Method for purifying retinal pigment epithelial cells

Abstract

Provided is a method for purifying retinal pigment epithelial cells, with which high-purity retinal pigment epithelial cells can be obtained from a cell population obtained by inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells through a simple and easy operation in a short time. The purification method comprises a step of introducing a cell population containing retinal pigment epithelial cells onto a filter and obtaining a cell population passing through the filter, the cell population being obtained by inducing differentiation of pluripotent stem cells on laminin or fragments thereof.

Description

[0001] This application is a divisional application of an invention patent application filed on October 9, 2014, with application number 201480055588.9 and invention name “Method for purifying retinal pigment epithelial cells”. Technical Field

[0002] The present invention relates to a method for purifying retinal pigment epithelial cells from a cell population obtained by inducing pluripotent stem cells to differentiate into retinal pigment epithelial (RPE) cells, a method for producing retinal pigment epithelial cells using the above method, and the like. Background Art

[0003] As a method for producing retinal pigment epithelial cells from pluripotent stem cells, the following methods are known: the method comprising the ES cell (patent documentation 1 etc.) called SFEB method cultivated as suspension aggregates in serum-free medium, the method (non-patent documentation 1 etc.) of inducing the differentiation of pluripotent stem cells in the presence of differentiation inducing factors on a culture substrate coated with a weak cell adhesion coating agent etc. However, due to low differentiation induction efficiency, these methods need to combine the multiple steps of adhesion culture and suspension culture to obtain a cell colony of highly concentrated retinal pigment epithelial cells, and have the following problems, such as needing to have a purification step with high workload and for a long time, including the colony of selectively picking pigment cells under an optical microscope. These methods can only obtain a part of induced RPE cells in a culture vessel, and the purity of the obtained cells is greatly affected by the technique of the experimenter, which makes these methods problematically unsuitable for large-scale production. Therefore, it has been required that even when large-scale production of RPE cells, a method for stably obtaining highly purified retinal pigment epithelial cells can be obtained by a simple and easy method.

[0004] For the maintenance culture of human pluripotent stem cells, a method using an extracellular matrix instead of feeder cells has been widely used. Among them, laminin is preferably used, and, for example, Non-Patent Document 2 reports the long-term successful maintenance culture of human ES cells on laminin-511. As for the E8 fragment of laminin called the modified one with improved cell adhesion activity, for example, Patent Document 2 and Non-Patent Document 3 disclose a method for culturing human pluripotent stem cells, which uses the E8 fragment of human laminin-α5β1γ1 (laminin-511E8, hereinafter indicated in the same manner) and human laminin-322E8. Non-Patent Document 4 records that laminin-511E8 maintains the binding activity to α6β1 integrin, which is the same level as the full-length laminin-511, and Patent Document 2 describes that by using the laminin-511E8, pluripotent stem cells can be stably immobilized on a culture dish, with the result that the cells can be maintained and cultured while maintaining differentiation pluripotency. However, there are no reports describing the use of such a laminin E8 fragment other than for pluripotent stem cell culture, for example, differentiation induction of pluripotent stem cells.

[0005] On the other hand, as a method for inducing human pluripotent stem cells to differentiate into retinal pigment epithelial cells in the absence of feeder cells, a method using laminin is known. For example, non-patent literature 5 describes that by making pluripotent stem cells adhere to culture on laminin-111 and matrigel, the differentiation induction efficiency of differentiation into retinal pigment epithelial cells is significantly increased. However, there is no report recording the use of the E8 fragment of laminin for pluripotent stem cells to be induced to differentiate into retinal pigment epithelial cells.

[0006] [Reference list] Patent Literature Patent Document 1: WO 2005 / 123902 Patent Document 2: WO 2011 / 043405 [Non-patent literature] Non-patent literature 1: PLoS One. 2012; 7(5): e37342. Non-patent literature 2: Nature Biotech. June 2010; 28(6): 611-5 Non-patent literature 3: Nat. Commun. 3:1236 doi: 10.1038 / ncomms2231 Non-patent literature 4: J Biol Chem. 284:7820-7831, 2009 Non-patent literature 5: J. Tissue Eng Regen Med 2013; 7: 642-653. Summary of the invention

[0007] Technical Problems to be Solved by the Invention An object of the present invention is to provide a method for purifying highly pure retinal pigment epithelial cells from a cell population obtained by inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells in a short time by a simple and easy operation.

[0008] Solutions to technical problems The inventor has conducted in-depth research to attempt to achieve the above purpose, and found that when human pluripotent stem cells are cultured on a culture matrix coated with laminin-E8, the inoculated pluripotent stem cells rapidly adhere to the culture matrix, and a large amount of pigment cells are produced from the early stage, and the yield of retinal pigment epithelial cells can be significantly improved, and not only retinal pigment epithelial cells, but also other visual cell lineage cells are produced together with matrix components. The inventor has further found that retinal pigment epithelial cells can be effectively purified from the retinal pigment epithelial cells produced on laminin, even if they are buried in other cells and matrix components, by introducing all these simple operations on the filter. They have further found that low recovery, which is a problem in inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells, can be significantly improved, and required retinal pigment epithelial cells can be conveniently and stably purified. The inventor has since conducted in-depth research and completed the present invention.

[0009] That is, the present invention relates to the following contents.

[0010] [1] A method for purifying retinal pigment epithelial cells, comprising the steps of introducing a cell population containing retinal pigment epithelial cells onto a filter and obtaining a cell population that passes through the filter, wherein the cell population is obtained by inducing differentiation of pluripotent stem cells on laminin or a fragment thereof.

[0011] [2] The method of [1] above, wherein the laminin or its fragment is a laminin-E8 fragment.

[0012] [3] The method of [1] or [2] above, wherein the cell population containing the retinal pigment epithelial cells is recovered by treating with a cell separation solution after differentiation induction.

[0013] [4] The method of the above-mentioned [3], wherein the cell detachment solution contains trypsin.

[0014] [5] The method of any one of [1] to [4] above, wherein the filter has a pore size of 20-70 μm.

[0015] [6] A method for producing retinal pigment epithelial cells from pluripotent stem cells, comprising: (1) a step of obtaining a cell population containing retinal pigment epithelial cells by inducing differentiation of pluripotent stem cells on laminin or a fragment thereof; and (2) A step of introducing the cell population obtained in (1) onto a filter to obtain a cell population that has passed through the filter.

[0016] [7] The method of [6] above, wherein the laminin or its fragment is a laminin-E8 fragment.

[0017] [8] The method of [6] or [7] above, wherein the cell population obtained in step (1) is recovered by treating the cell population containing retinal pigment epithelial cells with a cell separation solution.

[0018] [9] The method of the above-mentioned [8], wherein the cell detachment solution contains trypsin.

[0019]

[10] The method of any one of [6] to [9] above, wherein the filter has a pore size of 20-70 μm.

[0020]

[11] Retinal pigment epithelial cells obtained by the method of any one of [6] to

[10] above.

[0021]

[12] A retinal pigment epithelial cell sheet obtained by the method of any one of [6] to

[10] above.

[0022] Effects of the Invention According to the present invention, retinal pigment epithelial cells induced from pluripotent stem cells can be conveniently purified with high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram showing the structure of laminin-E8.

[0024] Figure 2 Shown are the expressions of RPE-related genes of RPE cells induced from human iPS cells (201B7 and 1120C7).

[0025] Figure 3 The status of cells cultured immediately before purification is shown.

