Will, Malcolm B.
Gene expression profiling of mesenchymal stem cells aged in vitro.
PhD thesis, University of Glasgow.
Full text available as:
Mesenchymal Stem Cells (MSC’s) have shown promise as a cell-based therapy for myocardial repair. However, MSC’s have a finite replicative lifespan and lose proliferative and differentiation capacity during expansion in vitro. Therefore, understanding the molecular mechanisms that regulate ageing and senescence of MSC’s should enhance our ability to use these cells in cell-based approaches and give insight into mechanisms of tissue ageing.
We established MSC cultures from the sternal bone marrow of eight donors undergoing coronary artery bypass surgery. After thirty population doublings (nine passages) MSC’s displayed morphological abnormalities, expression of senescence associated β-galactosidase, telomere erosion and decreased adipogenic and osteogenic differentiation capacity. Using serial analysis of gene expression (SAGE) we identified 243 known genes differentially expressed between MSC’s at passages two and nine. Analysis of known direct interactions between genes revealed a regulatory signaling network centered on down-regulation of the transcription factor, activator protein 1 (AP-1). Transcriptional changes in MSC’s at passage nine included genes associated with inflammation, regulation of cell cycle, metabolism and extracellular matrix re-modelling. The validation studies corroborated the SAGE results and eighteen genes were identified as differentially expressed in late passage MSC’s from multiple donors. Furthermore, caveolin 1, cyclin D1, tissue plasminogen activator and olfactomedin-like 3 were able to discriminate MSC’s of different culture age.
In addition, we show evidence that the p38 MAPK signalling pathway contributes to the decline in proliferation and differentiation of MSC’s during expansion and is critical for the maintenance of genomic stability. The results provide further evidence that MSC’s senesce prematurely in response to undefined culture stresses. Our studies have provided novel markers that identify MSC ageing in vitro and suggest that identifying factors that activate p38 MAPK signalling should enhance our ability to use MSC’s in cell-based therapies.
Actions (login required)