BREATHING LIFE INTO ORGANOIDS: GENERATING PRE-BOTZINGER COMPLEX NEURONS TO STUDY RESPIRATORY DEPRESSION IN OPIOID OVERDOSE

The breathing signal originates from the preBotzinger complex (preBotC), the breathing centre core in the brainstem. The preBotC has an intrinsic central pattern generator activity to maintain a breathing rhythm. Opioids targeting the preBotC can cause respiratory depression and overdose fatalities, seen with the US opioid crisis and growing outbreaks in Europe. However, it is difficult to study the interactions between opioids and the preBotC in humans. We aimed to generate preBotC neurons using human pluripotent stem cells (hPSCs) for in vitro/organoid and in vivo/neural transplantation modelling of the breathing centre.
We differentiated hPSCs into respiratory neural progenitors and then preBotC neurons by 70days. Neuronal activity was assessed on 3D high-density micro-electrode arrays (HD-MEA) and treatment with potent opioid agonist DAMGO and opioid antagonist Naloxone, modelling opioid overdose/treatment. In vivo, progenitors were injected intracerebrally into mice and grafts histologically assessed after 4months of maturation.
Neural progenitors acquired HOXB4+HOXB5– rhombomere 7 hindbrain and DBX1+ V0 subplate identity, consistent with the preBotC's developmental origin. Mature neurons expressed HOXB4 and NK1R, appropriate for preBotC. Consistent with preBotC's intrinsic central pattern generator feature, neurons exhibited regular, spontaneous bursting activity, and activity suppression by DAMGO was combatted by antagonist naloxone. Transplanted respiratory progenitors produced HOXB4+NK1R+HuD+ neurons that innervated ipsilateral and contralateral host brain structures, demonstrating capacity to form a bilateral network, critical for coordinating bilateral breathing movements.
We demonstrated hPSC-derived preBotC neurons can model CNS breathing disorders, like opioid overdose/treatment in vitro, and be transplanted for disease modelling.