Abstract
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.
Highlights
To gain a profound understanding of diseases at the molecular level, cell models are employed for research
Neural induction was initiated by dual SMAD inhibition via the administration of SB-431542 and LDN-193189, inhibiting the transforming growth factor-beta (TGF-β), activin/nodal, and bone morphogenetic protein (BMP) signaling (Figure 2D; Chambers et al, 2009; Qi et al, 2017)
Morphogens were added to the medium to achieve a rostro-ventral patterning [for rostralization the small molecule XAV-939 (XAV); for ventralization either purmorphamine (Pu) or the recombinant protein Sonic hedgehog (SHH)] (Figure 2D)
Summary
To gain a profound understanding of diseases at the molecular level, cell models are employed for research. To obtain specified regional identities along the rostro-caudal and dorsoventral neural axis (Figure 1A), signaling peptides (so-called morphogens) or their small-molecule counterparts need to be administered (Petros et al, 2011) By their addition, NPCs can be patterned into desired neuronal lineages, by mimicking the neurogenesis of specific brain regions, resulting in dopaminergic, glutamatergic, and GABAergic neurons (Wang et al, 2015; Sun et al, 2016; Cao et al, 2017). We focused on the most ventral part of the telencephalon – the medial ganglionic eminence (MGE) (Figure 1B) – from which basal forebrain cholinergic neurons (BFCNs) and GABAergic interneurons arise (Sussel et al, 1999) Alterations in these cell types are associated with sporadic Alzheimer’s disease (Whitehouse et al, 1982; Coyle et al, 1983) and isolated dystonia (Levy and Hallett, 2002)
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