Volume 12, Issue 5, Pages 520-530 (May 2013) Next-Generation Regenerative Medicine: Organogenesis from Stem Cells in 3D Culture  Yoshiki Sasai  Cell Stem Cell  Volume 12, Issue 5, Pages 520-530 (May 2013) DOI: 10.1016/j.stem.2013.04.009 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Self-Formation of Compartmentalized Tissue Structures in Teratoma A teratoma formed by injection of pluripotent stem cells (human ESCs) into an immunodeficient mouse testis (reproduced from Watanabe et al., 2007 and our unpublished data). ESCs gave rise to various tissues of three germ-layer derivatives such as brain tissue (ectodermal), cartilage (mesodermal), and gut/bronchial epithelia (endodermal). Importantly, the teratoma contains compartmentalized tissues whose identities are readily recognizable by H&E staining, demonstrating that they convey typical morphological structures of corresponding tissues. Thus, in the testis, where central signaling centers for patterning instructions are, mesoscopic-sized tissues can self-form from nonprepatterned ESCs. Cell Stem Cell 2013 12, 520-530DOI: (10.1016/j.stem.2013.04.009) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Self-Organization of Ectodermal Tissue Structures in 3D ESC Culture (A) Schematic of SFEBq culture (serum-free floating culture of embryoid body-like aggregates with quick reaggregation) for various ectodermal tissue generation from ESCs/iPSCs (modified from Eiraku et al., 2008; Suga et al., 2011; Nakano et al., 2012). Typically, 3,000 mouse or 9,000 human ESCs/iPSCs per well are used to make an aggregate in the 96-well plate that has a special surface coating to avoid cell-plate adhesion. In this culture, hollow neuroectodermal spheres self-form from aggregates and they acquire different fates according to the culture conditions. Cortical, retinal, and pituitary tissues can form 3D structures by self-organization (orange). Cerebellar tissue generation involves secondary induction by isthmic organizer tissues that first form in the aggregate by the influence of Fgf signals. (B) Three basic modes of self-organization. Self-assembly is defined by spontaneous appearance of spatial arrangements in the cell populations, mostly by selective adhesion and self-sorting. Self-patterning is defined by spontaneous emergence of spatial patterns from the nonprepatterned cell populations. It often involves symmetry breaking. Self-driven morphogenesis is defined by spontaneous deformation of tissue structures by internal mechanisms. In self-organizing phenomena, these three modes often work together. Cell Stem Cell 2013 12, 520-530DOI: (10.1016/j.stem.2013.04.009) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Endodermal Tissue Formation in 3D Stem Cell Culture Schematic of endodermal tissue formation in stem cell culture. PSC, pluripotent stem cells; DEP, definitive endodermal progenitors; FE, foregut endodermal progenitors; HP, hepatic progenitors; PP, pancreatic progenitors; IsEP, islet endocrine progenitors; PhEP, pharyngeal endodermal progenitors; PTP, pulmonary and tracheal progenitors; ThP, thyroid progenitors; IP, intestinal endodermal progenitors; ISC, intestinal stem cells. Cell Stem Cell 2013 12, 520-530DOI: (10.1016/j.stem.2013.04.009) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Manipulation of Multiplex Intercellular Interactions In 3D self-organizing culture, progenitors induced by differentiation conditions (e.g., neural induction and retinal positional information) undergo multiple cellular interactions. These local interactions start to build up emergent collective behaviors leading to self-organization of complex structures as a whole. Future studies of 4D stem cell biology should aim at controlling these complex cellular interactions, and thereby manipulating the characteristics in emergent phenomena such as pattern, shape, and size. A long-term goal may be a synthesis or de novo design of novel types of miniorgans. Emergence biology is certainly an attractive new field of biology that is also relevant to many areas of life sciences beside stem cell biology. Cell Stem Cell 2013 12, 520-530DOI: (10.1016/j.stem.2013.04.009) Copyright © 2013 Elsevier Inc. Terms and Conditions