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Definitions

• the present invention relates to a method for manufacturing a retinal tissue.
• Non-Patent Literature 1 Non-Patent Literature 1
• Non-Patent Literature 2 and Patent Literature 1 a method for obtaining a multilayered retinal tissue from pluripotent stem cells
• Patent Literature 3 and Patent Literature 2 a method for obtaining a multilayered retinal tissue by forming an aggregate of homogeneous pluripotent stem cells in a serum-free medium containing a Wnt signaling pathway inhibitor, and suspension-culturing the obtained aggregate in the presence of a basal membrane preparation and then in a serum medium
• Non-Patent Literature 4 and Patent Literature 3 a method for obtaining retinal tissue by suspension-culturing an aggregate of pluripotent stem cells in a culture medium containing a BMP signaling pathway agonist
• Non-Patent Literature 5 Non-Patent Literature 5
• an object of the present invention is to provide a method for reforming the neuroepithelial structure of retinal tissue from a retinal cell and a method for manufacturing a retinal tissue by applying the method.
• the present inventors have found that a neuroepithelial structure can be reformed using dispersed retinal cells as starting cells by adding a Wnt signaling pathway agonist to the dispersed retinal cells to manufacture a retinal tissue.
• impure cells such as retinal pigment epithelial cells (RPE) are removed by (1) further adding a ROCK inhibitor, a SHH signaling pathway agonist and/or a fibroblast growth factor, (2) performing culture on a culture plate coated with an extracellular matrix serving as a scaffold of cell adhesion, particularly, in order to reform a favorable sheet-shaped neuroepithelial structure, and (3) improving the purity of retinal progenitor cells as starting cells.
• RPE retinal pigment epithelial cells
• the present invention relates to each of the following aspects.
• the present invention it becomes possible to provide a method for reforming the layer structure of retinal tissue from a retinal cell and a method for manufacturing a retinal tissue by applying the method, and a retinal tissue.
• the “stem cells” refer to undifferentiated cells having differentiation potency and proliferation potency (particularly, self-renewal ability).
• stem cells subgroups of pluripotent stem cells, multipotent stem cells and unipotent stem cells, are included according to the differentiation potency.
• the pluripotent stem cells refer to stem cells that can be cultured in vitro and has an ability (pluripotency) to be able to differentiate into three germ layers (ectoderm, mesoderm, endoderm) and/or all cell lineages belonging to the extraembryonic tissue.
• the multipotent stem cells refer to stem cells having an ability to differentiate into a plurality of tissues or cells, although the definition is not applied to all of them.
• the unipotent stem cells refer to stem cells having an ability to be able to differentiate into a predetermined tissue or cells.
• the "pluripotent stem cells” can be induced from, e.g., a fertilized egg, a cloned embryo, germline stem cells, tissue stem cells and somatic cells.
• the pluripotent stem cells can include embryonic stem cells (ES cells), embryonic germ cells (EG cells) and induced pluripotent stem cells (iPS cells).
• Muse cells Multi-lineage differentiating stress enduring cells obtained from the mesenchymal stem cells (MSC) and GS cells prepared from germ cells (for example, testis) are included in the pluripotent stem cells.
• Human embryonic stem cells were established in 1998 and have been used also for regenerative medicine.
• the embryonic stem cells can be produced by culturing inner cell aggregate on feeder cells or a culture medium containing bFGF.
• the method for producing embryonic stem cells is described, for example, in WO96/22362 , WO02/101057 , US5,843,780 , US6,200,806 , US6,280,718 .
• the embryonic stem cells are available from a predetermined institution and also, commercially available.
• human embryonic stem cells such as KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Life and Medical Sciences, Kyoto University.
• Human embryonic stem cells such as human ES cells genetically engineered so as to have Crx::Venus strain and Rx::Venus strain (both are derived from KhES-1) are available from RIKEN.
• induced pluripotent stem cells refers to cells having pluripotency, which is induced by reprogramming somatic cells by a method known in the art.
• the induced pluripotent stem cells were established in mouse cells by Yamanaka et al., in 2006 (Cell, 2006, 126 (4), pp. 663-676 ). The induced pluripotent stem cells were also established in human fibroblasts in 2007. The induced pluripotent stem cells have pluripotency and self-renewal ability similarly to embryonic stem cells ( Cell, 2007, 131 (5), pp. 861-872 ; Science, 2007, 318 (5858), pp. 1917-1920 ; Nat. Biotechnol., 2008, 26 (1), pp. 101-106 ).
• the induced pluripotent stem cells more specifically refer to cells which are induced to be pluripotent by reprogramming somatic cells differentiated into, for example, fibroblasts and peripheral blood mononuclear cells, by allowing any one of sets of a plurality of genes selected from a reprogramming gene group containing Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28 and Esrrb to express.
• Examples of a preferable set of reprogramming factors may include (1) Oct3/4, Sox2, Klf4, and Myc (c-Myc or L-Myc) and (2) Oct3/4, Sox2, Klf4, Lin28 and L-Myc ( Stem Cells, 2013; 31: 458-466 ).
• the pluripotent stem cells can be artificially induced from somatic cells, for example, by adding a chemical compound ( Science, 2013, 341, pp. 651-654 ).
• an induced pluripotent stem cell strain is available.
• human induced pluripotent cell strains established by Kyoto University such as 201B7 cell, 201B7-Ff cell, 253G1 cell, 253G4 cell, 1201C1 cell, 1205D1 cell, 1210B2 cell and 1231A3 cell, are available form Kyoto University and iPS Academia Japan, Inc.
• the induced pluripotent stem cells for example, Ff-I01 cell, Ff-I14 cell and QHJI01s04 cell established by Kyoto University, are available from Kyoto University.
• the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, more preferably induced pluripotent stem cells.
• the pluripotent stem cells are human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).
• iPS cells human induced pluripotent stem cells
• ES cells human embryonic stem cells
• Pluripotent stem cells such as human iPS cells can be subjected to maintenance culture and expansion culture performed by methods known to those skilled in the art.
• neural tissue refers to a tissue constituted by neural cells of the cerebrum, the midbrain, the cerebellum, the spinal cord, a retina, peripheral nerve, the forebrain, the hindbrain, the telencephalon, the diencephalon, and the like of a developing stage or an adult stage.
• the neural tissue may form an epithelial structure (neural epithelium) having a layer structure, and the abundance of neural epithelium in a cell aggregate can be evaluated by bright field observation using an optical microscope.
• neural cells refer to cells other than epidermal cells in ectoderm-derived tissue. Specifically, they include cells such as neural precursor cells, neurons (neuronal cells), glia, neural stem cells, neuronal progenitor cells and glial progenitor cells.
• the neural cells also encompass cells constituting retinal tissue mentioned below (retina cells), retinal progenitor cells, retinal layer-specific neuronal cells, neural retina cells, and retinal pigment epithelial cells.
• the neural cells can be identified with nestin, TuJ1, PSA-NCAM, N-cadherin and the like as markers.
• neuronal cells are functional cells that form a neural circuit and contribute to signal transduction, and can be identified with the expression of immature neuronal cell markers such as TuJ1, Dcx, and HuC/D and/or mature neuronal cell markers such as Map2 and NeuN as an index.
• neural precursor cells are a population of precursor cells including neural stem cells, neuronal progenitor cells and glial progenitor cells and have proliferation potency and the ability to produce neurons and glia.
• the neural precursor cells can be identified with nestin, GLAST, Sox2, Sox1, Musashi, Pax6 and the like as markers.
• neural cell marker-positive and proliferation marker Ki67, pH3, MCM-positive cells can also be identified as the neural precursor cells.
• the "retinal tissue” means a tissue in which a single type or a plurality of types of retinal cells constituting each retinal layer in a retina in vivo are present according to a predetermined order.
• the "neural retina” is a retinal tissue and means a tissue containing an inside neural retinal layer that does not contain a retinal pigment epithelial layer among retinal layers mentioned later.
• the "retinal cells” mean cells constituting each retinal layer in a retina in vivo or progenitor cells thereof.
• cells such as photoreceptor cells (rod photoreceptor cell, cone photoreceptor cell), horizontal cells, amacrine cells, intermediate neuronal cells, retinal ganglion cells (ganglion cell), bipolar cells (rod bipolar cell, cone bipolar cell), Muller glial cells, retinal pigment epithelial (RPE) cells, ciliary body, their progenitor cells (e.g., photoreceptor progenitor cell, bipolar progenitor cell, retinal pigment epithelial progenitor cells), neural retinal progenitor cells and retinal progenitor cells are included, though not limited thereto.
• examples of cells constituting a neural retinal layer specifically include cells such as photoreceptor cells (rod photoreceptor cell, cone photoreceptor cell), horizontal cells, amacrine cells, intermediate neuronal cells, retinal ganglion cells (ganglion cell), bipolar cells (rod bipolar cell, cone bipolar cell), Muller glial cells, and their progenitor cells (e.g., photoreceptor progenitor cell, bipolar progenitor cell).
• the neural retina-related cells neither retinal pigment epithelial cells nor ciliary body cells are included.
• the "matured retinal cells” mean cells that may be contained in the retinal tissue of a human adult, and specifically mean differentiated cells such as photoreceptor cells (rod photoreceptor cell, cone photoreceptor cell), horizontal cells, amacrine cells, intermediate neuronal cells, retinal ganglion cells (ganglion cell), bipolar cells (rod bipolar cell, cone bipolar cell), Muller glial cells, retinal pigment epithelial (RPE) cells, and ciliary body cells.
• the "immature retinal cells” mean progenitor cells (e.g., photoreceptor progenitor cell, bipolar progenitor cell, retinal progenitor cell) destined for differentiation into matured retinal cells.
• the photoreceptor progenitor cells, the horizontal progenitor cells, the bipolar progenitor cells, the amacrine progenitor cells, the retinal ganglion progenitor cells, the Muller glial progenitor cells, and the retinal pigment epithelial progenitor cells refer to progenitor cells destined for differentiation into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Muller glial cells, and retinal pigment epithelial progenitor cells, respectively.
• the "retinal progenitor cells” are progenitor cells capable of differentiating into any one of the immature retinal cells such as photoreceptor progenitor cells, horizontal progenitor cells, bipolar progenitor cells, amacrine progenitor cells, retinal ganglion progenitor cells, Muller glial cells, and retinal pigment epithelial progenitor cells, and refer to progenitor cells also capable of eventually differentiating into any one of the matured retinal cells such as photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells.
• the "neural retinal progenitor cell” are progenitor cells capable of differentiating into any one of the immature neural retinal cells such as photoreceptor progenitor cells, horizontal progenitor cells, bipolar progenitor cells, amacrine progenitor cells, retinal ganglion progenitor cells, and Muller glial cells, and refer to progenitor cells also capable of eventually differentiating into any one of the matured neural retinal cells such as photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells.
• the neural retinal progenitor cells have no differentiation capacity into retinal pigment epithelial cells.
• the "photoreceptor cells” are present in the photoreceptor layer of a retina in vivo and plays a role in absorbing light stimuli and converting them to electrical signals.
• the photoreceptor cells have two types, cones which function in the light and rods which function in the dark (referred to as cone photoreceptor cells and rod photoreceptor cells, respectively).
• cone photoreceptor cells can include S cone photoreceptor cells which express S-opsin and receive blue light, L cone photoreceptor cells which express L-opsin and receive red light, and M cone photoreceptor cells which express M-opsin and receive green light.
• the photoreceptor cells are matured after differentiation from photoreceptor progenitor cells.
• cells are photoreceptor cells or photoreceptor progenitor cells
• cell markers Crx and Blimp1 expressed in photoreceptor progenitor cells, recoverin expressed in photoreceptor cells, rhodopsin, S-opsin and M/L-opsin expressed in mature photoreceptor cells, etc.
• the photoreceptor progenitor cells are Crx-positive cells
• the photoreceptor cells are rhodopsin-, S-opsin- and M/L-opsin-positive cells.
• the rod photoreceptor cells are NRL- and rhodopsin-positive cells.
• the S cone photoreceptor cells are S-opsin-positive cells
• the L cone photoreceptor cells are L-opsin-positive cells
• the M cone photoreceptor cells are M-opsin-positive cells.
• neural retina-related cells can be confirmed from the presence or absence of expression of a neural retina-related cell-related gene (hereinafter, also referred to as "neural retina-related cell marker” or “neural retina marker”).
• neural retina-related cell marker also referred to as "neural retina-related cell marker” or “neural retina marker”
• the presence or absence of expression of the neural retina-related cell marker, or the percentage of neural retina-related cell marker-positive cells in a cell population or a tissue can be readily confirmed by those skilled in the art. Examples thereof include an approach using an antibody, an approach using nucleic acid primers, and an approach using sequencing reaction.
• the expression of a protein of the neural retina-related cell marker can be confirmed, for example, by dividing the number of predetermined neural retina-related cell marker-positive cells by the total number of cells in accordance with an approach such as flow cytometry (FACS) or immunostaining using a commercially available antibody.
• FACS flow cytometry
• nucleic acid primers the expression of RNA of the neural retina-related cell marker can be confirmed by, for example, PCR, semiquantitative PCR, or quantitative PCR (e.g., real-time PCR).
• the expression of RNA of the neural retina-related cell marker can be confirmed using, for example, a nucleic acid sequencer (e.g., next-generation sequencer).
• neural retina-related cell marker examples include Rx (also referred to as Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6 and Chx10 (also referred to as Vsx2) expressed in neural retinal progenitor cells, and Crx and Blimp1 expressed in photoreceptor progenitor cells.
• Rx also referred to as Rax
• PAX6 expressed in retinal progenitor cells
• Rx, PAX6 and Chx10 also referred to as Vsx2
• Crx and Blimp1 expressed in photoreceptor progenitor cells.
• Examples thereof also include Chx10 strongly expressed in bipolar cells, PKC ⁇ , Go ⁇ , VSX1 and L7 expressed in bipolar cells, TuJ1 and Brn3 expressed in retinal ganglion cells, calretinin and HPC-1 expressed in amacrine cells, calbindin expressed in horizontal cells, recoverin expressed in photoreceptor cells and photoreceptor progenitor cells, rhodopsin expressed in rod cells, Nrl expressed in rod photoreceptor cells and rod photoreceptor progenitor cells, S-opsin and LM-opsin expressed in cone photoreceptor cells, RXR- ⁇ expressed in cone cells, cone photoreceptor progenitor cells and ganglion cells, TR ⁇ 2, OTX2 and OC2 expressed in cone photoreceptor cells that appear at the early phase of differentiation among cone photoreceptor cells, or progenitor cells thereof, and Pax6 commonly expressed in horizontal cells, amacrine cells and ganglion cells.
• the "positive cells” mean cells expressing a predetermined marker on the cell surfaces or within the cells.
• the "Chx10-positive cells” mean cells expressing Chx10 protein.
• retinal pigment epithelial cells mean epithelial cells present outside the neural retina in a retina in vivo. Whether or not cells are retinal pigment epithelial cells can be readily confirmed by those skilled in the art, for example, through the expression of cell markers (MITF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (brown-black), intercellular tight junctions, or polygonal/flagstone-like characteristic cell morphology.
• cell markers MIRF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.
• retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.
• the "retinal pigment epithelial cell sheet” refers to a single-layer or multilayer sheet-shaped structure constituted from single or a plurality of cells in which retinal pigment epithelial cells adhere to each other through a biological bond at least in the two-dimensional direction.
• the "retinal layer” means individual layers constituting the retina, and examples thereof can specifically include retinal pigment epithelial layer, photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer and inner limiting membrane.
• the "neural retinal layer” means individual layers constituting the neural retina, and examples thereof can specifically include photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer and inner limiting membrane.
• the "photoreceptor layer” means a retinal layer that is formed in the outermost of the neural retina and is rich in one or more cells selected from the group consisting of a photoreceptor cell (rod photoreceptor cell, cone photoreceptor cell), a photoreceptor progenitor cell and a retinal progenitor cell. Each layer other than the photoreceptor layer is referred to as an inner layer. Which retinal layer the individual cells constitute can be confirmed by a known method, for example, by determining the presence or absence of expression or expression level of a cell marker.
• a layer containing proliferating neural retinal progenitor cells is referred to as "neuroblastic layer” and includes inner neuroblatic layer and outer neuroblastic layer.
• neutral layer a layer containing proliferating neural retinal progenitor cells
• Those skilled in the art can make a judgment from the shade of color (the outer neuroblastic layer is light, and the inner neuroblatic layer is dark) by a known method, for example, under a bright field microscope.
• the "ciliary body” includes “ciliary body” and "ciliarymarginal zone” in the process of development and of an adult.
• a marker of the "ciliary body” include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1 and AQP4.
• Examples of the "ciliarymarginal zone (CMZ)” can include a tissue that is present in a boundary region between the neural retina and the retinal pigment epithelium in a retina in vivo, and is a region containing tissue stem cells of the retina (retinal stem cells).
• the ciliarymarginal zone is also called ciliarymargin or retinalmargin, and the ciliarymarginal zone, the ciliarymargin and the retinalmargin are equivalent tissues.
• the ciliarymarginal zone is known to play an important role in the supply of retinal progenitor cells or differentiated cells to retinal tissue, the maintenance of a retinal tissue structure, etc. Examples of a marker gene of the ciliarymarginal zone can include Rdh10 gene (positive), Otx1 gene (positive) and Zic1 (positive).
• the "ciliarymarginal zone-like structure" is a structure similar to the ciliarymarginal zone.
• the "cerebral tissue” is a tissue in which one type or at least a plurality of types of cells constituting the cerebrum of a fetal stage or an adult (e.g., cortical neural precursor cells, dorsal cerebral neural precursor cells, ventral cerebral neural precursor cells, cerebral layer structure-specific neuronal cells (neurons), layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, glial cells (astrocytes and oligodendrocytes), and their progenitor cells) are three-dimensionally arranged in layers.
• the cerebrum of a fetal stage is also called forebrain or telencephalon.
• the presence of each cell can be confirmed by a known method, for example, the presence or absence of expression of a cell marker or the level thereof.
• the “cerebral layer” refers to each layer constituting the adult cerebrum or the fetal stage cerebrum, and examples can specifically include molecular layer, outer nuclear layer, external pyramidal layer, inner nuclear layer, neuronal cell layer (internal pyramidal layer), polymorphic cell layer, layer 1, layer 2, layer 3, layer 4, layer 5, layer 6, cortex zone, intermediate zone, subventricular zone, and ventricular zone.
• cortical neural precursor cells can include neuronal progenitor cells, layer 1 neuronal progenitor cells, layer 2 neuronal progenitor cells, layer 3 neuronal progenitor cells, layer 4 neuronal progenitor cells, layer 5 neuronal progenitor cells, layer 6 neuronal progenitor cells, astrocyte progenitor cells, and oligodendrocyte progenitor cells. These cells are progenitor cells committed to differentiate into layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocyte progenitor cells, respectively.
• the “cortical neural precursor cells” include multipotent stem cells (multipotent neural stem cells) having differentiation capacity (multipotency) into at least a plurality of differentiation lineages among layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocytes.
• the “cerebral layer-specific neuronal cells” are cells constituting the cerebral layer and refer to neuronal cells specific for the cerebral layer.
• Examples of the cerebral layer-specific neuronal cells can include layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, cerebral excitatory neurons, and cerebral inhibitory neurons.
• Examples of the cerebral cell marker include FoxG1 (also called Bf1) expressed in cerebral cells, Sox2 and nestin expressed in cortical neural precursor cells, Pax6 and Emx2 expressed in dorsal cortical neural precursor cells, Dlx1, Dlx2 and Nkx2.1 expressed in ventral cortical neural precursor cells, Tbr2, Nex, and Svet1 expressed in neuronal progenitor cells, Tbr1 expressed in layer 6 neurons, Ctip2 expressed in layer 5 neurons, ROR ⁇ expressed in layer 4 neurons, Cux1 or Brn2 expressed in layer 3 neurons or layer 2 neurons, and reelin expressed in layer 1 neurons.
