Fig 1: Generation and characterization of human midbrain-like organoids (hMLOs) and GBA1 +/-, GBA1 -/-, and SNCA overexpressing (O/E) isogenic embryonic stem cells (ESCs). (A) Schematic diagrams of a differentiation protocol to generate hMLOs from starting population of human pluripotent stem cells (hPSCs). (B) Immunostaining of day 60 hMLOs using antibodies against tyrosine hydroxylase (TH) and dopamine transporter (DAT), markers of mature dopaminergic neurons, with quantification (mean ± standard error of the mean [SEM]; n = 3). Scale bar = 10µm. (C) Neurons immunostained with antibodies against TH and calbindin (top panels) or TH and GIRK2 (bottom panels) in day 90 hMLOs. Scale bar = 10µm. (D) A representative voltage trace obtained from TH+ neurons in response to hyperpolarizing current pulse. TH+ neurons displayed rebound depolarization as well as a typical sag. Scale bar = 10µm. (E) Representative traces of spontaneous action potentials from TH+ neurons. (F) Glucocerebrosidase (GCase) activity in day 30 wild-type (W/T), GBA1 +/-, and GBA1 -/- hMLOs (mean ± SEM; **p = 0.0015, ****p < 0.0001; n = 3). (G) a-Iduronate-2-sulfatase (a-i-2-sulf) activity in day 30 W/T, GBA1 +/-, and GBA1 -/- hMLOs (mean ± SEM; n.s. = no significance; n = 3). (H) Principal component (PC) analysis of lipidomics data from W/T and GBA1 -/- hMLOs at days 30, 60, and 90. (I) Measurement of galactosyl/glucosylceramide in W/T and GBA1 -/- hMLOs demonstrating the specificity of GBA1 loss-of-function (mean ± SEM; *p < 0.05, **p < 0.01 ***p < 0.001; n = 3). (J) Quantitative measurements of ceramide, dihydrosphingomyelin (DHSM), and sphingomyelin in W/T and GBA1 -/- hMLOs (mean ± SEM; *p < 0.05; n = 3). (K) Schematic of the use of lentiviral constructs to generate a doxycycline-inducible SNCA O/E ESC line. (L) Western blot validation of a-synuclein (a-syn) overexpression in ESCs with and without doxycycline (Dox) treatment using specific antibodies against a-syn (17 and 19kDa, indicating endogenous and exogenous a-syn, respectively) and hemagglutinin (HA; 19kDa). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (M) Semiquantitative analysis of the Western blot data for Dox-induced a-syn expression (mean ± SEM; *p = 0.04; n = 3). BDNF = brain-derived neurotrophic factor; DAPI = 4,6-diamidino-2-phenylindole; FGF = fibroblast growth factor; GDNF = glia-derived neurotrophic factorl pGK = phosphoglycerate kinase; rtTA = reverse tetracycline-controlled transactivator; SHH = sonic hedgehog; SMAD = a name of a structurally similar protein family. The abbreviation refers to the homologies to the C. elegans SMA ("small" worm phenotype) and MAD family ("Mothers Against Decapentaplegic") of genes in Drosophila (from Wikipedia); tetO = tetracycline operator; UbC = Ubiquitin C; WNT = wingless-related integration site; WPRE = Woodchuck hepatitis virus posttranslational regulatory element. [Color figure can be viewed at www.annalsofneurology.org]