Intrinsic and extrinsic factors controlling the differentiation of human midbrain progenitor cells

Sammanfattning: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor deficits. Levodopa treatment provides marked symptomatic relief. However, within 5-10 years after the start of Levodopa treatment, most PD patients display a gradual loss of drug efficacy. Importantly, the neuropathology of PD is dominated by a marked loss of dopaminergic neurons located in the substantia nigra. This makes PD a suitable target for cell replacement therapy strategies. Clinical grafting trials have proved that cell replacement therapies may be effective in PD. So far, dopaminergic progenitors (and neurons) harvested from the embryonic ventral midbrain have been used to replace degenerated ventral midbrain dopaminergic neurons. The limited availability of tissue from embryos and the associated technical and ethical difficulties have lead to a search for alternative sources of transplantable cells. The search has identified stem and neural progenitor cells as possible sources. The idea is that these can be expanded in vitro for banking and then differentiated into dopaminergic neurons just prior to implantation into patients. This thesis focuses on the characterization of three potential human cell sources for the generation of transplantable midbrain dopaminergic progenitors and neurons. First, we studied neural stem cells obtained from the human embryonic and fetal forebrain. We observed induction of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in human fetal forebrain-derived neural stem cells differentiated under defined conditions in vitro. These tyrosine hydroxylase-expressing neurons were lost upon transplantation into a rat model of PD. To further characterize dopaminergic differentiation of human cells, we designed and tested a focused stem cell microarray(NeuroStem Chip) and performed gene expression profiling of human embryonic stem cells. Using this platform, we identified several genes expressed during proliferation of human embryonic stem cells and their early differentiation into dopaminergic neurons. We also employed the focused microarray to characterize immortalized cell lines derived from the human embryonic ventral midbrain. We observed significant up-regulation of mRNA levels of multiple dopaminergic neuron-related markers as well as novel secreted growth factors in one of the cell lines (MesC2.10 cells) undergoing differentiation. When grafting MesC2.10 cells into a rat model of PD, these cells ceased to express tyrosine hydroxylase. Work in this thesis also focused on the downstream targets of a known neuroprotective secreted growth factor (glial cell line-derived neurotrophic factor, GDNF) in midbrain dopaminergic neurons. We found that the protein Delta-like-1 homologue was highly up-regulated in the ventral midbrain following intrastriatal delivery of GDNF, and that it is also expressed in the midbrain during normal development. In conclusion, the studies included in this thesis provide new insights into the use of different human cell sources i) for cell replacement therapy in PD and ii) as platforms to identify genes expressed during normal development of midbrain dopaminergic neurons.