P4-278: Characterization of Basal Forebrain Cholinergic Neurons From Induced Pluripotent Stem Cells Harboring Familial Alzheimer’S MUTATION PSEN2 N141I

The differentiation of human induced pluripotent stem cells (hiPSCs) into specific cell types for disease modeling and restorative therapies is a key research agenda and offers the possibility to obtain patient-specific cells of interest for a wide range of diseases. Basal forebrain cholinergic neurons (BFCNs) play a particular role in the pathophysiology of Alzheimer’s dementia and isolated dystonias. In this work, various directed differentiation protocols based on monolayer neural induction were tested for their effectiveness in promoting a ventral telencephalic phenotype and generating BFCN. Ventralizing factors [i.e., purmorphamine and Sonic hedgehog (SHH)] were applied at different time points, time intervals, and concentrations. In addition, caudal identity was prevented by the use of a small molecule XAV-939 that inhibits the Wnt-pathway. After patterning, gene expression profiles were analyzed by quantitative PCR (qPCR). Rostro-ventral patterning is most effective when initiated simultaneously with neural induction. The most promising combination of patterning factors was 0.5 μM of purmorphamine and 1 μM of XAV-939, which induces the highest expression of transcription factors specific for the medial ganglionic eminence, the source of GABAergic inter- and cholinergic neurons in the telencephalon. Upon maturation of cells, the immune phenotype, as well as electrophysiological properties were investigated showing the presence of marker proteins specific for BFCN (choline acetyltransferase, ISL1, p75, and NKX2.1) and GABAergic neurons. Moreover, a considerable fraction of measured cells displayed mature electrophysiological properties. Synaptic boutons containing the vesicular acetylcholine transporter (VACHT) could be observed in the vicinity of the cells. This work will help to generate basal forebrain interneurons from hiPSCs, providing a promising platform for modeling neurological diseases, such as Alzheimer’s disease or Dystonia.

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A Simple Differentiation Protocol for Generation of Induced Pluripotent Stem Cell-derived Basal Forebrain Cholinergic Neurons for Alzheimer’s Disease and Frontotemporal Dementia Disease Modeling

10.20944/preprints202008.0091.v1 ◽

2020 ◽

Author(s):

Sonia Sanz Muñoz ◽

Martin Engel ◽

Rachelle Balez ◽

Dzung Do-Ha ◽

Mauricio Castro Cabral-Da-Silva ◽

...

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Frontotemporal Dementia ◽

Small Molecule ◽

Pluripotent Stem Cells ◽

Basal Forebrain ◽

Cholinergic Neurons ◽

Basal Forebrain Cholinergic Neurons ◽

Differentiation Protocol ◽

Induced Pluripotent

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 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.

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