MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

BACKGROUND: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. METHODS: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). RESULTS: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. CONCLUSION: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.

The Effect of Belantamab Mafodotin on Primary Myeloma-Stroma Co-Cultures: Asymmetrical Mitochondrial Transfer between Myeloma Cells and Autologous Bone Marrow Stromal Cells.

Belantamab mafodotin (belamaf) is an afucosylated monoclonal antibody conjugated to the microtubule disrupter monomethyl auristatin-F (MMAF) that targets B cell maturation antigen (BCMA) on the surface of malignant plasma cells. Belamaf can eliminate myeloma cells (MMs) through several mechanisms. On the one hand, in addition to inhibiting BCMA-receptor signaling and cell survival, intracellularly released MMAF disrupts tubulin polymerization and causes cell cycle arrest. On the other hand, belamaf induces effector cell-mediated tumor cell lysis via antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In our in vitro co-culture model, the consequences of the first mentioned mechanism can be investigated: belamaf binds to BCMA, reduces the proliferation and survival of MMs, and then enters the lysosomes of malignant cells, where MMAF is released. The MMAF payload causes a cell cycle arrest at the DNA damage checkpoint between the G2 and M phases, resulting in caspase-3-dependent apoptosis. Here, we show that primary MMs isolated from different patients can vary widely in terms of BCMA expression level, and inadequate expression is associated with extremely high resistance to belamaf according to our cytotoxicity assay. We also reveal that primary MMs respond to increasing concentrations of belamaf by enhancing the incorporation of mitochondria from autologous bone marrow stromal cells (BM-MSCs), and as a consequence, MMs become more resistant to belamaf in this way, which is similar to other medications we have analyzed previously in this regard, such as proteasome inhibitor carfilzomib or the BCL-2 inhibitor venetoclax. The remarkable resistance against belamaf observed in the case of certain primary myeloma cell cultures is a cause for concern and points towards the use of combination therapies to overcome the risk of antigen escape.

Osteoblastic differentiation of bone marrow mesenchymal stromal cells in Bruck Syndrome.

BACKGROUND: Osteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus. METHODS: We analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. RESULTS: No mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different. CONCLUSIONS: Our work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro.

Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects.

Immune response treated with bone marrow mesenchymal stromal cells after stroke.

Stroke is a leading cause of death and long-term disability worldwide. Tissue plasminogen activator (tPA) is an effective treatment for ischemic stroke. However, only a small part of patients could benefit from it. Therefore, finding a new treatment is necessary. Bone marrow mesenchymal stromal cells (BMSCs) provide a novel strategy for stroke patients. Now, many patients take stem cells to treat stroke. However, the researches of the precise inflammatory mechanism of cell replacement treatment are still rare. In this review, we summarize the immune response of BMSCs treated to stroke and may provide a new perspective for stem cell therapy.

Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties.

PURPOSE: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems. METHOD: Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for ß3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites. RESULTS: The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (ß3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 µm vs. 1860.5 µm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05). CONCLUSION: This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

PTEN-induced kinase 1 enhances the reparative effects of bone marrow mesenchymal stromal cells on mice with renal ischaemia/reperfusion-induced acute kidney injury.

Acute kidney injury (AKI) is a common severe acute syndrome caused by multiple factors and is characterized by a rapid decline in renal function during a short period. Bone marrow mesenchymal stromal cells (BMSCs) are effective in treating AKI. However, the mechanism of their beneficial effects remains unclear. PTEN-induced kinase 1 (PINK1) may play an important role in kidney tissue repair. In this study, we explored the effect of PINK1 overexpression on enhancing BMSC-mediated repair of AKI. In this study, ischaemia/reperfusion-induced AKI (IRI-AKI) in mice and a hypoxia-reoxygenation model in cells were established, and the indices were examined by pathology and immunology experiments. After ischaemia/reperfusion, PINK1 overexpression reduced apoptosis in injured kidney tissue cell, decreased T lymphocyte infiltration, increased macrophage infiltration, and alleviated the inflammatory response. PINK1 relieved the stress response of BMSCs and renal tubular epithelial cells (RTECs), reduced apoptosis, altered the release of inflammatory factors, and reduced the proliferation of peripheral blood mononuclear cells (PBMCs). In conclusion, BMSCs and RTECs undergo stress responses in response to hypoxia, inflammation and other conditions, and overexpressing PINK1 in BMSCs could enhance their ability to resist these stress reactions. Furthermore, PINK1 overexpression can regulate the distribution of immune cells and improve the inflammatory response. The regulation of mitochondrial autophagy during IRI-AKI maintains mitochondrial homeostasis and protects renal function. The results of this study provide new strategies and experimental evidence for BMSC-mediated repair of IRI-AKI.

