Developing a Human Cell Model for Fragile X Syndrome Using Direct Reprogramming

Abstract

A comprehensive view of the mechanisms underpinning the neurodevelopmental disorder Fragile X Syndrome (FXS) is lacking owing to limited access to relevant human cell models. Therefore, the development of tractable and representative models of human neurodevelopmental disorders is crucial to our ability to draw physiologically relevant conclusions about the mechanisms of FXS pathogenesis. Ideally, experimental models of this disorder would recapitulate the cellular and molecular mechanisms underlying the phenotypic characteristics of FXS as they are manifested in humans. Additionally, both the genetic and phenotypic characteristics of this disorder would be represented within FXS-associated cell types at defined stages of neuronal development that are relevant to FXS progression. The use of cell reprogramming to generate FXS patient-derived human neural stem/precursor cells provides a powerful platform to elucidate the mechanistic underpinnings of aberrant neurodevelopmental phenotypes as they occur in humans. Therefore, the aim of this study was to establish our capability to generate FXS patientderived neural progenitor cells (iNPs) and subsequently neurons by direct reprogramming to a neural progenitor state. Furthermore, direct reprogramming of FXS patient-derived fibroblasts would be achieved using a novel chemically modified mRNA gene delivery system advanced by the Neural Reprogramming and Repair laboratory. This study demonstrated that diverse cell populations positive for both neural progenitor and neuronal markers can be derived directly from somatic cells by SOX2/PAX6-SNIM® RNA mediated reprogramming. Importantly, reprogrammed FXS cell lines exhibited transcriptional and phenotypic differences, which may be suggestive of early FXS-associated mechanisms occurring during progenitor and neuronal development in vitro. This is significant as it provides proof-of-concept data that this model may be useful in future efforts to characterise the effects of the loss of FMRP on the development and function of neurons and uncover possible therapeutic targets for early intervention strategies in the future. To the best of our knowledge, the present study is the first to derive live human neural progenitors and differentiated neuron-like cells by direct-to-iNP reprogramming of FXS-patient derived somatic cells.