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BRAIN Publication-derived

Fusion of Regionally Specified hPSC-Derived Organoids: Thalamic

Source Xiang et al. · Harvard Medical School / Memorial Sloan Kettering Cancer Center, New York, USA · 10.1016/j.stem.2018.12.015

👤 Xiang Y, Tanaka Y, Patterson B, Kang YJ, Govindaiah G, Roselaar N, Cakir B, Kim KY, Lomber AP, Bhatt SM, Bhatt RM, Bhatt KJ, Park IH ⏱ 100 days 📋 5 phases 🧫 Human ESC / Human iPSC

Abstract

Xiang et al. developed a protocol for generating thalamic organoids from human pluripotent stem cells using insulin and SHH signaling, which when fused with cortical organoids form thalamocortical assembloids with reciprocal axonal projections. This Cell Stem Cell 2019 study established the first model for studying thalamocortical circuit formation and related neurological disorders.

Cell source
Human ESC / Human iPSC
Application
Thalamocortical Circuit Modeling

Protocol overview

20 steps across 5 phases

hESC Dissociation and Embryoid Body Formation Day 0–2
  1. 1 Dissociate hESCs into single cells
  2. 2 Plate cells in induction media with dual SMAD inhibition
  3. 3 First media change on Day 2
  4. 4 Second media change on Day 4
  5. 5 Third media change on Day 6
Transition to Spinning Culture and Thalamic Patterning Day 8–16
  1. 6 Transfer organoids to spinning culture
  2. 7 Initiate thalamic patterning with BMP7 and PD0325901
Neural Differentiation and Maturation Day 16 onwards
  1. 8 Switch to differentiation media with neurotrophic factors
  2. 9 Media replenishment schedule for maturation phase
Organoid Fusion (hThO + hCO) Day 18–22 (fusion start) onwards
  1. 10 Select organoids for fusion
  2. 11 Co-culture organoid pairs
  3. 12 First media change post-fusion (Day 20)
  4. 13 Transfer fused organoids to larger wells
  5. 14 Continue culture with standard differentiation media
Characterization and Analysis Day 18–100+
  1. 15 Perform qPCR analysis for regional markers
  2. 16 Perform immunostaining for regional and neuronal markers
  3. 17 Live imaging of axon projections in fused organoids
  4. 18 Quantify axon targeting by range index analysis
  5. 19 Perform patch-clamp electrophysiology
  6. 20 Perform calcium imaging for spontaneous activity

Full SOP

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Attribution

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

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