iPSC-DERIVED NEURAL STEM CELLS CULTURED ON ENGINEERED SUBSTRATES AS AN IN VITRO MODEL FOR AICARDI-GOUTIÈRES SYNDROME
R.M. Ferraro1, P.S. Ginestra2, D.Vairo1, G. Lanzi1, S. Masneri1, C. Barisani1, G. Piovani3, G.Savio3, J. Galli4, S.
Orcesi5, E. Fazzi4, E. Ceret2, S. Giliani1
1 Institute of Molecular Medicine "Angelo Nocivelli", Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy. 2 Department of Mechanical and Industrial Engineering, University of Brescia, Brescia, Italy. 3 Biology and Genetics Division, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy. 4 Unit of Child Neurology and Psychiatry, University of Brescia, Brescia, Italy. 5 Unit of Child Neurology and Psychiatry, IRCCS "C. Mondino National Institute of Neurology" Foundation, Pavia, Italy
Aicardi–Goutières Syndrome (AGS) is a rare monogenic inflammatory encephalopathy with neurological impairment and cerebrospinal fluid lymphocytosis with elevated levels of proinflammatory cytokines such as IFNα and CXCL10. AGS exhibits locus heterogeneity in enzymes involved in nucleic acids metabolism to prevent activation of cell intrinsic responses to immunostimulatory DNA and RNA. The main
neuropathological feature of the disease is abnormal myelination, probably caused by increased expression of proteases like cathepsin D, able to degrade myelin and brain tissue matrix. The unavailability of human model systems able to recapitulate the neurological disease in vitro makes it difficult to investigate the pathophysiological mechanisms. A valid experimental tool to study this disorder could be offered by genetically stable neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs). We
generated iPSCs from fibroblasts of three AGS patients with mutations in TREX1, RNASEH2B, IFIH1, one age matched healthy donor and one strain BJ, as positive transduction control. After obtaining stable and characterized iPSCs lines we performed the induction to neural stem cells (NSCs). Immunofluorescence and real-time quantitative PCR analysis suggest that cells derived from iPSCs possess the NSC phenotype and could be used for the differentiation to the three major types of central nervous system cells: neurons, astrocytes and oligodendrocytes.
In order to provide to the NSCs the best growth support, we cultured cells on engineered substrates used in the biomedical industry that are designed to promote cellular adhesion, proliferation and differentiation. A specific configuration was design to pattern a silicon wafer with the SU-8 polymer. In particular, the features of the patterns were produced by the cross-linking of the polymer during the photolithography process. Moreover, the polymer was used as a precursor in a pyrolysis treatment to obtain glassy-carbon features. Preliminary results show that the patterned substrate is able to guide the NSCs orientation along a specific direction; in particular Nestin and Actin immunostaining showed that the cytoskeletal arrangement was modified by the interaction with the substrates highlighting a different morphology of the cells. Future studies will be focused on the analysis of the performances of these substrates in terms of cells differentiation.
