Generation of heterozygous and homozygous NF1 lines from human-induced pluripotent stem cells using CRISPR/Cas9 to investigate bone defects associated with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders caused by heterozygous germline mutations. NF1 affects many systems, including the skeletal system. To date, no curative therapies are available for skeletal manifestations such as scoliosis and tibial dysplasia, mainly due to the lack of knowledge about the mechanisms that underlie this process. By using CRISPR/Cas9-mediated gene editing in human-induced pluripotent stem cells (hiPSCs) to minimize the variability due to genetic background and epigenetic factors, we generated isogenic heterozygous and homozygous -deficient hiPSC lines to investigate the consequences of neurofibromin inactivation on osteoblastic differentiation. Here, we demonstrate that loss of one or both copies of does not alter the potential of isogenic hiPSCs to differentiate into mesenchymal stem cells (hiPSC-MSCs). However, (+/-) and (-/-) hiPSC-MSCs show a defect in osteogenic differentiation and mineralization. In addition, we show that a mono-allelic deletion in in an isogenic context is sufficient to impair cell differentiation into osteoblasts. Overall this study highlights the relevance of generating isogenic lines, which may help in genotype-phenotype correlation and provide a human cellular model to understand the molecular mechanisms underlying NF1 and, thus, discover new therapeutic strategies.