Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.

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. CONCLUSION: 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.