Fragile X syndrome (FXS) is an inherited form of human mental retardation caused by epigenetic silencing in the FMR1 gene and the consequent loss of the fragile X mental retardation protein (FMRP) is directly linked with alterations in dendritic spine morphology, synaptogenesis and connectivity in the developing brain leading to neurological, cognitive and behavioral defects.
Our study aims to create a 3D model based on human induced pluripotent stem cells (hiPSCs) with the purpose of deciphering the neurobiological phenotypes associated with FXS. In order to study the electrophysiological properties of FXS iPSC-derived neurons in-vitro, we firstly optimized a 2D differentiation protocol for obtaining a mixed cortical neuron culture. Although preliminary patch-clamp experiments showed no differences, in terms of Na+ and K+ currents and ability to evoke action potentials, between control and FXS neural cultures, calcium imaging analysis showed that the FXS lines appear to display a reduced frequency of spontaneous calcium events, with significantly reduced synchronicity at day 68, thus suggesting an alteration in the neuronal network development.
Moreover, in order to better characterize the alteration due to the absence of FMRP and reduce the genetic variability between iPSC lines, we generated an FMR1 knockout iPSC line, isogenic to the control one, by using a CRISPR gene-editing approach. These two populations will be then used for generating up an iPSC-derived 3D cell model based on the whole-brain organoids culture method.
Currently, we report the efficient production of brain organoids from three control hiPSC lines and we aim to further study how the absence of FMRP can morphologically and functionally invalidate the formation of cortical plates during early neurodevelopment events.
This project aims to generate 2D and 3D hiPSC-based cellular models to investigate the mechanisms and the pathological functioning of the Fragile X syndrome (FXS), focusing on a brain region particularly affected by the disease: the cerebral cortex. These models, being built with patient-derived cells, best represent the neurodevelopmental events of the pathology and constitute reliable platforms for pharmacological screening, applying as new models for translational research.
Several animal models have been developed in order to provide a significant understanding of FXS and various targets for potential pharmaceutical treatments have been identified, many of which have been shown to be efficient in preclinical studies. However, all attempts to turn these findings into a therapy for patients did not give the desired results.
In 2006 Yamanaka and his colleagues revolutionized disease modeling by successfully reprogramming murine fibroblast to a pluripotent cell type, so called induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). One year later they succeeded to reproduce the protocol for human fibroblasts (Takahashi et al., 2007) and in the past ten years technologies to generate iPSCs have vastly improved.
Human iPSCs can be converted into any cell type and they finally make possible the in-vitro generation and maintenance of human neurons and other brain cell types. Moreover, the possibility to obtain iPSCs carrying pathogenic mutation makes this cell system a great resource for regenerative medicine, disease modeling and drug screening (Shi et al., 2017).
Two types of neurons could be used as 2D in-vitro models of FXS: cortical neurons and hippocampal neurons and we have set up conditions for cortical neuron differentiation from human iPSCs lines with the FXS mutation and one line with the FMR1 premutation (range from 55 to 200 repeats).
We studied the electrophysiological properties of FXS iPSC-derived neurons revealing an alteration in the FXS neuronal network development in terms of frequency and synchronicity of calcium events. In this project, we will further investigate these preliminary results by studying the electrophysiological properties of cortical neurons obtained from a FMR1 knockout iPSC line, isogenic to the control one, and by challenging these neuronal with different plasticity protocols.
Traditionally cells have been cultured using 2D conventional tissue culture techniques; however, because the human brain is extremely complex 3D arrangements of different kinds of cells, 2D cell cultures risk to be a reductionist approach. In this regard, new techniques are proving capable to accelerate the study of the human brain representing also a necessary improvement for the production of more physiological in-vitro models of human development and disease.
On this point, iPSC-derived 3D brain organoids are proving to be very useful models for the study of the first events of neurodevelopment both in physiological and pathological conditions. These ¿mini-organs¿ contain several areas resembling different regions of the brain and they are able to reproduce in-vitro their complex 3D architecture. For this reason, brain organoids have already been used to model developmental diseases like microcephaly (Lancaster et al., 2013) and the production of radial glial cells, intermediate progenitors and deep- and superficial-layer neurons in an ordered temporal fashion has been reported by studies using all the protocols currently in use (Lancaster et al., 2013) making human cerebral organoids a realistic model of cortical development. Currently, we set up iPSCS-derived 3D cell cultures using whole-brain organoids protocols (Lancaster and Knoblich., 2014) (Lancaster et al., 2017) and we report the efficient production of cerebral organoids from three control hiPSC lines already available in our laboratory, revealing the presence of a cortical stratification containing both TBR1 and CTIP2 positive early-born deep-layers neurons. In this project we propose to generate cerebral organoids starting also from FXS and FMR1 knockout iPSCs lines thus reproducing for the first time in-vitro the early neurodevelopment events that underlie the FXS and we aim to further study how the absence of FMRP can morphologically and functionally invalidate the formation of cortical plates in this 3D model of neurodevelopment.
In conclusion, this work will be able to provide both 2D and 3D models useful for the study of FXS and for therapeutic testing. These tools will be able to in-vitro recapitulate the cellular components and the complex 3D cytoarchitecture of the human brain furthermore bypassing the use of animal models which turned out to be insufficiently adequate to reproduce the FXS neurodevelopmental events.