Elongation of Caudalized Human Organoids Mimics Neural Tube Development

Abstract Axial elongation of the neural tube is critical during mammalian embryogenesis to establish the anterior-posterior body axis1, but this process is difficult to interrogate directly because it occurs post-implantation2,3. Here we report an organoid model of neural tube extension by caudalized human pluripotent stem cell (hPSC) aggregates that recapitulates the morphologic and temporal gene expression patterns of neural tube development. Axially extending organoids consisting largely of longitudinally elongated neuroepithelial compartments also contained TBXT(+)SOX2(+) neuromesodermal progenitors, PAX6(+)nestin(+) neural progenitor populations, and MEOX1(+) paraxial mesoderm populations. Wnt agonism stimulated singular axial extensions in a dose-dependent manner, and elongated organoids displayed regionalized rostral-caudal HOX gene expression, with spatially distinct hindbrain (HOXB1) expression from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR-interference-mediated silencing of the TBXT, a downstream Wnt target, increased neuroepithelial compartmentalization and resulted in multiple extensions per aggregate. Further, knock-down of BMP inhibitors, Noggin and Chordin, induced elongation phenotypes that mimicked murine knockout models. These results indicate the potent morphogenic capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early nervous system development in humans.