The isolation, proliferation and differentiation of adult neural progenitors

The relatively recent discovery of adult neural stem cells has provided hope to sufferers of a wide range of central nervous system (CNS) ailments. A wide range of therapies that would harness the potential of endogenous stem cells or use cultured cells as transplant material have been proposed. Before these therapies come to fruition however, a more detailed understanding of the basic biology of these cells is necessary. This thesis examines the in vitro isolation, proliferation and differentiation of rat adult neural progenitor cells. With a few exceptions, the current methods of isolating progenitor cells rely on the disassociation of brain regions followed by culture in a selective medium. A novel explant protocol for the culture of progenitor cells is described here which has the advantages of greater simplicity and anatomical resolution. Using this protocol it was possible to precisely isolate progenitors from diverse CNS regions including the spinal cord, thalamus and cortex. Once they have been isolated, a key factor in driving the proliferation of progenitors is fibroblast growth factor-2 (FGF-2). This growth factor signals proliferation through its cognate receptors in concert with the extracellular matrix molecule, heparan sulfate. It is shown that both specific and non-specific varieties of heparan sulfate can profoundly influence progenitor proliferation in a specific and competitive manner. This is then explained with a model in which different receptor isoforms perform different signalling functions in progenitor cells. The intracellular pathways controlling proliferation were then examined using signalling pathway inhibitors. It was found that inhibiting p42/44 signalling had no significant effect on proliferation, whilst inhibiting p38, and possibly JNK, reduced proliferation and produced a distinct morphological change. The effectiveness of the inhibitors was confirmed using western blotting. Finally different strategies for differentiating the progenitor cells were examined. It was found that hippocampal progenitors could become electrically active neurons that respond to a variety of neurotransmitters. A significant proportion of cortical progenitors also differentiated into neurons in response to foetal calf serum (FCS). This finding has important implications for the nature of adult stem cells and their potential to differentiate in vitro. A model of growing the progenitors on fresh frozen sections of peripheral nerve showed that the differentiating neurons respond to physiologically relevant substrates. The results detailed here provide useful techniques for the investigation of the potential neural progenitors throughout the CNS. They also make a significant contribution to the understanding of the signalling pathways that govern the proliferation of adult neural progenitors.