FIGURE 5.

Presence of Lewy body–like inclusions (LBLIs) in human midbrain‐like organoids (hMLOs) from GBA1 ^−/−::SNCA overexpressing (O/E) embryonic stem cells (ESCs) in the H1 background as well as Parkinson disease patient‐derived induced pluripotent stem cells (iPSCs). (A) Schematic showing CRISPR/Cas9 genome editing to generate H1‐GBA1 ^−/−::SNCA O/E clones. The gRNA targeting sequence is in green; the protospacer adjacent motif (PAM) sequence is in red. Genomic DNA sequencing of the GBA1 ^−/− ESC line showed a homozygous 7‐base pair (bp) deletion. (B) Western blot validation of α‐synuclein (α‐syn) protein levels in the H1‐GBA1 ^−/−::SNCA O/E ESC line upon doxycycline (Dox) treatment using specific antibodies against α‐syn (17 and 19kDa, indicating endogenous and exogenous α‐syn, respectively), glucocerebrosidase (GCase; 60kDa), and hemagglutinin (HA; 19kDa). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Immunostaining of H1‐GBA1 ^−/−::SNCA O/E ESC clones upon Dox treatment with specific antibodies against α‐syn and HA. Scale bar = 100μm. (D) Immunostaining of LBLIs exhibiting halolike α‐syn⁺ immunoreactivity in tyrosine hydroxylase (TH)⁺ neurons of day 90 hMLOs derived from H1‐GBA1 ^−/−::SNCA O/E ESCs (G::S), SNCA triplication iPSCs or control iPSCs with conduritol‐b‐epoxide (CBE) treatment, and GBA1‐iPSCs with and without SNCA overexpression. White arrows indicate LBLIs. Scale bar = 5μm. (E) Immunohistochemical analyses of day 90 hMLOs derived from H1‐GBA1 ^−/−::SNCA O/E ESCs, SNCA triplication iPSCs with CBE treatment, and GBA1‐iPSCs with SNCA overexpression using specific antibodies against α‐syn and ubiquitin (UBQ). The white and black arrows indicate LBLIs and nuclei, respectively. Scale bar = 5μm. (F) Quantification of the diameters of the identified LBLIs (n = 8). (G) Frequency of LBLI occurrence in cryosections of day 90 hMLOs based on α‐syn immunoreactivity. DAPI = 4,6‐diamidino‐2‐phenylindole. [Color figure can be viewed at www.annalsofneurology.org]