Isolation and Characterisation of Mesenchymal Stem Cells

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Title: Isolation and Characterisation of Mesenchymal Stem Cells
Authors: Vaghjiani, Rasilaben Jethalal
Item Type: Thesis or dissertation
Abstract: MSCs have great therapeutic potential and are currently used in various clinical trials. However, the extremely low frequency of MSCs and the absence of a known cell-specific marker have made their purification and identification a highly challenging goal. Our hypothesis is that identification of a MSC specific cell surface marker would facilitate isolation of a pure population of MSCs, which in clinical studies may enhance the subsequent regenerative effect in comparison to a heterogeneous MSC population. Our aim therefore was to attempt to identify such MSC-specific markers using a transcriptomics approach. Individual clones were isolated after seeding bone marrow mononuclear cells from BALB/b and BALB/c mice in 5% oxygen tension. All clones had the ability to differentiate into adipocytes, chondrocytes, and osteoblasts, and to self-renew, and were therefore functionally characterised as MSCs. All murine MSC clones consistently expressed very high levels of Sca-1. Cytogenetic analysis of clones revealed an abnormal karyotype of 69 chromosomes and transplantation of cells into immuno-compromised SCID mice revealed no evidence of tumour formation after 7 months indicating that cells were not malignant. A rigorous genome-wide supervised microarray analysis revealed six genes were differentially expressed on the MSC clones in comparison to various controls - STEAP1, STEAP2, ly6f, versican, vitamin D receptor, and H2-M9. Two-dimensional hierarchical clustering of 28 different arrays revealed both STEAP genes and vitamin D receptor also had a similar expression pattern to Sca-1. Thus STEAP1 and STEAP2 were the only two cell membrane protein encoding genes identified by both analysis methods. Importantly, flow cytometry analysis revealed STEAP2 was differentially expressed in normal diploid multipotent human bone marrow stromal cells (hBMSCs) compared to fibroblasts and freshly isolated bone marrow cells. Furthermore, western blot analysis revealed STEAP1 was significantly expressed in hBMSCs, but not by human fibroblasts or human chondrocytes. STEAP1 depletion by RNA interference resulted in decreased cell adherence to tissue culture plastic. Results suggest STEAP1 and STEAP2 may be novel MSC markers in murine and in human cells. Further work is needed to elucidate their role in MSCs and to establish their usefulness as potential cell-specific markers.
Issue Date: Sep-2008
Date Awarded: Jan-2009
URI: http://hdl.handle.net/10044/1/4416
Supervisor: Murphy, Chris
Saklatvala, Jeremy
Author: Vaghjiani, Rasilaben Jethalal
Department: The Kennedy Institute of Rheumatology
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses



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