Generation of 2D and 3D iPSC-derived neuronal models for the study of Congenital Central Hypoventilation Syndrome (CCHS)

The biggest limitation in the study of the pathogenesis of neurodevelopmental disorder is the generation of a valid model that can recapitulate the complex adult human neurological phenotype and possible defects occurring during embryonic neurodevelopment. Congenital Central Hypoventilation Syndrome (CCHS) is a rare genetic disorder affecting the autonomic nervous system (ANS) and central chemosensitivity. It is caused by mutations on PHOX2B, a master transcription factor whose role is essential during development of the ANS and central structures that participate in breathing control. Given PHOX2B’s crucial role, obtaining a viable animal model has not been possible yet and only conditional models that recapitulate partial CCHS phenotypes have been generated. Here, we used reprogramming technology to generate induced pluripotent stem cells (iPSC) from patient’s fibroblasts carrying different PHOX2B mutations. We conducted a characterization of all iPSC lines by karyotyping, immunocytochemistry, and qPCR analysis to confirm the expression of pluripotency markers (Nanog, OCT4 and SSEA4) [Previously published in: Cuadros et al. (2022) Stem Cell Res. 61:102781]. To give insight into possible developmental defects at both peripheral and central level, we generated 2D autonomic neurons and 3D cerebral organoids with cytoarchitectures resembling central chemoreceptors from CCHS patients and controls. We adapted a protocol for the differentiation to autonomic neurons based on the generation of intermediate cell populations, neural crest cells and sympathoadrenal progenitors, mimicking the embryonic development and demonstrated that the protocol can be successfully applied to CCHS patients iPSCs. These new personalized disease-in-a-dish models of CCHS open numerous possibilities to identify molecular and cellular defects induced by the mutations as well as modelling for drug discovery/screening for therapeutic perspectives.