Generation of Midbrain-like Organoids (hMLOs) from Human Pluripotent Stem Cells
Source Jo et al. · National University of Singapore; National Institute of Mental Health, USA
Abstract
This protocol describes the generation of midbrain-like organoids (hMLOs) from human pluripotent stem cells that develop functional dopaminergic and neuromelanin-producing neurons. hMLOs are patterned using SHH and FGF8 signaling, embedded in Matrigel, and cultured in differentiation medium to produce mature midbrain tissue with dopaminergic neurons expressing TH, MAP2, and neuromelanin by day 60-146.
Protocol overview
16 steps across 3 phases
- 1 Dissociate hPSCs to single cells
- 2 Plate cells in low-adhesion 96-well plates
- 3 Add ROCK inhibitor Y27632
- 4 Change neuronal induction medium
- 5 Add midbrain patterning factors
- 1 Monitor neuroectodermal bud extrusion
- 2 Remove culture medium completely
- 3 Embed in reduced growth factor Matrigel
- 4 Allow Matrigel to solidify
- 5 Culture in tissue growth induction medium
- 6 Transfer organoids to ultra-low-attachment plates
- 1 Culture in final differentiation medium
- 2 Use orbital shaker for culture
- 3 Include antibiotics to prevent contamination
- 4 Change medium every 3 days
- 5 Culture until day 60 or beyond
Full SOP
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Attribution
This SOP was authored by Organthis based on the published method in Jo et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.
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