Effect of bone marrow–derived mesenchymal stromal cells on hepatoma

Background aimsThe aim of the study was to evaluate the effect of mesenchymal stromal cells (MSCs) on tumor cell growth in vitro and in vivo and to elucidate the apoptotic and anti-proliferative mechanisms of MSCs on a hepatocellular carcinoma (HCC) murine model.MethodsThe growth-inhibitory effect of MSCs on the Hepa 1–6 cell line was tested by means of methyl thiazolyl diphenyl-tetrazolium assay. Eighty female mice were randomized into four groups: group 1 consisted of 20 mice that received MSCs only by intrahepatic injection; group 2 consisted of 20 HCC mice induced by inoculation of Hepa 1–6 cells into livers without MSC treatment; group 3 consisted of 20 mice that received MSCs after induction of liver cancer; group 4 consisted of 20 mice that received MSCs after induction of liver cancer on top of induced biliary cirrhosis.ResultsMSCs exhibited a growth-inhibitory effect on Hepa 1–6 murine cell line in vitro. Concerning in vivo study, decreases of serum alanine transaminase, aspartate transaminase and albumin levels after MSC transplantation in groups 2 and 3 were found. Gene expression of α-fetoprotein was significantly downregulated after MSC injection in the HCC groups. We found that gene expression of caspase 3, P21 and P53 was significantly upregulated, whereas gene expression of Bcl-2 and survivin was downregulated in the HCC groups after MSC injection. Liver specimens of the HCC groups confirmed the presence of dysplasia. The histopathological picture was improved after administration of MSCs to groups 2 and 3.ConclusionsMSCs upregulated genes that help apoptosis and downregulated genes that reduce apoptosis. Therefore, MSCs could inhibit cell division of HCC and potentiate their death.     

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.     

Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells

Background

Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the ‘licensing’ of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ.

Methods

2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins.

Results

In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types.

Conclusions

This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

     

Pancreatic cancer growth promoted by bone marrow mesenchymal stromal cell–derived IL-6 is reversed predominantly by IL-6 blockade

Pancreatic cancer is a highly lethal cancer characterized by local invasiveness, early metastasis, recurrence and high resistance to current therapies. Extensive stroma or desmoplasia is a key histological feature of the disease, and interactions between cancer and stromal cells are critical for pancreatic cancer development and progression. Mesenchymal stromal cells [MSCs] exhibit preferential tropism to primary and metastatic tumor sites and may either suppress or support tumor growth. Although MSCs represent a potential source of pancreatic cancer stroma, their contribution to pancreatic tumor growth remains poorly known. Here, we show that bone marrow MSCs significantly contribute to pancreatic cancer growth in vitro and in vivo. Furthermore, MSCs create a pro-carcinogenic microenvironment through the release of key factors mediating growth and angiogenesis, including interleukin (IL)-6, IL-8, vascular endothelial growth factor and activation of STAT3 signaling in tumor cells. IL-6 released by MSCs was largely responsible for the pro-tumorigenic effects of MSCs. Knockdown of IL-6 expression in MSCs by small interfering RNA (siRNA) abolished the MSC growth-promoting effect in vitro, reducing tumor cell proliferation and clonogenic potential. In addition, in a heterotopic nude mouse model of human pancreatic tumor xenografts, blockade of IL-6 with the anti-IL-6 receptor antibody, tocilizumab, or of its downstream effector STAT3 with the small molecule STAT3 inhibitor S3I-201, abrogated MSC-mediated tumor promotion and delayed tumor formation significantly. Our data demonstrate that MSCs promote pancreatic cancer growth, with IL-6 produced by MSCs playing a pivotal role.     

Challenges of manufacturing mesenchymal stromal cell–derived extracellular vesicles in regenerative medicine

The field of regenerative medicine has expanded greatly in the past decade, with more than 1000 current clinical trials involving mesenchymal stromal cell (MSC) treatment. Multiple recent publications have demonstrated that the beneficial effects from MSCs are not simply due to engraftment into the target organ as classically thought but rather are largely attributable to the release of paracrine factors including cytokines, growth factors and extracellular vesicles (EVs). These EVs contain miRNAs, free fatty acids and proteins that promote regeneration, proliferation and cell function and improve inflammation. Although EVs have shown promising results in animal studies, there are many obstacles to the manufacturing of EVs for clinical applications. This review discusses challenges associated with the manufacturing of clinical-grade EVs in regard to identity, purity, reproducibility, sterility, storage, potency and safety. We discuss currently employed methods and approaches for developing clinical Good Manufacturing Practices (GMP)-grade EVs and the limitations for each. We further discuss the best approaches to overcome the current hurdles in developing clinical GMP-grade EVs.     

