Directed differentiation of neural-stem cells and subtype-specific neurons from hESCs

Abstract

We describe a chemically defined protocol for efficient differentiation of human embryonic stem cells (hESCs) to neural epithelial cells and then to functional spinal motor neurons. This protocol comprises four major steps. Human ESCs are differentiated without morphogens into neuroepithelial cells that form neural tube-like rosettes in the first 2 weeks. The neuroepithelial cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) in the following 2 weeks. These OLIG2 progenitors generate postmitotic, HB9 expressing motoneurons at the fifth week and mature to functional motor neurons thereafter. The protein factor SHH can be replaced by a small molecule purmorphamine in the entire process, which may facilitate potential clinical applications. This protocol has been shown equally effective in generating motor neurons from human induced pluropotent stem (iPS) cells.

Figure 1

Scheme of differentiation of spinal cord motoneurons from hESCs. The hESCs are directed to neuroepithelial cells in the first 2 weeks. These neureopithelial cells are patterned to OLIG2-expressing motoneuron progenitors in the subsequent 2 weeks in response to RA and SHH (or purmorphamine). Finally, the progenitors differentiate to post-mitotic motoneurons in the presence of neurotrophic factors. The process employs a simple serum-free neural differentiation medium for motoneuron differentiation. The adherent culture during the neural induction phase is uniquely designed for directly observing neuroepithelial differentiation and for purifying the neuroepithelial cells by removing the non-neural colonies.

Figure 2

Differentiation of motor neurons from hESCs. (A) hESCs growing on MEF feeder as a uniform colonies. (B) After lifting the hESCs from the MEF and growing in suspension, the hESCs aggregate to spheres. (C) From day 10, columnar epithelial cells appear within the hESC colonies. The columnar cells are starting to organize into rosettes in the colony. (D) At day 15, neural tube-like rosettes are obvious. (E) The motoneruon progenitors are cultured in suspension. (F) Extensive axonal projections come out from the clusters a week after adherent culture. (G) At the 4^th week, HB9-expressing motoneurons present among the OLIG2-expressing progenitors. (H) HB9 motorneurons are also positively stained for neuronal marker Tuj1+ at the 5^th week.