WO2022239868A1 - 網膜組織の製造方法 - Google Patents
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Definitions
- the present invention relates to a method for producing retinal cells or retinal tissue.
- the present invention also relates to a sphere-like cell aggregate comprising a multi-layer structure of an outer structure containing retinal pigment epithelial cells and an inner structure containing neural retina produced by the production method.
- Patent Document 1 a method for producing multilayered retinal tissue from pluripotent stem cells has been reported (Patent Document 1 and Non-Patent Document 1).
- uniform pluripotent stem cell aggregates are formed in a serum-free medium containing a Wnt signaling pathway inhibitor, suspended in the presence of a basement membrane preparation, and then placed in a serum medium.
- a method of obtaining multilayered retinal tissue by suspension culture (Patent Document 2 and Non-Patent Document 2), forming uniform pluripotent stem cell aggregates in a medium containing a BMP4 signaling pathway activator,
- a method for obtaining a multilayered retinal tissue (Patent Document 3 and Non-Patent Document 3), a method for obtaining a multilayered retinal tissue by naturally differentiating adhered pluripotent stem cells and separating the retinal tissue contained in a part thereof. (Non-Patent Document 4) is known.
- the quality of retinal tissue has a certain amount of variation, and a certain percentage of non-target cells may be induced. Strict quality control is particularly required when these are used as tissues for transplantation.
- PD407824 (CHK1: Checkpoint kinase 1 inhibitor) is known as a BMP sensitizer.
- CHK1 Checkpoint kinase 1 inhibitor
- BMP BMP sensitizer
- the present inventors found that the amount of BMP to be added can be reduced to about 1/10 of the normal concentration by using a CHK1 signaling pathway inhibitor in combination with a method for producing retinal tissue using BMP. . Furthermore, by combining BMP and a CHK1 signal transduction pathway inhibitor, compared with the case of using BMP alone, it was found that the ratio of non-target cells can be reduced and retinal tissue with a certain shape can be produced. Furthermore, the present inventors have found that, by using a normal concentration of BMP and a CHK1 signaling pathway inhibitor in combination, the retinal pigment epithelium (RPE) is localized on the outside and the retinal tissue (neural retinal tissue) is localized on the inside. We have found that it is possible to selectively produce spherical cell aggregates having a layered structure.
- RPE retinal pigment epithelium
- the present invention relates to each of the following inventions.
- A suspension culture of pluripotent stem cells to form cell aggregates of pluripotent stem cells;
- B the cell aggregate obtained in step (A) is subjected to suspension culture in the presence of a BMP signaling pathway agonist and a CHK1 signaling pathway inhibitor to obtain a cell aggregate containing retinal cells;
- a method for producing retinal cells or retinal tissue comprising: [2] In step (B), the BMP signaling pathway agent is present at a concentration such that cell aggregates close to true spheres are formed and induction of differentiation into retinal pigment epithelial cells is suppressed [1] Method of manufacture as described.
- the cell aggregate obtained in step (B) is a spherical cell aggregate having a multi-layer structure of an outer structure containing retinal pigment epithelial cells and an inner structure containing neural retina, and in step (B), The production method according to [1], wherein the BMP signaling pathway active substance is present at a concentration such that the sphere-like cell aggregate with the multi-layered structure is formed.
- the BMP signaling pathway agent is one or more proteins selected from the group consisting of BMP2, BMP4, BMP7 and GDF7.
- step (B) Any of [1] to [5], wherein in step (B), the BMP signaling pathway agent is added to the medium between days 2 and 9 after the start of suspension culture in step (A). Method of manufacture as described.
- step (B) The production method according to any one of [1] to [6], wherein in step (B), the CHK1 signaling pathway inhibitor is added to the medium at the same time as the BMP signaling pathway active substance.
- step (B) the concentration of the CHK1 signaling pathway inhibitor is a concentration at which 0.1 ⁇ M to 10 ⁇ M of PD407824 exerts a CHK1 signaling pathway inhibitory effect equivalent to that of PD407824.
- the neural retina comprising one or more cells that (2) in the inner structure, the neural retina is folded and present; (3) the retinal pigment epithelial cells in the outer structure are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells, and (4) the cell aggregates are lens, vitreous, cornea and blood vessels Spherical cell aggregates, characterized in that they do not contain [11]
- the retinal pigment epithelial cells and the neural retina are connected as an epithelial structure, and the spherical cell aggregate further forms a ciliary body rim-like structure between the retinal pigment epithelial cells and the neural retina.
- a pharmaceutical composition comprising the spherical cell aggregate or a part thereof according to any one of [10] to [15].
- disorders of retinal cells or retinal tissue or damage to retinal tissue including transplanting the spherical cell aggregates or a portion thereof according to any one of [10] to [15] to a subject in need of transplantation Methods of treatment of diseases based on [19]
- the present invention by combining BMP and PD407824, it is possible to provide a method for producing retinal cells or retinal tissue in which the ratio of non-target cells is reduced compared to the case of using BMP alone. Also, it becomes possible to manufacture a retinal tissue with a good shape.
- FIG. 1 is a fluorescence microscope photograph showing the state of aggregates 17 days after the start of suspension culture by adding 1 ⁇ M of PD407824 (“PD” in the figure) together with BMP4 (0.15 nM or 1.5 nM) in Example 1.
- FIG. 1 is a fluorescence microscope photograph showing the state of aggregates 15 days after the start of suspension culture with the addition of 1.5 nM BMP4 alone or 0.15 nM BMP4 and 1 ⁇ M PD407824 (“PD” in the figure) in Example 2. .
- Example 1 is a fluorescence microscope photograph showing the state of aggregates 15 days after the start of suspension culture with the addition of 1.5 nM BMP4 alone or 0.15 nM BMP4 and 1 ⁇ M PD407824 (“PD” in the figure) in Example 2.
- 1.5 nM BMP4 alone, or 0.15 nM BMP4 and 1 ⁇ M PD407824 ("PD" in the figure) were added, and the state of aggregates was observed 36 days after the start of suspension culture.
- It is a photomicrograph. 1 is bright-field microscope and fluorescence microscope photographs of the state of aggregates 21 days after the start of suspension culture with the addition of 1 ⁇ M PD407824 (“PD” in the figure) together with 1.5 nM BMP4 in Example 3.
- Example 3 1 ⁇ M PD407824 (“PD” in the figure) was added together with 1.5 nM BMP4, and sections of aggregates were immunostained 25 days after the start of suspension culture, and observed with a confocal fluorescence microscope.
- 1 is a photograph of a section of aggregates immunostained 25 days after the start of suspension culture by adding 1 ⁇ M PD407824 together with 1.5 nM BMP4 in Example 3, and observed with a confocal fluorescence microscope.
- FIG. 10 is a fluorescence microscope photograph showing the state of aggregates on the 9th day after the start of suspension culture by adding various concentrations of PD407824 together with various concentrations of BMP4 in Example 4.
- FIG. 1 is a photograph of immunostaining sections of aggregates 60 days after the start of suspension culture with the addition of 1 ⁇ M PD407824 together with 1.5 nM BMP4 in Example 5, and observed with a confocal fluorescence microscope.
- a “stem cell” means an undifferentiated cell having differentiation potential and proliferation potential (especially self-renewal potential).
- Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential.
- Pluripotent stem cells are capable of being cultured in vitro and capable of differentiating into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, endoderm) and/or extra-embryonic tissues (pluripotent stem cells). It refers to stem cells that have pluripotency.
- Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple, but not all, types of tissues and cells.
- a unipotent stem cell means a stem cell that has the ability to differentiate into a specific tissue or cell.
- pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germ stem cells, tissue stem cells, somatic cells, etc.
- pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and the like. can be done.
- Pluripotent stem cells also include Muse cells (multi-lineage differentiating stress-ending cells) obtained from mesenchymal stem cells (MSCs) and GS cells produced from germ cells (eg, testis).
- MSCs mesenchymal stem cells
- GS cells produced from germ cells (eg, testis).
- Human embryonic stem cells are those established from human embryos within 14 days of fertilization.
- Embryonic stem cells Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell aggregates on feeder cells or in a medium containing bFGF. Methods for producing embryonic stem cells are described, for example, in WO96/22362, WO02/101057, US5,843,780, US6,200,806, US6,280,718 and the like. Embryonic stem cells can be obtained from designated institutions or can be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2 and KhES-3 are available from Institute for Frontier Medical Sciences, Kyoto University.
- “Induced pluripotent stem cells” are cells in which pluripotency is induced by reprogramming somatic cells by known methods.
- Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. in 2006 (Cell, 2006, 126 (4), pp. 663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007, and have pluripotency and self-renewal ability like 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).
- induced pluripotent stem cells are fibroblasts, peripheral blood mononuclear cells and other differentiated somatic cells such as Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Examples include cells that have been reprogrammed to induce pluripotency by expressing any combination of a plurality of genes selected from the reprogramming gene group including Glis1, Nanog, Sall4, lin28, Esrrb, and the like.
- Preferred combinations of reprogramming factors include (1) Oct3/4, Sox2, Klf4 and Myc (c-Myc or L-Myc), (2) Oct3/4, Sox2, Klf4, Lin28 and L-Myc (Stem Cells, 2013;31:458-466).
- induced pluripotent stem cells can be induced from somatic cells by addition of compounds, etc., in addition to the method of producing by direct reprogramming by gene expression (Science, 2013, 341, pp. 651- 654).
- induced pluripotent stem cell lines for example, 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, and 1210B2 cells established at Kyoto University.
- 1231A3 cells are available from Kyoto University and iPS Academia Japan.
- established induced pluripotent stem cell lines for example, Ff-I01 cells, Ff-I14 cells and QHJI01s04 cells established at Kyoto University are available from Kyoto University.
- pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, more preferably induced pluripotent stem cells.
- 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 by methods well known to those skilled in the art.
- Retinal tissue means a tissue in which one or more types of retinal cells that make up each retinal layer in the living retina exist according to a certain order
- Neurona (NR) ) means a retinal tissue that includes the inner neural retinal layer, which does not include the retinal pigment epithelial layer, among the retinal layers described below.
- Retinal cells means cells that constitute each retinal layer in the living retina or their progenitor cells.
- Retinal cells include photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone photoreceptors) Bipolar cells), Muller glial cells, retinal pigment epithelial (RPE) cells, ciliary bodies, their progenitor cells (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, etc.), retinal progenitor cells, etc.
- neural retinal cells cells constituting the neural retinal layer (also referred to as neural retinal cells or neural-related cells), specifically, photoreceptor cells (rod photoreceptors, cone vision cells) cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, and their progenitor cells (e.g., optic cell progenitor cells, bipolar cell progenitor cells, etc.). That is, neuroretinal cells do not include retinal pigment epithelial cells and ciliary body cells.
- mature retinal lineage cells refers to cells that can be contained in the retinal tissue of an adult human, and specifically includes photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, interneurons Differentiated cells such as nerve cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelial (RPE) cells, ciliary cells, etc. do.
- Immature retinal cells means progenitor cells committed to differentiate into mature retinal cells (eg, photoreceptor progenitor cells, bipolar cell progenitor cells, retinal progenitor cells, etc.).
- Photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, retinal ganglion cell progenitor cells, Müller glial progenitor cells, and retinal pigment epithelial progenitor cells are, respectively, photoreceptor cells, horizontal cells, and bipolar cells. , amacrine cells, retinal ganglion cells, Müller glial cells, and retinal pigment epithelial cell-committed progenitor cells.
- Retinal progenitor cell means any immature photoreceptor cell, horizontal cell progenitor, bipolar cell progenitor, amacrine cell progenitor, retinal ganglion cell progenitor, Müller glial cell, retinal pigment epithelial progenitor, etc.
- Progenitor cells capable of differentiating into various retinal lineage cells, and finally, photoreceptors, rod photoreceptors, cone photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, retinal pigment epithelial cells It refers to progenitor cells that can differentiate into any mature retinal lineage cell such as.
- Photoreceptor cells exist in the photoreceptor layer of the retina in vivo and have the role of absorbing light stimuli and converting them into electrical signals.
- cone photoreceptors cone photoreceptors that function in bright light and rods that function in darkness
- rod photoreceptors respectively.
- S cone photoreceptors that express S-opsin and receive blue light
- L cone photoreceptors that express L-opsin and receive red light
- M-opsin are expressed. Mention may be made of M cone photoreceptors that receive green light. Photoreceptors differentiate from photoreceptor precursor cells and mature.
