Generation of a telencephalon-eye organoid composed of concentric zones of anterior ectodermal progenitors (CONCEPT). (A)

A scheme of the procedure. (B) A diagram of CONCEPT organoids showing concentric zones of the anterior ectodermal progenitors. See also Figs.1, 2, 3, 5, S1, S10. (C) Morphology of cysts at day 2 showing the epithelial structure indicated by the apical reporter TJ::GFP at the lumen. (D) Morphology of CONCEPT organoids at day 26. (E-G) Expression of telencephalon (Tel) marker Foxg1, neuroretinal (NR) markers Vsx2 and Pax6 in mouse eyes at E10-10.5. Rostral optic stalk (OS) connected the telencephalic vesicle to the optic cup. (H-O) FOXG1+ telencephalic progenitors, VSX2+ and/or PAX6+ retinal progenitors formed concentric zones in CONCEPT organoids. N > 5 experiments. (P-W) In CONCEPT organoids, morphogens FGF8, BMP4, and BMP7 mRNA expression started at early stages and subsequently formed circular gradients. N > 5 experiments. Scale bars, 100 µm (C, E, M, O, P, S, T), 200 µm (I, K), 500 µm (Q, U), 1 mm (D, H, J, L, N, R, V, W).

Retinal ganglion cells (RGCs) grow axons toward and then along a path defined by PAX2+ cell populations in CONCEPT telencephalon-eye organoids.

N > 5 experiments. (A- D) POU4F2+ RGCs grew TUBB3+ axons toward and then along a path with a circular or a portion of circular shape. (E, F) In mice, Pax2 was expressed in central regions of the retina and optic stalk at E10.5 (E) and in the optic disc and optic stalk at E13.5 (F). Tubb3+ axons from the initial RGCs grew toward the optic disc, exited the eye, and navigated within the optic stalk (G). (H-L) In CONCEPT organoids at day 26, TUBB3+ RGC axons grew toward and then along a path defined by an adjacent PAX2+ VSX2+ cell population (arrowhead in H, brackets in I, J); the PAX2+ VSX2- cell population sets up an inner boundary of RGC axon growth. (L) A diagram summarizing RGC axon growth, PAX2+ optic disc (OD), and PAX2+ optic stalk (OS) in CONCEPT organoids. The area labeled by the asterisk may appear as false signals in a low- resolution printout but it is clearly a background in digital display. (M) A count of CONCEPT organoids showing directional retinal ganglion cell axons. Scale bars, 50 µm (E, F, G), 100 µm (B), 200 µm (A, H, I).

CONCEPT telencephalon-eye organoids contain lens cells that undergo terminal differentiation.

N > 5 experiments. (A-H) In CONCEPT organoids, lens markers CRYAA and CRY B were expressed at day 22 (A, B) and day 39 (C, D). Lens cells were not stained by DAPI (E, F); they exhibited a crystal-like shape (G). A count of CONCEPT organoids with these lens cells is shown (H). (I, J) When CONCEPT organoids were detached using Dispase at around day 28 and grown in suspension, crystal-like clusters were found (I) and survived for months (J). (K-L) Crystal-like lens clusters were free of organelles and exhibited ball-and-socket structures (K, L), as revealed by electron microscopy. Scale bars, 100 µm (A, B, C, D, I), 200 µm (J), 5 µm (K), 200 nm (L).

Cell clustering analysis identifies telencephalic cells, ocular cells, and two PAX2+ cell populations that mimic the optic disc and optic stalk, respectively.

See also suppl. Figs. S2-S8. CONCEPT organoids at day 24 were used for single-cell RNA sequencing and analyzed using Seurat (v3.2.0). (A) Identification of 14 cell clusters. (B) Cell cycle phases as shown as cell cycle scores. (C) FOXG1 expression marked telencephalic cells. (D, E) The expression of PAX6 and/or VSX2 marked retinal cells. (F) PAX2+ cells were found in two major cell populations: PAX2+ VSX2+ cells were assigned as the optic disc (OD), whereas PAX2+ FOXG1+ VSX2- cells were assigned as the optic stalk (OS). (G) The expression of the optic disc/stalk marker SEMA5A. (H-L) The expression of major DEGs in cluster 2, the major cell population that mimic the optic disc. (M-P) The expression of major gene markers for PAX2+ VSX2- optic stalk cells. (Q-T) Identification of glycosylphosphatidylinositol (GPI)-anchored cell membrane protein CNTN2 as a specific marker for developing human RGCs. Cluster 11 differentially expressed RGC markers, including CNTN2.

Cell counts of clusters in the scRNA-seq dataset.

Abbreviations: Idents, assigned cell identities; # Cells, cell number; Tel, telencephalon; R, retina; OD, optic disc; OS, optic stalk; RPE, retina pigment epithelium; RGC, retinal ganglion cell; N, early neurons; UD, undetermined.