Autophagy induction is a survival response against oxidative stress in bone marrow–derived mesenchymal stromal cells

Background aimsBone marrow–derived mesenchymal stromal cells (BMSCs) are being extensively investigated as cellular therapeutics for many diseases, including cardiovascular diseases. Although preclinical studies indicated that BMSC transplantation into infarcted hearts improved heart function, there are problems to be resolved, such as the low survival rate of BMSCs during the transplantation process and in the ischemic region with extreme oxidative stress. Autophagy plays pivotal roles in maintaining cellular homeostasis and defending against environmental stresses. However, the precise roles of autophagy in BMSCs under oxidative stress remain largely uncharacterized.MethodsBMSCs were treated with H₂O₂, and autophagic flux was examined by means of microtubule-associated protein 1A/1B-light chain 3 II/I ratio (LC3 II/I), autophagosome formation and p62 expression. Cytotoxicity and cell death assays were performed after co-treatment of BMSCs by autophagy inhibitor (3-methyladenine) or autophagy activator (rapamycin) together with H₂O₂.ResultsWe show that short exposure (1 h) of BMSCs to H₂O₂ dramatically elevates autophagic flux (2- to 4-fold), whereas 6-h prolonged oxidative treatment reduces autophagy but enhances caspase-3 and caspase-6–associated apoptosis. Furthermore, we show that pre- and co-treatment with rapamycin ameliorates H₂O₂-induced caspase-3 and caspase-6 activation and cell toxicity but that 3-methyladenine exacerbates H₂O₂-induced cell apoptotic cell death.ConclusionsOur results demonstrate that autophagy is critical for the survival of BMSCs under oxidative conditions. Importantly, we also suggest that the early induction of autophagic flux is possibly a self-defensive mechanism common in oxidant-tolerant cells.     

Biomarkers of bone healing induced by a regenerative approach based on expanded bone marrow–derived mesenchymal stromal cells

BackgroundSafety and feasibility of a regenerative strategy based on the use of culture-expanded mesenchymal stromal cells (MSCs) have been investigated in phase 2 trials for the treatment of nonunion and osteonecrosis of the femoral head (ONFH). As part of the clinical study, we aimed to evaluate if bone turnover markers (BTMs) could be useful for predicting the regenerative ability of the cell therapy product.Materials and MethodsThe bone defects of 39 patients (nonunion: n = 26; ONFH: n = 13) were treated with bone marrow–derived MSCs, expanded using a clinical-grade protocol and combined with biphasic calcium phosphate before implantation. Bone formation markers, bone-resorption markers and osteoclast regulatory proteins were measured before treatment (baseline) and after 12 and 24 weeks from surgery. At the same time-points, clinical and radiological controls were performed to evaluate the bone-healing progression.ResultsWe found that C-Propeptide of Type I Procollagen (CICP) and C-terminal telopeptide of type-I collagen (CTX) varied significantly, not only over time, but also according to clinical results. In patients with a good outcome, CICP increased and CTX decreased, and this trend was observed in both nonunion and ONFH. Moreover, collagen biomarkers were able to discriminate healed patients from non-responsive patients with a good diagnostic accuracy.DiscussionCICP and CTX could be valuable biomarkers for monitoring and predicting the regenerative ability of cell products used to stimulate the repair of refractory bone diseases. To be translated in a clinical setting, these results are under validation in a currently ongoing phase 3 clinical trial.     

The use of unlicensed bone marrow–derived platelet lysate–expanded mesenchymal stromal cells in colitis: a pre-clinical study

Background

Mesenchymal stromal cells (MSCs) are a promising candidate for treatment of inflammatory disorders, but their efficacy in human inflammatory bowel diseases (IBDs) has been inconsistent. Comparing the results from various pre-clinical and clinical IBD studies is also challenging due to a large variation in study designs.

Isolation and characterization of mesenchymal stem cells derived from bone marrow of patients with Parkinson’s disease

Mesenchymal stem cells (MSCs) are capable of self-renewing and differentiating into multiple tissues; they are expected to become a source of cells for regenerative therapy. Compared to allogeneic MSCs, autologous MSCs from patients needing cell-based therapy may be an ideal alternative stem cell source. However, characterizations of MSCs from a disease state remains extremely limited. Therefore, we have isolated and characterized MSCs from Parkinson's disease (PD) patients and compared them with MSCs derived from normal adult bone marrow. Our results show that PD-derived MSCs are similar to normal MSCs in phenotype, morphology, and multidifferentiation capacity. Moreover, PD-derived MSCs are capable of differentiating into neurons in a specific medium with up to 30% having the characteristics of dopamine cells. At last, PD-derived MSCs could inhibit T-lymphocyte proliferation induced by mitogens. These findings indicate that MSCs derived from PD patients' bone marrow may be a promising cell type for cellular therapy and somatic gene therapy applications.     

