CARLO BRIGHI

Dottore di ricerca

ciclo: XXXIII


supervisore: Silvia Di Angelantonio
relatore: Silvia Di Angelantonio
co-supervisore: Silvia Di Angelantonio

Titolo della tesi: iPSC-derived cortical neurons and 3D whole-brain organoids to closely model human brain development in Fragile X syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by an aberrant expansion of the CGG repeat motif in the fragile X mental retardation-1 gene (FMR1) promoter, which leads to methylation, transcriptional silencing and eventually irreversible loss of the fragile X mental retardation protein (FMRP). FMRP plays a pivotal role in the proper development of synaptic connections and its absence is directly linked with alterations in dendritic spine morphology, synaptogenesis and connectivity in the developing brain leading to neurological, cognitive and behavioural defects (Salcedo-Arellano et al., 2020). Human brain development and disease, including FXS, have been modelled in vitro using human-induced pluripotent stem cells (hiPSCs) (Liu et al., 2018). However, the lack of appropriate in vitro models mimicking the real complexity of human brain hinders the deep understanding of pathological mechanisms and consequently the development of effective and safe drugs for neurological disorders. Therefore, the general aim of my PhD study was to develop and characterize at the molecular, morphological and functional level, both two-dimensional (2D) and three-dimensional (3D) neuronal culture models derived from hiPSCs for the study of the FXS early neurodevelopment events in vitro in a more physiologically relevant brain model. Firstly, we developed a 2D culture model of mixed cortical population adapting the method described by Livesey lab (Shi et al., 2012). A control (WT) iPSC line and an isogenic house-made FMR1 Knockout (FMR1 KO) iPSC line were used for this project and the differentiation protocol allowed to obtain a complex network of cortical neurons, including TBR1-positive deep layer neurons and GFAP-positive astrocytes. The 2D networks were characterized using calcium imaging experiments, which confirmed the full maturation of the generated neurons as a function of their ability to integrate the spontaneous electrophysiological activity. Moreover, with immunofluorescence assays, we also studied the different cell populations present in these 2D mixed cultures discovering that 2D FMR1 KO cultures displayed increased astrocytic markers after 70 days in vitro and, deepening the study of the synaptic contacts of these two neuronal populations, we demonstrated that 2D FMR1 KO cortical neurons showed an increase in glutamatergic synapses after 54 days in culture and both WT and Fmr1 KO neurons were able to respond to synaptic plasticity treatments. Furthermore, since self-organizing cerebral organoids excel at recapitulating the complex 3D cytoarchitecture of the human brain, proving to be very useful models for the study of the first events of neurodevelopment, we set up iPSC-derived 3D cell cultures using whole-brain organoids protocols (Lancaster and Knoblich., 2014) for the production of more physiological in vitro models of human development and disease. We applied this innovative technology to the study of FXS creating a new in vitro model not yet used to study this pathology and examining how the absence of FMRP can morphologically and functionally invalidate the proper first neurodevelopment events in this pathological context. Therefore, we obtained brain organoids from both WT and Fmr1 KO iPSCs lines demonstrating that Fmr1 KO cerebral organoids displayed a larger size and increased expression of the GFAP astrocytic marker respect to WT. In conclusion in this project we provide novel, physiologically relevant 2D and 3D hiPSC-based cellular models to investigate the mechanisms and the pathological features of the FXS, focusing on a brain region particularly affected by the disease: the cerebral cortex. These models recapitulate the cellular components and the complex 3D cytoarchitecture of the human brain and, being built with patient-derived cells, can be used to study FXS in a time frame that is relevant to the disease, understand its mechanisms and allow for therapeutic testing, all in 2D and 3D cellular models carrying the genetic background of individual patients. Lancaster MA, Knoblic JA (2014) Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014 Oct; 9 (10): 2329-40. Liu C, Oikonomopoulos A, Sayed N, Wu JC. Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond. Development. 2018;145(5):dev156166. Published 2018 Mar 8. doi:10.1242/dev.156166. Salcedo-Arellano MJ, Dufour B, McLennan Y, Martinez-Cerdeno V, Hagerman R. Fragile X syndrome and associated disorders: Clinical aspects and pathology. Neurobiol Dis. 2020;136:104740. doi:10.1016/j.nbd.2020.104740. Shi Y, Kirwan P, Livesey FJ. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc. 2012 Oct;7(10):1836-46.

