Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells

¹ Faculty of Life Sciences, Bar-Ilan University, Israel
² Human Embryonic Stem Cell Research Center, Hadassah University Hospital, Israel

^* To whom correspondence should be addressed. E-mail: goldst{at}mail.biu.ac.il.

	Abstract

Human embryonic stem cells (hESC) have been directed to differentiate into neuronal cells using many cell culture techniques. Central nervous system cells with clinical importance have been produced from hESC. However, to date, there have not been any definitive reports of generation of peripheral neurons from hESC. We have used a modification of the method of Sasai et.al. for mouse and primate embryonic stem cells to elicit neuronal differentiation from hESC. When hESC cells are co-cultured with the mouse stromal line PA6 for 3 weeks, neurons are induced that co-express: 1) peripherin and Brn3a, and 2) peripherin and tyrosine hydroxylase, combinations characteristic of sympathetic and peripheral sensory neurons respectively. In-vivo, peripheral sensory and sympathetic neurons develop from the neural crest (NC). Analysis of expression of mRNAs identified in other species as NC markers reveals that the PA6 cells induce NC-like cells before neuronal differentiation takes place. Several neural crest markers including SNAIL, dHAND, and Sox9 are increased at 1 week of co-culture relative to naïve cells. Furthermore, the expression of several NC marker genes known to be down-regulated upon in-vivo differentiation of NC derivatives, was observed to be present at lower levels at three weeks of PA6-hESC co-culture than at 1 week. Our report is the first of the expression of molecular markers of NC-like cells in primates, in general, and in humans, specifically. Our results suggest that this system can be used for study of molecular and cellular events in the almost inaccessible human NC , as well as producing normal human peripheral neurons for developing therapies for diseases such as Familial Dysautonomia.