Ventral Telencephalic Patterning Protocols for Induced Pluripotent Stem Cells

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.

Plantainoside B in <i>Bacopa monniera</i> Binds to Aβ Aggregates Attenuating Neuronal Damage and Memory Deficits Induced by Aβ

“…Recently, several methods for differentiating cholinergic neurons from pluripotent stem cells have been reported. [3][4][5] Similarly, we independently developed a feeder-free differentiation protocol for basal forebrain and cholinergic neurons using low molecular weight compounds (small molecules) and confirmed the differentiation process (Figs. 2A, B).…”

“…24) With respect to the hiPSC technologies, differentiation protocols of cholinergic neurons from mouse and human pluripotent stem cells have been reported. [3][4][5] In the present study, we also independently developed a method for the differentiation of hiPSCs into cholinergic neurons targeting the basal forebrain using small molecules under the feeder-free condition. While previous reports have confirmed the expression of ChAT, a cholinergic neuronal marker, at more than Plantainoside B has been shown to reduce plasma triglycerides levels in mice.…”

“…Differentiation methods from human induced pluripotent stem cells (hiPSCs) toward basal forebrain cholinergic neurons have recently been developed. [3][4][5] These hiPSC-derived basal forebrain cholinergic neurons could be used as a reasonable AD neuronal model. The hiPSC technology allows us to conduct research on actual human cells, which was previously limited to alternative analyses using transformed cell lines and model animals.…”

Patterning ganglionic eminences in developing human brain organoids using a morphogen-gradient-inducing device

“… 65 , 66 Furthermore, protocols attempting to produce LGE-derived cell types will typically use lower concentrations of Pur, ranging from 0.5 to 0.65 μM. 39 , 67 , 68 , 69 , 70 Therefore, it is likely that the Pur gradient created by our MIBO device was more conducive for the generation of dGE tissue in general.…”

Mining the literature for ethics statements: a step towards standardizing research ethics

“…Nuessle et al (2021), Gugatschka et al (2021) -as well as in English -e.g. Krajka et al (2021), Leuenberger et al (2021.…”