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BRAIN Publication-derived

Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A

Source Sen et al., 2020 · Department of Chemical and Biomolecular Engineering, North Carolina State University · 10.1016/j.stemcr.2020.08.006

👤 Dilara Sen, Alexis Voulgaropoulos, Zuzana Drobna, Albert J. Keung ⏱ 17 days 📋 5 phases 🧫 Human ESC (H9, H1), Human iPSC, Patient-Derived iPSC (Angelman syndrome)

Abstract

This protocol generates human cerebral organoids (hCOs) from neurotypical and Angelman syndrome-derived pluripotent stem cells to model UBE3A expression dynamics during early human neurodevelopment. The organoids reveal spatiotemporal patterns of UBE3A nuclear localization in neurons and track paternal UBE3A imprinting, enabling testing of topoisomerase inhibitors as potential therapeutics for restoring UBE3A levels.

Cell source
Human ESC (H9, H1), Human iPSC, Patient-Derived iPSC (Angelman syndrome)
Application
Disease modeling

Protocol overview

19 steps across 5 phases

Cell culture and organoid generation 0-12 weeks
  1. 1 Maintain feeder-independent pluripotent stem cells
  2. 2 Generate cerebral organoids using whole-brain protocol
Characterization of UBE3A subcellular localization 1–12 weeks post-generation
  1. 1 Prepare organoids for immunofluorescence staining
  2. 2 Perform immunofluorescence staining for UBE3A and cell-type markers
  3. 3 Image and quantify nuclear UBE3A localization
  4. 4 Perform subcellular fractionation and western blot
Imprinting status and UBE3A-ATS expression analysis 1–17 weeks post-generation
  1. 1 Extract RNA from hCOs at multiple timepoints
  2. 2 Perform RT-qPCR for UBE3A and UBE3A-ATS transcripts
  3. 3 Correlate UBE3A expression pattern to fetal developmental stage
Topoisomerase inhibitor treatment and functional response 4–17 weeks post-generation
  1. 1 Treat AS hCOs with topoisomerase inhibitors
  2. 2 Test dose-response and age-dependent effects
  3. 3 Assess persistence of therapeutic effect following single dose
  4. 4 Test repeated daily dosing
  5. 5 Quantify nuclear UBE3A by immunofluorescence in treated organoids
Live calcium imaging and functional assessment 14 days post-drug treatment
  1. 1 Treat AS hCOs with indotecan or vehicle for downstream functional analysis
  2. 2 Dissociate organoids and prepare single cells for imaging
  3. 3 Perform live calcium imaging
  4. 4 Analyze calcium transient parameters
  5. 5 Compare calcium transient parameters between treatment groups

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sen et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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