Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species.

INTRODUCTION: Administration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes. METHODS: We set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates. RESULTS: Our analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150-200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression. CONCLUSIONS: We demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.

V-ATPase as an effective therapeutic target for sarcomas.

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9­6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment.

MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31.

Osteogenesis is the result of a complex sequence of events that involve the differentiation of mesenchymal stem cells (MSC) into osteoblasts. MSCs are multipotent adult stem cells that can give rise to different cell types of the mesenchymal germ layer. The differentiation fate of MSCs depends on the microenvironmental signals received by these cells and is tightly regulated by multiple pathways that lead to the activation of specific transcription factors. Among the transcription factors involved in osteogenic differentiation Osterix (Sp7) plays a key role and has been shown to be fundamental for bone homeostasis. However, the molecular events governing the expression of this transcription factor are not fully understood. In this study we set out to investigate the changes in the microRNA (miRNA) expression that occur during the osteogenic differentiation of bone marrow-derived MSCs. To this purpose, we analyzed the miRNA expression profile of MSCs deriving from 3 donors during the differentiation and mineralization processes by microarray. 29 miRNAs were significantly and consistently modulated during the osteogenic differentiation and 5 during the mineralization process. Interestingly, most of the differentially expressed miRNAs have been reported to be implicated in stemness maintenance, differentiation and/or oncogenesis. Subsequently, we focused our attention on the regulation of Osterix by miRNAs and demonstrated that one of the miRNAs differentially modulated during osteogenic differentiation, miR-31, controls Osterix expression through association to the 3' untranslated region of this transcription factor. By analyzing miR-31 and Osterix expression levels we found an inverse miRNA-target expression trend during osteogenic differentiation and in osteosarcoma cell lines. Moreover, the inhibition of the microRNA activity led to an increase in the endogenous expression of Osterix. Our results define a miRNA signature characterizing the osteogenic differentiation of MSCs and provide evidence for the involvement of miR-31 in the regulation of the bone-specific transcription factor Osterix.

Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy.

Mesenchymal stem cells (MSCs) are adult multipotent cells that give rise to various cell types of the mesodermal germ layer. MSCs are of great interest in the field of regenerative medicine and cancer therapy because of their unique ability to home to damaged and cancerous tissue. These cells also regulate the immune response and contribute to reparative processes in different pathological conditions, including musculoskeletal and cardiovascular diseases. The use of MSCs for tissue repair was initially based on the hypothesis that these cells home to and differentiate within the injured tissue into specialized cells. However, it now appears that only a small proportion of transplanted MSCs actually integrate and survive in host tissues. Thus, the predominant mechanism by which MSCs participate in tissue repair seems to be related to their paracrine activity. Indeed, MSCs provide the microenvironment with a multitude of trophic and survival signals including growth factors and cytokines. Recent discoveries suggest that lipid microvesicles released by MSCs may also be important in the physiological function of these cells. Over the past few years the biological relevance of micro- and nano-vesicles released by cells in intercellular communication has been established. Alongside the conventional mediators of cell secretome, these sophisticated nanovesicles transfer proteins, lipids and, most importantly, various forms of RNAs to neighboring cells, thereby mediating a variety of biological responses. The physiological role of MSC-derived vesicles (MSC-MVs) is currently not well understood. Nevertheless, encouraging results indicate that MSC-MVs have similar protective and reparative properties as their cellular counterparts in tissue repair and possibly anti-cancer therapy. Thus, MSC-MVs represent a promising opportunity to develop novel cell-free therapy approaches that might overcome the obstacles and risks associated with the use of native or engineered stem cells.

A regenerative approach for bone repair in congenital pseudarthrosis of the tibia associated or not associated with type 1 neurofibromatosis: correlation between laboratory findings and clinical outcome.