- a person skilled in the art can determine whether a cell is a photoreceptor cell or a photoreceptor progenitor cell, for example, using the cell markers described later (Crx and Blimp1 expressed in photoreceptor progenitor cells, Recoverin expressed in photoreceptor cells, mature It can be easily confirmed by the expression of rhodopsin, S-Opsin, M/L-Opsin, etc. expressed in photoreceptor cells, the formation of an outer segment structure, and the like.
- the photoreceptor precursor cells are Crx-positive cells and the photoreceptor cells are rhodopsin, S-Opsin and M/L-Opsin positive cells.
- the rod photoreceptors are NRL and Rhodopsin positive cells.
- S cone photoreceptors are S-opsin positive cells
- L cone photoreceptors are L-opsin positive cells
- M cone photoreceptors are M-opsin positive cells.
- neuroretinal cells can be confirmed by the presence or absence of expression of neuroretinal cell-related genes (hereinafter sometimes referred to as “neuroretinal cell markers” or “neuroretinal markers”).
- neuroretinal cell markers or “neuroretinal markers”
- a person skilled in the art can easily confirm the presence or absence of expression of a neuroretinal cell marker or the ratio of neuroretinal cell marker-positive cells in a cell population or tissue. Examples thereof include a technique using an antibody, a technique using a nucleic acid primer, and a technique using a sequence reaction.
- the expression of a neuroretinal cell marker protein can be measured by, for example, flow cytometry using a commercially available antibody, immunostaining, etc., to determine the number of specific neuroretinal cell marker-positive cells. This can be confirmed by dividing by the total number of cells.
- the expression of RNA of neuroretinal cell markers can be confirmed by, for example, PCR, semi-quantitative PCR, or quantitative PCR (eg, real-time PCR).
- a technique using a sequence reaction the expression of RNA of a neuroretinal cell marker can be confirmed using, for example, a nucleic acid sequencer (eg, next-generation sequencer).
- Neuroretinal cell markers 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, Crx expressed in photoreceptor progenitor cells and Blimp 1 and the like.
- 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, and horizontal cells expressed Calbindin, Recoverin expressed in photoreceptors and photoreceptor precursor cells, Rhodopsin expressed in rod cells, Nrl expressed in rod photoreceptors and rod photoreceptor precursor cells, S-opsin and LM expressed in cone photoreceptors -opsin, RXR- ⁇ expressed in pyramidal cells, pyramidal photoreceptor progenitor cells and ganglion cells, among cone photoreceptors, TR ⁇ 2 and OTX2 expressed in pyramidal photoreceptors that appear at the early stage of differentiation or their progenitor cells and Pax6, which are commonly expressed in OC2, horizontal cells, amacrine cells and ganglion cells.
- “Positive cells” means cells that express a specific marker on the cell surface or inside the cell.
- a “Chx10-positive cell” means a cell expressing Chx10 protein.
- Retinal pigment epithelial cells means epithelial cells that exist outside the neural retina in the living retina.
- a person skilled in the art can determine whether a cell is a retinal pigment epithelial cell by, for example, expression of a cell marker (MITF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.). Also, it can be easily confirmed by the presence of melanin granules (dark brown), tight junctions between cells, polygonal/cobblestone-like characteristic cell morphology, and the like.
- the retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.
- Retinal layer means each layer that constitutes the retina, specifically, the retinal pigment epithelium layer, the photoreceptor layer, the outer limiting membrane, the outer nuclear layer, the outer reticular layer, the inner nuclear layer, the inner reticular layer, Mention may be made of the ganglion cell layer, the nerve fiber layer and the inner limiting membrane.
- neural retinal layer means each layer that constitutes the neural retina, and specifically includes photoreceptor layer, outer limiting membrane, outer nuclear layer, outer reticular layer, inner nuclear layer, inner plexiform layer, ganglion cell. Layers, nerve fiber layers and inner limiting membranes may be mentioned.
- photoreceptor layer means a retinal layer formed on the outermost side of the neural retina and containing many photoreceptors (rod photoreceptors, cone photoreceptors), photoreceptor progenitor cells and retinal progenitor cells. Each layer other than the photoreceptor layer is called the inner layer. Whether each cell constitutes one of the retinal layers can be confirmed by a known method, for example, the presence or absence or degree of expression of cell markers.
- the layer containing proliferating neuroretinal progenitor cells is called the "neuroblastic layer", and the inner neuroblastic layer and the outer neuroblastic layer are called.
- the neutral layer the layer containing proliferating neuroretinal progenitor cells
- the inner neuroblastic layer and the outer neuroblastic layer are called.
- It can be determined by a method well known to those skilled in the art, for example, by color gradation (the outer neuroblastic layer is light and the inner neuroblastic layer is dark) under a bright-field microscope.
- Ciliary body includes developmental processes and adult “ciliary body”, “ciliary body margin”, and “Ciliary body”. Markers of the "ciliary body” include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1 and AQP4.
- the "ciliary marginal zone (CMZ)" is, for example, a tissue present in the boundary region between the neural retina and the retinal pigment epithelium in the living retina, and retinal tissue stem cells (retinal stem cells). Areas containing The ciliary margin is also called the ciliary margin or the retinal margin and the ciliary margin, ciliary margin and retinal margin are equivalent tissues.
- ciliary body periphery plays an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, and the like.
- Marker genes for the ciliary body periphery include, for example, Rdh10 gene (positive), Otx1 gene (positive) and Zic1 (positive).
- a "ciliary rim-like structure" is a structure similar to the ciliary rim.
- Cell Aggregate is not particularly limited as long as a plurality of cells adhere to each other to form a three-dimensional structure, for example, cells dispersed in a medium such as a medium It refers to a cluster formed by aggregation, a cluster of cells formed through cell division, or the like. Cell aggregates also include those forming a specific tissue.
- a "sphere-like cell aggregate” means a cell aggregate having a three-dimensional shape close to a sphere.
- a three-dimensional shape close to a sphere is a shape that has a three-dimensional structure, for example, a spherical shape that exhibits a circular or elliptical shape when projected onto a two-dimensional surface, and a plurality of spherical shapes that are overlapped.
- a shape (for example, a shape formed by overlapping two to four circles or ellipses when projected two-dimensionally, also referred to as a “clover shape”) can be mentioned.
- the core portion of the aggregate has a vesicular layered structure, and is characterized in that the central portion is dark and the outer edge portion is bright under a bright field microscope.
- Epithelial tissue is a tissue formed by cells that cover the surface of the body surface, lumen (such as the digestive tract), and body cavity (such as the pericardial cavity) without gaps. Cells forming epithelial tissue are called epithelial cells. Epithelial cells have an apical-basal cell polarity. Epithelial cells can form a layer of cells by forming strong connections between epithelial cells through adherence junctions and/or tight junctions. Epithelial tissue is a tissue made up of one to ten and several layers of these cell layers. Tissues that can form epithelial tissue also include fetal and/or adult retinal tissue, cerebrospinal tissue, ocular tissue, nerve tissue, and the like. The neural retina as used herein is also an epithelial tissue. By “epithelial structure” is meant a structure characteristic of epithelial tissue, such as the apical surface or basement membrane.
- a “continuous epithelial tissue” is a tissue having a continuous epithelial structure.
- a continuous epithelial structure is a state in which the epithelial tissue is continuous.
- a continuous epithelial tissue means, for example, 10 to 10 7 cells tangentially to the epithelial tissue, preferably 30 to 10 7 cells tangentially, more preferably 10 2 to 10 7 cells side by side. It is the state of being.
- the continuous epithelial structure formed in the retinal tissue has an apical surface peculiar to the epithelial tissue, and the apical surface forms the neural retinal layer. They are formed generally parallel and continuously on the surface of the retinal tissue.
- an apical surface is formed on the surface of the aggregate, and 10 cells or more, preferably 30 cells or more, more preferably tangential to the surface. 100 cells or more, more preferably 400 cells or more of photoreceptor cells or photoreceptor precursor cells are arranged regularly and continuously.
- the epithelial tissue is polarized into an "apical surface” and a “basement membrane.”
- Basement membrane refers to the basal layer produced by epithelial cells, which is rich in laminin and type IV collagen and has a thickness of 50-100 nm.
- the “apical surface” refers to the surface (superficial surface) formed on the side opposite to the “basement membrane”.
- the “apical surface” is a photoreceptor in which an outer limiting membrane is formed and photoreceptors and photoreceptor precursor cells are present in retinal tissue in which the developmental stage has progressed to the extent that photoreceptors or photoreceptor precursor cells are observed.
- apical surface refers to the surface in contact with the layer (outer nuclear layer).
- apical surface markers e.g., atypical-PKC (hereinafter abbreviated as "aPKC"), E-cadherin, N-cadherin, which is well known to those skilled in the art. can be identified by the law, etc.
- One aspect of the present invention provides a method for producing retinal lineage cells or retinal tissue.
- the manufacturing method includes the following steps: (A) suspension culture of pluripotent stem cells to form cell aggregates of pluripotent stem cells; (B) A step of culturing the cell aggregates obtained in step (A) in suspension in the presence of a BMP signaling pathway agonist and a CHK1 signaling pathway inhibitor to obtain cell aggregates containing retinal cells.
- the production method of the present invention is a method for producing retinal cells or retinal tissue by inducing differentiation of pluripotent stem cells using a combination of a BMP signaling pathway agent and a CHK1 signaling pathway inhibitor.
- Methods for inducing differentiation from pluripotent stem cells to retinal cells or retinal tissues are described in WO2011/055855, WO2013/077425, WO2015/025967, WO2016/063985, WO2016/063986, WO2017/183732, PLoS One. 2010 Jan 20;5(1):e8763. , Stem Cells. 2011 Aug;29(8):1206-18. , Proc Natl Acad Sci USA. 2014 Jun 10;111(23):8518-23, or Nat Commun. 2014 Jun 10;5:4047, but there was no method for producing retinal lineage cells or retinal tissue using a CHK1 signaling pathway inhibitor.
- Pluripotent stem cells are as described above, and preferred pluripotent stem cells include induced pluripotent stem cells or ES cells, more preferably human induced pluripotent stem cells or human ES cells.
- the method for producing induced pluripotent stem cells is not particularly limited, and they can be produced by a method well known to those skilled in the art. establishment step) is also desirably performed under feeder-free conditions.
- Step (A) is a step of culturing pluripotent stem cells in suspension to form cell aggregates of pluripotent stem cells.
- Pluripotent stem cells used in step (A) can be obtained by maintenance/expansion culture. That is, the step (A) consists of (A-1) a step of maintaining and expanding the culture of the pluripotent stem cells, and (A-2) a suspension culture of the pluripotent stem cells obtained in the step (A-1). and forming cell aggregates of pluripotent stem cells.
- Maintenance culture/expansion culture for obtaining pluripotent stem cells can be performed by methods well known to those skilled in the art.
- the maintenance culture/expansion culture of pluripotent stem cells can be carried out by either adherent culture or suspension culture, but is preferably carried out by adherent culture.
- the maintenance/expansion culture of pluripotent stem cells may be carried out in the presence of feeders or under feeder-free conditions, preferably under feeder-free conditions. Under feeder-free conditions, a medium containing an undifferentiated maintenance factor, which will be described later, can be used.
- the medium in step (A-1) may further contain a TGF ⁇ family signaling pathway inhibitor and/or Sonic hedgehog signaling pathway active substance.
- the medium in step (A-2) may contain a Sonic hedgehog signaling pathway active substance and/or a Wnt signaling pathway inhibitor, as described later.
- This method is also disclosed, for example, in WO2015/025967, WO2016/063985, and WO2017/183732, and for more details, it is possible to refer to WO2015/025967, WO2016/063985, and WO2017/183732.
- a basal medium for cell growth (also called a basal medium) can be used as the medium used to prepare cell aggregates.
- the basal medium for cell growth is not particularly limited as long as the cells can be cultured, and a basal medium commercially available as a medium for cell growth can be used as appropriate.
- a culture medium can be mentioned.
- a medium supplemented with N2 medium which is a supplementary medium, may be used.
- the TGF ⁇ family signaling pathway inhibitor refers to a substance that inhibits the TGF ⁇ family signaling pathway, that is, a substance that inhibits the signaling pathway transmitted by the Smad family. -364947, SB505124, A-83-01 etc.), Nodal/Activin signaling pathway inhibitors (e.g. SB431542, A-83-01 etc.) and BMP signaling pathway inhibitors (e.g. LDN193189, Dorsomorphin etc.) be able to. These substances are commercially available.
