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BMP, MEK and WNT modulation with NGN2 expression for rapid generation of hiPSC-derived neurons amenable to regional patterning [snRNA-seq]

ABSTRACT: hiPSC are a promising tool for studying neurological diseases and developing therapies for neurodegenerative diseases. Differentiation of hiPSC into neurons can be achieved by dual SMAD inhibition (dSMADi) or induced NGN2 overexpression (“iNGN2”). Starting directly from hiPSC, iNGN2 shortens the time to a neuronal stage but leads to neurons partially resembling peripheral or posterior fates while dSMADi more faithfully recapitulates forebrain development. To modify the iNGN2 paradigm, we applied an accelerated induction paradigm that is dependent on inhibition of BMP, MEK and WNT pathways (“BMWi”), to commit hiPSC into a rostral neuroectoderm fate before iNGN2. The resulting neurons showed strong expression of telencephalic and cortical markers, with decreased levels of peripheral and posterior marker genes compared to iNGN2 alone. The resulting cortical neurons are suitable for a tau aggregation assay. Furthermore, we could demonstrate that during BMWi treatment, the cells are amenable to additional regional patterning cues. This allowed the generation of neurons from different regions of the CNS and PNS, which will significantly facilitate in vitro modeling of a range of neurodevelopmental and neurodegenerative disorders.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279591 | GEO | 2025/06/19

REPOSITORIES: GEO

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Project description:Trans-differentiation of human induced pluripotent stem cells into neurons (hiPSC-N) via Ngn2-induction has become an efficient system to quickly generate neurons for disease modeling and in vitro assay development, a significant step up from previously used neoplastic and other cell lines. Recent single-cell interrogation of Ngn2-induced neurons however, has revealed some similarities to unexpected neuronal lineages. Similarly, a straightforward method to generate hiPSC derived astrocytes (hiPSC-A) for the study of neuropsychiatric disorders has also been described. Here we examine the homogeneity and similarity of hiPSC-N and hiPSC-A to their in vivo counterparts, the impact of different lengths of time post Ngn2 induction on hiPSC-N (15 or 21 days) and of hiPSC-N / hiPSC-A co-culture. We explore how often genes differentially expressed between conditions relate to genetic risk for neuropsychiatric disease. Leveraging the wealth of existing public single-cell RNA-seq (scRNA-seq) data in Ngn2-induced neurons and in vivo data from the developing brain, we provide perspectives on the lineage origins and maturation of hiPSC-N and hiPSC-A. Both show heterogeneity and share similarity with multiple in vivo cell fates, and both cell types more precisely approximate their in vivo counterparts when co-cultured. hiPSC-A show more heterogeneity and similarities to other non-neural cell types, especially when cultured in isolation. Gene expression data from the hiPSC-N show excess of genes linked to schizophrenia (SZ) and autism spectrum disorders (ASD) as has been previously shown for neural stem cells and neurons. These overrepresentations of disease genes are strongest in our system at early times (day 15) in Ngn2-induction/maturation of neurons, which together with our observation of similarities with in vivo neurons earlier in development suggest they may be a better model for neurodevelopmental disorders. We have assembled this new scRNA-seq data along with the public data explored here as an integrated biologist-friendly web-resource for researchers seeking to understand this system more deeply: https://nemoanalytics.org/p?l=DasEtAl2022 .

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BMP, MEK and WNT modulation with NGN2 expression for rapid generation of hiPSC-derived neurons amenable to regional patterning [snRNA-seq]

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