Normal Marrow Mesenchymal Stem Cells Research Articles

Sequential gene expression analysis of myelodysplastic syndrome transformation identifies HOXB3 and HOXB7 as the novel targets for mesenchymal cells in disease

BackgroundMyelodysplastic syndrome (MDS) is known to arise through the pathogenic bone marrow mesenchymal stem cells (MSC) by interacting with hematopoietic stem cells (HSC). However, due to the strong heterogeneity of MDS patients, it is difficult to find common targets in studies with limited sample sizes. This study aimed to describe sequential molecular changes and identify biomarkers in MSC of MDS transformation.MethodsMultidimensional data from three publicly available microarray and TCGA datasets were analyzed. MDS-MSC was further isolated and cultured in vitro to determine the potential diagnostic and prognostic value of the identified biomarkers.ResultsWe demonstrated that normal MSCs presented greater molecular homogeneity than MDS-MSC. Biological process (embryonic skeletal system morphogenesis and angiogenesis) and pathways (p53 and MAPK) were enriched according to the differential gene expression. Furthermore, we identified HOXB3 and HOXB7 as potential causative genes gradually upregulated during the normal-MDS-AML transition. Blocking the HOXB3 and HOXB7 in MSCs could enhance the cell proliferation and differentiation, inhibit cell apoptosis and restore the function that supports hematopoietic differentiation in HSCs.ConclusionOur comprehensive study of gene expression profiling has identified dysregulated genes and biological processes in MSCs during MDS. HOXB3 and HOXB7 are proposed as novel surrogate targets for therapeutic and diagnostic applications in MDS.

Growth and dormancy control of myeloma cells by mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) may have contrasting impacts on the progression of multiple myeloma (MM). Priming normal MSCs, by culturing them with MM cells, mimics the MSC-induced MM growth. We studied the contrasting effects of conditioned medium (CM) from unprimed or primed MSCs on growth of MM cells from newly diagnosed cases. We elucidated potential molecular pathways using global gene expression profiling and focused on the role of the mTOR2 component, RICTOR, as a novel mediator of dormancy in MM. Primed MSCs CM consistently increased proportions of proliferating cells and supported MM growth in 3-day (n = 20) and 10-day (n = 12) cultures, effects that were partially mediated through the IGF1 axis. In contrast, unprimed MSCs CM inhibited growth of MM cells in cases mainly from stages I/II MM. The genes most overexpressed in MM cells treated with primed MSCs CM were associated with cell cycle, DNA-damage repair, and proliferation; genes most overexpressed in MM cells treated with unprimed MSCs CM were associated with dormancy pathways including RICTOR (mTOR2 pathway), CXCR4, and BCL2. RICTOR protein level was induced by unprimed MSCs CM and was lower in KI67+ proliferating MM cells treated with primed MSCs CM. RICTOR was underexpressed in clinical relapse samples compared with baseline samples of the same patients. Inhibiting RICTOR expression in primary MM cells promoted their growth, and enforced expression of RICTOR in MM cell lines inhibited their growth. Our findings suggest that, after prolonged interactions with MM cells, bone marrow MSCs shift from MM-repressive to MM-permissive. Availability of Data and MaterialsOur institutional GEP data of MM cells from newly diagnosed patients used to show RICTOR expression have been deposited at Gene Expression Omnibus (GEO: GSE2658, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2658)

Exosome-Derived miR21/SMAD7 Derived from Donor Osteoporosis Mesenchymal Stem Cells Inhibits Osteogenic Differentiation of Reconstituted Hosts

