception (Illness perception scale) of the BE diagnosis. Outcome parameters of patients from expert centers were compared to non-expert centers. RESULTS: A total of 859 NDBE patients (mean age 63.6% and 74.5% male), of which 640 from BE expert centers were included. BE patients scored similar or higher means (i.e. better) on generic HRQoL in comparison with a Dutch norm population. The multivariable regression model showed that cancer worry, GERD symptoms, signs of anxiety and depression, and female gender were associated with a negative illness perception of BE. GERD symptoms were reported in the minority (22.4%) of BE patients. Levels of anxiety symptoms were comparable to a Dutch norm population (mean 3.7 vs. 3.9 p 0.183) and lower for depression symptoms (mean 6.8 vs. 7.6 p < 0.001). Overall, there were no differences found on outcomes between expert centers and non-expert centers. CONCLUSION: NDBE patients scored similar or better on generic HRQoL, anxiety and depression than an age and gender matched norm population. The presence of cancer worry, gastrointestinal symptoms, anxiety and depression, and female gender are factors associated with a negative illness perception of the diagnosis BE.

Characterization of Induction and Targeting of Senescent Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) from older donors have limited potential for bone tissue formation compared with cells from younger donors, and cellular senescence has been postulated as an underlying cause. There is a critical need for methods to induce premature senescence to study this phenomenon efficiently and reproducibly. However, the field lacks consensus on the appropriate method to induce and characterize senescence. Moreover, we have a limited understanding of the effects of commonly used induction methods on senescent phenotype. To address this significant challenge, we assessed the effect of replicative, hydrogen peroxide, etoposide, and irradiation-induced senescence on human MSCs using a battery of senescent cell characteristics. All methods arrested proliferation and resulted in increased cell spreading compared with low passage controls. Etoposide and irradiation increased expression of senescence-related genes in MSCs at early time points, proinflammatory cytokine secretion, DNA damage, and production of senescence-associated ß-galactosidase. We then evaluated the effect of fisetin, a flavonoid and candidate senolytic agent, to clear senescent cells and promote osteogenic differentiation of MSCs entrapped in gelatin methacryloyl (GelMA) hydrogels in vitro. When studying a mixture of nonsenescent and senescent MSCs, we did not observe decreases in senescent markers or increases in osteogenesis with fisetin treatment. However, the application of the same treatment toward a heterogeneous population of human bone marrow-derived cells entrapped in GelMA decreased senescent markers and increased osteogenesis after 14 days in culture. These results identify best practices for inducing prematurely senescent MSCs and motivate the need for further study of fisetin as a senolytic agent. Impact Statement The accumulation of senescent cells within the body has detrimental effects on tissue homeostasis. To study the role of senescent cells on tissue repair and regeneration, there is a need for effective means to induce premature cell senescence. Herein, we characterized the influence of common stressors to induce premature senescence in human mesenchymal stromal cells (MSCs). Irradiation of MSCs resulted in a phenotype most similar to quiescent, high-passage cells. These studies establish key biomarkers for evaluation when studying senescent cells in vitro.

Web-Based Educational Intervention for Patients With Uninvestigated Dyspepsia Referred for Upper Gastrointestinal Tract Endoscopy: A Randomized Clinical Trial.

