ABSTRACT
Background Chromodomain helicase DNA-binding protein 8 (CHD8), which encodes a chromatin remodeling protein that regulates Wnt/β-catenin mediated gene expression, is one of the most strongly associated genes with autism spectrum disorder (ASD). Characterization of ASD patients with CHD8 disrupting mutations and animal and stem cell models of CHD8 deficiency suggest that CHD8 plays a role in neurodevelopment.
Methods We generated iPSCs from the T-lymphocytes of a healthy, typically-developing human male and iPSC lines from the same source which were subjected to CRISPR/Cas9-mediated knockdown (KD) of CHD8. We subsequently derived neural progenitor cells (NPCs) and neural cells and examined the effects of CHD8 deficiency on cell proliferation and neural differentiation.
Results We observed that, compared to WT, CHD8 KD: (1) increased the number of iPSC colonies formed, (2) suppressed spontaneous differentiation along the edges of iPSC colonies, (3) increased the proliferation of NPCs, (4) delayed the formation of neural rosettes, (5) delayed neurite outgrowth, (6) decreased the percentage of cells in the G0/G1 phase of the cell cycle, (7) increased the percentage of cells in the G2/M phase of the cell cycle, (8) decreased presence of the neuronal marker MAP2 although not the glial marker GFAP, (9) decreased presence for the excitatory neuronal marker VGLUT1, and (10) decreased presence of the synaptic marker SYN1.
Conclusions Our results suggest that CHD8 deficiency causes alterations in the cell cycle. More specifically, CHD8 KD appears to increase cell proliferation and delay neural differentiation. This may contribute to the pathophysiology of ASD.
ABBREVIATIONS
- ASD
- Autism spectrum disorder
- ALP
- Alkaline phosphatase
- CHD8
- Chromodomain helicase DNA-binding protein 8
- CRISPR/Cas9
- Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9
- EB
- Embryoid body
- FACS
- Fluorescence-activated cell sorting
- GFAP
- Glial fibrillary acidic protein
- GI
- Gastrointestinal
- iPSC
- Induced pluripotent stem cell
- KD
- Knockdown
- KO
- Knockout
- MAP2
- Microtubule-associated protein 2
- MEF
- Mouse embryonic fibroblast
- NPC
- Neural progenitor cell
- PBMC
- Peripheral blood mononuclear cell
- SYN1
- Synapsin 1
- VGLUT1
- Vesicular glutamate transporter 1
- WES
- Whole-exome sequencing
- WT
- Wild-type