In vitro Differentiation of Transplantable Neural Precursors from Human Embryonic Stem Cells
Source Zhang et al., 2001 · University of Wisconsin
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.
Protocol overview
18 steps across 6 phases
- 1 Culture ES cells on feeder layer
- 2 Detach ES cell colonies
- 3 Form embryoid bodies (EBs)
- 4 Plate EBs in chemically defined medium with FGF-2
- 5 Monitor rosette formation
- 6 Enzymatic separation of rosettes from flat cells
- 7 Collect rosette clumps
- 8 Further purification by adhesion separation
- 9 Culture as floating neurospheres
- 10 Verify FGF-2 dependency
- 11 Induce differentiation by FGF-2 withdrawal and substrate plating
- 12 Monitor neuronal differentiation
- 13 Monitor astrocyte differentiation
- 14 Promote oligodendrocyte differentiation (optional)
- 15 Prepare cells for transplantation
- 16 Intracerebroventricular transplantation
- 17 Immunosuppression
- 18 Harvest brains for analysis
Full SOP
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Attribution
This SOP was authored by Organthis based on the published method in Zhang et al., 2001. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.
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