Single-cell Transcriptomics Reveals Correct Developmental Dynamics and High-Quality Midbrain Cell Types by Improved Hesc Differentiation

Stem cell technologies provide new opportunities for modeling cells in health and disease and for regenerative medicine. In both cases, developmental knowledge and defining the molecular properties and quality of the cell types is essential. In this study, we identify devel-opmental factors important for the differentiation of human embryonic stem cells (hESCs) into functional midbrain dopaminergic (mDA) neurons. We found that laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3b inhibition plus FGF8b, improved midbrain patterning. In addition, neurogenesis and differentiation were enhanced by activation of liver X recep-tors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dy-namics similar to that of the endogenous human ventral midbrain and the emergence of high-quality molecularly defined midbrain cell types, including mDA neurons. Our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly defined hESC-derived cell types in Parkinson disease.