Research projects

Objectives:

The overall goal of this proposal was to elucidate the role of the purinergic signaling in the human cortical development in order to contribute to the future design of therapeutic strategies against cortical malformations.

The Project aimed to employ the ground-breaking research possibilities opened by organoids to perform a complete phenotypic study as well as to clarify which signaling pathways and target genes are involved in the human cortical scenario:

Specifically, we aimed to i) characterize the expression and function of the purinergic system during the human cortex development both at single cell and at whole organ structure level. Moreover, we ii) pursued to improve the organoid culture method. 

Overall summary of the progress of the project:

Throughout the progression of this project we achieved the following goals:

-Improvement and consolidation of the organoid culture method.

-Analysis of the expression of purinergic receptors and VNUT by immunohistochemistry, Western blot and mRNA in both the neural progenitor and neuronal domain of the developing cortex.

-Analysis of the mRNA and protein levels along the organoid formation (hiPSCs, Embryonic Bodies…). 

-Preliminary experiments of electroporation of the organoids with vectors encoding for gain and loss of function of the P2X7 receptor.

-Establishment of the 2D protocols that allows for a detailed functional assessment of the purinergic system along the organoid formation. We performed this functional analysis of the purinergic receptors by both calcium imaging and patch clamp techniques.

-We performed a set of experiments with organoids being cultured in presence or absence of purinergic agonist and antagonist to evaluate the impact of the purinergic system on the organoid formation.