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BRAIN Publication-derived

Generation and Transplantation of Human Cerebral Organoids for Stroke Recovery

Source Cao et al., 2023 · Department of Pharmacology, School of Pharmacy, Nanjing Medical University · 10.1038/s41536-023-00301-7

👤 Shi-Ying Cao, Di Yang, Zhen-Quan Huang, Yu-Hui Lin, Hai-Yin Wu, Lei Chang, Chun-Xia Luo, Yun Xu, Yan Liu, Dong-Ya Zhu ⏱ 180 days 📋 9 phases 🧫 Human iPSC (IMR-90-4), Human ESC (H9), Human iPSC (H9-CAG-ChR2-EYFP)

Abstract

This protocol describes the generation of human cerebral organoids from pluripotent stem cells and their transplantation into photothrombotic stroke-injured mouse brain. Grafted organoids survive long-term in the infarcted core, differentiate into cortical neurons, repair tissue structure, establish synaptic connections with host circuits, and restore sensorimotor function.

Cell source
Human iPSC (IMR-90-4), Human ESC (H9), Human iPSC (H9-CAG-ChR2-EYFP)
Application
Disease modeling (stroke), Tissue repair, Functional restoration

Protocol overview

34 steps across 9 phases

Pluripotent Stem Cell Culture Days -30 to 0 (preparation)
  1. 1 Culture human pluripotent stem cells
Embryoid Body Formation and Rosette Induction Days 0–15 in vitro
  1. 1 Detach pluripotent stem cells and form embryoid bodies
  2. 2 Culture embryoid bodies in neural induction medium
  3. 3 Allow EBs to attach and form rosettes
Cerebral Organoid Formation Days 16–20+ in vitro
  1. 1 Dissect and culture rosette structures
  2. 2 Allow organoids to develop neural tube structures
  3. 3 Continue long-term culture and medium refresh
Lentivirus Transduction of Organoids Days 40–50 in vitro (10 days prior to transplantation)
  1. 1 Prepare lentiviral infection solution for EGFP labeling
  2. 2 Incubate organoids with lentiviral solution
  3. 3 Transfer infected organoids to culture flask
  4. 4 Culture infected organoids before transplantation
Photothrombotic Stroke Model in Mice Surgical day (day 0)
  1. 1 Prepare mouse for surgery
  2. 2 Inject rose bengal dye
  3. 3 Illuminate brain to induce focal stroke
  4. 4 Close surgical site and recover animal
Organoid Transplantation Day 7 post-stroke
  1. 1 Prepare mouse for stereotaxic transplantation
  2. 2 Transplant three cerebral organoids
  3. 3 Close surgical site and recover animal
Long-term Assessment and Functional Studies Days 30–180 post-transplantation
  1. 1 Perform behavioral testing
  2. 2 Prepare acute brain slices for electrophysiology
  3. 3 Recover and equilibrate brain slices
  4. 4 Perform whole-cell patch-clamp electrophysiology on organoid neurons
  5. 5 Test organoid neuron responses to optogenetic stimulation
  6. 6 Perform local field potential (LFP) recordings in distant brain targets
Immunohistochemistry and Cell Composition Analysis Days 45–180 post-transplantation
  1. 1 Prepare organoids or brain tissue for immunostaining
  2. 2 Section tissue and prepare slides
  3. 3 Block nonspecific binding and permeabilize
  4. 4 Incubate sections with primary antibodies
  5. 5 Apply secondary antibodies and fluorescent detection
  6. 6 Mount and image sections
  7. 7 Perform cell counting and quantitative analysis
Chemogenetic Modulation Studies Days 150–180 post-transplantation
  1. 1 Transplant chemogenetically-modified organoids
  2. 2 Validate hM4Di/CNO system in vitro
  3. 3 Perform behavioral testing with CNO treatment

Full SOP

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Attribution

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

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