Spinal Cord Organoids from Human Amniotic Fluid iPSC Recapitulate the Diversity of Cell Phenotypes During Fetal Neural Tube Morphogenesis

Myelomeningocele (MMC) is a severe form of spina bifida associated with substantial neurologic morbidity. In vitro modeling systems of human spinal cord development may help to elucidate the underlying pathophysiology of the MMC spinal cord. To that end, we developed spinal cord organoids (SCO), defined as self-organized, three-dimensional clusters of spinal tissue, that were derived from human amniotic fluid–induced pluripotent stem cells. Here, we used a variety of analyses, including immunofluorescent and single-cell transcriptomic approaches, to characterize SCOs from healthy and MMC fetuses. Organoids contained a diverse range of neural and mesodermal phenotypes when cultured for up to 130 days in vitro. Multielectrode arrays revealed functional activity with evidence of emerging neuronal networks. Fetal spina bifida environment modeling was successfully established by culturing SCOs in second- and third-trimester amniotic fluid for 3 weeks. Taken together, we show that functional SCOs can recapitulate the cellular identity of the fetal spinal cord and represent a novel research platform to study the interplay between cellular, biochemical, and mechanical cues during human MMC neural tube morphogenesis.