BACKGROUND AND AIMS: Congenital pseudarthrosis of the tibia (CPT) is a rare orthopedic disease presenting spontaneous fractures that do not heal. The treatment of CPT is characterized by repeated surgical procedures that often fail, with the inevitable outcome of severe disability and amputation. We tested the hypothesis that CPT may benefit from regenerative strategies based on mesenchymal stromal cells (MSC) combined with platelet-rich fibrin (PRF) as a source of growth factors. The aim of the study was to verify whether laboratory testing to assess the osteogenic properties of MSC and the osteo-inductive activity of PRF correlated with the clinical outcome. METHODS: Ten patients affected by refractory CPT were treated by using MSC derived from the iliac crest (IC-MSC), PRF and lyophilized bone. In six patients, CPT was associated with type 1 neurofibromatosis (NF1). Biochemical, functional and molecular assays were performed to assess the intrinsic osteogenic potential of IC-MSC (cells cultured with fetal calf serum) and the osteo-inductive properties of PRF (cells cultured with autologous serum). RESULTS: Bone consolidation was obtained in three patients who had CPT and NF1. In these patients, the IC-MSC exposed to autologous serum were able to form mineral nodules in vitro, while the mineralizing ability was totally abrogated in patients with a poor clinical outcome. CONCLUSIONS: Cell therapy may be a useful tool for the treatment of refractory CPT because it increases the opportunity to achieve effective bone tissue regeneration. Our data suggest that the presence of pro-osteogenic growth factors is an essential requirement for bone healing.

Neuroblastoma and bone metastases: clinical significance and prognostic value of Dickkopf 1 plasma levels.

The critical role of the Wnt pathway inhibition in sustaining the onset of bone lesions has been demonstrated in a variety of bone diseases and tumors, and it has been associated with cancer aggressiveness. We have previously demonstrated that neuroblastoma cells express Dickkopf 1 (Dkk1), an inhibitor of the canonical Wnt pathway which prevents the differentiation of bone-forming cells. Since Dkk1 is a secreted factor, it could have potential clinical application as tumor marker for detecting bone metastasis and monitoring of disease. In this study, we investigated the diagnostic and prognostic value of Dkk1 plasma levels in 92 children affected by neuroblastoma, including 32 with bone metastases. Fifty-seven children hospitalized for minor surgical problems served as control group. Circulating levels of Dkk1 were higher in healthy children than in normal adults and were comparable to those found in adult patients with aggressive tumors. No significant differences were found between neuroblastoma patients and controls and between patients with and without bone metastases. However, when only patients with metastatic neuroblastoma were considered, the highest Dkk1 levels were detected in patients that poorly responded to induction chemotherapy and in subjects with unamplified MYCN and three or more different metastatic sites. The 'Receiver Operating Characteristic' curve enabled us to identify a threshold value to distinguish patients who were unresponsive to induction treatment. The relationship between Dkk1 and drug resistance was supported by in vitro experiments, since an increased sensitivity to doxorubicin was found in neuroblastoma cells releasing low Dkk1 levels, either constitutively or experimentally following the treatment with specific siRNA. In conclusion, Dkk1 is released by neuroblastoma cells and is able to affect the balance between osteoblastogenesis and osteoclastogenesis, thus favoring the onset of osteolytic metastases. Nevertheless, Dkk1 plasma levels do not allow the detection of bone lesions in neuroblastoma but seem to have a predictive value with regard to the severity and the prognosis of the disease in a subset of patients with metastatic tumor. New knowledge on the biological role of Dkk1 in driving the natural history of neuroblastoma has to be further investigated and could help to establish specific therapeutic strategies able to target key factors of tumor progression.

Recent highlights on bone stem cells: a report from Bone Stem Cells 2009, and not only .

The use of stem cells has opened new prospects for the treatment of orthopaedic conditions characterized by large bone defects. However, many issues still exist to which answers are needed before routine, large-scale application becomes possible. Bone marrow stromal cells (MSC), which are clonogenic, multipotential precursors present in the bone marrow stroma, are generally employed for bone regeneration. Stem cells with multilineage differentiation similar to MSC have also been demonstrated in adipose tissue, peripheral blood, umbilical cord and amniotic fluid. Each source presents its own advantages and drawbacks. Unfortunately, no unique surface antigen is expressed by MSC, and this hampers simple MSC enrichment from heterogeneous populations. MSC are identified through a combination of physical, morphological and functional assays. Different in vitro and in vivo models have been described for the research on bone stem cells. These models should predict the in vivo bone healing capacity of MSC and if the induced osteogenesis is similar to the physiological one. Although stem cells offer an exciting possibility of a renewable source of cells and tissues for replacement, orthopaedic applications often represent case reports whereas controlled randomized trials are still lacking. Further biological aspects of bone stem cells should be elucidated and a general consensus on the best models, protocols and proper use of scaffolds and growth factors should be achieved.

Biological basis for the use of autologous bone marrow stromal cells in the treatment of congenital pseudarthrosis of the tibia.

