Generation of patient-specific, iPSC-derived brainstem organoids to study Congenital Central Hypoventilation Syndrome (CCHS)

Human induced pluripotent stem cells (hiPSC) technology enables in vitro modeling of the human brain through the generation of three-dimensional organoids. This new approach is particularly valuable for investigating those diseases for which in vivo and in vitro models are to date limited or inadequate, such as Congenital Central Hypoventilation Syndrome (CCHS). CCHS is a rare neurodevelopmental disorder of the autonomic nervous system (ANS), characterized by alveolar hypoventilation, due to an impaired autonomic response to hypoxia and hypercapnia, especially during non-REM sleep. The causative gene has been identified in PHOX2B, which encodes for a master regulator of the ANS development and is expressed also in the brainstem centers involved in the autonomic control of breathing. To generate an appropriate disease model, we first established hiPSC lines from CCHS patients with different PHOX2B mutations and clinical presentations. Then, we differentiated them, alongside control hiPSC lines, into brainstem organoids (hBSO), following a 60 day-long, guided protocol. In this process, cells aggregate into embryoid bodies and undergo neural induction via dual SMAD inhibition, followed by ventralization, caudalization and a final phase of maturation. Our preliminary results from transcriptomic analyses and fluorescent IHC revealed abnormalities in patient-derived hBSOs, including defects in regional patterning and impaired neuronal maturation. In conclusion, this personalized organoid model provides a powerful tool to investigate CCHS pathogenesis and offers a promising platform for future therapeutic screening and development.