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BRAIN Publication-derived

Brain Assembloids for Developmental Studies

Full SOP with Academic or a plan

Source Andersen et al. · Stanford University School of Medicine, Stanford, CA, USA · 10.1016/j.cell.2020.11.017

Authors: Andersen J, Revah O, Miura Y, Thom N, Amin ND, Kelley KW, Singh M, Chen X, Thete MV, Walczer EM, Bhatt SM, Bhatt AH, Paşca SP Duration: 70 days 8 phases Cell source: Human iPSC

Abstract

Andersen et al. created a three-component assembloid system combining cortical, spinal, and skeletal muscle spheroids to model the complete voluntary motor pathway. Cortical stimulation evoked muscle contractions through spinal motor neuron relay, demonstrating functional corticospinal-neuromuscular connectivity in human tissue for the first time.

Cell source
Human iPSC
Application
Corticospinal Tract Development

Protocol overview

35 steps across 8 phases

Phase 1 · Generation of human cortical spheroids (hCS) Days 0-42+
  1. a Culture hiPS cells on feeder layers
  2. b Initiate spheroid formation
  3. c Transition to neural medium
  4. d Add growth factors for maturation
  5. e Maintain mature hCS
Phase 2 · Generation of human hindbrain/cervical spinal cord spheroids (hSpS) Days 0-50+
  1. a Initiate neural induction with dual SMAD inhibition
  2. b Add WNT activator and establish rostro-caudal patterning
  3. c Add SHH modulator for ventral specification
  4. d Switch to maturation medium and apply Notch inhibitor
  5. e Maintain long-term hSpS culture
Phase 3 · Generation of 3D human skeletal muscle (hSkM) Days 0-25
  1. a Culture human skeletal myoblasts in growth medium
  2. b Dissociate myoblasts for 3D culture
  3. c Seed 3D hSkM in silicone wells and initiate gelation
  4. d Transfer to ultra-low attachment plates for differentiation
  5. e Switch to differentiation medium
Phase 4 · Assembly of cortico-spinal assembloids (hCS-hSpS) Days 0-30+
  1. a Prepare hCS and hSpS for assembly
  2. b Fuse spheroids in close proximity
  3. c Change medium on day 2 of assembly
  4. d Transfer to culture plates on day 3
  5. e Maintain long-term culture
Phase 5 · Generation of spinal-muscle assembloids (hSpS-hSkM) Days 0-20+
  1. a Prepare 3D hSkM for assembly
  2. b Prepare transwell insert setup
  3. c Place hSpS on insert in contact with hSkM
  4. d Change medium and maintain culture
Phase 6 · Assembly of three-part cortico-motor assembloids (hCS-hSpS-hSkM) Days 0-70+
  1. a Pre-assemble hSpS-hSkM
  2. b Add hCS to established hSpS-hSkM assembloid
  3. c Maintain three-part assembloid
Phase 7 · Viral labeling and transduction Days -7 to +0 (relative to assembly)
  1. a Prepare spheroid for viral infection
  2. b Add fresh medium and transfer to culture plate
  3. c Assess viral expression before assembly
Phase 8 · Functional analysis—Imaging and electrophysiology Days 5-70 post-assembly (variable based on analysis type)
  1. a Live imaging of eYFP projections in intact assembloids
  2. b Optogenetic stimulation with GCaMP imaging
  3. c Patch-clamp electrophysiology in sliced assembloids
  4. d Glutamate uncaging on hSkM
  5. e Optogenetic stimulation of hCS with muscle contraction readout

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Attribution

This SOP was written by Organthis based on the published method in Andersen et al.. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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