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Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential.

ABSTRACT: Human induced pluripotent stem cells (hiPSCs) are invaluable to study developmental processes and disease mechanisms particularly in the brain. hiPSCs can be differentiated into mature and functional dopaminergic (DA) neurons. Having robust protocols for the generation of differentiated DA neurons from pluripotent cells is a prerequisite for the use of hiPSCs to study disease mechanisms, for drug discovery, and eventually for cell replacement therapy. Here, we describe a protocol for generating and expanding large numbers of homogeneous midbrain floor plate progenitors (mFPPs) that retain efficient DA neurogenic potential over multiple passages and can be cryobanked. We demonstrate that expanded mFPPs have increased DA neuron potential and differentiate more efficiently and rapidly than progenitors generated by standard protocols. In addition, this novel method results in increased numbers of DA neurons that in vitro show characteristic electrophysiological properties of nigrostriatal DA neurons, produce high levels of dopamine, and integrate into host mice when grafted in vivo. Thus, we describe a robust method for producing human mesencephalic DA neurons from hiPSCs.

SUBMITTER: Fedele S

PROVIDER: S-EPMC5519680 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential.

Fedele Stefania S Collo Ginetta G Behr Katharina K Bischofberger Josef J Müller Stephan S Kunath Tilo T Christensen Klaus K Gündner Anna Lisa AL Graf Martin M Jagasia Ravi R Taylor Verdon V

Scientific reports 20170720 1

Human induced pluripotent stem cells (hiPSCs) are invaluable to study developmental processes and disease mechanisms particularly in the brain. hiPSCs can be differentiated into mature and functional dopaminergic (DA) neurons. Having robust protocols for the generation of differentiated DA neurons from pluripotent cells is a prerequisite for the use of hiPSCs to study disease mechanisms, for drug discovery, and eventually for cell replacement therapy. Here, we describe a protocol for generating ...[more]

PMID: 28729666

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Project description:The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta-catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta-catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta-catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta-catenin is conditionally stabilized in the Shh+domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta-catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta-catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia Nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta-catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta-catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson's disease.

Dataset Information

Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential.

Publications

Expansion of human midbrain floor plate progenitors from induced pluripotent stem cells increases dopaminergic neuron differentiation potential.

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