Exogenous bone marrow mesenchymal stem cells (MSCs) can regulate osteogenic differentiation. MicroRNA-21 has been shown to target SMAD7. This study explored the mechanism by which miR-21/SMAD7 inhibits osteogenic differentiation from exosomes secreted by osteoporosis patients-derived MSCs. Exosomes were obtained from MSCs and miR-21 expression was detected. Normal MSCs were treated with exosomes secreted by MSCs from different sources for osteogenic differentiation followed by detection of ALP, Bglap and Runx2 level and ALP activity. Normal MSCs were divided into three groups, which were treated with exosomes from normal adult MSCs, osteoporosis patients-derived MSCs and osteoporosis patients-derived MSCs + SMAD7 overexpression followed by analysis of the mRNA expression of ALP, Bglap and Runx2 by qRT-PCR and ALP activity. miR-21 expression in exosomes from osteoporosis patients-derived MSCs was significantly higher than that from normal adults MSCs. After treatment with exosomes from osteoporosis patients-derived MSCs, Runx2 expression and ALP activity was significantly decreased. SMAD7 expression in osteoporosis patients was significantly lower than that in normal adults. The expression of ALP, Bglap and Runx2 is significantly decreased after overexpression of SMAD7. SMAD7 is a target gene of miR-21 and plays a role in inhibiting osteogenic differentiation induced by exosomes from osteoporosis-derived MSCs.

Role of microRNA 146a on the healing of cornea alkali burn treated with mesenchymal stem cells

The aim of the present study was to investigate the effect of microRNA 146a (miR146a) on promoting the repair of corneal alkali burn with bone marrow mesenchymal stem cells (MSCs). A total of 24 Sprague-Dawley female rats were divided into a normal group (Control), a normal MSC treatment group (Normal MSCs), an miR146a knockout MSC treatment group (miR146a-low MSCs) and an miR146a high-expression MSC treatment group (miR146a-high MSCs) according to the random number table. Quantitative polymerase chain reaction was used to evaluate the expression levels of miR146a. MTT assay was performed to measure the cell viability of mesenchymal stem cells (MSCs) and apoptosis was measured by flow cytometry. The expression levels of p65 nuclear factor (NF)-κB, proliferating cell nuclear antigen (PCNA) and Fas proteins were analyzed by western blotting. MSCs were tested for the secretion levels of vascular endothelial growth factor (VEGF), CD45, interferon (IFN)-γ and interleukin (IL)-10 by ELISA. The miR146a-high MSCs improved cell viability of MSCs and inhibited apoptosis of MSCs following alkali burn. miR146a-high MSCs decreased the expression levels of p65NF-κB and PCNA, and enhanced the expression level of Fas. Furthermore, miR146a-high MSCs improved the cornea opacity and enhanced the inhibition of neovascularization in the rats following alkali burn. miR146a-high MSCs inhibit the expression of VEGF, CD45, IFN-γ, while enhanced the expression of IL-10. Therefore, miR146a promotes the repair of corneal alkali burn in rats treated with MSCs.

Effect of Bone Marrow Mesenchymal Stem Cells in Patients with Multiple Myelima on Migration of Myeloma Cells In Vitro

To explore the effect of bone marrow mesenchymal stem cells (MSC) in patients with multiple myeloma(MM) on chemotactic migration of myeloma cells in vitro. By in vitro co-culture with diffferent MSC, the myeloma cell U266 was divided into 2 groups: group A in which the U266 cells were co-cultured with normal person MSC (N-MSC) and group B in which the U266 cells were co-cultured with MM-MSC. The expression level of CCR1 in U266 cells, migration rate of U266 cells in Transwell, and the effect of supernantant from co-culture of U266 cells with N-MSC and MM-MSC on the migration in Transwell were compared in condition with or without bortezomib. After co-culture of U266 cells with N-MSC or MM-MSC, the migration rate of U266 cells in Transwell in B group was higher than that in A group(P<0.05). The difference between 2 groups could not be eliminated after treatment of U266 cells with bortezomib. The CCR1 expression level of U266 cells in B group was higher than that in A group (P<0.05). The culture supernatant of bone marrow MSC showed that in condition without bortezomib the culture supernatant of MSC in MM patients and normal persons both possessed more strong chemotactic ability and enhanced the migration rate of cells in Transwell, compared with SDF-1, meanswhile the culture supernatant in 3 groups reduced the migration rate of cells in condition with bortezomib (P<0.05), but there were no statistical difference in migration rate of U266 cells in Transwell between supernatant of N-MSC and MM-MSC culture (P>0.05), no matter the bortezonib was used or not. The bone marrow MSC in MM patients have same intrinsic defects that affect the chemotaxis of cells in vitro by directly interacting with myeloma cells.