The extracellular matrix of human bone marrow adipocytes and glucose concentration differentially alter mineralization quality without impairing osteoblastogenesis.

Bone marrow adipocytes (BMAds) accrue in various states of osteoporosis and interfere with bone remodeling through the secretion of various factors. However, involvement of the extracellular matrix (ECM) produced by BMAds in the impairment of bone marrow mesenchymal stromal cell (BM-MSC) osteoblastogenesis has received little attention. In type 2 diabetes (T2D), skeletal fragility is associated with several changes in bone quality that are incompletely understood, and BMAd quantity increases in relationship to poor glycemic control. Considering their altered phenotype in this pathophysiological context, we aimed to determine the contribution of the ECM of mature BMAds to osteoblastogenesis and mineralization quality in the context of chronic hyperglycemia. Human BM-MSCs were differentiated for 21 days in adipogenic medium containing either a normoglycemic (LG, 5.5 mM) or a high glucose concentration (HG, 25 mM). The ECM laid down by BMAds were devitalized through cell removal to examine their impact on the proliferation and differentiation of BM-MSCs toward osteoblastogenesis in LG and HG conditions. Compared to control plates, both adipocyte ECMs promoted cell adhesion and proliferation. As shown by the unmodified RUNX2 and osteocalcin mRNA levels, BM-MSC commitment in osteoblastogenesis was hampered by neither the hyperglycemic condition nor the adipocyte matrices. However, adipocyte ECMs or HG condition altered the mineralization phase with perturbed expression levels of type 1 collagen, MGP and osteopontin. Despite higher ALP activity, mineralization levels per cell were decreased for osteoblasts grown on adipocyte ECMs compared to controls. Raman spectrometry revealed that culturing on adipocyte matrices specifically prevents type-B carbonate substitution and favors collagen crosslinking, in contrast to exposure to HG concentration alone. Moreover, the mineral to organic ratio was disrupted according to the presence of adipocyte ECM and the glucose concentration used for adipocyte or osteoblast culture. HG concentration and adipocyte ECM lead to different defects in mineralization quality, recapitulating contradictory changes reported in T2D osteoporosis. Our study shows that ECMs from BMAds do not impair osteoblastogenesis but alter both the quantity and quality of mineralization partly in a glucose concentration-dependent manner. This finding sheds light on the involvement of BMAds, which should be considered in the compromised bone quality of T2D and osteoporosis patients more generally.

Stromal Cells Serve Drug Resistance for Multiple Myeloma via Mitochondrial Transfer: A Study on Primary Myeloma and Stromal Cells.

Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation.

LRP5-Mediated Lipid Uptake Modulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells.

Nutritional microenvironment determines the specification of progenitor cells, and lipid availability was found to modulate osteogenesis in skeletal progenitors. Here, we investigated the implications of lipid scarcity in the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and the role of low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor transducing canonical Wnt/beta-catenin signals, in BMSC lipid uptake during osteogenesis. The osteogenic differentiation of murine BMSCs was suppressed by lipid scarcity and partially rescued by additional fatty acid treatment with oleate. The enhancement of osteogenesis by oleate was found to be dosage-dependent, along with the enhanced activation of beta-catenin and Wnt target genes. Conditional knockout (CKO) of Lrp5 gene in murine mesenchymal lineage using Lrp5 fl/fl ;Prrx1-cre mice led to decreased bone quality and altered fat distribution in vivo. After Lrp5 ablation using adenoviral Cre-recombinase, the accumulation of lipid droplets in BMSC cytoplasm was significantly reduced, and the osteogenesis of BMSCs was suppressed. Moreover, the impaired osteogenesis due to either lipid scarcity or Lrp5 ablation could be rescued by recombinant Wnt3a protein, indicating that the osteogenesis induced by Wnt/beta-catenin signaling was independent of LRP5-mediated lipid uptake. In conclusion, lipid scarcity suppresses BMSC osteogenic differentiation. LRP5 plays a role in the uptake of lipids in BMSCs and therefore mediates osteogenic specification.

