Single Cerebral Organoid Mass Spectrometry of Cell-Specific Protein and Glycosphingolipid Traits

Abstract

Cerebral organoids are a prolific research topic and an emerging model system for neurological diseases in human neurobiology. However, the batch-to-batch reproducibility of current cultivation protocols is challenging and thus requires a high-throughput methodology to comprehensively characterize cerebral organoid cytoarchitecture and neural development. We report a mass spectrometry-based protocol to quantify neural tissue cell markers, cell surface lipids, and housekeeping proteins in a single organoid. Profiled traits probe the development of neural stem cells, radial glial cells, neurons, and astrocytes. We assessed the cell population heterogeneity in individually profiled organoids in the early and late neurogenesis stages. Here, we present a unifying view of cell-type specificity of profiled protein and lipid traits in neural tissue. Our workflow characterizes the cytoarchitecture, differentiation stage, and batch cultivation variation on an individual cerebral organoid level.

Apolipoprotein-based regulation of ganglioside metabolism upon secretase activity inhibition in iPSC-derived cerebral organoids

“…All iPSC lines were 1 maintained under standard culture conditions as previously described 20 , using mTESR medium (Stem Cell Technologies) on cell culture plates coated with Geltrex (Thermo Fisher Scientific). Organoids for the experiments described in this study were generated following previously published protocols 12,15,20,21 . To treat the organoids with β and γ-Secretase inhibitors, we followed a previously published protocol with minor modifications 13 .…”

“…Lipid extraction followed by sequential protein extraction was performed, as previously reported by our group 21 . Briefly, the harvested organoids were freeze-dried and homogenized.…”

Domino-like Effect of C112R Mutation on ApoE4 Aggregation and Its Reduction by Alzheimer’s Disease Drug Candidate

“…Cerebral organoids were generated using the protocol described previously [73,74]. Briefly, for spheroid formation, cells were plated at day 0 into non-adherent V-shaped 96-well plates at 2000-3000 cells in 150 μl of mTeSR TM 1 medium with 50 μM ROCK inhibitor (S1049, Selleckchem).…”

“…Non-adherent cell culture plates were prepared with poly(2-hydroxyethyl methacrylate) (poly-HEMA; P3932, Merck) coating; 3) On day 2, the cell culture medium was exchanged for fresh mTeSR TM 1 without ROCK inhibitor. When spheroids reached the size of 400 - 600 µm, fresh Neural Induction Medium [74] was added every day for six days (usually from day 3 to day 8). The next day, twelve organoids were transferred to one 6 cm cell culture dish.…”

“…Geltrex TM was left to solidify as hanging drops on the inverted cell culture dish for 10 min at 37 °C. Solidified Geltrex TM drops with organoids were gently detached from the bottom and cultured without shaking in Cerebral Organoid Differentiation Medium (CODM) [74] without vitamin A for seven days. Subsequently, organoids were cultured in CODM with vitamin A and were moved on an orbital shaker on day 26 (± two days).…”