Epac-Rap1-activated mesenchymal stem cells improve cardiac function in rat model of myocardial infarction.

Rap1, a member of Ras superfamily of small GTP-binding proteins, is involved in cardiovascular biology in numerous ways. It is an evolutionary conserved regulator of adhesion, polarity, differentiation and growth. Our aim was to analyze Rap1-activated rat bone marrow mesenchymal stem cells (MSCs) for their potential role in adhesion and cardiac differentiation. Myocardial infarction (MI) was produced in Sprague Dawley (SD) rats through occlusion of the left anterior descending coronary artery. MSCs were treated with 8-pCPT-2'-O-Me-cAMP (CPT) to activate Rap1. Normal (untreated) and CPT-treated MSCs were transplanted through intramyocardial injection in respective groups. Cardiac function was assessed by echocardiography at 2 and 4weeks after cell transplantation. Histological analysis was performed to observe changes at tissue level. Homing of CPT-treated MSCs was significantly (***P<.001) higher as compared to normal MSCs in the infarcted hearts. This may be due to increase in the gene expression of some of the cell adhesion molecules as evident by qRT-PCR analysis. Significant (***P<.001) improvement in the restoration of heart function in terms of left ventricular diastolic and systolic internal diameters (LVIDd, LVIDs), % ejection fraction, % fraction shortening and end-systolic and end-diastolic volumes were observed in CPT-treated MSCs as compared to the MI model. Histological analyses showed significant (***P<.001) reduction in scar formation in the CPT-treated group. Differentiation of treated MSCs into functional cardiomyocytes was evident through immunohistochemical staining. LV wall thickness was also preserved significantly (***P<.001). Blood vessel formation was more pronounced in CPT-treated group although both cell therapy groups showed significant increase as compared to MI model. Our findings showed that pharmacological activation of Epac-Rap1 improves cardiac function through better survival, adhesion and differentiation of transplanted cells. Transplantation of these MSCs in the infarct area restored functional myocardium.

Mesenchymal Stem Cells Support Survival and Proliferation of Primary Human Acute Myeloid Leukemia Cells through Heterogeneous Molecular Mechanisms.

Acute myeloid leukemia (AML) is a bone marrow malignancy, and various bone marrow stromal cells seem to support leukemogenesis, including osteoblasts and endothelial cells. We have investigated how normal bone marrow mesenchymal stem cells (MSCs) support the in vitro proliferation of primary human AML cells. Both MSCs and primary AML cells show constitutive release of several soluble mediators, and the mediator repertoires of the two cell types are partly overlapping. The two cell populations were cocultured on transwell plates, and MSC effects on AML cells mediated through the local cytokine/soluble mediator network could thus be evaluated. The presence of normal MSCs had an antiapoptotic and growth-enhancing effect on primary human AML cells when investigating a group of 51 unselected AML patients; this was associated with increased phosphorylation of mTOR and its downstream targets, and the effect was independent of cytogenetic or molecular-genetic abnormalities. The MSCs also supported the long-term proliferation of the AML cells. A subset of the patients also showed an altered cytokine network with supra-additive levels for several cytokines. The presence of cytokine-neutralizing antibodies or receptor inhibitors demonstrated that AML cells derived from different patients were heterogeneous with regard to effects of various cytokines on AML cell proliferation or regulation of apoptosis. We conclude that even though the effects of single cytokines derived from bone marrow MSCs on human AML cells differ among patients, the final cytokine-mediated effects of the MSCs during coculture is growth enhancement and inhibition of apoptosis.