[0026] Figure 4 The expression of retinal pigment epithelial cell marker genes, progenitor cell and neural cell marker genes before and after purification with filters is shown. The values ​​are relative values ​​± standard deviation (n = 1, 3 replicates), and the expression level before filtering is 1. Specific implementation plan

[0027] 1. Purification Method and Production Method of Retinal Pigment Epithelial Cells The present invention relates to a method for purifying retinal pigment epithelial cells (hereinafter also referred to as the purification method of the present invention), comprising introducing a cell population containing retinal pigment epithelial cells onto a filter, wherein the cell population is obtained by differentiation induction of pluripotent stem cells on laminin or a fragment thereof. The present invention also relates to a method for producing retinal pigment epithelial cells from pluripotent stem cells (hereinafter also referred to as the production method of the present invention), characterized in that the method is used. The production method of the present invention comprises the following two main separate steps: (1) a step of obtaining a "cell population containing retinal pigment epithelial cells" by inducing differentiation of pluripotent stem cells on laminin or a fragment thereof; and (2) A step of introducing the "cell population containing retinal pigment epithelial cells" obtained in (1) into a filter to purify the retinal pigment epithelial cells. The purification method of the present invention is directed to the step (2). Each step is explained in detail below.

[0028] (1) Steps for obtaining a cell population containing retinal pigment epithelial cells In step (1), pluripotent stem cells are induced to differentiate by adhesion culture using a culture substrate coated with laminin or a fragment thereof, and a cell population containing retinal pigment epithelial cells is obtained.

[0029] The "pluripotent stem cells" in the present invention refer to stem cells that have the ability to self-replicate and differentiate into pluripotent stem cells, and are not particularly limited. For example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. are widely used. Preferably, human ES cells or human iPS cells are used, and more preferably, human iPS cells are used.

[0030] The "iPS cells" in the present invention refer to cells that have artificially acquired self-replication ability and differentiation pluripotency by contacting nuclear reprogramming factors with somatic cells (e.g., fibroblasts, skin cells, lymphocytes, etc.). The method for producing iPS cells in the present invention is not particularly limited.

[0031] In the present invention, as pluripotent stem cells, pluripotent stem cells derived from mammals can be used. Although the mammals are not particularly limited, humans are preferred for clinical use.

[0032] "Retinal pigment epithelial cells" in the present invention refer to epithelial cells constituting the retinal pigment epithelium, and their progenitor cells. Whether it is a retinal pigment epithelial cell can be confirmed by, for example, the expression of cell markers (RPE65, CRALBP, MERTK, BEST1, etc.), cell morphology (intracellular melanin dye deposition, polygonal and flat cell morphology, polygonal actin bundles, etc.). The progenitor cells of retinal pigment epithelial cells refer to cells that are directional induced to differentiate into retinal cells, and whether they are progenitor cells can be confirmed by the expression of cell markers (Mitf (pigment cells, progenitor cells), Pax6 (progenitor cells), Rx (retinal progenitor cells), Crx (photoreceptor precursor cells), Chx10 (bipolar cells), etc.). The functional evaluation of retinal pigment epithelial cells can be confirmed using, for example, the secretion ability of cytokines (VEGF, PEDF, etc.), phagocytic ability, etc. as indicators. These functional evaluation and confirmation operations can be performed by those skilled in the art by setting appropriate conditions.

[0033] In the present invention, "laminin" is a heterotrimeric molecule composed of α, β, and γ chains, and is an extracellular matrix protein containing subtypes having different subunit chain compositions. Specifically, laminin has about 15 subtypes, including heterotrimers of combinations of 5 α chains, 4 β chains, and 3 γ chains. The name of laminin is determined by combining the respective numbers of α chains (α1-α5), β chains (β1-β4), and γ chains (γ1-γ3). For example, laminin composed of a combination of α1 chain, β1 chain, and γ1 chain is named laminin 111, laminin composed of a combination of α5 chain, β1 chain, and γ1 chain is named laminin-511, and laminin composed of a combination of α5 chain, β2 chain, and γ1 chain is named laminin-521. As laminin, for example, mammalian laminin can be used. The example of mammal comprises mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cattle, horse, goat, monkey and people.When producing retinal pigment epithelial cell for transplanting into the purpose of people, preferably use human laminin etc.At present stage, known human laminin comprises 15 kinds of subtypes.

[0034] As the laminin in the present invention, any subtype can be used. For example, it is preferred to use a laminin subtype that shows binding specificity to at least one of the integrins expressed on the surface of human pluripotent stem cells / or human retinal pigment epithelial cells, preferably a laminin subtype that shows binding specificity to integrins expressed on the surface of human pluripotent stem cells and human retinal pigment epithelial cells and integrins expressed on the surface of human retinal pigment epithelial cells. Examples of integrins expressed on the surface of human pluripotent stem cells include α6β1 integrin, etc., and examples of integrins expressed on the surface of human retinal pigment epithelial cells include α6β1 integrin, α3β1 integrin, α7β1 integrin, etc. According to the above, as a preferred laminin, it is preferred to use a laminin that shows binding specificity to α6β1 integrin and can stably adhere to pluripotent stem cells in the initial stage of differentiation induction and retinal pigment epithelial cells or their progenitor cells in the later stage of differentiation induction. According to such an aspect, laminin-511 and laminin-521 are preferred among the laminin subtypes. Laminin-511 has binding specificity to α6β1 integrin as well as α3β1 integrin and α7β1 integrin, and laminin-521 has binding specificity to α6β1 integrin and stronger binding specificity to α3β1 integrin. Therefore, by improving the adhesion activity to retinal pigment epithelial cells, these subtypes contribute to the improvement of the induction efficiency of pluripotent stem cells to differentiate into retinal pigment epithelial cells, or the stabilization of the maintenance culture of retinal pigment epithelial cells.

[0035] The laminin fragments in the present invention may be any, as long as they retain the functions of the respective laminins. That is, in the present invention, the "laminin fragments" are not limited in terms of the length of the respective chains, as long as they are molecules having laminin α chain, β chain and γ chain constituting heterotrimers, retain binding activity to integrins, and maintain cell adhesion activity. The laminin fragments show integrin binding specificity, which varies with the original laminin subtype, and can exert adhesion activity to cells expressing the corresponding integrins. As such a laminin fragment, a laminin-E8 fragment is preferred.

[0036] The laminin-E8 fragment was originally one of the fragments obtained by digesting mouse laminin-111 with elastase, and was identified as a fragment having strong cell adhesion activity (EMBO J., 3: 1463-1468, 1984., J. Cell Biol., 105: 589-598, 1987). When digested with elastase, it is assumed that in addition to mouse laminin-111, a fragment corresponding to the E8 fragment of mouse laminin-111 is present in laminin. However, except for mouse laminin-111, the separation and identification of the E8 fragment by laminin digestion with elastase has not been reported so far. Therefore, the laminin E8 fragment used in the present invention does not need to be a product of elastase digestion of each laminin, but may be recombinant as long as it is a laminin fragment having a cell adhesion activity similar to that of each corresponding laminin and having a structure corresponding to the E8 fragment digested with elastase. That is, the "laminin-E8 fragment (hereinafter sometimes referred to as "laminin-E8")" in the present invention refers to a molecule constituting a heterotrimer at each C-terminal region of the α chain, β chain, and γ chain, which retains binding activity to integrin, and retains cell adhesion activity. Laminin-E8 shows integrin binding specificity that varies for each laminin subtype, and can exert a strong adhesion activity to cells expressing the corresponding integrin.

