Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors

Abstract

Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.

Keywords: cone photoreceptors; differentiation; photoreceptor; retina and transplantation; stem cells.

Graphical abstract

Figure 1

Efficient Photoreceptor Differentiation in hPSC 2D/3D Cultures (A) Schematic of retinal 2D/3D differentiation protocol. (B) Representative bright-field images of differentiation stages in culture. At 4 weeks of differentiation RPE (white arrows) and neuroretinal regions (black arrowheads) are present. (C) Quantification of NRVs in 3D (mean ± SD; n > 80 vesicles from N = 10 differentiations; ^∗∗∗∗p < 0.0001, paired t test). (C′ and C″) Representative image of retinal and non-retinal vesicles at 10 weeks. (D and E) Images of 15 week NRVs showing RECOVERIN+ and NRL+ photoreceptors. (F and F′) High-magnification images of NRL+ photoreceptor precursors. (G) Photoreceptors at week 12 of differentiation co-expressing CRX and RECOVERIN. (H) Flow cytometry analyses showing 36% of RECOVERIN+ photoreceptors at 17 weeks of culture (n = 20 NRVs from N = 4 differentiations). Scale bars,
 25 μm (F, F′, and G), 50 μm (B, panel 2), 70 μm (B, panels 4, 5, and 6, and C and C′), 100 μm (D and E), 200 μm (B, panels 1 and 3). NRV, neuroretinal vesicles.

Figure 2

Time Course of Photoreceptor Development in 2D/3D Differentiation Cultures IHC of neuroepithelial regions in hESC-derived NRVs (A–D). Staining for CRX (A), RECOVERIN (B), NRL (C), and RHODOPSIN (E) at various time points. (D) Summary of temporal expression of photoreceptor markers during human eye development at indicated fetal week (Fwk). Scale bars, 25 μm (A–C, and E).

Figure 3

Photoreceptor Maturation in 2D/3D Differentiation Cultures (A and B) Bright-field images of hESC-derived NRVs in suspension at late stages. (A) Neuroepithelium at 17 weeks showing two distinct layers (white bars); high-magnification panel showing presumptive IS buds (white arrow). (B) NRV at 28 weeks. (B′) High-magnification image of neuroepithelium containing protrusion-like structures at the apical border. (C and D) IHC analysis of GNAT1+ rod photoreceptors (C) and L/Mopsin stained cell body and IS of cones full of mitochondria at 20 weeks of culture (D). (E) OLM protein ZO-1. (F and F′) Rootlet markers, ROOTLETIN, in the IS, basal to OS-specific RETGC. (G) CC protein, RPGR, shows punctate localization at week 27. (H) RHODOPSIN and PRPH2 at 17 weeks. (I–K) RHODOPSIN changes localization from cell body to OS region by 27 weeks (I), photoreceptors showing elongated PRPH2+ (J), and ABCA4+ (K) OS-like structures at 33 weeks. (L–N) Ultrastructural images of hESC-derived neuroepithelium. (L) 17 week photoreceptors showing OLM, ISs, and CC, terminating in OS-like structure. (M) Images of week 27 photoreceptors showing transverse sections through two OS-like structures, CC, and ISs containing mitochondria. (N) 33 week old nascent OS with highlighted panel showing disorganized membraneous discs. Scale bars, 10 μm (D) 25 μm (C and E–K), 50 μm (A and B′), and 200 μm (B). IS, inner segment; OLM, outer limiting membrane; OS, outer segment; CC, connecting cilia.

Figure 4

Generation and Characterization of Cone Photoreceptor Precursors (A, D, and E) IHC of neuroepithelia at 7, 9, 12, and 20 weeks in culture for ONECUT1, OLIG2, and OTX2. At 7 and 9 weeks ONECUT1+ cells were present throughout the neuroblastic layers (A). At later stages ONECUT1 and OLIG2 cells became localized to the presumptive INL (A and D), and OTX2 cells were present in the ONL and INL (E). (B–C′) Photoreceptor precursors co-expressing ONECUT1 and CRX-positive (arrowheads) at 7 weeks in culture (B and B′). (F) RXRγ+ cone photoreceptors were present at the apical surface of the weeks 8 and 15 neuroblastic layers. (G) RT-PCR analysis at 6 and 10 weeks of differentiation showing the expression of retinal progenitor markers (RPC), cone-biased retinal progenitors (OC1, OLIG2, and OTX2), cone photoreceptor precursors (RXRγ and TRΒ2), and CRX and RCVRN. Positive control was a Fwk 6 human retina. Scale bars, 25 μm (A–F). ONL, outer nuclear layer; INL, inner nuclear layer.

