Dataset Information

Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells.

ABSTRACT: Three-dimensional in vitro cell culture models, particularly for the central nervous system, allow for the exploration of mechanisms of organ development, cellular interactions, and disease progression within defined environments. Here we describe the development and characterization of three-dimensional tissue models that promote the differentiation and long-term survival of functional neural networks. These tissue cultures show diverse cell populations including neurons and glial cells (astrocytes) interacting in 3D with spontaneous neural activity confirmed through electrophysiological recordings and calcium imaging over at least 8 months. This approach allows for the direct integration of pluripotent stem cells into the 3D construct bypassing early neural differentiation steps (embryoid bodies and neural rosettes), which streamlines the process while also providing a system that can be manipulated to support a variety of experimental applications. This tissue model has been tested in stem cells derived from healthy individuals as well as Alzheimer's and Parkinson's disease patients, with similar growth and gene expression responses indicating potential use in the modeling of disease states related to neurodegenerative diseases.

SUBMITTER: Cantley W

PROVIDER: S-EPMC7725274 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells.

Cantley William W Du Chuang C Lomoio Selene S DePalma Thomas T Peirent Emily E Kleinknecht Dominic D Hunter Martin M Tang-Schomer Min M Tesco Giuseppina G Kaplan David L DL

ACS biomaterials science & engineering 20181001 12

Three-dimensional in vitro cell culture models, particularly for the central nervous system, allow for the exploration of mechanisms of organ development, cellular interactions, and disease progression within defined environments. Here we describe the development and characterization of three-dimensional tissue models that promote the differentiation and long-term survival of functional neural networks. These tissue cultures show diverse cell populations including neurons and glial cells (astroc ...[more]

PMID: 33304995

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Project description:Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Dataset Information

Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells.

Publications

Functional and Sustainable 3D Human Neural Network Models from Pluripotent Stem Cells.

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