[0037] When specifically stated, laminin-E8 in the present invention is a laminin fragment having the following: (1) functionally, at least equivalent to full-length laminin in terms of cell adhesion activity and at least equivalent integrin binding activity, and (2) Structurally, it corresponds to the structure of mouse laminin-E8, specifically, the structure from the coiled-coil C-terminal region to the 1st to 3rd G domains of the laminin trimer. The laminin fragment, particularly laminin-E8, is described in more detail below from the functional aspects of (1) and the structural aspects of (2).

[0038] (1) Functions of laminin fragments Examples of laminin fragments of the present invention include molecules that show binding specificity to at least one of the integrins expressed on the surface of human pluripotent stem cells and / or human retinal pigment epithelial cells, preferably molecules that show binding specificity to integrins expressed on the surface of human pluripotent stem cells and human retinal pigment epithelial cells and integrins expressed on the surface of human retinal pigment epithelial cells. These integrins are as described above.

[0039] The laminin fragments in the present invention show a binding specificity for integrins, preferably a binding specificity at least equivalent to each corresponding laminin. Preference is given to using laminin fragments that show a particularly strong affinity for integrins. "Laminin fragments that show a particularly strong affinity for integrins" are laminin fragments that show a significantly low dissociation constant, as measured by known methods, and for example by, for example, the dissociation constants measured by the method shown in Table 1 of The Journal of Biological Chemistry (2009) 284, pp. 7820-7831.

[0040] As the laminin fragment of the present invention, a laminin fragment having cell adhesion activity, preferably a strong cell adhesion activity is used. A "laminin fragment having strong cell adhesion activity" is a laminin fragment that shows significantly strong adhesion activity in a cell adhesion test, the test using measurement by a known method, and the coating concentration of the fragment not exceeding 10 nM shows not less than 400 cells / mm 2 When performing a cell adhesion assay as described in The Journal of Biological Chemistry (2007) 282, pp. 11144-11154, for example.

[0041] In the present invention, as the laminin fragment, it is preferred to use a laminin fragment that shows binding specificity to α6β1 integrin, can stably adhere to pluripotent stem cells in the initial stage of differentiation induction, and can stably adhere to retinal pigment epithelial cells or progenitor cells thereof in the later stage of differentiation induction. From such an aspect, fragments of laminin-511 or laminin-521 or the like are preferred among the laminin fragments, and laminin-511 E8 and laminin-521 E8 are particularly preferred. In addition to α6β1 integrin, laminin-511 E8 also has binding specificity to α3β1 integrin and α7β1 integrin, and laminin-521 E8 has binding specificity to α6β1 integrin and stronger binding specificity to α3β1 integrin in addition to α6β1 integrin. Therefore, such laminin-E8 can promote the improvement of differentiation induction efficiency of pluripotent stem cells into retinal pigment epithelial cells by enhancing adhesion activity to retinal pigment epithelial cells. Alternatively, such laminin-E8 can promote the stabilization of maintenance culture of retinal pigment epithelial cells.

[0042] (2) Structure of laminin fragments In the present invention, the "laminin fragment" is not limited in terms of the length of each chain, as long as it is a molecule having laminin α chain, β chain and γ chain constituting a heterotrimer, retains binding activity to integrin, and maintains cell adhesion activity. Such a laminin fragment can be appropriately designed by a person skilled in the art who understands the structure of each domain of laminin, etc. Regarding such a laminin fragment, as described above, preferably, as described above or as Figure 1 As shown, the structure corresponds to a fragment of laminin (E8 fragment) having cell adhesion activity in the elastase digestion product of mouse laminin-111. That is, it retains a portion of domain II (three-stranded coiled-coil domain) of full-length laminin (in Figure 1 The E8 fragment depicted in FIG. 1 does not show such a pattern, but actually maintains a coiled coil structure), and forms a short coiled coil structure with the corresponding fragment of the β chain and the corresponding fragment of the γ chain on the N-terminal side of E8. On the C-terminal side of E8, the G1-G3 domain structure of the α chain is maintained. The β chain and the γ chain are bound to each other by forming a disulfide bond via a cysteine ​​residue on each C-terminal side.

[0043] As described above with respect to laminin-E8, the laminin fragment in the present invention may be an enzyme-treated product obtained by treating natural laminin with elastase, or a recombinant produced by gene recombination.

[0044] When the laminin or its fragment of the present invention is a recombinant, a tag may be bonded to the N-terminus for purification purposes, etc., as long as the binding activity of the corresponding full-length (native) laminin to integrin is maintained and the cell adhesion is not impaired. Such a tag is not particularly limited, and for example, His tag, Flag tag, HA tag, etc. can be mentioned. In addition, the sequence of the linker region between the tag and the laminin or its fragment is not particularly limited, as long as the binding activity of the corresponding full-length (native) laminin is maintained and the cell adhesion is not impaired.

[0045] In the laminin or fragments thereof of the present invention, a part of the amino acid sequence may be deleted, added or substituted as long as the binding activity of the corresponding laminin to integrin is maintained and the cell adhesion is not impaired.

[0046] Although the E8 fragment generally lacks two G domains (G4 and G5) on the C-terminal side of the α chain, the G5 and G5 domains may be partially or completely included in the laminin-E8 of the present invention, as long as the binding activity of the corresponding full-length (native) laminin to integrin is maintained and its cell adhesion is not impaired. For example, the G4 and G5 domains may be partially or completely included in the laminin-511 E8, as long as the binding activity of laminin-511 to integrin α6β1 at an equivalent level is maintained and the cell adhesion activity is not impaired.

[0047] Similar to full-length laminin, the β chain and γ chain of laminin-E8 are bonded via cysteine ​​on the C-terminal side of the coiled coil portion. Since cysteine ​​affects integrin binding activity, it is desirable that it is not substituted or deleted. In addition, since the C-terminal amino acid after the cysteine ​​in the γ chain also affects integrin binding activity, it is also desirable that it is at least not deleted (J Biol Chem. 2007 Apr 13; 282(15): 11144-54.).

[0048] Specific examples of such laminin-E8 include rhLM511E8 produced in Example (3) of WO2011 / 043405. In the present invention, the laminin-511E8 can be preferably used as laminin-E8.

[0049] "Cultivation matrix" used in the present invention can be produced by coating the surface of the culture vessel with the laminin or its fragment in the present invention. As used herein, the surface of the "coated" culture vessel means that laminin or its fragment is adsorbed on the culture vessel surface by some interactions between laminin or its fragment and the culture vessel surface, wherein the orientation of the laminin or its fragment does not cause special problems for realizing the effect of the present invention. The culture vessel is not particularly limited, as long as it can be used for cell culture, and for example, dish (also referred to as culture dish), covered culture dish and plate (microtiter plates, microplates, deep well plates, etc. of 6 holes, 24 holes, 48 ​​holes, 96 holes, 384 holes, 9600 holes, etc.), bottle, slide, pipe, cell factory, roller bottle, rotating bottle, hollow fiber, microcarrier, pearl, etc. The culture matrix in the present invention can be applied with appropriate surface treatment, as long as the cell adhesion of laminin or its fragment is not damaged.

