Fetal mouse mesencephalic NPCs generate dopaminergic neurons from post-mitotic precursors and maintain long-term neural but not dopaminergic potential in vitro

Abstract

Stem cells have one major advantage over primary cells for regenerative therapies in neurodegenerative diseases. They are able to self-renew making sufficient quantities of cells available for transplantation. Embryonic stem cells and fetal neural progenitor cells (NPCs) have been transplanted into models for PD with functional recovery of motor deficits. However, their precise characteristics are still unknown and ideal conditions for their long-term expansion and differentiation into dopamine neurons remain to be explored. Mouse fetal NPCs are commonly grown as characteristic neurospheres, but they also proliferate under monolayer culture conditions. We investigated the proliferative behavior and dopaminergic differentiation capacity of fetal mouse midbrain NPCs derived from E10 to E14 embryos expanded either as neurosphere or monolayer culture. We found similar proliferation capacities in NPCs of all embryonic stages. Neuronal differentiation capacity is higher in neurosphere cultures compared to monolayer NPCs and persists in long-term cultures. We did not find dopaminergic differentiation in long-term expanded mouse NPC types, which is in contrast to rat and human fetal midbrain NPCs. Mouse NPCs generate dopaminergic neurons until up to three weeks in vitro but they do not incorporate BrdU. Quantitative analysis showed that they were not just primary neurons from the isolation process but formed to a great extent in vitro during differentiation suggesting that they are formed by promotion of post-mitotic neuroblasts. A detailed transcription profile reveals de-specification processes during in vitro cultivation, which matches their NPC behavior. We provide the constitutive work for studies using fetal midbrain NPCs of mouse including transplantation studies and transgenic models.