Human iPSC-Derived Spinal Cord Organoids – The First Electrophysiological Assessment of Neuronal Maturation

Abstract

The spinal cord exhibits a complex cytoarchitecture and neuronal circuitry organization that challenges in vitro replication of its functional integrity. Here we employed a previously published protocol to generate spinal cord organoids (SCOs) from human-induced pluripotent stem cells (hiPSCs). We characterized phenotypic changes in functional neuronal properties in SCO neurons at early developmental stages (15–32 days in culture) using a quantitative electrophysiological approach. Using the whole-cell patch-clamp technique to evaluate electrophysiological properties, we found that SCO neurons exhibited progressive maturation, as evidenced by hyperpolarized resting membrane potentials, increased inward current amplitude, refined action potential kinetics, and the early emergence of mature-type firing patterns. In particular, we show that the spike frequency adaptation phenomenon, which prevails in motor neurons, appears at early stages of SCO neuron development. Immunohistochemical assessment confirmed the expression of key transcription factors in motor neurons (ISLET1 and HB9) and immature spinal interneurons (LHX1/5 and PAX2). Collectively, our findings demonstrate that neurons in hiPSC-derived SCOs exhibit physiological differentiation, which is important for using SCOs to investigate human spinal cord development and advance translational research in CNS disorders and cell replacement therapies.