• FoxG1 also called Bf1
• Sox2 and nestin expressed in cortical neural precursor cells Pax6 and Emx2 expressed in dorsal cortical neural precursor cells
• Dlx1, Dlx2 and Nkx2.1 expressed in ventral cortical neural precursor cells Tbr2, Nex, and Svet1 expressed in neuronal progenitor cells
• the "cell aggregate” is not particularly limited as long as a plurality of cells mutually adhere to form a three-dimensional structure, and refers to, for example, a mass formed by the aggregation of cells dispersed in a vehicle such as a culture medium, or a mass of cells formed through cell division. In the cell aggregate, the case of forming a predetermined tissue is also included.
• epithelial tissue is a tissue formed by covering the body surface or the surface of a lumen (digestive tract, etc.), body cavity (pericardial cavity, etc.) or the like with cells without any space.
• the cells forming the epithelial tissue are referred to as epithelial cells.
• the epithelial cells have a polarity in the apical-basal direction.
• the epithelial cells can mutually and firmly join via adherence junction and/or tight junction to form a layer of the cells.
• a tissue formed from a single layer or dozen layers overlapping of this layer of the cells is the epithelial tissue.
• epithelial structure means a structure characteristic of the epithelial tissue (e.g., having polarities of a basal surface and an apical surface).
• the “continuous epithelial tissue” is a tissue having a continuous epithelium structure.
• the continuous epithelium structure is a structure where the epithelial tissue is continuously formed.
• the epithelium tissue continuously formed is a state in which 10 cells to 10 7 cells, for example, in the tangent direction of the epithelial tissue, preferably 30 cells to 10 7 cells, further preferably 10 2 cells to 10 7 cells, in the tangent direction, are aligned.
• the retinal tissue has an apical surface intrinsic to the epithelial tissue.
• the apical surface is formed almost in parallel to, for example, at least photoreceptor layer (outer nuclear layer) among the layers forming a neural retinal layer and continuously on the surface of the retinal tissue.
• the apical surface is formed on the surface of the aggregate, and 10 cells or more, preferably 30 cells or more, more preferably 100 cells or more, further preferably 400 cells or more of photoreceptor cells or photoreceptor progenitor cells are regularly and continuously arranged in a row in the tangent direction of the surface.
• epithelial tissue is polarized so that "apical surface” and “basal surface” and “basal membrane” are formed.
• the “basal membrane” refers to a basal side layer (basal membrane) produced by epithelial cells, is rich in laminin and IV-type collagen, and has a thickness of 50 to 100 nm.
• the “basal surface” refers to the surface (upper surface layer) formed on the “basal membrane” side.
• the “apical surface” refers to the surface (upper surface layer) formed on the opposite side to the "basal membrane”.
• the "apical surface” refers to a surface in contact with photoreceptor layer (outer nuclear layer) in which outer limiting membrane is formed and photoreceptor cells and photoreceptor progenitor cells are present.
• an apical surface can be identified by, for example, immunostaining (known to those skilled in the art) using an antibody against an apical surface marker (e.g., atypical PKC (hereinafter, abbreviated to "aPKC”), tight junction marker (Zo-1), ERM protein ezrin, E-cadherin, N-cadherin).
• aPKC atypical PKC
• Zo-1 tight junction marker
• ERM protein ezrin E-cadherin
• N-cadherin N-cadherin
• An aspect of the present invention is a manufacturing method for manufacturing a cell aggregate comprising retinal tissue having an epithelial structure (or a multilayered structure) (in the present specification, the "cell aggregate comprising retinal tissue” is also simply referred to as "retinal tissue”) from a dispersed retinal cell population.
• the method comprises suspension-culturing or adhesion-culturing a dispersed retinal cell population in a culture medium containing a Wnt signaling pathway agonist.
• the retinal cell is as defined above, and in an embodiment, the retinal cell includes one or more cells selected from the group consisting of a retinal progenitor cell and a photoreceptor progenitor cell and may additionally include cells such as photoreceptor cells (rod photoreceptor cell, cone photoreceptor cell), horizontal cells, amacrine cells, retinal ganglion cells (ganglion cell), bipolar cells (rod bipolar cell, cone bipolar cell), and Muller glial cells. It is preferable that the retinal cell should be a neural retinal cell.
• Pluripotent stem cell-derived retinal cells as starting cells in the manufacturing method of the present invention can be obtained by differentiating pluripotent stem cells.
• the pluripotent stem cell-derived retinal cells as starting cells can be obtained by a method comprising steps of
• the region A is a region that can maintain pluripotent stem cells in adhesive culture.
• the region B is a region having cell adhesiveness lower than that of the region A so that, when pluripotent stem cells are seeded on the region A, the cells are prevented from extending from the region A to the region B adjacent to the region A during the induction of retinal tissue differentiation (i.e. the period until differentiation to a retinal tissue is confirmed).
• the cell adhesiveness can be compared by seeding pluripotent stem cells of an excess amount relative to an area (e.g. 5 ⁇ 10 5 to 10 ⁇ 10 5 cells/cm 2 ) and comparing the percentage of the area to which the cells adhere after 24 hours culture at 37° C and 5% CO 2 .
• the cell adhesiveness is typically evaluated after the medium is removed after the 24 hours culture and the cells are washed with a medium or buffer to remove unadhered cells.
• the region A may be, for example, a region where, when pluripotent stem cells of an excess amount relative to its area (e.g. 5 ⁇ 10 5 to 10 ⁇ 10 5 cells/cm 2 ) are seeded and cultured at 37°C and 5% CO 2 for 24 hours, the cells adhere to 70% or more, 80% or more, or 90% or more of the area after the 24 hours.
• pluripotent stem cells of an excess amount relative to its area e.g. 5 ⁇ 10 5 to 10 ⁇ 10 5 cells/cm 2
• the cells adhere to 70% or more, 80% or more, or 90% or more of the area after the 24 hours.
• the region B is a region having no cell adhesiveness.
• the region having no cell adhesiveness refers to a region that does not have sufficient cell adhesiveness to maintain pluripotent stem cells in adhesive culture.
• the region having no cell adhesiveness may be, for example, a region where, when pluripotent stem cells of an excess amount relative to its area (e.g. 5 ⁇ 10 5 to 10 ⁇ 10 5 cells/cm 2 ) are seeded and cultured at 37°C and 5% CO 2 for 24 hours, the cells adhere to 30% or less, 20% or less, or 10% or less of the area after the 24 hours.
• the culture substrate may be made of any material as long as it can have the region A and the region B on its surface.
• the culture substrate may be made of an inorganic material such as metal, glass, or silicone, or an organic material such as plastic (e.g., polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin, polytetrafluoroethylene tetrafluoroethylene resin).
• the culture substrate is made of glass or plastic, especially polystyrene resin.
• the culture substrate may have any shape commonly used for cell culture.
• the culture substrate may be, for example, a culture vessel such as a petri dish, plate, bottle, chamber, or multi-well plate (e.g., 6, 12, 24, 48, 96, or 384 well plate), or may be a film or porous membrane.
• the culture substrate typically has the region A and the region B on the surface that is horizontal to the direction of gravity (on the bottom surface, when the culture substrate is a culture vessel). In an embodiment, the culture substrate has the region A and the region B on the same plane. In an embodiment, when a culture vessel has the region A and the region B on the plane of the bottom surface, the inner wall surface of the culture vessel is not the region B.
• the region B is the side of the convex portion adjacent to the upper surface of the convex portion; In the present disclosure, however, the region A on the upper surface of the convex portion and the region B on the side of the convex portion are considered to be in the same plane.
• the region A may be a region of the surface of a cell-adhesive culture substrate, or a region of the surface of a culture substrate coated with a cell-adhesive material to make the surface cell-adhesive or to enhance the cell-adhesiveness of the surface.
• cell-adhesive materials include positively charged polymers such as poly L-lysine and poly L-ornithine; laminin; collagens such as type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, and type VII collagen; tenascin; fibrillin; fibronectin; vitronectin; elastin; entactin; proteoglycans composed of sulfated glucosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate and core proteins; glucosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, dermatan sulfate, and hyaluronic acid; Synthemax ® (a vitronectin derivative) and Matrigel ® .
• positively charged polymers such as
• the region A is coated with laminin.
• Laminin is a heterotrimeric molecule comprising three subunit chains, ⁇ , ⁇ , and ⁇ . There are five types of ⁇ chains, ⁇ 1- ⁇ 5, three types of ⁇ chains, ⁇ 1- ⁇ 3, and three types of ⁇ chains, ⁇ 1- ⁇ 3, and each laminin isoform is represented by the numbers indicating its constituent subunits (for example, laminin 111 is composed of ⁇ 1 chain, ⁇ 1 chain, and ⁇ 1 chain).
• laminins examples include laminin 111, laminin 121, laminin 211, laminin 213, laminin 222, laminin 311 (laminin 3A11), laminin 332 (laminin 3A32), laminin 321 (laminin 3A21), laminin 3B32, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, laminin 522, laminin 523, or fragments thereof.
• fragments of laminins include E8 fragments, which are fragments of integrin binding sites, such as laminin 211-E8, laminin 311-E8, laminin 411-E8, and laminin 511-E8.
• laminin is laminin 511 or a fragment thereof. In a further embodiment, laminin is the laminin 511-E8 fragment.
• a commercial product such as iMatrix-511 (Nippi. Inc.) may be used. iMatrix-511 (Nippi. Inc.) contains the laminin 511-E8 fragment.
• the region B may be a region of the surface of a culture substrate having cell adhesiveness lower than that of the region A, or a region of the surface of a culture substrate coated with a material to make the region less cell-adhesive than the region A.
• the region B is coated with a cell non-adhesive material.
• non-cell-adhesive materials include MPC (2-methacryloyloxyethyl phosphorylcholine) polymers; celluloses such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl cellulose; polyethylene oxide; carboxyvinyl polymers; polyvinylpyrrolidone; polyethylene glycol; polyamides such as polyacrylamide and poly-N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, pectin, carrageenan, guar gum, gum arabic, and dextran; albumin and their derivatives.
• the non-cell-adhesive material is an MPC polymer.
• the region A may have any shape, and for example, may have, a circular, oval, or polygonal (e.g., triangle, tetragonal, pentagonal, hexagonal, octagonal, decagonal, or dodecagonal) shape.
• the region A has a circular shape.
• a circular shape refers to a shape that is recognized as substantially circular in the art, including a perfect circle.
• the area of the region A may be, but is not limited to, 0.01 to 100 cm 2 , 0.01 to 30 cm 2 , 0.01 to 10 cm 2 , 0.03 to 30 cm 2 , 0.03 to 10 cm 2 , or 0.1 to 10 cm 2 .
• the area of the region A is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm 2 or more, and 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm 2 or less (the upper and lower limits are independently selected).
• the area of the region A may be 0.5 to 10 cm 2 , 0.7 to 10 cm 2 , 1 to 10 cm 2 , 0.5 to 5 cm 2 , 0.7 to 5 cm 2 , 1 to 5 cm 2 , 0.5 to 2 cm 2 , 0.7 to 2 cm 2 , 0.5 to 1 cm 2 , or 0.7 to 1 cm 2 .
• the region A may have a circular shape with a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm or more, and 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm or less (the upper and lower limits are independently selected), or a polygonal shape having an equal area to the circular shape.
• the region A has a circular shape with a diameter of 0.1 to 10 cm, 0.1 to 5 cm, 0.1 to 3 cm or 0.2 to 1 cm.
• the region A has a circular shape with a diameter of 1 to 10 cm, 1 to 5 cm, or 1 to 3 cm.
• the culture substrate may have a plurality of regions A and/or a plurality of regions B on its surface.
• the distance between two regions A flanking the region B may be, but is not limited to, about 1 mm or more.
• the regions A and B may be formed on the surface of a culture substrate by any of methods used for cell patterning.
• the regions A and B can be formed on the surface of a culture substrate by techniques such as soft lithography, photolithography, and 3D printing, for example.
• the regions A and B may be formed by treating a portion of the surface of a culture substrate to make the treated and non-treated regions have different cell adhesive properties.
• the region A and B may be formed, for example, by coating a portion of the surface of the culture substrate with a cell-adhesive or non-cell-adhesive material.
• these regions may be formed by masking a portion of the surface of a culture substrate and coating the unmasked area with a cell-adhesive or non-cell-adhesive material.
• these regions may be formed by providing a layer of a cell-adhesive or non-cell-adhesive material on a culture substrate and treating a portion of the layer to alter the cell adhesiveness.
• the treated portion can be a portion where the surface of the culture substrate is exposed or a portion where the property of the non-cell-adhesive material or cell-adhesive material has been changed by the treatment.
• the regions A and B may be formed on a culture substrate by preparing a sheet with a hole of the shape and size of the region A using a 3D printed mold, covering the surface of the culture substrate with the sheet, coating the portion of the surface not covered with the sheet with a cell-adhesive material, and then removing the sheet.
• the regions A and B may be formed on a culture substrate by preparing a sheet of the shape and size of the region A, placing the sheet on the surface of the culture substrate, coating the portion of the surface not covered with the sheet with a non-cell-adhesive material, removing the sheet, and coating the portion of the surface covered with the sheet with a cell-adhesive material.
• the sheet may be prepared from a biocompatible material such as polydimethylsiloxane (PDMS), polyethylene glycol hydrogel, or agarose gel.
• PDMS polydimethylsiloxane
• the coating can be performed, for example, by bringing the culture substrate into contact with a solution of a cell-adhesive or non-cell-adhesive material and allowing it to react with the solution at 37°C or room temperature for the required time (e.g., 1 hour or more).
• the concentration of the cell-adhesive or non-cell-adhesive material can be appropriately determined by those skilled in the art, and, for example, for laminin, may be 0.1 to 1 ⁇ g/cm 2 , 0.1 to 0.5 ⁇ g/cm 2 , or about 0.25 ⁇ g/cm 2 .
• the regions A and B may also be formed on the culture substrate without using the sheet as described above, by making a droplet of a solution of a cell-adhesive material on the culture substrate and allowing the culture substrate to react with the solution.
• the culture substrate may have a convex portion (also called a pillar) on the surface and the convex portion may have the region A on its upper surface.
• the region B is the side of the convex portion adjacent to the upper surface of the convex portion.
• the shape of the convex portion may be, but is not limited to, a circular cylinder or a square cylinder.
• a circular cylinder refers to a column whose cross section is recognized as a substantially circular shape in the art, including a perfect circle.
• the convex portion is in the form of a circular cylinder.
• the height of the convex portion may be, but is not limited to, 0.1 mm to 10 mm, 0.1 mm to 5 mm, 1 mm to 5 mm, or 3 mm to 5 mm, for example 4 mm.
• the material of the convex portion may be the same as or different from that of the culture substrate.
• examples of materials for the convex portion include polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyamide (PA), and polymethylglutarimide (PMGI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene vinyl acetate (PEVA), and polyethylene oxide (PEO).
• PDMS polydimethylsiloxane
• PMMA polymethylmethacrylate
• PET polyethylene terephthalate
• PA polyamide
• PMGI polymethylglutarimide
• PVA polyvinyl alcohol
• PEG polyethylene glycol
• PEVA polyethylene vinyl acetate
• PEO polyethylene oxide
• the cells may be seeded only in the region A or on the entire region of the culture substrate including the region A and the region B.
• the cells can be seeded on the entire region of the culture substrate.
• the pluripotent stem cells are seeded on a culture substrate at a number of cells so that the cells occupy 70% or more of region A.
• the pluripotent stem cells are seeded on a culture substrate at 0.5 ⁇ 10 5 , 0.6 ⁇ 10 5 , 0.7 ⁇ 10 5 , 0.8 ⁇ 10 5 , 0.9 ⁇ 10 5 , 1.0 ⁇ 10 5 , 1.1 ⁇ 10 5 , 1.2 ⁇ 10 5 , 1.3 ⁇ 10 5 , 1.4 ⁇ 10 5 , 1.5 ⁇ 10 5 , 1.6 ⁇ 10 5 , 1.7 ⁇ 10 5 , 1.8 ⁇ 10 5 , 1.9 ⁇ 10 5 , 2.0 ⁇ 10 5 , 2.1 ⁇ 10 5 , 2.2 ⁇ 10 5 , 2.3 ⁇ 10 5 , or 2.4 ⁇ 10 5 cells/cm 2 or more, and 2.5 ⁇ 10 5 , 2.4 ⁇ 10 5 , 2.3 ⁇ 10 5 , 2.2 ⁇ 10 5 , 2.1 ⁇ 10 5 , 2.0 ⁇ 10 5 , 1.9 ⁇ 10 5 , 1.8 ⁇ 10 5 , 1.7 ⁇ 10 5 ,
• the pluripotent stem cells may be seeded at 0.5 ⁇ 10 5 to 2.5 ⁇ 10 5 cells/cm 2 , 1.0 ⁇ 10 5 to 2.5 ⁇ 10 5 cells/cm 2 , 1.5 ⁇ 10 5 to 2.5 ⁇ 10 5 cells/cm 2 , 0.5 ⁇ 10 5 to 2.0 ⁇ 10 5 cells/cm 2 , 1.0 ⁇ 10 5 to 2.0 ⁇ 10 5 cells/cm, or 0.5 ⁇ 1 0 5 to 1.5 ⁇ 10 5 cells/cm 2 .
• pluripotent stem cells are collected and dispersed with a cell dispersing solution containing an enzyme (e.g., trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, or papain) and/or a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)), and suspended in a desired medium. Then, the resulting cell suspension is added onto the culture substrate.
• an enzyme e.g., trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, or papain
• EDTA ethylenediaminetetraacetic acid
• a commercial product such as TrypLE TM Select (Thermo Fisher Scientific) or TrypLE TM Express (Thermo Fisher Scientific) may be used, for example.
• Differentiation induction of pluripotent stem cells into retinal cells is performed by adhesion culture on the culture substrate described above.
• the differentiation induction of pluripotent stem cells into retinal cells may be started after pluripotent stem cells seeded on a culture substrate are cultured for a certain period of time in a medium containing a factor for maintaining undifferentiated state.
• the time when the culturing in a medium that does not contain a factor for maintaining undifferentiated state is started is defined as the time when the differentiation induction into retinal cells is started.
• the inducing differentiation of pluripotent stem cells into retinal cells comprises culturing pluripotent stem cells in a culture medium containing a BMP signaling pathway agonist.
• the culturing the cells in a culture medium containing a BMP signaling pathway agonist may be started at the start of differentiation induction or after a certain period of time (e.g., 4 to 6 days) after the start of differentiation induction.
• Differentiation induction of pluripotent stem cells into retinal cells may include culturing pluripotent stem cells in a culture medium containing a BMP signaling pathway inhibitor prior to culturing the cells in a culture medium containing a BMP signaling pathway agonist.
• the culturing the cells in the presence of a BMP signaling pathway inhibitor before adding a BMP signaling pathway agonist can increase the efficiency of differentiation into a retinal tissue.
• the medium used in the differentiation of pluripotent stem cells into retinal cells can be prepared by using a medium commonly used for culturing animal cells as a basal medium.
• the basal medium may be IMDM, DMEM, F-12, DMEM/F12, IMDM/F12, BME, BGJb, CMRL 1066, Glasgow MEM (GMEM), Improved MEM Zinc Option, Medium 199, Eagle MEM, alpha MEM, Ham's medium, RPMI 1640, Fischer's medium, or a mixture thereof.
• the medium may contain one or more components selected from serum, serum substitutes, growth factors, factors for maintaining undifferentiated state, proteins (e.g., cytokines, insulin), fatty acids, lipids, vitamins, amino acids (e.g., nonessential amino acids, retinoids, glutamine, taurine), antioxidants, 2-mercaptoethanol, 1-thioglycerol, antibiotics, buffers, and inorganic salts, which are added to a basal medium as needed.
• serum substitutes include, for example, albumin such as bovine serum albumin (BSA), transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 1-thioglycerol, and their equivalents, and mixtures thereof.
• the medium may contain a commercially available serum substitute such as KnockOut TM Serum Replacement (Thermo Fisher Scientific), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), GlutaMAX TM Supplement (Thermo Fisher Scientific), Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement (Thermo Fisher Scientific), B27 Supplement (Thermo Fisher Scientific), N2 Supplement (Thermo Fisher Scientific) or ITS Supplement (Thermo Fisher Scientific).