[0050] "Adhesion culture" in the present invention refers to culture in a state in which the target cells adhere to the bottom of the culture vessel via laminin or a fragment thereof and do not float in the culture medium even when the culture vessel is gently shaken during culture. Since the laminin or a fragment thereof used in the present invention can show very excellent cell adhesion, the cells after cell inoculation are preferably uniformly dispersed by a method including rapidly shaking the culture vessel, etc. The target cells may be subjected to suspension culture in a culture vessel containing laminin or a fragment thereof before or after adhesion culture, as long as the purpose of the present invention can be achieved.

[0051] The culture medium is composed of a basal medium, serum and / or serum replacement, and other components. As the basal medium, one or more synthetic culture media commonly used for culturing mammalian cells can be used in combination, and for example, commercially available products such as DMEM, GMEM, etc. can be obtained.

[0052] As the serum, serum derived from mammals such as cattle, humans, pigs, etc. can be used. Serum substitutes are low-protein substitutes for replacing serum such as fetal bovine serum, etc. for cell culture, and commercially available products such as knockout serum substitute (KSR), chemically defined lipid concentrate (manufactured by Gibco), Glutamax (manufactured by Gibco), etc., as well as serum substitutes N2, B27, etc. for neural cell culture can be obtained. Preferably, an appropriate amount of purified or recombinantly produced albumin and cytokines, further lipid mixtures, etc. can also be independently combined with the basal medium. In the present invention, serum substitutes are preferred, and KSR is particularly preferred from the aspect of quality management of target cells.

[0053] The concentration of serum or serum substitute can be appropriately set within the range of, for example, 0.5-30% (volume / volume). The concentration may be constant, or gradually changed. For example, the concentration may be reduced in stages at intervals of about 1-3 days (preferably 2 days). For example, serum or serum substitute may be added in 3 stages of concentrations of 20%, 15% and 10%.

[0054] As other components constituting the culture medium, Rho kinase inhibitors such as Y-27632 can be used to inhibit cell death of human pluripotent stem cells dispersed in the culture medium. The Rho kinase inhibitor can be added during a portion of the differentiation induction step or the entire period. For example, unnecessary cells that have not differentiated into target cells can be removed by cell death in the later stage of the differentiation induction step by using a culture medium that does not contain a Rho kinase inhibitor.

[0055] In addition to the above components, the culture medium may contain other components commonly used for culturing mammalian cells.

[0056] The concentration of human pluripotent stem cells used in the production method of the present invention is not particularly limited as long as the pluripotent stem cells can be uniformly seeded and adhesion culture is possible. For example, when a 10 cm dish is used, it is 1×10 5 -1×10 8 cells, preferably 2×10 6 -5×10 7 cells, more preferably 5×10 5 -9×10 6 cells.

[0057] Adhesion culture in the production method of the present invention can also be carried out in the presence of a differentiation inducing factor. As the differentiation inducing factor, a factor known as a factor promoting differentiation induction into a target cell can be utilized. Since the production method of the present invention includes differentiation induction into retinal pigment epithelial cells, it is desirable to use a differentiation inducing factor that differentiates into retinal pigment epithelial cells. Examples of differentiation inducing factors that differentiate into retinal pigment epithelial cells include Nodal signal inhibitors, Wnt signal inhibitors, Sonic hedgehog signal inhibitors, and activin signal promoters, etc.

[0058] The Nodal signal inhibitor is not particularly limited as long as it can inhibit signal transduction mediated by Nodal, and proteins, nucleic acids, low molecular weight compounds, etc. can be used. Examples of Nodal signal inhibitors include proteins, peptides or nucleic acids such as Lefty-A, soluble Nodal receptors, anti-Nodal antibodies, Nodal receptor inhibitors, etc.; low molecular weight compounds such as SB431542, etc. In particular, low molecular weight compounds such as SB431542, etc., which are easily available and show less difference between batches, are preferred.

[0059] The Wnt signal inhibitor is not particularly limited, and as long as it can inhibit signal transduction mediated by Wnt, proteins, nucleic acids, low molecular weight compounds, etc. can be used. Examples of Wnt signal inhibitors include proteins, peptides or nucleic acids such as DKK1, Cerberus protein, Wnt signal receptor inhibitors, soluble Wnt receptors, Wnt antibodies, casein kinase inhibitors, dominant negative Wnt proteins, etc.; and low molecular weight compounds such as CKI-7 (N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide), D4476 (4-{4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide), IWR-1-endo (IWR1e), IWP-2, etc. Specifically, low molecular weight compounds that are easily available and show less variation between batches are preferred. Among them, low molecular weight compounds having selective casein kinase I inhibitory activity are preferred, and for example, CKI-7, D4476 and the like can be used.

[0060] Examples of the activin signaling promoter include proteins belonging to the activin family, activin receptors, activin receptor agonists, and the like.

[0061] The concentration of these differentiation-inducing factors can be appropriately selected according to the type of differentiation-inducing factors. Specifically, when SB431542 is used as a Nodal signaling inhibitor, the concentration is, for example, 0.01-50 μM, preferably 0.1 - 10 μM, more preferably 5 μM; when CKI-7 is used as a Wnt signaling inhibitor, it is added at a concentration of 0.01 - 30 μM, preferably 0.1 - 30 μM, more preferably 3 μM.

[0062] In the production method of the present invention, it is preferable to use a combination of a Nodal signaling inhibitor (eg, SB431542) and a Wnt signaling inhibitor (eg, CKI-7) as the differentiation-inducing factor.

[0063] According to the culture induction of the above method, pluripotent stem cells are differentiated into retinal pigment epithelial cells, so that retinal pigment epithelial cells can be generated usually at the 25th-45th day of inoculation of pluripotent stem cells. The generation of retinal pigment epithelial cells can be confirmed according to the above method. When the generation of retinal pigment epithelial cells is confirmed, the culture medium is exchanged with the culture medium for maintaining retinal pigment epithelial cells, and, for example, the cells are preferably further cultivated for 5-10 days, while the total amount of the culture medium is exchanged at a frequency of no more than once in 3 days. As a result, the cell colony and the polygonal flat cell colony of the melanin dye deposition adhering to the basal laminin can be more clearly observed.

[0064] As maintenance medium for retinal pigment epithelial cells, for example, those described in IOVS, March 2004, Vol. 45, No. 3, Masatoshi Haruta, et al., IOVS, November 2011, Vol. 52, No. 12, Okamoto and Takahashi, J. Cell Science 122 (17), Fumitaka Osakada, et al., IOVS, February 2008, Vol. 49, No. 2, Gamm, et al., which consist of a basal medium, serum and / or serum substitute, and other components. As a basal medium, one or more synthetic culture media commonly used for culturing mammalian cells can be used in combination, and for example, commercially available products such as DMEM, GMEM, etc. can be obtained.

[0065] As the serum, serum derived from mammals such as cattle, humans, pigs, etc. can be used. Serum substitutes are low-protein substitutes for replacing serum such as fetal bovine serum for cell culture, and commercial products such as knockout serum substitute (KSR), chemically defined lipid concentrate (manufactured by Gibco), Glutamax (manufactured by Gibco) and the like, as well as serum substitutes N2 and B27 for neural cell culture, etc. can be obtained. In the present invention, serum substitutes are preferred, and B27 is particularly preferred from the aspect of quality management of target cells.

[0066] Examples of other components include L-glutamine, sodium penicillin, streptomycin sulfate and the like.

