Background: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development.
Methods: We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols.
Results: Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors.
Conclusions: The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.
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http://dx.doi.org/10.1186/s13287-018-0812-6 | DOI Listing |
bioRxiv
October 2024
Department of Pharmacology, University of Washington, Seattle, WA 98195.
Copy number variation (CNV) in the 16p11.2 (BP4-BP5) genomic locus is strongly associated with autism. Carriers of 16p11.
View Article and Find Full Text PDFBackground: Bryant-Li-Bhoj neurodevelopmental syndrome (BLBS) is neurogenetic disorder caused by variants in and the two genes that encode the histone H3.3 protein. Ninety-nine percent of individuals with BLBS show developmental delay/intellectual disability, but the mechanism by which variants in H3.
View Article and Find Full Text PDFNeuroscience
November 2024
Department of Functional Anatomy and Neuroscience, Asahikawa Medical University, Asahikawa, Hokkaido 078-8510, Japan.
It has been widely established that neural stem cells (NSCs) exist in the adult mammalian brain. The area postrema (AP) and the ependymal cell layer of the central canal (CC) in the medulla were recently identified as NSC niches. There are two types of NSCs: astrocyte-like cells in the AP and tanycyte-like cells in the CC.
View Article and Find Full Text PDFCell Mol Life Sci
March 2024
Key Laboratory of Molecular Epigenetics, Ministry of Education, Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China.
Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation.
View Article and Find Full Text PDFSci Adv
March 2024
Epigenetics and Stem Cell Biology Laboratory, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Cell fate decisions are achieved with gene expression changes driven by lineage-specific transcription factors (TFs). These TFs depend on chromatin remodelers including the Brahma-related gene 1 (BRG1)-associated factor (BAF) complex to activate target genes. BAF complex subunits are essential for development and frequently mutated in cancer.
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