Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use

Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson’s disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson’s disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies. [Display omitted] •Scalable and reproducible mDA neuron differentiation via biphasic WNT activation•EN1 is required to mediate effects of biphasic WNT activation•Protocol minimizes diencephalic, hindbrain, and non-neural contaminants•Cryopreserved mDA precursors yield A9 mDA neurons in vivo and rescue PD-rat model Studer, Tabar, and colleagues present a midbrain dopamine neuron differentiation protocol that uses biphasic WNT activation to optimize the induction of midbrain identity and to avoid inappropriate neural and non-neural contaminants. The resulting cryopreserved, off-the-shelf product induces functional recovery in parkinsonian rats and may be suitable for human translational use.