TAZ DOWNREGULATES ANXA1 EXPRESSION TO MODULATE MYELOMA CELL INTERACTION WITH BONE MARROW MESENCHYMAL STROMAL CELLS.

We and others have previously shown that TAZ plays a tumor suppressive role in multiple myeloma. However, recent reports suggest that molecular crosstalk between the myeloma cells and bone marrow stromal components contributes to the myeloma cell survival and drug resistance. These reports further point to reciprocal interaction via adhesion molecules as the most prominent mechanism of intercellular crosstalk between myeloma cells and BM-MSCs. YAP/TAZ silencing/expression has been shown to correlate across all cancers with a set of adhesion/extracellular matrix proteins. Therefore, we hypothesized that TAZ may regulate myeloma cell interaction with BM stromal cells by influencing the expression of distinct cell adhesion signatures. We used previously established TAZ myeloma cell line models: DELTA47-pLENTI or TAZ knockout DELTA47 cells cocultured with or without BM-MSCs as our study models. Using RNA sequencing analysis, we performed the first comprehensive screen for cell adhesion-related transcriptional targets of TAZ in MM. In doing so, we uncovered an enrichment of cell adhesion-related genes in TAZ knockout DELTA47 cells relatively to pLENTI-DELTA47 cells, including 11 genes with log2 fold change>2 (P<0.05): ANXA1, ADGRL2, NCAM1, NCAM2, ADGRL3, CXADR, ALCAM, JAM2, KIRREL1, KIRREL2, and ADGRG7, suggesting possible relationship with TAZ. We validated ANXA1 as a bona fide target of TAZ in MM. We show that TAZ represses myeloma cell migration and interaction with BM-MSCs by transcriptionally downregulating ANXA1 expression via TEAD-dependent mechanism. Our data provide new insights into the understanding of the role of TAZ in the intercellular communication signals between myeloma cells and BM-MSCs. Our findings also suggest that ANXA1 represents a putative cell adhesion target to attenuate BM-MSC driven, tumour-promoting interaction with myeloma cells.

The effect of bone marrow mesenchymal stromal cell exosomes on acute myeloid leukemia's biological functions: a focus on the potential role of LncRNAs.

Acute myeloid leukemia represents a group of malignant blood disorders that originate from clonal over-proliferation and the differentiation failure of hematopoietic precursors, resulting in the accumulation of blasts in the bone marrow. Mesenchymal stromal cells (MSCs) have been shown to exert diverse effects on tumor cells through direct and indirect interaction. Exosomes, as one of the means of indirect intercellular communication, are released from different types of cells, including MSCs, and their various contents, such as lncRNAs, enable them to exert significant impacts on target cells. Our study aims to investigate the effects of BM-MSC exosomes on the cellular and molecular characterization of HL-60 AML cells, particularly detecting the alterations in the expression of lncRNAs involved in AML leukemogenesis, cell growth, drug resistance, and poor prognosis. BM-MSCs were cultured with serum-free culture media to isolate exosomes from their supernatants. The validation of exosomes was performed in three stages: morphological analysis using TEM, size evaluation using DLS, and CD marker identification using flow cytometry. Subsequently, the HL-60 AML cells were treated with isolated BM-MSC exosomes to determine the impact of their contents on leukemic cells. Cell metabolic activity was evaluated by the MTT assay, while cell cycle progression, apoptosis, ROS levels, and proliferation were assessed by flow cytometry. Furthermore, RT-qPCR was conducted to determine the expression levels of lncRNAs and apoptosis-, ROS-, and cell cycle-related genes. MTT assay and flow cytometry analysis revealed that BM-MSC exosomes considerably suppressed cell metabolic activity, proliferation, and cell cycle progression. Also, these exosomes could effectively increase apoptosis and ROS levels in HL-60 cells. The expression levels of p53, p21, BAX, and FOXO4 were increased, while the BCL2 and c-Myc levels decreased. MALAT1, HOTAIR, and H19 expression levels were also significantly decreased in treated HL-60 cells compared to their untreated counterparts. BM-MSC exosomes suppress cell cycle progression, proliferation, and metabolic activity while simultaneously elevating the ROS index and apoptosis ratio in HL-60 cells, likely by reducing the expression levels of MALAT1, HOTAIR, and H19. These findings suggest that BM-MSC exosomes might serve as potential supportive therapies for leukemia.