[0067] The cell colony containing the retinal pigment epithelial cells obtained by culture according to the present invention may include, in addition to the retinal pigment epithelial cells, cells such as visual cell-like cells, neuron-like cells, glial cells, Muller cells, amacrine cells, bipolar cells, horizontal cells, ganglion cells, and various matrix components such as polysaccharides, phospholipids and adhesion proteins. More specifically, in the culture according to the present invention, after the inoculation of pluripotent stem cells, the cells are rapidly adhered and fixed to the culture vessel via laminin-E8, which is excellent in cell adhesion, and the cells are layered and cover the cells adhered to the culture vessel in large quantities to form a cell layer showing various forms, that is, a composite cell layer structure is formed about 20 days from the inoculation of pluripotent stem cells, in which the cell layer with a slight elevation and covering the whole is the base, and a tubular and cable structure or a spider web or round cell group is formed thereon. After the structure is formed, by further continuing the culture, retinal pigment epithelial cells can be induced and generated, while being buried in a viscous substance (jelly-like substance), which apparently seems to be a mixture of visual cells and extracellular matrix components such as various matrix proteins, polysaccharides and phospholipids.

[0068] (2) A step of purifying retinal pigment epithelial cells by introducing the "cell population containing retinal pigment epithelial cells" obtained in (1) onto a filter In this step, in order to purify retinal pigment epithelial cells from the "cell population containing retinal pigment epithelial cells" obtained in (1), the cell population is introduced onto a filter as described below.

[0069] It is desirable to obtain a cell population containing retinal pigment epithelial cells by differentiation induction of pluripotent stem cells as described above, and the cell population may also contain, in addition to the target retinal pigment epithelial cells, unwanted retinal progenitor cells, visual cells and neural cells at different stages of differentiation induction, as well as extracellular matrix components assumed to have been produced by these cells.

[0070] For example, in the SFEB method described in WO 2005 / 123902, it is necessary to culture stem cells in a suspended state in a dedicated serum-free medium without supporting cells to form aggregates, perform differentiation induction of immature neural progenitor cells and recover retinal epithelial cell clusters from non-adherent cell aggregates formed by these cells under an optical microscope. Even in a method similar to this method using weak cell adhesion coating agents such as poly-L-lysine and gelatin (modified SFEB method (described in US20130224156 A1)), a cell population containing retinal pigment epithelial cells is dissociated to a certain extent after differentiation induction, and cell aggregates are formed by suspension culture once, and cell aggregates with strong melanin productivity are selected under a microscope according to the color development level or black-brown distribution, and the cell aggregates are further subjected to adhesion culture, and cultured until sufficient purity and cell number are reached, while irregularly shaped or heterologous cells are manually removed under a microscope when they grow, and then recovered. In this traditional method without using laminin or its fragment as a matrix, the occurrence rate of retinal pigment epithelial cells is extremely low. Even when they are subjected to suspension culture and grown to form cell aggregates, it is necessary to select cells other than target cells to be manually removed from each cell aggregate because the target retinal pigment epithelial cells integrate with cells other than target cells to form tissues.

[0071] Therefore, by conventional known methods, when inducing differentiation of retinal pigment epithelial cells from pluripotent stem cells, not only the target retinal pigment epithelial cells, but also cells other than the target cells are usually obtained at the same time. In addition, the complex and specialized steps of directly taking out and purifying retinal pigment epithelial cells by hand, because the retinal pigment epithelial cell colony (cell aggregates showing melanin black-brown) can be visually confirmed, but the amount is small, and it is integrated with the cell aggregates to form tissue.

[0072] In the purification method or production method of the present invention, since laminin or its fragment is used as a matrix for differentiation induction, the adhesion of iPS cells to the matrix is ​​obviously dense. However, in the final stage of the differentiation induction step, not only retinal pigment epithelial cells but also cells other than the target cells are obtained in large quantities. By observing under an optical microscope, a cell population containing retinal pigment epithelial cells and a mixture of visual cells other than the target cells and extracellular matrix components assumed to have been produced by these cells (the above-mentioned jelly-like substance) are obtained at the same time, and the cell population is obviously buried in these mixtures ( Figure 3 ). Therefore, as in the above-mentioned modified SFEB method, they also need to be directly harvested under a microscope, and it is not possible to conveniently and efficiently obtain high-purity retinal pigment epithelial cells, where it is not conceivable to purify retinal pigment epithelial cells by simply introducing them on a filter.

[0073] However, by careful observation by the inventor, it was found that due to the strong adhesion to the laminin or its fragment as the matrix, the retinal pigment epithelial cells of differentiation induction were not simply buried in the multilayer cell mixture, but were present below the jelly-like material (i.e. on the laminin or its fragment) containing other visual cells, and did not form a mixture with the jelly-like material. According to such an existence mode of the cell colony, the idea of ​​the purification method by introducing the retinal pigment cells into the filter was obtained. This purification method does not require complicated, specialized and technical purification steps as conventional methods, and is convenient, efficient and extremely useful because it can be used for general purposes.

[0074] For introduction on the filter, the obtained cell colony is detached from the incubator. The detachment can be carried out by physically scraping the cells from the contact surface with a pipette or cell scraper, etc., or by treating with proteases such as trypsin, collagenase, dispase, etc. Preferably, the detachment is carried out by treating with proteases such as trypsin, collagenase, dispase, etc. These treatments can be carried out according to methods known per se.

[0075] The cell colony after the above-mentioned breakaway is preferably processed, to dissociate the adhesion between the cells to a certain extent, and then introduced into the filter, so that the jelly-like material covered will not fill the hole of the filter. Can for example, by reciprocating suction beat the cell colony several times to process. Excessive cell dissociation processing is undesirable, because it destroys the jelly-like material. In addition, it is desirable to remove the protease solution and its residual impurities in the cell mixture, and matrix components etc. together with the supernatant by centrifugation.

[0076] The cell colony obtained as described above is recovered and introduced on the filter. According to the method of the present invention, the retinal pigment epithelial cells pass through the filter, a mixture containing other cells (e.g., visual cells, etc.) and various matrix components can be captured on the filter, and the retinal pigment epithelial cells can be purified. The filter treatment in the present invention is not particularly limited to the form and steps of the filter, etc., as long as it can achieve the goal. The filter treatment is explained in detail below.

[0077] As the material of the filter, those materials commonly used for filtering cell culture media can be used. For example, it is a synthetic polymer selected from at least one of the following: polyester, polypropylene, polystyrene, acrylic acid, rayon, polyolefin, vinylon, polyethylene, nylon, polyurethane, etc. Nylon, etc., which show low polarity and little protein adsorption, is preferred. It is difficult to use materials that show high polarity and high ionic charge, or strong hydrophobicity, because the surface of the filter is easily covered with jelly-like substances.

[0078] Although the form of the filter may be a porous form having a continuous pore structure, a fiber assembly, a fabric, etc., a nonwoven fabric is more preferred. Since a jelly-like substance covers the filter surface to cause clogging, it is undesirable to use twisted fibers.

[0079] The fiber diameter of the filter is not particularly limited as long as unnecessary components can be captured and retinal pigment epithelial cells can pass through efficiently. Considering the passage of retinal pigment epithelial cells, it is smaller than the pore size of the filter and is, for example, 5 to 20 μm.

[0080] Although the pore size of the filter is not particularly limited as long as the unnecessary components can be captured and the retinal pigment epithelial cells can pass efficiently, it is generally 15-100 μm. Considering the size of the cells and the capture effect of the unnecessary components, 20-70 μm is preferred, and 20-40 μm is more preferred. When the pore size is less than 20 μm, the passing rate of the retinal pigment epithelial cells may be reduced, and when it is greater than 100 μm, it may cause a decrease in the capture effect of the unnecessary components in terms of capture efficiency.

