Dottorato in SCIENZE DELLA VITA

Titolo della tesi: 3D Bioprinted Human Neural Constructs Derived from Induced Pluripotent Stem Cells as an innovative and versatile tool for Fragile X Syndrome modeling

Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder caused by a CGG repeat expansion in the 5’UTR region of Fragile X Mental Retardation 1 (FMR1) gene, which leads to methylation, transcriptional silencing and loss of the encoded Fragile X Mental Retardation Protein (FMRP). FMRP is an RNA-binding protein involved in the regulation of a large number of mRNAs implicated in synaptic function and maturation. Human induced pluripotent stem cells (iPSCs) can be obtained from any individual by reprogramming body cells (e.g. skin, blood) and then differentiated in a wide range of neurons and glia, providing an ideal tool for modelling the human nervous system. However, conventional 2D cell cultures fail to represent the complexity of the brain and novel 3D systems are emerging as more realistic and representative models. While some 3D brain models (the so-called “organoids”) show variability and reproducibility issues due to the “self-assembling” process, 3D bioprinting, an additive manufacturing technique, has emerged as an alternative approach to ensure high control on scaffold composition and cell deposition. Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and several human tissues have been recently produced using cells derived by differentiation of iPSCs. We have set up an isogenic FXS model by generating a FMR1 knock out (FMR1 KO) iPSC line using CRISPR/Cas9 gene editing technology. In particular, we have inserted a transcriptional stop in FMR1 by the introduction of an exogenous cassette containing a premature polyadenylation signal. FMR1 KO iPSC clones were then validated and differentiated towards cortical neurons, the most affected neural subtype by FXS. Even though FXS main features were recapitulated over time, we observed high degree of variability with conventional monolayer differentiation methods, leading us to shift to a more physiological and reproducible model. We thus adopted 3D bioprinting technology to realize neural scaffolds containing iPSC-derived cortical neurons and glial precursors, in order to promote cell deposition in a controlled manner and with micrometric resolution. We show that the extrusion-based printing process does not impair cell viability in the short and long term. Bioprinted cells can be further differentiated within the construct and properly express neuronal and astrocytic markers. Functional analysis of 3D bioprinted cells highlights an early stage of maturation and the establishment of early network activity behaviors. Development and optimization of differentiation protocols and printing conditions will allow to reduce variability and generate a reliable and reproducible 3D bioprinted FXS model. This will greatly facilitate the investigation of FXS molecular and functional features in a more physiological context compared to monolayer cultures. In the long term, our goal is to generate a large scale platform that can be exploited as customized, standardized and scalable pre-clinical models for drug safety and toxicity studies.