• the medium may be a commercial product that contains any of the above components in a basal medium, such as Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement (Thermo Fisher Scientific), DMEM/F-12, GlutaMAX TM supplement (Thermo Fisher Scientific).
• the medium is preferably a serum-free medium.
• the serum-free medium refers to a medium not containing unmodified or unpurified serum.
• a medium containing a purified blood-derived component or animal tissue-derived component is also included in the serum-free medium as long as it does not contain unmodified or unpurified serum.
• the serum-free medium may contain a serum substitute.
• the differentiation induction is preferably performed in the absence of feeder cells (also described as under the feeder-free condition).
• the differentiation induction is also preferably performed under the xeno-free condition.
• xeno-free refers to being free of components that are derived from a species different from the species of the cells to be cultured.
• factors for maintaining undifferentiated state include FGF signaling pathway agonists, TGF ⁇ family signaling agonists, and insulin.
• FGF signaling pathway agonists include FGF (e.g., bFGF, FGF4, FGF8).
• TGF ⁇ family signaling agonists include TGF ⁇ signaling agonists and Nodal/Activin signaling agonists.
• TGF ⁇ signaling agonists include TGF ⁇ 1 and TGF ⁇ 2.
• Nodal/Activin signaling agonists include Nodal, ActivinA, and ActivinB.
• factors for maintaining undifferentiated state preferably include bFGF.
• the concentration of a factor for maintaining undifferentiated state can be appropriately determined by a person skilled in the art, as long as it can maintain undifferentiated state of pluripotent stem cells.
• the concentration of bFGF may be 4 ng to 500 ng/mL, 10 ng to 200 ng/mL, or 30 ng to 150 ng/mL.
• a medium containing a factor for maintaining undifferentiated state may be a medium such as StemFit ® AK02N (Ajinomoto Co., Inc.), StemFit ® AK03N (Ajinomoto Co., Inc.), S-medium (DS Pharma Biomedical Co.), StemPro TM (Thermo Fisher Scientific), mTeSR1 TM (STEMCELL Technologies), mTeSR2 TM (STEMCELL Technologies), TeSR TM -E8 TM (STEMCELL Technologies), or hESF9 ( Proc. Natl. Acad. Sci. USA. 2008 Sep 9;105(36):13409-14 ).
• the medium containing a factor for maintaining undifferentiated state is StemFit ® AK02N (Ajinomoto Co., Inc.).
• the period of culturing in a medium containing a factor for maintaining undifferentiated state may be, but is not limited to, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, or 2 to 3 days.
• Pluripotent stem cells are cultured in a culture medium containing a ROCK inhibitor before the cells are cultured in a culture medium containing a BMP signaling pathway agonist.
• the ROCK inhibitor may be added to the culture medium containing a factor for maintaining undifferentiated state.
• ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152.
• the concentration of the ROCK inhibitor is appropriately determined according to the inhibitor to be used, and may be 1 to 100 ⁇ M, 5 to 50 ⁇ M, or about 10 ⁇ M, or a concentration providing an activity equivalent to that of Y-27632 at the concentration as mentioned above.
• the period of culturing in a culture medium containing a ROCK inhibitor is 1 to 16 hours, 1 to 14 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, 2 to 16 hours, 2 to 14 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, or 2 to 3 hours.
• the period of culturing in a culture medium containing a ROCK inhibitor is 1 to 3 hours.
• the period of culturing in a culture medium containing a factor for maintaining undifferentiated state and a ROCK inhibitor may be a part or the whole of the period of culturing in a culture medium containing a factor for maintaining undifferentiated state.
• the cells may be cultured in a culture medium containing a factor for maintaining undifferentiated state and a ROCK inhibitor for 1 to 16 hours, and then cultured in a culture medium containing a factor for maintaining undifferentiated state but not a ROCK inhibitor until the period of culturing in a culture medium containing a factor for maintaining undifferentiated state reaches a predetermined length.
• BMP bone morphogenetic protein
• BMP4 BMP7
• BMP12 BMP12
• the BMP signaling pathway agonist and inhibitor may be an agonist and an inhibitor of one or more of these BMPs, respectively.
• a BMP signaling pathway inhibitor is a substance that inhibits signaling mediated by BMP, and includes substances that act on BMP or its receptor, substances that suppress gene expression of BMP or its receptor, and substances that inhibit binding between BMP and its receptor.
• BMP signaling pathway inhibitors include LDN-193189 4-[6-(4-Piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), Dorsomorphin (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), and DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).
• the BMP signaling pathway inhibitor is LDN-193189.
• the concentration of a BMP signaling pathway inhibitor is determined according to the inhibitor to be used, and may be 1 to 1000 nM, 10 to 500 nM, 30 to 300 nM, or about 100 nM, or a concentration providing an activity equivalent to that of LDN-193189 at the concentration as mentioned above.
• the period of culturing in a culture medium containing a BMP signaling pathway inhibitor may be, but is not limited, 1 to 10 days, 2 to 9 days, 3 to 7 days, 4 to 6 days, or about 5 days.
• a BMP signaling pathway agonist is a substance that activates signaling mediated by BMP, and includes substances that act on BMP or its receptor, substances that enhance gene expression of BMP or its receptor, and substances that promote binding between BMP and its receptor.
• BMP signaling pathway agonists include BMP2, BMP4, BMP7, BMP12 (GDF7) and fragments thereof, and anti-BMP receptor antibodies.
• the BMP signaling pathway agonist is BMP4.
• the concentration of a BMP signaling pathway agonist is determined according to the agonist to be used, and may be 0.01 to 1000 nM, 0.1 to 100 nM, 1 to 10 nM, 1 to 3 nM, or about 1.5 nM, or a concentration providing an activity equivalent to that of BMP4 at the concentration as mentioned above.
• the concentration of a BMP signaling pathway agonist is 3 to 15 nM or 3 to 12 nM, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nM, or a concentration providing an activity equivalent to that of BMP4 at the concentration as mentioned above.
• the cells are cultured in a culture medium containing a BMP signaling pathway agonist for a period necessary for differentiation into retinal cells.
• the period of culturing may be, but is not limited to, 1 to 30 days, 2 to 20 days, 3 to 15 days, 4 to 12 days, or 5 to 10 days (e.g., 5, 6, 7, 8, 9, or 10 days).
• the concentration of a BMP signaling pathway agonist may be constant or varied during the culturing period. For example, the concentration of a BMP signaling pathway agonist may be decreased stepwise at a rate of 40 to 60% reduction per 2 to 4 days.
• the concentration of a BMP signaling pathway agonist in a medium may be decreased stepwise by replacing a portion (e.g., half) of the culture medium containing the BMP signaling pathway agonist with a medium not containing the BMP signaling pathway agonist every 2, 3, or 4 days after the start of culturing in the culture medium containing the BMP signaling pathway agonist.
• the culture medium containing a BMP signaling pathway inhibitor or BMP signaling pathway agonist is prepared from a 1:1 mixture of F-12 medium and IMDM medium containing KnockOut TM Serum Replacement (Thermo Fisher Scientific) (e.g., 0.5% to 30%, 1% to 20%, or 10%), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), BSA, and 1-thioglycerol.
• Thermo Fisher Scientific KnockOut TM Serum Replacement
• the culture medium may be replaced with a culture medium containing no BMP signaling pathway agonist to continue the culturing.
• This period of culturing may be, but is not limited to, 1 to 100 days, 10 to 90 days, 20 to 80 days, 30 to 70 days, 40 to 60 days, or about 50 days.
• the cells may be cultured in a culture medium containing a Wnt signaling pathway inhibitor during a part or the whole of the period of culturing in a culture medium containing a BMP signaling pathway agonist.
• a Wnt signaling pathway inhibitor is a substance that inhibits signaling mediated by Wnt, and includes substances that act on Wnt or its receptor, substances that suppress gene expression of Wnt or its receptor, and substances that inhibit binding between Wnt and its receptor.
• Wnt signaling pathway inhibitors examples include CKI-7 (N-(2-Aminoethyl)-5-chloro-8-isoquinolinesulfonamide, D4476 (4-(4-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide), IWR-1-endo (IWR1e) (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide) and IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide
• the medium may be replaced as needed. For example, a part or the whole of the medium may be replaced every 1 to 4 days.
• the whole of the medium may be replaced with a medium containing the component at a desired concentration, or a part of the medium may be replaced to achieve the desired final concentration of the component (e.g., half of the medium may be replaced with a medium at twice the final concentration).
• the concentration of the component may be constant or varied during the culturing period in a medium containing the specific component.
• Culture conditions such as culture temperature and CO 2 concentration can be appropriately determined.
• the culture temperature may be, for example, 30°C to 40°C or about 37°C.
• the CO 2 concentration may be, for example, 1% to 10% or about 5%.
• Differentiation into a retinal tissue can be confirmed by detecting expression of a neural retina-related cell marker in cells of the tissue.
• the neural retina-related cell marker is described above.
• the expression of a marker may be confirmed, for example, on any of Days 10 to 100 of differentiation induction (e.g., Day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100), or later.
• the expression of a marker is confirmed on any of Days 16 to 22 of differentiation induction.
• the retinal tissue as starting cells obtained by the method described herein can be called as a retina sheet, which generally contains retinal progenitor cells and may be referred to as "a retinal progenitor cell sheet".
• the retinal tissue (retina sheet) as starting cells contains Chx10-positive cells or Chx10-positive and Rx-positive cells at a percentage of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
• the percentage of the marker-positive cells may be the percentage at Day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of differentiation induction or later.
• the retinal tissue contains Chx10-positive and Rx-positive cells at a percentage of 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more at any of Days 16 to 22 (e.g., Day 16, 18, or 20) of differentiation induction. In a further embodiment, the retinal tissue contains Chx10-positive and Rx-positive cells at a percentage of 90% or more or 95% or more at any of Days 16 to 22 (e.g., Day 16, 18, or 20) of differentiation induction.
• the retinal tissue (retina sheet) as starting cells contains neural retina cells at a percentage of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
• the percentage of the cells may be the percentage at any of Days 16 to 100 (e.g., Day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100) of differentiation induction or later.
• the retinal tissue preferably has a layered structure of cells.
• the retinal tissue (retina sheet) as starting cells may have a thickness of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ⁇ m or more, and 500, 400, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210 or 220 ⁇ m or less (the upper and lower limits are independenty selected).
• the retinal tissue has a thickness of 50 ⁇ m to 300 ⁇ m.
• the retinal tissue need not have the same thickness throughout the tissue. When a retinal tissue is defined with a range of thickness, the retinal tissue may have a thickness within that range at any portion of the retinal tissue.
• the retinal tissue (retina sheet) as starting cells usually contains about 30 cells or less in a direction perpendicular to the culture substrate, or has a thickness of 500 ⁇ m or less.
• the retinal tissue (retina sheet) as starting cells is obtained by a method comprising steps of
• the method of this embodiment can produce a uniform retinal tissue that has few holes or voids (i.e., areas where no cells exist) on the region A.
• the retinal tissue (retina sheet) as starting cells covers more than 95%, 96%, 97%, 98%, 99% or more of the region A.
• the percentage of area covered by a retinal tissue on the region A may be measured by any methods, and for example, can be measured by staining the retinal tissue. For example, it is measured by staining expression of the neural retina-related cell marker described above (such as Chx10) (e.g., by immunostaining with a fluorescently labeled anti-Chx10 antibody).
• the method of this embodiment can also efficiently produce a retinal tissue with a low percentage of unintended cells and a high percentage of retinal cells. Specifically, the method of this embodiment can produce a retinal tissue with high expression of genes expressed in neural retina-related cells and low expression of genes expressed in unintended cells.
• the neural retina-related cells and their markers are described above.
• Unintended cells include eye-related cells (e.g., ciliary body, lens, retinal pigment epithelium) and cerebrospinal-related cells (e.g., telencephalon, midbrain, spinal cord), and their markers are described above.
• eye-related cells e.g., ciliary body, lens, retinal pigment epithelium
• cerebrospinal-related cells e.g., telencephalon, midbrain, spinal cord
• the method of producing a retinal tissue (retina sheet) as starting cells comprises
• the method of producing a retinal tissue (retina sheet) as starting cells comprises
• the retinal tissue (retina sheet) obtained by the steps (1) to (3) is dispersed as described below.
• the retinal tissue (retina sheet) may be dispersed without collection from the culture substrate, or may be dispersed after collection from the culture substrate.
• the retinal tissue (retina sheet) can be collected from the culture substrate in a usual manner.
• the retinal tissue may be collected with an instrument such as a pair of tweezers.
• a stimulus-responsive polymer e.g., a temperature-responsive polymer or light-responsive polymer
• the retinal tissue can be collected by applying the corresponding stimulus.
• the retinal tissue can be collected by placing the culture substrate at the temperature.
• the "dispersion” refers to separating cells or tissues into a small cell clot or cell clump (2 cells or more and 100 cells or less, preferably 50 cells or less, 30 cells or less, 20 cells or less, 10 cells or less, 5 cells or less; for example, a clump of 2 to 5 cells) or single cells by dispersion treatment such as enzymatic treatment or physical treatment.
• the dispersed cell population refers to a cell clot or a cell clump, a collection of a given number of single cells.
• the "dispersed retinal cell population” refers to a cell population in a dispersed state and can be obtained by dispersing living tissue or a cell clump such as a cell aggregate. It is preferable that the dispersed retinal cell population should be obtained by dispersing the aggregate of retinal cells described above.
• a dispersion method can be a method that can disperse cells alive.
• examples thereof include mechanical dispersion treatment, cell dispersion solution treatment, and cytoprotective agent addition treatment. These treatments may be performed in combination.
• cell dispersion solution treatment is performed, and subsequently, mechanical dispersion treatment can be performed.
• Examples of a method of the mechanical dispersion treatment include pipetting treatment and scraping operation with a scraper.
• Examples of the cell dispersion solution that is used in the cell dispersion solution treatment can include solutions containing any of enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, and papain, and chelating agents such as ethylenediaminetetraacetic acid.
• a commercially available cell dispersion solution for example, TrypLE Select (manufactured by Life Technologies Corp.), TrypLE Express (manufactured by Life Technologies Corp.), or a neuronal cell dispersion solution (FUJIFILM Holdings Corp.) can also be used.
• cytoprotective agent When cells are dispersed, cell death of the cells may be suppressed by treating with a cytoprotective agent.
• the cytoprotective agent that is used in the cytoprotective agent treatment can include FGF signaling pathway agonists, heparin, IGF signaling pathway agonists, serum, and serum replacements.
• an inhibitor of Rho-associated coiled-coil containing protein kinase (ROCK” or "Rho kinase") (ROCK inhibitor) or an inhibitor of myosin
• ROCK inhibitor can include Y-27632, fasudil (HA1077), and H-1152.
• the inhibitor of myosin can include blebbistatin.
• Preferable examples of the cytoprotective agent include ROCK inhibitors.
• the dispersed retinal cell population includes a state in which single cells are included, for example, 70% or more, preferably 80% or more, with respect to the total number of cells of the cell population is single cells, and a mass of 2 to 50 cells is present at 30% or less, preferably 20% or less, with respect to the total number of cells of the cell population.
• the mutual adhesion e.g., surface adhesion
• the dispersed retinal cell population should consist of single cells as much as possible.
• Such a dispersed retinal cell population can be obtained by removing a mass of cells that are not single cells after dispersion treatment.
• Examples of the method for removing a mass of cells include, but are not particularly limited to, the removal of the mass through a membrane filter (cell strainer).
• the dispersed cells include a state cell-cell junction (e.g., adherence junction) has been mostly lost.
• retinal cell population In the case of preparing a retinal cell population as starting cells from retinal tissue, unnecessary cells such as retinal pigment epithelial cells may be contained in the retinal tissue. In this case, the retinal cell population can be dispersed after a region where unnecessary cells such as retinal pigment epithelial cells are present are isolated. Retinal pigment epithelial cells are identifiable from morphology or a pigment and can be readily resected by those skilled in the art.
• the step of increasing the purity of a retinal progenitor cell or a neural retinal progenitor cell (purification step) as mentioned later may be carried out, and the maintenance and/or expansion culture of the dispersed retinal cell population may be further carried out.
• the culture medium is not particularly limited as long as it is a culture medium (DMEM medium, etc.) in which retinal cells are capable of surviving and proliferating. Since a frozen and thawed retinal cell population can also be used as starting cells in the manufacturing method of the present invention, the dispersed retinal cell population may be cryopreserved.
• a cryopreservation solution is not particularly limited, and a commercially available cryopreservation solution can be used.
• the step of increasing the percentage (purity) of a retinal progenitor cell, preferably the percentage (purity) of a neural retinal progenitor cell may be carried out for the dispersed retinal cell population.
• an operation such as cell sorting using a specific marker expressed in the retinal progenitor cell and/or the neural retinal progenitor cell may be carried out.
• the cell sorting can be carried out using a well-known technique such as FACS or MACS.
• the purification step for a retinal progenitor cell and/or a neural retinal progenitor cell it is possible to reduce contamination with RPE cells or non-target cells in the manufacturing method described in the present specification.
• the step of increasing the percentage of a retinal progenitor cell contained in the dispersed retinal cell population the production of retinal pigment epithelial progenitor cells and/or retinal pigment epithelial cells is suppressed. Whether or not the production of retinal pigment epithelial progenitor cells and/or retinal pigment epithelial cells has been suppressed can be determined from whether or not the retinal pigment epithelial cells have been produced on the basis of a marker, the morphology, the properties, etc.
• the production of retinal pigment epithelial progenitor cells and/or retinal pigment epithelial cells has been suppressed can mean that the percentage of the retinal pigment epithelial cells to the total number of cells after the culture is suppressed as compared with the case where the step of increasing the percentage of a retinal progenitor cell has not been carried out. Provided that the percentage of the retinal pigment epithelial cells is a level described in the paragraph 0151, it can be determined that the production of retinal pigment epithelial progenitor cells and/or retinal pigment epithelial cells has been suppressed.
• Rx, Chx10, and the like are well known as cell markers for retinal progenitor cells (positive markers).
• these genes are intracellularly expressed, for example, a device such as use of cells in which such a gene is linked to a fluorescent protein by a gene recombination technique, or cells in which the gene is replaced with a fluorescent protein is necessary (e.g., Rx::Venus cells).
• CD9 GenBank ID: NM_001769.4, NM_001330312.2
• CD24 GenBank ID: NM_001291737.1, NM_001291738.1, NM_001291739.1, NM_001359084.1, NM_013230.3
• CD29 GenBank ID: NM_002211.4, NM_033668.2, NM_133376.2
• CD39 GenBank ID: NM_001098175.2, NM_001164178.1, NM_001164179.2, NM_001164181.1, NM_001164182.2, NM_001164183.2, NM_001312654.1, NM_001320916.1, NM_001776.6
• CD47 GenBank ID: NM_001777.3, NM_198793.2
• CD49b GenBank ID: NM_002203.4
• a method for increasing the percentage of a retinal progenitor cell in a cell population comprising the step of contacting the cell population containing the retinal progenitor cell with substance(s) (e.g., antibody or peptide) that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90 and CXCR4 to separate positive fractions may be carried out before the aforementioned culture in a culture medium containing a Wnt signaling pathway agonist is started.
• substance(s) e.g., antibody or peptide
• SSEA1 GenBank ID: NM_002033.3
• CD66b GeneBank ID: NM_001816.4
• CD69 GeneBank ID: NM_001781.2
• CD84 GeneBank ID: NM_001184879.2, NM_001184881.2, NM_001184882.1, NM_001330742.2, NM_003874.4
• the step of contacting the dispersed retinal cell population with substance(s) (e.g., antibody) that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84 to separate negative fractions of these markers is the step of obtaining a cell population having expression levels of the antigens that are equal to or less than a reference.
• substance(s) e.g., antibody
• the reference can be arbitrarily set by those skilled in the art.
• negative fractions can be separated by obtaining a cell population treated with fluorescently labeled antibodies against the target antigens having intensity equivalent to fluorescence intensity obtained when the cell population is treated with a fluorescently labeled isotype control antibody.