Astragalus polysaccharides augment BMSC homing via SDF-1/CXCR4 modulation: a novel approach to counteract peritoneal mesenchymal transformation and fibrosis.

PURPOSE: This study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms. METHODS: Forty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts. RESULTS: In vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis's downstream pathways, and partially reversed the AMD3100-induced effects. CONCLUSION: APS augments the SDF-1/CXCR4 axis's downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.

O-linked ß-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells.

Cumulative evidence suggests that O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) plays an important regulatory role in pathophysiological processes. Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated, the potential mechanisms of O-GlcNAcylation in bone metabolism, particularly, in the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) remains unexplored. In this study, the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed, assuming that it could trigger more scholars to focus on research related to O-GlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

[Chidamide Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells from Patients with Myelodysplastic Syndromes].

OBJECTIVE: To explore the effects and mechanisms of chidamide on the osteogenic differentiation of bone marrow mesenchymal stromal cells (MSC) from myelodysplastic syndromes (MDS). METHODS: MSC were isolated and cultured from bone marrow of MDS patients and healthy donors. CCK-8 assay was used to detect the effects of chidamide on the proliferation of MSC. The effects of chidamide on the activity of histone deacetylase (HDAC) in MSC was measured by a fluorescence assay kit and Western blot. Alkaline phosphatase (ALP) activity was detected on day 3 and calcium nodule formation was observed by Alizarin Red staining on day 21 after osteogenic differentiation. The expression of early and late osteogenic genes was detected on day 7 and day 21, respectively. RT-PCR and Western blot were used to detect the effects of chidamide on mRNA and protein expression of RUNX2 which is the key transcription factor during osteogenesis. RESULTS: As the concentration of chidamide increased, the proliferation of MSC was inhibited. However, at a low concentration (1 µmol/L), chidamide had no significant inhibitory effect on MSC proliferation but significantly inhibited HDAC activity. In MSC from both MDS patients and healthy donors, chidamide (1 µmol/L) significantly increased ALP activity, calcium nodule formation, thereby mRNA expression of osteogenic genes, and restored the reduced osteogenic differentiation ability of MDS-MSC compared to normal MSC. Mechanistic studies showed that the osteogenic-promoting effect of chidamide may be related to the upregulation of RUNX2 . CONCLUSION: Chidamide can inhibit HDAC activity in MSC, upregulate the expression of the osteogenic transcription factor RUNX2, and promote the osteogenic differentiation of MDS-MSC.

Functionalization of Ceramic Scaffolds with Exosomes from Bone Marrow Mesenchymal Stromal Cells for Bone Tissue Engineering.

The functionalization of bone substitutes with exosomes appears to be a promising technique to enhance bone tissue formation. This study investigates the potential of exosomes derived from bone marrow mesenchymal stromal cells (BMSCs) to improve bone healing and bone augmentation when incorporated into wide open-porous 3D-printed ceramic Gyroid scaffolds. We demonstrated the multipotent characteristics of BMSCs and characterized the extracted exosomes using nanoparticle tracking analysis and proteomic profiling. Through cell culture experimentation, we demonstrated that BMSC-derived exosomes possess the ability to attract cells and significantly facilitate their differentiation into the osteogenic lineage. Furthermore, we observed that scaffold architecture influences exosome release kinetics, with Gyroid scaffolds exhibiting slower release rates compared to Lattice scaffolds. Nevertheless, in vivo implantation did not show increased bone ingrowth in scaffolds loaded with exosomes, suggesting that the scaffold microarchitecture and material were already optimized for osteoconduction and bone augmentation. These findings highlight the lack of understanding about the optimal delivery of exosomes for osteoconduction and bone augmentation by advanced ceramic scaffolds.

Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells.

