Directed Differentiation of Dopaminergic Neuronal Subtypes from Human Embryonic Stem Cells

Abstract

How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC-derived Sox1 + neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)-positive neurons that were mostly bipolar cells, coexpression with γ-aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH + neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitrogenerated DA neurons were electrophysiologically active and released DA in an activity-dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system. Stem Cells 2005;23:781-790

Derivation of neural precursor cells from human ES cells at 3% O2 is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

“…There was no significant difference between the efficiency of neural conversion at 3 and 20% O 2 , with a neural identity acquired by D14 in both instances (Figure 1c), consistent with previous reports of a 2-week timescale for neural conversion of hESCs at 20% O 2 . 14,33,35,36 Neural conversion at 3% O 2 was robust, highly reliable and reproducible across two independent hESC Figure 1A). Growth curves confirmed a significant increase in numbers of NPCs generated at 3% compared with 20% O 2 , under basal culture conditions (Figure 2a Figure 1B).…”

A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo

“…In earlier studies, DA differentiation of hES cells required incubation in culture with other cell types (PA6 mouse stromal line; human HepG2 liver line) or in cell-CM, usually containing fetal calf serum and/or other undefined components (4,5,16,17,18,22,26). In several recent studies, efforts have been made to move to a serum-free defined protocol for neuronal (9,10,13) or DA differentiation (19,22) of hES cells. With only one exception (19), however, media was supplemented with serum substitutes, such as B27, and often cells were grown on Matrigel, both of which contain undefined proprietary components and growth substances of animal origin.…”

“…While the differentiation of DA traits in some hES lines has been described previously (4,5,16,17,18,26), with the exception of one study (19) these protocols involve the prolonged use of complex media containing serum or other undefined reagents and/or cell conditioned media (CM) or co-culture with these cells (ie. PA6 mouse stromal cells) (4,5,10,13,16,17,18,22,26). Since animal cells contain immunogenic antigens that can be incorporated into hES cells (14), they ultimately can cause immune rejection after their transplantation into the brain.…”

Dopamine Cell Transplantation for Parkinson's Disease: The Importance of Controlled Clinical Trials

“…The most promising source of an unlimited supply of dopamine neurons is from differentiated human embryonic stem cells or induced pluripotent stem cells [64][65][66][67]. While strategies have differed in details, the basic concept has been to differentiate embryonic stem cells to neuroprogenitor cells, then expand that cell population, and finally guide differentiation to dopamine neurons using known differentiation factors, such as sonic hedgehog and FGF8.…”