Student electives for Cemile Kalkan and Randal Stronge. (360G-Wellcome-081118_Z_06_A)

Characterisation of sensory neurons produced by pluripotent mouse neural crest-like stem cells The regulation of cell determination is a central issue in developmental biology. There has been much recent interest in postnatal stem cells and their unexpectedly broad differentiation capacity because of the potential therapeutic implications. We established from neonatal mouse skin three immortal, pluripotent stem cell lines that resemble neural crest stem cells and are capable of producing at least three cell lineages (melanoblasts/melanocytes, neurons and Schwann precursor cells).They promise to be valuable tools for study of cell determination. Neuronal differentiation of these cell lines is the least well characterised. We plan to expose pluripotent neural crest-like stem cells to growth/differentiation factors known to control determination of sensory neurons in short-term cultures: brain derived neurotrophic factor (BDNF), neurotrophic factor (NT3), fibroblast growth factor 2 (FGF2) and nerve growth factor (NGF). We have preliminary data showing that in the presence of these factors very long bipolar neuron-like cells and large, dendritic cells resembling sensory neurons appear in the cultures. The student will repeat and confirm these observations, analysing and recording changes of cell morphology using photo microscopy. He will also study effect of these factors on cell proliferation by counting cell numbers before and some time after growing the cells in the presence of these growth actors by haemocytometer. To test whether morphologically different cells are indeed sensory neurons he will perform immunocytochemical analysis of these cells by using a marker of sensory neurons (substance P) and neuronal markers (neuronal tubulin beta 3 and a neurofilament subunit).