BACKGROUND: Bone marrow mesenchymal stromal cells (BMSCs) are the commonly used seed cells in tissue engineering. Aryl hydrocarbon receptor (AhR) is a transcription factor involved in various cellular processes. However, the function of constitutive AhR in BMSCs remains unclear. AIM: To investigate the role of AhR in the osteogenic and macrophage-modulating potential of mouse BMSCs (mBMSCs) and the underlying mechanism. METHODS: Immunochemistry and immunofluorescent staining were used to observe the expression of AhR in mouse bone marrow tissue and mBMSCs. The overexpression or knockdown of AhR was achieved by lentivirus-mediated plasmid. The osteogenic potential was observed by alkaline phosphatase and alizarin red staining. The mRNA and protein levels of osteogenic markers were detected by quantitative polymerase chain reaction (qPCR) and western blot. After coculture with different mBMSCs, the cluster of differentiation (CD) 86 and CD206 expressions levels in RAW 264.7 cells were analyzed by flow cytometry. To explore the underlying molecular mechanism, the interaction of AhR with signal transducer and activator of transcription 3 (STAT3) was observed by co-immunoprecipitation and phosphorylation of STAT3 was detected by western blot. RESULTS: AhR expressions in mouse bone marrow tissue and isolated mBMSCs were detected. AhR overexpression enhanced the osteogenic potential of mBMSCs while AhR knockdown suppressed it. The ratio of CD86+ RAW 264.7 cells cocultured with AhR-overexpressed mBMSCs was reduced and that of CD206+ cells was increased. AhR directly interacted with STAT3. AhR overexpression increased the phosphorylation of STAT3. After inhibition of STAT3 via stattic, the promotive effects of AhR overexpression on the osteogenic differentiation and macrophage-modulating were partially counteracted. CONCLUSION: AhR plays a beneficial role in the regenerative potential of mBMSCs partially by increasing phosphorylation of STAT3.

Macromolecular crowding in equine bone marrow mesenchymal stromal cell cultures using single and double hyaluronic acid macromolecules.

Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition in eukaryotic cell culture. Single hyaluronic acid (HA) molecules have not induced a notable increase in the amount and rate of deposited ECM. Thus, herein we assessed the physicochemical properties and biological consequences in equine bone marrow mesenchymal stromal cell cultures of single and mixed HA molecules and correlated them to the most widely used MMC agents, the FicollⓇ cocktail (FC) and carrageenan (CR). Dynamic light scattering analysis revealed that all HA cocktails had significantly higher hydrodynamic radius than the FC and CR; the FC and the 0.5 mg/ml 100 kDa and 500 kDa single HA molecules had the highest charge; and, in general, all molecules had high polydispersity index. Biological analyses revealed that none of the MMC agents affected cell morphology and basic cell functions; in general, CR outperformed all other macromolecules in collagen type I and V deposition; FC, the individual HA molecules and the HA cocktails outperformed CR in collagen type III deposition; FC outperformed CR and the individual HA molecules and the HA cocktails outperformed their constituent HA molecules in collagen type IV deposition; FC and certain HA cocktails outperformed CR and constituent HA molecules in collagen type VI deposition; and all individual HA molecules outperformed FC and CR and the HA cocktails outperformed their constituent HA molecules in laminin deposition. With respect to tri-lineage analysis, CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. This work opens new avenues in mixed MMC in eukaryotic cell culture. STATEMENT OF SIGNIFICANCE: Mixed macromolecular crowding (MMC) in eukaryotic cell culture is still under-investigated. Herein, single and double hyaluronic acid (HA) macromolecules, along with the traditional MMC agents FicollⓇ cocktail (FC) and carrageenan (CR), were used as MMC agents in equine mesenchymal stromal cell cultures. Biological analysis showed that none of the MMC agents affected cell morphology and basic cell functions. Protein deposition analysis made apparent that CR outperformed all other macromolecules in collagen type I and collagen type V deposition, whilst FC, the individual HA macromolecules and the HA cocktails outperformed CR in collagen type III deposition. Tri-lineage analysis revealed that CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. These data illustrate that MMC agents are not inert macromolecules.

ASPN Synergizes with HAPLN1 to Inhibit the Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells and Extracellular Matrix Mineralization of Osteoblasts.

