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BRAIN Publication-derived

Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids

Source Harbuzariu et al., 2019 · Morehouse School of Medicine, Department of Microbiology, Biochemistry and Immunology · 10.1038/s41598-019-55631-8

👤 Adriana Harbuzariu, Sidney Pitts, Juan Carlos Cespedes, Keri Oxendine Harp, Annette Nti, Andrew P. Shaw, Mingli Liu, Jonathan K. Stiles ⏱ 40 days 📋 10 phases 🧫 Human iPSC (CD34+ umbilical cord blood-derived)

Abstract

This protocol describes the generation and characterization of human brain cortical organoids derived from CD34+ umbilical cord blood-derived iPSCs to model heme-mediated brain injury associated with cerebral malaria. The organoids are exposed to heme to recapitulate pathogenic mechanisms and tested for neuroprotective effects of neuregulin-1 (NRG-1) treatment.

Cell source
Human iPSC (CD34+ umbilical cord blood-derived)
Application
Disease modeling

Protocol overview

41 steps across 10 phases

iPSC Characterization and Culture Passage 3-4
  1. 1 iPSC Morphology Assessment
  2. 2 Flow Cytometry Assessment of Pluripotency Markers
  3. 3 iPSC Culture Maintenance
Embryoid Body Formation and Neural Induction Days 0-10
  1. 1 Embryoid Body (EB) Initiation
  2. 2 EB Maturation
  3. 3 Neural Induction
Neuroepithelial Expansion in Matrigel Days 10-14
  1. 1 Matrigel Embedding
  2. 2 Transfer to Culture Plates
  3. 3 Formation of Ventricular-like Cavities
Organoid Maturation Days 14-40+
  1. 1 Matrix Removal and Transfer
  2. 2 Orbital Shaker Culture
  3. 3 Medium Changes
  4. 4 Developmental Assessment
Heme and NRG-1 Treatment of Organoids Days 20-40 (treatment window)
  1. 1 Heme Treatment Preparation
  2. 2 Organoid Heme Exposure
  3. 3 NRG-1 Co-treatment
  4. 4 Control Treatments
Apoptosis and Cell Viability Assessment in Organoids After heme and NRG-1 treatment (hours 26-28)
  1. 1 Real-Time Glo Annexin V Apoptosis/Necrosis Assay Setup
  2. 2 Apoptosis Detection (Chemiluminescence Readout)
  3. 3 Necrosis Detection (Fluorescence Readout)
  4. 4 Fluorescence Microscopy of Necrotic Cells
Histological Analysis and Immunohistochemistry After treatment (day 20 or 40)
  1. 1 Organoid Fixation
  2. 2 Tissue Sectioning and Preparation
  3. 3 Hematoxylin and Eosin (H&E) Staining
  4. 4 Epitope Unmasking
  5. 5 Blocking and Primary Antibody Incubation
  6. 6 Secondary Antibody and Fluorescence Staining
  7. 7 Mounting and Confocal Imaging
  8. 8 Quantification of Marker Expression
iPSC Viability and Apoptosis Studies Single timepoint experiments
  1. 1 CCK-8 Cell Viability Assay
  2. 2 Optical Density Measurement
  3. 3 Annexin V Apoptosis/Necrosis Assay
  4. 4 Flow Cytometry Data Analysis
Pluripotency Marker Analysis Single timepoint experiments
  1. 1 Heme-Induced Differentiation Assay Setup
  2. 2 Flow Cytometry Assessment of Pluripotency Markers
  3. 3 Flow Cytometry Data Acquisition and Analysis
ERBB4 and NRG-1 Expression Analysis in iPSCs Single timepoint experiments
  1. 1 ERBB4 Flow Cytometry Assessment
  2. 2 Western Blot Sample Preparation
  3. 3 SDS-PAGE Separation
  4. 4 Antibody Incubation
  5. 5 Chemiluminescent Detection and Quantification

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Harbuzariu et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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