[0081] The pore size of the filter can be measured by photographing the filter with a scanning electron microscope, measuring the pores (maximum length) of substantial pores formed by intersections of two or more different fibers at 50 random points with an image analyzer and determining the average value.

[0082] The filter can be used in any form such as a sphere, container, box, bag, tube, column, etc. Specific preferred examples include a transparent or translucent cylindrical container with a volume of about 0.1-1000 ml and a diameter of about 0.1-24 cm, or a quadrangular prism with a side length of about 0.1 cm-20 cm, and a thickness of about 0.1 cm-5 cm, etc. having a square or rectangular shape.

[0083] The passage of the solution through the filter may be by natural fall from a bag containing the cell population (cell suspension), or by the use of a syringe or pump.

[0084] By aforesaid operation, unnecessary components are captured by the filter and the retinal pigment epithelial cells selectively pass through the filter in the cell culture medium introduced on the filter, thereby can be purified by the cell.For the number of cells in the liquid through the filter, it is possible to obtain, for example, the purity of the retinal pigment epithelial cells being not less than 80%, preferably not less than 90%, more preferably not less than 95%.For purity, carry out Pax6, Bestrophin or Mitf immunostaining, when the cell is used any dyeing therein, the cell is determined to be the retinal pigment epithelial cells, when no fluorescence is seen in the cell, the dark brown-black coloration of whether there is melanin in the test cell, and when confirming the generation of pigment, it can be determined that the cell is the retinal pigment epithelial cells.In addition, by inoculating the cell colony obtained in the new culture container coated with laminin or its fragment, and carrying out the purification operation again, purity can be further improved.The productive rate of the retinal epithelial pigment cells obtained in this stage is about 50 to 100 times by the SFEB improvement method.

[0085] According to production method of the present invention, human pluripotent stem cells can be rapidly adhered to culture vessel via laminin or its fragment excellent in cell adhesion, and differentiation induction efficiency is significantly improved with the cultivation of immobilized state, and in addition, cell loss during culture medium exchange can be suppressed. In addition, the cell colony of retinal pigment epithelial cells of high concentration can be obtained in a large number by the above-mentioned purification step in a short time by simple and easy operation very effectively. In addition, according to production method of the present invention, retinal pigment epithelial cells can adhere to each other, to form a sheet-like structure. Therefore, the sheet of retinal pigment epithelial cells can be produced by production method of the present invention. As described in detail below, as cell colony used as the cell transplantation therapeutic medicine for the treatment of retinal diseases, the sheet of retinal pigment epithelial cells is useful.

[0086] The retinal pigment epithelial cell of producing as mentioned above also can carry out further amplification culture.Amplification culture can be by carrying out as follows, similar in the aforesaid method, inoculate retinal pigment epithelial cell on the culture vessel of coating laminin or its fragment, to allow cell adhesion to laminin or its fragment, and carry out adhesion culture.Go down to posterity and amplification culture selection like this, only select to specifically adhere to laminin or its fragmentary cell as matrix.The result is, because the monolayer structure of leading retinal pigment epithelial cell stably keeps, and the cell (it can not induce differentiation) included in the retinal pigment epithelial cell of differentiation induction can reduce relatively, it also can be used as the further purification method of retinal pigment epithelial cell.

[0087] The concentration of the inoculated retinal pigment epithelial cells is not particularly limited as long as uniform adhesion culture of the retinal pigment epithelial cells can be performed. For example, when a 10 cm dish is used, it is 1×10 per dish. 5 -1×10 8 cells, preferably 2×10 6 -5×10 7 cells, more preferably 5×10 5 -1×10 7 cells.

[0088] In the expansion culture, laminin or a fragment thereof, and the coating method for the culture substrate are the same as those described above, and are therefore preferred embodiments.

[0089] As the culture medium, the above-mentioned maintenance medium for retinal pigment epithelial cells can be used.

[0090] Although the culture period is not particularly limited, after the retinal pigment epithelial cells are inoculated, it is preferred to culture while exchanging the total amount of the culture medium with the maintenance medium, not less than once every 3 days, for about 3 weeks. The cell culture medium after culture is preferably subjected to a treatment similar to the above step (2) (i.e., detachment from the culture vessel, optional cell dissociation treatment, and filtration treatment) to further purify the retinal pigment epithelial cells. In addition, using the retinal pigment epithelial cells thus obtained, it is preferred to carry out an expansion culture similar to the above for about 2 weeks, and the obtained cell culture medium is again subjected to a treatment similar to the above step (2) for further purification. For the number of cells in the liquid passed through the filter by the above operation, it is possible to obtain, for example, a purity of retinal pigment epithelial cells of not less than 85%, preferably not less than 95%, and more preferably not less than 99%.

[0091] According to the expansion method of the present invention, since the retinal pigment epithelial cells are quickly fixed to the culture vessel via laminin or its fragments having excellent cell adhesion, cell loss during the culture medium exchange process can be suppressed, and deformation of the cell morphology due to passage can be suppressed, and the maintenance culture and culture growth of the retinal pigment epithelial cells can be stably performed. In addition, according to the expansion method of the present invention, the membranous retinal pigment epithelial cell clusters can also be used directly or in the form of a suspension, and the membranous retinal pigment epithelial cell clusters are separated and recovered from the culture matrix, used to be fixed to a new matrix or support material (biodegradable, porous, mesh structure, etc.) to form a shape suitable for the affected area on which the cells are transplanted, and used as a therapeutic drug for the retinal diseases shown below.

[0092] 2. Retinal Pigment Epithelial Cells The retinal pigment epithelial cells obtained by the purification method or production method of the present invention have high purity and excellent properties whether used as cells alone or as sheets.

[0093] 3. Drugs for the treatment of retinal diseases The retinal pigment epithelial cells obtained by the purification method or production method of the present invention can be used as a cell transplantation therapeutic drug and transplanted into a living organism in the form of a suspension or a sheet for the treatment of retinal diseases. Retinal diseases are ophthalmic diseases related to the retina and also include complications of other diseases such as diabetes.

[0094] 4. Toxicity and efficacy evaluation of drugs The retinal pigment epithelial cells produced by the amplification method or production method of the present invention can be used as normal or disease model cells for screening therapeutic drugs for retinal diseases and therapeutic drugs for other complications, such as efficacy evaluation of diabetes, etc., or its preventive drugs, safety tests of chemical substances, etc., stress tests, toxicity tests, side effect tests, infection / pollution tests. On the other hand, they can also be used for toxicity studies, toxicity tests, etc. of phototoxicity specific to retinal cells, retinal excitotoxicity, etc. Its evaluation method includes evaluation by stimulation, toxicity tests such as apoptosis, etc., and evaluation tests for the effects of normal differentiation from progenitor cells to retinal pigment epithelial cells and visual cells (expressed protein analysis and phagocytic ability test, RT-PCR by various gene markers, ELISA of cytokines, etc.), toxicity tests of phototoxicity, etc., retinal potential and transepithelial impedance for visual function, cell damage tests caused by autoimmune reactions, etc. As cells for these tests, not only retinal pigment epithelial cells but also progenitor cells thereof can be used, and for example, plates, cell suspensions, sheets or compacts thereof on which cells adhere by inoculation can be provided. They can be used as extrapolation tests for human and animal tests.

[0095] The disclosures disclosed in any publication cited in this specification, including patents and patent applications, are hereby incorporated by reference in their entirety to the extent that they have been disclosed herein. The present invention is explained in more detail below by reference to examples, which should not be construed as limiting.

