Kyubin Seo ¹, Subin Cho ², Hyogeun Shin ³, Aeri Shin ¹, Ju-Hyun Lee ¹, June Hoan Kim ¹, Boram Lee ¹, Hwanseok Jang ³, Youngju Kim ¹, Hyo Min Cho ¹, Yongdoo Park ³, Hee Youn Kim ⁴, Taeseob Lee ⁴, Woong-Yang Park ^4,5, Yong Jun Kim ⁶, Esther Yang ¹, Dongho Geum ³, Hyun Kim ¹, Il-Joo Cho ^1,3, Sanghyuk Lee ^2,7, Jae Ryun Ryu ¹, Woong Sun ¹

¹Department of Anatomy, Korea University College of Medicine, Seoul, 02841, Republic of Korea.
²Department of Bio-Information Science, Ewha Womans University, Seoul, 03760, Republic of Korea.
³Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea.
⁴Geninus Inc., Seoul, 05836, Republic of Korea.
⁵Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University, Seoul, 06351, Republic of Korea.
⁶Department of Pathology, College of Medicine, Kyung Hee University, Seoul, 02447, Republic of Korea.
⁷Department of Life Science, Ewha Womans University, Seoul, 03760, Republic of Korea.

K.S. and S.C. contributed equally to the study. 
CORRESPONDING AUTHORS: Jae Ryun Ryu, Woong Sun

Axis formation and related spatial patterning are initiated by symmetry breaking during development. A geometrically confined culture of human pluripotent stem cells (hPSCs) mimics symmetry breaking and cell patterning. Using this, polarized spinal cord organoids (pSCOs) with a self-organized dorsoventral (DV) organization are generated. The application of caudalization signals promoted regionalized cell differentiation along the radial axis and protrusion morphogenesis in confined hPSC colonies. These detached colonies grew into extended spinal cord-like organoids, which established self-ordered DV patterning along the long axis through the spontaneous expression of polarized DV patterning morphogens. The proportions of dorsal/ventral domains in the pSCOs can be controlled by the changes in the initial size of micropatterns, which altered the ratio of center-edge cells in 2D. In mature pSCOs, highly synchronized neural activity is separately detected in the dorsal and ventral side, indicating functional as well as structural patterning established in the organoids. This study provides a simple and precisely controllable method to generate spatially ordered organoids for the understanding of the biological principles of cell patterning and axis formation during neural development.