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BRAIN Publication-derived

Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids

Source Trujillo et al., 2021 · University of California San Diego, Department of Pediatrics/Rady Children's Hospital · 10.15252/emmm.202012523

👤 Cleber A Trujillo, Jason W Adams, Priscilla D Negraes, Cassiano Carromeu, Leon Tejwani, Allan Acab, Ben Tsuda, Charles A Thomas, Neha Sodhi, Katherine M Fichter, Sarah Romero, Fabian Zanella, Terrence J Sejnowski, Henning Ulrich, Alysson R Muotri ⏱ 90 days 📋 10 phases 🧫 Human pluripotent stem cells (PSC); Patient-derived iPSC (Rett syndrome MECP2 mutations) and CRISPR/Cas9-generated isogenic hESC lines

Abstract

This protocol establishes a tiered drug-screening pipeline using human pluripotent stem cells differentiated into neurons, MECP2-mosaic neurospheres, and cortical organoids to identify pharmacological compounds that reverse synaptic and network pathology caused by MECP2 deficiency. Two lead compounds, Nefiracetam and PHA 543613, were validated to specifically reverse MECP2-knockout neuropathology without affecting control neurons, supporting clinical trial development for MECP2-related neurodevelopmental disorders.

Cell source
Human pluripotent stem cells (PSC); Patient-derived iPSC (Rett syndrome MECP2 mutations) and CRISPR/Cas9-generated isogenic hESC lines
Application
Disease modeling and drug screening for Rett syndrome

Protocol overview

44 steps across 10 phases

PSC maintenance and MECP2-KO line generation Day -30 to Day 0
  1. 1 Culture pluripotent stem cells
  2. 2 Verify pluripotency with teratoma assay
Neural differentiation of PSCs to neurons Day 0 to Day 28
  1. 1 Initiate neural induction
  2. 2 Form embryoid bodies
  3. 3 Isolate neural progenitor cells
  4. 4 Expand and maintain NPCs
  5. 5 Induce neuronal differentiation
Characterization of MECP2-KO neurons Day 14 to Day 28
  1. 1 Perform RT–qPCR array for gene expression
  2. 2 Conduct single-cell qRT–PCR analysis
  3. 3 Perform immunofluorescence for neuronal markers
  4. 4 Quantify neuronal morphology (soma and spine)
Calcium imaging in monolayer neurons Day 28 to Day 42
  1. 1 Transduce neurons with Syn::RFP lentiviral reporter
  2. 2 Load neurons with Fluo-4AM calcium indicator
  3. 3 Perform calcium imaging
  4. 4 Analyze calcium transient data
Generation and characterization of MECP2-mosaic neurospheres Day 0 to Day 60
  1. 1 Generate 50/50 control/MECP2-KO neural progenitor mixtures
  2. 2 Culture neurospheres to 4–8 weeks of age
  3. 3 Verify mosaic composition via immunofluorescence
  4. 4 Measure neurosphere diameter
Drug screening in monolayer MECP2-KO neurons Day 28 to Day 42
  1. 1 Select and prepare pharmacological compounds
  2. 2 Treat neurons with pharmacological compounds
  3. 3 Screen for effects on synaptogenesis (Synapsin 1 and PSD-95 levels)
  4. 4 Quantify co-localized synaptic puncta
  5. 5 Assess effects on calcium transient frequency and neuronal activity
  6. 6 Perform multi-electrode array (MEA) electrophysiology
Generation and characterization of cortical organoids Day 0 to Day 90
  1. 1 Initiate cortical organoid formation
  2. 2 Feed organoids with neural induction media (Media1)
  3. 3 Feed organoids with growth factor-supplemented media (Media2)
  4. 4 Maintain organoids long-term
Drug treatment in MECP2-mosaic neurospheres Day 28 to Day 56
  1. 1 Treat 4-week-old neurospheres with lead compounds
  2. 2 Assess neurosphere viability
  3. 3 Measure neurosphere diameter
  4. 4 Measure calcium transient frequency and amplitude
  5. 5 Perform MEA electrophysiology on neurospheres
Drug treatment in cortical organoids Day 30 to Day 60
  1. 1 Treat 1-month-old cortical organoids with lead compounds
  2. 2 Measure organoid diameter
  3. 3 Quantify Ki67+ proliferation
  4. 4 Extract RNA for sequencing and gene ontology analysis
  5. 5 Quantify synaptic puncta (Synapsin1+)
  6. 6 Verify neuronal composition and cortical layer identity
  7. 7 Observe spine-like protrusions
Multi-electrode array (MEA) electrophysiology in cortical organoids Day 30 to Day 60
  1. 1 Prepare cortical organoids for MEA plating
  2. 2 Record population spiking activity
  3. 3 Analyze population spiking data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Trujillo et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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