Importance: Diagnostic yield of upper gastrointestinal (GI) tract endoscopy for uninvestigated dyspepsia is low, and its clinical implications are limited. There is an unmet need for better strategies to reduce the volume of upper GI tract endoscopic procedures for dyspepsia. Objective: To study the effectiveness of a web-based educational intervention as a tool to reduce upper GI tract endoscopy in uninvestigated dyspepsia. Design, Setting, and Participants: This open-label, multicenter, randomized clinical trial enrolled participants between November 1, 2017, and March 31, 2019, with follow-up 52 weeks after randomization, at 4 teaching hospitals in the Netherlands. Participants included patients with uninvestigated dyspeptic symptoms who were referred for upper GI tract endoscopy by their general health care clinician without prior consultation of a gastroenterologist. A total of 119 patients, aged 18 to 69 years, were included. Patients were excluded if any of the following red flag symptoms were present: (indirect) signs of upper GI tract hemorrhage (hematemesis, melena, hematochezia, or anemia), unintentional weight loss of 5% or higher of normal body weight during a period of 6 to 12 months, persistent vomiting, dysphagia, or jaundice. Interventions: Patients were randomly assigned (1:1) to education (intervention) or upper GI tract endoscopy (control). Education consisted of a self-managed web-based educational intervention, containing information on gastric function, dyspepsia, and upper GI tract endoscopy. Main Outcomes and Measures: Difference in the proportion of upper GI tract endoscopy procedures between those who received access to the web-based educational intervention and those who did not at 12 weeks and 52 weeks after randomization, analyzed in the intention-to-treat population. Secondary outcomes included quality of life (Nepean Dyspepsia Index) and symptom severity (Patient Assessment of Gastrointestinal Disorders Symptom Severity Index) measured at baseline and 12 weeks. Results: Of 119 patients included (median age, 48 years [interquartile range, 37-56 years]; 48 men [40%]), 62 were randomized to web-based education (intervention) and 57 to upper GI tract endoscopy (control). Significantly fewer patients compared with controls underwent upper GI tract endoscopy after using the web-based educational intervention: 24 (39%) vs 47 (82%) (relative risk, 0.46; 95% CI, 0.33-0.64; P < .001). Symptom severity and quality of life improved equivalently in both groups. One additional patient in the intervention group required upper GI tract endoscopy during follow-up. Conclusions and Relevance: Findings of this study indicate that web-based patient education is an effective tool to decrease the need for upper GI tract endoscopy in uninvestigated dyspepsia. Trial Registration: ClinicalTrials.gov Identifier: NCT03205319.

Growth factor delivery using extracellular matrix-mimicking substrates for musculoskeletal tissue engineering and repair.

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors (GFs) to influence neighboring cells and promote migration, proliferation, or differentiation. Despite promising results in preclinical models, the use of inductive biomacromolecules has achieved limited success in translation to the clinic. The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages, which commonly result in detrimental side effects. Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix (ECM), which acts as a reservoir for GFs via electrostatic interactions. Advances in the manipulation of extracellular proteins, decellularized tissues, and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs. Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules. This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone, cartilage, and muscle.

Impact of multidisciplinary tumor board discussion on palliation of patients with esophageal or gastro-esophageal junction cancer: a population-based study.

Background: The Dutch guidelines for esophageal and gastro-esophageal junction (GEJ) cancer recommend discussion of patients by a multidisciplinary tumor board (MDT). Despite this recommendation, one previous study in the Netherlands suggested that therapeutic guidance was missing for palliative care of patients with esophageal cancer. The aim of the current study was therefore to assess the impact of an MDT discussion on initial palliative treatment and outcome of patients with esophageal or GEJ cancer.Material and methods: The population-based Netherlands Cancer Registry was used to identify patients treated for esophageal or GEJ cancer with palliative intent between 2010 and 2017 in 7 hospitals. We compared patients discussed by the MDT with patients not discussed by the MDT in a multivariate analysis. Primary outcome was type of initial palliative treatment. Secondary outcome was overall survival.Results: A total of 389/948 (41%) patients with esophageal or GEJ cancer were discussed by the MDT before initial palliative treatment. MDT discussion compared to non-MDT discussion was associated with more patients treated with palliative intent external beam radiotherapy (38% vs. 21%, OR 2.7 [95% CI 1.8-3.9]) and systemic therapy (30% vs. 23%, OR 1.6 [95% CI 1.0-2.5]), and fewer patients treated with stent placement (4% vs. 12%, OR 0.3 [95% CI 0.1-0.6]) and best supportive care alone (12% vs. 33%, OR 0.2 [95% CI 0.1-0.3]). MDT discussion was also associated with improved survival (169 days vs. 107 days, HR 1.3 [95% CI 1.1-1.6]).Conclusion: Our study shows that MDT discussion of patients with esophageal or GEJ cancer resulted in more patients treated with initial palliative radiotherapy and chemotherapy compared with patients not discussed by the MDT. Moreover, MDT discussion may have a positive effect on survival, highlighting the importance of MDT meetings at all stages of treatment.

Non-adherence to Anti-TNF Therapy is Associated with Illness Perceptions and Clinical Outcomes in Outpatients with Inflammatory Bowel Disease: Results from a Prospective Multicentre Study.