OBJECTIVE: Bone marrow mesenchymal stromal cells (BMSCs) are major sources of osteogenic precursor cells in bone remodeling, which directly participate in osteoporosis (OP) progression. However, the involved specific mechanisms of BMSCs in OP warrant mass investigations. Initially, our bioinformatics analysis uncovered the prominent up-regulation of Asporin (ASPN) and proteoglycan link protein 1 (HAPLN1) in osteoblasts (OBs) of OP patients and their possible protein interaction. Hence, this study aimed to explore the effects of ASPN and HAPLN1 on osteogenic differentiation of BMSCs, extracellular matrix (ECM) mineralization of OBs, and osteoclastogenesis, hoping to offer research basis for OP treatment. METHODS: GSE156508 dataset was used for analysis and screening to acquire the differentially expressed genes in OBs of OP patients, followed by the predicative analysis via STRING. OP mouse models were induced by ovariectomy (OVX), and ASPN and HAPLN1 expression was determined. BMSCs and bone marrow macrophages (BMMs) were isolated from OVX mice and induced for osteogenic differentiation and osteoclastogenesis, respectively. After knockdown experiments, we assessed adipogenic differentiation and osteogenic differentiation in BMSCs. Osteogenic (OPN, OCN, and COL1A1) and osteoclast (Nfatc1 and c-Fos) marker protein expression was determined. The binding of ASPN to HAPLN1 was analyzed. RESULTS: High expression of ASPN and HAPLN1 and their protein interaction were observed in OBs of OP patients via bioinformatics and in bone tissues of OVX mice. ASPN interacted with HAPLN1 in BMSCs of OVX mice. ASPN/HAPLN1 knockdown increased ALP, OPN, OCN, and COL1A1 protein expression and ECM mineralization in BMSCs while decreasing Nfatc1 and c-Fos expression in BMMs. These effects were aggravated by the simultaneous knockdown of ASPN and HAPLN1. CONCLUSION: Our results indicate that ASPN synergises with HAPLN1 to suppress the osteogenic differentiation of BMSCs and ECM mineralization of OBs and promote the osteoclastogenesis in OP.

Titanium surface interacting with blood clot enhanced migration and osteogenic differentiation of bone marrow mesenchymal stem cells.

Introduction: Blood clot formation is the initial phase upon implantation, and the feature of blood clot orchestrates the following complement system activation, coagulation cascade, and bone marrow mesenchymal stromal cells (BMSCs) recruitment. This study aimed to investigate the effect of implant surface on blood-material interactions and subsequent BMSC cellular behaviors. Methods: This study was established to imitate the physiological process of implantation in vivo and in vitro. Whole blood was incubated with polished titanium (PT) surfaces and sandblasted and double acid-etching (SLA) surfaces for 10 min or 2 h, then seeded with BMSCs. The adhesion, proliferation, migration, and differentiation of cells were studied at specific time points. Titanium implants were implanted into the tibia in vivo and were screwed out after implantation. The activation of the coagulation cascade, platelets, complement system, and clot networks were assessed and further quantitatively analyzed. Results: Compared with the PT surface, the SLA surface induced the earlier and stronger blood coagulation cascade and formed a more stratified clots network with fibrinogen, platelets, and CD14 positive cell. The adhesion, proliferation, and migration of BMSCs were enhanced by pre-incubated surfaces. The higher levels of the osteogenic-related genes, ALP activity, and calcium nodule formation were showed on SLA surfaces with blood incubation. Conclusion: SLA titanium surfaces play a role in influencing the formation of blood clots and coordinating surface-blood interactions and cell biological processes. These findings provide the idea of modifying the blood clots formed on the implant surface by biomaterials modification and thus has implications for the development of better osteogenic biomaterials.