High level of reactive oxygen species impaired mesenchymal stem cell migration via overpolymerization of F-actin cytoskeleton in systemic lupus erythematosus

Some lines of evidence have demonstrated abnormalities of bone marrow mesenchymal stem cells (MSCs) in systemic lupus erythematosus (SLE) patients, characterized by defective phenotype of MSCs and slower growth with enhanced apoptosis and senescence. However, whether SLE MSCs demonstrate aberrant migration capacity or abnormalities in cytoskeleton are issues that remain poorly understood. In this study, we found that MSCs from SLE patients did show impairment in migration capacity as well as abnormalities in F-actin cytoskeleton, accompanied by a high level of intracellular reactive oxygen species (ROS). When normal MSCs were treated in vitro with H2O2, which increases intracellular ROS level as an oxidant, both reorganization of F-actin cytoskeleton and impairment of migration capability were observed. On the other hand, treatment with N-acetylcysteine (NAC), as an exogenous antioxidant, made F-actin more orderly and increased migration ratio in SLE MSCs. In addition, oral administration of NAC markedly reduced serum autoantibody levels and ameliorated lupus nephritis (LN) in MRL/lpr mice, partially reversing the abnormalities of MSCs. These results indicate that overpolymerization of F-actin cytoskeleton, which may be associated with high levels of ROS, causes impairment in the migration capacity of SLE MSCs and that oral administration of NAC may have potential therapeutic effects on MRL/lpr mice.

THU0052 The Treg/Th17 Imbalance of Lupus Patients Were Mediated by MIR-663 Through Down-Regulating TGF-B1 Secretion of Bone Marrow-Derived Mesenchymal Stem Cells

BackgroundSystemic lupus erythematosus (SLE) patients showed an imbalance between CD4+CD25+FoxP3+ T regulatory (Treg) and IL-17-producing cells (Th17), as well as the deficient activity of bone marrow mesenchymal stem cells (MSCs)....

Protective effects of bone marrow mesenchymal stem cells with stable expression of Bcl-2 on ischemia- hypoxia of hippocampal neurons

The protective effect of bone marrow mesenchymal stem cells (MSCs) with stable expression of Bcl-2 on the in vitro ischemia-hypoxia of hippocampal neurons was investigated in the present study. Mouse Bcl-2 cDNA was amplified by RT-PCR followed by Lentivirus-mediated gene transfection into MSCs. Puromycin resistance was detected to screen MSCs undergoing successful transfection, and the mRNA expression of Bcl-2 was determined by RT-PCR. Serum and oxygen deprivation was performed in normal MSCs and those undergoing transfection aiming to mimic ischemia injury in vitro, and flow cytometry was used to detect the apoptosis rate of these cells. Neurogenic induction of Bcl-2 expressing MSCs was carried out followed by detection of neuron markers. The rat hippocampal neurons were isolated from neonate rats. The Bcl-2 expressing MSCs and hippocampal neurons were cocultured, and the protective effect of Bcl-2 expressing MSCs on the ischemia-hypoxia of hippocampal neurons was observed. Following transfection, MSCs had stable and efficient expression of Bcl-2 after screening. When compared with normal MSCs, the number of apoptotic Bcl-2 expressing cells was significantly decreased and that of survival cells markedly increased after serum and oxygen deprivation. In addition, the Bcl-2 expressing MSCs had the potent differentiation into neurons. In the coculture system, Bcl-2 expressing MSCs could remarkably reduce the apoptosis rate of hippocampal neurons after ischemia-hypoxia (P < 0.01). The MSCs undergoing Bcl-2 transfection had stable Bcl-2 expression, which can protect them from serum and oxygen deprivation induced apoptosis and protect hippocampal neuron against ischemia-hypoxia induced apoptosis in vitro. Furthermore, the Bcl-2 transfecion does not affect the differentiation of MSCs into neurons.