Medium-Intensity Treadmill Exercise Exerts Beneficial Effects on Bone Modeling Through Bone Marrow Mesenchymal Stromal Cells.

As a type of multipotential cells, bone marrow mesenchymal stromal cells (BMMSCs) can differentiate into chondrocytes, osteoblasts, and adipocytes under different loading condition or specific microenvironment. Previous studies have shown that BMMSCs and their lineage-differentiated progeny (for example, osteoblasts), and osteocytes are mechanosensitive in bone. The appropriate physical activity and exercise could help attenuate bone loss, effectively stimulate bone formation, increase bone mineral density (BMD), prevent the progression of osteoporosis, and reduce the risk of bone fractures. Bone morphogenetic protein (BMP) is originally discovered as a protein with heterotopic bone-inducing activity in the bone matrix that exerts a critical role in multiple stages of bone metabolism. In the present study, the medium-intensity treadmill exercise enhanced bone formation and increased osteocalcin (OCN) and osteopontin (OPN) mRNA expression as well as activation of the BMP-Smad signaling pathway in vivo. In order to investigate the effect of a BMP-Smad signaling pathway, we injected mice with activated enzyme inhibitors (LDN-193189HCL) and subjected the mice to treadmill exercise intervention. LDN-193189HCL attenuated the BMD and bone mass mediated by medium-intensity exercise and BMP-Smad signaling pathway.

Close interaction with bone marrow mesenchymal stromal cells induces the development of cancer stem cell-like immunophenotype in B cell precursor acute lymphoblastic leukemia cells.

Minimal residual disease of leukemia may reside in the bone marrow (BM) microenvironment and escape the effects of chemotherapeutic agents. This study investigated interactions between B cell precursor (BCP)-acute lymphoblastic leukemia (ALL) cells and BM mesenchymal stromal cells (BM-MSCs) in vitro. Five BCP-ALL cell lines established from pediatric patients and primary samples from a BCP-ALL patient were examined by flow cytometry and immunocytochemistry for expression of specific cell surface markers and cell adhesion proteins. The cell lines developed chemoresistance to commonly used anti-leukemic agents through adhesion to MSC-TERT cells in long-term culture. The change in chemosensitivity after adhering to BM-MSCs was associated with the expression of CD34, CD133, P-glycoprotein and BCRP/ABCG2, and downregulation of CD38. Similar phenotypic changes were observed in primary samples obtained by marrow aspiration or biopsy from a BCP-ALL patient. BM-MSC-adhering leukemia cells also showed deceleration of cell proliferation and expressed proteins in the Cadherin and Integrin pathways. These results suggest that BCP-ALL cells residing in the BM microenvironment may acquire chemoresistance by altering their phenotype to resemble that of cancer stem cells. Our results indicate that cell adhesion could be potentially targeted to improve the chemosensitivity of residual BCP-ALL cells in the BM microenvironment.

Defining osteoblast and adipocyte lineages in the bone marrow.

Bone is a complex endocrine organ that facilitates structural support, protection to vital organs, sites for hematopoiesis, and calcium homeostasis. The bone marrow microenvironment is a heterogeneous niche consisting of multipotent musculoskeletal and hematopoietic progenitors and their derivative terminal cell types. Amongst these progenitors, bone marrow mesenchymal stem/stromal cells (BMSCs) may differentiate into osteogenic, adipogenic, myogenic, and chondrogenic lineages to support musculoskeletal development as well as tissue homeostasis, regeneration and repair during adulthood. With age, the commitment of BMSCs to osteogenesis slows, bone formation decreases, fracture risk rises, and marrow adiposity increases. An unresolved question is whether osteogenesis and adipogenesis are co-regulated in the bone marrow. Osteogenesis and adipogenesis are controlled by specific signaling mechanisms, circulating cytokines, and transcription factors such as Runx2 and Pparγ, respectively. One hypothesis is that adipogenesis is the default pathway if osteogenic stimuli are absent. However, recent work revealed that Runx2 and Osx1-expressing preosteoblasts form lipid droplets under pathological and aging conditions. Histone deacetylase 3 (Hdac3) and other epigenetic regulators suppress lipid storage in preosteoblasts and/or control marrow adiposity. Establishing a better understanding of fat storage in bone marrow cells, as well as the osteoblast-adipocyte relationship within the bone marrow niche is necessary to understand the mechanisms underlying disease- and aging-related marrow fat storage and may lead to the development of new therapeutic targets for "fatty bone" and osteoporosis.