Dexamethasone enhances venetoclax-induced apoptosis in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) therapies have been significantly improved by the development of medicines that can target BCL-2. On the other hand, non-recurrent alterations in oncogenic pathways and gene expression patterns have already been linked to therapeutic resistance to venetoclax therapy. Bone marrow mesenchymal stromal cells (BM-MSCs) support leukemic cells in preventing chemotherapy-induced apoptosis by mitochondrial transfer in leukemic microenvironment. In this study, we investigated the enhancement of the antitumor effect of BCL-2 inhibitor venetoclax by dexamethasone. In particular, dexamethasone had no significant effect on the viability of AML cells, but dexamethasone combined with venetoclax could significantly increase the apoptosis of AML cells induced by venetoclax. When AML cells were co-cultured with BM-MSCs, dexamethasone combined with venetoclax showed additional anti-tumor effect compared to venetoclax alone. Venetoclax increased reactive oxygen species level in co-cultured AML cells, contributed to transfer more mitochondria from BM-MSCs to AML cells and protect AML cells from apoptosis. Dexamethasone combined with venetoclax induced more apoptosis, but dexamethasone reduced the venetoclax-induced reactive oxygen species level in AML cells and reduced the transfer of mitochondria from BM-MSCs to AML cells. This may lead to a diminished protective effect of BM-MSCs on AML cells. Together, our findings indicated that venetoclax in combination with dexamethasone could be a promising therapy in AML.

Long non-coding RNAs in osteoporosis: from mechanisms of action to therapeutic potential.

Osteoporosis is a clinical disease characterized by decreased bone density due to a disrupted balance between bone formation and resorption, which increases fracture risk and negatively affects the quality of life of a patient. LncRNAs are RNA molecules over 200 nucleotides in length with non-coding potential. Many studies have demonstrated that numerous biological processes involved in bone metabolism are affected. However, the complex mechanisms of action of lncRNAs and their clinical applications in osteoporosis have not yet been fully elucidated. LncRNAs, as epigenetic regulators, are widely involved in the regulation of gene expression during osteogenic and osteoclast differentiation. LncRNAs affect bone homeostasis and osteoporosis development through different signaling pathways and regulatory networks. Additionally, researchers have found that lncRNAs have great potential for clinical application in the treatment of osteoporosis. In this review, we summarize the research results on lncRNAs for clinical prevention, rehabilitation treatment, drug development, and targeted therapy for osteoporosis. Moreover, we summarize the regulatory modes of various signaling pathways through which lncRNAs affect the development of osteoporosis. Overall, these studies suggest that lncRNAs can be used as novel targeted molecular drugs for the clinical treatment of osteoporosis to improve symptoms.

Human Bone Marrow-Derived Mesenchymal Stromal Cells Reduce the Severity of Experimental Necrotizing Enterocolitis in a Concentration-Dependent Manner.

Necrotizing enterocolitis (NEC) is a devastating gut disease in preterm neonates. In NEC animal models, mesenchymal stromal cells (MSCs) administration has reduced the incidence and severity of NEC. We developed and characterized a novel mouse model of NEC to evaluate the effect of human bone marrow-derived MSCs (hBM-MSCs) in tissue regeneration and epithelial gut repair. NEC was induced in C57BL/6 mouse pups at postnatal days (PND) 3-6 by (A) gavage feeding term infant formula, (B) hypoxia/hypothermia, and (C) lipopolysaccharide. Intraperitoneal injections of PBS or two hBM-MSCs doses (0.5 × 106 or 1 × 106) were given on PND2. At PND 6, we harvested intestine samples from all groups. The NEC group showed an incidence of NEC of 50% compared with controls (p < 0.001). Severity of bowel damage was reduced by hBM-MSCs compared to the PBS-treated NEC group in a concentration-dependent manner, with hBM-MSCs (1 × 106) inducing a NEC incidence reduction of up to 0% (p < 0.001). We showed that hBM-MSCs enhanced intestinal cell survival, preserving intestinal barrier integrity and decreasing mucosal inflammation and apoptosis. In conclusion, we established a novel NEC animal model and demonstrated that hBM-MSCs administration reduced the NEC incidence and severity in a concentration-dependent manner, enhancing intestinal barrier integrity.

SDF-1α-Releasing Microspheres Effectively Extend Stem Cell Homing after Myocardial Infarction.