• the method for increasing the percentage of a retinal progenitor cell and/or a neural retinal progenitor cell in a cell population using substances (e.g., antibodies) that bind to the positive markers and the negative markers mentioned above is not limited to a method as one step of the manufacturing method described in the present specification.
• the method for increasing the percentage of a retinal progenitor cell and/or a neural retinal progenitor cell in a cell population using the cell surface markers can also be used as, for example, one step of other methods for manufacturing retinal tissue.
• the retinal progenitor cell and/or the neural retinal progenitor cell can occupy, for example, 30% or more of the total number of cells. It is preferable to occupy 50% or more of the total number of cells, it is more preferable to occupy 80% or more of the total number of cells, and it is further preferable to occupy 90% or more of the total number of cells.
• the dispersed retinal cell population comprises, for example, 50% or more of a cell (retinal progenitor cell or neural retinal progenitor cell) that is positive to at least one marker selected from the group consisting of CD9, CD39, CD90 and CXCR4 and is Rx- and/or Chx10-positive with respect to the total number of cells in the cell population, and preferably comprises 80% or more, 85% or more, 90% or more, or 95% or more thereof. It is preferable that the cell population should be negative to one or more, preferably two or more, three or more or all antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84. In this context, the negativity can be equal to or less than the reference mentioned above.
• the method for manufacturing retinal tissue according to the present invention comprises adhesion-culturing or suspension-culturing a dispersed retinal cell population in a culture medium containing a Wnt signaling pathway agonist.
• a culture medium containing a Wnt signaling pathway agonist By any of the culture methods, it is possible to reform an epithelial structure (multilayered structure) from the dispersed retinal cell population.
• Adhesion culture is preferable for manufacturing a sheet-shaped retinal tissue having an epithelial structure (or a multilayered structure).
• the culture medium that is used in the culture of the dispersed retinal cell population is not particularly limited as long as it is a culture medium in which retinal cells are capable of surviving and proliferating.
• examples of the culture medium that is used in the culture of the dispersed retinal cell population include culture media for continuous epithelial tissue maintenance.
• One example of the culture medium for continuous epithelial tissue maintenance can include a medium in which Neurobasal medium (e.g., manufactured by Thermo Fisher Scientific Inc., 21103049) is blended with B27 supplement (e.g., Thermo Fisher Scientific Inc., 12587010).
• the Wnt signaling pathway agonist contained in a culture medium is not particularly limited as long as it is capable of enhancing signal transduction mediated by Wnt.
• Examples of a specific Wnt signaling pathway agonist can include GSK3 ⁇ inhibitors (e.g., 6-bromoindirubin-3'-oxime (BIO), CHIR99021, kenpaullone), Wnt proteins Wnt2b and Wnt3a, and partial peptides thereof.
• GSK3 ⁇ inhibitors e.g., 6-bromoindirubin-3'-oxime (BIO), CHIR99021, kenpaullone
• Wnt proteins Wnt2b and Wnt3a and partial peptides thereof.
• One substance may be used, or two or more substances may be used.
• the Wnt signaling pathway agonist is preferably one or more substances, two or more substances, or three or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b and Wnt
• the Wnt signaling pathway agonist can reform an epithelial structure (or a multilayered structure) having a polarity of apical surface/basal membrane while increasing the size of an aggregate in the process of reaggregating the dispersed retinal cell population.
• rosette-like structure formation can also be suppressed.
• the concentration of the Wnt signaling pathway agonist can be a concentration capable of inducing the formation of the desired cell aggregate (e.g., the reforming of an epithelial structure (or a multilayered structure)).
• the concentration of the Wnt signaling pathway agonist can be 0.01 ⁇ M to 100 ⁇ M and is preferably 0.1 ⁇ M to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M, more preferably 3 ⁇ M to 6 ⁇ M.
• the concentration can exhibit a Wnt signal-activating effect equivalent to that of CHIR99021 with the concentration mentioned above.
• the Wnt signal-activating effect is measurable by those skilled in the art through a method, for example, the confirmation of expression of ⁇ -catenin.
• the timing of adding the Wnt signaling pathway agonist is not particularly limited. It is preferable to add it at as early timing as possible after the start of suspension culture or adhesion culture for preparation into a sheet again. In an embodiment, it is preferable to perform culture in the culture medium containing the Wnt signaling pathway agonist from the start of the culture.
• the number of culture days in the culture medium containing the Wnt signaling pathway agonist is not particularly limited within a range in which an effect of reforming an epithelial structure (or a multilayered structure) having a polarity of apical surface/basal membrane is found, and is, for example, 1 day to 14 days.
• the culture medium may further contain one or more substances selected from the group consisting of a ROCK inhibitor, a SHH (sonic hedgehog) signaling pathway agonist and a fibroblast growth factors (FGF) signaling pathway agonist.
• a ROCK inhibitor an effect of promoting the reaggregation of a dispersed retinal progenitor cell population is found.
• SHH signaling pathway agonist an effect of promoting the proliferation of a cell aggregate and increasing the size of the cell aggregate is found.
• FGF signaling pathway agonist e.g., FGF2, FGF8
• the ROCK inhibitor is not particularly limited as long as a function of Rho kinase (ROCK) can be suppressed.
• ROCK Rho kinase
• examples thereof include Y-27632 (see e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000 ); Narumiya et al., Methods Enzymol. 325, 273-284 (2000 )), fasudil/HA1077 (see e.g., Uenata et al., Nature 389: 990-994 (1997 )), H-1152 (see e.g., Sasaki et al., Pharmacol. Ther.
• Wf-536 see e.g., Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003 )
• Wf-536 see e.g., Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003 )
• antisense nucleic acids against ROCK e.g., Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003 )
• RNA interference-inducing nucleic acids e.g., siRNA
• dominant negative mutants e.g., RNA interference-inducing nucleic acids
• other low-molecular compounds are also known as ROCK inhibitors, such compounds or their derivatives can also be used in the present invention (see e.g., U.S. Patent Application Publication Nos.
• the ROCK inhibitor can employ one or two or more ROCK inhibitors.
• the ROCK inhibitor preferably includes one or more substances selected from the group consisting of Y-27632, fasudil (HA1077), and H-1152.
• the concentration of the ROCK inhibitor can be capable of promoting the reaggregation of a dispersed retinal progenitor cell population.
• the concentration in the case of using Y-27632, it may be 0.1 ⁇ M to 1 mM and is preferably 1 ⁇ M to 100 ⁇ M, more preferably 5 ⁇ M to 20 ⁇ M.
• the concentration in the case of using a ROCK inhibitor other than Y-27632, the concentration can exhibit a ROCK inhibitory effect equivalent to that of Y-27632 with the concentration mentioned above.
• the ROCK inhibitory effect is measurable by those skilled in the art through a method, for example, expression analysis on the phosphorylation of MLC2.
• the SHH (sonic hedgehog) signaling pathway agonist is a substance capable of enhancing signal transduction mediated by SHH (also referred to as Shh).
• SHH signaling pathway agonist include proteins belonging to the hedgehog family (e.g., Shh and Ihh), SHH receptor, Shh receptor agonists, PMA (purmorphamine; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine) and SAG (smoothened agonist; N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane).
• the Shh signaling pathway agonist is preferably one or more substances selected from the group consisting of Shh (GenBank accession No: NM_000193, NP_000184), SAG and PMA.
• the concentration of the SHH signaling pathway agonist can be within a range in which an effect of increasing the size of a cell aggregate is found.
• SAG is used at a concentration of usually 1 to 2000 nM, preferably 10 to 700 nM.
• PMA is used at a concentration of usually 0.002 to 20 ⁇ M, preferably 0.02 to 2 ⁇ M.
• SHH is used at a concentration of usually 4 to 500 ng/mL, preferably 10 to 200 ng/mL.
• the concentration can exhibit a SHH signal-activating effect equivalent to that of SAG with the concentration mentioned above.
• the SHH signal-activating effect is measurable by those skilled in the art through a method, for example, the expression analysis of a downstream signal (SMO, GLI).
• the FGF signaling pathway agonist is not particularly limited as long as it is a substance capable of enhancing signal transduction mediated by FGF.
• the FGF signaling pathway agonist specifically include fibroblast growth factors (e.g., bFGF, FGF4, FGF8 and FGF9).
• the FGF signaling pathway agonist is preferably one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF8.
• the concentration of the FGF signaling pathway agonist can be within a range in which an effect of suppressing differentiation into RPE cells is found.
• FGF2 FGF4 or FGF8
• FGF8 it is on the order of 4 to 500 ng/mL, preferably on the order of 10 to 200 ng/mL, more preferably on the order of 25 to 100 ng/mL.
• the concentration can exhibit an FGF signal-activating effect equivalent to that of FGF8 or the like with the concentration mentioned above.
• the FGF signal-activating effect is measurable by those skilled in the art through a method, for example, the expression analysis of a downstream signal (Akt, MEK).
• the timing of adding the ROCK inhibitor, the SHH (sonic hedgehog) signaling pathway agonist and/or the FGF signaling pathway agonist is not particularly limited. It is preferable to perform culture in the culture medium containing the ROCK inhibitor, the SHH (sonic hedgehog) signaling pathway agonist and/or the FGF signaling pathway agonist from the start of the culture. It is preferable that these substances should be simultaneously added to the culture medium, and further, it is preferable to add them to the culture medium simultaneously with the Wnt signaling pathway agonist. In an embodiment, the dispersed retinal cells can be cultured in the culture medium containing these substances for 1 day to 14 days.
• the suspension culture refers to culturing cells in a state non-adhesive to a culture container and is not particularly limited. It can be performed using a culture container that has not undergone artificial treatment (e.g., coating treatment with an extracellular matrix or the like) for the purpose of improving adhesiveness to cells, or a culture container treated by coating using a treatment that artificially suppresses adhesion (e.g., polyhydroxyethyl methacrylate (poly-HEMA), nonionic surface activating polyol (Pluronic F-127, etc.) or a phospholipid-analogous substance (e.g., water-soluble polymer (Lipidure) having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit).
• a treatment that artificially suppresses adhesion e.g., polyhydroxyethyl methacrylate (poly-HEMA), nonionic surface activating polyol (Pluronic F-127, etc.
• the suspension culture can be performed with dispersed retinal cells as starting cells, for example, by using SFEB (serum-free floating culture of embryoid bodies-like aggregates) ( WO2005/12390 ) or SFEBq ( WO2009/148170 ).
• SFEB serum-free floating culture of embryoid bodies-like aggregates
• SFEBq WO2009/148170
• the adhesion culture refers to culturing cells in a state of adhesion to a culture container and is not particularly limited. It can be performed using a culture container or the like artificially treated for the purpose of improving adhesiveness to cells. It is preferable that the adhesion culture should be performed using a culture container or the like coated with an extracellular matrix and/or a temperature-responsive polymer.
• the method for manufacturing retinal tissue of an embodiment comprises the step of exposing a culture container coated with a temperature-responsive polymer to a temperature that changes the properties of the temperature-responsive polymer to thereby detach a sheet-shaped retinal tissue from the culture container.
• a sheet-shaped retinal tissue mentioned later can be manufactured by performing adhesion culture on a culture container coated with an extracellular matrix.
• a culture container or the like coated with an extracellular matrix and/or a temperature-responsive polymer particularly, both an extracellular matrix and a temperature-responsive polymer.
• examples thereof include the case where a culture surface of the culture container is coated with the temperature-responsive polymer, and an upper surface of the polymer is coated with the extracellular matrix.
• the culture surface refers to a surface to which cells adhere in the culture container
• the upper surface of the polymer refers to a surface opposite to a surface in contact with the polymer-coated culture surface.
• the extracellular matrix refers to a biopolymer constituting the external space of a cell.
• cell-adhesive proteins such as fibronectin, vitronectin, and laminin
• fibrous proteins such as collagen and elastin, fragments of these proteins, hyaluronic acid, glycosaminoglycan or proteoglycan such as chondroitin sulfate, and Matrigel.
• the extracellular matrix is preferably one or more substances selected from the group consisting of collagen, laminin, fibronectin, Matrigel, vitronectin and fragments of these proteins.
• the laminin fragment include commercially available products such as iMatrix-511, iMatrix-411, and iMatrix-221.
• Matrigel is a basal membrane preparation derived from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared by, for example, a method disclosed in US Patent No. 4829000 , and a commercially available product can also be purchased. Main components of Matrigel are laminin, IV-type collagen, heparan sulfate proteoglycan and entactin.
• EHS Engelbreth Holm Swarn
• the temperature-responsive polymer is a polymer in which the properties of the polymers vary depending on change in temperature. Specifically, it has a lower critical solution temperature (LCST) in water and exhibits phase transition behavior in which, with a certain temperature as a threshold, an intramolecular or intermolecular hydrophobic bond thereof is strengthened at a temperature higher than it so that the polymer chain aggregates, whereas the polymer chain is hydrated by binding to a water molecule at a lower temperature.
• LCST critical solution temperature
• examples thereof include temperature-responsive polymers that maintain the hydrophobic state of equipment surface at a culture temperature (about 37°C) while the equipment surface shifts to hydrophilicity at a temperature lower than the culture temperature, for example, on the order of 20 to 30°C, so that cultured cells become easy to detach.
• a culture temperature about 37°C
• the equipment surface shifts to hydrophilicity at a temperature lower than the culture temperature, for example, on the order of 20 to 30°C, so that cultured cells become easy to detach.
• enzymatic treatment is not necessary for cell detachment, and it is also possible to recover one large sheet without disrupting proteins present between cells due to enzymatic treatment.
• PIPAAm poly-N-isopropylacrylamide
• LCST 32°C
• cell culture is performed under a condition of about 37°C, and a temperature-responsive polymer whose LCST is in the range of about 20°C to 35°C is preferable with consideration also given to the damage of a low temperature on cells.
• Temperature-responsive cell culture equipment for cell sheet recovery (CellSeed Inc.: UpCell(R)) or the like in which the temperature-responsive polymer is fixed to the surface is also obtainable as a commercially available product.
• the culture temperature is not particularly limited and is about 30 to 40°C, preferably about 37°C.
• Culture is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%.
• a Wnt signaling pathway agonist is necessary for the formation of a layer structure and, particularly, apical surface/basal surface (apical/basal polarity), in a sheet-shaped retinal tissue.
• the concentration of the Wnt signaling pathway agonist can be a concentration capable of inducing a sheet-shaped retinal tissue having a layer structure and an apical surface.
• the concentration of the Wnt signaling pathway agonist can be 0.01 ⁇ M to 100 ⁇ M and is preferably 0.1 ⁇ M to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M.
• the concentration can exhibit a Wnt signal-activating effect equivalent to that of CHIR99021 with the concentration mentioned above.
• Whether or not the layer structure has been formed can be readily determined by those skilled in the art, for example, by observation under a microscope or measuring a thickness using an apparatus such as OCT. Whether the apical surface has been formed can be confirmed, for example, by staining using an anti-Zo-1 antibody, an anti-ezrin antibody, or an anti-atypical-PKC antibody.
• the timing of adding the Wnt signaling pathway agonist is not particularly limited. It is preferable to add it at as early timing as possible after the start of adhesion culture for preparation into a sheet again. In an embodiment, it is preferable to perform culture in the culture medium containing the Wnt signaling pathway agonist from the start of the culture.
• the number of culture days in the culture medium containing the Wnt signaling pathway agonist is not particularly limited within a range in which an effect of reforming an epithelial structure (or a multilayered structure) having a polarity of apical surface/basal membrane is found, and is, for example, 1 day to 15 days, preferably 1 day to 9 days.
• Culture may be continued in a culture medium from which the Wnt signaling pathway agonist or the like has been removed. By continuing culture, the layer structure is thickened, and the differentiation of retinal cells proceeds.
• the culture period is not particularly limited and can be a period for which a seeded dispersed retinal cell population proliferates and at least an epithelial structure (or a multilayered structure) is formed. Culture can be performed until a sheet-shaped retinal tissue at a targeted stage of differentiation is manufactured. It is desirable that culture should be performed for at least 7 days from the viewpoint of the formation of an epithelial structure (or a multilayered structure).
• the culture period can be, for example, 7 days to 60 days or less and may be 40 days or less, 30 days or less, 20 days or less, or 16 days or less (e.g., 16 days).
• culture may be further continued in order to allow cells to proliferate, differentiate or mature.
• the culture medium that is used in the further culture may or may not contain a Wnt signaling pathway agonist, a ROCK inhibitor, a SHH (sonic hedgehog) signaling pathway agonist and/or an integrin signaling pathway agonist.
• the "multilayered structure” is a structure where two or more cell layers in which cells are arranged in the same direction are stacked in a tissue having polarities on the basal membrane side and the apical side (i.e., having an epithelial structure), and is a structure where the tangent directions of the respective surfaces of different layers are roughly in parallel. It is preferable that the multilayered structure should have polarities of a basal surface and an apical surface.
• Retinal tissue having a basal membrane and in an embodiment, the multilayered structure can be a sheet-shaped retinal tissue, and a cell aggregate comprising retinal tissue having the multilayered structure can be a sheet-shaped cell aggregate.
• the cell layers can be neural retinal progenitor cell layers, ganglion cell layers, or photoreceptor cell layers.
• the orientation of cells in the multilayered structure can be a direction roughly perpendicular to the layer direction.
• the "orientation of cells” refers to a direction in which the shape of nuclei and the orientation of cell bodies extend on the basal membrane side and the apical side.
• the direction roughly perpendicular to the layer direction refers to a direction orthogonal to a direction in which individual cells are arranged in contact in layers of the multilayered structure (i.e., the tangent directions of the surfaces of layers), and refers to a perpendicular direction or a longitudinal direction with respect to the layers.
• the step of dissecting a size necessary for transplantation from the retinal tissue (particularly, sheet-shaped retinal tissue) having an epithelial structure (or a multilayered structure) obtained by the suspension culture or the adhesion culture may be further included.
• Dissection can be performed using, for example, tweezers, a knife, or scissors.
• RPE cells are manufactured at a given percentage and cause contamination. These RPE cells can be visually confirmed and can also be removed. It is preferable that the RPE cells should not be contained from the beginning from the viewpoint of quality and manufacturing efficiency. Accordingly, as a result of conducting diligent studies to solve the problem, it has become possible to drastically reduce contamination with RPE cells by using a method disclosed below.
• An aspect of the present invention is a sheet-shaped retinal (neural retinal) tissue having an epithelial structure.
• An embodiment of the sheet-shaped retinal tissue consists of a retinal cell layer having a multilayered structure, wherein the multilayered structure has polarities of a basal surface and an apical surface, the retinal cell layer having a multilayered structure comprises one or more cells selected from the group consisting of a retinal progenitor cell, a photoreceptor progenitor cell and a photoreceptor cell, and in the retinal cell layer, the orientation of cells is a direction roughly perpendicular to the layer direction.
• the "sheet shape” refers to a single-layer or multilayer structure constituted by single or a plurality of cells having a biological bond at least in the two-dimensional direction.
• One of the advantages of the present invention is that a sheet-shaped retinal tissue having an arbitrary size can be manufactured according to the culture equipment used. Specifically, it is possible to manufacture a sheet-shaped retinal tissue having a size that has been impossible to manufacture so far. When a disease spans a wide range, treatment becomes possible by the transplantation of one sheet.
• the major axis (also referred to as diameter) of the sheet-shaped retinal tissue according to the present invention is, for example, 2 mm or more, 4 mm or more, 5 mm or more, 7.5 mm or more, or 10 mm or more.
• the minor axis of the sheet-shaped retinal tissue according to the present invention is, for example, 2 mm or more, 3 mm or more, 4 mm or more, or 5 mm or more.
• the major axis and the minor axis have no upper limit and are limited by the size of a dish or the like that is used in culture.
• the major axis can be 10 cm or less, 5 cm or less, 4 cm or less, 3 cm or less, 2 cm or less, or 1 cm or less.
• the height of the sheet-shaped retinal tissue according to the present invention can be, for example, 50 ⁇ m to 1500 ⁇ m, and is preferably 200 ⁇ m to 700 ⁇ m.
