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Plexiform neurofibromas (pNFs) are developmental tumors that appear in neurofibromatosis type 1 individuals, constituting a major source of morbidity and potentially transforming into a highly metastatic sarcoma (MPNST). pNFs arise after NF1 inactivation in a cell of the neural crest (NC)-Schwann cell (SC) lineage. Here, we develop an iPSC-based NC-SC in vitro differentiation system and construct a lineage expression roadmap for the analysis of different 2D and 3D NF models. The best model consists of generating heterotypic spheroids (neurofibromaspheres) composed of iPSC-derived differentiating NF1(-/-) SCs and NF1(+/-) pNF-derived fibroblasts (Fbs). Neurofibromaspheres form by maintaining highly proliferative NF1(-/-) cells committed to the NC-SC axis due to SC-SC and SC-Fb interactions, resulting in SC linage cells at different maturation points. Upon engraftment on the mouse sciatic nerve, neurofibromaspheres consistently generate human NF-like tumors. Analysis of expression roadmap genes in human pNF single-cell RNA-seq data uncovers the presence of SC subpopulations at distinct differentiation states.
Neurofibromas are benign peripheral nervous system tumors associated with neurofibromatosis type 1, which originate from NF1(-/-) Schwann cell precursors. We describe a protocol to generate neurofibromaspheres by differentiating NF1(-/-) Schwann cells from induced pluripotent stem cells and combining them with neurofibroma primary fibroblasts. We also describe the development of neurofibroma-like tumors when neurofibromaspheres are engrafted in the sciatic nerve of nude mice. This model constitutes a versatile platform for drug screening and the study of neurofibroma biology. For complete details on the use and execution of this protocol, please refer to Mazuelas et al. (2022).1.
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