Human iPSC-Derived Neurons and Cerebral Organoids Establish Differential Effects of Germline NF1 Gene Mutations

Abstract

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability.

NGS Gene Panel Analysis Revealed Novel Mutations in Patients with Rare Congenital Diarrheal Disorders

“…Currently, the classification of these VUS as pathogenic involves the guidelines indicated by the American College of Medical Genetics, which are based on several criteria, including family studies, type of mutation, protein residue affected by the variants, genetic association studies, posterior-probabilities analysis, and functional studies [ 12 ]. Although these criteria are widely accepted and used, the classification of VUS is still a challenge due to their low frequency, the lack of family information, and the difficulty of performing functional studies using ex-vivo cell models [ 20 ], enteroids, or stem cells [ 21 ] in a routine context. In the present study, we assessed the pathogenic role of the novel variants by using the ACMG classification first, and then, by the main prediction tools currently used, as described in the methods section.…”

Humanized neurofibroma model from induced pluripotent stem cells delineates tumor pathogenesis and developmental origins

“…For this reason, we differentiated patient-derived isogenic hiPSCs (20) directly into SCPs using DMEM/F12-Neural Basal-N2B27 media supplemented with SB431542 (Activin and TGF-β inhibitor to prevent SMAD signaling, suppress pluripotency, and prevent mesoderm/endoderm induction), a GSK3 inhibitor (CHIR99021; to activate WNT signaling), and higher concentrations of neuregulin β-1 to support glial fate differentiation (21). Before differentiation, the cells formed colonies ( Figure 1A and Supplemental Figure 1A), which are characteristic of hiPSCs and were immunopositive for pluripotent markers, including NANOG, SOX2, Oct3/4, and TRA1-60 ( Figure 1A).…”

“…Commercially available BJFF.6 healthy human fibroblasts were reprogrammed into iPSCs (NF1 +/+ ) and were subsequently CRISPR/Cas9 engineered by the Washington University Genome Engineering and iPSC Core (GEiC) to harbor a heterozygous (NF1 +/-) or homozygous (NF1 -/-) NF1 mutation found in NF1 patients: 6619C>T; c.6513T>A or c.2041C>T (20). hiPSCs were maintained in feeder-free, Matrigel-coated plates in mTeSR Plus medium (STEMCELL Technologies).…”

Modeling cancer progression using human pluripotent stem cell-derived cells and organoids

“…Modeling glioma has been made possible by the recapitulation of glioma tumor-initiating cells (GTICs) with iPSC-derived neural progenitor cells (NPCs) ( Sancho-Martinez et al, 2016 ). iPSCs and iPSC-derived brain organoid models to study neurofibromatosis type 1 (NF1), a related cancer predisposition syndrome, have also been developed ( Anastasaki et al, 2020 ). Bi-allelic inactivation of the NF1 gene gives rise to optic pathway gliomas (OPGs) in about 15–20% children with NF1 and additionally, other brainstem gliomas ( Wegscheid et al, 2018 ).…”