Abstract
Here, we present a protocol for the generation of functional midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hESCs), which mimics the development of the human ventral midbrain. We describe steps for hESC proliferation, induction of mDA progenitors, freezing stocks of mDA progenitors as an intermediate starting point to reduce the time to make mDA neurons, and maturation of mDA neurons. The entire protocol is feeder-free and uses chemically defined materials. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2023).1.