The study was designed to establish the biological basis for the use of autologous bone-marrow stromal cells (MSC) in order to improve the curing opportunities of congenital pseudarthrosis of the tibia (CPT). The investigation was planned by taking into account that the pathophysiology of bone healing mainly depends on the osteogenic potential of the resident cells, although several factors play a crucial role in restoring the normal bone structure. Bone marrow samples were collected from the lesion site (P) and the iliac crest (IC) of 7 patients affected by CPT and type 1 neurofibromatosis (NF1+) and 6 patients affected by CPT without NF1 (NF1-). Four patients without CPT served as control group. Biochemical, functional and molecular assays showed that the ability to generate bone-forming cells was higher in IC-MSC than in P-MSC, but lower in CPT patients than in control group. We evaluated whether host factors, such as autologous serum and the microenvironment surrounding the pseudarthrosis lesion, could impair the osteogenic differentiation of IC-MSC. Autologous serum was less effective than FBS in promoting the IC-MSC differentiation, but the damage was more evident in NF1- than in NF1+ patients. Additionally, the supernatant of osteoblast cultures obtained from bone fragments close to the lesion site favoured the differentiation of IC-MSC in NF1- patients. In summary, our results suggest that MSC transplantation could be a promising strategy for the therapy of CPT. Further studies are warranted to confirm the clinical effectiveness in comparison to standard surgical treatment.

Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.

Osteogenic properties of late adherent subpopulations of human bone marrow stromal cells.

The nonadherent (NA) population of bone-marrow-derived mononuclear cells (MNC) has been demonstrated to be a source of osteogenic precursors in addition to the plastic-adherent mesenchymal stromal cells (MSC). In the current study, two subpopulations of late adherent (LA) osteoprogenitors were obtained by subsequent replating of NA cells, and their phenotypic, functional, and molecular properties were compared with those of early adherent (EA) MSC. Approximately 35% of MNC were LA cells, and they acquired a homogeneous expression of MSC antigens later than EA cells. In EA-MSC, the alkaline phosphatase (ALP) activity increased significantly from time of seeding to the first confluence, whereas in LA cells it raised later, after the addition of mineralization medium. All subpopulations were able to produce type I collagen and to deposit extracellular matrix with organized collagen fibrils. The proportion of large colonies with more than 50% of ALP positive cells as well as the calcium content was higher in LA than in EA cells. Molecular analysis highlighted the upregulation of bone-related genes in LA-MSC, especially after the addition of mineralization medium. Our results confirm that bone marrow contains LA osteoprogenitors which exhibit a delay in the differentiation process, despite an osteogenic potential similar to or better than EA-MSC. LA cells represent a reservoir of osteoprogenitors to be recruited to gain an adequate bone tissue repair and regeneration when a depletion of the most differentiated component occurs. Bone tissue engineering and cell therapy strategies could take advantage of LA cells, since an adequate amount of osteogenic MSCs may be obtained while avoiding bone marrow manipulation and cell culture expansion.

Paracrine inhibition of osteoblast differentiation induced by neuroblastoma cells.

The aim of our study was to investigate whether the defective function of osteogenic cells induced by neuroblastoma might play a role in the development of skeletal metastases. This mechanism has been extensively demonstrated for multiple myeloma, in which the blockage of osteoblast differentiation has been ascribed to the inhibitors of canonical Wingless pathway (Wnt), namely Dickkopf 1 (Dkk1). Our purpose was to verify if neuroblastoma cells derived from bone marrow metastases (SH-SY5Y, LAN1) or primaries (NB100, CHP212) hamper the differentiation of mesenchymal stem cells (hMSCs) into osteoblasts in a paracrine manner, and to test whether this ability depends on Dkk1 activity. We found that all neuroblastoma cells increased the proliferation of hMSCs collected from pediatric-aged donors, with a corresponding decrease in osteoblast differentiation markers, including alkaline phosphatase (ALP), analyzed as gene expression, enzymatic activity and number of ALP-positive colony forming units, osteoprotegerin (OPG) release, OPG and osteocalcin gene-expression. Dkk1 mRNA and protein were detectable in all cell lines, and the use of neutralizing anti-Dkk1 antibody reversed the effects induced by SH-SY5Y cells. Taken together, our results confirm that neuroblastoma hinders osteoblastogenesis, and that Dkk1 release seems to play a crucial role in blocking the differentiation of osteoprogenitor cells, though the ability to promote osteoclast activation remains an essential requirement for the development of skeletal metastases. Finally, our findings suggest that strategies regulating Wnt signaling and Dkk1 activity could be considered for adjuvant therapies in neuroblastoma metastasizing to the skeleton.