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BRAIN Publication-derived

Developing human pluripotent stem cell-based cerebral organoids with a controllable microglia ratio for modeling brain development and pathology

Source Xu et al., 2021 · Rutgers University · 10.1016/j.stemcr.2021.06.011

👤 Ranjie Xu, Andrew J. Boreland, Xiaoxi Li, Caroline Erickson, Mengmeng Jin, Colm Atkins, Zhiping P. Pang, Brian P. Daniels, Peng Jiang ⏱ 40 days 📋 5 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol generates microglia-containing cerebral organoids by co-culturing hPSC-derived primitive neural progenitor cells (pNPCs) and primitive macrophage progenitors (PMPs) in 3D culture. The resulting organoids contain ~8% microglia (mimicking in vivo ratios), develop functional neurons and astrocytes, and enable study of microglial functions including phagocytosis, synaptic pruning, and responses to viral infection such as Zika virus.

Cell source
Human iPSC, Human ESC
Application
Disease modeling (Zika virus infection), developmental neurobiology, pathology studies

Protocol overview

23 steps across 5 phases

Neural progenitor cell (pNPC) generation and expansion Day 0–21
  1. 1 Dual SMAD inhibition for neural induction
  2. 2 Plate EBs on Matrigel-coated dishes
  3. 3 Manual isolation of neural rosettes
  4. 4 Expand pNPCs in proliferation medium
  5. 5 Dissociate pNPCs into single cells
Primitive macrophage progenitor (PMP) generation and collection Day 0–35
  1. 1 Generate yolk sac embryoid bodies (YS-EBs)
  2. 2 Plate YS-EBs and initiate myeloid differentiation
  3. 3 Collect PMPs from supernatant
  4. 4 Assess PMP identity and proliferation
Co-culture of pNPCs and PMPs: organoid initiation and proliferation stage Day 24–35
  1. 1 Co-plate pNPCs and PMPs in ultra-low-attachment plates
  2. 2 Culture in 96-well plates for initial aggregation
  3. 3 Transfer organoids to 6-well plates
  4. 4 Initiate orbital shaking and patterning (optional)
  5. 5 Maintain proliferation stage through day 35
Neural differentiation and microglia maturation stage Day 35 onwards
  1. 1 Switch to neural differentiation medium
  2. 2 Maintain organoids in differentiation medium
  3. 3 Optional: Enhance neuronal maturation with BrainPhys medium
  4. 4 Assess microglia and neural markers
Functional assessment and downstream applications Day 40 onwards (dependent on application)
  1. 1 Assess microglia phagocytic function
  2. 2 Assess synaptic pruning by microglia
  3. 3 Perform whole-cell patch-clamp recordings
  4. 4 Perform Zika virus infection (optional disease model)
  5. 5 Measure gene expression by qRT-PCR

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Xu et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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