Single cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation

Summary Stem cell technologies provide new opportunities for modeling cells in the healthy and diseased states and for regenerative medicine. In both cases developmental knowledge as well as the quality and molecular properties of the cells are essential for their future application. In this study we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons. We found that Laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3β inhibition plus FGF8b, improved midbrain patterning. In addition, mDA neurogenesis and differentiation was enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high quality molecularly-defined midbrain cell types, including mDA neurons that become functional. Thus, our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly-defined hESC-derived midbrain cell types in Parkinson’s disease.