BACKGROUND AND AIMS: Non-adherence to anti-tumour necrosis factor [TNF] agents in patients with inflammatory bowel disease [IBD] is a serious problem. In this study, we assessed risk factors for non-adherence and examined the association between adherence to anti-TNF agents and loss of response [LOR]. METHODS: In this multicentre, 12-month observational study, outpatients with IBD were included. Demographic and clinical characteristics were recorded. Adherence was measured with the Modified Morisky Adherence Scale-8 [MMAS-8] and 12-month pharmacy refills [medication possession ratio, MPR]. Risk factors included demographic and clinical characteristics, medication beliefs, and illness perceptions. Cox regression analysis was performed to determine the association between MPR and LOR to anti-TNF, IBD-related surgery or hospitalisation, dose intensification, or discontinuation of anti-TNF. RESULTS: In total, 128 patients were included [67 infliximab, 61 adalimumab], mean age 37 ( ± standard deviation [SD] 14) years, 71 [56%] female. Median disease duration was 8 (interquartile range [IQR] 4-14) years. Clinical disease activity was present in 41/128 [32%] patients, 36/127 [28%] patients had an MMAS-8 < 6 ['low adherence'], and 25/99 [25%] patients had an MPR < 80% [non-adherence]. Risk factors for non-adherence included adalimumab use (odds ratio [OR] 10.1, 95% confidence interval [CI] 2.62-40.00), stronger emotional response [OR 1.16, 95% CI 1.02-1.31], and shorter timeline perception, i.e. short perceived illness duration [OR 0.60, 95% CI 0.38-0.96]. Adherence is linearly and negatively [OR 0.14, 95% CI 0.03-0.63] associated with LOR. CONCLUSION: Non-adherence to anti-TNF agents is strongly associated with LOR to anti-TNF agents, adalimumab use, and illness perceptions. The latter may provide an important target for interventions aimed at improving adherence and health outcomes.

Sca-1+Lin-CD117- mesenchymal stem/stromal cells induce the generation of novel IRF8-controlled regulatory dendritic cells through Notch-RBP-J signaling.

Mesenchymal stem/stromal cells (MSCs) can influence the destiny of hematopoietic stem/progenitor cells (HSCs) and exert broadly immunomodulatory effects on immune cells. However, how MSCs regulate the differentiation of regulatory dendritic cells (regDCs) from HSCs remains incompletely understood. In this study, we show that mouse bone marrow-derived Sca-1(+)Lin(-)CD117(-) MSCs can drive HSCs to differentiate into a novel IFN regulatory factor (IRF)8-controlled regDC population (Sca(+) BM-MSC-driven DC [sBM-DCs]) when cocultured without exogenous cytokines. The Notch pathway plays a critical role in the generation of the sBM-DCs by controlling IRF8 expression in an RBP-J-dependent way. We observed a high level of H3K27me3 methylation and a low level of H3K4me3 methylation at the Irf8 promoter during sBM-DC induction. Importantly, infusion of sBM-DCs could alleviate colitis in mice with inflammatory bowel disease by inhibiting lymphocyte proliferation and increasing the numbers of CD4(+)CD25(+) regulatory T cells. Thus, these data infer a possible mechanism for the development of regDCs and further support the role of MSCs in treating immune disorders.

A new fully covered metal stent with anti-migration features for the treatment of malignant dysphagia.

BACKGROUND AND STUDY AIMS: A new esophageal stent with two anti-migration features was developed to minimize migration. The aim of this study was to evaluate the clinical efficacy and safety of this stent in patients with malignant dysphagia. PATIENTS AND METHODS: A total of 40 patients with dysphagia due to a malignant obstruction of the esophagus were prospectively enrolled in this cohort study. RESULTS: Stent placement was technically successful in 39 patients (98 %). The median dysphagia-free time after stent placement was 220 days (95 % confidence interval 94 - 345 days). Nine patients (23 %) experienced recurrent dysphagia due to tissue overgrowth (n = 2), stent fracture (n = 1), and partial (n = 5) or complete (n = 1) stent migration. A total of 16 serious adverse events occurred in 14 patients (36 %), with hemorrhage (n = 3) and severe nausea or vomiting (n = 3) being the most common causes. CONCLUSIONS: This new stent design was effective for the palliation of malignant dysphagia and had a low rate of recurrent dysphagia. However, despite the anti-migration features, stent migration was still a major cause of recurrent dysphagia. Furthermore, treatment was associated with a high adverse event rate. Dutch Trial Registration (NTR 3313).

The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke.

