Knockdown of TERC with siRNA in normal bone marrow stromal cells (BMSCs) recapitulates the defective BMSC phenotype in patients with telomere biology disorders
ABSTRACT: Dyskeratosis congenita (DC) is an inherited multi-system disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the bone marrow stromal cell population (BMSCs, also known as bone marrow-derived mesenchymal stem cells), may contribute to the hematological phenotype. TBD-BMSCs exhibited reduced clonogenicity, reduced telomerase activity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by siTERC-RNA recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the mRNA level, and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to bone marrow failure in TBD. RNA (5 mg) isolated from N-BMSCs, siNC-BMSCs and siTERC-BMSCs 72hrs after transfection using an RNeasy Mini kit (Qiagen), was reverse transcribed and hybridized to an Affymetrix GeneChip Human Genome U133 Plus 2.0 array (LMT, NCI-Frederick). Three independent replicates for each experimental condition were carried out to control for intra-sample variation. Genes that were under/over-represented by >2-fold were analyzed using GeneSpring software. Signal intensity values were normalized using RMA (Robust Multi-array Analysis) summarization and baseline transformation to median of all samples was performed. Entities were filtered based on their signal intensity values. Hierarchical clustering was performed on filtered signal intensity (>20.0), non-averaged, fold change >2. A fold change analysis (>10-fold) was performed to generate a list of top genes under/over represented between the groups.
Project description:Dyskeratosis congenita (DC) is an inherited multi-system disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the bone marrow stromal cell population (BMSCs, also known as bone marrow-derived mesenchymal stem cells), may contribute to the hematological phenotype. At least 5x105 cells from each SCDS were homogenized in Trizol (Invitrogen) for RNA extraction. RNA was purified by using a combination of chloroform phase separation and RNeasy Mini Kits (Qiagen) according to the manufacturer's protocol. RNA was quantified and 5ug was processed for microarray analysis (LMT, NCI, Frederick, MD). RNA was reverse transcribed to form cDNA, and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Three independent replicates for each of the experimental conditions were carried out and analyzed to control for intra-sample variation.
Project description:The aging of bone marrow stromal cells (BMSCs) lead to decreased ability to maintain hematopoiesis, however, effects of aging on BMSC-derived exosomes in bone marrow microenvironment remain unclear. The aim of this study is therefore to determine the age-related change of BMSC-derived exosomal miRNAs. Human BMSCs of young (yBMSC s, age of donors: 19 and 20 years) and elderly (eBMSC s, age of donors: 68 and 72 years) donors were purchased from Lonza. BM samples were obtained from MM patients (age of donors: 62 and 77 years) in accordance with the Declaration of Helsinki and using protocols approved by the research Ethics Committee of Tokyo Medical University (IRB No. 2648), and BMSCs derived from MM patients (mmBMSCs) were isolated using the classical plastic adhesion method. The exosomes from culture medium of BM-MSCs were isolated by Total Exosome Isolation Reagent (Invitrogen). Exosomal miRNA profiling was done using a TaqMan low-density array (ABI), and Student’s t-test was used to determine statistical significance for comparisons between young and old groups using R software.
Project description:Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, post-natally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.
Project description:The aging of bone marrow stromal cells (BMSCs) lead to decreased ability to maintain hematopoiesis. The aim of this study is therefore to determine the age-related change of cellular miRNAs in BMSCs. Human BMSCs of young (yBMSC s, age of donors: 19 and 20 years) and elderly (eBMSC s, age of donors: 68 and 72 years) donors were purchased from Lonza. BM samples were obtained from MM patients (age of donors: 62 and 77 years) in accordance with the Declaration of Helsinki and using protocols approved by the research Ethics Committee of Tokyo Medical University (IRB No. 2648), and BMSCs derived from MM patients (mmBMSCs) were isolated using the classical plastic adhesion method. The Cellular miRNA profiling was done using a TaqMan low-density array (ABI), and Student’s t-test was used to determine statistical significance for comparisons between young and old groups using R software.