- a sonic hedgehog (hereinafter sometimes referred to as "Shh") signal transduction pathway agonist is a substance capable of enhancing signal transduction mediated by Shh.
- Shh signaling pathway active substances include, for example, SHH, partial peptides of SHH, PMA (Purmorphamine), SAG (Smoothened Agonist) and the like.
- the concentration of the TGF ⁇ family signaling pathway inhibitor and Sonic hedgehog signaling pathway active substance may be any concentration that can induce differentiation into retinal cells.
- SB431542 is usually used at a concentration of 0.1-200 ⁇ M, preferably 2-50 ⁇ M.
- A-83-01 is usually used at a concentration of 0.05-50 ⁇ M, preferably 0.5-5 ⁇ M.
- LDN193189 is usually used at a concentration of 1-2000 nM, preferably 10-300 nM.
- SAG is usually used at a concentration of 1-2000 nM, preferably 10-700 nM.
- PMA is usually used at a concentration of 0.002-20 ⁇ M, preferably 0.02-2 ⁇ M.
- the undifferentiated maintenance factor is not particularly limited as long as it is a substance that suppresses the differentiation of pluripotent stem cells.
- Undifferentiation maintenance factors commonly used by those skilled in the art include FGF signal transduction pathway agents, TGF ⁇ family signal transduction pathway agents, insulin and the like.
- FGF signaling pathway agents include fibroblast growth factors (eg, bFGF, FGF4 and FGF8, more preferably bFGF).
- TGF ⁇ family signaling pathway agonists include TGF ⁇ signaling pathway agonists and Nodal/Activin signaling pathway agonists.
- TGF ⁇ signaling pathway agonists include, for example, TGF ⁇ 1 and TGF ⁇ 2.
- Nodal/Activin signaling pathway agonists include, for example, Nodal, ActivinA, and ActivinB.
- the medium in step (A-1) preferably contains bFGF as an undifferentiated maintenance factor.
- the concentration of the undifferentiated maintenance factor in the medium used in step (A-1) is a concentration that can maintain the undifferentiated state of the cultured pluripotent stem cells, and can be appropriately set by a person skilled in the art.
- concentration is usually about 4 ng to 500 ng/mL, preferably about 10 ng to 200 ng/mL, more preferably 30 ng to 150 ng. /mL.
- Essential 8 medium is DMEM/F12 medium with L-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14 ⁇ g/L), insulin (19.4 mg/L), NaHCO3 (543 mg/L) as additives. /L), transferrin (10.7 mg/L), bFGF (100 ng/mL), and TGF ⁇ family signaling pathway agonists (TGF ⁇ 1 (2 ng/mL) or Nodal (100 ng/mL)) (Nature Methods, 8, 424-429 (2011)).
- feeder-free media include S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Life Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9; 105(36): 13409-14), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), or StemFit (manufactured by Ajinomoto).
- S-medium manufactured by DS Pharma Biomedical
- StemPro manufactured by Life Technologies
- hESF9 Proc. Natl. Acad. Sci. USA. 2008 Sep 9; 105(36): 13409-14
- mTeSR1 manufactured by STEMCELL Technologies
- mTeSR2 manufactured by STEMCELL Technologies
- TeSR-E8 manufactured by STEMCELL Technologies
- StemFit manufactured by Ajinomoto
- an appropriate matrix may be used as a scaffold to provide pluripotent stem cells with a scaffold that replaces feeder cells.
- Matrices that can be used as scaffolds include laminin (Nat Biotechnol 28, 611-615, (2010)), laminin fragments (Nat Commun 3, 1236, (2012)), basement membrane preparations (Nat Biotechnol 19, 971- 974, (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, vitronectin, and the like.
- the culture time of pluripotent stem cells in step (A-1) is, when cultured in the presence of a TGF ⁇ family signaling pathway inhibitor and/or Sonic hedgehog signaling pathway agonist (e.g., 100 nM to 700 nM), although there is no particular limitation as long as the effect of improving the quality of the cell aggregates formed in step (A-2) can be achieved, it is usually 0.5 to 144 hours. In one aspect, preferably 2-96 hours, more preferably 6-48 hours, even more preferably 12-48 hours, even more preferably 18-28 hours (eg, 24 hours).
- the cells obtained in step (A-1) have pluripotency-like properties (pluripotent-like state) is maintained, and pluripotent-like properties are maintained through step (A-1).
- a pluripotent-like property means a state of maintaining at least part of the traits unique to pluripotent stem cells that are common to pluripotent stem cells, including pluripotency. Strict pluripotency is not required for pluripotency-like properties.
- the "pluripotent state” includes a state in which all or part of a marker indicative of a pluripotent state is expressed.
- Markers of pluripotency-like properties include Oct3/4 positivity, alkaline phosphatase positivity, and the like.
- the cells with maintained pluripotency-like properties are Oct3/4 positive. Even if the expression level of Nanog is lower than that of ES cells or iPS cells, they fall under "cells exhibiting pluripotency-like properties".
- the cells obtained in step (A-1) contain Oct3/4-positive stem cells at 60% or more, for example 90% or more.
- step (A-2) the pluripotent stem cells obtained in step (A-1) are cultured in suspension to form cell aggregates of pluripotent stem cells.
- the term "cell aggregate of pluripotent stem cells” as used herein does not necessarily mean that all cells are pluripotent stem cells, , 80% or more, 90% or more) includes pluripotent stem cells.
- step (A-1) when step (A-1) is cultured in the presence of a TGF ⁇ family signaling pathway inhibitor and/or Sonic hedgehog signaling pathway agonist (e.g., 100 nM to 700 nM), step (A-2) In the above-described "cells exhibiting pluripotency-like properties" (e.g., containing 60% or more, for example, 90% or more Oct3/4-positive stem cells) cell aggregates are formed, which is also "pluripotent stem cell aggregates”.
- a TGF ⁇ family signaling pathway inhibitor and/or Sonic hedgehog signaling pathway agonist e.g., 100 nM to 700 nM
- step (A-2) In the above-described "cells exhibiting pluripotency-like properties” (e.g., containing 60% or more, for example, 90% or more Oct3/4-positive stem cells) cell aggregates are formed, which is also "pluripotent stem cell aggregates”.
- Suspension culture is to culture cells in a non-adherent state to a culture vessel, and is not particularly limited, but is artificially treated for the purpose of improving adhesion to cells (for example, coating with extracellular matrix etc. treatment) untreated culture vessel, or treatment to artificially suppress adhesion (e.g., polyhydroxyethyl methacrylate (poly-HEMA), nonionic surfactant polyol (Pluronic F-127, etc.), or phospholipid-like It can be carried out using a culture vessel coated with a structure (for example, a water-soluble polymer (Lipidure) having 2-methacryloyloxyethylphosphorylcholine as a structural unit).
- a structure for example, a water-soluble polymer (Lipidure) having 2-methacryloyloxyethylphosphorylcholine as a structural unit.
- Floating culture can be performed, for example, by using the SFEB (Serum-free Floating culture of Embryoid Bodies-like aggregates) method (WO2005/12390) or the SFEBq method (WO2009/148170).
- SFEB Silicon-free Floating culture of Embryoid Bodies-like aggregates
- the medium used in step (A-2) can be serum-containing medium or serum-free medium.
- a serum-free medium is preferably used.
- a serum-free medium supplemented with an appropriate amount of serum substitute such as commercially available KSR can be used.
- the amount of KSR added to the serum-free medium is usually about 1% to about 30%, preferably about 2% to about 20%.
- dispersed cells are prepared by dispersing the cells obtained in step (A-1).
- the "dispersed cells” obtained by the dispersing operation are, for example, 70% (preferably 80% or more) single cells and 2-50 cell clusters of 30% or less (preferably 20% or less). state to do.
- Dispersed cells include a state in which cell-to-cell adhesion (for example, surface adhesion) is almost lost.
- a suspension of dispersed cells is sown in a culture vessel, and the dispersed cells are cultured under non-adhesive conditions to the culture vessel, whereby a plurality of cells aggregate to form aggregates. do.
- a certain number of dispersed stem cells are placed in each well of a multi-well plate (U bottom, V bottom) such as a 96-well plate, and the stem cells are statically cultured, the cells rapidly aggregate. , one aggregate is formed in each well (SFEBq method).
- step (A) contains a Sonic hedgehog signaling pathway agonist. That is, as a specific aspect, step (A) includes the following steps (A1) and (A2): (A1) Pluripotent stem cells in the absence of feeder cells, and optionally a TGF ⁇ family signaling pathway inhibitor and/or a medium containing an undifferentiated maintenance factor, which may contain an active substance of the Sonic hedgehog signaling pathway culturing in (A2) A step of floating-cultivating the cells obtained in step (A1) in a medium containing an active substance of the Sonic hedgehog signaling pathway to form cell aggregates.
- the active substance of the Sonic hedgehog signaling pathway in step (A-2) can be used at the concentrations described above (eg, 10 nM to 300 nM).
- the Sonic hedgehog signaling pathway agent is preferably contained in the medium from the start of suspension culture.
- the medium may be supplemented with a ROCK inhibitor (eg Y-27632).
- the culture time is, for example, 12 hours to 6 days.
- the medium used in step (A-2) is selected from the group consisting of BMP signaling pathway agonists, Wnt signaling pathway agonists, TGF ⁇ family signaling pathway inhibitors, and TGF ⁇ family signaling pathway agonists. It is a medium to which one or more (preferably all) are not added.
- step (B) the cell aggregate obtained in step (A) is subjected to suspension culture in the presence of a BMP signaling pathway agonist and a CHK1 signaling pathway inhibitor to obtain a cell aggregate containing retinal cells. It is a process.
- the medium used in step (B) includes, for example, a serum-free medium or a serum medium (preferably serum-free medium) supplemented with a BMP signaling pathway active substance and a CHK1 signaling pathway inhibitor. Serum-free medium and serum medium can be prepared as described above.
- the medium used in step (B) contains one or more (preferably all) selected from the group consisting of Wnt signaling pathway agents, TGF ⁇ family signaling pathway inhibitors, and TGF ⁇ family signaling pathway agents. Medium without additions.
- the medium used in step (B) is a medium to which no active substance of the Sonic hedgehog signaling pathway is added.
- the medium used in step (B) is a medium to which a Wnt signaling pathway active substance may be added.
- a BMP signaling pathway agonist is a substance that can enhance the signaling pathway mediated by BMP.
- BMP signal transduction pathway agents include BMP proteins such as BMP2, BMP4 or BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides.
- BMP2 protein, BMP4 protein and BMP7 protein are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Wako Pure Chemical Industries.
- a BMP signaling pathway agonist is preferably one or more proteins selected from the group consisting of BMP2, BMP4, BMP7 and GDF7.
- the normal concentration of the BMP signaling pathway agent in the medium should be a concentration that can induce differentiation into retinal cells.
- the normal concentration capable of inducing differentiation into retinal cells is about 0.1 nM or more (more than 0.15 nM), preferably about 0 .5 nM or more, more preferably about 1 nM or more, more preferably about 1.5 nM (55 ng/mL), or about 1 ⁇ M or less, preferably about 100 nM or less, more preferably about 10 nM or less, more preferably about 5 nM or less. concentration.
- a normal concentration of other BMP signaling pathway active substance may be a concentration that has the same BMP signaling pathway activation effect as human BMP4 protein at the above concentration.
- the concentration can be easily set by those skilled in the art.
- BMP activity can be measured by measuring the ability to induce alkaline phosphatase production in ATDC-5 cells, which are mouse cartilage progenitor cells.
- the concentration of human BMP4 protein when used in combination with a CHK1 signaling pathway inhibitor forms cell aggregates close to true spheres, and inhibits induction of differentiation into retinal pigment epithelial cells.
- concentration Specifically, the concentration of human BMP4 protein in the medium may be as low as about 1/10 of the normal concentration described above, i.e.
- the concentration of human BMP4 when used in combination with a CHK1 signaling pathway inhibitor is a sphere-like cell having a multi-layered structure of an outer structure containing retinal pigment epithelial cells and an inner structure containing the neural retina.
- the concentrations are such that aggregates are formed.
- the concentration of human BMP4 protein in the medium is, for example, about 0.01 nM or higher, preferably about 0.1 nM or higher, more preferably about 0.5 nM or higher, even more preferably about 1 nM or higher, most preferably about It may be 1.5 nM (55 ng/mL), or about 1 ⁇ M or less, preferably about 100 nM or less, more preferably about 10 nM or less, even more preferably about 5 nM or less.
