Another question is the heterogeneity within and between org

Another question is the heterogeneity within and between organoids, which seems common to all the protocols developed so far but has not yet been studied in detail. Several processes, such as progenitor proliferation, cell differentiation, and ontogenetic cell death, could be potential sources of organoid variation. Transgenic animals with fluorescently labeled win 55 212-2 have been instrumental in visualizing major processes in the developing and adult retina. However, it is unknown whether reporter expression is comparable between retinal organoids and in vivo. Thus, we investigated PAX6 transgenic reporter expression to gain an insight into retinal organoidogenesis. PAX6 is a highly conserved master regulator of neurogenesis (Shaham et al., 2012), playing several roles in eye and retinal development, e.g., eyefield specification, stemness control, and cell-fate specification. PAX6 reporter expression might also provide an insight into the formation of retinal structure because it remains expressed in postmitotic horizontal and amacrine cells, whose synaptic processes are part of the outer and inner plexiform layer, respectively. Here, we have developed a protocol to facilitate efficient organoidogenesis of large, complex, 3D retinas derived from wild-type mESCs without requiring the formation and isolation of OV/OC-like structures. Gene-expression profile analyses of individual organoids and retinal cell birthdating experiments indicate efficient, reproducible, and temporally regulated retinogenesis. We have established retinal organoidogenesis from mESC and hESC lines carrying a human PAX6 transgenic GFP reporter, hPAX6GFP BAC, and respective transgenic mice to assess GFP-expressing cells in a comparative approach. Our results suggest that our protocol is a valuable addition to the existing organoid technologies, and will facilitate future retina research and regenerative medicine.
Results
Discussion We developed an efficient approach for retinal organoidogenesis stemming from the pioneering protocol (Eiraku et al., 2011) which makes use of the power of PSC to generate self-organized, complex, stratified 3D retinal tissue. The original protocol utilizes the generation of OC-like structures, but requires the OV-like evagination of the neuroepithelium and its manual dissection. We observed that about 80% of organoid neuroepithelia developed into eyefields, comparable with previous publications (Table S1). However, using E14TG2a mESC, or the original reported RAX-GFP mESC line (kindly provided by Y. Sasai, Japan [Eiraku et al., 2011]), others (Hiler et al., 2015) and our group (RAX-GFP data not shown) observed that OV-like structures form irregularly and OC-like structures infrequently. The fact that evagination of the eyefield neuroepithelium occurs rather inefficiently in about 20% of aggregates, and varies between PSC lines (Hiler et al., 2015; see also Figure 2G and Table S1), which might depend on the intrinsic capacity of the PSC line or on culture conditions, currently limits retinal organoidogenesis. Notably, eyefield domains achieved with E14TG2a mESCs are rather large (covering up to 50% of the organoid at D10). OV size has been reported to be a critical factor influencing OC formation (Decembrini et al., 2014; Eiraku et al., 2011), suggesting that this may be a major factor preventing OC formation. Thus, factors affecting eyefield restriction and expansion, such as sonic hedgehog, may be differentially regulated in different mESC lines. By merely isolating the aggregate evaginations, the majority of eyefield tissues were discarded. Furthermore, our data show that evaginations frequently lack eyefields, and are thus not reliable predictors of prospective retinas. By trisecting an unbiased simple organoid neuroepithelia, we overcame these limitations. The trisection protocol utilizes all starting aggregates to improve the retinal organoid yield and enables robust generation of large, stratified 3D retinal organoids derived from wild-type mESC.