Bone marrow–mesenchymal stromal cell infusion in patients with chronic kidney disease: A safety study with 18 months of follow-up

Background

Chronic kidney disease (CKD) is a progressive loss of kidney function and structure that affects approximately 13% of the population worldwide. A recent meta-analysis revealed that cell-based therapies improve impaired renal function and structure in preclinical models of CKD. We assessed the safety and tolerability of bone marrow–mesenchymal stromal cell (MSC) infusion in patients with CKD.

Downregulation of microRNA-214 improves therapeutic potential of allogeneic bone marrow–derived mesenchymal stem cell by targeting PIM-1 in rats with acute liver failure

Acute liver failure (ALF) is a disease resulted from diverse etiology, which generally leads to a rapid degenerated hepatic function. However, transplantation bone marrow–derived mesenchymal stem cells (BMSCs) transplantation has been suggested to relieve ALF. Interestingly, microRNA-214 (miR-214) could potentially regulate differentiation and migration of BMSCs. The present study aims to inquire whether miR-214 affects therapeutic potential of BMSCs transplantation by targeting PIM-1 in ALF. 120 male Wistar rats were induced as ALF model rats and transplanted with BMSCs post-alteration of miR-214 or PIM-1 expression. Further experiments were performed to detect biochemical index (alanine aminotransferase [ALT], aspartate transaminase [AST], total bilirubin [TBiL]), and expression of miR-214, PIM-1, hepatocyte growth factor (HGF), caspase 3, tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in rat serum. Apart from the above detection, apoptosis of hepatocytes and Ki67 protein expression in hepatic tissues of rats were additionally assessed. After BMSCs transplantation with miR-214 inhibition, a decreased expression of ALT, AST, and TBiL yet an increased expression of HGF was shown, coupled with a decline in the expression of caspase 3, TNF-α, and IL-10. Meanwhile, alleviated hepatic injury and decreased apoptotic index of hepatic cells were observed and the positive rate of Ki67 protein expression was significantly increased. Moreover, miR-214 and caspase 3, TNF-α, and IL-10 decreased notably, while PIM-1 was upregulated in response to miR-214 inhibition. Strikingly, the inhibition of PIM-1 reversed effects triggered by miR-214 inhibition. These findings indicated that downregulation of miR-214 improves therapeutic potential of BMSCs transplantation by upregulating PIM-1 for ALF.     

Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow−/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O₂) and hypoxic (5% O₂) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

     

Independent human mesenchymal stromal cell–derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model

Background aimsExtracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application.MethodsIndependent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized.ResultsThe functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers.ConclusionsStandardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.     

Bone marrow–derived mesenchymal stromal cells ameliorate severe acute pancreatitis in rats via hemeoxygenase-1–mediated anti-oxidant and anti-inflammatory effects

Background and aims

It has been previously verified that mesenchymal stromal cells (MSCs) have a good therapeutic effect on severe acute pancreatitis (SAP) and the potential for regeneration of damaged pancreatic tissue, but the exact molecular mechanism remains unclear. In this study, we demonstrated the therapeutic effect of bone morrow MSCs (BMSCs) on SAP, probably by targeting heme oxygenase-1 (HO-1).

Umbilical cord–derived mesenchymal stromal cells in cardiovascular disease: review of preclinical and clinical data

The human umbilical cord has recently emerged as an attractive potential source of mesenchymal stromal cells (MSCs) to be adopted for use in regenerative medicine. Umbilical cord MSCs (UC-MSCs) not only share the same features of all MSCs such as multi-lineage differentiation, paracrine functions and immunomodulatory properties, they also have additional advantages, such as no need for bone marrow aspiration and higher self-renewal capacities. They can be isolated from various compartments of the umbilical cord (UC) and can be used for autologous or allogeneic purposes. In the past decade, they have been adopted in cardiovascular disease and have shown promising results mainly due to their pro-angiogenic and anti-inflammatory properties. This review offers an overview of the biological properties of UC-MSCs describing available pre-clinical and clinical data with respect to their potential therapeutic use in cardiovascular regeneration, with current challenges and future directions discussed.