- the concentrations may be sufficient as long as they have the same level of BMP signal transduction pathway activating action as the human BMP4 protein at the concentrations described above.
- a CHK1 signaling pathway inhibitor is a substance that inhibits the signaling pathway mediated by CHK1 (checkpoint kinase 1).
- the signaling pathway mediated by CHK1 is a signaling pathway mediated by CHK1-p21-CDK9 (Cyclin-dependent kinase 9).
- CHK1 activates p21
- the activated p21 inhibits CDK9, inhibiting phosphorylation of SMAD2/3 and suppressing degradation of SMAD2/3. is the route.
- CHK1 signaling pathway inhibitors include CHK1 inhibitors, p21 inhibitors or CDK9 activators.
- CHK1 signaling pathway inhibitor is a CHK1 inhibitor that binds to CHK1 and inhibits the activity of CHK1.
- Specific CHK1 inhibitors include PD407824 (CAS622864-54-4), CHIR-124 (CAS405168-58-3), Debromohymenialdisine (CAS75593-17-8), SB 218078 (CAS135897-06-2), Chk2 inhibitor (CAS724708-21-8) LY2603618 (CAS911222-45-2), SCH 900776 (CAS891494-63-6), TCS 2312 (CAS838823-32-8), PF 477736 (CAS952021-60-2), UCN-01 ( CAS112953-11-4), AZD7762 (CAS860352-01-8), XL844 (CAS: NONE), CBP501 (CAS565434-85-7), anti-CHK1 antibody and the like, but are not limited thereto.
- CHK1 inhibitors also include siRNA against CHK1 that degrades CHK1 mRNA.
- CHK1 signaling pathway inhibitor is a p21 inhibitor that binds to p21 and inhibits p21 activity.
- Specific p21 inhibitors include anti-p21 antibodies and siRNA against p21.
- One aspect of the CHK1 signaling pathway inhibitor is a CDK9 activator that binds to CDK9 and enhances the activity of CDK9.
- the concentration of the CHK1 signaling pathway inhibitor may be a concentration that can enhance the differentiation-inducing action of the BMP signaling pathway active substance. , about 0.5 ⁇ M to about 10 ⁇ M, about 0.5 ⁇ M to about 5 ⁇ M, or about 1 ⁇ M to about 5 ⁇ M.
- Concentrations of other CHK1 signaling pathway inhibitors include about 0.1 ⁇ M to about 10 ⁇ M, about 0.3 ⁇ M to about 10 ⁇ M, about 0.5 ⁇ M to about 10 ⁇ M, about 0.5 ⁇ M to about 5 ⁇ M, or about 1 ⁇ M to about
- the concentration may be a concentration that exhibits a CHK1 signaling pathway inhibitory effect (eg, CHK1 inhibitory effect) comparable to that of 5 ⁇ M PD407824.
- the CHK1 inhibitory action can be examined by a person skilled in the art using a known technique or a commercially available reagent or kit (eg, Promega Co., Ltd., catalog number: V1941) or the like.
- a human CDC25C-derived peptide (KKKVSRSGLYRSPSMPENLNRPR (SEQ ID NO: 1)
- CHK1 a human CDC25C-derived peptide
- an evaluation target substance a reaction vessel containing ATP
- the degree of phosphorylation of the peptide is detected.
- the degree of phosphorylation of SMAD2/3 existing downstream of the pathway may be detected using a commercially available anti-phosphorylated SMAD2/3 antibody or the like.
- the BMP signaling pathway agent may be added after about 24 hours from the start of suspension culture in step (A), and within several days (for example, within 15 days) after the start of suspension culture. good too.
- the BMP signal transduction pathway agent is added between days 1 to 15 after the start of the suspension culture in step (A), more preferably between days 1 to 9 or days 2 to 9. Add to medium during day 3, most preferably day 3.
- the BMP signaling pathway agonist is added to the medium and the differentiation of the aggregate-forming cells into retinal cells is initiated, there is no need to add the BMP signaling pathway agonist to the medium, and the BMP signaling is initiated.
- Media exchanges may be performed with serum-free or serum media without pathway agonists.
- the concentration of the BMP signaling pathway agonist in the medium is reduced to 2 to Gradual or stepwise reduction at a rate of 40-60% reduction every 4 days.
- Cells that have initiated differentiation induction into retinal cells can be confirmed, for example, by detecting the expression of retinal progenitor cell marker genes (e.g., Rx gene (also known as Rax), Pax6 gene, Chx10 gene) in the cells. can. Aggregates formed in step (2) using pluripotent stem cells in which a fluorescent reporter protein gene such as GFP has been knocked into the Rx locus are subjected to the action of the BMP signaling pathway at a concentration necessary for inducing differentiation into retinal cells. By carrying out suspension culture in the presence of a substance and detecting the fluorescence emitted from the expressed fluorescent reporter protein, it is also possible to confirm the time at which the induction of differentiation into retinal cells was initiated.
- retinal progenitor cell marker genes e.g., Rx gene (also known as Rax), Pax6 gene, Chx10 gene
- step (3) aggregates formed in step (2) are treated until cells expressing retinal progenitor cell marker genes (e.g., Rx gene, Pax6 gene, Chx10 gene) begin to appear.
- retinal progenitor cell marker genes e.g., Rx gene, Pax6 gene, Chx10 gene.
- suspension culture in a serum-free medium or serum medium containing a BMP signaling pathway agent at a concentration required to induce differentiation into retinal cells to obtain aggregates containing retinal progenitor cells.
- the BMP signaling pathway active substance is added to the medium, for example, between days 1 and 9, preferably between days 2 and 9, after the start of suspension culture in step (B).
- the BMP signaling pathway active substance is BMP4
- part or all of the medium is replaced with a medium containing BMP4 during 2 to 9 days after the start of suspension culture in step (B)
- the final concentration of BMP4 is It can be adjusted to about 1 to 10 nM and cultured in the presence of BMP4 for, for example, 1 to 12 days, preferably 2 to 9 days, more preferably 2 to 5 days.
- part or all of the medium can be replaced with a medium containing BMP4 about once or twice to maintain the same concentration of BMP4.
- the concentration of BMP4 can be reduced stepwise. For example, after maintaining the concentration of the BMP signaling pathway agent (BMP4) until 2 to 10 days after the start of suspension culture in step (B), stepwise until 6 to 20 days after the start of suspension culture in step (B) Alternatively, the concentration of BMP signaling pathway agonist (BMP4) may be reduced.
- the CHK1 signaling pathway inhibitor may coexist with the BMP signaling pathway agonist in the medium for a certain period of time in the step (B), and is preferably added to the medium at the same time as the BMP signaling pathway agonist, preferably It coexists with the BMP signaling pathway agonist for the same period as the addition period of the BMP signaling pathway agonist.
- Culture conditions such as culture temperature and CO 2 concentration in the above steps (A) and (B) can be appropriately set.
- the culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C.
- the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
- retinal cells at various stages of differentiation can be produced as retinal cells contained in the cell aggregates. That is, manufacturing retinal cells in cell aggregates containing immature retinal cells (e.g., retinal progenitor cells, photoreceptor progenitor cells) and mature retinal cells (e.g., photoreceptors) in various proportions can do.
- immature retinal cells e.g., retinal progenitor cells, photoreceptor progenitor cells
- mature retinal cells e.g., photoreceptors
- the percentage of mature retinal cells can be increased.
- a Wnt signaling pathway inhibitor may be further added to the medium.
- the Wnt signaling pathway inhibitor used in step (A) and/or step (B) is not particularly limited as long as it can suppress signal transduction mediated by Wnt, and includes proteins, nucleic acids, and low-molecular-weight compounds. and so on. Wnt-mediated signals are transmitted through Wnt receptors that exist as heterodimers of Frizzled (Fz) and LRP5/6 (low-density lipoprotein receptor-related protein 5/6).
- Wnt signaling pathway inhibitors include, for example, substances that directly act on Wnt or Wnt receptors (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), and expression of genes encoding Wnt or Wnt receptors.
- inhibitory substances e.g., antisense oligonucleotides, siRNA, etc.
- substances that inhibit the binding of Wnt receptors and Wnt soluble Wnt receptors, dominant-negative Wnt receptors, etc., Wnt antagonists, Dkk1, Cerberus protein, etc.
- Wnt receptors soluble Wnt receptors, dominant-negative Wnt receptors, etc., Wnt antagonists, Dkk1, Cerberus protein, etc.
- Substances that inhibit physiological activity resulting from signal transduction by Wnt receptors [CKI-7 (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2, 3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IWR-1-endo (IWR1e) (4-[(3aR, 4S
- Wnt signaling pathway inhibitors CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2 and the like are known Wnt signaling pathway inhibitors, and commercially available products and the like are available as appropriate.
- IWR1e is preferably used as the Wnt signaling pathway inhibitor.
- the concentration of the Wnt signaling pathway inhibitor in step (A) may be any concentration that can induce good formation of cell aggregates.
- the concentration is about 0.1 ⁇ M to about 100 ⁇ M, preferably about 0.3 ⁇ M to about 30 ⁇ M, more preferably about 1 ⁇ M to about 10 ⁇ M, still more preferably about 3 ⁇ M.
- a Wnt signaling pathway inhibitor other than IWR-1-endo it is preferably used at a concentration exhibiting Wnt signaling pathway inhibitory activity equivalent to that of IWR-1-endo.
- step (A) the earlier the timing of adding the Wnt signaling pathway inhibitor to the medium, the better.
- the Wnt signaling pathway inhibitor is usually added within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, and more preferably within 12 hours from the start of the suspension culture in step (A).
- the period during which the cells obtained by maintenance culture/expansion culture are allowed to act on the Wnt signaling pathway inhibitor is not particularly limited, but preferably added to the medium at the start of suspension culture in step (A).
- step (A) After that, it is allowed to act until the end of step (A) (immediately before addition of the BMP signal transduction pathway active substance). More preferably, exposure to the Wnt signaling pathway inhibitor is continued even after completion of step (A) (that is, during step (B)), as described later. In one aspect, as described later, even after the step (A) is completed (that is, during the step (B)), the Wnt signaling pathway inhibitor is continuously acted until retinal tissue is formed. good too.
- step (B) any of the Wnt signaling pathway inhibitors described above can be used as the Wnt signaling pathway inhibitor.
- the Wnt signaling pathway inhibitor used in step (A) and The same type is used in step (B).
- the concentration of the Wnt signaling pathway inhibitor in step (B) may be any concentration that can induce retinal progenitor cells and retinal tissue.
- the concentration is about 0.1 ⁇ M to about 100 ⁇ M, preferably about 0.3 ⁇ M to about 30 ⁇ M, more preferably about 1 ⁇ M to about 10 ⁇ M, still more preferably about 3 ⁇ M.
- a Wnt signaling pathway inhibitor other than IWR-1-endo it is preferably used at a concentration exhibiting Wnt signaling pathway inhibitory activity equivalent to that of IWR-1-endo.
- the concentration of the Wnt signaling pathway inhibitor in the medium of step (B) is preferably 50 to 150, more preferably 80, when the concentration of the Wnt signaling pathway inhibitor in the medium of step (A) is 100. ⁇ 120, more preferably 90-110, more preferably equivalent to the concentration of the Wnt signaling pathway inhibitor in the medium in step (B).
- a Wnt signaling pathway inhibitor is added to the medium at the start of step (B). More preferably, after the Wnt signaling pathway inhibitor is added in step (A), it is continuously contained in the medium in step (B) (that is, from the start of step (A)). More preferably, after the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (A), it is continuously contained in the medium in step (B).
- a BMP signaling pathway agonist eg, BMP4 may be added to the culture obtained in step (A) (aggregate suspension in medium containing Wnt signaling pathway inhibitor).
- the period for which the Wnt signaling pathway inhibitor is allowed to act is not particularly limited. It is 2 to 30 days, more preferably 6 to 20 days, 8 to 18 days, 10 to 18 days, or 10 to 17 days (for example, 10 days) from time.
- the period for which the Wnt signaling pathway inhibitor is allowed to act is calculated from the start of the suspension culture in the step (A) when the Wnt signaling pathway inhibitor is added at the start of the suspension culture in the step (A). As a point, it is preferably 3 days to 15 days (eg, 5 days, 6 days, 7 days), more preferably 6 days to 10 days (eg, 6 days).
