Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642477 |
Date | January 2014 |
Creators | Fellows, Christopher R. |
Publisher | Cardiff University |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://orca.cf.ac.uk/70446/ |
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