• Methods for measuring the major axis, minor axis and height of the sheet-shaped retinal tissue are not particularly limited, and they can be measured, for example, from an image taken under a microscope.
• a front image taken with an apical surface turned to an objective lens side, and a side image taken with the cut surface inclined so as to be perpendicular to an objective lens are taken under a stereo microscope as to the sheet-shaped retinal tissue, and they can be measured from the taken images.
• the major axis means the longest line segment among line segments connecting two end points on the sheet cross section in the front image, and the length thereof.
• the minor axis means the longest line segment among line segments connecting two end points on the sheet cross section in the front image and orthogonal to the major axis, and the length thereof.
• the height means the longest line segment among line segments orthogonal to the sheet cross section and having a point intersecting the sheet cross section and the apex of the retina sheet as end points, and the length thereof.
• the basal surface and the apical surface are as described in the definitions mentioned above.
• the "multilayered structure has polarities of a basal surface and an apical surface” means that the basal surface is present on one side of the multilayered structure and the apical surface is present on the other side.
• a basal membrane may be present on the basal surface.
• the "orientation of cells is a direction roughly perpendicular to the layer direction" means that the major axis of cells present in each layer of the retinal cell layer faces a direction roughly perpendicular to the layer direction.
• an acute angle formed by the layer direction and the major axis of cells is roughly 75° (or 80°) or more and 90° or less.
• the orientation of cells is a direction roughly perpendicular to the layer direction.
• the sheet-shaped retinal tissue should not contain RPE cells.
• the percentage of the number of RPE cells to the total number of cells in the sheet-shaped retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
• FACS flow cytometry
• the percentage of the area of RPE cells to the total area of the sheet-shaped retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Since RPE cells assume black color, the percentage of the area of RPE cells to the total area of the sheet-shaped retinal tissue is calculable from the percentage of an area that exhibits black color under a microscope. It is also possible to detect cells expressing the markers for RPE cells by PCR or the like. A method for decreasing the percentage of RPE cells in the sheet-shaped retinal (neural retinal) tissue is as mentioned above.
• the retinal tissue may comprise the retinal cell layer having a multilayered structure and a sheet-shaped retinal pigment epithelial cell joined to the retinal cell layer, and may be a complex (complex sheet) in which the tangent directions of the respective surfaces of the retinal cell layer and the sheet-shaped retinal pigment epithelial cell are roughly in parallel, the apical surface of the retinal cell layer and the apical surface of the sheet-shaped retinal pigment epithelial cell face each other, and the retinal cell layer and the sheet-shaped retinal pigment epithelial cell are joined through an adhesion factor present therebetween.
• a sheet-shaped neural retina is also referred to as a neural retina sheet or a NR sheet
• the sheet-shaped retinal pigment epithelial cell is also referred to as a retinal pigment epithelial cell sheet or an RPE cell sheet (RPE sheet).
• RPE sheet retinal pigment epithelial cell sheet
• an aspect of the present invention also provides a complex (complex sheet) of a neural retina and RPE cells.
• Another aspect of the present invention also includes a complex (complex sheet) in which dispersed retinal pigment epithelial cells are joined to the apical surface side of a sheet-shaped retinal cell layer (sheet-shaped neural retina) through an adhesion factor.
• the respective tangent directions of the neural retina sheet and the retinal pigment epithelial cell sheet are roughly in parallel.
• the "tangent directions are roughly in parallel” means that the tangent directions of the facing surfaces of the neural retina and the retinal pigment epithelial cell sheet are in parallel.
• the apical surface of the neural retina and the apical surface of the retinal pigment epithelial cell face each other. Specifically, the apical surface of the neural retina and the apical surface of the retinal pigment epithelial cell are adjacently present.
• the complex (complex sheet) of a neural retina and RPE cells can be prepared by joining a sheet-shaped retinal cell layer (sheet-shaped neural retina) to an RPE cell sheet or dispersed RPE cells in the presence of an adhesion factor, as mentioned later.
• the sheet-shaped retinal cell layer (sheet-shaped neural retina) can be manufactured by the aforementioned method for manufacturing a cell aggregate comprising retinal tissue having an epithelial structure (or a multilayered structure).
• the RPE cell sheet can be manufactured by a method for manufacturing a retinal pigment epithelial cell sheet mentioned later.
• the retinal pigment epithelial (RPE) cells are derived from pluripotent stem cells and, specifically, can be obtained by differentiating pluripotent stem cells.
• Examples of the method for manufacturing retinal pigment epithelial cells include, but are not particularly limited to, methods disclosed in WO2005/070011 , WO2006/080952 , WO2011/063005 , WO2012/173207 , WO2015/053375 , WO2015/053376 , WO2015/068505 , WO2017/043605 , Stem Cell Reports, 2 (2), 205-218 (2014 ) and Cell Stem Cell, 10 (6), 771-785 (2012 ).
• the retinal pigment epithelial cells may be manufactured as a cell sheet or a sphere-like cell aggregate.
• the RPE cell sheet is preparable by cutting open the cell aggregate using, for example, tweezers, a knife, or scissors.
• pluripotent stem cells are cultured in the absence of feeder cells under conditions involving 1) performing a treatment with a TGF ⁇ family signaling pathway inhibitor and a sonic hedgehog signaling pathway agonist 1 day before differentiation and 2) performing no treatment with a sonic hedgehog signaling pathway agonist at the start of differentiation in the method mentioned above. Then, the steps (B) and (C) mentioned above are performed. Further, it is preferable to accelerate the timing of start of the step (D). Specifically, the step (D) is started around 9 days after (e.g., 7 days, 8 days, 9 days, 10 days, or 11 days after) the start of suspension culture of the step (B).
• 9 days after e.g., 7 days, 8 days, 9 days, 10 days, or 11 days after
• a sphere-like cell aggregate of RPE cells is obtained.
• the cell aggregate may be dispersed to prepare a cell suspension, and the RPE cell sheet can also be prepared by cutting open the cell aggregate using tweezers, a knife, scissors, or the like.
• the RPE cell sheet can also be prepared by culturing the dispersed cell suspension by adhesion culture.
• Dispersed RPE cells can also be obtained by the dispersion of the RPE cell sheet or the aggregate of RPE cells.
• the retinal pigment epithelial cell sheet may be further cultured until having polygonal/flagstone-like cell morphology before being contacted with a cell aggregate of a neural retina.
• the culture medium in this case is not particularly limited, and culture can also be further performed by replacement with a maintenance medium for retinal pigment epithelial cells (hereinafter, also referred to as a RPE maintenance medium).
• a maintenance medium for retinal pigment epithelial cells hereinafter, also referred to as a RPE maintenance medium.
• a melamine pigmented cell group or a cell group having polygonal flat morphology adhering to a basal membrane can thereby be observed further clearly.
• Culture in a RPE maintenance medium is not limited as long as a colony that proliferates is formed while the properties of retinal pigment epithelial cells are maintained.
• the maintenance medium for retinal pigment epithelial cells can employ one described in, for example, IOVS, March 2004, Vol. 45, No. 3 , Masatoshi Haruta et al., IOVS, November 2011, Vol. 52, No. 12 , Okamoto et al., Cell Science 122 (17 ), Fumitaka Osakadar et al., or February 2008, Vol. 49, No. 2, Gamm et al.
• the major axis of the retinal pigment epithelial cell sheet may be the same as the major axis of the sheet-shaped retinal tissue (neural retina sheet).
• the major axis of the retinal pigment epithelial cell sheet can be in the range of, for example, 3 mm to 50 mm, 5 mm to 30 mm, or 10 mm to 20 mm.
• the minor axis of the retinal pigment epithelial cell sheet may be the same as the minor axis of the sheet-shaped retinal tissue (neural retina sheet).
• the minor axis of the retinal pigment epithelial cell sheet can be in the range of, for example, 2 mm to 40 mm, 5 mm to 30 mm, or 10 mm to 20 mm.
• the degree of melanin pigmentation of the retinal pigment epithelial cell sheet is not particularly limited. It is preferable that the degree of melanin pigmentation of retinal pigment epithelial cells contained in the retinal pigment epithelial cell sheet should be the same level among the cells.
• the average melanin content of the retinal pigment epithelial cell sheet can be less than 20 pg/cell, less than 15 pg/cell, less than 10 pg/cell, less than 8 pg/cell, less than 7 pg/cell, less than 6 pg/cell, less than 5 pg/cell, less than 4 pg/cell, less than 3 pg/cell, less than 2 pg/cell, or less than 1 pg/cell.
• the average melanin content of the retinal pigment epithelial cell sheet may be 0.1 pg/cell or more, 0.5 pg/cell or more, 1 pg/cell or more, 2 pg/cell or more, or 5 pg/cell or more.
• the melanin content of the retinal pigment epithelial cell sheet can be measured using, for example, cell extracts obtained with NaOH or the like after dispersing the retinal pigment epithelial cell sheet, and using a spectrophotometer or the like.
• the average melanin content can be determined by dividing the melanin content by the total number of cells contained in the retinal pigment epithelial cell sheet.
• the complex mentioned above can be manufactured by joining a neural retina sheet to dispersed RPE cells or a RPE cell sheet.
• a complex sheet in which a neural retina sheet is joined to a RPE cell sheet is preferable.
• the neural retina sheet and the RPE cell sheet mentioned above can be easily taken out of culture equipment by using tweezers, a knife, scissors, or the like. Both the sheets taken out may be transferred to a fresh container (culture equipment, etc.). While one of the sheets is allowed to remain in the culture equipment, the other sheet may be transferred into the culture equipment.
• the sizes of the sheet-shaped tissues to be joined can be uniformed by manufacturing them in culture equipment having the same size. In the case of using a neural retina sheet and retinal pigment epithelial cells with different numbers of culture days in the manufacturing method mentioned above as to the neural retina sheet and the retinal pigment epithelial cells to be contacted, the days when manufacturing is started can be changed.
• the adhesion factor refers to a substance having the action of allowing cells to adhere to each other. Examples thereof include, but are not particularly limited to, the extracellular matrixes mentioned above and artificial hydrogels.
• the adhesion factor does not have to be an isolated single substance and also includes, for example, Matrigel, an inter-photoreceptor cell matrix, and a preparation from a living body or cells, such as serum.
• Matrigel is a basal membrane preparation derived from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared by, for example, a method disclosed in US Patent No.
• Matrigel Main components of Matrigel are laminin, IV-type collagen, heparan sulfate proteoglycan and entactin.
• the inter-photoreceptor cell matrix is a generic name for extracellular matrixes present between retinal cells such as photoreceptor cells in a retina in vivo, and, for example, hyaluronic acid is included.
• Those skilled in the art are capable of collecting the inter-photoreceptor cell matrix from a retina in vivo by, for example, a method of placing the retina in distilled water so as to swell, followed by separation, and a commercially available product can also be purchased.
• An extracellular matrix or a hydrogel is preferable as the adhesion factor.
• One or more extracellular matrixes selected from the group consisting of hyaluronic acid, fibrin, laminin, IV-type collagen, heparan sulfate proteoglycan, and entactin are preferable as the extracellular matrix.
• One or more hydrogels selected from the group consisting of gelatin, fibrin, collagen, pectin, hyaluronic acid, and alginic acid are preferable as the hydrogel.
• a substance that is classified into both an extracellular matrix and a hydrogel is present and is treated as a hydrogel in the present specification when used as a gel-like adhesion factor.
• the adhesion factor can be one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, IV-type collagen, heparan sulfate proteoglycan and entactin, and it is particularly preferable to be gelatin or fibrin.
• Fibrin gel is gel-like fibrin that is obtained by reacting a fibrinogen solution with a thrombin solution.
• a fibrinogen solution with a thrombin solution.
• Bolheal(R) for tissue adhesion can also be used.
• the fibrinogen solution and the thrombin solution are contacted or mixed and both can thereby be reacted.
• the "gelatin” is a solubilized form of water-insoluble collagen, for example, by pretreatment with an acid or an alkali and thermal hydrolysis. It is also possible to obtain commercially available gelatin. Examples thereof include gelatin LS-H (Nitta Gelatin Inc., alkali-treated porcine skin gelatin, no-heat-treated gelatin, high jelly strength) and gelatin LS-W (Nitta Gelatin Inc., alkali-treated porcine skin gelatin, heat-treated gelatin, low jelly strength). Alkali-treated (limed) gelatin (B-type gelatin) is preferable, and heat-treated gelatin is preferable.
• the hydrogel is heated or cooled so that the solution changes a phase from gel to sol or from sol to gel.
• the hydrogel is converted to gel (jelly) by losing fluidity through cooling and converted to sol (aqueous solution) by acquiring fluidity through heating.
• the "melting point” means a temperature at which solation occurs under a predetermined pressure
• the "freezing point” means a temperature at which gelation occurs under a predetermined pressure. It is preferable that the hydrogel should be biodegradable.
• a hydrogel e.g., gelatin whose melting point is a temperature (25°C to 40°C) near a body temperature is preferable.
• the hydrogel in the present specification may have a melting point of 20°C to 40°C (e.g., 20°C to 35°C, 25°C to 35°C, 30°C to 40°C, or 35°C to 40°C).
• the melting point of gel is a measure for the strength of a network.
• the melting point of the hydrogel e.g., gelatin
• the melting point of the hydrogel is elevated with the elevation of the concentration and molecular weight of the hydrogel.
• the melting point and the freezing point tend to be elevated as a solid content is increased with a saccharide.
• a method for measuring the melting point of the hydrogel is not particularly limited, and it can be measured by, for example, a method prescribed in JIS K6503.
• the strength of the hydrogel can be to an extent that the hydrogel does not collapse in operation for transplantation.
• the "jelly strength” serves as an index for the strength of the hydrogel.
• the “jelly strength” of the hydrogel means the mechanical strength of an object that has formed gel. It is usually expressed as force required for deforming gel in a predetermined shape or force required for breaking gel (unit: g, dyne(s)/cm 2 or g/cm 2 ), and is typically a measure for the hardness of gel. 1 dyne is defined as force which when acting on a body of mass 1 g produces an acceleration of 1 cm/s 2 in that direction.
• the jelly strength of gelatin can be measured by, for example, a method prescribed in JIS K6503.
• the jelly strength of the hydrogel (e.g., gelatin) gel can be, for example, 50 g or more, 100 g or more, 200 g or more, 500 g or more, 1000 g or more, 1200 g or more, 1300 g or more, 1400 g or more, or 1500 g or more.
• the jelly strength of the hydrogel (gelatin) may be 3000 g or less, 2500 g or less or 2000 g or less.
• the concentration of the adhesion factor differs depending on the size of the neural retina sheet or the retinal pigment epithelial cell sheet or the number of cells of retinal pigment epithelial cells. Those skilled in the art are capable of readily setting it by confirming the state of adhesion of RPE cells. For example, it is preferable to add Matrigel at a concentration of a ready-made (Corning, Inc.) diluted 200-to 10000-fold or iMatrix511 at a concentration of 0.1 to 5 ⁇ g/mL.
• culture for allowing the neural retina sheet to adhere to the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet can be performed in a vehicle containing the adhesion factor (extracellular matrix).
• vehicle used include, but are not particularly limited to, culture media that are used in the culture of retinal pigment epithelial cells or neural retinas (e.g., DMEM/F12 medium, neurobasal medium, a mixture of these culture media, RPE maintenance medium, etc.).
• Culture for adhesion may be performed in the presence of other components such as a growth factor (e.g., EGF) together with the extracellular matrix.
• EGF growth factor
• culture may be continuously performed in the vehicle containing the adhesion factor, or culture may be continuously performed by culture in the vehicle containing the adhesion factor for a given period (e.g., 1 day to 10 days), followed by replacement with a vehicle containing no adhesion factor.
• the neural retina sheet or the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet Before culture for allowing the neural retina sheet to adhere to the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet is performed, the neural retina sheet or the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet may be coated with the adhesion factor.
• the neural retina sheet or the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet can be cultured in the vehicle containing the adhesion factor.
• Those skilled in the art are appropriately capable of setting a culture time. Culture can be performed for a time on the order of 10 minutes to 5 hours (e.g., 10 minutes to 60 minutes). After culture, washing may be performed with a vehicle such as PBS.
• the adhesion factor, the hydrogel or the matrix gel is preferably fibrin gel.
• the fibrin gel is gel-like fibrin that is obtained by reacting a fibrinogen solution with a thrombin solution.
• a substance having a property of forming gel through reaction is referred to as a matrix precursor, and, for example, thrombin and fibrinogen which react to form fibrin gel are one example of the matrix precursor.
• the fibrinogen solution can be prepared by dissolving a fibrinogen powder or the like in a dissolving liquid that contains aprotinin and can dissolve fibrinogen, and its concentration is not particularly limited, but is, for example, from 40 to 480 mg/ml, preferably from 80 mg/mL to 320 mg/mL (e.g., 160 mg/mL).
• concentration is not particularly limited, but is, for example, from 40 to 480 mg/ml, preferably from 80 mg/mL to 320 mg/mL (e.g., 160 mg/mL).
• the activity of blood coagulation factor VIII contained in 1 ml of normal human plasma is defined as 1 unit, it can be from 37.5 units/mL to 225 units/mL (e.g., 75 units/mL).
• the thrombin solution can be prepared by dissolving a thrombin powder or the like in a thrombin-dissolving liquid containing calcium chloride hydrate, and its concentration is not particularly limited, but is, for example, from 125 units/mL to 750 units/mL (e.g., 75 units/mL).
• the fibrinogen solution and the thrombin solution are contacted or mixed so that they can be reacted with each other.
• the fibrinogen solution and the thrombin solution should be used in the range of 1:1 to 1:9, preferably 1:3 to 1:4, in terms of an activity ratio.
• a method for manufacturing the complex using fibrin gel will be given below as an embodiment of the method for manufacturing the complex using a hydrogel.
• the neural retina sheet is allowed to adhere to the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet through fibrin gel that is produced by reacting fibrinogen and thrombin.
• one tissue (neural retina or retinal pigment epithelial cells) contacted with a fibrinogen solution and the other tissue (retinal pigment epithelial cells or neural retina) contacted with a thrombin solution are contacted with each other so that fibrinogen and thrombin may be reacted for gelation.
• the contact with a solution means that at least an adhesion surface (a surface that faces another tissue when adhering to another tissue through fibrin gel) of a tissue is contacted with a fibrinogen solution or a thrombin solution to the extent that attachment to the adhesion surface of the tissue is caused by the solution.
• the neural retina sheet in the case of allowing a neural retina sheet to adhere to a retinal pigment epithelial cell sheet, the neural retina sheet can be added in a fibrinogen solution, and the retinal pigment epithelial cell sheet can be added in a thrombin solution (the solutions may be reversed). It is preferable to use the fibrinogen solution and the thrombin solution in the range of 3:1 to 1:3 in terms of a volume ratio. Before adhesion, extra fibrinogen or thrombin attached to the tissue may be removed. By this operation, the thickness of the fibrin gel can be adjusted.
• both may be contacted.
• contacting the tissues refers to contacting, i.e., overlapping, the surface attached to the fibrinogen solution and the surface attached to the thrombin solution. It is preferable to allow the retinal pigment epithelial cell sheet to adhere to the neural retina such that the tangent directions of their respective surfaces are roughly in parallel and the apical surface of the neural retina and the apical surface of the retinal pigment epithelial cell sheet face each other.
• fibrinogen and thrombin may be reacted by contacting the tissues described above with a fibrinogen solution, and subsequently adding a thrombin solution to the fibrinogen solution.
• the tissues are embedded in fibrin gel.
• the manufacturing method of the present invention may further comprise the step of dissecting a complex having a size necessary for transplantation from the complex. Since two or more tissues have been allowed to adhere firmly through fibrin gel, a graft, when dissected from the complex, can be easily dissected into the desired size without the tissues coming off from the complex. It is possible to use tweezers, a knife, scissors, or the like in dissection.
• An aspect of the present invention includes a pharmaceutical composition comprising a retinal tissue (for example, a sheet-shaped retinal tissue).
• the pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier, in addition to the retinal tissue of the present invention.
• the pharmaceutical composition can be used in the treatment of a disease caused by the damage of a neural retinal cell or a neural retina or the injury of a neural retina.