- Cell aggregates obtained by the above-described method are cultured in a serum-free medium or serum medium containing a Wnt signaling pathway agonist and/or an FGF signaling pathway inhibitor for a period of about 2 to 4 days (Step (C )), then in a serum-free medium or serum medium containing no Wnt signaling pathway agonists and FGF signaling pathway inhibitors for about 30 to 200 days (30 to 150 days, 50 to 120 days, 60 days to By culturing (step (D)) for 90 days, a neural retina containing ciliary rim-like structures can be produced.
- the Wnt signaling pathway agonist is not particularly limited as long as it can enhance signal transduction mediated by Wnt.
- Specific Wnt signaling pathway agonists include, for example, GSK3 ⁇ inhibitors (eg, 6-bromoindirubin-3'-oxime (BIO), CHIR99021, Kenpaullone).
- GSK3 ⁇ inhibitors eg, 6-bromoindirubin-3'-oxime (BIO)
- CHIR99021 e.g, 6-bromoindirubin-3'-oxime (BIO)
- BIO 6-bromoindirubin-3'-oxime
- CHIR99021 Kenpaullone
- the range is about 0.1 ⁇ M to about 100 ⁇ M, preferably about 1 ⁇ M to about 30 ⁇ M.
- the FGF signaling pathway inhibitor is not particularly limited as long as it can inhibit signal transduction mediated by FGF.
- FGF signaling pathway inhibitors include, for example, SU-5402, AZD4547, BGJ398 and the like.
- SU-5402 is added at a concentration of about 0.1 ⁇ M to about 100 ⁇ M, preferably about 1 ⁇ M to about 30 ⁇ M, more preferably about 5 ⁇ M.
- the medium used in step (C) is selected from BMP signaling pathway agonists, Wnt signaling pathway inhibitors, SHH signaling pathway agonists, TGF ⁇ family signaling pathway inhibitors, and TGF ⁇ family signaling pathway agonists. It is a medium to which one or more (preferably all) selected from the group consisting of has not been added.
- a part or all of the above step (D) can be cultured using the continuous epithelial tissue maintenance medium disclosed in WO2019/017492. That is, the continuous epithelial structure of the neural retina can be maintained by culturing using the medium for maintaining continuous epithelial tissue.
- a neurobasal medium eg, Thermo Fisher Scientific, 21103049
- a B27 supplement eg, Thermo Fisher Scientific, 12587010
- the culture in the step (D) should be replaced step by step with a continuous epithelial tissue maintenance medium in order to achieve both differentiation and/or maturation of retinal cells (especially photoreceptors) and maintenance of a continuous epithelial structure. is preferred.
- the basal medium for cell growth e.g., DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine
- the next 10 to 40 days for the first 10 to 30 days.
- the basal medium for cell growth e.g., DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine
- a medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 ⁇ M taurine can be used for culture.
- Thyroid hormone signaling transduction even when using any of a cell growth basal medium, a continuous epithelial tissue maintenance medium, or a mixed medium thereof in part or all of the above step (D)
- a pathway agent may further be included.
- thyroid hormone signaling pathway agonists include, for example, triiodothyronine (hereinafter sometimes abbreviated as T3), thyroxine (hereinafter sometimes abbreviated as T4), thyroid hormone receptor (preferably TR ⁇ receptor). Agonist etc. are mentioned.
- T3 When used as an active substance of the thyroid hormone signaling pathway, it can be added to the medium in a range of, for example, 0.1 to 1000 nM. More preferably 1 to 500 nM; more preferably 10 to 100 nM; still more preferably 30 to 90 nM; still more preferably concentrations around 60 nM that have thyroid hormone signaling enhancing activity equivalent to T3.
- T4 When used as the thyroid hormone signaling pathway agonist, it can be added to the medium in a range of, for example, 1 nM to 500 ⁇ M. Preferably it ranges from 50 nM to 50 ⁇ M; more preferably from 500 nM to 5 ⁇ M.
- any concentration may be used as long as the agonist activity is comparable to that of T3 or T4 at the concentrations described above.
- the medium used in step (D) may contain L-glutamine, taurine, serum, etc. as appropriate.
- the medium used in step (D) is a BMP signaling pathway agonist, FGF signaling pathway inhibitor, Wnt signaling pathway agonist, Wnt signaling pathway inhibitor, SHH signaling pathway agonist, TGF ⁇ family A medium to which one or more (preferably all) selected from the group consisting of signal transduction pathway inhibitors and TGF ⁇ family signal transduction pathway active substances are not added.
- a cell aggregate containing retinal cells or retinal tissue can be prepared by a method comprising the following steps (A) to (E): (A) A culture medium containing a factor for maintaining undifferentiated pluripotent stem cells in the absence of feeder cells and optionally containing a TGF ⁇ family signaling pathway inhibitor and/or a sonic hedgehog signaling pathway agonist culturing in (B) The cells obtained in step (A) are subjected to suspension culture in a medium that may contain a Wnt signaling pathway inhibitor and/or Sonic hedgehog signaling pathway active substance to form cell aggregates.
- step (C) The cell aggregate obtained in step (B) is further suspended in a medium containing a BMP signaling pathway agent and a CHK1 signaling pathway inhibitor (e.g., CHK1 inhibitor), and retinal cells or obtaining a cell aggregate containing retinal tissue;
- step (D) The cell aggregates obtained in step (C) are treated in a serum-free or serum medium containing Wnt signaling pathway agonists and/or FGF signaling pathway inhibitors for about 2 to 4 days. culturing for a period of time, and (E) applying the cell aggregates obtained in step (D) to a serum-free medium or A step of culturing in a serum medium for about 30 to 200 days.
- a cell aggregate containing retinal cells or retinal tissue can be prepared by a method comprising the following steps (A) to (E): (A) In the absence of feeder cells, pluripotent stem cells are treated in a medium containing undifferentiated maintenance factors and containing TGF ⁇ family signaling pathway inhibitors and/or Sonic hedgehog signaling pathway agonists for 12 hours or more.
- step (B) the cells obtained in step (A) in a medium containing Wnt signaling pathway inhibitors and/or Sonic hedgehog signaling pathway agonists for 12 hours to 72 days (24 hours to 48 hours); forming cell aggregates by suspension culture;
- step (C) the cell aggregates obtained in step (B) in a medium containing a BMP signaling pathway agonist and a CHK1 signaling pathway inhibitor (e.g., CHK1 inhibitor) for 8 to 15 days (10 days ⁇ 13 days) suspension culture to obtain cell aggregates containing retinal cells or retinal tissue
- step (D) The cell aggregate obtained in step (C) is cultured for 2 to 4 days in a serum-free or serum medium containing a Wnt signaling pathway agonist and/or an FGF signaling pathway inhibitor.
- step (E) applying the cell aggregates obtained in step (D) to a serum-free medium or A step of culturing in a serum medium for about 10 to 200 days.
- the step (D) is culturing in a cell growth basal medium for 10 to 30 days, and then a mixed medium of a cell growth basal medium and a continuous epithelial tissue maintenance medium containing a thyroid hormone signaling agent for 10 to 40 days. and culturing in continuous epithelial tissue maintenance medium containing a thyroid hormone signaling agent for an additional 20 to 140 days.
- step (D) comprises culturing for 20 to 60 days (30 to 50 days) in the presence of a thyroid hormone signaling pathway agonist.
- the culture period from step (A) to step (D) is 70 to 100 days (80 to 90 days).
- a good retinal tissue can be produced by using the signal transduction agent explicitly described in each step. is possible. In conditions that interfere with the purpose of each step (eg, when tissue other than retinal tissue is induced), it is better not to include other signaling agents.
- the culture medium in each step includes Sonic hedgehog signaling pathway activator/inhibitor, TGF ⁇ family signaling pathway activator/ inhibitor, BMP signaling pathway activator/inhibitor, CHK1 signaling pathway inhibitor, Wnt signaling pathway activator/inhibitor, FGF signaling pathway activator/inhibitor, Nodal signaling pathway activator/ inhibitors, MEK (ERK) activators/inhibitors, PI3k/Akt signaling pathway activators/inhibitors, platelet-derived growth factor (PDGF) signaling pathway activators/inhibitors, vascular endothelial growth factor (VEGF ) signaling pathway activator/inhibitor, epidermal growth factor (EGF) signaling pathway activator/inhibitor, Notch signaling pathway activator/inhibitor, integrin signaling pathway activator/inhibitor, retinoin It can be acid free. Naturally, other signaling agents may be included as long as they do not interfere with the purpose of each step (eg, when
- the cell aggregate obtained by the production method of the present invention is preferably a spherical cell aggregate, more preferably a cell aggregate having a shape close to a true sphere.
- the cell aggregates contain retinal cells, and in one embodiment, the cell aggregates are spherical cell aggregates comprising a multi-layer structure of an outer structure containing retinal pigment epithelial cells and an inner structure containing neural retina.
- a sphere-like cell aggregate means a cell aggregate having a three-dimensional shape close to a sphere.
- a three-dimensional shape close to a sphere is a shape that has a three-dimensional structure, for example, a spherical shape that exhibits a circular or elliptical shape when projected onto a two-dimensional surface, and a plurality of spherical shapes that are overlapped. Shapes (for example, representing a shape formed by two to four overlapping circles or ellipses when projected into two dimensions).
- a sphere-like cell aggregate may have a shape close to a true sphere.
- true sphere refers to a perfect sphere with a constant distance from the center to the outer circumference.
- a “nearly spherical shape” means a sphere that is nearly perfectly spherical.
- the difference between the longest distance and the shortest distance from the center to the outer circumference may be within 10%, 5%, or 3% of the longest distance.
- determination may be made based on a circle projected onto a two-dimensional surface by observation with a microscope or the like. For example, cases in which the difference between the longest distance and the shortest distance from the center to the outer circumference of a circle projected onto a two-dimensional surface is within 10%, 5%, or 3% of the longest distance are included.
- cell aggregates are “true spheres” or “close to true spheres”.
- cell aggregates that are “true spheres” or “shapes close to true spheres” in which multiple shapes do not overlap can be obtained at a high rate.
- a cell aggregate having a nearly spherical shape that contains retinal tissue and does not contain non-target cells (eg, RPE cells).
- non-target cells eg, RPE cells
- One aspect of the invention also provides a culture of cell aggregates comprising retinal tissue.
- a culture of cell aggregates containing retinal tissue (1) a cell aggregate having a nearly spherical shape that contains retinal tissue and does not contain non-target cells (e.g., RPE cells); (2) a medium necessary to maintain the viability of the cell aggregates;
- a culture comprising:
- culture is usually performed using a multi-well plate (U-bottom, V-bottom) such as a 96-well plate. Include one cell aggregate per well of the plate. There may be wells that do not contain cell aggregates. For example, the outermost wells of the plate may contain only medium and no cell aggregates in order to prevent the effects of medium evaporation.
- a culture of cell aggregates containing retinal tissue (1) multiwell plates (e.g. 192 wells, 96 wells, 48 wells, 24 wells, 12 wells); (2) Each well of the plate (at least 50%, 60%, 70%, 80%, 90% or 95% or more of all wells) contains (A) retinal tissue and cells outside the purpose (e.g. : RPE cells) and a cell aggregate having a shape close to a true sphere, (B) a medium necessary to maintain the viability of the cell population for transplantation;
- a culture comprising:
- the ratio of the number of wells containing cell aggregates having a shape close to true spheres containing retinal tissue to the number of wells containing cell aggregates is, for example, 50% or more, 60% or more, 70% or more, 80% or more, It may be 90% or more, 95% or more, 97% or more, or 98% or more.
- non-target cells e.g RPE cells
- the ratio of non-target cells (e.g., RPE cells) to the total number of cell aggregates is about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, About 0.5% or less, about 0.1% or less.
- non-target cells means cells other than cells contained in the neural retina.
- non-target cells examples include cerebrospinal tissues and eyeball-related tissues
- cerebrospinal tissues include telencephalon (cerebrum), diencephalon (including hypothalamus), midbrain, and spinal cord cells
- eyeball-related Tissues include the retinal pigment epithelium (RPE), ciliary body, lens and optic stalk (eye stalk and optic nerve tissue).
- RPE retinal pigment epithelium
- “Culture” in the present invention includes a medium and cell aggregates necessary to maintain viability, and may further contain biological substances added or produced from cell aggregates.
- biological substances include, but are not limited to, cytokines, chemokines, and the like.
- the "medium necessary to maintain viability" in the present invention includes medium, physiological buffer solution, etc., but is not particularly limited as long as the cell aggregate containing retinal tissue survives. If any, it can be selected as appropriate.
