MERLIN-DEFICIENT iPSCs AS AN IN VITRO MODEL SYSTEM FOR STUDIYNG NF2 PATHOGENESIS

Background: The appearance of bilateral vestibular schwannomas (VS) is one of the most characteristic features of NF2-related schwannomatosis (NF2-related SWN), an autosomal dominant syndrome that predisposes to the development of tumours of the nervous system. VS are caused by the bi-allelic inactivation of the NF2 gene in a cell of the Schwann cell lineage. Our current understanding of VS initiation and progression as well as the development of new effective therapies is hampered by the absence of human non-perishable cell-based models. Principal Findings: We generated and characterized induced pluripotent stem cell (iPSC) lines with single or bi-allelic inactivation of NF2 by combining the direct reprogramming of VS cells with the use of CRISPR/Cas9 editing. Despite the difficulty of maintaining merlin-deficient iPSCs, we were able to differentiate them into neural crest (NC) cells. At this stage, these cells showed spontaneous expression of the SC marker S100B and the impossibility of generating Schwann cells in 2D cultures. Nevertheless, by applying a 3D Schwann cell differentiation protocol, we successfully generated NF2(+/-) and NF2(-/-) spheroids homogeneously expressing classical markers of the NC-SC axis. Conclusions: Our results show a critical function of NF2 for both reprograming and maintaining a stable pluripotent state. In addition, merlin-deficient cultures also denoted an altered differentiation capacity of merlin-deficient cells towards the NC-SC axis, in the in vitro conditions used. Finally, the generated NF2(+/-) and NF2(-/-) spheroids show potential as a genuine in vitro model of NF2-related tumours.