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 NF1 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 NF1-deficient hiPSC lines to investigate the consequences of neurofibromin inactivation on osteoblastic differentiation. Here, we demonstrate that loss of one or both copies of NF1 does not alter the potential of isogenic hiPSCs to differentiate into mesenchymal stem cells (hiPSC-MSCs). However, NF1 (+/-) and NF1 (-/-) hiPSC-MSCs show a defect in osteogenic differentiation and mineralization. In addition, we show that a mono-allelic deletion in NF1 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.

Generation of highly pure pluripotent stem cell-derived myogenic progenitor cells and myotubes.

Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7+ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches in vitro.

Reducing Timp3 or vitronectin ameliorates disease manifestations in CADASIL mice.

OBJECTIVE: CADASIL is a genetic paradigm of cerebral small vessel disease caused by NOTCH3 mutations that stereotypically lead to the extracellular deposition of NOTCH3 ectodomain (Notch3(ECD) ) on the vessels. TIMP3 and vitronectin are 2 extracellular matrix proteins that abnormally accumulate in Notch3(ECD) -containing deposits on brain vessels of mice and patients with CADASIL. Herein, we investigated whether increased levels of TIMP3 and vitronectin are responsible for aspects of CADASIL disease phenotypes. METHODS: Timp3 and vitronectin expression were genetically reduced in TgNotch3(R169C) mice, a well-established preclinical model of CADASIL. A mouse overexpressing human TIMP3 (TgBAC-TIMP3) was developed. Disease-related phenotypes, including cerebral blood flow (CBF) deficits, white matter lesions, and Notch3(ECD) deposition, were evaluated between 6 and 20 months of age. RESULTS: CBF responses to neural activity (functional hyperemia), topical application of vasodilators, and decreases in blood pressure (CBF autoregulation) were similarly reduced in TgNotch3(R169C) and TgBAC-TIMP3 mice, and myogenic responses of brain arteries were likewise attenuated. These defects were rescued in TgNotch3(R169C) mice by haploinsufficiency of Timp3, although the number of white matter lesions was unaffected. In contrast, haploinsufficiency or loss of vitronectin in TgNotch3(R169C) mice ameliorated white matter lesions, although CBF responses were unchanged. Amelioration of cerebrovascular reactivity or white matter lesions in these mice was not associated with reduced Notch3(ECD) deposition in brain vessels. INTERPRETATION: Elevated levels of TIMP3 and vitronectin, acting downstream of Notch3(ECD) deposition, play a role in CADASIL, producing divergent influences on early CBF deficits and later white matter lesions.