Senescence of Cultured Bone Marrow Stromal Cells (2011)

Bone marrow stromal cell (BMSC) therapy is effective for treating acute-Graft versus Host Disease. Clinical BMSCs are produced by serial passage of adherent cells from marrow aspirates. Multiple passages allow for the production of large quantities of human BMSCs, but expansion is limited by cell senescence. We assessed the nature of BMSC changes associated with multiple passages in order to better understand the nature and impact of senescence on human BMSCs and to identify potential senescence associated biomarkers. BMSCs from marrow aspirates of 5 healthy subjects were cultured in flasks and passed serially until cell growth stopped between the 8th to 12th passages. Early (passage 1 and 2, n10) and late passage cells (passages 5 through 11, n15) were selected based on their replicative lifespan and compared. Senescence was associated with a change to a flattened morphology and increased senescence associated beta-galactosidase (SA β-gal) staining. The BMSCs were analyzed by global transcriptome analysis with an oligonucleotide microarray with >44,000 probes. Unsupervised hierarchical clustering analysis separated the 25 samples into two clusters, one with all early and a second with all late passage BMSCs and a total of 1,739 genes were differentially expressed. Ingenuity pathway analysis revealed that genes highly expressed in the early passage BMSCs belonged to several immune pathways: cell-mediated immune response, humoral immune responses, hypersensitive responses, and lymphoid tissue structure and development. Genes highly expressed in the late passage BMSCs belonged to the DNA replicative, recombination and repair and nucleic acid metabolism pathways. Hierarchical clustering analysis of 347 differentially expressed cell cycle and apoptosis genes again separated the early and late passage cells into two groups and a node of genes up-regulated in late passages cells included two genes involved with replication senescence, cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (CDKN2A) and one that inhibits TRAIL mediated apoptosis, tumor necrosis factor receptor superfamily, member 10d (TNFRSF10D, DCR2, CD264). Flow cytometry analysis confirmed greater expression of TNFRS10D on late passage cells. BMSC senescence is associated with distinct molecular changes. Early passage BMSCs may have more immune modulatory properties. Bone marrow stromal cell (BMSC) therapy is effective for treating acute-Graft versus Host Disease. Clinical BMSCs are produced by serial passage of adherent cells from marrow aspirates. Multiple passages allow for the production of large quantities of human BMSCs, but expansion is limited by cell senescence. We assessed the nature of BMSC changes associated with multiple passages in order to better understand the nature and impact of senescence on human BMSCs and to identify potential senescence associated biomarkers. BMSCs from marrow aspirates of 5 healthy subjects were cultured in flasks and passed serially until cell growth stopped between the 8th to 12th passages. Early (passage 1 and 2, n10) and late passage cells (passages 5 through 11, n15) were selected based on their replicative lifespan and compared. Senescence was associated with a change to a flattened morphology and increased senescence associated beta-galactosidase (SA β-gal) staining. The BMSCs were analyzed by global transcriptome analysis with an oligonucleotide microarray with >44,000 probes. Unsupervised hierarchical clustering analysis separated the 25 samples into two clusters, one with all early and a second with all late passage BMSCs and a total of 1,739 genes were differentially expressed. Ingenuity pathway analysis revealed that genes highly expressed in the early passage BMSCs belonged to several immune pathways: cell-mediated immune response, humoral immune responses, hypersensitive responses, and lymphoid tissue structure and development. Genes highly expressed in the late passage BMSCs belonged to the DNA replicative, recombination and repair and nucleic acid metabolism pathways. Hierarchical clustering analysis of 347 differentially expressed cell cycle and apoptosis genes again separated the early and late passage cells into two groups and a node of genes up-regulated in late passages cells included two genes involved with replication senescence, cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (CDKN2A) and one that inhibits TRAIL mediated apoptosis, tumor necrosis factor receptor superfamily, member 10d (TNFRSF10D, DCR2, CD264). Flow cytometry analysis confirmed greater expression of TNFRS10D on late passage cells. BMSC senescence is associated with distinct molecular changes. Early passage BMSCs may have more immune modulatory properties.