Figure 5

Time Course of hPSC-Derived Cone Photoreceptor Development in 2D/3D Differentiation Cultures (A–C″) IHC analysis showing time course of differentiation for cone-specific markers. (A–A″) S OPSIN, (B–B″) L/M OPSIN, and (C–C″) ARRESTIN3. (D) Percent of ARRESTIN3+ cones at 12 and 20 weeks in culture (mean ± SD; n = 30 images from N = 3 differentiations; ^∗∗∗p < 0.001, unpaired t test). (E) Schematic summarizing temporal expression of cone markers. (F and F′) 3D view of an NRV showing the distribution of ARRESTIN3+ cones and RHODOPSIN+ rods (green and red, respectively). (F′) High-magnification image showing distribution of ARRESTIN3+ cone and RHODOPSIN+ rods. High-magnification panel highlights ISs of both cells. (G) Cross-section image showing typical morphology of rod and cone photoreceptors. (G′ and G″) High-magnification image showing large ARRESTIN3+ cone ISs (G′) and thinner RHODOPSIN+ rod (G″) ISs. (H) Peanut agglutinin (PNA) staining of 24 week ARRESTIN3+ cones. (I) CNGB3 localized to OS-like region of the neuroepithelia. (J) Relative expression of cone-specific phototransduction markers (mean ± SD; n = 15 NRVs from N = 3 differentiations). Scale bars, 10 μm (G′ and G″), 25 μm (A–C″, F′, and G–I), and 100 μm (F).

Figure 6

Incorporation of hPSC-Derived Cone Photoreceptors into Nrl^−/− Mouse Model of Retinal Degeneration (A) Low-magnification confocal image of transplanted eye showing spread of L/Mopsin.GFP+ cones in the subretinal space. Inserts, high-magnification images showing cell masses in close proximity to, but not integrated into, host ONL. (B–B″) Incorporation of hPSC-derived L/Mopsin.GFP+/hNUCLEI+ photoreceptors into the Nrl^−/− adult retina. Inserts: high-magnification images of incorporated cell showing pedicle in the OPL (B′, arrowhead). (C–C″) Confocal projection showing a small cluster of incorporated cells (C) and single confocal images showing process extension and pedicle formation in the OPL (C’) (arrowhead) and IS oriented toward the subretinal space (C’’) (arrow). (D) Number of L/Mopsin.GFP+/hNUCLEI+ hESC-derived incorporated cones/eye (mean ± SD; n = 9 eyes; N > 4 experiments). (E) Nuclei size of L/Mopsin.GFP+/hNUCLEI+ hPSC-derived cones, L/Mopsin.GFP+/hNUCLEI– cells, endogenous mouse photoreceptor nuclei, and hESC-derived cone hNUCLEI in NRVs (mean ± SD; n > 30 nuclei measured N = 3 samples; ^∗∗∗∗p > 0.0001, one-way ANOVA). (F and F′) Incorporated L/Mopsin.GFP+ cone cell extending pedicle to the OPL (F) (arrowhead) shows localized punctate RIBEYE (F′) (arrowhead). (G and G′). Incorporated L/Mopsin.GFP+/hNUCLEI+ cone co-expressing ARRESTIN3 and showing pedicle in the OPL (arrowhead). (H and H′). Incorporated L/Mopsin.GFP+/hNUCLEI+ cone co-expressing M/L OPSIN (H′) (arrow and arrowhead). (I and I′) Incorporated L/Mopsin.GFP+/hNUCLEI+ cone photoreceptors showing typical large ISs positive for M/L OPSIN protein (arrows). Single confocal image is shown in (I′). (J) Maximum projection image showing FISH for mouse Y chromosome (red) in male Nrl^−/− eyes and examples of incorporated cells extending processes toward the OPL (arrowhead). (J′ and J″) Single confocal images showing that hESC-derived L/Mopsin.GFP+ cells are negative for Y chromosome DNA probe (red, arrows). Scale bars, 5 μm (J′ and J″), 10 μm (C′, C″, F–G′, and I–J) 25 μm (inserts in A, B–B″, C, H, and H′), and 100 μm (A). INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

Figure 7

Transplantation of hPSC-Derived Cone Photoreceptors into Aipl1^−/− Mouse Model of Retinal Degeneration (A and A′) In vivo fundus image of a transplanted Aipl1^−/− eye showing L/Mopsin.GFP+ cells (arrowheads). (B) Low-magnification image showing hPSC-derived L/Mopsin.GFP+ cones in the subretinal space between the INL and the RPE (arrow). (C–E′) IHC of L/Mopsin.GFP cones cell mass (white bar) with ISs bearing human mitochondria (C and C′), ARRESTIN3 (D and D′), and L/M OPSIN (E and E′). (F–G′) High-magnification images of ARRESTIN3+ (F and F′) and L/M OPSIN+ (G and G′) cones detailing IS containing hMITOCHONDRIA. (H) L/Mopsin.GFP cone showing a PRPH2+ process (arrowhead). (I) PKC+ host bipolar cells in close apposition to L/Mopsin.GFP cones. (J) CALBINDIN+ horizontal cell neurites shown in close apposition to L/Mopsin.GFP cones. (K–L′) L/Mopsin.GFP cells extending neurites showing punctate RIBEYE+ ribbon synapses. (M) GFAP+ activated Müller glial cells surround L/Mopsin.GFP cones. Scale bars, 10 μm (F–H, L, and L′), 25 μm (C–E′ and I–K′), 50 μm (M), and 100 μm (B).RGC, retinal ganglion cell layer; INL, inner nuclear layer; SRS, subretinal space.