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BRAIN Publication-derived

Generation of Spatially Arranged Ventral Midbrain–Striatum–Cortex Assembloids (MISCOs) from Human Pluripotent Stem Cells

Source Reumann et al., 2023 · Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences · 10.1038/s41592-023-02080-x

👤 Daniel Reumann, Christian Krauditsch, Maria Novatchkova, Edoardo Sozzi, Sakurako Nagumo Wong, Michael Zabolocki, Marthe Priouret, Balint Doleschall, Kaja I. Ritzau-Reid, Marielle Piber, Ilaria Morassut, Charles Fieseler, Alessandro Fiorenzano, Molly M. Stevens, Manuel Zimmer, Cedric Bardy, Malin Parmar, Jürgen A. Knoblich ⏱ 130 days 📋 10 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol describes the generation of midbrain-striatum-cortex organoid assembloids (MISCOs) from human pluripotent stem cells, which recapitulate key morphological and functional features of the human dopaminergic system. MISCOs develop functional dopaminergic innervation between spatially organized ventral midbrain, striatal, and cortical tissues, enabling study of dopaminergic circuit formation, maturation, and responses to perturbations relevant for Parkinson's disease and addiction research.

Cell source
Human iPSC, Human ESC
Application
Disease modeling, Developmental study, Cell replacement therapy screening

Protocol overview

50 steps across 10 phases

Cell Maintenance and Preparation Day -7 to Day 0
  1. 1 Culture hPSCs on Matrigel
  2. 2 Passage hPSCs
  3. 3 Prepare cells for organoid generation
Ventral Midbrain Organoid Generation Day 0 to Day 8
  1. 1 Initiate ventral midbrain patterning
  2. 2 Medium exchange on day 2
  3. 3 Add ventralization and caudalizing factors on day 4
  4. 4 Medium exchange on day 6
  5. 5 Transfer to 10 cm plate on day 8
Striatal Organoid Generation Day 0 to Day 8
  1. 1 Initiate striatal patterning
  2. 2 Medium exchange on day 2
  3. 3 Medium exchange on day 4
  4. 4 Medium exchange on day 6
  5. 5 Transfer to 10 cm plate on day 8
Cortical Organoid Generation Day 0 to Day 13
  1. 1 Initiate cortical patterning
  2. 2 Medium exchange on day 3
  3. 3 Sequential neural induction medium additions (days 5, 7, 9)
  4. 4 Transfer to 10 cm plate on day 11
  5. 5 Add CHIR99021 on day 12
  6. 6 Medium exchange on day 13
Common Organoid Maturation (All Types) Day 16 to Day 60+
  1. 1 Switch to Improved+A medium around day 16
  2. 2 Transfer to orbital shaker on day 20
  3. 3 Continue culture to day 60
  4. 4 Transition to Brainphys medium at day 60
PDMS Mold Preparation for Fusion Prior to day 20-25
  1. 1 Design and 3D print negative molds
  2. 2 Smooth PLA mold surface
  3. 3 Cast PDMS positive mold
  4. 4 Remove and clean PDMS mold
Organoid Fusion into MISCOs Day 20-25
  1. 1 Coat mold with anti-adherence solution
  2. 2 Position cortical organoids in mold
  3. 3 Position striatal organoids in mold
  4. 4 Position ventral midbrain organoids in mold
  5. 5 Remove residual medium and add Matrigel
  6. 6 Incubate fusions in incubator
  7. 7 Remove fused organoids from mold
  8. 8 Transfer MISCOs to orbital shaker
Optional: Ventral Midbrain Progenitor Injection into MISCOs Day 40-50
  1. 1 Prepare ventral midbrain progenitor cells
  2. 2 Prepare injection solution
  3. 3 Prepare glass capillaries
  4. 4 Transfer MISCO to injection plate and remove medium
  5. 5 Inject ventral midbrain progenitors
  6. 6 Allow organoid recovery
  7. 7 Culture injected MISCOs without shaking
Optional: Cocaine Treatment Day 40 to Day 130
  1. 1 Prepare cocaine treatment solution
  2. 2 Apply cocaine treatment
  3. 3 Perform medium exchange and cocaine removal
Analysis and Characterization Day 20 onwards
  1. 1 Perform qPCR for FOXA2 expression
  2. 2 Perform immunohistochemistry
  3. 3 Perform tissue clearing and 3D imaging
  4. 4 Perform bulk RNA sequencing
  5. 5 Perform single-cell RNA sequencing

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Reumann et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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