- One example is a medium prepared using a medium commonly used for culturing animal cells as a basal medium.
- basal media examples include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, F-12 Medium, DMEM/F12 medium, IMDM/F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, mixed medium of these mediums, and the like, which can be used for culturing animal cells.
- GMEM Glasgow MEM
- IMDM Zinc Option medium IMDM medium
- Medium 199 medium Eagle MEM medium
- ⁇ MEM medium DMEM medium
- F-12 Medium DMEM/F12 medium
- IMDM/F12 medium Ham's medium
- RPMI 1640 medium Fischer's medium, mixed medium of these mediums, and the like, which can be used for culturing animal cells.
- the production method may further include a step of cutting out a retinal tissue having a size necessary for transplantation from the obtained cell aggregate. For example, it can be cut out using tweezers, a knife, scissors, or the like.
- a spherical cell aggregate as one aspect of the present invention comprises a multi-layer structure of an outer structure containing retinal pigment epithelial cells and an inner structure containing neural retina.
- the spherical cell aggregate is (1)
- a neural retinal layer containing at least a photoreceptor layer is formed, and the photoreceptor layer is at least selected from the group consisting of photoreceptors, photoreceptor precursor cells, and retinal precursor cells.
- the neural retina comprising one or more cells that (2) in the inner structure, the neural retina is folded and present; (3) the retinal pigment epithelial cells in the outer structure are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells, and (4) the cell aggregates are lens, vitreous, cornea and blood vessels characterized by not containing
- the outer structure containing retinal pigment epithelial cells in the spherical cell aggregate is the outermost layer structure of the spherical cell aggregate, and at least part of the surface of the inner structure containing the neural retina is continuous. or intermittently.
- the outer structure preferably covers 30% or more, more preferably 50% or more of the surface area of the inner structure.
- Continuously covering the surface of the internal structure means that the external structure exists as a continuous mass on the surface of the internal structure, and intermittently covering the surface of the internal structure , the external structure exists as two or more continuous masses or layers on the surface of the internal structure, and the continuous masses are not connected to each other.
- each continuous mass preferably continuously covers 10% or more, or 20% or more of the surface area of the core.
- a neural retinal layer including at least a photoreceptor layer is formed, and the photoreceptor layer is selected from the group consisting of at least photoreceptors, photoreceptor progenitor cells and retinal progenitor cells. It includes one or more cells (hereinafter sometimes referred to as “photoreceptors, etc.”).
- Photoreceptor cells include rod photoreceptor cells and cone photoreceptor cells, and photoreceptor cells account for 70% or more, preferably 80% or more, more preferably 80% or more of all cells present in the photoreceptor layer based on the number of nuclei. accounts for more than 90%.
- the inner structure is the neural retina.
- the neural retina is folded.
- the location and number of times the neural retina is folded is not particularly limited, and it may be folded only in a part of the inner structure.
- Some of the inner structures may include retinal pigment epithelial cells, structures for the ciliary rim and voids.
- the neural retina may form one continuous epithelial structure or may form multiple epithelial structures.
- the photoreceptor layer in the neural retina of the inner structure is formed on the outermost side (the surface, the surface in contact with the outer structure) in at least part of the inner structure of the cell aggregate.
- a photoreceptor layer may also be formed on the inner side.
- the photoreceptors and the like are continuous in the tangential direction of the surface of the epithelial structure with a continuous inner structure, that is, they are present while adhering to each other.
- a photoreceptor layer containing photoreceptors and the like is formed.
- the tangential direction refers to the tangential direction to the surface of each epithelial tissue present in the inner structure of the spherical cell aggregate, that is, the direction in which the photoreceptor cells, etc. are arranged in the photoreceptor layer. parallel or transverse direction.
- the outer structure comprises retinal pigment epithelial (RPE) cells in contact with each other, where RPE cells also comprise retinal pigment epithelial progenitor cells, RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells; is preferably RPE cells "in contact with each other" means that one RPE cell is in contact with another RPE cell in the external structure, and an independent single RPE cell that does not contact other RPE cells is in the external structure. do not configure
- the basal surface of the retinal pigment epithelial cells may face the inner structure, and the basal surface of the neural retina may face the outer structure. That is, the basal surface of the retinal pigment epithelial cells faces the basal surface of the neural retina.
- This structure is also a different feature from the living retina, since in the living retina the basal plane of the layer of retinal pigment epithelial cells faces the apical plane of the neural retina.
- Living retinas including fetal retinas, contain lens, vitreous, cornea, and blood vessels, whereas cell aggregates of the present invention do not contain lens, vitreous, cornea, and blood vessels.
- the "lens” is a tissue that plays the role of a lens that refracts light entering the eyeball from the outside and focuses it on the retina. Partial structures of the lens include the lens epithelium, lens nucleus, lens capsule, and the like. Precursor tissues of the lens include lens placode, lens vesicle, and the like.
- the lens placode is a lens precursor tissue consisting of a thickened epidermal ectodermal cell layer. During embryogenesis, contact of the optic vesicle with the epidermal ectoderm forms the contact area by thickening.
- a lens vesicle is a vesicle formed by invagination of the lens placode.
- Markers of the lens, its substructures, or its precursor tissues include, but are not limited to, L-Maf (lens precursor tissue), ⁇ , ⁇ , and ⁇ crystallins (lens), and the like.
- the "vitreous body” is a transparent jelly-like tissue that is located behind the lens and fills the lumen. It maintains the shape of the eyeball and at the same time has the effect of dispersing external force.
- the vitreous is made up of water and protein (collagen). The presence of vitreous can be confirmed by its jelly-like shape.
- the "cornea” is a transparent watch-glass-shaped tissue that occupies about the front 1/6 of the outer layer of the eyeball wall.
- the partial structure of the cornea includes corneal epithelium, Bowman's membrane, corneal stroma, Descemet's membrane, corneal endothelium, and the like.
- the cornea is normally composed of five layers, which are the corneal epithelium, Bowman's membrane, corneal stroma, Descemet's membrane, and corneal endothelium, in this order from the body surface side. The presence of the cornea, its partial structure, or its progenitor tissue can be confirmed by the expression of the marker.
- Markers of the cornea, its partial structure, or its precursor tissue include pan-cytokeratin (corneal epithelial precursor tissue), E-cadherin (corneal epithelial precursor tissue), cytokeratin 3 (corneal epithelium), cytokeratin 12 (corneal epithelium).
- cytokeratin 14 (corneal epithelium), p63 (corneal epithelium), ZO-1 (corneal epithelium), PDGFR- ⁇ (corneal stroma, corneal endothelium, or its precursor tissue), Pitx2 (corneal stroma and corneal endothelium precursor tissue ), ABCG2 (precursor tissue of corneal stroma and corneal endothelium), and the like.
- the lens, etc. When the lens, etc. is removed from the fetal retina, a hole is created in that part, and the tissue is divided by the void.
- cells present in the inner layer (the part excluding the outermost photoreceptor layer) that constitutes the neural retinal layer in the core of the spherical cell aggregates e.g., horizontal cells, amacrine cells, bipolar cells) cells
- a low number of ganglion cells Specifically, it is 80% or less, 70% or less, 60% or less, or 50% or less of the number of cells present in the inner layer of the human fetal retina, for example.
- a part of the fetal retina is cut and cultured, it becomes a sheet structure with a two-dimensional thickness, but it cannot become a three-dimensional spherical cell aggregate.
- the spherical cell aggregate may contain a ciliary margin-like structure.
- a ciliary rim-like structure may be included in the outer structure and/or the inner structure.
- the retinal pigment epithelial cells and the neural retina may be connected as an epithelial structure, and may be contained in a ciliary body rim-like structure between the retinal pigment epithelial cells and the neural retina. “Connected as an epithelial structure” means, for example, a state in which the Apical-Basal polarities are continuously connected.
- the spherical cell aggregate may contain, for example, 2 or more, 3 or more, 4 or more, 5 or more ciliary margin-like structures.
- a ciliary rim-like structure is a structure similar to the ciliary rim.
- the "ciliary marginal zone (CMZ)" is, for example, a tissue present in the boundary region between the neural retina and the retinal pigment epithelium in the living retina, and retinal tissue stem cells (retinal stem cells). Areas containing The ciliary margin, also called the ciliary margin or the retinal margin, is the equivalent tissue. It is known that the ciliary body periphery plays an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, and the like. Marker genes for the ciliary body periphery include, for example, Rdh10 gene (positive), Otx1 gene (positive) and Zic1 (positive). That is, the ciliary margin-like structure may contain Rdh10-positive cells, Otx1-positive cells, and/or Zic1-positive cells.
- the diameter (maximum diameter) of the spherical cell aggregates may be, for example, 0.1 mm or more, 0.2 mm or more, 0.5 mm or more, 1 mm or more, or 2 mm or more. .5 mm to 2 mm, or 0.5 to 1 mm.
- the diameter of the spherical cell aggregate is measured as the longest distance from the center of the cell aggregate to the surface.
- the spherical cell aggregate of the present invention can also be transplanted after being cut into an appropriate size using tweezers, a knife, scissors, or the like.
- sheet preparations containing neural retina and retinal pigment epithelial cells also referred to as cell sheets or NR-RPE cell sheets
- one cell sheet e.g., 300 ⁇ m in diameter and 50 ⁇ m in height
- one cell sheet is transplanted in one or more sheets depending on the area of the region where photoreceptor cells and retinal pigment epithelial cells are degenerated. Things are mentioned.
- a person skilled in the art can select the number of cell sheets according to the degenerated and dead region.
- One aspect of the present invention also provides a method for producing spherical cell aggregates, comprising the following steps.
- (B) The cell aggregate obtained in step (A) is subjected to suspension culture in the presence of a BMP signaling pathway agonist and a CHK1 signaling pathway inhibitor, and an outer structure containing retinal pigment epithelial cells and a neural retina are formed.
- obtaining a spherical cell aggregate comprising a multi-layered structure with an inner structure comprising;
- steps (A) and (B), etc. are as described in the method for producing retinal cells or retinal tissue described above.
- a pharmaceutical composition (a composition for transplantation, a tissue for transplantation or a Transplant) comprising the cell aggregate of the present invention (paragraphs [0108], [0111] or [0120], etc.) or a portion thereof mentioned.
- a pharmaceutical composition preferably contains a pharmaceutically acceptable carrier in addition to the cell aggregate of the present invention or a portion thereof.
- a part of the cell aggregate is a part of the cell aggregate that can be used for the pharmaceutical composition, and can be obtained by excising a retinal tissue having a size necessary for transplantation from the cell aggregate.
- the pharmaceutical composition can be used to treat neural retinal cell or neural retinal disorders or diseases based on neural retinal damage.
- Diseases based on disorders of neural retinal cells or neural retina include, for example, retinal degenerative diseases, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous chorioretinopathy, Ophthalmic diseases such as cone dystrophy and cone-rod dystrophy can be mentioned.
- Damaged states of the neural retina include, for example, a state in which photoreceptor cells are degenerated and dead.
- a physiological aqueous solvent can be used as a pharmaceutically acceptable carrier.
- the pharmaceutical composition may contain preservatives, stabilizers, reducing agents, tonicity agents, etc., which are commonly used in medicines containing tissues or cells to be transplanted in transplantation medicine.
- a therapeutic agent for diseases based on neural retinal disorders containing the cell aggregate of the present invention or a portion thereof.
- the cell aggregate of the present invention or a portion thereof is transplanted to a subject in need of transplantation (e.g., subretinal in an eye suffering from an ophthalmic disease). Included are methods of treating lineage or neural retinal disorders or diseases based on neural retinal damage.
- the cell aggregates of the present invention, or portions thereof can be used as therapeutic agents for diseases based on neuroretinal damage, or for replenishment of relevant damaged sites in conditions of neuroretinal damage.
- the cell aggregate of the present invention or a portion thereof is transplanted into a patient having a disease based on neuroretinal cells or neural retinal damage, or a patient with neural retinal damage, in need of transplantation, and the neuroretinal system Diseases based on neuroretinal cell or neural retinal damage or neural retinal damage conditions can be treated by replenishing the cells or the damaged neural retina.
- the transplantation method includes, for example, a method of transplanting a sheet-like retinal tissue under the retina of the injured site by incision of the eyeball or the like. Examples of the method of transplantation include a method of injecting using a thin tube and a method of transplanting by clamping with tweezers, and examples of the thin tube include injection needles and the like.