• diseases caused by the damage of a neural retinal cell or neural retina include ophthalmic diseases such as retinal degenerative diseases, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous chorioretinopathy, cone dystrophy, and cone rod dystrophy.
• Examples of the injury state of neural retina include a state in which photoreceptor cells die of degeneration.
• a physiological aqueous solvent physiological saline, buffer, serum-free medium, etc.
• the pharmaceutical composition may be blended with a preservative, a stabilizer, a reducing agent, a tonicity agent, and the like which are usually used in a medicine containing tissues or cells to be transplanted in medical transplantation.
• An aspect of the present invention provides a therapeutic product for a disease caused by the damage of a neural retina, comprising a retinal tissue (for example, a sheet-shaped retinal tissue) obtained in the present invention.
• An aspect of the present invention includes a method for treating a disease caused by the damage of a neural retinal cell or a neural retina or the injury of a neural retina, comprising transplanting a retinal tissue obtained in the present invention to a subject in need of transplantation (e.g., subretinally to an eye having the ophthalmic disease).
• the retinal tissue of the present invention can be used as the therapeutic product for a disease caused by the damage of a neural retina, or in order to make up for a corresponding injured site in the injury state of the neural retina.
• the disease caused by the damage of a neural retinal cell or a neural retina or the injury state of a neural retina can be treated by transplanting the sheet-shaped retinal tissue of the present invention to a patient having the disease caused by the damage of a neural retinal cell or a neural retina, or a patient with the injury state of a neural retina, in need of transplantation, and making up for the neural retinal cell or the damaged neural retina.
• a transplantation method include a method of subretinally transplanting the retinal tissue to an injured site through an incision to an eyeball.
• Examples of a method for transplantation include a method of performing infusion using a thin tube, and a method of performing transplantation by sandwiching between tweezers, and examples of the thin tube include injection needles.
• An aspect of the present invention provides a retinal tissue (for example, a sheet-shaped retinal tissue) obtained in the present invention for use in the treatment of a disease caused by the damage of a retinal cell or retinal tissue or the injury of retinal tissue.
• a retinal tissue obtained in the present invention in the manufacturing of a therapeutic product for a disease caused by the damage of a retinal cell or retinal tissue or the injury of retinal tissue.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) and human iPS cells (1231A3 strain, Kyoto University) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in "Scientific Reports, 4, 3594 (2014)".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold as an alternative to feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• human ES/iPS cells specific operation of maintenance culture for human ES and human iPS cells (human ES/iPS cells) was performed as follows: First, human ES/iPS cells that reached sub-confluency (state where about 60% of the culture area was covered by cells) were washed with PBS and separated into single cells by use of TrypLE Select (trade name, manufactured by Life Technologies).
• the dispersion into single cells refers to performing dispersion so as to become single cells, and a cell population dispersed into single cells is capable of containing a mass of 2 to 50 cells, in addition to single cells.
• the separated human ES single cells were seeded in plastic culture dishes coated with Laminin 511-E8 and cultured under feeder-free conditions in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M).
• Y-27632 ROCK inhibitor, 10 ⁇ M
• the number of separated human ES/iPS single cells to be seeded was specified as 0.4 to 1.2 ⁇ 10 4 cells per well.
• the medium was exchanged with StemFit medium not containing Y-27632. Thereafter, the medium was exchanged with Y27632-free StemFit medium once every 1 to 2 days.
• the human ES cells were cultured under feeder-free conditions until 1 day before reaching sub-confluency.
• the human ES cells the 1 day before the sub-confluency were cultured for 1 day (preconditioning treatment) under feeder-free conditions in the presence of SB431542 (TGF ⁇ signaling pathway inhibitor, 5 ⁇ M) and SAG (Shh signaling pathway agonist, 300 nM).
• SB431542 TGF ⁇ signaling pathway inhibitor, 5 ⁇ M
• SAG Shh signaling pathway agonist
• the human ES/iPS cells were washed with PBS, then treated for cell dispersions using TrypLE Select, and further separated into single cells by pipetting, and then the separated human ES single cells were suspended in 100 ⁇ L of a serum-free medium such that the density of cells per well of a non-cell adhesive 96-well culture plate (trade name: PrimeSurface, 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) was 1.2 ⁇ 10 4 cells, and subjected to suspension culture in the conditions of 37°C and 5% CO 2 .
• a non-cell adhesive 96-well culture plate trade name: PrimeSurface, 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.
• the serum-free medium (gfCDM + KSR) used herein is a serum-free medium prepared by adding 10% KSR and 450 ⁇ M 1-monothioglycerol and 1X Chemically defined lipid concentrate to a mixture of culture fluids containing F-12 medium and IMDM medium in a ratio of 1:1.
• Y-27632 (ROCK inhibitor, final concentration: 10 ⁇ M or 20 ⁇ M) and SAG (Shh signaling pathway agonist, 300 nM, 30 nM or 0 nM) were added to the serum-free medium.
• Day 3 from initiation of the suspension culture 50 ⁇ L of a medium was used which did not contain Y-27632 or SAG and contained exogenous human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Systems, Inc.) at a final concentration of 1.5 nM.
• BMP4 trade name: Recombinant Human BMP-4, manufactured by R&D Systems, Inc.
• the aggregates of Day 14 to Day 18 from initiation of the suspension culture were transferred to a 90-mm low adhesive plate (petri dish 90 ⁇ (deep type) for suspension culture, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured at 37°C and 5% CO 2 for 3 to 4 days in a serum-free medium (DMEM/F12 medium supplemented with 1% N2 Supplement) containing a Wnt signaling pathway agonist (CHIR99021, 3 ⁇ M) and a FGF signaling pathway inhibitor (SU5402, 5 ⁇ M).
• DMEM/F12 medium supplemented with 1% N2 Supplement
• the aggregates of Day 16 (1231A3-derived) or Day 27 (KhES-1-derived) from initiation of the suspension culture were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C for 20 to 30 minutes, they were dispersed into single cells by pipetting.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports, 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 20 (1231A3) and Day 27 (KhES-1) from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• the reformed states of the aggregates were observed under a bright field microscope and a fluorescence microscope (BZ-X810 manufactured by Keyence Corp.) on Day 1, Day 7, and Day 14 after suspension culture for aggregate reforming.
• the results of the KhES-1-derived cells are shown in Figures 3 to 5 .
• the areas of the aggregates were measured in Image J.
• the results of the KhES-1-derived cells are shown in Figure 6 (an average value from 12 cells measured).
• the results of the 1231A3-derived cells in which the reformed states of the aggregates of Day 1 after suspension culture were observed are shown in Figure 7 .
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports, 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold as an alternative to feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 26 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting. These cells were suspended in 100 ⁇ L of a serum-free medium so as to become 3.0 ⁇ 10 4 cells per well of a non-cell adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.), and suspension-cultured at 37°C and 5% CO 2 .
• a non-cell adhesive 96-well culture plate trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.
• the aggregates of Day 15 (corresponding to Differentiation Day 41) and Day 28 (corresponding to Differentiation Day 54) after suspension culture for aggregate reforming were fixed in 4% PFA, and frozen sections were prepared. These frozen sections were subjected to immunostaining using DAPI and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, Exalpha Biologicals Inc.), anti- ⁇ -catenin antibody (R&D Systems, Inc.), anti-collagen IV antibody (Abcam plc), anti-Zo-1 antibody (Invitrogen Corp.), anti-Ki67 antibody (Becton, Dickinson and Company), anti-Pax6 antibody (BioLegend, Inc.), anti-RxR-y (RxRg) antibody (Spring Bioscience Corp.), anti-CRX antibody (Abnova Corp.), anti-NRL antibody (R&D Systems, Inc.), anti-recoverin antibody (manufactured by Proteintech Group, Inc.), anti-Islet-1 antibody (Developmental Studies Hybridoma Bank (
• retinal tissue as epithelial tissue having an apical-basal polarity can be reorganized and differentiated into a retina by once adding CHIR99021 to a single cell suspension of dispersed retinal cells.
• StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 18, Day 25, Day 40, Day 61, and Day 75 (dd18, dd25, dd40, dd61, dd75) from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports, 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• a medium which did not contain Y-27632 or SAG and contained exogenous human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Systems, Inc.) at a final concentration of 1.5 nM.
• BMP4 trade name: Recombinant Human BMP-4, manufactured by R&D Systems, Inc.
• a group that was not supplemented with BMP4 was established.
• Day 6 or later from initiation of the suspension culture a half of the medium was exchanged with a culture medium, which did not contain Y-27632, SAG or human recombinant BMP4, once every 3 days.
• the aggregates of Day 40 from initiation of the suspension culture prepared in this way were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 31 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• a non-frozen group was suspended in 100 ⁇ L of a serum-free medium containing 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.), 300 nM SAG (Enzo Life Sciences, Inc.) and 3 ⁇ M CHIR99021 (FUJIFILM Wako Pure Chemical Corp.) so as to become 2.0 ⁇ 10 5 cells per well of a non-cell adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.), and suspension-cultured at 37°C and 5% CO 2 .
• a serum-free medium containing 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.), 300 nM SAG (Enzo Life Sciences, Inc.) and 3 ⁇ M CHIR99021 (FUJIFILM Wako Pure Chemical Corp.) so as to become 2.0 ⁇ 10 5 cells per well of a non-cell adhesive 96-well culture plate (
• the single cells were suspended at 1.0 ⁇ 10 6 cells/mL using a negative control PBS (Gibco) and the following cryopreservation solutions, Cell Banker 1 (Takara Bio Inc.), Stem Cell Banker (Takara Bio Inc.), CultureSure(R) cryopreserved solution (CultureSure(R) Freezing Medium; FUJIFILM Wako Pure Chemical Corp.), STEMdiff(TM) Neural Progenitor Freezing Medium (STEMCELL Technologies Inc.), StemSure(R) cryopreserved solution (FUJIFILM Wako Pure Chemical Corp.), and Bambanker (Nippon Genetics Co., Ltd.), and cryopreserved at -80°C using BiCell.
• Cell Banker 1 Takara Bio Inc.
• Stem Cell Banker Takara Bio Inc.
• CultureSure(R) cryopreserved solution CultureSure(R) Freezing Medium
• retinal tissue having a layer structure and having a polarity can be reorganized by dispersing retinal cells, differentiating pluripotent stem cells into retinas to obtain cell aggregates containing retinal cells, dispersing the cell aggregates to obtain a single cell suspension, adding Y-27632 (FUJIFILM Wako Pure Chemical Corp.), SAG (Enzo Life Sciences, Inc.) and CHIR99021 (FUJIFILM Wako Pure Chemical Corp.) at the time of seeding of the single cell suspension, and performing suspension culture.
• Y-27632 (FUJIFILM Wako Pure Chemical Corp.), SAG (Enzo Life Sciences, Inc.) and CHIR99021 (FUJIFILM Wako Pure Chemical Corp.)
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports, 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of 25 to 35 days after the start of the suspension culture prepared as in Reference example 1-1 were fixed in 4% PFA, and frozen sections were prepared. These frozen sections were subjected to immunostaining using DAPI and anti-laminin antibody (trade name: Anti Laminin Antibody, Abcam plc), anti-fibronectin antibody (R&D systems, Inc.), and anti-collagen IV antibody (Abcam plc). These immunostained sections were observed under a confocal laser microscope. As a result, it was confirmed that the aggregates expressed at least laminin, fibronectin, and collagen IV, which are constituents of a basal membrane ( Figure 25 ). Specifically, it was found that in this self-organization culture system, human retinal tissue itself forms a basal membrane and expresses laminin, fibronectin, and collagen IV.
• Laminin511, Laminin521, Laminin411, fibronectin, collagen IV, and the like are useful as an extracellular matrix for reforming a sheet-shaped retina sheet by adhesion culture.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 16 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• Matrigel (Corning, Inc.) and Laminin511-E8 (trade name: iMatrix511, manufactured by Nippi, Inc.) were used.
• the single cells of the dispersed retinal cells were suspended in 300 ⁇ L of a serum-free medium containing 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.), 300 nM SAG (Enzo Life Sciences, Inc.) and 3 ⁇ M CHIR99021 (FUJIFILM Wako Pure Chemical Corp.) and seeded so as to become 0.5 to 4 ⁇ 10 5 cells per well of 24-well Transwell (Corning, Inc.) under three conditions given below. 3 days or later from seeding, the medium was replaced with a serum-free medium (gfCDM), which did not contain Y-27632, SAG or CHIR99021, once every 3 to 4 days.
• gfCDM serum-free medium
• a sheet-shaped retinal tissue having an apical-basal polarity can be prepared again (prepared into a sheet again) by coating retinal cells (NR) dispersed into single cells with Laminin511-E8 or Matrigel as a scaffold beforehand.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Days 15 to 30 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• the cells were suspended in 300 ⁇ L of a serum-free medium containing (1) 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.), (2) 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.) and 300 nM SAG (Enzo Life Sciences, Inc.), (3) 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.) and 5 ⁇ M CHIR99021 (FUJIFILM Wako Pure Chemical Corp.), or (4) 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.), 300 nM SAG (Enzo Life Sciences, Inc.) and 3 ⁇ M CHIR99021 (FUJIFILM Wako Pure Chemical Corp.) so as to become 8 ⁇ 10 5 cells per well of 12-well Transwell coated with Laminin511-E8, and adhesion-cultured at 37°C and 5%CO 2 . 3 days or later from seeding, the medium was replaced
• a retina sheet having an apical-basal polarity can be prepared again by adding CHIR99021 to retinal cells (NR) dispersed into single cells.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of 33 days (dd33) after the start of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• the cells were seeded so as to become 2 to 4 ⁇ 10 5 cells per well of 24-well Transwell coated with Laminin511-E8, and 300 ⁇ L of a serum-free medium containing 1 ⁇ M, 3 ⁇ M or 9 ⁇ M CHIR99021 (FUJIFILM Wako Pure Chemical Corp.) in addition to 10 ⁇ M Y-27632 (FUJIFILM Wako Pure Chemical Corp.) and 300 nM SAG (Enzo Life Sciences, Inc.) was added, followed by adhesion culture at 37°C and 5% CO 2 for 3 days (Days 0 to 3) or 6 days (Days 0 to 6) or 9 days (Days 0 to 9). 3 days or later from seeding, the medium was replaced once every 3 to 4 days.
• a retina sheet can be prepared again by adding CHIR99021 at least 1 to 9 ⁇ M for 3 to 9 days to retinal cells (NR) dispersed into single cells.
• NR retinal cells
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of 24 days after the start of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• the cells were seeded so as to become 2.0 ⁇ 10 5 cells per well of 24-well Transwell (1) without coating (Non Coat) or coated with (2) 2 ⁇ L of iMatrix511 (trade name, manufactured by Nippi, Inc.), (3) 2 ⁇ L of iMatrix411 (trade name, manufactured by Nippi, Inc.), (4) 2 ⁇ L of iMatrix211 (trade name, manufactured by Nippi, Inc.), (5) 2 ⁇ L Vitronectin (VTN-N) Recombinant Human Protein (Thermo Fisher Scientific Inc.), (6) 2 ⁇ L of Cell Start (Thermo Fisher Scientific Inc.), (7) 5 ⁇ L of Laminin111, 5 ⁇ L of Laminin121, 5 ⁇ L of Laminin211, 5 ⁇ L of Laminin221, 5 ⁇ L of Laminin411, 5 ⁇ L of Laminin421, 5 ⁇ L of Laminin511, and 5 ⁇ L of Laminin5
• a retina sheet can be prepared again using not only iMatrix511 or Matrigel but various scaffold proteins.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Crx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 15 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 200 ⁇ L of a serum-free medium containing 10 ⁇ M Y-27632, 300 nM SAG and 3 ⁇ M CHIR99021 so as to become 2.0 ⁇ 10 5 cells per well of 24-well Transwell coated with Laminin511-E8, and cultured at 37°C and 5% CO 2 for 3 days. 3 days or later from seeding, the medium was replaced with a serum-free medium (NucT0), which did not contain Y-27632, SAG or CHIR99021, once every 3 to 4 days.
• a serum-free medium NucT0
• the cell sheets of Day 57 were fixed in 4% PFA and then subjected to whole immunostaining without preparing sections. Immunostaining was performed using DAPI and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, Exalpha Biologicals Inc.), anti-Sox2 antibody (BD Pharmingen), anti-Ki67 antibody (Becton, Dickinson and Company), anti-Pax6 antibody (BioLegend, Inc.), anti-Brn3 antibody (Santa Cruz Biotechnology), anti-TUJ1 antibody (Merck Millipore), anti-Islet-1 antibody (R&D Systems, Inc.), anti-Crx antibody (Abnova Corp.), anti-recoverin antibody (Proteintech Group, Inc.), anti-Zo-1 antibody (Invitrogen Corp.), and anti-collagen IV antibody (Abcam plc).
• DAPI and anti-Chx10 antibody trade name: Anti CHX10 Antibody, Exalpha Biologicals Inc.
• anti-Sox2 antibody BD
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 31 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting.
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• the cells were cultured at 37°C and 5% CO 2 for 3 days so as to become 2.0 ⁇ 10 5 cells per well of 12-well Transwell coated with 10 ⁇ L of Laminin511-E8, by adding (1) a control, (2) 20 ng/mL FGF2, (3) 20 ng/mL EGF, (4) 100 nM SAG, (5) 1 unit of LIF, (6) 10 ng/mL IGF-1, (7) 100 ng/mL PDGF-AA, (8) 100 ng/mL PDGF-AB, (9) 10 ⁇ g/mL GDNF, (10) 20 ⁇ g/mL BDNF, (11) 2 ⁇ M Pyrintegrin, or (12) 1 ⁇ M BMP4 to a culture medium in which N2 was added to 500 ⁇ L of serum-free DMEM/F12 containing 10 ⁇ M Y-27632, 300 nM SAG and 3 ⁇ M CHIR99021. 3 days or later from seeding, the medium was replaced with a serum
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Crx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Day 27 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 500 ⁇ L of the following culture medium containing 10 ⁇ M Y-27632, 300 nM SAG and 3 ⁇ M CHIR99021 and seeded so as to become 8.0 ⁇ 10 5 cells/well of 12-well Transwell coated with 10 ⁇ L of Laminin511-E8.
• culture was performed for 1 month by adding 20 ng/mL FGF2 and 20 ng/mL EGF to a culture medium of (1) NeuroCult NS-A (STEMCELL Technologies, Inc.), (2) STEMdiff Neural Progneitor (STEMCELL Technologies, Inc.), (3) StemPro NSC SFM (Thermo Fisher Scientific Inc.), or (4) RHB-A (Takara Bio Inc.).
• NucT0 medium was established. 3 days or later from seeding, the medium was replaced with a culture medium, which did not contain Y-27632, SAG or CHIR99021, once every 3 to 4 days.
• NeuroCult NS-A and RHB-A media and EGF or FGF in preparation into a sheet again has an effect of maintaining retinal progenitor cells and delaying differentiation.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• Collagen gel was prepared using beMatrix low-endotoxin collagen solution (Nitta Gelatin Inc.). As specific operation, collagen AT, 5 x DME, and a buffer solution for reconstitution were mixed at a ratio of 7:2:1, and a mesh of Transwell was coated and incubated at 37°C for 30 minutes in a 5% CO 2 incubator. After incubation, a culture medium was added to the inside and the outside of the insert.
• the aggregates (NR) of Day 26 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting. After dispersion, the cells were seeded at 8.0 ⁇ 10 5 cells/well onto the collagen gel of 12-well Transwell. For the first 3 days, culture was performed in the presence of 10 ⁇ M Y-27632, 300 nM SAG and 3 ⁇ M CHIR99021. 3 days or later from seeding, the medium was replaced with a serum-free medium (NucT0 medium), which did not contain Y-27632, SAG or CHIR99021, once every 3 to 4 days.
