Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of primary human bone marrow stromal cells (BMSCs) and telomerase-immortalised single cell derived clonal BMSC lines

ABSTRACT: 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.

INSTRUMENT(S): Agilent DNA microarray scanner G2565CA

ORGANISM(S): Homo sapiens

SUBMITTER: Sally James

PROVIDER: E-MTAB-3511 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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. 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.

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Gene expression profiling of primary human bone marrow stromal cells (BMSCs) and telomerase-immortalised single cell derived clonal BMSC lines

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