Phenotypic Characterization of 3D Human Spinal Cord Organoids Derived from Fetuses with Neural Tube Defects

Background: Myelomeningocele (MMC) is the most common neural tube defect characterized by failure of neural tube closure during embryonic development, resulting in exposure of the unprotected fetal spinal cord to the intrauterine environment. Despite neurosurgical closure of the defect in the fetal or early postnatal period, affected children still suffer substantial and permanent disabilities, including leg weakness and paralysis, hydrocephalus, and bladder/bowel dysfunction. In the present study, we describe the generation of 3D dorsalized spinal cord organoids from human MMC amniotic fluid-derived induced pluripotent stem cells (iPSCs) as a tool for patient-specific diagnostic and therapeutic use.

Methods: Under IRB approved protocol (IRB00202082), human amniotic fluid (HAF) samples were harvested from two MMC fetuses and one healthy fetus during the second trimester. Mesenchymal cells were isolated and reprogrammed into iPSCs by transduction with non-integrating Sendai virus. IPSC clones were fully characterized and subsequently differentiated into 3D spinal cord organoid tissue, based on a modified protocol as described by the Takahashi group. Statistical analyses were performed using non-parametric tests as appropriate (p < 0.05).

Results: MMC and control iPSCs were successfully generated by reprogramming HAF mesenchymal cells as demonstrated by characteristic morphology, alkaline phosphatase staining, positive expression of markers of undifferentiation (e.g., NANOG, OCT4, SOX2, SSEA3) and pluripotency, through the formation of embryoid bodies expressing markers of the three germ layers. Differentiation into 3D spinal cord organoids was shown by quantitative gene expression, demonstrating neuroectoderm (SOX1, PAX6), roof plate (Lmx1a), dorsal (Olig3, PAX7), and ventral (Olig2) spinal cord progenitors. Following 9 days of dorsalization with BMP4 morphogen, day 24 organoids expressed Brn3a and Lbx1, markers of dorsal interneurons. Lhx9 and Islet1 gene expression identifies specifically dl1 and dl3 subtypes of neurons, whereas Pax2 and Lmx1b dl4-dl6 domains. On the ventral half of the spinal cord, expression of Lhx3 (V2a and MN), GATA2 (V2b) and Isl2 (MN) account for neurons of the ventral domains (Figure). Overall, MMC cell lines had significantly fewer neuroectodermal cells than control line at day 9 of differentiation, and progenitors/neurons were more restricted to the dorsal spinal cord as observed at day 15 and 24. However, a subset of cells in MMC organoids become motoneurons as detected at 24 days of differentiation

Conclusion: Human 3D dorsal spinal cord organoids can be successfully generated in as little as 3 months from amniotic fluid harvested from prenatally diagnosed MMC patients. Dorsal spinal cord organoids will provide a novel in vitro platform to study the disease pathogenesis of neural tube defects. Additionally, these cells may represent an important autologous source for neural progenitors aimed at facilitating spinal cord regeneration at the time of fetal or neonatal MMC repair.