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

Iacovitti, Lorraine; Donaldson, Angela E.; Marshall, Cheryl E.; Suon, Sokreine; Yang, Ming

doi:10.1016/j.brainres.2006.10.022

Cited by 101 publications

(98 citation statements)

References 26 publications

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“…hESCs (line H9) were differentiated using the dbcAMPbased DA differentiation protocol [22] divided into 5 stages (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…H9 cells were differentiated using a dbcAMP protocol [21,22]. The differentiation process consisted of 5 stages, starting with undifferentiated hESCs ( Fig.…”

Section: Neuronal Differentiation Of Hescsmentioning

confidence: 99%

“…One of the simplest differentiation protocols for deriving dopaminergic neurons from human embryonic stem cells (hESCs) uses dibutyryl cAMP (dbcAMP) during the final differentiation stage [20][21][22]. Drugs, such as forskolin and isobutylmethylxanthine that activate the cAMP signaling pathway (similar to dbcAMP) have also been used successfully in dopaminergic differentiation protocols [23,24].…”

Section: Introductionmentioning

confidence: 99%

“…Dopaminergic signaling is not entirely based on the cAMP pathway. DA binds to multiple receptors that can stimulate several G proteins [25,26] and thus, activate intracellular pathways different from those activated by dbcAMP alone [22,27]. The current study investigates whether DA treatment can improve the yield and quality of hESC-derived DA neurons.…”

Section: Introductionmentioning

confidence: 99%

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Dopamine Receptors in Human Embryonic Stem Cell Neurodifferentiation

Belinsky

Sirois

Rich

et al. 2013

Stem Cells and Development

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We tested whether dopaminergic drugs can improve the protocol for in vitro differentiation of H9 human embryonic stem cells (hESCs) into dopaminergic neurons. The expression of 5 dopamine (DA) receptor subtypes (mRNA and protein) was analyzed at each protocol stage (1, undifferentiated hESCs; 2, embryoid bodies [EBs]; 3, neuroepithelial rosettes; 4, expanding neuroepithelium; and 5, differentiating neurons) and compared to human fetal brain (gestational week 17-19). D2-like DA receptors (D2, D3, and D4) predominate over the D1-like receptors (D1 and D5) during derivation of neurons from hESCs. D1 was the receptor subtype with the lowest representation in each protocol stage (Stages 1-5). D1/D5-agonist SKF38393 and D2/D3/D4-agonist quinpirole (either alone or combined) evoked Ca 2 + responses, indicating functional receptors in hESCs. To identify when receptor activation causes a striking effect on hESC neurodifferentiation, and what ligands and endpoints are most interesting, we varied the timing, duration, and drug in the culture media. Dopaminergic agonists or antagonists were administered either early (Stages 1-3) or late (Stages 4-5). Early DA exposure resulted in more neuroepithelial colonies, more neuronal clusters, and more TH + clusters. The D1/D5 antagonist SKF83566 had a strong effect on EB morphology and the expression of midbrain markers. Late exposure to DA resulted in a modest increase in TH + neuron clusters (*75%). The increase caused by DA did not occur in the presence of dibutyryl cAMP (dbcAMP), suggesting that DA acts through the cAMP pathway. However, a D2-antagonist (L741) decreased TH + cluster counts. Electrophysiological parameters of the postmitotic neurons were not significantly affected by late DA treatment (Stages 4-5). The mRNA of mature neurons (VGLUT1 and GAD1) and the midbrain markers (GIRK2, LMX1A, and MSX1) were lower in hESCs treated by DA or a D2-antagonist. When hESCs were neurodifferentiated on PA6 stromal cells, DA also increased expression of tyrosine hydroxylase. Although these results are consistent with DA's role in potentiating DA neurodifferentiation, dopaminergic treatments are generally less efficient than dbcAMP alone.

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“…hESCs (line H9) were differentiated using the dbcAMPbased DA differentiation protocol [22] divided into 5 stages (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…H9 cells were differentiated using a dbcAMP protocol [21,22]. The differentiation process consisted of 5 stages, starting with undifferentiated hESCs ( Fig.…”

Section: Neuronal Differentiation Of Hescsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Dopamine Receptors in Human Embryonic Stem Cell Neurodifferentiation

Belinsky

Sirois

Rich

et al. 2013

Stem Cells and Development

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“…It has become evident that there are many combinations of neural differentiation agents that can produce a similar, marker-defined, output. For example, TH+ DA neurons have also been induced by culture in the presence of noggin and dibutyryl cAMP (dbcAMP) [43]. Transplantation of these neuronal progenitors into the striata of 6-hydroxydopamine treated rats produced functionally differentiated DA cells in vivo 2−3 weeks post-transplantation.…”

Section: ) Neuronal/glial Differentiationmentioning

confidence: 99%

Differentiation of neural lineage cells from human pluripotent stem cells

Schwartz

Brick

Stover

et al. 2008

Methods

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Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: 1) starting material, 2) induction of loss of pluripotency, 3) neural induction, 4) neural maintenance and expansion, and 5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

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