Project description:Bone marrow stromal cells (BMSCs) provide hematopoietic support, immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. A heterogeneous population of primary bone marrow BMSCs were isolated from a single human donor (FH181), and a lentiviral expression system was used to overexpress human telomerase reverse transcriptase (hTERT) and generate single cell-derived immortalised BMSC lines for multi-level analysis of functional markers for BMSC subsets. All clones expressed typical BMSC cell surface antigens, however clones Y101 and Y201 displayed typical BMSC tri-lineage differentiation capacity where clones Y102 and Y202 lacked significant tri-lineage differentiation potential. High quality RNA samples were isolated from all lines, and global gene expression analysis was performed in triplicate arrays (using Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays) in order to identify distinguishing characteristics of these lines, compared to the parental primary BMSCs from which they were derived.
Project description:Evidence suggests that the bone marrow microenvironment (niches) support hematopoietic stem cells (HSCs). The cell-cell interaction between bone marrow mesenchymal stromal cells (BMSCs) and HSCs plays a crucial role in hematopoiesis. The aging of BMSCs lead to decreased ability to maintain hematopoiesis. We used microarray to determine the age-related change of BMSCs. Human BMSCs of young (yBMSC s, age of donors: 19 and 20 years) and elderly (eBMSC s, age of donors: 68 and 72 years) donors were purchased from Lonza. BM samples were obtained from MM patients (age of donors: 62 and 77 years) in accordance with the Declaration of Helsinki and using protocols approved by the research Ethics Committee of Tokyo Medical University (IRB No. 2648). BMSCs derived from MM patients (mmBMSCs) were isolated using the classical plastic adhesion method. Total RNA from each sample (yBMSCs, eBMSCs and mmBMSCs) was processed for hybridization onto GeneChip® human Gene 1.0 ST Array (Affymetrix) according to the manufacturer's protocol and scanned.
Project description:Bone marrow derived stromal cells (BMSCs) are a multipotent population that supports angiogenesis, wound healing, immunomodulation and plays an active role in the hematopoietic niche. On the other hand, they are also involved in the nurturing of bone marrow tumors and metastasis, showing a pro-tumorigenic behavior. BMSCs secrete a wide range of cytokines, growth factors and matrix proteins that are likely responsible for many of these effects. However, it is not clear whether this pro-tumorigenic behavior of BMSCs is induced by the tumor cells, neither in what extent the tumor cells affect the type and quantity of factors produced by BMSCs. To determine how tumor cells that arise from bone marrow affect the BMSCs, we selected three myeloid leukemia cell lines (TF-1, TF-1alpha and K562) and co-cultured them with BMSCs from healthy donors. We found that, under co-culture condition, the gene expression profiling of BMSCs revealed up-regulation of many pro-inflammatory signaling related genes, mainly IL-17 signaling-related genes. Moreover, IL-17 signaling-related cytokines CCL2 and IL8, were increased in co-culture supernatants. We conclude that BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profile. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable to leukemia stem cells. BMSCs from healthy donors were transwell co-cultured with three different myeloid leukemia cell lines: TF-1 (n=3), TF-1alpha (n=3) and K562 (n=3). A 1-um Transwell system (BDBiosciences, San Jose, CA USA) was used to maintain the cultured BMSC and leukemia cell populations separate from each other. As a control BMSCs were also transwell co-cultured under the same conditions with CD34+ cells (n=9) isolated from G-CSF-mobilized peripheral blood stem cells from healthy donors. An alternative co-culture method was used to analyze BMSCs and leukemia cells in direct contact: TF-1 (n=3), TF-alpha (n=3) and K562 (n=3). The two populations were cultured together in the same well without any membrane separation. BMSCs (n=18), TF-1 (n=3), TF-1alpha (n=3), K562 (n=3) and CD34+ (n=9) cells cultured alone (mono-cultures) were used as controls. Cells from both mono- and co-culture conditions were harvested at 4h, 10h, and 24h.
