Characterization of NF1 (+/+), (+/-), and (-/-) iPSC lines generated through genetic editing using CRISPR/Cas9

Abstract

The use of pluripotent stem cell lines from oocytes and embryos has been a cause of ethical discussion. For that reason, reprogramming somatic cells to produce induced pluripotent stem cells (iPSCs) has been an alternative to avoid this issue and an opportunity to develop new therapeutic strategies and disease models. In the last few years, researchers of the ‘Hereditary Cancer’ group in the ‘Germans Trias i Pujol Research Institute (IGTP)’ have been using this iPSC technology to study Neurofibromatosis type 1, an autosomal dominant genetic condition caused by mutations in the NF1 gene and capable of developing tumors associated with the peripheral nervous system called neurofibromas. In Eduard Serra’s lab, they have developed an NF1 iPSC-based cellular model by reprogramming NF1 (+/-) and (-/-) cells from plexifrom neurofibromas, generating NF1 (+/-) and (-/-) different iPSC lines. These iPSC cell lines were capable of capturing the genomic status of the original tumors and maintaining the characteristics of stem cells, as they were able to differentiate toward neural crest stem cells and further toward Schwann cells, the main cell type of the tumors. Moreover, when Schwann cells NF1 (-/-) are injected into the sciatic nerve of a nude mice, neurofibromas are formed. In addition, they have also used the technology of CRISPR/Cas9 to genetically edit exon 2 of the NF1 in NF1(+/+) control iPSC lines, with the aim of generating mutations in one or both NF1 alleles to obtain extra clones with the genotypes NF1 (+/-) and (-/-). In this project, we have identified three different clones from this editing experiment: one of them carrying a mutation only in one allele of the NF1 gene (NF1(+/-)); another one carrying mutations in both alleles (NF1(-/-)); and the third one without any mutation (NF1 (+/+)) as a control line. These clones have been characterized to confirm their NF1 genetic status and evaluate their stem cell abilities. Mutations in exon 2 of the NF1 gene were confirmed by determining the sequence of each clone; in vitro pluripotency capacity was tested by the analysis of the expression of specific markers; and the ability to differentiate into cells of the NC lineage was demonstrated by analyzing the expression of transcription factors and membrane markers specific to the lineage. These newly characterized cell lines will be useful to expand the number of NF1 iPSC line in the lab to create NF1 cell models and establish non-perishable cell lines to test new therapeutic strategies before preclinical in vivo models