# US 2020/0399590 A1 · Methods for forming three-dimensional human retinal tissue in vitro > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399590 A1 - Document ID: US20200399590A1 - Status: Published application - Kind: A1 - Application number: 16/839,345 - Filed: 2020-04-03 - Published: 2020-12-24 - Earliest claimed: 2014-01-16 - Assignees: The Johns Hopkins University - Applicants: The Johns Hopkins University - Inventors: Maria Valeria Canto-Soler; Xiufeng Zhong - Web page: https://patentgrep.com/patent/US20200399590A1 ## CPC - C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain} - C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR] - C12N 2506/45: from artificially induced pluripotent stem cells - C12N 2509/00: Methods for the dissociation of cells, e.g. specific use of enzymes - C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to the field of stem cells. More specifically, the invention provides methods and compositions useful for forming three-dimensional human retinal tissue in vitro. In a specific embodiment, an in vitro method for differentiating hiPSCs into three-dimensional retinal tissue comprising functional photoreceptors comprises the steps of (a) culturing the hiPSCs to form aggregates; (b) transitioning the aggregates into a neural induction medium; (c) seeding the aggregates on to extracellular matrix coated cell culture substrates; (d) replacing NIM with a chemically-defined differentiation medium; (e) detaching NR domains; (f) culturing in suspension; and (g) adding animal serum or plasma component and retinoic acid. ## Claims ### Claim at position 1 1-42. (canceled) ### Claim 43 A retinal tissue composition comprising three-dimensional retinal cups comprising neural retina (NR) epithelium and retinal pigmented epithelium (RPE) bundled at the tip. ### Claim 44 (depends on 43) The retinal tissue composition of claim 43, wherein the NR epithelium comprises undifferentiated retinal progenitors. ### Claim 45 (depends on 44) The retinal tissue composition of claim 44, wherein the NR epithelium further comprises differentiating retinal precursors. ### Claim 46 (depends on 43) The retinal tissue composition of claim 43, wherein the NR epithelium comprises laminated retinal tissue comprising at least two retinal layers. ### Claim 47 (depends on 46) The retinal tissue composition of claim 46, wherein the at least two retinal layers are selected from the group consisting of outer nuclear layer, outer inner plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, and nerve fiber layer. ### Claim 48 (depends on 43) The retinal tissue composition of claim 43, wherein the NR epithelium comprises photoreceptors with outer-segment disc formation. ### Claim 49 (depends on 48) The retinal tissue composition of claim 48, wherein the photoreceptors exhibit response to light. ### Claim 50 A retinal tissue composition comprising three-dimensional retinal cups comprising neural retina (NR) epithelium. ### Claim 51 (depends on 50) The retinal tissue composition of claim 50, wherein the NR epithelium comprises undifferentiated retinal progenitors. ### Claim 52 (depends on 51) The retinal tissue composition of claim 51, wherein the NR epithelium further comprises differentiating retinal precursors. ### Claim 53 (depends on 50) The retinal tissue composition of claim 50, wherein the NR epithelium comprises laminated retinal tissue comprising at least two retinal layers. ### Claim 54 (depends on 53) The retinal tissue composition of claim 53, wherein the at least two retinal layers are selected from the group consisting of outer nuclear layer, outer inner plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, and nerve fiber layer. ### Claim 55 (depends on 50) The retinal tissue composition of claim 50, wherein the NR epithelium comprises photoreceptors with outer-segment disc formation. ### Claim 56 (depends on 55) The retinal tissue composition of claim 55, wherein the photoreceptors exhibit response to light. ### Claim 57 A retinal tissue composition comprising (a) three-dimensional retinal cups comprising neural retina (NR) epithelium and retinal pigmented epithelium (RPE) bundled at the tip; and (b) three dimensional retinal cups comprising NR epithelium. ### Claim 58 (depends on 57) The retinal tissue composition of claim 57, wherein the NR epithelium comprises undifferentiated retinal progenitors. ### Claim 59 (depends on 58) The retinal tissue composition of claim 58, wherein the NR epithelium further comprises differentiating retinal precursors. ### Claim 60 (depends on 57) The retinal tissue composition of claim 57, wherein the NR epithelium comprises laminated retinal tissue comprising at least two retinal layers. ### Claim 61 (depends on 60) The retinal tissue composition of claim 60, wherein the at least two retinal layers are selected from the group consisting of outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, and nerve fiber layer. ### Claim 62 (depends on 57) The retinal tissue composition of claim 57, wherein the NR epithelium comprises photoreceptors with outer-segment disc formation. ### Claim 63 (depends on 63) The retinal tissue composition of claim 63, wherein the photoreceptors exhibit response to light. ### Claim 64 An in vitro method for differentiating human induced pluripotent stem cells (hiPSCs) into three-dimensional retinal tissue comprising the steps of: (a) culturing hiPSCs to form aggregates; (b) transitioning the aggregates into culture medium to induce neural differentiation; (c) seeding the aggregates on to cell culture substrates; (d) detaching neural retina (NR) domains; and (e) culturing the NR domains in suspension, wherein the NR domains form three-dimensional retinal tissue. ### Claim 65 (depends on 64) The method of claim 64, wherein the culture medium of step (b) is replaced by a chemically-defined medium prior to step (d). ### Claim 66 (depends on 64) The method of claim 64, wherein the cell culture substrates comprise extracellular matrix coated cell culture substrates. ### Claim 67 (depends on 64) The method of claim 64, wherein serum or plasma component is added during step (e). ### Claim 68 (depends on 64) The method of claim 64, wherein all-trans retinoic acid is added during step (e). ### Claim 69 (depends on 65) The method of claim 65, wherein serum or plasma component is added during step (e). ### Claim 70 (depends on 65) The method of claim 65, wherein all-trans retinoic acid is added during step (e). ### Claim 71 (depends on 67) The method of claim 67, wherein all-trans retinoic acid is added during step (e). ### Claim 72 (depends on 69) The method of claim 69, wherein all-trans retinoic acid is added during step (e).