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BRAIN Publication-derived

Apolipoprotein E regulates lipid metabolism and α-synuclein pathology in human iPSC-derived cerebral organoids

Source Zhao et al., 2021 · Mayo Clinic · 10.1007/s00401-021-02361-9

👤 Jing Zhao, Wenyan Lu, Yingxue Ren, Yuan Fu, Yuka A. Martens, Francis Shue, Mary D. Davis, Xue Wang, Kai Chen, Fuyao Li, Chia-Chen Liu, Neill R. Graff-Radford, Zbigniew K. Wszolek, Steven G. Younkin, David A. Brafman, Nilüfer Ertekin-Taner, Yan W. Asmann, Dennis W. Dickson, Ziying Xu, Meixia Pan, Xianlin Han, Takahisa Kanekiyo, Guojun Bu ⏱ 90 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the generation and characterization of human iPSC-derived cerebral organoids to study apolipoprotein E (apoE) function in lipid metabolism and α-synuclein pathology. ApoE-deficient cerebral organoids display increased α-synuclein accumulation, lipid droplet formation, and dysregulation of intracellular organelles, phenotypes that are partially rescued by exogenous apoE2 and apoE3 but not apoE4, providing insights into APOE4 as a genetic risk factor for synucleinopathies.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

94 steps across 10 phases

Generation of iPSCs from human skin fibroblasts Days 0–21
  1. 1 Prepare human skin fibroblasts
  2. 2 Electroporate fibroblasts with episomal vectors
  3. 3 Plate transfected fibroblasts
  4. 4 Replace fibroblast medium with pluripotency medium
  5. 5 Isolate and expand iPSC colonies
  6. 6 Passage iPSC colonies with rock inhibitor
Generation of APOE−/− iPSCs via gene editing Days 0–28
  1. 1 Generate APOE−/− iPSCs via Zinc Finger Nucleases (ZFN)
  2. 2 Culture MC0192 iPSCs in mTeSR1 medium
  3. 3 Design and prepare CRISPR gRNA/Cas9 constructs
  4. 4 Transfect MC0192 iPSCs with gRNA/Cas9 plasmids
  5. 5 Screen for APOE knockout efficiency by PCR
  6. 6 Plate single cells in 96-well plates
  7. 7 Extract genomic DNA and perform PCR analysis
  8. 8 Confirm knockouts by DNA sequencing
Cerebral organoid generation Days 0–90
  1. 1 Dissociate iPSC colonies to single cells
  2. 2 Seed cells into U-bottom ultra-low-attachment plates (Day 0)
  3. 3 Add medium A (Day 2)
  4. 4 Add medium A (Day 4)
  5. 5 Transfer EBs to neural induction medium (Day 5)
  6. 6 Embed EBs in Matrigel and transfer to neural expansion medium
  7. 7 Transfer organoids to 10 cm dishes on orbital shaker
  8. 8 Culture organoids for 4 weeks in medium E
  9. 9 Replace medium E with neuronal maturation medium (Day 28)
  10. 10 Continue culture until Day 90
Tissue processing for biochemical analysis Day 30–90
  1. 1 Harvest cerebral organoids
  2. 2 Lyse organoids in RIPA buffer
  3. 3 Sonicate samples
  4. 4 Ultracentrifuge at high speed to obtain soluble fraction
  5. 5 Collect soluble fraction supernatant
  6. 6 Resuspend pellet and sonicate in SDS buffer
  7. 7 Centrifuge SDS samples
  8. 8 Collect SDS fraction supernatant
  9. 9 Determine total protein concentration
Immunostaining and imaging Day 30–90
  1. 1 Harvest and fix cerebral organoids
  2. 2 Wash organoids with PBS
  3. 3 Dehydrate organoids in sucrose solution
  4. 4 Embed organoids in OCT compound
  5. 5 Freeze organoids on dry ice
  6. 6 Section organoids with cryostat
  7. 7 Store frozen sections
  8. 8 Permeabilize sections
  9. 9 Block non-specific binding
  10. 10 Incubate with primary antibodies
  11. 11 Wash sections with PBS
  12. 12 Incubate with secondary antibodies
  13. 13 Final wash and mounting
  14. 14 Image sections
BODIPY staining of lipid droplets Day 30–90
  1. 1 Prepare organoid sections
  2. 2 Wash sections with PBS
  3. 3 Prepare BODIPY and DAPI staining solution
  4. 4 Incubate sections with BODIPY and DAPI
  5. 5 Wash sections with PBS
  6. 6 Mount sections with Vectashield
  7. 7 Image lipid droplets
Western blotting Day 30–90
  1. 1 Prepare RIPA and SDS fractions
  2. 2 Prepare sodium dodecyl sulfate–polyacrylamide gel
  3. 3 Transfer proteins to PVDF membrane
  4. 4 Block membranes
  5. 5 Incubate with primary antibodies
  6. 6 Wash membranes
  7. 7 Incubate with secondary antibodies
  8. 8 Detect signal
  9. 9 Quantify band intensities
  10. 10 Validate antibody specificity with blocking peptides
RT-qPCR analysis Day 30–90
  1. 1 Extract RNA from organoids
  2. 2 Treat RNA with DNase
  3. 3 Purify RNA
  4. 4 Assess RNA quantity and quality
  5. 5 Synthesize cDNA
  6. 6 Prepare qPCR reaction mixture
  7. 7 Perform real-time qPCR
  8. 8 Calculate relative gene expression
  9. 9 Validate results
Glucocerebrosidase (GCase) activity assay Day 30–90
  1. 1 Homogenize cerebral organoids
  2. 2 Prepare cell lysate
  3. 3 Centrifuge lysate
  4. 4 Quantify protein concentration
  5. 5 Prepare assay plate
  6. 6 Add substrate
  7. 7 Incubate reaction
  8. 8 Stop reaction
  9. 9 Read fluorescence
  10. 10 Normalize to protein content
Treatment of cerebral organoids with conditioned media Day 85–90
  1. 1 Prepare conditioned medium from immortalized astrocytes
  2. 2 Replace with serum-free medium
  3. 3 Collect conditioned medium
  4. 4 Normalize apoE concentration
  5. 5 Concentrate conditioned medium
  6. 6 Aliquot and store conditioned medium
  7. 7 Prepare cerebral organoids at Day 90
  8. 8 Perform full medium change
  9. 9 Add conditioned medium
  10. 10 Culture with conditioned medium
  11. 11 Harvest organoids for analysis

Full SOP

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Attribution

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

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