Postdoctoral Researcher University of Sydney Sydney, New South Wales, Australia
Abstract: The rhythm of breathing is vital for mammalian life, required for daily activities and during sleep. The central nervous system (CNS) cells responsible for initiating and autonomously maintaining breathing rhythm are the preBotzinger complex (preBotC) neurons, residing in the brainstem. In rodents, preBotC development requires an orchestrated rostro-caudal signalling to achieve the correct rhombomeric regional identity within the hindbrain, as well as the correct dorso-ventral patterning. Although the biology of these neurons has been investigated in rodent studies, very little information exists from investigations in human systems. Here, we aimed to establish a novel, small molecule-based, human pluripotent stem cell (hPSC) protocol to differentiate regionally-specified preBotC neurons for advancing our understanding of human-specific features of the preBotC.
Following dual-SMAD inhibition for neural induction in vitro, we found that retinoic acid was necessary to upregulate the expression of HOX4 gene, required for the correct rostro-caudal rhombomeric patterning. Finetuning of dorso-ventral patterning of our cells successfully generated progenitors that expressed DBX1, a key marker of respiratory neural progenitors in the hindbrain. Maturation of these progenitors gave rise to neurons expressing markers consistent with preBotC identity and are electrophysiologically functional as demonstrated by multi-electrode array recordings.
This protocol represents one of the first to generate human preBotC neurons by modulating rostro-caudal and dorso-ventral patterning of human PSCs. Establishing this hPSC protocol of preBotC neuron differentiation will facilitate future gene editing and disease modelling studies to further understand breathing and the rhythm of breathing in humans, as well as therapeutically supporting patients living with CNS-related respiratory disorders.