BACKGROUND AND PURPOSE: Intravascular injection of mesenchymal stem cells (MSCs) has been found to cause considerable vascular obstructions which may lead to serious outcomes, particularly after intra-arterial injection. However, the underlying mechanisms have been poorly understood. METHODS: In this study, we fractionated MSCs that had been cultured in monolayer for six passages into small (average diameter = 17.9 µm) and large (average diameter 30.4 µm) populations according to their sizes, and examined their vascular obstructions after intra-internal carotid artery injection in rats and mice in comparison with MSCs derived from 3D spheroids which were uniformly smaller in size (average diameter 12.6 µm). RESULTS: We found that 3D MSCs did not cause detectable infarct in the brain as evidenced by MRI scan and TTC stain, 2D MSCs in small size caused a microinfarct in one of five animals, which was co-localized to the area of entrapped MSCs (labeled with DiI), while 2D MSCs in large size caused much larger infarcts in all five animals, and substantial amounts of DiI-positive MSCs were found in the infarct. Meanwhile, corresponding neurological defects were observed in the animals with stroke. In consistence, injection of 2D MSCs (average diameter 26.5) caused a marked loss of cortical neurons and their axons in Thy1-GFP transgenic mice and the activation of microglia in CX3CR1-GFP transgenic mice in the area with MSC entrapment. CONCLUSIONS: Our results suggest that the size of MSCs is a significant cause of MSC caused vascular obstructions and stroke.

Three-dimensional spheroid-cultured mesenchymal stem cells devoid of embolism attenuate brain stroke injury after intra-arterial injection.

The therapeutic effect of mesenchymal stem cells (MSCs) in tissue repair/regeneration is substantially dampened by the loss of primitive properties and poor engraftment to target organs. In this study, the multipotency and cell sizes of human MSCs, which had been expanded in monolayer culture for several passages, were dramatically restored after an episode of three-dimensional (3D) spheroid culture. Unlike MSCs derived from monolayer, which caused embolism and blindness, MSCs derived from 3D spheroids did not cause vascular obstructions, after intra-carotid artery infusion in rats. Importantly, intra-carotid infusion of 1 million 3D spheroid MSCs in rats 24 h after middle cerebral artery occlusion and reperfusion resulted in engraftment of the cells into the lesion and significant (over 70%) reduction of infarct size along with restoration of neurologic function. Moreover, the enhanced effect of spheroid MSCs was coincided with significantly increased differentiation of the MSCs into neurons and markedly increased number of endogenous glial fibrillary acidic protein-positive neural progenitors in the peri-infarct boundary zone. However, the similarly administered monolayer MSCs resulted in a modest functional improvement. Our results suggest that 3D MSCs, in combination with intra-carotid delivery, may represent a novel therapeutic approach of MSCs for stroke.

miR-21 modulates the ERK-MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation.

The ERK-MAPK signaling pathway plays a pivotal role during mesenchymal stem cell (MSC) differentiation. Studies have demonstrated that ERK-MAPK promotes adipogenesis and osteogenesis through the phosphorylation of differentiation-associated transcription factors and that it is the only active signaling in all three lineages (adipogenic, chondrogenic, and osteogenic) during MSC differentiation. Recent studies pointed to the significant roles of microRNA-21 (miR-21) during several physiological and pathological processes, especially stem cell fate determination. The miR-21 expression pattern is also correlated with ERK-MAPK activity. Here, we found that miR-21 expression is elevated and associated with an increased differentiation potential in MSCs during adipogenesis and osteogenesis. The overexpression of miR-21 elevated the expression level of the differentiation-associated genes PPARγ and Cbfa-1 during MSC differentiation, whereas miR-21 knockdown reduced the expression level of both genes. The ERK-MAPK signaling pathway activity had an increasing tendency to respond to miR-21 upregulation and a decreasing tendency to respond to miR-21 down-regulation during the first 4 days of adipogenesis and osteogenesis. Our data indicate that miR-21 modulated ERK-MAPK signaling activity by repressing SPRY2 expression, a known regulator of the receptor tyrosine kinase (RTK) signaling pathway, to affect the duration and magnitude of ERK-MAPK activity. The ERK-MAPK signaling pathway was regulated by Sprouty2 (SPRY2) expression via a miR-21-mediated mechanism during MSC differentiation.

Enhancer of zeste homolog 2 is overexpressed and contributes to epigenetic inactivation of p21 and phosphatase and tensin homolog in B-cell acute lymphoblastic leukemia.