Ischemic heart disease (IHD) is one of the main focuses in today's healthcare due to its implications and complications, and it is predicted to be increasing in prevalence due to the ageing population. Although the conventional pharmacological and interventional methods for the treatment of IHD presents with success in the clinical setting, the long-term complications of cardiac insufficiency are on a continual incline as a result of post-infarction remodeling of the cardiac tissue. The migration and involvement of stem cells to the cardiac muscle, followed by differentiation into cardiac myocytes, has been proven to be the natural process, though at a slow rate. SDF-1α is a novel candidate to mobilize stem cells homing to the ischemic heart. Endogenous SDF-1α levels are elevated after myocardial infarction, but their presence gradually decreases after approximately seven days. Additional administration of SDF-1α-releasing microspheres could be a tool for the extension of the time the stem cells are in the cardiac tissue after myocardial infarction. This, in turn, could constitute a novel therapy for more efficient regeneration of the heart muscle after injury. Through this practical study, it has been shown that the controlled release of SDF-1α from biodegradable microspheres into the pericardial sac fourteen days after myocardial infarction increases the concentration of exogenous SDF-1α, which persists in the tissue much longer than the level of endogenous SDF-1α. In addition, administration of SDF-1α-releasing microspheres increased the expression of the factors potentially involved in the involvement and retention of myocardial stem cells, which constitutes vascular endothelial growth factor A (VEGFA), stem cell factor (SCF), and vascular cell adhesion molecules (VCAMs) at the site of damaged tissue. This exhibits the possibility of combating the basic limitations of cell therapy, including ineffective stem cell implantation and the ability to induce the migration of endogenous stem cells to the ischemic cardiac tissue and promote heart repair.

Genome-Wide Methylation Changes Associated with Replicative Senescence and Differentiation in Endothelial and Bone Marrow Mesenchymal Stromal Cells.

Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/ß-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent cells.

Over expression of ubiquitin-conjugating enzyme E2O in bone marrow mesenchymal stromal cells partially attenuates acute myeloid leukaemia progression.

Bone marrow mesenchymal stromal cells (BM-MSCs) are implicated in the pathogenesis of acute myeloid leukaemia (AML). However, due to the high heterogeneity of AML the mechanism underlying the cross-talk between MSCs and leukaemia cells is not well understood. We found that mixed-lineage leukaemia-AF9 (MLL-AF9)-induced AML mice-derived MSCs had higher proliferative viability compared to wild-type mice-derived MSCs with ubiquitin-conjugating enzyme E2O (Ube2o) down-regulation. After overexpression of UBE2O in AML-derived MSCs, the growth capacity of MSCs was reduced with nuclear factor kappa B subunit 1 (NF-κB) pathway deactivation. In vitro co-culture assay revealed that UBE2O-overexpression MSCs suppressed the proliferation and promoted apoptosis of AML cells by direct contact. In vivo results revealed that the leukaemia burden was reduced and the overall survival of AML mice was prolonged, with decreased dissemination of leukaemia cells in BM, spleen, liver and peripheral blood. Additionally, subcutaneous tumorigenesis revealed that tumour growth was also suppressed in the UBE2O-overexpression MSCs group. In conclusion, UBE2O was expressed at a low level in MLL-AF9-induced AML mice-derived MSCs. Overexpression of UBE2O in MSCs suppressed their proliferation through NF-κB pathway deactivation, which resulted in AML suppression. Our study provides a theoretical basis for a BM microenvironment-based therapeutic strategy to control disease progression.

Controlled Release of Encapsuled Stromal-Derived Factor 1α Improves Bone Marrow Mesenchymal Stromal Cells Migration.

Stem cell treatment is a promising method of therapy for the group of patients whose conventional options for treatment have been limited or rejected. Stem cells have the potential to repair, replace, restore and regenerate cells. Moreover, their proliferation level is high. Owing to these features, they can be used in the treatment of numerous diseases, such as cancer, lung diseases or ischemic heart diseases. In recent years, stem cell therapy has greatly developed, shedding light on stromal-derived factor 1α (SDF-1α). SDF-1α is a mobilizing chemokine for application of endogenous stem cells to injury sites. Unfortunately, SDF-1α presented short-term results in stem cell treatment trials. Considering the tremendous benefits of this therapy, we developed biodegradable polymeric microspheres for the release of SDF-1α in a controlled and long-lasting manner. The microspheres were designed from poly(L-lactide/glycolide/trimethylene carbonate) (PLA/GA/TMC). The effect of controlled release of SDF-1α from microspheres was investigated on the migration level of bone marrow Mesenchymal Stromal Cells (bmMSCs) derived from a pig. The study showed that SDF-1α, released from the microspheres, is more efficient at attracting bmMSCs than SDF-1α alone. This may enable the controlled delivery of selected and labeled MSCs to the destination in the future.