RGCs grow CNTN2+ axons toward and then along a defined path in CONCEPT telencephalon-eye organoids and can be isolated in one step via CNTN2 in a native condition. N > 5 experiments.

(A-F) PAX2+ cells formed two concentric zones mimicking the optic stalk (OS) and optic disc (OD), respectively (A-C; high magnifications in D, E). POU4F2+ RGCs grew CNTN2+ axons toward and then along a path defined by adjacent PAX2+ optic-disc cells (A-E). RGCs at a few hundreds of micrometers away from PAX2+ optic-disc cells grew axons centrifugally (arrow in C). At regions where there was a gap in PAX2+ optic-disc cells, CNTN2+ RGC axons exited the circular path and grew centrifugally (diamond arrowhead in A, double arrowheads in B and F). PAX2+ optic-stalk cells set up an inner boundary for RGC axon growth. (G, H) PAX2+ optic-disc cells did not express ALDH1A3; the cells that set up the boundaries of the path highly expressed ALDH1A3. (I, J) Cells that set up the inner boundary for RGC axon growth expressed VAX1/VAX2. (K) In E13.5 mouse eye, Aldh1a3 was highly expressed in the peripheral retina and a small region in the optic disc. Aldh1a3 expression was at low or nearly absent in the central retina. (L-P) One-step isolation of RGCs. RGCs from floating retinal organoids at day 41 (L, M) and day 70 (N-O) were dissociated into single cells using Accutase and then isolated using MACS via CNTN2 for 10-day growth. Isolated RGCs expressed POU4F2 and grew TUBB3+ neurites in random directions (L). RGCs also expressed CNTN2 (M), ISL1 (N), RBPMS (N), and SNCG (O); positive cells were counted (P). Scale bars, 200 µm (A,G,I), 100 µm (D-F,H,J,K), 50 µm (L).

Electrophysiological features of RGCs.

RGCs from retinal organoids on day 48 were isolated using MACS via a CNTN2 antibody and then grown on polymer coverslips in a chamber slide for 20-25 days before whole-cell patch clamp recordings. (A) Resting membrane potential of RGCs. Black triangle: average; black line: median; box: interquartile range, n=9. (B) RGCs can fire action potentials. Cells were patched in current-clamp mode. A steady current (Im) was injected to maintain the membrane potential at -70mV and depolarizing current steps of 10ms (left), 100ms (middle) or 1s (right) were injected to elicit action potentials (Vm). (C) RGCs show functional voltage-gated currents. Cells were recorded in voltage-clamp (holding=-80mV) and depolarizing voltage steps (200ms, +10mV steps up to 80mV, Vm) were applied to record inward and outward voltage-gated currents (Im). Inset: zoom on inward currents. (D) Outward currents are primarily due to voltage-gated potassium channels. Left, representative example of a current-voltage experiment performed in presence of 20mM Tetraethylammonium (TEA), a blocker of voltage-gated potassium channels. Inset: zoom on inward currents. Right, amplitude of potassium current as a function of membrane potential (mean±SEM; ncontrol=5, nTEA=5). (E) Inward currents result from activity of voltage-gated sodium channels. Left, representative example of a current-voltage experiment performed in presence of 1µM Tetrodotoxin (TTX), a blocker of voltage-gated sodium channels. Inset: zoom on inward currents. Right, amplitude of sodium current as a function of membrane potential (mean±SEM; ncontrol=5, nTTX=3).

FGF signaling mediated by FGFRs is required for early RGC differentiation and directional axon growth. (A)

PAX2, FGF9, and FGF8 are differentially expressed in cluster 2, the major component of PAX2+ optic-disc cells. (B) PAX2 mRNA expression in CONCEPT organoids on day 25. Two PAX2+ concentric zones corresponding to the optic stalk (OS) and optic disc (OD) are labeled. (C) Dual-color immunocytochemistry indicates the co-localization of FGF8 and PAX2 in the optic-disc zone of CONCEPT organoids on day 25. (D-E) TUBB3+ axons grew towards and then along the cells that expressed high levels of FGF8 (D) and FGF9 mRNA (E) in CONCEPT organoids on day 25. Immunocytochemistry of TUBB3 was performed after in situ hybridization. (F-K) After the inhibition of FGF signaling with FGFR inhibitor PD 161570 during days 17-24, the number of RGC somas drastically reduced, and directional axon growth of RGCs was nearly absent, whereas FGF8 protein expression largely remained. CNTN2 immunocytochemistry before (F, H) and after FGF8 immunocytochemistry (G, I, J, K) are shown. N = 3/3 experiments. Scale bar, 250 µm (B), 100 µm (C-E), 1 mm (F), 200 µm (K).