Exploring iPSCs, NESCs, and Striatum Organoids: A Comprehensive Guide

iPSCs and NESCs

Striatum Organoids

Striatum organoids (StrOs) are formed using an adapted protocol (C4) from Miura et al. (2020). iPSCs, approximately 70% confluent, undergo spheroid formation after overnight treatment with 1% DMSO in Essential 8 medium. On the day before spheroid formation (D -1), cells are incubated in Accutase, resuspended in Essential 8 medium with 20 μM Ri, and counted. For C4 condition, 10,000 cells are added to U-bottom BIOFLOAT™ 96-well plates, centrifuged, and incubated at 37°C with 5% CO2. Spheroids are formed over two days, with medium changes starting at D1.

From D6, spheroids start differentiating into StrOs. The differentiation medium includes Neurobasal-A, B-27 without Vitamin A, Penicillin/Streptomycin, GlutaMAX, IWP-2, and Activin A. After D17, medium changes every 3-4 days promote neuronal differentiation with BDNF, NT-3, AA, and cAMP.

Other conditions (RA and SR) use similar protocols with adjusted media compositions. Cells are plated in U-bottom plates at 9000 cells per well, with steps for induction and differentiation.

Midbrain Organoids

Midbrain organoids (MOs) are derived from NESCs following a modified method from Monzel et al. (2017). At D0, 9000 NESCs are added to U-bottom BIOFLOAT™ plates, cultured for two days, and switched to a differentiation medium containing purmorphamine.

Assembloids

Midbrain and striatum organoids combine to form assembloids. The co-culture medium is optimized through testing various formulations. The ideal co-culture medium, N2B27, contains DMEM F12/Neurobasal, N2 supplement, B-27, GlutaMAX, and growth factors (BDNF, GDNF, NT-3, AA, cAMP). At Day 0 of assembloid creation, StrOs and MOs are merged, receiving the co-culture medium. After four days, they are transferred to ultra-low attachment plates for continued culture.

For inducing progerin overexpression, assemblies from progerin-expressing cell lines are treated with doxycycline.

ATP and LDH Assays

ATP levels in assembloids are measured using CellTiter-Glo® 3D assays. LDH toxicity is assessed from media samples frozen at collection. Both assays involve luminescence measurement and normalization to assembloid area.

Western Blotting

Samples for Western blotting are lysed in RIPA buffer, sonicated, and centrifuged. Protein concentrations are standardized, and samples are subjected to SDS-PAGE and transferred to PVDF membranes for antibody incubation, followed by detection with imaging systems.

Flow Cytometry

Flow cytometry assesses live GFF+ cells in embedded assembloids. Samples are dissociated using papain and accutase, followed by staining and analysis with a flow cytometer.

Immunofluorescence Staining

Immunofluorescence for iPSCs and NESCs involves cell fixation, permeabilization, blocking, and incubation with primary and secondary antibodies. Organoids and assembloids are similarly processed before imaging.

Light Sheet Fluorescence Expansion Microscopy

Organoids undergo preparation to expand before imaging. Samples are analyzed using light sheet microscopy to observe three-dimensional structures.

Microelectrode Array (MEA) Analysis

Basal activity of assembloids is recorded using MEAs. Plates are pre-treated, and assembloids placed at electrode centers for electrical activity recording. Signals are analyzed for connectivity and propagation characteristics between different areas.

Summary

This structured approach, focusing on detailed methodologies regarding organoid generation, assays, and imaging techniques, enhances our understanding of cellular dynamics in neural tissue.