Produzione scientifica

11573/1565955 - 2022 - Stem cell-based 3D brain organoids for mimicking, investigating, and challenging Alzheimer's diseases
Cordella, F.; Brighi, C.; Soloperto, A.; Di Angelantonio, S. - 01f Lettera, Nota
rivista: NEURAL REGENERATION RESEARCH (Shenyang : Publishing House of Neural Regeneration Research.) pp. 330-332 - issn: 1673-5374 - wos: WOS:000678125500025 (6) - scopus: 2-s2.0-85111025094 (8)

11573/1661240 - 2022 - Human iPSC-Derived Cortical Neurons Display Homeostatic Plasticity
Cordella, Federica; Ferrucci, Laura; D'antoni, Chiara; Ghirga, Silvia; Brighi, Carlo; Soloperto, Alessandro; Gigante, Ylenia; Ragozzino, Davide Antonio; Bezzi, Paola; Di Angelantonio, Silvia - 01a Articolo in rivista
rivista: LIFE (Basel: MDPI) pp. 1884- - issn: 2075-1729 - wos: WOS:000895626200001 (4) - scopus: 2-s2.0-85149560233 (4)

11573/1426014 - 2020 - Retinal and brain organoids: bridging the gap between in vivo physiology and in vitro micro-physiology for the study of Alzheimer’s diseases
Brighi, Carlo; Cordella, Federica; Chiriatti, Luigi; Soloperto, Alessandro; Di Angelantonio, Silvia - 01g Articolo di rassegna (Review)
rivista: FRONTIERS IN NEUROSCIENCE (Lausanne : EPFL : Frontiers Research Foundation, 2007-) pp. - - issn: 1662-453X - wos: WOS:000546797500001 (13) - scopus: 2-s2.0-85087179680 (16)

11573/1276468 - 2019 - Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors
Garone, Maria G; De Turris, Valeria; Soloperto, Alessandro; Brighi, Carlo; De Santis, Riccardo; Pagani, Francesca; Di Angelantonio, Silvia; Rosa, Alessandro - 01a Articolo in rivista
rivista: JOURNAL OF VISUALIZED EXPERIMENTS ([Boston, Mass. : MYJoVE Corp., 2006]-) pp. - - issn: 1940-087X - wos: WOS:000469977600054 (12) - scopus: 2-s2.0-85066930236 (16)

11573/1318360 - 2019 - 3D bioprinted human cortical neural constructs derived from induced pluripotent stem cells
Salaris, Federico; Colosi, Cristina; Brighi, Carlo; Soloperto, Alessandro; Turris, Valeria De; Benedetti, Maria Cristina; Ghirga, Silvia; Rosito, Maria; Di Angelantonio, Silvia; Rosa, Alessandro - 01a Articolo in rivista
rivista: JOURNAL OF CLINICAL MEDICINE (MDPI Publishing, Basel, Switzerland) pp. - - issn: 2077-0383 - wos: WOS:000498398500093 (37) - scopus: 2-s2.0-85084064985 (44)

11573/1124761 - 2018 - Inflammation, neurodegeneration and protein aggregation in the retina as ocular biomarkers for Alzheimer’s Disease in the 3xTg-AD mouse model
Grimaldi, A; Brighi, Carlo; Peruzzi, G; Ragozzino, D; Bonanni, V; Limatola, C; Ruocco, G; Di Angelantonio, S. - 01a Articolo in rivista
rivista: CELL DEATH & DISEASE (Nature Group) pp. - - issn: 2041-4889 - wos: WOS:000434774800009 (107) - scopus: 2-s2.0-85048237301 (107)

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