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BRAIN Publication-derived

An in vivo neuroimmune organoid model to study human microglia phenotypes

Source Schafer et al., 2023 · The Salk Institute for Biological Studies, La Jolla, CA · 10.1016/j.cell.2023.04.022

👤 Simon T. Schafer, Abed A. Mansour, Johannes C.M. Schlachetzki, Monique Pena, Saeed Ghassemzadeh, Lisa Mitchell, Amanda Mar, Daphne Quang, Sarah Stumpf, Irene Santisteban Ortiz, Addison J. Lana, Clara Baek, Raghad Zaghal, Christopher K. Glass, Axel Nimmerjahn, Fred H. Gage ⏱ 42 days 📋 6 phases 🧫 Human iPSC, Human ESC, Patient-Derived iPSC (Autism with Macrocephaly)

Abstract

This protocol describes an in vivo xenotransplantation approach to generate immunocompetent human brain organoids (iHBO) containing functionally mature human microglia (hMG) that can be studied within a vascularized, physiologically relevant human brain environment. The transplanted organoids allow investigation of human microglia phenotypes in health and disease, including disease-specific immune responses in patient-derived models of autism with macrocephaly.

Cell source
Human iPSC, Human ESC, Patient-Derived iPSC (Autism with Macrocephaly)
Application
Disease modeling, Developmental study, Neuroimmune interaction research

Protocol overview

28 steps across 6 phases

Generation of forebrain organoids and fluorescent reporter lines Days 0–42
  1. 1 Maintenance and lentiviral infection of hESC/iPSC lines
  2. 2 Initiate forebrain organoid differentiation
  3. 3 Apply neural induction
  4. 4 Embed organoids in Matrigel and mature
  5. 5 Continue organoid maturation and media changes
Generation and isolation of erythromyeloid progenitors (EMPs) Days 0–16
  1. 1 Hematopoietic differentiation initiation
  2. 2 First media change and growth factor supplementation
  3. 3 Transition to normoxic conditions and second growth factor supplementation
  4. 4 Harvest and isolate EMPs by FACS
EMP colonization of forebrain organoids and in vitro co-culture Days 35–42 (organoid age)
  1. 1 Prepare organoids for EMP co-culture
  2. 2 Co-culture EMPs with organoids
  3. 3 Monitor EMP colonization and morphology during in vitro culture
Surgical transplantation of EMP-containing organoids into mouse brain Day 10 post-EMP integration
  1. 1 Animal preparation and anesthesia
  2. 2 Surgical site preparation and craniotomy
  3. 3 Position and transplant the organoid
  4. 4 Create cranial window and seal
  5. 5 Post-operative care
In vivo characterization of human microglia (weeks 1–24 post-transplantation) Weeks 6–24 post-transplantation
  1. 1 Immunohistochemical analysis of microglia maturation
  2. 2 Isolate human microglia for single-cell RNA-sequencing
  3. 3 Perform single-cell RNA-sequencing
  4. 4 In vivo two-photon imaging of microglial dynamics
  5. 5 Induce focal laser lesions and assess microglial response
  6. 6 Systemic LPS challenge and morphological assessment
Patient-specific disease modeling: ASD with macrocephaly Days 0–12 weeks post-transplantation
  1. 1 Generate isogenic iPSC lines from ASD and control subjects
  2. 2 Generate GFP+ cortical organoids and tdT+ EMPs from subject-derived lines
  3. 3 Transplant subject-specific iHBOs and assess microglia phenotype
  4. 4 Generate cross-subject 'sensor' models to establish environment-dependence
  5. 5 Harvest and analyze subject-specific iHBOs

Full SOP

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Attribution

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

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