Enhancer of zeste homolog 2 (EZH2) is crucially involved in epigenetic silencing by acting as a histone methyltransferase. Although EZH2 is overexpressed in many solid cancers, the role of EZH2 in B-cell acute lymphoblastic leukemia (B-ALL) remains largely unexplored. In a microarray experiment, we found that EZH2 was significantly upregulated in Nalm-6 cells and this was associated with the silencing of tumor suppressor genes p21, p53 and phosphatase and tensin homolog (PTEN). The abnormal expression of these genes was further confirmed by quantitative realtime polymerase chain reaction and Western blot analysis on Nalm-6 cells. Chromatin immunoprecipitation assay showed that EZH2 and H3K27me3 were both enriched in the promoter region of PTEN and p21 in Nalm-6 cells but not in normal B cells. Functional analysis showed that siRNA-mediated EZH2 knockdown led to decreased proliferation and increased apoptosis of Nalm-6 cells, accompanied by the reactivation of PTEN and p21 expression. Furthermore, we found that EZH2 inhibitor deazaneplanocin A promoted vincristine sulfate-induced apoptosis of Nalm-6 cells. Taken together, our data suggest that EZH2 is overexpressed in B-ALL and promotes the progression of B-ALL by directly mediating the inactivation of tumor suppressor genes p21 and PTEN, and could serve as a potential epigenetic target for B-ALL therapy.

Mesenchymal stem/stromal cells induce the generation of novel IL-10-dependent regulatory dendritic cells by SOCS3 activation.

Suppression of immune response by mesenchymal stem/stromal cells (MSCs) is well documented. However, their regulatory effects on immune cells, especially regulatory dendritic cells, are not fully understood. We have identified a novel Sca-1(+)Lin(-)CD117(-) MSC population isolated from mouse embryonic fibroblasts (MEF) that suppressed lymphocyte proliferation in vitro. Moreover, the Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced hematopoietic stem/progenitor cells to differentiate into novel regulatory dendritic cells (DCs) (Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs) when cocultured in the absence of exogenous cytokines. Small interfering RNA silencing showed that Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced the generation of Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs via IL-10-activated SOCS3, whose expression was regulated by the JAK-STAT pathway. We observed a high degree of H3K4me3 modification mediated by MLL1 and a relatively low degree of H3K27me3 modification regulated by SUZ12 on the promoter of SOCS3 during SOCS3 activation. Importantly, infusion of Sca-1(+)CD117(-)Lin(-) MEF-MSCs suppressed the inflammatory response by increasing DCs with a regulatory phenotype. Thus, our results shed new light on the role of MSCs in modulating regulatory DC production and support the clinical application of MSCs to reduce the inflammatory response in numerous disease states.

Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion.

Recent findings indicate that mesenchymal stem cells (MSCs) may act as a regulator of Th17 cell differentiation, however, the underlying mechanism is still under debate. To investigate the underlying mechanisms of MSCs' regulatory effect, mouse bone marrow-derived MSCs were cocultured with mouse CD4(+)CD25(low)CD44(low)CD62L(high) T cells in vitro, and the proportion of induced Th17 cells, cytokines secretion, and transcription factors expression were examined by flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and Western blotting. For the first time, our results showed that bone marrow-derived MSCs were able to inhibit Th17 cell differentiation via interleukin (IL)-10 secretion as the Th17 cell proportion was significantly regained when IL-10 was neutralized, or expression of IL-10 by bone marrow-derived MSCs was downregulated by RNA interference technique. Furthermore, IL-10 may suppress expression of Rorγt, the key transcription factor for Th17 cells, both by activating suppressor of cytokine signaling 3 through signal transducers and activators of transcription 5 phosphorylation, and decreasing signal transducers and activators of transcription 3 binding, which is at the promoter of Rorγt. Thus, our results demonstrate the inhibitory effect of MSCs on Th17 cells differentiation, and suggest increased IL-10 secretion might be the key factor.

The role of chemokines in mesenchymal stem cell homing to myocardium.

A growing body of preclinical evidence suggests that mesenchymal stem cells (MSCs) are effective for the structural and functional recovery of the infracted heart. Accordingly, clinical trials are underway to determine the benefit of MSC-based therapies. While systemic administration of MSCs is an attractive strategy, and is the route currently used for the administration of MSCs in clinical studies for myocardial infarction, the majority of infused cells do not appear to localize to infracted myocardium in animal studies. Recently, important progress has been made in identifying chemokine receptors critical for the migration and homing of MSCs. Here, we review recent literature regarding mechanisms of MSC homing and recruitment to the ischemic myocardium, and discuss potential influences of low engraftment rates of systemically administered MSCs to the infracted heart tissue on the effects of MSC-based therapies on myocardial infarction.