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BRAIN Publication-derived

Structural and functional integration of human forebrain organoids with the injured adult rat visual system

Source Jgamadze et al., 2023 · Perelman School of Medicine, University of Pennsylvania · 10.1016/j.stem.2023.01.004

👤 Dennis Jgamadze, James Lim, Zhijian Zhang, Paul Harary, James Germi, Kobina Mensah-Brown, Chris Adam, Ehsan Mirzakhalili, Shikha Singh, Jiahe Ben Gu, Rachel Blue, Mehek Dedhia, Marissa Fu, Fadi Jacob, Xuyu Qian, Kimberly Gagnon, Matthew Sergison, Oceane Fruchet, Imon Rahaman, Huadong Wang, Fuqiang Xu, Rui Xiao, Diego Contreras, John A. Wolf, Hongjun Song, Guo-li Ming, Han-Chiao Isaac Chen ⏱ 88 days 📋 7 phases 🧫 Human iPSC (C1.2, AICS); Human ESC (H9)

Abstract

This protocol describes the generation of dorsal forebrain organoids from human pluripotent stem cells (at day 80–88) and their transplantation into aspiration cavities in the visual cortex of adult rats. The organoid grafts integrate structurally and functionally with the injured host brain, forming synaptic connections with the visual system and responding to visual stimuli with feature selectivity.

Cell source
Human iPSC (C1.2, AICS); Human ESC (H9)
Application
Disease modeling; Functional integration of neural transplants; Brain repair

Protocol overview

52 steps across 7 phases

Maintenance of pluripotent stem cell lines Ongoing, passages every 4–6 days
  1. 1 Culture stem cells on feeder layer
  2. 2 Prepare hES cell media
  3. 3 Passage stem cell cultures
Generation of dorsal forebrain organoids (in vitro) Day 0 to day 80–88
  1. 1 Create embryoid bodies (EBs)
  2. 2 Half-media change to induction media (days 5–6)
  3. 3 Embed EBs in Matrigel (day 7)
  4. 4 Culture embedded EBs in stationary conditions (days 7–14)
  5. 5 Transfer organoids to orbital shaker (day 14)
  6. 6 Culture organoids in differentiation media (days 14–71)
  7. 7 Switch to maturation media (day 71 onwards)
  8. 8 Quality control assessment
  9. 9 Pre-transplantation incubation with necrostatin-1
Animal preparation and organoid transplantation Surgery day (2 days before transplant through post-operative recovery)
  1. 1 Immunosuppression initiation
  2. 2 Pre-operative anesthesia induction
  3. 3 Administer pre-operative medications
  4. 4 Maintain anesthesia and body temperature
  5. 5 Perform craniotomy
  6. 6 Achieve hemostasis and insert organoid
  7. 7 Seal craniotomy with cranioplasty cap
  8. 8 Close surgical incision
  9. 9 Post-operative analgesia and immunosuppression
Immunohistochemistry and histological analysis At 1, 2, or 3 months post-transplantation (mpt)
  1. 1 Perfuse and extract brain tissue
  2. 2 Post-fix and cryoprotect tissue
  3. 3 Prepare frozen sections
  4. 4 Prepare sections for immunostaining
  5. 5 Block and permeabilize tissue
  6. 6 Apply primary antibodies
  7. 7 Wash and apply secondary antibodies
  8. 8 Final wash and mounting
  9. 9 Confocal imaging
Virus-based trans-synaptic tracing (RABV and HSV) At 2 months post-transplantation (7–10 days before sacrifice for RABV; 2 months before sacrifice for HSV)
  1. 1 Modified rabies virus (RABV) retrograde tracing—organoid microinjection
  2. 2 RABV post-injection closure and recovery
  3. 3 Herpes simplex virus (HSV) anterograde tracing—intravitreal injection
  4. 4 HSV expression and tissue harvesting
In vivo extracellular electrophysiology and visual stimulation At 1, 2, or 3 months post-transplantation
  1. 1 Prepare anesthetized animal for recording
  2. 2 Insert reference and ground electrodes
  3. 3 Insert host brain silicon probe
  4. 4 Establish recording plane and reduce isoflurane
  5. 5 Insert organoid silicon probe
  6. 6 Record baseline spontaneous activity
  7. 7 Perform whole-screen flashing light stimulation
  8. 8 Present drifting grating stimuli (orientation tuning)
  9. 9 Chemogenetic validation (optional)
Spike sorting and neural signal analysis Post-recording (offline analysis)
  1. 1 Merge multi-timepoint recordings
  2. 2 Automated spike sorting (Klustakwik)
  3. 3 Manual cluster refinement
  4. 4 Unit quality filtering
  5. 5 Extract single-unit firing parameters
  6. 6 Determine unit depths and exclude border units
  7. 7 Identify light-responsive units
  8. 8 Extract event-related potentials (ERPs)
  9. 9 Calculate orientation selectivity index (OSI)

Full SOP

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Attribution

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

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