This article describes a protocol for differentiating human embryonic stem cells into neural progenitor cells using specific inhibitors and growth factors. The process involves maintaining cells in an undifferentiated state before inducing neural differentiation.
Begin with a multi-well plate coated with a basement membrane matrix that includes extracellular polymers and adhesion proteins.
Seed the wells with a single-cell suspension of human embryonic stem cells in a growth medium containing a Rho-kinase inhibitor.
Incubate to allow stem cell attachment to the matrix.
Cells absorb the inhibitor, which blocks the Rho-kinase enzymes and prevents apoptotic cell death.
This promotes the cells' survival and maintains them in an undifferentiated state.
Replace the growth medium with a neural induction medium rich in growth factors and containing inhibitors that target specific ALK receptors.
These inhibitors interact with their receptors and block the BMP and TGF-β signaling pathways, preventing the cell differentiation into endodermal or mesodermal precursors.
This leads to selective differentiation of stem cells into ectodermal precursors.
These ectodermal precursors utilize nutrients and growth factors from the medium to multiply and later differentiate into neural progenitor cells.
Plate the cells on a basement membrane matrix coated 6-well plate at a density of 2 x 105 cells per well in 2 milliliters of mTeSR1 with 10 micromolar ROCK inhibitor. After 24 hours, replace the culture medium with neural induction medium supplemented with 1 micromolar dorsomorphin and 5 micromolar SB431542. Change the medium every other day during the first four days of neural induction, then every day, until confluence is reached at day seven.
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