Generation of Spontaneously Beating Neuromyocardial Organoids with Neural Crest-Derived Proprioceptive-Biased Neurons and Real-Time Respirometry.

The neural crest gives rise to diverse cell types, including the proprioceptive sensory neurons that relay limb and organ position to the central nervous system. Studying how these neurons interact with their peripheral targets in a human context has been constrained by the lack of integrated in vitro models that combine neural crest derivatives with functional effector tissue. Here, we present a protocol for generating spontaneously beating neuromyocardial organoids from human pluripotent stem cells (hPSCs), incorporating neural crest-derived proprioceptive-biased neurons. Differentiation proceeds under defined conditions that reproducibly co-specify cardiac and neural crest lineages, yielding organoids that contract rhythmically within 7-8 days and harbor proprioceptive-biased sensory neuron populations. We further describe how to integrate these organoids with continuous oxygen consumption rate (OCR) monitoring, enabling non-invasive measurement of mitochondrial respiration over days to weeks. This longitudinal readout captures dynamic shifts in respiration that are inaccessible to endpoint assays, providing a sensitive functional metric of metabolic health and stress. The combined model is broadly applicable to questions in human cardiac and neural crest development, neuromuscular and sensorimotor integration, and the metabolic basis of disease. We illustrate its utility in Friedreich's ataxia (FRDA), a mitochondrial disorder affecting both proprioceptive neurons and cardiac tissue, in which real-time OCR monitoring reveals respiratory deficits. The protocol offers a reproducible, scalable system for interrogating neural crest biology alongside cardiac function and for evaluating metabolic phenotypes and pharmacological interventions in a physiologically relevant, multi-lineage human context. Notably, the entire differentiation sequence runs on chemically defined, animal-free media, and organoids form directly in standard U-bottom 96-well plates, without ultra-low-attachment surfaces, matrix embedding, pre-coating, or orbital shaking, and each organoid is formed and maintained in 100 µL of medium, keeping reagent, consumable and equipment costs low and the workflow straightforward to adopt.