[0096] EXAMPLES Study Example 1 Production of RPE cells derived from iPS cells Reagents Differentiation induction basic medium (GMEM medium (Invitrogen), KSR (Invitrogen), 0.1 mM MEM non-essential amino acid solution (Invitrogen), 1 mM sodium pyruvate (SIGMA), 0.1 M 2-mercaptoethanol (WakoPure Chemical Industries, Ltd.), 100 U / ml penicillin-100 μg / ml streptomycin (Invitrogen)) Primary differentiation induction medium (differentiation induction basic medium containing 20% ​​KSR, 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA)) Secondary differentiation induction medium (differentiation induction basic medium containing 15% KSR, 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA)) Tertiary differentiation induction medium (differentiation induction basic medium containing 10% KSR, 10 μM Y-27632 (Wako Pure Chemical Industries, Ltd.), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA)) ·Fourth-stage differentiation induction medium (differentiation induction basic medium containing 10% KSR) RPE maintenance medium (67% DMEM low glucose (SIGMA), 29% F12 (SIGMA), 1.9 mM L-glutamine (Invitrogen), 1.9% B-27 supplement (Invitrogen), 96 U / ml penicillin sodium, 96 μg / mL streptomycin sulfate).

[0097] Production of retinal pigment epithelial cells (differentiation induction) iPS cells derived from human peripheral blood (monocytes) (1120C7, provided by Kyoto University) were grown at 9x10 6 cells / 9 cm dish were seeded in a laminin-coated dish (manufactured by Sumitomo Bakelite Co., Ltd.). 2An aqueous solution of the laminin-511 E8 fragment (protein disclosed in Example (3) of WO 2011043405, (manufactured by Nippi (iMatrix-511, NIP-8920-02)) was coated on a 9 cm culture dish (BD FALCON) at 37°C for not less than 1 hour to produce a laminin-coated culture dish. iPS cells adhered to the culture dish quickly, and the formation of suspended aggregates was not confirmed.

[0098] With the first day of culture as day 0, the total amount of the medium was exchanged every day from the start of culture (day 1) to about day 40 (when pigment cells were confirmed). The composition of the medium was changed in stages as shown below. That is, the primary differentiation induction medium (20% KSR) was used for days 1-4, the secondary differentiation induction medium (15% KSR) was used for days 5-8, the tertiary differentiation induction medium (10% KSR) was used for days 9-12, and the quaternary differentiation induction medium (10% KSR) was used from day 13 to about day 40 (when pigment cells were confirmed).

[0099] After about 40 days, when confirming pigment cell, the total amount of culture medium and RPE maintained culture medium with exchange not less than once every 3 days, up to 47 days.Along with cultivation, cytochrome becomes darker, and at the 47th day, observe a lot of dark pigment cell groups.At the 47th day, reclaim the cell colony containing pigment cell.

[0100] Using laminin-511 E8 fragment as a coating agent, the seeded iPS cells quickly adhered to the culture dish at a high density and stably maintained the adhesion state even during culture in the differentiation induction medium. Therefore, even when the total amount of the culture medium is repeatedly exchanged, cell loss can be suppressed to a low level. In addition, compared to when iPS cells were seeded on a culture vessel coated with collagen, the generation rate of pigment cells was significantly increased and the differentiation induction efficiency was significantly improved.

[0101] Research Example 2 Production of RPE cells (other iPS cells) Pigment cells were obtained by a method similar to that of Study Example 1, except that iPS cells derived from human skin (fibroblasts) (201B7, provided by Kyoto University) were used instead of iPS cells derived from human peripheral blood (monocytes) (1120C7, provided by Kyoto University).

[0102] As a result, like Study Example 1, the generation rate of pigment cells was significantly increased and the differentiation induction efficiency was significantly increased compared to the seeded iPS cells.

[0103] Example 1 Purification and expansion of RPE cells derived from iPS cells On day 47, a cell population comprising pigment cells was subjected to adhesion culture in the culture dishes of Study Example 1 and Study Example 2 ( Figure 3 ), treated with 0.01% trypsin-0.53 mM EDTA, and cell aggregates were detached from the culture dish. Then, cell adhesions were separated by gentle pipetting. The protease liquid and its residual impurities in the cell mixture were removed together with the centrifugation supernatant, and then, unnecessary cells were separated by filter filtration separation through a cell strainer (DBFalcon Cell Strainer40 μm Nylon), and the cell population containing RPE cells (48 days) was recovered.

[0104] The obtained cells were divided into 9x10 6 10 cells / 9 cm dish were seeded in the RPE maintenance medium described in Study Example 1, in the same 5 dishes coated with laminin-511 E8 as in Study Example 1, and static culture was performed until about 50 days, when adhesion of retinal pigment epithelial cell colonies was confirmed.

[0105] From 51 days to 71 days, the total amount of culture medium was exchanged with RPE maintenance medium for no less than 3 days once for 3 weeks, and then filtered and separated (purified) using a filter, and seeded into each of the same 5 culture dishes coated with laminin-511 E8 and similarly cultured for 2 weeks. As a result, retinal pigment epithelial cells were expanded to a yield of 25 dishes (10 cm dishes) from any cell colony of Research Example 1 and Research Example 2. The purity (n = 4) of the obtained cell colony was 96.4%, 100%, 98.6% or 99.6%.

[0106] As an example, purity calculation data for a dish having a purity of 98.6% is shown.

[0107] For purity, immunostaining is carried out for Pax6, Bestrophin and Mitf, and when any dyeing, cell is judged to be RPE cell.When not observing fluorescence, the existence of melanin in the test cell or not, and cell is judged to be RPE cell (because some pigments suppress fluorescence observation, so even when not observing fluorescence, cell is also judged to be RPE cell) based on the confirmation of pigment.Determine purity by comprising the method that will be added as positive cell separately.

[0108] This example provides similar results even when a different line of iPS cells (201B7) is used. In addition, even when purification similar to this example is performed using a filter having a pore size of 70 μm or 20 μm, RPE cells of similar high purity are obtained.

[0109] Comparative Example 1: A differentiation induction step was performed by a method similar to that of Study Example 1, except that in the differentiation induction step of Study Example 1, suspension culture was performed using a non-adhesive culture dish (Nunc) treated with MPC (2-methacryloylphosphocholine) instead of adhesion culture using a culture dish coated with laminin-511 E8 (BD FALCON).

[0110] As a result, almost all pigment cells were lost during the medium exchange during the differentiation induction step, and pigment cells could not be recovered on day 47.

[0111] Comparative Example 2: Differentiation induction was performed by a method similar to that of Study Example 1, except that in the differentiation induction step of Study Example 1, a culture dish coated with poly-D-lysine and gelatin was used instead of a culture dish coated with laminin-511 E8 for adhesion culture.

[0112] Compared with the culture dish coated with laminin-511 E8, the adhesion of cells to the culture dish coated with poly-D-lysine / gelatin was weak, and the cells were easily lost during the medium exchange process. Therefore, by visually observing the number of pigment cells relative to the total cells in the culture dish, the ratio of pigment cells on the 47th day after the start of culture was no more than 1 / 20 of that in Study Example 1, and the differentiation induction efficiency was also significantly lower.

