Directed differentiation and functional characterization of embryonic stem cell-derived motoneurons

Motoneurons are specialized types of neurons essential for the control of body movement. Motoneuron loss is the cause of a wide range of neurological disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy. Embryonic stem cells (ESCs) are promising cell source for the study and potential treatment of such motoneuron diseases. The mechanisms that govern the development of central nervous system and the pathways that specify motoneurons in vertebrates were already quite well characterized. However, there was a complete lack of strategies for the directed differentiation of mouse (m) and human (h) ESCs towards motoneuron progeny at the time these studies were initiated.; We first developed a co-culture based neural differentiation system for mESCs on MS5 stromal feeders. The MS5 protocol allows the efficient derivation of a wide range of neural subtypes from mESCs. My own studies demonstrated in particular that spinal motoneurons can be derived at high efficiencies from mESCs on MS5 stroma upon exposure to sonic hedgehog (SHH) and retinoic acid (RA), two key patterning factors for motoneuron development in vivo. Next, we applied the MS5 protocol to hESCs for the derivation of human motoneurons. Neural induction of hESCs on MS5 feeders resulted in the formation of neural rosettes, neuroepithelial structures that can be directed to differentiate into specific neuronal subtypes. Neural rosettes progressively adopted forebrain fates by default but retained the potential for regional respecification including the adoption of caudal fates. The neural rosettes could be efficiently directed towards spinal motoneurons in response to SHH and RA. The hESC-derived motoneurons showed appropriate morphological, physiological and biochemical properties of spinal motoneurons in vitro. Furthermore, transplantation of hESC-derived motoneuron progeny into the developing chick embryo resulted in robust engraftment, maintenance of motoneuron phenotype, and long-distance axonal projections into peripheral host tissues. Transplantation into the adult rat spinal cord resulted in neural grafts comprised of large number of surviving human motoneurons and outgrowth of choline acetyltransferase positive fibers. These data provide a key step in the development of hESC-based strategies for the study and potential treatment of human motoneuron disorders.