The Analgesic Efficacy of Intradiscal Injection of Bone Marrow Aspirate Concentrate and Culture-Expanded Bone Marrow Mesenchymal Stromal Cells in Discogenic Pain: A Systematic Review.

Pain originating from the intervertebral disc (discogenic pain) is a prevalent manifestation of low back pain and is often challenging to treat. Of recent interest, regenerative medicine options with injectable biologics have been trialed in discogenic pain and a wide variety of other painful musculoskeletal conditions. In particular, the role of bone marrow aspirate concentrate (BMAC) and culture-expanded bone marrow derived mesenchymal stromal cells (BM-MSCs) in treating discogenic pain remains unclear. The primary objective of this systematic review was to appraise the evidence of intradiscal injection with BMAC and culture-expanded BM-MSCs in alleviating pain intensity from discogenic pain. Secondary outcomes included changes in physical function after intradiscal injection, correlation between stromal cell count and pain intensity, and anatomical changes of the disc assessed by radiographic imaging after intradiscal injection. Overall, 16 studies consisting of 607 participants were included in qualitative synthesis without pooling. Our synthesis revealed that generally intradiscal autologous or allogeneic BMAC and culture-expanded BM-MSCs improved discogenic pain compared to baseline. Intradiscal injection was also associated with improvements in physical functioning and positive anatomical changes on spine magnetic resonance imaging (improved disc height, disc water content, Pfirrmann grading) although anatomical findings were inconsistent across studies. However, the overall GRADEscore for this study was very low due to heterogeneity and poor generalizability. There were no serious adverse events reported post intradiscal injection except for a case of discitis.

Role of mesenchymal stromal cells derivatives in diabetic foot ulcers: a controlled randomized phase 1/2 clinical trial.

BACKGROUND: Diabetes-related foot complications have been identified as the most common isolated cause of morbidity among patients with diabetes and the leading cause of amputation. Therefore, new strategies to stimulate skin regeneration may provide a novel therapeutic approach to reduce non-healing ulcer disease. Recently, we demonstrated in proof-of-concept in humans that administration of allogeneic bone marrow mesenchymal stromal cellss derivatives (allo-hBM-MSCDs) is effective in a similar way to the use of allogeneic bone marrow mesenchymal stromal cellss (allo-hBM-MSCs) in grade 2 diabetic foot ulcers (DFUs). AIM: To assess the safety and efficacy profile of the allo-hBM-MSCDs relative to the conventional approach (PolyMen® dressing) in 1/2 clinical trial phases in patients with grade 1 and 2 DFUs. METHODS: In the present study, we used 2 doses of allo-hBM-MSCDs (1 mL) or 1 dose of allo-hBM-MSCs (1 × 106 cells) intradermally injected around wounds and assessed their safety and effectiveness, relative to the conventional approach (PolyMem dressing). Allo-hBM-MSCDs and allo-hBM-MSCs were produced in a certified Good Manufacturing Practice-type Laboratory. Patients with grade 1 and 2 DFUs were randomized to receive allo-hBM-MSCDs (n=12), allo-hBM-MSCs (n=6) or conventional treatment (PolyMem dressing) (n=10). The wound-healing process was macroscopically evaluated until the complete closure of the ulcers. RESULTS: No adverse events were reported. Patients with grade 1 and 2 DFUs treated with either allo-hBM-MSCDs or allo-hBM-MSCs, achieved greater percentages of wound closure, enhanced skin regeneration in shorter times and a greater ulcer-free survival relative to the patients who received conventional treatment. Finally, through proteomic analysis, we elucidated the proteins and growth factors that are secreted by allo-hBM-MSCs and relevant to the wound-healing process. In addition, by combining proteomics with Gene Ontology analysis, we comprehensively classified secreted proteins on both biological process and molecular function. CONCLUSIONS: In this phase 1/2 trial, our cumulative results suggest that 2 doses of allo-hBM-MSCDs combined with a wound dressing are a safe and effective treatment for grade 1 and 2 DFUs.