BMP/SMAD pathway promotes neurogenesis of midbrain dopaminergic neuronsin vivoand in human induced pluripotent and neural stem cells

The embryonic formation of midbrain dopaminergic (mDA) neurons in vivo provides critical guidelines for the in vitro differentiation of mDA neurons from stem cells, currently being developed for Parkinson9s disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway for in vivo specification of mammalian mDA neurons is virtually unknown. We report here that BMP5/7 deficient mice ( Bmp5 -/- ; Bmp7 -/- ) lack mDA neurons, caused by reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations in Bmp5 -/- ; Bmp7 -/- mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivating SMAD1 in neural stem cells of mice in vivo ( Smad1 Nes ) hampered the differentiation of progenitor cells into mDA neurons by preventing cell cycle exit, especially of TH + SOX6 + (tyrosine hydroxylase, SRY-box 6) and TH + GIRK2 + (potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. Notably, BMP5/7 robustly increased the in vitro differentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to 3-fold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesis in vivo that balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem cell-derived mDA neurons in vitro , highlighting the importance of sequential BMP/SMAD inhibition and activation in this process. SIGNIFICANCE STATEMENT We identify BMP/SMAD signaling as novel essential pathway regulating the development of mammalian mDA neurons in vivo and provide insights into the molecular mechanisms of this process. BMP5/7 regulate MSX1/2 and SHH expression to direct mDA neurogenesis. Moreover, the BMP signaling component SMAD1 controls the differentiation of mDA progenitors, particularly to substantia nigra neurons, by directing their cell cycle exit. Importantly, BMP5/7 increase robustly the differentiation of human induced pluripotent and induced neural stem cells to mDA neurons. BMP/SMAD are routinely inhibited in initial stages of stem cell differentiation protocols currently being developed for Parkinson9s disease cell replacement therapies. Thus, our findings on opposing roles of the BMP/SMAD pathway during in vitro mDA neurogenesis might significantly improve these procedures.