Toward a 3D in vitro model based on decellularized thymus to maintain adult thymic ephitelial cells functionality

During my PhD,I have been develop an innovative technique to reproduce in vitro the 3D thymic microenvironment, to be used for growth and differentiation of thymocytes, and possible transplantation replacement in conditions of depressed thymic immune regulation. The work has been developed in the laboratory of Tissue Engineering at the University Hospital in Basel, Switzerland, under the tutorship of Prof.Ivan Martin. Since a number of studies have suggested that the 3D structure of the thymic microenvironment might play a key role in regulating the survival and functional competence of thymocytes, I’ve focused my effort on the isolation and purification of the extracellular matrix of the mouse thymus. Specifically, based on the assumption that TEC can favour the differentiation of pre-T lymphocytes, I’ve developed a specific decellularization protocol to obtain the intact, DNA-free extracellular matrix of the adult mouse thymus. Two different protocols satisfied the main characteristics of a decellularized matrix, according to qualitative and quantitative assays. In particular, the quantity of DNA was less than 10% in absolute value, no positive staining for cells was found and the 3D structure and composition of the ECM were maintained. In addition, I was able to prove that the decellularized matrixes were not cytotoxic for the cells themselves, and were able to increase expression of MHC II antigens compared to control cells grown in standard conditions. I was able to prove that TECs grow and proliferate up to ten days on top the decellularized matrix. After a complete characterization of the culture system, these innovative natural scaffolds could be used to improve the standard culture conditions of TEC, to study in vitro the action of different factors on their differentiation genes, and to test the ability of TECs to induce in vitro maturation of seeded T lymphocytes.

Altered transcriptional and epigenetic regulation affects brain cells proliferation and differentiation in the ultra-rare genetic demyelinating disease AGC1 deficiency: a study on in vitro models

AGC1 deficiency is a rare demyelinating disease caused by mutations in the SLC25A12 gene, which encodes for the mitochondrial glutamate-aspartate carrier 1 (AGC1/Alarar), highly expressed in the central nervous system. In neurons, impairment in AGC1 activity leads to reduction in N-acetyl-aspartate, the main lipid precursor for myelin synthesis (Profilo et al., 2017); in oligodendrocytes progenitors cells, AGC1 down regulation has been related to early arrest proliferation and premature differentiation (Petralla et al., 2019). Additionally, in vivo AGC1 deficiency models i.e., heterozygous mice for AGC1 knock-out and neurospheres from their subventricular zone, respectively, showed a global decrease in cells proliferation and a switch in neural stem cells (NSCs) commitment, with specific reduction in OPCs number and increase in neural and astrocytic pools (Petralla et al., 2019). Therefore, the present study aims to investigate the transcriptional and epigenetic regulation underlying the alterations observed in OPCs and NSCs biological mechanisms, in either AGC1 deficiency models of Oli-neu cells (murine immortalized oligodendrocytes precursors cells), partially silenced by a shRNA for SLC25A12 gene, and SVZ-derived neurospheres from AGC1+/- mice. Western blot and immunofluorescence analysis revealed significant variations in the expression of transcription factors involved in brain cells’ proliferation and differentiation, in association with altered histone post-translational modifications, as well as histone acetylases (HATs) and deacetylases (HDACs) activity/expression, suggesting an improper transcriptional and epigenetic regulation affecting both AGC1 deficiency in vitro models. Furthermore, given the large role of acetylation in controlling in specific time-windows OPC maturation (Hernandez and Casaccia; 2015), pharmacological HATs/HDACs inhibitions were performed, confirming the involvement of chromatin remodelling enzymes in the altered proliferation and early differentiation observed in the AGC1 deficiency models of siAGC1 Oli-neu cells and AGC1+/- mice-derived neurospheres.