Engineering Human CNS Morphogenesis: Controlled Induction of Singular Neural Rosette Emergence

Abstract Human pluripotent stem cell (hPSC)-derived neural organoids have revolutionized in vitro modelling of human neurological disorders. Cell-intrinsic morphogenesis processes displayed within these tissues could serve as the basis for ex vivo manufacture of brain and spinal cord tissues with biomimetic anatomy and physiology. However, we must first understand how to control their emergent properties starting at the genesis of neural organoid formation, i.e. emergence of polarized neuroepithelium. In vivo, all CNS tissues develop from a singular neuroepithelial tube. Yet, current protocols yield organoids with multiple neuroepithelial rings, a.k.a. neural rosettes, each acting as independent centers of morphogenesis and thereby impeding coordinate tissue development. We discovered that the morphology of hPSC-derived neural tissues is a critical biophysical parameter for inducing singular neural rosette emergence. Tissue morphology screens conducted using micropatterned array substrates and an automated image analysis determined that circular morphologies of 200-250 and 150μm diameter are optimal for inducing singular neural rosette emergence within 80-85% forebrain and 73.5% spinal tissues, respectively. The discrepancy in optimal circular morphology for Pax6 + /N-cadherin + neuroepithelial forebrain versus spinal tissues was due to previously unknown differences in ROCK-mediated cell contractility. The singular neuroepithelium induced within geometrically confined tissues persisted as the tissues morphed from a 2-D monolayer to multilayered 3-D hemispherical aggregate. Upon confinement release using clickable micropatterned substrates, the tissue displayed radial outgrowth with maintenance of a singular neuroepithelium and peripheral neuronal differentiation. Thus, we have quantitatively defined a pertinent biophysical parameter for effectively inducing a singular neuroepithelium emergence within morphing hPSC-derived neural tissues. Significance Statement Human pluripotent stem cell (hPSC)-derived neural organoids display emergent properties that, if harnessed, could serve as the basis for ex vivo manufacture of brain and spinal cord tissues with biomimetic macroscale anatomy and physiology. Their chaotic terminal structure arises from uncontrolled morphogenesis at their genesis, resulting in spontaneous induction of multiple neuroepithelial morphogenesis centers,a.k.a. neural rosettes. Here, we determined that neural tissue morphology is a pertinent biophysical parameter for controlling subsequent morphogenesis, and identified discrete circular tissue morphologies as optimal and effective at inducing singular neural rosette emergence within forebrain and spinal neural tissues. Thus, we developed an approach to reproducibly control the initial stage of hPSC-derived neural tissue morphogenesis enabling their manufacture with a biomimetic nascent CNS anatomy.