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BRAIN Publication-derived

TSC1/TSC2 mTOR Dysregulation

Source Blair et al. · University of Michigan, Ann Arbor, MI, USA · 10.1038/s41591-018-0139-y

👤 Blair JD, Hockemeyer D, Bhatt SM, Bhatt AH ⏱ 150 days 📋 10 phases 🧫 Patient-Derived iPSC (TSC2+/-) / CRISPR TSC1/TSC2 Knockout

Abstract

Blair et al. modeled Tuberous Sclerosis Complex using cerebral organoids from TSC2-heterozygous patient iPSCs and CRISPR-engineered biallelic knockouts, recapitulating cortical tuber-like lesions with mTOR hyperactivation and balloon-like cells. Rapamycin treatment rescued the phenotype, validating the model for mTOR-targeted therapy screening.

Cell source
Patient-Derived iPSC (TSC2+/-) / CRISPR TSC1/TSC2 Knockout
Application
Tuberous Sclerosis Modeling

Protocol overview

36 steps across 10 phases

hESC culture and maintenance Ongoing
  1. 1 Maintain hESCs on MEF feeder layer
  2. 2 Passage hESCs every 7 days
CRISPR/Cas9 genome editing for TSC1 and TSC2 Days 1-30
  1. 1 Prepare CRISPR/Cas9 constructs
  2. 2 Select GFP-positive cells by FACS
  3. 3 Clone and genotype single colonies
  4. 4 Generate conditional TSC2 allele (for second-hit modeling)
  5. 5 Remove puromycin cassette via Flp recombinase
  6. 6 Generate tdTomato Cre-reporter allele
Two-dimensional neural induction and NPC expansion Days 1–35
  1. 1 Initiate neural induction
  2. 2 Transition induction media composition
  3. 3 Dissociate and expand NPCs
Neuronal differentiation from NPCs (2D) Days 1–100
  1. 1 Initiate neuronal differentiation at passage 8
  2. 2 Plate neurons for analysis
  3. 3 Mature neurons until harvest
Three-dimensional cortical spheroid differentiation Days 0–150
  1. 1 Generate cortical spheroids from hESCs
  2. 2 Days 1–5: Initial spheroid formation
  3. 3 Days 6–25: Neural induction phase
  4. 4 Days 25–43: Neurogenic phase with neurotrophic factors
  5. 5 Days 43–150: Maturation phase
Lentiviral infection for Cre-mediated second-hit modeling Days 12–110 (spheroid time)
  1. 1 Add Cre-GFP or GFP lentivirus at day 12
  2. 2 Visualize Cre expression via tdTomato reporter
Rapamycin treatment for mTOR inhibition studies Indicated timepoints during spheroid development
  1. 1 Prepare rapamycin stock
  2. 2 Add rapamycin to spheroids at final concentration
  3. 3 Test treatment windows
Immunohistochemistry and analysis of cortical spheroids At harvest timepoints
  1. 1 Fix and cryosection spheroids
  2. 2 Immunostain sections
  3. 3 Image and quantify cell phenotypes
Western blotting for protein analysis At harvest timepoints
  1. 1 Harvest cells/spheroids
  2. 2 Quantify total protein
  3. 3 Transfer and block membranes
  4. 4 Incubate with primary antibodies
  5. 5 Incubate with secondary antibodies and detect
  6. 6 Strip and re-blot
Quantitative RT-PCR for gene expression At harvest timepoints
  1. 1 Extract RNA from spheroids
  2. 2 Reverse transcription
  3. 3 Perform real-time qPCR

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Blair et al.. 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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