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BRAIN Publication-derived

Generation of midbrain-like organoids (hMLOs) and mDA neuron differentiation from human embryonic stem cells with DNAJC6 mutations

Source Wulansari et al., 2021 · Hanyang University · 10.1126/sciadv.abb1540

👤 Noviana Wulansari, Wahyu Handoko Wibowo Darsono, Hye-Ji Woo, Mi-Yoon Chang, Jinil Kim, Eun-Jin Bae, Woong Sun, Ju-Hyun Lee, Il-Joo Cho, Hyogeun Shin, Seung-Jae Lee, Sang-Hun Lee ⏱ 50 days 📋 8 phases 🧫 Human ESC

Abstract

This protocol describes the generation of human midbrain-like organoids (hMLOs) and ventral midbrain (VM) neural stem cell cultures from human embryonic stem cells (hESCs), including those with DNAJC6 mutations. The hMLOs recapitulate Parkinson's disease pathology including midbrain dopamine neuron degeneration, α-synuclein aggregation, mitochondrial dysfunction, and impaired lysosomal degradation, serving as a platform for studying DNAJC6-linked juvenile Parkinson's disease and testing therapeutic interventions.

Cell source
Human ESC
Application
Disease modeling

Protocol overview

42 steps across 8 phases

hESC maintenance and CRISPR-Cas9-mediated gene editing to generate DNAJC6 mutations Prior to organoid generation
  1. 1 Culture human ESCs on Matrigel-coated plates
  2. 2 Dissociate hESCs and prepare for transfection
  3. 3 Cotransfect hESCs with Cas9 and sgRNA plasmids
  4. 4 Select transfected colonies with puromycin
  5. 5 Expand single-cell clones in 96-well plates
  6. 6 Perform T7E1 assay and Sanger sequencing
  7. 7 Confirm chromosomal integrity by karyotyping
Formation of embryoid bodies (EBs) and initial neural induction DIV0–DIV4
  1. 1 Prepare hESCs for EB formation
  2. 2 Seed cells in ultra-low-attachment 96-well plates to form uniform EBs
  3. 3 Culture EBs for 4 days
  4. 4 Add ventral midbrain (VM) patterning factors on day 4
hMLO expansion and maturation DIV8–DIV50+
  1. 1 Embed organoids in Matrigel and transfer to orbital shaker culture
  2. 2 Transfer organoids to terminal differentiation medium on DIV9
  3. 3 Change medium every 3 days
  4. 4 Continue culture for 50–130 days for full maturation
Derivation of VM-type neural stem cell (NSC) cultures from hMLOs DIV11–DIV15
  1. 1 Prepare hMLOs for NSC isolation
  2. 2 Chop and dissociate hMLOs with Accutase
  3. 3 Plate dissociated cells on vitronectin-coated plates
  4. 4 Culture NSCs with ROCK inhibitor for 24 hours only
  5. 5 Expand NSCs with serial passages at 5–7-day intervals
Differentiation of VM-NSCs into mature dopamine neurons Differentiation day 0–15+
  1. 1 Initiate NSC differentiation by mitogen withdrawal
  2. 2 Culture in differentiation medium for 7–15 days
  3. 3 Analyze dopaminergic differentiation
Molecular characterization and phenotypic analyses Concurrent with organoid and NSC cultures
  1. 1 Perform RNA extraction and qRT-PCR
  2. 2 Perform Western blot analysis
  3. 3 Perform immunofluorescence (ICC) on cultured cells
  4. 4 Perform immunofluorescence (IHC) on hMLO sections
  5. 5 Perform CLARITY imaging for deep tissue visualization
  6. 6 Measure α-synuclein aggregation by Western blot fractionation
  7. 7 Assess α-synuclein aggregation by bimolecular fluorescence complementation (BiFC)
  8. 8 Measure mitochondrial function
  9. 9 Assess autophagy and lysosomal function
  10. 10 Measure dopamine release
  11. 11 Measure reactive oxygen species (ROS)
  12. 12 Perform multielectrode array (MEA) recording for neural activity
  13. 13 Perform RNA-sequencing (RNA-seq)
  14. 14 Perform chromatin immunoprecipitation–qPCR (ChIP-qPCR)
Functional rescue experiments (optional) Concurrent with characterization
  1. 1 Prepare lentiviral vectors expressing DNAJC6 or LMX1A
  2. 2 Transduce mutant NSCs or hMLOs with rescue lentiviruses
  3. 3 Analyze rescue of developmental markers and disease phenotypes
Optional: CRISPR-interference (CRISPRi) gene knockdown validation Concurrent with characterization
  1. 1 Prepare lentiviral CRISPRi vectors targeting DNAJC6 or LMX1A
  2. 2 Transduce WT NSCs with CRISPRi vectors and assess knockdown

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wulansari et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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