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BRAIN Publication-derived

Generation of telencephalon cortical organoids from hESCs and JEV infection modeling

Source Zhang et al., 2018 · Department of Anatomy, Histology and Embryology, Shanghai Medical College, Fudan University · 10.1038/s41419-018-0763-y

👤 Boya Zhang, Yangzhige He, Yanpeng Xu, Fan Mo, Tingwei Mi, Qing Sunny Shen, Chunfeng Li, Yali Li, Jing Liu, Yihui Wu, Guilai Chen, Wenliang Zhu, Chengfeng Qin, Baoyang Hu, Guomin Zhou ⏱ 120 days 📋 10 phases 🧫 Human ESC (H9/WA09, Q-CTS-hESC-1)

Abstract

This protocol generates telencephalon cortical organoids from human embryonic stem cells (hESCs) that recapitulate developing human cortex, including multi-layer progenitor zones and all six neuronal subtypes. The organoids are then infected with Japanese encephalitis virus (JEV) to model viral infection, cell death, proliferation suppression, and differential antiviral immunity depending on organoid developmental stage.

Cell source
Human ESC (H9/WA09, Q-CTS-hESC-1)
Application
Disease modeling - Japanese encephalitis virus infection and antiviral immunity

Protocol overview

35 steps across 10 phases

hESC Maintenance and Preparation Ongoing until use
  1. 1 Culture hESC lines on Matrigel
  2. 2 Manual passaging of hESC colonies
Organoid Formation and Neural Patterning (Days 0–22) Day 0 – Day 22
  1. 1 Create cellular aggregates and initiate differentiation
  2. 2 Add ROCK inhibitor (Day 0, first 24 h)
  3. 3 Apply dorsal telencephalon patterning cues (Days 1–7)
  4. 4 Transfer to neural medium with growth factors (Days 8–22)
Organoid Maturation and Neuronal Differentiation (Days 23–120) Day 23 – Day 120
  1. 1 Initiate neuronal differentiation (Day 23 onwards)
  2. 2 Monitor organoid growth and morphology
JEV Virus Preparation and Infection As needed; infection experiments typically at Days 24, 55, and 90+
  1. 1 Prepare JEV stock (SA14 strain)
  2. 2 Determine viral titer by plaque-forming assay
  3. 3 Infect cortical organoids with JEV
  4. 4 Collect supernatant for viral titer measurement
Sample Preparation for Immunofluorescence and Molecular Analysis Upon harvest (typically 8 days post-infection or at specified time points)
  1. 1 Fix organoids with paraformaldehyde
  2. 2 Cryoprotect with sucrose overnight
  3. 3 Embed in O.C.T. compound and freeze
  4. 4 Cryosection organoid blocks
Immunofluorescence and Microscopy Same day as cryosectioning or within 2–3 days
  1. 1 Wash cryosections with Triton-X-100 buffer
  2. 2 Re-fix sections with paraformaldehyde
  3. 3 Block non-specific binding
  4. 4 Incubate with primary antibodies overnight
  5. 5 Incubate with secondary antibodies and stain nuclei
  6. 6 Mount slides and image with confocal microscopy
Gene Expression Analysis by qRT-PCR Same day as infection endpoint or within 1–2 days
  1. 1 Extract total RNA with Trizol
  2. 2 Perform one-step SYBR green qRT-PCR
Protein Analysis by Western Blotting Same day as infection endpoint or within 1–2 days
  1. 1 Lyse organoid samples in RIPA buffer
  2. 2 Quantify protein concentration
  3. 3 Load and run SDS-PAGE gel
  4. 4 Transfer proteins to nitrocellulose membrane
  5. 5 Block and probe with primary antibodies
  6. 6 Incubate with secondary antibodies and visualize
Interferon-β Detection by ELISA Same day as infection endpoint or within 1 day
  1. 1 Collect supernatant from infected organoids
  2. 2 Perform IFN-β ELISA
Electrophysiology (Optional) At specified developmental stages (Day 35 onwards)
  1. 1 Prepare organoid slices in agarose
  2. 2 Recover slices in ACSF
  3. 3 Perform whole-cell patch-clamp recordings

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhang et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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