- a specific maintenance culture operation of human ES cells was performed as follows. First, human ES cells that have become subconfluent (about 60% of the culture area is covered with cells) are washed with PBS, and then converted into single cells using TrypLE Select (trade name, manufactured by Life Technologies). Dispersed. Thereafter, the human ES cells dispersed into single cells were seeded on a plastic culture dish coated with Laminin511-E8, and in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M) in StemFit medium under feeder-free conditions. cultured.
- Y-27632 Y-27632
- the number of seeded human ES cells dispersed in the single cells was 0.00 per well. 4-1.2 ⁇ 10 4 cells.
- the medium was changed to StemFit medium without Y-27632. Thereafter, the medium was replaced with StemFit medium containing no Y-27632 once every 1 to 2 days. Thereafter, the cells were cultured under feeder-free conditions until one day before subconfluency.
- the human ES cells one day before the subconfluency were treated with SB431542 (TGF ⁇ signaling pathway inhibitor, 5 ⁇ M) and SAG (Shh signaling pathway agonist, 300 nM) in the presence (preconditioning treatment) under feeder-free conditions for 1 day. cultured in
- the human ES cells were washed with PBS, treated with a cell dispersion using TrypLE Select, and further dispersed into single cells by pipetting. Thereafter, the human ES cells dispersed into single cells were added to 1.2 ⁇ 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). The cells were suspended in 100 ⁇ L of serum-free medium and cultured in suspension under conditions of 37° C. and 5% CO 2 .
- the serum-free medium (gfCDM + KSR) at that time is a 1:1 mixture of F-12 medium and IMDM medium, 10% KSR, 450 ⁇ M 1-monothioglycerol, 1 ⁇
- a serum-free medium supplemented with a chemically defined lipid concentrate was used.
- Y-27632 (ROCK inhibitor, final concentration 10 ⁇ M) was added to the serum-free medium.
- exogenous human recombinant BMP4 (trade name: Recombinant Human BMP-4, 50 ⁇ L of medium containing R&D) was added.
- PD407824 (TOCRIS) was added at the same time as BMP4 to a final concentration of 1 ⁇ M. After 6 days from the start of suspension culture, half of the medium was exchanged with medium containing none of Y-27632, human recombinant BMP4 and PD407824 once every 3 days.
- the aggregates (cell aggregates) 14 days after the start of the suspension culture were transferred to a 90 mm low-adhesion culture dish (manufactured by Sumitomo Bakelite), and the Wnt signaling pathway agonist (CHIR99021, 3 ⁇ M) and FGF signaling pathway inhibition
- the cells were cultured in a serum-free medium (DMEM/F12 medium supplemented with 1% N2 supplement) containing the substance (SU5402, 5 ⁇ M) under conditions of 37° C. and 5% CO 2 for 3 days.
- DMEM/F12 medium supplemented with 1% N2 supplement containing the substance (SU5402, 5 ⁇ M) under conditions of 37° C. and 5% CO 2 for 3 days.
- the above method was also carried out in the following examples, unless otherwise specified.
- PD407824 when PD407824 was added to 1.5 nM BMP4, sphere-like cell aggregates with a multi-layered structure in which the layer of retinal pigment epithelium (RPE) cells on the outside and the layer of neuroretinal tissue (NR) on the inside were localized. was observed to be produced (Fig. 1).
- RPE retinal pigment epithelium
- NR neuroretinal tissue
- Example 2 Confirmation of promotion of Rx::Venus-positive retinal differentiation induction>
- 3 days after the start of suspension culture (1) 1.5 nM BMP4 alone, or (2) 1 ⁇ M PD407824 in addition to 0.15 nM BMP4 was added to prepare aggregates. After culturing for 15 days (Day 15) or 36 days (Day 36), aggregates were observed.
- Example 3 Method for inducing differentiation of neural retinal tissue into spherical cell aggregates of retinal pigment epithelial cells>
- aggregates prepared by adding 1 ⁇ M PD407824 in addition to 1.5 nM BMP4 three days after the start of suspension culture were cultured for 21 days (Day 21) after the start of suspension culture and observed.
- RPE retinal pigment epithelium
- NR neural retinal tissue
- Ciliary Marginal Zone Ciliary Marginal Zone (CMZ) structures characteristic of the boundary between the MITF-positive RPE layer and the Chx10-positive neural retina layer (indicated by "[" in FIG. 7) Approximately 3 to 10 per site and one aggregate) were observed (Fig. 7).
- Example 4 Study of concentration of BMP4 and PD>
- 0.15 nM, 0.5 nM or 1.5 nM plus 1 ⁇ M or 3 ⁇ M PD407824 was added and cultured for 9 days after the start of suspension culture (Day 9) to form aggregates.
- 1 ⁇ M PD in addition to 0.15 nM BMP4 differentiated the Rx::Venus-positive neural retina in the entire area of the aggregates.
- BMP4 at 0.5 nM or 1.5 nM plus PD407824 at 1 ⁇ M or 3 ⁇ M resulted in multiple layers of outer RPE cells and inner layers of CMZ-rich, polarized neuroretinal tissue. It was observed that spheroidal cell aggregates with structure were produced (Fig. 8).
- Example 5 Confirmation of photoreceptor differentiation by long-term culture> Aggregates prepared by culturing for 60 days after the start of suspension culture under the conditions of adding 1 ⁇ M PD407824 in addition to 1.5 nM BMP4 as in Example 1 were fixed with 4% PFA and replaced with 20% sucrose. , cryosections were made. These frozen sections were subjected to antigen retrieval treatment using microwaves, and then treated with DAPI, anti-CHX10 antibody (trade name: Anti CHX10 Antibody, manufactured by EX Alpha) and anti-CRX antibody (manufactured by TaKaRa). Immunostaining was performed.
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Abstract
Description
[1]
(A)多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程と、
(B)工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜系細胞を含む細胞凝集体を得る工程と
を含む、網膜系細胞又は網膜組織の製造方法。
[2]
工程(B)において、BMPシグナル伝達経路作用物質が、真球に近い細胞凝集体が形成され、かつ、網膜色素上皮細胞への分化誘導が抑制されるような濃度で存在する、[1]に記載の製造方法。
[3]
工程(B)で得られた細胞凝集体が、網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体であり、工程(B)において、BMPシグナル伝達経路作用物質が、上記複層構造を備えるスフェア状細胞凝集体が形成されるような濃度で存在している、[1]に記載の製造方法。
[4]
上記CHK1シグナル伝達経路阻害物質がPD407824である、[1]~[3]のいずれかに記載の製造方法。
[5]
上記BMPシグナル伝達経路作用物質が、BMP2、BMP4、BMP7及びGDF7からなる群から選択される1以上のタンパク質である、[1]~[4]のいずれかに記載の製造方法。
[6]
工程(B)において、工程(A)の浮遊培養開始後2日目から9日目の間に上記BMPシグナル伝達経路作用物質が培地に添加される、[1]~[5]のいずれかに記載の製造方法。
[7]
工程(B)において、上記CHK1シグナル伝達経路阻害物質が、上記BMPシグナル伝達経路作用物質と同時に培地に添加される、[1]~[6]のいずれかに記載の製造方法。
[8]
上記CHK1シグナル伝達経路阻害物質の濃度が、0.1μMから10μMのPD407824と同程度のCHK1シグナル伝達経路阻害作用を奏する濃度である、[1]~[7]のいずれかに記載の製造方法。
[9]
工程(B)で得られた細胞凝集体から、移植に必要な大きさの網膜組織を切り出す工程をさらに含む、[1]~[8]のいずれかに記載の製造方法。
[10]
網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体であって、
(1)上記内側構造における上記神経網膜において、少なくとも視細胞層を含む神経網膜層が形成されており、上記視細胞層は少なくとも視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞を含み、
(2)上記内側構造において、上記神経網膜が折り重なって存在しており、
(3)上記外側構造における上記網膜色素上皮細胞がRPE65陽性細胞、MITF陽性細胞、又は、RPE65陽性かつMITF陽性細胞であり、かつ
(4)上記細胞凝集体は、水晶体、硝子体、角膜及び血管を含まない
ことを特徴とするスフェア状細胞凝集体。
[11]
上記スフェア状細胞凝集体の少なくとも一部において、上記網膜色素上皮細胞の基底面が上記内側構造に向き、上記神経網膜の基底面が上記外側構造に向いている、[10]に記載のスフェア状細胞凝集体。
[12]
さらに、上記網膜色素上皮細胞と上記神経網膜が上皮構造としてつながっており、上記スフェア状細胞凝集体が、上記網膜色素上皮細胞と上記神経網膜との間に毛様体周縁部様構造体をさらに含む、[10]又は[11]に記載のスフェア状細胞凝集体。
[13]
上記毛様体周縁部様構造体が、Rdh10陽性細胞、Otx1陽性細胞、及び/又はZic1陽性細胞を含む、[12]に記載のスフェア状細胞凝集体。
[14]
上記内側構造の30%以上が神経網膜である、[10]~[13]のいずれかに記載のスフェア状細胞凝集体。
[15]
直径が0.2mm~2mmである、[10]~[14]のいずれかに記載のスフェア状細胞凝集体。
[16]
(A)多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程と、
(B)工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体を得る工程と