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques. The aim of this study is to investigate the effect of connective tissue growth factor (CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs). A CTGF-expressing plasmid (pCTGF) was constructed and transfected into MSCs. Then expressions of bone morphogenesis-related genes, proliferation rate, alkaline phosphatase activity, and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs. Overexpression of CTGF was confirmed in pCTGF-MSCs. pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs (P<0.05). CTGF induced a 7.5-fold increase in cell migration over control (P<0.05). pCTGF transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, and collagen type I in MSCs. The levels of alkaline phosphatase (ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0- and 3.0-fold higher than those of MSCs cultured in OS-medium, significantly higher than those of mock-MSCs and normal control MSCs (P<0.05). Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules. Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs, and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.

Characterization of Mesenchymal Stem Cells Abnormalities in Multiple Myeloma.

Introduction: The bone marrow microenvironnement (BMMe) play a significant role in the physiopathology of the multiple myeloma (MM). However, its abnormality still remains controversial. To address this question, we studied bone marrow mesenchymal stem cells (MSCs), the only long-lived cells of the BMMe. We compared, at a genomic and functional level, the MSCs isolated from patients with MM, to MSCs isolated from healthy subjects and those with monoclonal gammopathy of unknown significance (MGUS).Material and methods : Bone marrow samples from 26 MM patients, 7 MGUS patients and 11 healthy individuals were compared. The MSCs were selected by their adherence on plastic and were cultured in alpha-MEM medium + 10% SVF and antibiotics during 2 passages (primo-culture = P0 and first passage = P1). The gene expression profiling was carried out by Affymetrix GeneChip microarrays (U133 plus 2.0). The expression of interesting differentially expressed genes was validated by ELISA or qRT-PCR. The phenotype was studied by flow cytometry (CD45, CD90, CD73, CD13, CD14). The CFU-F frequencies in BM samples and in cell suspensions after P0 and P1 were studied as well as the cell productions after P0 and P1. The osteoblastic differentiation was evaluated both by alkaline phosphatase dosing and matrix mineralization quantification. We also carried out co-cultures of the MSCs with CD34+ cells to quantify their hematopoietic supportive potential. Finally XG1 and Molp-6, respectively stroma independent and stroma dependent cell lines, were co-cultured with MSCs to check the capacity of the MSCs to support malignant plasma cell growth.Results: Gene expression profile independently classified the MSCs in a normal and in a MM group. MGUS MSCs were interspersed between those 2 groups. 145 distinct genes were differentially expressed in MM and normal MSCs. Among them, 46% could be involved in tumor-microenvironment cross-talk. Known soluble factors involved in MM physiopathologic features, such as IL-6, IL-1ß, DKK1 and amphiregulin, were identified and new ones found. In particular growth and differentiation factor-15 (GDF-15), already described as a accurate biomarker of numerous tumours, was significantly overexpressed (p<0.001) in MM MSCs both at mRNA and protein levels (183.5 ± 64.9 vs 749 ± 90.9 for mRNA, 1 10−4 pg/cell ± 1.9 10−5 pg/cell vs 4.3 10−4 pg/cell ± 1.4 10−4 pg/cell for protein respectively for normal and MM MSCs). It was also able to induce dose-dependant growth of Molp-6, in the absence of a supportive stroma. The phenotype and the CFU-F frequencies and the cell productions were similar in the 3 groups of MSCs and their hematopoietic supportive capacity was maintained. The MM MSCs complete differentiation towards the osteoblastic lineage, evaluated quantitatively, was faded. And very importantly, MM MSCs constituted a better supportive feeder layer for the Molp-6 cell line as compared to normal MSCs (cell expansion after 7 days : 2.1 ± 0.3 vs 3.3 ± 0.4, p = 0.04, respectively for normal and MM MSCs). On the other hand, the growth of XG1 was not influence by the subject origin of the MSCs.Conclusion: Our results show that the MSCs, like the malignant plasma cells, are abnormal in MM. This confirms the place of microenvironnement in the physiopathology of the MM and makes it possible to identify new potential therapeutic targets.

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.