Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum‐Free Suspension Culture

Abstract

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.

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.…”

“…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. Only in the case where hES cells were grown as spheres in suspension was a simple unsupplemented serum-free media sufficient to support the differentiation of DA neurons (19), possibly due to the local production of necessary factors in the spheres.…”

“…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. Only in the case where hES cells were grown as spheres in suspension was a simple unsupplemented serum-free media sufficient to support the differentiation of DA neurons (19), possibly due to the local production of necessary factors in the spheres. However, DA neurons differentiated in this manner did not survive in adherent culture and only rarely survived engraftment in vivo (19).…”

“…All of these cell lines were originally propagated on mouse feeder layers, although several have recently been transferred to feeder free support systems (1,3,7,9,12,21). 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).…”

“…During this period, whether cells are attached to a substrate or differentiated in suspension culture, they can elicit an extensive highly branched network of processes that make harvest untenable without irreparable mechanical damage to cells. Ultimately, few, if any, of these cells survive transplantation into the brain (19,24).…”