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BRAIN Publication-derived

Alzheimer's Disease Brain Organoids with Microglia

Full SOP with Academic or a plan

Source Abud et al., 2017 · University of California Irvine · 10.1016/j.neuron.2017.03.042

Authors: Edsel M. Abud, Ricardo N. Ramirez, Eric S. Martinez, Luke M. Healy, Cecilia H.H. Nguyen, Sean A. Newman, Andriy V. Yeromin, Vanessa M. Scarfone, Samuel E. Marsh, Cristhian Fimbres, Chad A. Caraway, Gianna M. Fote, Abdullah M. Madany, Anshu Agrawal, Rakez Kayed, Karen H. Gylys, Michael D. Cahalan, Brian J. Cummings, Jack P. Antel, Ali Mortazavi, Monica J. Carson, Wayne W. Poon, Mathew Blurton-Jones Duration: 38 days 5 phases Cell source: Human iPSC

Abstract

This protocol generates iPSC-derived microglia-like cells (iMGLs) through a fully-defined, two-step differentiation approach. First, iPSCs are differentiated to hematopoietic progenitor cells (iHPCs) over 10 days, then iHPCs are differentiated to iMGLs over 28 days in serum-free media containing CSF-1, IL-34, and TGF-β1, yielding highly pure (>97%) functional microglia-like cells suitable for studying Alzheimer's disease and other neurological disorders.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

20 steps across 5 phases

Phase 1 · iPSC Maintenance and Preparation Ongoing until day -1
  1. a Maintain human pluripotent stem cells on Matrigel in TeSR-E8 medium
  2. b Confirm pluripotency and sterility of iPSC lines
Phase 2 · Differentiation of iPSCs to Hematopoietic Progenitor Cells (iHPCs) Day -1 to Day 10
  1. a Prepare iPSCs for hematopoietic differentiation
  2. b Culture iPSCs in E8 medium with ROCK inhibitor for 24 hours
  3. c Initiate hematopoietic induction on Day 0
  4. d Media change on Day 2
  5. e Media change on Day 4 and transition to normoxia
  6. f Media supplementation on Days 6 and 8
  7. g FACS isolation of CD43+ iHPCs on Day 10
Phase 3 · Differentiation of iHPCs to iMGLs (Phase 1: Days 10-12) Day 10 (or Day 0 iMGL differentiation) to Day 12
  1. a Prepare iMGL differentiation medium and plate sorted iHPCs
  2. b Culture iHPCs in microglial differentiation medium
  3. c First media supplementation on Day 12 (Day 2 iMGL)
Phase 4 · Differentiation of iHPCs to iMGLs (Phase 2: Days 12-38) Day 12 to Day 38 (Day 2-28 iMGL culture)
  1. a Continue every-two-days media supplementation
  2. b Optional early iMGL characterization (Day 14 or Day 22)
  3. c Complete 50% media change at Day 22 (Day 12 iMGL)
  4. d Continue every-two-days supplementation through Day 35
Phase 5 · iMGL Maturation and Preparation for Use (Days 35-38) Day 35 to Day 38
  1. a Add CD200 and CX3CL1 to promote homeostatic maturation
  2. b Continue every-two-days media supplementation with CD200/CX3CL1
  3. c Final FACS quality control on Day 38
  4. d Prepare iMGLs for functional assays or transplantation

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Attribution

This SOP was written by Organthis based on the published method in Abud et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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