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BRAIN Publication-derived

Neural cell engraftment therapy for sporadic Creutzfeldt-Jakob disease restores neuroelectrophysiological parameters in a cerebral organoid model

Source Williams et al., 2023 · National Institute of Allergy and Infectious Diseases, National Institutes of Health · 10.1186/s13287-023-03591-2

👤 Katie Williams, Simote T. Foliaki, Brent Race, Anna Smith, Tina Thomas, Bradley R. Groveman, Cathryn L. Haigh ⏱ 180 days 📋 12 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human cerebral organoids from iPSCs, their infection with sporadic Creutzfeldt-Jakob disease (sCJD) prions, and subsequent seeding with neural progenitor cells (NPCs) to assess therapeutic effects on neuroelectrophysiological function and disease parameters. NPCs successfully integrated into infected organoids and restored impaired neuronal spike rates despite only modest reductions in prion accumulation.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

49 steps across 12 phases

iPSC culture and maintenance Ongoing
  1. 1 Routine culture of hu-iPSCs
  2. 2 Passage iPSC colonies
Embryoid body (EB) production Days 0–4
  1. 1 Seed cells for EB formation
  2. 2 Add Y27632 on day 0
  3. 3 Feed EBs on days 2 and 4
Neural induction and Matrigel embedding Days 5–10
  1. 1 Transfer to neural induction medium
  2. 2 Embed in Matrigel
  3. 3 Transfer organoids to maintenance culture
  4. 4 Switch to B27 plus retinoic acid on day 10
  5. 5 Establish agitated long-term culture
NPC differentiation and preparation 11 days prior to CO embedding
  1. 1 Thaw NPCs and passage
  2. 2 Collect and count NPCs
  3. 3 Prepare NPC-Matrigel mixture
NPC seeding into COs Day 90 post-infection (dpi)
  1. 1 Prepare organoids for seeding
  2. 2 Apply NPC-Matrigel mixture
  3. 3 Coat organoids in Matrigel
  4. 4 Allow Matrigel to solidify
  5. 5 Transfer seeded organoids to culture
  6. 6 Prepare mock-seeded controls
sCJD prion infection Day 0 of infection
  1. 1 Prepare inoculum
  2. 2 Inoculate organoids
  3. 3 Add fresh media at 24 hours
  4. 4 Perform full media exchange at day 7
  5. 5 Maintain infected organoids
Organoid viability assessment Days 0–180 dpi
  1. 1 Prestoblue metabolism assay
  2. 2 LDH cytotoxicity assay
Prion infection parameter assessment Day 180 dpi
  1. 1 Prepare organoid homogenates
  2. 2 RT-QuIC assay
  3. 3 Proteinase-K digestion and Western blotting
Neuroelectrophysiology recording Day 180 dpi
  1. 1 Coat MEA wells with PEI and laminin
  2. 2 Plate organoids on MEA
  3. 3 Incubate before recording
  4. 4 Record local field potential
Immunohistochemistry and immunofluorescence Day 180 dpi
  1. 1 Fix organoids for histology
  2. 2 Routine H&E staining
  3. 3 Immunohistochemistry for PrP
  4. 4 Immunofluorescence for NPC characterization
  5. 5 MAP2 staining of organoids
  6. 6 General organoid characterization IHC
  7. 7 Immunofluorescence on frozen sections
Cell signaling analysis Day 180 dpi
  1. 1 Prepare samples for BioPlex
  2. 2 Quantify total protein
  3. 3 Run BioPlex cell signaling assays
Western blotting for neuronal and glial markers Day 180 dpi
  1. 1 Prepare samples (non-PK treated)
  2. 2 Run gels and transfer
  3. 3 Probe with primary antibodies
  4. 4 Probe with secondary antibodies
  5. 5 Visualize bands
  6. 6 Quantification

Full SOP

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Attribution

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

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