Modeling Parkinson’s disease in midbrain-like organoids

Generation of midbrain-specific organoids. a Illustration of the conditions used to differentiate spherical midbrain floor plate neural progenitor cells (mfNPCs) into human midbrain-specific organoids (hMOs). mfNPCs are stably expandable up to passage 15; day X indicates the start of dopaminergic differentiation. SB？=？SB-431542, LDN？=？LDN-193189, SAG？=？sonic hedgehog agonist, BD？=？brain-derived neurotrophic factor, GD？=？glial cell-derived neurotrophic factor. b Maximum intensity projection of midbrain dopaminergic neuron (mDAN) markers TH and FOXA2 in 70-day-old hMO sections. Scale bar is 100？μm. hMOs derived from mfNPC lines H1–3 (image shows line H3). c Immunohistological stainings of mDAN markers TH, FOXA2, EN1, and LMX1A in the center of hMO sections. Scale bar is 50？μm. hMOs derived from mfNPC lines H1–3 (images shows line H2). d Bright-field image showing neurons obtained from three-dimensional (3D) culture of line H4 where a recorded cell is attached to the patch-clamp pipette tip (×10 magnification). Scale bar is 20？μm. Representative current-clamp trace of the pace-making firing activity obtained from the recorded mDAN is shown. Experiment conducted with mfNPC lines H4, P2-GC, and P3-GC, in total 26 cells have been recorded, 12 of these showed a similar pattern; representative images and traces of line H4. e Representative immunohistological stainings of mDAN markers FOXA2, TH, dopamine decarboxylase (DDC), and dopamine transporter (DAT) at the edge of an organoid. Scale bar is 20？μm. hMOs were derived from mfNPC lines H1–4 (images show lines H3 and H1). f Appearance of dark granules in hMOs at day 255, derived from mfNPC line H1. Scale bar is 500？μm. Fontana-Masson staining reveals neuromelanin-like granules at the edge of the organoid section after 100 days in culture, derived from mfNPCs line H4. Scale bar is 50？μm. g Representative immunohistological staining of spherical maintenance mfNPCs and 70-day hMO sections (50？μm thickness, taken at the edge of a section) for TH and neurotransmitter dopamine (DA). Scale bar is 50？μm. hMOs derived from mfNPC lines H1–3 (images show line H3). h Quantitative analysis of DA extracted from the supernatant of mfNPCs and hMOs, *p <？0.05. The two time points analyzed in all the experiments were 35 and 70 days of differentiation. Data are presented as mean？±？SEM (mfNPC line H3, 4 different passages, n？=？4