Numerous morphogenetic events are explained by the cell surf

Numerous morphogenetic events are explained by the cell surface tension model. In equilibrium, surface tension is minimized via coordinated forces generated by cell surface tension and actomyosin-mediated cortical tension, resulting in cell sorting and self-organization (Beysens et al., 2000; Heisenberg and Bellaiche, 2013; Lecuit and Lenne, 2007; Steinberg, 1963). Rho kinase (ROCK) regulates actomyosin networks and cell polarity (Amano et al., 2010). Myosin activation and cell contraction depend on the phosphorylation of myosin regulatory light chains (pMYL) by apical ROCK. Significant progress has been made in generating retinal structures from hESC or hiPSC cultures in which a regime of extrinsic factors with or without Matrigel are used (Boucherie et al., 2013; Lamba et al., 2009; Meyer et al., 2011; Nakano et al., 2012; Reichman et al., 2014; Zhong et al., 2014; Zhu et al., 2013). However, mechanistic understanding of the in vitro process is only beginning. Deeper insight into retinal differentiation in vitro will help develop a better procedure for generating retinal ap-1 in stem cell-based research. Here we dissected retinal organoid morphogenesis in hESC cultures and established a Dispase-mediated method for isolating large quantities of retinal organoids comprising patterned NR, RPE, and CM. Our findings demonstrate that intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for self-organization of retinal organoids, and support a hypothesis that newly specified VSX2+ RPCs form characteristic structures in equilibrium via minimization of cell surface tension. In long-term culture, the retinal organoids generate stratified retinal tissues, including photoreceptors that contain the ultrastructure of outer segments. Our system requires minimal manual manipulation, has been validated in two lines of hPSCs, and thus has multiple applications in modeling human retinal development and disease.
Results
Discussion With a culture system established here, we dissected retinal organoid morphogenesis in hESC-derived cultures. We demonstrate that intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces are required for self-formation of retinal organoids and propose a hypothesis that newly specified VSX2+ RPCs form characteristic structures in equilibrium via minimization of cell surface tension. Our Dispase-mediated method is convenient for isolation of large amounts of optic cup-like retinal organoids with minimal manual manipulation, has been validated in two lines of hPSCs, and differs substantially from the methods of manual dissection or mechanical scraping described previously (Meyer et al., 2009, 2011; Nakano et al., 2012; Reichman et al., 2014; Zhong et al., 2014). The retinal organoids autonomously generate stratified retinal tissues with all retinal cell types in cultures, and thus are powerful models for studying human retinal development and disease. The ECM provides cues for coordinated cell polarization and cell survival in the transformation from the initially amorphous epiblast into the cup-shaped epithelium in mouse blastocysts (Bedzhov and Zernicka-Goetz, 2014; Coucouvanis and Martin, 1995). It also underlies cell epithelialization of the eye field in zebrafish (Ivanovitch et al., 2013). Matrigel (56% laminin, 31% collagen IV, and 8% entactin), an ECM surrogate, is used in retinal differentiation by either being suspended in culture media (Boucherie et al., 2013; Nakano et al., 2012) or forming a solidified layer of Matrigel/cells onto culture surfaces (Zhu et al., 2013). Here, floating culture of Matrigel/hESC clumps generates epithelialized cysts. In our hands, cell epithelialization is more efficient via Matrigel-aided cyst formation than by culturing embryonic bodies in Matrigel-containing media. In contrast to the solidified layer on culture surfaces (Zhu et al., 2013), the floating clumps of Matrigel/hESCs described here permit the cells to have better access to medium, and cysts in the floating clumps spontaneously attach to culture surfaces and spread at later stages. Matrigel promotes cell survival and epithelialization in cyst formation likely through integrin signaling (Manninen, 2015). In addition, epithelial structure of the cysts could confer cell survival, since correct polarization of acini accounts for resistance to apoptosis in breast cancer cell lines (Weigelt and Bissell, 2008). In literature, inhibitors of ROCK or myosin are used in retinal differentiation to suppress undesirable apoptosis (Nakano et al., 2012; Zhong et al., 2014). It is possible, however, that the inhibition compromises cell epithelialization. In our system, ROCK inhibitor Y27632 is dispensable and disruptive in cyst formation. Spontaneous cell specification in the epithelialized cysts from the initial pluripotent fate to the cell fates of anterior ectoderm/neural plate, eye field, and optic vesicle resembles neural differentiation as default (Stern, 2006); the existence of both retinal cells and brain cells in the cultures reflects the multipotency and/or heterogeneity of the cysts. We conclude that 3D contact between Matrigel and small hESC sheets provides a cue similar to that in the blastocyst and the eye field, leading to efficient epithelialization of hESCs and retinal differentiation.