Project description:Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here we describe a novel survival signaling pathway activated in stromal cells by contact to B-cells from chronic lymphocytic leukemia (CLL) patients. The expression of PKC-βII and the subsequent activation of NF-κB in bone marrow stromal cells is a prerequisite to support the survival of malignant B-cells. PKC-β knockout mice are insusceptible to CLL-transplantations, underscoring the in vivo significance of the PKC-βII- NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from CLL, breast- and pancreatic- cancer patients suggest that this pathway may commonly be activated in a variety of malignancies. We used microarrays to determine Nemo/ IKK-gamma dependent gene expression changes in bone marrow stromal cells (BMSCs) induced by co-culturing with leukemic B-cells (CLL) We compared mouse BMSC cells from Nemo knockout cells (Nemo+CRE) and Nemo wild type cells (Nemo-CRE) co-cultured for 5 days with leukemic B-cells microarray analysis on Affymetrix MG-430_2.0 arrays..
Project description:Ex vivo expansion of Bone Marrow Stromal Cells (BMSC) is required to obtain clinical dose for cellular therapy, and different labs use different confluence in their practice, however, the impacts of 100% confluence on the biological properties of BMSC remain controversial. In this study, we detected the changes occurred to BMSCs when they reached 100% confluence, including viability, population doubling time (PDT), apoptosis, colony formation, immunosuppression, proteins in the culture supernatant, and surface markers; we also performed gene expression profiling and microRNA profiling on 50%, 80% and 100% confluent BMSCs. Our results showed that 100% confluent BMSCs had similar level of immunosuppression to 80% confluent BMSCs; they increased the expression of CD10, CD54, CD106, CD200, TLR4, but decreased CD49f and PODXL; we detected of 39 proteins in the culture supernatant, which displayed different patterns and an increase on the PEDF/VEGF ratio was observed with higher confluence. We identified 26 differentially expressed microRNAs, which were reported to be involved in the proliferation and differentiation of BMSC; and 2708 genes that were involved in extracellular matrix, cell adhesion, immune response and inflammatory response; particularly, angiogenesis inhibitor PEDF, and Wnt Singling inhibitors DKK1, DKK2, and DKK3 were up-regulated by 100% confluent BMSCs. These data suggest that 100% confluent BMSC may retain immunosuppressive activities but have comprised pro-angiogenesis effects. Gene expression profiling on BMSCs of 3 confluences (50%, 80% and 100%) from 5 healthy donors: 09FC37 (n=3), 09FC43 (n=3), 09FC44 (n=3), 09FC45 (n=3), 09FC49 (n=3) as biological repeats.
Project description:miRNA expression profiling was performed on MM.1S MM cells cultured 8 hours in control media or 50nM RGB-286638, with or without BMSCs. The emerging role of miRNAs in the pathogenesis of multiple myeloma (MM) led us to hypothesize that the miRNA network might be among the inducible transcriptional alterations consequent to MM-bone marrow stromal cell (BMSC) interactions. Our data suggests that BMSC induced MM transcription led to aberrant miRNA expression. We therefore hypothesized that agents interfering with RNAPII transcription might inhibit aberrant miRNA expression in MM. To test this hypothesis we used RGB-286638, a novel protein kinase inhibitor, which works primarily via RNAPII inhibition followed by transcriptional arrest in MM cells. miRNA profiling of RGB-286638-exposed MM cells resulted in RNAPII arrest associated with reduced miRNA levels. RGB-286638 abrogated BMSCs-induced miRNAs, which correlated with growth arrest in MM cells. Analysis of RGB-286638-induced differentially-expressed miRNAs in MM cells, in the presence or absence of BMSCs, revealed RNAPII regulation of expression of BMSC-inducible miRNAs with established oncogenic functions in MM Our findings demonstrate the role of RNAPII in regulating miRNA network, suggesting a new rationale for using agents interfering with RNAPII transcription in the treatment of MM. TaqMan Low-Density Array (TLDA) using human miRNA version 2.0A and version 3.0B cards (Applied Biosystems) were applied to examine the global change of miRNA expression levels in MM.1S cells when co-cultured with BMSCs, with or without RGB-286638 treatment. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions as previously described. miRNAs with Ct values higher than 37 were excluded from the analysis. Normalization was carried out with the mean of RNU44 and RNU48. Relative quantification of miRNA expression was calculated with the 2−ΔΔCt Ct method using the ddCt program (Shannon McCormack Advanced Molecular Diagnostics Laboratory Research Services). The data was presented as log10 of the relative quantity of each miRNA.