miR-34a promotes bone regeneration in irradiated bone defects by enhancing osteoblastic differentiation of mesenchymal stromal cells in rats.

BACKGROUND: Radiation exposure negatively affects the regenerative ability and makes reconstruction of bone defects after tumor section difficult. miR-34a is involved in radiation biology and bone metabolism. The aim of this study was to investigate whether miR-34a could contribute to bone regeneration in irradiated bone defects. METHODS: The expression of miR-34a was analyzed during the osteoblastic differentiation of irradiated BMSCs and bone formation in irradiated bone defects. miR-34a mimics and miR-34a inhibitor were used to upregulate or suppress the expression of miR-34a in BMSCs irradiated with 2 or 4 Gy X-ray radiation. In vitro osteogenesis and subcutaneous osteogenesis were used to assess the effects of miR-34a on the osteogenic ability of radiation-impaired BMSCs. Collagen-based hydrogel containing agomiR-34a or antagomiR-34a were placed into the 3-mm defects of irradiated rat tibias to test the effect of miR-34a on bone defect healing after irradiation. RESULTS: miR-34a was upregulated in the process of bone formation after irradiation. Transfecting radiation-impaired BMSCs with miR-34a mimics enhanced their osteoblastic differentiation in vitro by targeting NOTCH1. Overexpression of miR-34a enhanced the ectopic bone formation of irradiated BMSCs. In situ delivery of miR-34a promoted bone regeneration in irradiated bone defects. CONCLUSIONS: miR-34a promoted the osteoblastic differentiation of BMSCs and enhanced the ectopic bone formation after irradiation. miR-34a promoted bone defect healing in irradiated rat tibias. miR-34a-targeted therapy might be a promising strategy for promoting the reconstruction of bone defects after radiotherapy.

MicroRNA-26b-5p Inhibits Mouse Liver Fibrogenesis and Angiogenesis by Targeting PDGF Receptor-Beta.

Here microRNAs (miRNAs) with potentially therapeutic effects were screened and explored during liver fibrogenesis and angiogenesis via targeting the important mediators. Chimera mice with EGFP+ bone marrow mesenchymal stromal cells (BMSCs) were fed with methionine-choline-deficient and high-fat (MCDHF) diet to induce liver injury. Increased expression of platelet-derived growth factor receptor-beta (PDGFR-ß) was detected in MCDHF mice, with a positive correlation to fibrosis and angiogenesis markers. BMSCs contributed to the significant proportion of PDGFR-ß+ cells in the fibrotic liver. MicroRNA-26b-5p (miR-26b-5p) was predicted to target PDGFR-ß from three databases. The hepatic expression of miR-26b-5p was decreased in the fibrotic liver, with a negative correlation to PDGFR-ß and fibrosis and angiogenesis markers. miR-26b-5p directly targeted PDGFR-ß in TGF-ß1-treated BMSCs by pull-down and lucifer reporter assays, which can be sponged by long non-coding RNA (lncRNA) maternally expressed gene 3 (lncMEG3). Microarray analysis revealed that miR-26b-5p overexpression affected a list of genes associated with fibrosis and angiogenesis. In vivo miR-26b-5p negatively regulated PDGFR-ß expression and attenuated liver fibrosis and angiogenesis. Together, miR-26b-5p inhibits liver fibrogenesis and angiogenesis via directly targeting PDGFR-ß and interacting with lncMEG3, which may represent an effective therapeutic strategy for liver fibrosis.

Influence of the three-dimensional culture of human bone marrow mesenchymal stromal cells within a macroporous polysaccharides scaffold on Pannexin 1 and Pannexin 3.

Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγnull ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.