Abstract
The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
Keywords:
Alzheimer’s disease; cholinergic neurons; disease modelling; frontotemporal dementia; induced pluripotent stem cells; neuronal differentiation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alzheimer Disease / genetics
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Alzheimer Disease / metabolism
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Alzheimer Disease / pathology
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Amyotrophic Lateral Sclerosis / genetics
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Amyotrophic Lateral Sclerosis / metabolism
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Amyotrophic Lateral Sclerosis / pathology
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Basal Forebrain / metabolism
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Basal Forebrain / pathology
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Benzamides / pharmacology
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Brain-Derived Neurotrophic Factor / pharmacology
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Cell Differentiation / drug effects*
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Cell Line
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Cholinergic Neurons / cytology
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Cholinergic Neurons / drug effects*
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Cholinergic Neurons / metabolism
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Culture Media / chemistry
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Culture Media / pharmacology*
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Dioxoles / pharmacology
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Embryoid Bodies / cytology
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Embryoid Bodies / drug effects*
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Embryoid Bodies / metabolism
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Female
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Fibroblast Growth Factor 2 / pharmacology
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Frontotemporal Dementia / genetics
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Frontotemporal Dementia / metabolism
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Frontotemporal Dementia / pathology
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Growth Differentiation Factor 2 / pharmacology
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Hedgehog Proteins / pharmacology
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Humans
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Induced Pluripotent Stem Cells / cytology
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Induced Pluripotent Stem Cells / drug effects*
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Induced Pluripotent Stem Cells / metabolism
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Male
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Models, Biological
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Nerve Growth Factor / pharmacology
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Patch-Clamp Techniques
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Primary Cell Culture / methods*
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Pyrazoles / pharmacology
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Pyrimidines / pharmacology
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Transforming Growth Factor beta / pharmacology
Substances
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4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide
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Benzamides
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Brain-Derived Neurotrophic Factor
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Culture Media
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Dioxoles
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GDF2 protein, human
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Growth Differentiation Factor 2
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Hedgehog Proteins
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LDN 193189
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NGF protein, human
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Pyrazoles
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Pyrimidines
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SHH protein, human
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Transforming Growth Factor beta
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Fibroblast Growth Factor 2
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BDNF protein, human
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Nerve Growth Factor