• a serum-free medium NucT0 medium
• StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Day 26 were washed with PBS on the collagen gel prepared as in Reference example 1-15, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting. After dispersion, the cells were seeded at 8.0 ⁇ 10 5 cells/well onto the collagen gel of 12-well Transwell. For the first 3 days, culture was performed in the presence of 10 ⁇ M Y-27632, 300 nM SAG and 3 ⁇ M CHIR99021. 3 days or later from seeding, the medium was replaced with a serum-free medium (NucT0 medium), which did not contain Y-27632, SAG or CHIR99021, once every 3 to 4 days.
• a serum-free medium NucT0 medium
• Retina cell sheets of Culture Day 37 and Day 44 for aggregate reforming and Differentiation Day 63 and Day 70 were treated with collagenase (F. Hoffmann-La Roche, Ltd.) at 37°C for 30 minutes and detached. Strips of the detached retina sheets were dissected using tweezers and scissors to prepare grafts for transplantations having a length on the order of 1.8 cm ( Figure 47 ).
• the prepared grafts were subretinally transplanted to immunodeficient retina-deficient rats (SD Foxn) using a glass Pasteur pipette.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Days 14 to 25 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting, and a target cell population was gated with FSC and SSC using Cell Sorter ARIAII (Becton, Dickinson and Company). Then, Rx::Venus-positive fractions were separated ( Figure 49 ).
• the separated cells were seeded at 1.6 to 8.0 ⁇ 10 5 cells per well onto 12-well Transwell (Corning, Inc.) coated with Laminin511-E8. As a control, unsorted sheets were also prepared. For the first 3 days, culture was performed in the presence of 10 ⁇ M Y-27632, 300 nM SAG and 3 ⁇ M CHIR99021. 3 days or later from seeding, the medium was replaced with a serum-free medium (NucT0 medium), which did not contain Y-27632, SAG or CHIR99021, once every 3 to 4 days. Day 26 after seeding, observation was made under a fluorescence stereo microscope ( Figure 50 ).
• a majority of RPE cells can be removed by gating a target cell population with FSC and SSC and then performing sorting with Rx::Venus as an index, and retina cells were observed to be differentiated without problems when prepared into a sheet again.
• StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Day 25 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting, and sorting was carried out with Rx::Venus as an index using Cell sorter ARIAII (Becton, Dickinson and Company).
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• the cells after sorting were seeded at 5 ⁇ 10 4 cells per well to a 96-well glass-bottom plate (Greiner Bio-One International GmbH) coated with Laminin511-E8 using Easy iMatrix, by adding Y-27632, CHIR99021, and SAG to 100 ⁇ L of NucT0. Simultaneously with seeding, the proteins shown in Table 1 or the low-molecular compounds shown in Table 2 were added.
• StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Day 24 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting, and sorting was carried out with Rx::Venus as an index using Cell sorter ARIAII (Becton, Dickinson and Company). The cells after sorting were seeded at 2.0 to 5.0 ⁇ 10 4 cells per well to a low adhesive V plate. As controls, a group that was not supplemented with FGF8 (FGF8-) and a group that was not sorted (unsorted) were also established.
• FGF8- FGF8-
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Day 24 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting, and sorting was carried out with Rx::Venus as an index using Cell sorter ARIAII (Becton, Dickinson and Company). The cells after sorting were suspended and seeded at 2.0 ⁇ 10 5 cells per well of 24-well Transwell (Corning, Inc.) using NucT0 medium containing Y-27632, CHIR99021 and SAG, FGF8. As a control, a group that was not supplemented with FGF8 (FGF8-) was also established.
• FGF8- FGF8-
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates of Day 27 from initiation of the suspension culture prepared as in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 200 ⁇ L of a serum-free medium containing 10 ⁇ M Y-27632, 300 nM SAG, 3 ⁇ M CHIR99021 and 100 ng/mL FGF8 so as to become 2.0 ⁇ 10 5 cells per well of 24-well Transwell coated with Laminin511-E8, and cultured at 37°C and 5% CO 2 for 3 days. 3 days or later from seeding, the medium was replaced with a serum-free medium (NucT0), which did not contain Y-27632, SAG, CHIR99021, or FGF8, once every 3 to 4 days.
• a serum-free medium NucT0
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• Suspension culture for differentiation was started as described in Reference example 1-1 using the cells. 300 nM SAG was added at the start of differentiation. The aggregates (NR) of Days 19 and 26 from initiation of the suspension culture were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting, and surface antigen screening was carried out using surface antigen screening kit MACS(R) Marker Screen, human (Miltenyi Biotec) (Alexa 647 fluorescent dye conjugate antibody group). As a control, human ES cells (hESC) were stained. The number of markers used in screening was 371 in total ( Figures 64 to 69 ).
• hESC high means that in human ES cells, an expression level is high or expression is present
• hESC: low means that in human ES cells, an expression level is low or expression is absent
• NR high means that in neural retina cells, an expression level is high
• NR: low means that in neural retina cells, an expression level is low or expression is absent.
• CD39, CD73, and CD184 (CXCR4) on dd18, dd25, dd53, and dd81 was analyzed by FACS using Mouse IgG1-APC-conjugate antibody (Miltenyi Biotec), anti-CD39-APC-conjugate antibody (Miltenyi Biotec), anti-CD73-APC-conjugate antibody (Miltenyi Biotec), and anti-CD184(CXCR4)-APC-conjugate antibody (Miltenyi Biotec) ( Figure 68 ).
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• Sections were prepared and stained with DAPI and anti-FoxG1 antibody (Takara Bio Inc.), and observation was performed under a fluorescence microscope (BZ-X810 manufactured by Keyence Corp.). As a result, in the group supplemented with BMP4, FoxG1 was negative. On the other hand, in the group that was not supplemented with BMP4, FoxG1 positive was confirmed, and it was confirmed that brain organoid was differentiated ( Figure 71 ).
• the brain organoid of Day 25 from initiation of the suspension culture prepared in this way was washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, it was dispersed into single cells by pipetting and stained with anti-CD39-APC conjugate antibody (Miltenyi Biotec), anti-CD73-APC conjugate antibody (Miltenyi Biotec), and anti-CXCR4-APC conjugate antibody (Miltenyi Biotec). In FACS measurement, the percentage of an expressing cell population was measured using FACSCantoII (BD Biosciences), and analysis was conducted using FlowJo.
• FACSCantoII BD Biosciences
• CD39 or CD73 was not expressed in the brain organoid, whereas CXCR4 was observed to be expressed in a majority of cells in the brain organoid ( Figure 72 ).
• a population of Rx::Venus-positive cells can be distinguished and separated from brain organoid by using a CD39-positive, CD73-positive, and CXCR4-negative cell population as an index.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates were dispersed into single cells using a neuronal cell dispersion solution, and FACS analysis was conducted as to the expression of CD39 and CXCR4 using anti-CD39-APC conjugate antibody (Miltenyi Biotec) and anti-CXCR4-APC conjugate antibody (Miltenyi Biotec). The percentage of an expressing cell population was measured using FACSCantoII (BD Biosciences), and analysis was conducted using FlowJo. These aggregates were fixed in 4% PFA, and sections were prepared and then subjected to staining using anti-ALDH1A1 antibody (R&D Systems, Inc.), anti-CoupTF1 antibody (Perseus Proteomics Inc.
• PPMX anti-LHX2 antibody
• Anti-Chx10 antibody manufactured by Exalpha Biologicals Inc.
• anti-Pax2 antibody manufactured by Covance
• anti-NKX2.1 antibody manufactured by Leica
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• NR For aggregates (NR) prepared by adding 300 nM SAG at the start of differentiation (dd0), 10 neural retinal tissues were transferred to low adhesive 60-mm Petri dish for suspension culture (manufactured by Sumitomo Bakelite Co., Ltd.) on Day 17 from initiation of the suspension culture. 1 mM or 0.2 mM adenosine (A2A receptor agonist, manufactured by Sigma-Aldrich Co.
• A2A receptor agonist manufactured by Sigma-Aldrich Co.
• CD39 can be enhanced by adding ATP and an A2A receptor agonist.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene, in which Islet-1 gene was knocked out, were cultured under feeder-free conditions in accordance with the methods described in " Scientific Reports 4, 3594 (2014 )” and WO2018/097253 .
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Day 25 from initiation of the suspension culture were dispersed into single cells and stained at 37°C for 15 minutes using anti-CD39-APC conjugate antibody (Miltenyi Biotec). Then, sorting was carried out with CD39-APC as an index using Cell sorter ARIAII (Becton, Dickinson and Company). The cells after sorting were seeded at 8.0 ⁇ 10 5 cells per well into 12-well Transwell coated with collagen gel.
• FGF8- 50 ng/mL FGF8
• the sheets prepared in this way were recovered using collagenase on Day 88 from initiation of the suspension culture. Then, long grafts for transplantations were dissected using microscissors ( Figure 82 ), and subretinal transplantation to immunodeficient retina-deficient rats (SD Foxn) was carried out.
• retina sheets manufactured from cells sorted with CD39 were transplantable.
• StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• NR Aggregates (NR) of Days 24 to 26 from initiation of the suspension culture prepared by adding 30 nM SAG at the start of the suspension culture (at the start of differentiation, dd0) were dispersed into single cells using a neuronal cell dispersion solution (FUJIFILM Wako Pure Chemical Corp.) and stained using surface antigen screening kit MACS(R) Marker Screen, human (Miltenyi Biotec), and surface antigen screening was carried out (Alexa 647 fluorescent dye conjugate antibody group). As a control, brain organoid prepared without adding BMP4 on Day 3 from initiation of the differentiation was stained.
• CD9, CD24, CD49c, CD90, CXCR4, and EpCAM are useful as markers that distinguish Rx::Venus-positive cells from negative cells ( Figure 84 ).
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• hESC and aggregates (NR) of dd4, dd11, dd18, dd25, dd32, and dd46 prepared by adding 30 nM SAG at the start of suspension culture (at the start of differentiation, dd0) were dispersed into single cells using a neuronal cell dispersion solution (FUJIFILM Wako Pure Chemical Corp.) and stained with APC-conjugated anti-CD9 antibody (BioLegend, Inc.). After the antibody was washed, FACS analysis was conducted. In FACS analysis, measurement was performed using MACS Quant10 (Miltenyi Biotec), and analysis was conducted using FlowJo.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• NR Aggregates (NR) of Day 25 prepared by adding 30 nM SAG at the start of suspension culture (at the start of differentiation, dd0) were dispersed into single cells using a neuronal cell dispersion solution (FUJIFILM Wako Pure Chemical Corp.) and stained with BV421-conjugated anti-SSEA1 antibody (BioLegend, Inc.) in addition to APC-conjugated anti-CD9 antibody (BioLegend, Inc.). Measurement was performed using FACSCantoII (BD Biosciences), and analysis was conducted using FlowJo.
• Rx-positive cells were about 87% in the whole aggregates.
• CD9-positive cells were gated, the Rx-positive cells were found to be increased to 94%.
• CD9-positive and SSEA1-negative cells were further gated, the Rx-positive cells were found to be increased to about 96% ( Figure 86 ).
• retinal cells can be more purified by combining SSEA1 in addition to CD9.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• NR neuronal cell dispersion solution
• staining was performed using (1) non-staining (purification with Rx::Venus as an index), (2) anti-CD9-APC-conjugate antibody (BioLegend, Inc.), (3) anti-CD90-APC-conjugate antibody (Becton, Dickinson and Company), (4) anti-CD184 (CXCR4)-APC-conjugate antibody (Miltenyi Biotec), (5) anti-CD9-APC-conjugate antibody (BioLegend, Inc.) and anti-SSEA1-BV421-conjugate antibody (BioLegend, Inc.), (6) anti-CD9-APC-conjugate antibody (BioLegend, Inc.) and anti-CD90-BV421-conjugate antibody (BioLegend, Inc.), and (7) anti-CD90-APC-conjugate antibody (Becton, Dickinson and Company) and anti-SSEA1-BV421-conjugate
• retina sheets sorted with CD9 and with CD9 and SSEA1-negative or CD90-positive fractions can remove RPE fractions more than Rx::Venus-positive sorting and are better.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• hESC and aggregates (NR) of dd4, dd11, dd18, dd25, dd32, and dd46 prepared by adding 30 nM SAG at the start of suspension culture (at the start of differentiation, dd0) were dispersed into single cells using neuronal cell dispersion solution (FUJIFILM Wako Pure Chemical Corp.), stained with anti-CD9-APC-conjugate antibody (BioLegend, Inc.) and anti-SSEA1-BV421-conjugate antibody (BioLegend, Inc.), and subjected to FACS analysis. Measurement was performed using FACSCantoII (BD Biosciences), and analysis was conducted using FlowJo.
• dd11 (d11 in Figure 92 ) is preferable as the timing of purification and it is preferable to perform purification on dd18 (d18 in Figure 92 ) to dd32 (d32 in Figure 92 ) or later ( Figure 92 ).
• the retina sheets on 12-well Transwell prepared in this way by performing sorting with CD9 and SSEA1 as indexes and adding 10 ⁇ M Y-27632, 3 ⁇ M CHIR99021, 300 nM SAG and 100 ng/mL FGF8 were recovered using a scalpel or scissors on Days 48 and 62 from initiation of the suspension culture. Then, long grafts for transplantations were dissected using microscissors, and subretinal transplantation to immunodeficient retina-deficient rats (SD Foxn) was carried out (not shown).
• retina sheets manufactured using cells sorted with CD9 and SSEA1 are transplantable.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Days 18 to 30 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting and seeded at 4.0 to 8.0 ⁇ 10 5 cells per well into 12-well Transwell. Y-27632, SAG, and CHIR99021 were added to NucT0 medium at the time of seeding. Then, medium replacement was carried out with NucT0 medium, which did not contain Y-27632, SAG, or CHIR99021, once every 3 to 4 days, and culture was performed for 1 month or more.
• a neuronal cell dispersion solution manufactured by FUJIFILM Wako Pure Chemical Corp.
• the aggregates of 80 days or later from initiation of the suspension culture in which RPE was also simultaneously differentiated, prepared in Reference example 1-1 were seeded into a culture medium in which B27 (Gibco), L glutamine, 10 ng/mL FGF2, and SB431542 were added to DMEM (Sigma-Aldrich Co. LLC) and F12 Ham (Sigma-Aldrich Co. LLC) media in a dish coated with Laminin511-E8, and cultured. After RPE adhered, then proliferated and spread, only RPE was scraped off using a 1-mL tip, seeded into another dish coated with Laminin511-E8, and subjected to purification. Then, after expansion culture, 1.0 ⁇ 10 6 cells of RPE were seeded into 12-well Transwell coated with Laminin511-E8. Medium replacement was carried out once every 3 to 4 days, and culture was performed for 1 month or more.
• Dissection was performed in the retinal tissue-RPE complex sheet prepared in this way using tweezers and scissors. First, the complex sheet was halved, and strips were then dissected along the dissected surface ( Figure 99 ). As a result, dissection was achieved in a state in which both the sheets adhered via gelatin. When the cross section of this dissected complex sheet was observed, RPE having a black dye was confirmed on one side and a Rx::Venus-positive sheet-shaped retinal tissue was confirmed on the opposite side, between which gelatin was observed to be packed ( Figure 100 ).
• a transplantable retinal tissue-RPE complex sheet can be prepared by using a sheet-shaped retinal tissue, a RPE sheet, and gelatin.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Days 18 to 30 from initiation of the suspension culture prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting and seeded at 4.0 to 8.0 ⁇ 10 5 cells per well into 12-well Transwell. Y-27632, SAG, and CHIR99021 were added to NucT0 medium at the time of seeding. Then, medium replacement was carried out with NucT0 medium, which did not contain Y-27632, SAG, or CHIR99021, once every 3 to 4 days, and culture was performed for 1 month or more ( Figure 102 ).
• the aggregates of 80 days or later from initiation of the suspension culture in which RPE was also simultaneously differentiated, prepared in Reference example 1-1 were seeded into a culture medium in which B27 (Gibco), L glutamine, 10 ng/mL FGF2, and SB431542 were added to DMEM (Sigma-Aldrich Co. LLC) and F12 Ham (Sigma-Aldrich Co. LLC) media in a dish coated with iMatrix511, and cultured. After RPE adhered, then proliferated and spread, only RPE was scraped off using a tip, seeded into another dish coated with iMatrix511, and subjected to purification. Then, after expansion culture, 1.0 ⁇ 10 6 cells of RPE were seeded into 12-well Transwell coated with iMatrix511. Medium replacement was carried out once every 3 to 4 days, and culture was performed for 1 month or more ( Figure 102 ).
• the RPE sheet was placed on a culture dish such that the apical side was located on the upper side and the mesh side was located on the culture dish side.
• the sheet-shaped retinal tissue was placed from above such that the apical surface faced the RPE side ( Figure 105 ).
• solidification was performed by incubation at room temperature for 5 minutes, and a sheet that adhered was prepared by recovery ( Figures 106 and 107 ).
• the retinal tissue-RPE complex sheet prepared in this way was reversed using tweezers, placed such that the sheet-shaped retinal tissue faced the lower side and the retinal pigment epithelial sheet faced the upper side, and observed ( Figure 108 ).
• the mesh of Transwell that adhered was detached ( Figure 109 ).
• a retinal tissue-RPE complex sheet can be prepared by using a sheet-shaped retinal tissue, a RPE sheet, and fibrin.
• Two, four, or five levee-shaped passages were prepared on a glass slide using a silicon sheet, and two pieces of CellShifter (CellSeed Inc.) between which gelatin was added were used and pushed from above to observe whether gelatin ran out thereof.
• CellShifter CellSeed Inc.
• Human ES cells (KhES-1 strain (Non Patent Literature 3)) genetically engineered so as to have Rx::Venus reporter gene were cultured under feeder-free conditions in accordance with the method described in " Scientific Reports 4, 3594 (2014 )".
• a feeder-free medium StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used, and as a scaffold in place of feeder cells, Laminin511-E8 (trade name, manufactured by Nippi, Inc.) was used.
• the aggregates (NR) of Days 18 to 30 prepared in Reference example 1-1 were washed with PBS, and a neuronal cell dispersion solution (manufactured by FUJIFILM Wako Pure Chemical Corp.) was added. After incubation at 37°C, they were dispersed into single cells by pipetting, and the cells were seeded at 8.0 ⁇ 10 5 cells/well into a 24-well temperature-responsive culture dish (CellSeed Inc.) with 7 different degrees of detachment coated with Laminin511-E8. Medium replacement was carried out once every 3 to 4 days. 30 days or later after seeding, a sheet was formed with a thickness, then incubated with the temperature lowered to room temperature for 2 hours, and observed. As a result, in a plurality of wells of 2b, 2c, 3, and 4 in the temperature-responsive culture dish with 7 degrees of detachment, the retina sheet was observed to be detachable by creating room temperature ( Figure 111 ).
• a retina sheet can be recovered by performing culture in a temperature-responsive culture dish.
• SD Foxn immunodeficient retina-deficient rats
• a differentiation method by patterning culture has been known as a method for producing a cell aggregate containing retinal progenitor cells (adherent cell aggregate) ( WO2023/003025 ). Focusing on patterning culture, conditions for preparing retinal tissues from undifferentiated human iPS cells were compared.
• Human iPS cells (LPF11 and DSP-SQ lines established at Sumitomo Pharma Co., Ltd.) were established using a commercially available Sendai virus vector (four factors of Oct3/4, Sox2, KLF4 and c-Myc, IDPharma Cytotune Kit), based on the methods described in Thermo Fisher Scientific's published protocol (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and Kyoto University's published protocol (Establishment and maintenance culture of feeder-free human iPS cells, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyotou.ac.jp/j/research/protocol.html), using StemFit medium (AK03, Ajinomoto Co. Inc.) and Laminin511-E8 (Nippi. Inc.).
• Sendai virus vector four factors of Oct3/4, Sox2, KLF4 and c-Myc
• the human iPS cells (LPF11 and DSP-SQ lines) were cultured under feeder-free condition according to the methods described in Scientific Reports, 4, 3594 (2014 ).
• StemFit medium (AK03N, Ajinomoto Co. Inc.) was used as feeder-free medium and Laminin 511-E8 (Nippi. Inc.) as feeder-free scaffold.
