Tracking Senescence In Human Bone Marrow Stromal Cell (BMSC) Cultures.

Abstract 1179

Bone marrow stromal cell (BMSC) therapy is effective for treating acute-Graft versus Host Disease (aGVHD) and is being investigated as a therapy for graft failure and organ failure following allogeneic hematopoietic stem cell transplantation. Clinical BMSC products are typically produced by serial passage of adherent cells obtained from marrow aspirates. Multiple passages allow for the production of large quantities of human BMSCs, but BMSC 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 8^th to 12^th passages. Early and late passage cells were compared. The early passage BMSCs were passages 1 and 2; because the onset of senescence as assessed by growth characteristics and morphology varied among the 5 donors, the passages included in the late passage group were variable and ranged from passage 5 to 11. The samples (25 total; 10 early and 15 late) were selected based on the replicative lifespan. Senescence was associated with a change to a flattened morphology and increased senescence associated beta-galactosidase (SA β-gal) staining. Early and late passage BMSCs from each subject were also analyzed by flow cytometry and global transcriptome analysis with an oligonucleotide microarray with >44,000 probes. Unsupervised hierarchical clustering analysis separated the 25 BMSC samples into two clusters, one with all early passage and a second one with all late passage samples. A total of 1,739 genes were differentially expressed between the early and late passage BMSCs. Ingenuity pathway analysis of the differentially expressed genes found that those highly expressed in the early passage cells 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. Specific genes highly expressed in early passage BMSCs compared to late passage BMSCs included the chemokine receptor CXCR7, Secreted frizzle-related protein 4 (SFRP4), WNT1 induced signaling pathway protein 1 (WISP1), lymphocyte-specific protein 1 (LSP1), and insulin-like growth factor 1 (IGF1). Genes up-regulated in late passage cells included the thrombospondin receptor (CD36), coagulation factor III (F3), and tissue plasminogen activator (PLAT). Among the differentially expressed genes were 347 involved with the cell cycle and apoptosis. Hierarchical clustering analysis of these 347 genes again separated the early and late passage cells into two groups and a node of genes more highly expressed 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 TRALI 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. Further studies are needed to determine more precisely the onset of senescence-associated changes.