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BRAIN Publication-derived

Spinal Cord 2D Cell Cultures for Regenerative Medicine

Full SOP with Academic or a plan

Source Zhang et al., 2001 · University of Wisconsin

Authors: Su-Chun Zhang, Marius Wernig, Ian D. Duncan, Oliver Brüstle, James A. Thomson Duration: 70 days 6 phases Cell source: Human ESC

Abstract

This protocol describes the in vitro differentiation of human embryonic stem cells into neural precursor cells via formation of embryoid bodies and neural tube-like rosette structures in the presence of FGF-2. The isolated neural precursors can differentiate into neurons, astrocytes, and oligodendrocytes, and upon transplantation into neonatal mouse brain, integrate into various brain regions with no teratoma formation.

Cell source
Human ESC
Application
Neural precursor generation for transplantation therapy and nervous system repair

Protocol overview

18 steps across 6 phases

Phase 1 · ES Cell Culture and EB Formation Days 1-4
  1. a Culture ES cells on feeder layer
  2. b Detach ES cell colonies
  3. c Form embryoid bodies (EBs)
Phase 2 · Neural Induction and Rosette Formation Days 5-12
  1. a Plate EBs in chemically defined medium with FGF-2
  2. b Monitor rosette formation
Phase 3 · Isolation and Enrichment of Neural Rosette Clusters Days 8-10
  1. a Enzymatic separation of rosettes from flat cells
  2. b Collect rosette clumps
  3. c Further purification by adhesion separation
Phase 4 · Expansion of Neural Precursor Cells Days 11-56
  1. a Culture as floating neurospheres
  2. b Verify FGF-2 dependency
Phase 5 · In Vitro Differentiation Days 57-70
  1. a Induce differentiation by FGF-2 withdrawal and substrate plating
  2. b Monitor neuronal differentiation
  3. c Monitor astrocyte differentiation
  4. d Promote oligodendrocyte differentiation (optional)
Phase 6 · Transplantation Preparation and In Vivo Analysis Variable
  1. a Prepare cells for transplantation
  2. b Intracerebroventricular transplantation
  3. c Immunosuppression
  4. d Harvest brains for analysis

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Attribution

This SOP was written by Organthis based on the published method in Zhang et al., 2001. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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