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BRAIN Publication-derived

AstTau: A 3D iPSC-Derived Neuron-Astrocyte Assembloid System for Modeling Tau Oligomer-Induced Neurodegeneration

Source Rickner et al., 2022 · Boston University · 10.1038/s41467-022-34005-1

👤 Hannah Drew Rickner, Lulu Jiang, Rui Hong, Nicholas K. O'Neill, Chromewell A. Mojica, Benjamin J. Snyder, Lushuang Zhang, Dipan Shaw, Maria Medalla, Benjamin Wolozin, Christine S. Cheng ⏱ 21 days 📋 5 phases 🧫 Human iPSC

Abstract

The AstTau system combines iPSC-derived neurons and astrocytes in a 3D spheroidal culture to rapidly model tauopathy pathology induced by seeding with human tau oligomers. Over 21 days of culture, the system recapitulates key pathological features of human tau-mediated neurodegeneration including tau misfolding, phosphorylation, aggregation, neuronal loss, and reactive astrogliosis, enabling high-throughput mechanistic studies and drug screening.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

25 steps across 5 phases

Oligomeric Tau Extraction and Preparation Preparation before cell culture
  1. 1 Collect brain tissue from PS19 P301S transgenic mice
  2. 2 Homogenize tissue in extraction buffer
  3. 3 First centrifugation step
  4. 4 Second centrifugation step (S1p isolation)
  5. 5 Resuspend and aliquot S1p fraction
  6. 6 Quantify total tau concentration by immunoblot
  7. 7 Normalize and prepare oTau working stock
Neuronal and Astrocytic Differentiation (2D culture) Days 0–21 (2D phase)
  1. 1 Maintain and passage neural progenitor cells (NPCs)
  2. 2 Differentiate NPCs into neuronal cells (hiNCs) via NGN2 overexpression
  3. 3 Differentiate NPCs into astrocytic cells (hiACs) via small-molecule differentiation
  4. 4 Mature astrocyte precursor cells
  5. 5 Treat hiNCs with oligomeric tau (oTau) before 3D assembly
3D Asteroid Assembly and Culture (DIV3D) Days 0–21 (3D phase)
  1. 1 Prepare single-cell suspensions of hiNCs and hiACs
  2. 2 Prepare asteroid culture media and coat AggreWell plates
  3. 3 Seed cells into AggreWell microwells
  4. 4 Incubate asteroids in AggreWell plate
  5. 5 Transfer asteroids to ultra-low-attachment plates
  6. 6 Maintain asteroids in suspension culture
Optional Drug Treatment (PU-H71 or Control) Days 19–21 DIV3D
  1. 1 Prepare PU-H71 treatment stock
  2. 2 Apply PU-H71 treatment to asteroids
Sample Collection and Fixation At 7, 14, and 21 DIV3D
  1. 1 Collect asteroids for fixation (immunofluorescence)
  2. 2 Wash fixed asteroids
  3. 3 Collect asteroids for protein lysates (immunoblotting)
  4. 4 Collect conditioned media (LDH assay)
  5. 5 Collect asteroids for single-cell RNA-seq

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Rickner et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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