Directed neuronal differentiation of human embryonic stem cells.
BACKGROUND
We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).


RESULTS
A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7-10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons.


CONCLUSIONS
This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.
Department of Animal and Dairy Science, University of Georgia, Athens, 30605, USA. tjschulz@prodigy.net
Thomas C
TC
Schulz
Gail M
GM
Palmarini
Scott A
SA
Noggle
Deborah A
DA
Weiler
Maisam M
MM
Mitalipova
Brian G
BG
Condie
D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors
D000943:Antigens, Differentiation; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free