Generation of cells for cell-replacement therapy: Specification of neural precursors in vivo and in neural stem cell cultures

Abstract: Cell replacement therapy of neurodegenerative disorders aims to substitute the degenerating cells with new functional neurons. Clinical trails with patients suffering from Parkinson’s or Huntington’s disease have generated proof-of-principle results that neural precursors taken from the developing human brain can survive upon grafting to the diseased brain and provide long-lasting symptomatic relief. However, further development of this type of therapy critically depends on the generation of an unlimited and standardized source of neural precursors that after transplantation differentiate into the proper neuronal subtypes. This requires knowledge on the molecular mechanisms responsible for the specification of neurons during development, and how cells with the potential for regional specific neuronal differentiation can be expanded in culture. The work of this thesis has focused on the role of the proneural gene Neurogenin2 in specification of the midbrain dopaminergic (mesDA) neurons, the cell population that degenerate in Parkinson’s disease. Additionally, we have studied to what extent neural stem cells isolated from the developing brain and expanded under growth-factor stimulation in culture maintain their regional specification. We show that Neurogenin2 is required in vivo for proper development of the mesDA neuron system, more specifically for the immature mesDA neuron precursors to adopt a neuronal fate. Furthermore, we successfully applied a new culture system for expansion of neural stem cells, the neural stem cell (NS cell) cultures, to neural precursors from different regions of the developing brain. We showed that even after extensive expansion cells in the NS cell cultures retain their capacity to form neurons. Furthermore, the expanded cells harbor regional differences in their growth properties and to some extend in their gene expression profile. This show that the NS cell culture is an attractive alternative to the traditionally and more commonly used neurosphere culture system for expansion of fetal neural stem cells. Unfortunately, our investigations also showed that neither in the neurosphere nor in the NS cell culture system cells with the characteristic of mesDA neuron precursors are expandable. These results are valuable for further progression in neural stem cell research and particular for improvement of the existing protocols for generating mesDA neurons from expanded neural stem cells.