Dopaminergic Differentiation of Human Embryonic Stem Cells

Abstract

In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)–positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH‐positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine‐β‐hydroxylase, a marker of noradrenergic neurons. PA6‐induced neurons were able to release dopamine and 3,4‐dihydroxphe‐hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+. When transplanted into 6‐hydroxydopamine–treated animals, hES‐derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH‐positive cells were present but in very small numbers compared with the high frequency of TH‐positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.

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.…”

“…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).…”

“…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.…”

Electrophysiological evaluation of engrafted stem cell-derived neurons

“…These neurons can be tailored, in cell culture, to display striking morphological and immunophenotypic similarities to cells found in the adult brain, and even to cells lost during the course of brain disease [1][2][3][4][5][6][7][8][9][10] . Because stem cell-derived neurons are born in a culture dish, without the environmental cues of the developing brain, it has become a major quest to determine whether these cells are actually functional, that is, capable of interacting with established neural circuitry.…”

Constraining the Pluripotent Fate of Human Embryonic Stem Cells for Tissue Engineering and Cell Therapy – The Turning Point of Cell-Based Regenerative Medicine

“…Before further differentiation, those secondary hNSCs were tediously mechanically isolated or enriched from hESC-differentiating multi-lineage aggregates [2]. Previously, co-culturing with stromal cells or telomerase-immortalized midbrain astrocytes as well as exposing to FGF and sonic hedgehog (SHH) signaling have been used to improve the yield of β-III-tubulin- and TH-positive cells from hESC-derived hNSCs [83–85]. The signaling factors that operate along the rostrocaudal and dorsoventral axes of the neural tube to specify motor neuron fate in vivo have been used to direct hESCs differentiate into an early motor neuron phenotype through germ-layer induction in vitro , but with low efficiencies [86, 87].…”