を含む、[10]~[15]のいずれかに記載のスフェア状細胞凝集体の製造方法。
[17]
[10]~[15]のいずれかに記載のスフェア状細胞凝集体又はその一部を含む、医薬組成物(移植用組成物、移植用組織又はTransplant)。
[18]
[10]~[15]のいずれかに記載のスフェア状細胞凝集体又はその一部を、移植を必要とする対象に移植することを含む、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の、治療方法。
[19]
網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患を治療する医薬組成物の製造における、[10]~[15]のいずれかに記載のスフェア状細胞凝集体又はその一部の使用。
[20]
網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の治療における、[10]~[15]のいずれかに記載のスフェア状細胞凝集体又はその一部の使用。
「幹細胞」とは、分化能及び増殖能(特に自己複製能)を有する未分化な細胞を意味する。幹細胞には、分化能力に応じて、多能性幹細胞(pluripotent stem cell)、複能性幹細胞(multipotent stem cell)、単能性幹細胞(unipotent stem cell)等の亜集団が含まれる。多能性幹細胞とは、インビトロにおいて培養することが可能で、かつ、三胚葉(外胚葉、中胚葉、内胚葉)及び/又は胚体外組織に属する細胞系譜すべてに分化しうる能力(分化多能性(pluripotency))を有する幹細胞をいう。複能性幹細胞とは、全ての種類ではないが、複数種の組織や細胞へ分化し得る能力を有する幹細胞を意味する。単能性幹細胞とは、特定の組織や細胞へ分化し得る能力を有する幹細胞を意味する。
本発明の一態様は、網膜系細胞又は網膜組織の製造方法を提供する。該製造方法は、下記工程を含む:
(A)多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程、
(B)工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜系細胞を含む細胞凝集体を得る工程。
工程(A)は、多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程である。工程(A)に用いられる多能性幹細胞は、維持培養・拡大培養によって得ることができる。すなわち、工程(A)は、(A-1)多能性幹細胞を維持培養・拡大培養する工程と、(A-2)工程(A-1)で得られた多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程とを含んでもよい。多能性幹細胞を得るための維持培養・拡大培養は、当業者に周知の方法で実施することができる。多能性幹細胞の維持培養・拡大培養は、接着培養でも浮遊培養でも実施することができるが、好ましくは接着培養で実施される。多能性幹細胞の維持培養・拡大培養は、フィーダー存在下で実施してもよいしフィーダーフリー条件下で実施してもよいが、好ましくはフィーダーフリー条件下で実施される。フィーダーフリー条件下では、後述する未分化維持因子を含む培地を用いることができる。
(A1)多能性幹細胞を、フィーダー細胞非存在下で、かつ任意でTGFβファミリーシグナル伝達経路阻害物質及び/又はソニック・ヘッジホッグシグナル伝達経路作用物質を含んでもよい、未分化維持因子を含む培地で培養する工程、
(A2)工程(A1)で得られた細胞を、ソニック・ヘッジホッグシグナル伝達経路作用物質を含む培地中で浮遊培養し、細胞凝集体を形成させる工程。
工程(B)は、工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜系細胞を含む細胞凝集体を得る工程である。
(A)多能性幹細胞を、フィーダー細胞非存在下で、未分化維持因子を含み、かつ任意でTGFβファミリーシグナル伝達経路阻害物質及び/又はソニック・ヘッジホッグシグナル伝達経路作用物質を含んでもよい培地で培養する工程、
(B)工程(A)で得られた細胞を、Wntシグナル伝達経路阻害物質及び/又はソニック・ヘッジホッグシグナル伝達経路作用物質を含んでいてもよい培地中で浮遊培養することによって細胞凝集体を形成させる工程、
(C)工程(B)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質(例:CHK1阻害物質)を含む培地中でさらに浮遊培養し、網膜系細胞又は網膜組織を含む細胞凝集体を得る工程、
(D)工程(C)で得られた細胞凝集体を、Wntシグナル伝達経路作用物質、及び/又は、FGFシグナル伝達経路阻害物質を含む無血清培地又は血清培地中で2日間から4日間程度の期間培養する工程、及び、
(E)工程(D)で得られた細胞凝集体を、Wntシグナル伝達経路作用物質及びFGFシグナル伝達経路阻害物質を含まず、甲状腺ホルモンシグナル伝達経路作用物質を含んでいてもよい無血清培地又は血清培地中で30日間~200日間程度培養する工程。
(A)多能性幹細胞を、フィーダー細胞非存在下で、未分化維持因子を含み、かつTGFβファミリーシグナル伝達経路阻害物質及び/又はソニック・ヘッジホッグシグナル伝達経路作用物質を含む培地で12時間~48時間培養する工程、
(B)工程(A)で得られた細胞を、Wntシグナル伝達経路阻害物質及び/又はソニック・ヘッジホッグシグナル伝達経路作用物質を含む培地中で、12時間~72日間(24時間~48時間)浮遊培養することによって細胞凝集体を形成させる工程、
(C)工程(B)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質(例:CHK1阻害物質)を含む培地中でさらに8日間~15日間(10日間~13日間)浮遊培養し、網膜系細胞又は網膜組織を含む細胞凝集体を得る工程、
(D)工程(C)で得られた細胞凝集体を、Wntシグナル伝達経路作用物質、及び/又は、FGFシグナル伝達経路阻害物質を含む無血清培地又は血清培地中で2日間から4日間培養する工程、及び、
(E)工程(D)で得られた細胞凝集体を、Wntシグナル伝達経路作用物質及びFGFシグナル伝達経路阻害物質を含まず、甲状腺ホルモンシグナル伝達経路作用物質を含んでいてもよい無血清培地又は血清培地中で10日間~200日間程度培養する工程。
(1)網膜組織を含み、かつ、目的外細胞(例:RPE細胞)を含まない、真球に近い形状を有する細胞凝集体と、
(2)上記細胞凝集体の生存能力を維持するために必要な媒体と、
を含む、培養物を提供する。
(1)マルチウェルプレート(例:192ウェル、96ウェル、48ウェル、24ウェル、12ウェル)と、
(2)当該プレートの各ウェル(少なくとも、全ウェルの50%、60%、70%、80%、90%若しくは95%以上)中に
(A)網膜組織を含み、かつ、目的外細胞(例:RPE細胞)を含まない、真球に近い形状を有する細胞凝集体と、
(B)上記移植用細胞集団の生存能力を維持するために必要な媒体と、
を含む、培養物を提供する。
本発明の一態様としてのスフェア状細胞凝集体は、網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備える。該スフェア状細胞凝集体は、
(1)上記内側構造における上記神経網膜において、少なくとも視細胞層を含む神経網膜層が形成されており、上記視細胞層は少なくとも視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞を含み、
(2)上記内側構造において、上記神経網膜が折り重なって存在しており、
(3)上記外側構造における上記網膜色素上皮細胞がRPE65陽性細胞、MITF陽性細胞、又は、RPE65陽性かつMITF陽性細胞であり、かつ
(4)上記細胞凝集体は、水晶体、硝子体、角膜及び血管を含まない
ことを特徴とする。
(A)多能性幹細胞を浮遊培養し、細胞凝集体を形成させる工程、
(B)工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体を得る工程。
ここで、工程(A)及び工程(B)等は、上述の網膜系細胞又は網膜組織の製造方法に記載のとおりである。
本発明の一態様として、本発明の細胞凝集体(段落[0108]、[0111]又は[0120]など)又はその一部を含む医薬組成物(移植用組成物、移植用組織またはTransplant)が挙げられる。医薬組成物は好ましくは、本発明の細胞凝集体又はその一部の他に、さらに医薬として許容される担体を含む。細胞凝集体の一部とは、医薬組成物に利用できる細胞凝集体の一部であり、細胞凝集体から、移植に必要な大きさの網膜組織を切り出すことによって得ることができる。
Rx::Venusレポーター遺伝子を持つように遺伝子改変したヒトES細胞(KhES-1株(非特許文献3))を、「Scientific Reports,4,3594 (2014)」に記載の方法に準じてフィーダー細胞非存在下(フィーダーフリー条件下)で培養した。フィーダー細胞非存在下での培養に用いる培地(フィーダーフリー培地)としてはStemFit培地(商品名:AK03N、味の素社製)、フィーダー細胞に代わる足場としてLaminin511-E8(商品名、ニッピ社製)を用いた。
実施例1のように、浮遊培養開始後3日目に(1)1.5nM BMP4のみ、又は(2)0.15nM BMP4に加えて1μM PD407824を添加して作製した凝集塊を浮遊培養開始後15日間(Day15)又は36日間(Day36)培養し、凝集塊を観察した。その結果、1.5nM BMP4を添加した場合、Rx::Venus陽性の凝集塊にRx::Venus陰性の細胞の塊が付着して、目的外細胞が含まれているのに対し、0.15nM BMP4にPD407824を添加した場合、Rx::Venus陰性の塊が大幅に減少し、Rx::Venus陽性のNRの分化誘導効率が高いことがわかった(図2~4)。また、BMPとPD407824を組み合わせることにより、BMPを単独で用いる場合に比べて、良好な形状(真球に近い形状)の凝集塊(網膜組織)を製造可能であることがわかった。具体的には、BMPを単独で用いる場合には、96ウェル中、真球に近い形状を有する細胞凝集体が5ウェル、クローバー型の細胞凝集体が91ウェルで認められたのに対し、PD407824を併用した場合には、96ウェル中、真球に近い形状を有する細胞凝集体が95ウェル、凝集塊形成が不良であった細胞凝集体が1ウェルで認められた。従って、製造した細胞凝集体のほぼ100%(約99%(95/96)、凝集塊形成不良を除けば100%)が真球に近い形状を有する細胞凝集体であった。
実施例1のように、浮遊培養開始後3日目に1.5nM BMP4に加えて1μM PD407824を添加して作製した凝集塊を、浮遊培養開始後21日間(Day21)培養し、観察した。その結果、外側に網膜色素上皮(RPE)細胞の層と、内側に神経網膜組織(NR)の層が局在する複層構造を備えるスフェア状細胞凝集体が作製されていることが観察された。また、良好な形状(複数の真球が重なる形状(例:クローバー))の凝集塊(網膜組織)を製造可能であることがわかった(図5)。
実施例1のように浮遊培養開始後3日目に、0.15nM、0.5nM又は1.5nMに加えて1μM又は3μM PD407824を添加し浮遊培養開始後9日間(Day9)培養し、凝集塊を観察した。その結果、0.15nM BMP4に加えてPDを1μM添加すると、凝集塊の全領域においてRx::Venus陽性の神経網膜が分化されることがわかった。一方で、BMP4を0.5nM又は1.5nMに加えてPD407824を1μM又は3μM添加すると、外側のRPE細胞の層と、CMZを多数含む極性をもった、内側の神経網膜組織の層の複層構造を備えるスフェア状細胞凝集体が作製されていることが観察された(図8)。
実施例1のように1.5nM BMP4に加えて1μM PD407824を添加する条件で、浮遊培養開始後60日間培養して作製した凝集塊を、4%PFAで固定し、20%のスクロースで置換後、凍結切片を作製した。これらの凍結切片に関し、マイクロウェーブを用いて抗原賦活化処理を行った後、DAPI、抗CHX10抗体(商品名:Anti CHX10 Antibody、EX alpha社製)及び抗CRX抗体(TaKaRa社製)を用いて免疫染色を行った。
Claims (18)
- (A)多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程と、
(B)工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜系細胞を含む細胞凝集体を得る工程と
を含む、網膜系細胞又は網膜組織の製造方法。 - 工程(B)において、BMPシグナル伝達経路作用物質が、真球に近い細胞凝集体が形成され、かつ、網膜色素上皮細胞への分化誘導が抑制されるような濃度で存在する、請求項1に記載の製造方法。
- 工程(B)で得られた細胞凝集体が、網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体であり、工程(B)において、BMPシグナル伝達経路作用物質が、前記複層構造を備えるスフェア状細胞凝集体が形成されるような濃度で存在している、請求項1に記載の製造方法。
- 前記CHK1シグナル伝達経路阻害物質がPD407824である、請求項1~3のいずれか一項に記載の製造方法。
- 前記BMPシグナル伝達経路作用物質が、BMP2、BMP4、BMP7及びGDF7からなる群から選択される1以上のタンパク質である、請求項1~4のいずれか一項に記載の製造方法。
- 工程(B)において、工程(A)の浮遊培養開始後2日目から9日目の間に前記BMPシグナル伝達経路作用物質が培地に添加される、請求項1~5のいずれか一項に記載の製造方法。
- 工程(B)において、前記CHK1シグナル伝達経路阻害物質が、前記BMPシグナル伝達経路作用物質と同時に培地に添加される、請求項1~6のいずれか一項に記載の製造方法。
- 前記CHK1シグナル伝達経路阻害物質の濃度が、0.1μMから10μMのPD407824と同程度のCHK1シグナル伝達経路阻害作用を奏する濃度である、請求項1~7のいずれか一項に記載の製造方法。
- 工程(B)で得られた細胞凝集体から、移植に必要な大きさの網膜組織を切り出す工程をさらに含む、請求項1~8のいずれか一項に記載の製造方法。
- 網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体であって、
(1)前記内側構造における前記神経網膜において、少なくとも視細胞層を含む神経網膜層が形成されており、前記視細胞層は少なくとも視細胞、視細胞前駆細胞及び網膜前駆細胞からなる群から選択される1以上の細胞を含み、
(2)前記内側構造において、前記神経網膜が折り重なって存在しており、
(3)前記外側構造における前記網膜色素上皮細胞がRPE65陽性細胞、MITF陽性細胞、又は、RPE65陽性かつMITF陽性細胞であり、かつ
(4)前記細胞凝集体は、水晶体、硝子体、角膜及び血管を含まない
ことを特徴とするスフェア状細胞凝集体。 - 前記スフェア状細胞凝集体の少なくとも一部において、前記網膜色素上皮細胞の基底面が前記内側構造に向き、前記神経網膜の基底面が前記外側構造に向いている、請求項10に記載のスフェア状細胞凝集体。
- さらに、前記網膜色素上皮細胞と前記神経網膜が上皮構造としてつながっており、前記スフェア状細胞凝集体が、前記網膜色素上皮細胞と前記神経網膜との間に毛様体周縁部様構造体をさらに含む、請求項10又は11に記載のスフェア状細胞凝集体。
- 前記毛様体周縁部様構造体が、Rdh10陽性細胞、Otx1陽性細胞、及び/又はZic1陽性細胞を含む、請求項12に記載のスフェア状細胞凝集体。
- 前記内側構造の30%以上が神経網膜である、請求項10~13のいずれか一項に記載のスフェア状細胞凝集体。
- 直径が0.2mm~2mmである、請求項10~14のいずれか一項に記載のスフェア状細胞凝集体。
- (A)多能性幹細胞を浮遊培養し、多能性幹細胞の細胞凝集体を形成させる工程と、
(B)工程(A)で得られた細胞凝集体を、BMPシグナル伝達経路作用物質及びCHK1シグナル伝達経路阻害物質の存在下に浮遊培養し、網膜色素上皮細胞を含む外側構造と、神経網膜を含む内側構造との複層構造を備えるスフェア状細胞凝集体を得る工程と
を含む、請求項10~15のいずれか一項に記載のスフェア状細胞凝集体の製造方法。 - 請求項10~15のいずれか一項に記載のスフェア状細胞凝集体又はその一部を含む、医薬組成物。
- 請求項10~15のいずれか一項に記載のスフェア状細胞凝集体又はその一部を、移植を必要とする対象に移植することを含む、網膜系細胞若しくは網膜組織の障害又は網膜組織の損傷に基づく疾患の、治療方法。
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