(a–c) After W7, RCs progressively lost their histological organization due to increasing cell death (caspase 3-positive, a,b), which was avoidable by supplementation with fetal bovine serum, taurine and RA from the beginning of W7 onward (c). (d,e) Under these conditions, RCs maintained their shape, cellular organization and steady growth during long-term culture (bars: mean±s.d.; 15–20 RCs per time point). (f–i) By W13–W14, RCs showed distinguishable layers containing the precursors of most of the major neuronal cell types, including ganglion, amacrine and horizontal cells (HU C/D- and PAX6-positive), and photoreceptors (OTX2- and recoverin-positive); as in the developing human retina, cells expressing OTX2 and recoverin were observed in the developing outer nuclear layer as well as in the inner side of the retinal epithelium69,70. A conspicuous neuroblastic layer containing mitotic retinal progenitors (VSX2/MCM2-positive) was still present by W14 (i). (j) Müller cells expressing CRALBP were first seen by W17. Scale bars, 200 μm (d); 50 μm (a–c and f–j).