Directed Differentiation of Ventral Spinal Progenitors and Motor Neurons from Human Embryonic Stem Cells by Small Molecules

¹ Departments of Anatomy, Neurology, and the Stem Cell Research Program, Waisman Center, 1500 Highland Avenue, University of Wisconsin-Madison, WI 53705

^* To whom correspondence should be addressed. E-mail: zhang{at}waisman.wisc.edu.

Correspondence may also be addressed to Xue-Jun Li at xjli@uchc.edu.

	Abstract

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the non-target differentiated populations is also crucial, which is lacking in the field. Here we show an efficient differentiation of motor neurons (50%) by a simple sequential application of retinoid acid (RA) and sonic hedgehog (SHH) in a chemically defined suspension culture. We further discovered that purmorphamine, a small molecule that activates the SHH pathway, could substitute SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, Pax7-) besides motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies employing human motoneurons at a minimal cost.