• subconfluent human iPS cells (LPF11 and DSP-SQ lines) were first washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The human iPS cells dispersed into single cells were then seeded onto plastic culture dishes coated with Laminin511-E8 and cultured under feeder-free condition in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). When a 6-well plate (Iwaki, for cell culture, culture area 9.4 cm 2 ) was used as the plastic culture dish, the number of seeded human iPS cells dispersed into single cells was 1.0 ⁇ 10 4 . One day after seeding, the medium was replaced with StemFit medium without Y-27632. Thereafter, the medium was changed with StemFit medium without Y-27632 every 1 to 2 days. The cells were then cultured until 6 days after seeding.
• Y-27632 ROCK inhibitor, 10 ⁇ M
• a droplet (1 cm in diameter) of a solution containing 0.785 ⁇ l of Laminin511-E8 (Nippi. Inc.) in 100 ⁇ l of PBS (Laminin511-E8 concentration of 3.925 ng/ ⁇ l) was made in the center of the wells of a 12-well plate (Iwaki, for cell culture, culture area 3.8 cm 2 ) and then incubated at 37°C or room temperature for 1 to 3 hours to prepare 12-well-plates for patterning culture (Laminin511-E8: 0.5 ⁇ g/cm 2 ).
• Subconfluent human iPS cells (LPF11 line) were then washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The human iPS cells dispersed into single cells were then seeded into one well of the patterning culture plates at various cell densities from 0.5 ⁇ 10 5 to 5.0 ⁇ 10 5 cells/cm 2 and cultured in Stemfit medium under feeder-free condition in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M) ( Figure 112 ).
• ROCK inhibitor 10 ⁇ M
• the culture period with Y-27632 was varied from 2 to 32 hours, and then the medium was replaced with 2 ml of StemFit medium without Y-27632 (ROCK inhibitor, 0 ⁇ M) ( Figure 114 ).
• LPF11 line Human iPS cells (LPF11 line) were seeded onto the patterning culture plates at various cell densities (cells/cm 2 ) to examine optimal conditions.
• a 12-well plate for patterning culture prepared by the method described in Reference example 2-1 was provided, and subconfluent human iPS cells (LPF11 line) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies).
• the human iPS cells dispersed into single cells were then seeded into one well of the patterning culture plate at a density of 0.5 ⁇ 10 5 , 1.0 ⁇ 10 5 , 2.5 ⁇ 10 5 , or 5.0 ⁇ 10 5 cells/cm 2 and cultured under feeder-free condition in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). Two hours later, the medium was replaced with 2 ml of StemFit medium without Y-27632.
• ROCK inhibitor Y-27632
• FIG. 112 The results of bright-field microscopy observation of cells at 1 hour, 1, 2, or 3 days after seeding human iPS cells into patterning culture plates are shown in Figure 112 .
• Analysis of the images in Figure 112 showed that human iPS cells adhered heterogeneously to the cell-adhesive regions at a seeding density of 5.0 ⁇ 10 5 cells/cm 2 , while the cells adhered uniformly to the cell-adhesive regions at seeding densities of 0.5 ⁇ 10 5 to 2.5 ⁇ 10 5 cells/cm 2 .
• the cell density of undifferentiated human iPS cells to be seeded was preferably 1.0 ⁇ 10 5 to 2.5 ⁇ 10 5 cells/cm 2 .
• a 12-well plate for patterning culture prepared by the method described in Reference example 2-1 was provided, and subconfluent human iPS cells (LPF11 and DSP-SQ lines) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies).
• the human iPS cells dispersed into single cells were then seeded into one well of the patterning culture plate at a density of 2.0 ⁇ 10 5 cells/cm 2 and cultured under feeder-free condition in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). Then, after 2, 4, 8, 16, or 32 hours culture, the medium was replaced with 2 ml of StemFit medium without Y-27632.
• the patterning culture was then performed as described in Reference example 2-1, and on Day 20 of seeding, cells were fixed with 4% paraformaldehyde and bright-field images were obtained with a fluorescence microscope (Keyence).
• the results using human iPS cells (LPF11 line) are shown in Figure 114 .
• the results showed that the longer the duration of action of Y-27632 was, the more holes were created on the cell sheet surface, resulting in less efficient formation of uniformly differentiated retinal tissue.
• the percentage of Chx10-positive areas on the sheet surface was calculated using ImageJ software (NIH) and was 91% for sheets with Y-27632 for 32 hours, compared to 97% or more for sheets with Y-27632 for 16 hours or less (16 hours: 97%, 8 hours: 99%, 4 hours: 98%, 2 hours: 99%). Further, counting the number of Chx10-negative holes at each duration of action of Y-27632 revealed that the duration of action of Y-27632 for 16 hours or less could suppress formation of a large number of holes on the sheet surface. Similarly with human iPS cells (DSP-SQ line), shortening the duration of action of Y-27632 resulted in fewer Chx10-negative areas on the sheet surface ( Figure 115 ).
• a 12-well plate for patterning culture prepared by the method described in Reference example 2-1 was provided, and subconfluent human iPS cells (LPF11 line) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The human iPS cells dispersed into single cells were then seeded into one well of the patterning culture plate at a density of 2.0 ⁇ 10 5 cells/cm 2 and cultured under feeder-free condition in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). Two hours later, the medium was replaced with 2 ml of StemFit medium without Y-27632 and patterning culture was performed as described in Reference example 2-1.
• Y-27632 Y-27632
• the medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous human recombinant BMP4 (R&D) at a final concentration of 0, 1.5, 3, 6, or 12 nM. Thereafter, half of the medium was replaced with serum-free medium (gfCDM+KSR) without human recombinant BMP4 every 2 to 3 days. For changing half of the medium, the half amount of the medium in the culture vessel, i.e., 1 ml, was discarded, and 1 ml of new serum-free medium (gfCDM+KSR) was added, bringing the total volume of medium to 2 ml.
• serum-free medium gfCDM+KSR
• the results of the analysis of relative mRNA expression levels for each gene are shown in Figure 117 .
• the ⁇ Ct value was calculated from the difference between the Ct value of the target gene and the Ct value of GAPDH, the gene used as the internal standard, and the ratio of expression level of each gene was calculated from the difference in ⁇ Ct between samples ( ⁇ Ct).
• the results showed that, similar with the results of immunostaining for Rx expression, retinal tissues in which retinal differentiation was induced at BMP4 concentrations of 3 to 12 nM had higher expression of the retinal progenitor cell marker genes Chx10 and Rx, compared to those at BMP4 concentration of 1.5 nM.
• the study also found that retinal tissues in which retinal differentiation was induced at BMP4 concentrations of 3 to 12 nM had low and suppressed expression of Emx2, a marker gene for unintended cells.
• the final concentration of BMP4 for inducing retinal differentiation was preferably from 3 to 15 nM for preparing retinal tissues by patterning culture.
• a 12-well plate for patterning culture prepared by the method described in Reference example 2-1 was provided, and subconfluent human iPS cells (LPF11 line) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies).
• the human iPS cells dispersed into single cells were then seeded into one well of the patterning culture plate at a density of 5.0 ⁇ 10 5 cells/cm 2 in the comparative method and 2.0 ⁇ 10 5 cells/cm 2 in the optimized method, and cultured under feeder-free condition in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M).
• Y-27632 Y-27632
• the medium was replaced with 2 ml of StemFit medium without Y-27632 and patterning culture was continued as described in Reference example 2-1.
• the medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous human recombinant BMP4 (R&D) at a final concentration of 1.5 nM for the method of WO2023/003025 and 6 nM for the optimized method. Thereafter, half of the medium was replaced with serum-free medium (gfCDM+KSR) without human recombinant BMP4 every 2 to 3 days. For changing half of the medium, the half amount of the medium in the culture vessel, i.e., 1 ml, was discarded, and 1 ml of new serum-free medium (gfCDM+KSR) was added, bringing the total volume of medium to 2 ml.
• serum-free medium gfCDM+KSR
• Example 1 Production of a reorganized organoid using a retinal progenitor cell sheet>
• cell aggregates containing retinal progenitor cells produced by the SFEBq method can be used as a starting material for reorganization and re-sheeting.
• the differentiation method by patterning culture can be used to produce a cell aggregate containing retinal progenitor cells (adherent cell aggregate).
• retinal tissue a cell aggregate, containing retinal progenitor cells produced by patterning culture (also referred to herein as retina sheet or retinal progenitor cell sheet) has some similarities in cell composition with a cell aggregate containing retinal progenitor cells produced by the SFEBq method, but the state of cells under culture differs. Therefore, it was examined if the retinal progenitor cell sheet produced by patterning could be a starting material for reorganization and re-sheeting.
• Human iPS cells (LPF11 line established at Sumitomo Pharma Co., Ltd.) were established using a commercially available Sendai virus vector (four factors of Oct3/4, Sox2, KLF4 and c-Myc, IDPharma Cytotune kit) based on the methods described in Thermo Fisher Scientific's published protocol (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and Kyoto University's published protocol (Establishment and maintenance culture of feeder-free human iPS cells, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyoto-u.ac.jp/j/research/ protocol.html) in StemFit medium (AK03, Ajinomoto) and Laminin511-E8 (Nippi).
• Sendai virus vector four factors of Oct3/4, Sox2, KLF4 and c-Myc, IDPharma Cytotune kit
• the human iPS cells were cultured under feeder-free condition according to the methods described in Scientific Reports, 4, 3594 (2014 ).
• StemFit medium (AK03N, Ajinomoto Co. Inc.) was used as feeder-free medium and Laminin 511-E8 (Nippi. Inc.) as feeder-free scaffold.
• subconfluent human iPS cells (LPF11 line) were first washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The human iPS cells dispersed into single cells were then seeded onto plastic culture dishes coated with Laminin511-E8 and cultured under feeder-free condition in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). When a 6-well plate (Iwaki, for cell culture, culture area 9.4 cm 2 ) was used as the plastic culture dish, the number of seeded human iPS cells dispersed into single cells was 1.0 ⁇ 10 4 . One day after seeding, the medium was replaced with StemFit medium without Y-27632. Thereafter, the medium was changed with StemFit medium without Y-27632 (Rock inhibitor, 0 ⁇ M) every 1 to 2 days. The cells were then cultured until 6 days after seeding.
• Y-27632 ROCK inhibitor, 10 ⁇ M
• a droplet (1 cm in diameter) of a solution containing 0.785 ⁇ l of Laminin511-E8 (Nippi. Inc.) in 100 ⁇ l of PBS (Laminin511-E8 concentration of 3.925 ng/ ⁇ l) was made in the center of the wells of a 12-well plate (Iwaki, for cell culture, culture area 3.8 cm 2 ) and then incubated at 37°C or room temperature for 1 to 3 hours to prepare 12-well-plates for patterning culture (Laminin511-E8: 0.5 ⁇ g/cm 2 ).
• Subconfluent human iPS cells (LPF11 line) were then washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies).
• the human iPS cells dispersed into single cells were then seeded into one well of the patterning culture plates at a cell density of 1.0 ⁇ 10 5 cells/cm 2 and cultured in Stemfit medium under feeder-free condition in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M).
• the cells were cultured with Y-27632 for 2 hours, and then the medium was replaced with 2 ml of StemFit medium without Y-27632 (ROCK inhibitor, 0 ⁇ M).
• gfCDM+KSR serum-free medium
• R&D human recombinant BMP4
• half of the medium was replaced with gfCDM+KSR without human recombinant BMP4 every 2 to 3 days.
• the half amount of the medium in the culture vessel i.e., 1 ml
• 1 ml of new gfCDM+KSR was added, bringing the total volume of medium to 2 ml.
• DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine
• DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine
• the retinal progenitor cell sheet on Day 20 of patterning culture was dispersed into single cells with Neuron Dissociation Solution (WAKO) and cultured by 3D culture in non-cell-adherent 96-well plates (trade name: PrimeSurface 96-well V-bottom plate, Sumitomo Bakelite Co., Ltd.) at 1.2 ⁇ 10 4 cells per well, suspended in the control medium for maturation.
• WAKO Neuron Dissociation Solution
• Y27632 final concentration of 10 ⁇ M
• SAG final concentration of 300 nM
• CHIR99021 final concentration of 3 ⁇ M
• the reorganized organoid was fixed in 4% paraformaldehyde and immunostained for the retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha, Inc.), Pax6 (anti-Pax6 antibody, BD Biosciences, Inc.), and Rx (anti-Rx antibody, Takara Bio Inc.), the photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara Bio Inc.), and the apical polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen Corp.). These immunostained cells were observed with a confocal laser microscope (LSM880, ZEISS) ( Figure 121 ).
• LSM880, ZEISS confocal laser microscope
• the retinal progenitor cell sheet on Day 20 of patterning culture prepared by the method described in Example 1 was dispersed into single cells with Neuron Dissociation Solution (WAKO) and seeded on 24-well Transwell (Corning) previously coated with Laminin511-E8 (Nippi, Inc.) at 0.5 ⁇ 10 5 cells per well in the control medium for maturation, and cultured by 2D culture.
• WAKO Neuron Dissociation Solution
• Laminin511-E8 Nappi, Inc.
• Y27632 final concentration of 10 ⁇ M
• SAG final concentration of 300 nM
• CHIR99021 final concentration of 3 ⁇ M
• the reorganized sheet was fixed in 4% paraformaldehyde and immunostained for the retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha, Inc.), Pax6 (anti-Pax6 antibody, BD Biosciences, Inc.), and Rx (anti-Rx antibody, Takara Bio Inc.), the photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara Bio Inc.), and the apical polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen Corp.).
• Chx10 anti-Chx10 antibody, Exalpha, Inc.
• Pax6 anti-Pax6 antibody, BD Biosciences, Inc.
• Rx anti-Rx antibody, Takara Bio Inc.
• the photoreceptor progenitor cell marker Crx anti-Crx antibody, Takara Bio Inc.
• the apical polarity marker ZO-1 anti-ZO-1 antibody, Invitrogen Corp.
• the reorganized sheet like the reorganized organoid, was formed of Chx10, Pax6, and Rx-positive retinal progenitor cells, with interspersed Crx-positive photoreceptor progenitor cells. Further, the upper side of the sheet (side not in contact with the well membrane) was ZO-1 positive, indicating that this reorganized sheet was a polarized retinal tissue with an apical surface formed on the upper side.
• Example 3 Production of a reorganized organoid using retinal progenitor cells sorted from a retinal progenitor cell sheet>
• the retinal progenitor cell sheet on Day 20 of patterning culture prepared by the method described in Example 1 was dispersed into single cells with Neuron Dissociation Solution (WAKO), stained for surface antigen with APC-Conjugate CD9 antibody (BioLegend) at 4°C for 1 hour, and sorted by MACS Quant Tyto Cell Sorter (Miltenyi). Flow cytometric analysis of cells before and after sorting was performed with MACS Quant Analyzer 10 Flow Cytometer (Miltenyi) to confirm purification of a CD9-positive cell fraction ( Figure 126 ).
• Purified cells were cultured by 3D culture in non-cell-adherent 96-well plates (trade name: PrimeSurface 96-well V-bottom plate, Sumitomo Bakelite Co., Ltd.) at 1.2 ⁇ 10 4 cells per well, suspended in the control medium for maturation.
• Y27632 final concentration of 10 ⁇ M
• SAG final concentration of 300 nM
• CHIR99021 final concentration of 3 ⁇ M
• the reorganized organoid was fixed in 4% paraformaldehyde and immunostained for the retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha, Inc.), Pax6 (anti-Pax6 antibody, BD Biosciences, Inc.), and Rx (anti-Rx antibody, Takara Bio Inc.), the photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara Bio Inc.), and the apical polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen Corp.).
• Chx10 anti-Chx10 antibody, Exalpha, Inc.
• Pax6 anti-Pax6 antibody, BD Biosciences, Inc.
• Rx anti-Rx antibody, Takara Bio Inc.
• the photoreceptor progenitor cell marker Crx anti-Crx antibody, Takara Bio Inc.
• the apical polarity marker ZO-1 anti-ZO-1 antibody, Invitrogen Corp.
• Rx gene a neural retinal marker, and Emx2, a dorsal telencephalon marker, were measured by real-time PCR to confirm that unintended cells were removed in the reorganized organoid prepared from CD9-positive cells sorted from a retinal progenitor cell sheet.
• concentration of total RNA was measured by a measuring device (Nanodrop, Thermo scientific,) and then the RNA was reverse transcribed into cDNA using a reverse transcriptase and primers (Reverse Transcription Master Mix Kit, Fluidigm).
• a reverse transcriptase and primers Reverse Transcription Master Mix Kit, Fluidigm.
• multiplex-PCR reaction Pre-Run was performed using a PCR system (Veriti 96well thermal cycler, Applied Biosystems).
• Pre-Run reaction solutions were then injected into a flow-through multi-well (96. 96 Dynamic Array IFC, Fluidigm) using an IFC Controller HX (Fluidigm), and the expression levels of Rx and Emx2 genes were determined by real-time PCR using a multi-sample real-time PCR system (Biomark HD, Fluidigm).
• Example 4 Production of a reorganized sheet using retinal progenitor cells sorted from a retinal progenitor cell sheet
• the retinal progenitor cell sheet on Day 20 of patterning culture produced in Example 1 was dispersed into single cells with Neuron Dissociation Solution (WAKO), stained for surface antigen with APC-Conjugate CD9 antibody (BioLegend) at 4°C for 1 hour, and sorted by MACS Quant Tyto Cell Sorter (Miltenyi). Collected cells were seeded on 24-well Transwell (Corning) previously coated with Laminin511-E8 (Nippi, Inc.) at 0.5 ⁇ 10 5 cells per well. At cell seeding, Y27632 (final concentration of 10 ⁇ M), SAG (final concentration of 300 nM) and CHIR99021 (final concentration of 3 ⁇ M) were added simultaneously to promote reorganization.
• WAKO Neuron Dissociation Solution
• APC-Conjugate CD9 antibody BioLegend
• MACS Quant Tyto Cell Sorter MACS Quant Tyto Cell Sorter
• the reorganized sheet was fixed in 4% paraformaldehyde and immunostained for the retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha, Inc.), Pax6 (anti-Pax6 antibody, BD Biosciences, Inc.), and Rx (anti-Rx antibody, Takara Bio Inc.), the photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara Bio Inc.), and the apical polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen Corp.). These immunostained cells were observed with a confocal laser microscope (LSM880, ZEISS) ( Figure 130 ).
• LSM880, ZEISS confocal laser microscope
• the reorganized sheet prepared from CD9-positive cells sorted from a retinal progenitor cell sheet was also formed with Chx10, Pax6 and Rx-positive retinal progenitor cells with interspersed Crx-positive photoreceptor progenitor cells. Further, the upper side of the sheet (side not in contact with the well membrane) was ZO-1 positive, indicating that this reorganized organoid was a polarized retinal tissue with an apical surface formed on the outside.
• Optimal concentrations were investigated for a ROCK inhibitor, Wnt signaling pathway agonist, and Shh signaling pathway agonist, which are important for dispersion and reorganization of a retinal progenitor cell sheet produced by patterning culture.
• a retinal progenitor cell sheet on Day 20 of patterning culture prepared by the method described in Example 1 were dispersed into single cells using Neuron Dissociation Solution (WAKO) and cultured in non-cell-adherent 96-well plates (trade name: PrimeSurface 96-well V-bottom plate, Sumitomo Bakelite Co., Ltd.) at 1.2 ⁇ 10 4 cells per well, suspended in the control medium for maturation.
• WAKO Neuron Dissociation Solution
• Y27632 final concentration of 0 or 10 ⁇ M
• SAG final concentration of 0, 150, 300, or 600 nM
• CHIR99021 final concentration of 0, 1.5, 3, or 6 ⁇ M
• retinal progenitor cell sheets obtained by patterning culture can be used as a starting material for reorganization and re-sheeting, although they are attached to the dish and may have different properties from cell aggregates on suspension. These results also show optimal concentrations of reorganizing factors to be added in this process.
• Retinal progenitor cell sheets produced by patterning culture can be a useful starting material for reorganization and re-sheeting because they can be mass-produced in various forms and sizes, and the present application demonstrates the feasibility of this process.

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