[0113] (Evaluation 1) Expression of RPE cell markers RT-PCR analysis was performed on the pigment cells obtained in Study Examples 1 and 2, and Example 1 using primers having the following sequences according to the method described in Journal of Cell Science 2009 Sep 1 122 3169-79. As a result, RPE cell-specific genes (RPE65, CRALBP, MERTK, BEST1) were found to be expressed, similar to commercially available human RPE cell lines, thereby confirming RPE cells.

[0114] This example provides similar results even when a different line of iPS cells (201B7) was used. As a representative example, the results obtained using RPE65 are shown in Figure 2 middle.

[0115] (Evaluation 2) Cytokine secretion ability: The pigment cells obtained in Study Examples 1 and 2 and Example 1 were tested for the production amount of PEDF by ELISA according to the method described in IOVS. 2006 47 612-3624. As a result, it was confirmed that they had cytokine secretion ability similar to that of RPE cells of adult retina (Table 2).

[0116] Table 2: PEDF secretion .

[0117] This example provides similar results even when a different line of iPS cells (201B7) is used.

[0118] (Evaluation 3) Phagocytic ability According to the method described in J Cell Sci. 1993 104 37-49 and using FluoSpheres (registered trademark) fluorescent microspheres (Invitrogen, F13081), for phagocytic ability analysis in research examples 1 and 2, and the pigment cells obtained in embodiment 1. As a result, it was confirmed that the cell has the phagocytic ability at the same level as commercially available human RPE cell lines. This evaluation provides similar results, even when using the iPS cells (201B7) of different lines. In addition, even when according to the method described in TheLancet 2012 379 713-720, using the iPS cells (201B7) of different lines and the pHrod Green E. coli BioParticles (registered trademark) conjugate (Molecular Probes, P35366) for phagocytosis, similar results were also obtained.

[0119] (Evaluation 4) Analysis of cell population expression before and after purification The cell aggregates separated in Study Examples 1 and 2 were passed through a cell strainer (DB Falcon CellStrainer 40 ul Nylon), and the cell population retained on the filter and the cell population containing the isolated retinal epithelial cell clusters were subjected to gene expression analysis and evaluation of cell type and differentiation stage, respectively.

[0120] Specifically, the expression of specific genes was detected by quantitative RT-PCR and the effect of separation of retinal pigment epithelial cells was evaluated by the filter filtration method. RNA was extracted from the cells separated on and off the filter according to conventional methods (RNeasy Micro Kit, 79254, QIAGEN), and cDNA was synthesized using the extracted RNA as a template (SuperScript III Reverse Transcriptase Kit, 18080-044, Invitrogen, 0.5 μg / μL Oligo (dT)12-18 Primer, invitrogen, 18418-012, invitrogen, 10 mM dNTP Mix, 18427-013, invitrogen). The expression of the target gene was detected using the synthesized cDNA as a template under PCR conditions of 1) 95°C for 20 seconds, 2) 95°C for 1 second, and 3) 60°C for 20 seconds (2)-3) (20× TaqMan (registered trademark) Gene Expression Assay, Applied Biosystems, 2× TaqMan (registered trademark) Fast Advanced Master Mix, 4444557, Applied Biosystems). The expression level was normalized using GAPDH as an internal standard, and the relative value was calculated by comparing the expression level of the target gene before and after the filter filtration as in 1 by the Ct method ( Figure 4 ). The primer sequences used for gene amplification are shown below. 20 × TaqMan (registered trademark) Gene Expression Assay (RAX (Rx)) (Applied Biosystems) Hs00429459_m1 20 × TaqMan (registered trademark) Gene Expression Assay (Pax6) (Applied Biosystems) Hs00240871_m1 20 × TaqMan (registered trademark) gene expression assay (MITF) (Applied Biosystems) Hs01117294_m1 20 × TaqMan (registered trademark) Gene Expression Assay (RPE65) (Applied Biosystems) Hs01071462_m1 20 × TaqMan (registered trademark) Gene Expression Assay (CRX) (Applied Biosystems) Hs00230899_m1 20 × TaqMan (registered trademark) gene expression assay (VSX2 (Chx10)) (Applied Biosystems) Hs1584047_m1 20× TaqMan (registered trademark) gene expression assay (GAPDH) (Applied Biosystems) Hs02758991_g1.

[0121] As a result, it was confirmed that the retinal pigment epithelial cell marker (RPE65) and its progenitor cell marker Mitf (pigment epithelial cells, progenitor cells) were highly expressed under the filter, and markers expressed at the initial stage of differentiation induction such as Pax6 (progenitor cells), Rx (retinal progenitor cells), Crx (photoreceptor progenitor cells), Chx10 (bipolar cells), etc., and visual cell and neural cell markers other than target cells were highly expressed on the filter. Therefore, it is believed that the use of a purification method at the end of the differentiation induction step is suitable as a method for effectively isolating retinal pigment epithelial cells.

[0122] Industrial Applicability According to the purification method of the present invention, the retinal pigment epithelial cells induced from pluripotent stem cells can be easily purified with high yield. According to the production method using the purification method of the present invention, the retinal pigment epithelial cells can be effectively and with high purity, produced by using a simple and easy method of a culture matrix coated with laminin or its fragment. The production method of the present invention is excellent in differentiation induction efficiency, and the retinal pigment epithelial cells can be purified by simple and easy operation, and the retinal pigment epithelial cells can be produced with high yield by suppressing the cell loss during the step. The retinal pigment epithelial cells produced by the method of the present invention are not only useful for the treatment of retinal diseases, but also as the production or preparation method of normal and disease model cells.

[0123] This application is based on patent application No. 2013-212345 filed in Japan (filing date October 9, 2013), the contents of which are incorporated herein in their entirety.

Claims

1. A method for purifying retinal pigment epithelial cells, comprising: (1) a step of inducing pluripotent stem cells to differentiate into a cell population containing retinal pigment epithelial cells on a laminin-511 E8 fragment; (2) a step of recovering the cell population obtained in (1) by treating with a cell separation solution; (3) a step of dissociating adhesion between retinal pigment epithelial cells of the cell population obtained in (2); and (4) A step of introducing the cell population obtained in (3) into a filter to obtain a retinal pigment epithelial cell population that has passed through the filter.

2. The method according to claim 1, further comprising a step of removing the cell separation solution and its residual impurities, as well as the matrix components in the cell population together with the supernatant by centrifugation between (3) and (4).

3. The method according to claim 1 or 2, wherein the step of dissociating the adhesion between retinal pigment epithelial cells is performed by several reciprocating pipetting.

4. The method according to any one of claims 1 to 3, wherein the cell detachment solution comprises trypsin.

5. The method according to any one of claims 1 to 4, wherein the pore size of the filter is 20-70 μm.

6. A method for producing retinal pigment epithelial cells from pluripotent stem cells, comprising: (1) a step of inducing pluripotent stem cells to differentiate into a cell population containing retinal pigment epithelial cells on a laminin-511 E8 fragment; (2) a step of recovering the cell population obtained in (1) by treating with a cell separation solution; (3) a step of dissociating adhesion between retinal pigment epithelial cells of the cell population obtained in (2); and (4) A step of introducing the cell population obtained in (3) into a filter to obtain a retinal pigment epithelial cell population that has passed through the filter.

7. The method according to claim 6, further comprising a step of removing the cell separation solution and its residual impurities, and the matrix components in the cell population together with the supernatant by centrifugation between (3) and (4).

8. The method according to claim 6 or 7, wherein the step of dissociating the adhesion between retinal pigment epithelial cells is performed by several reciprocating pipetting.

9. The method according to any one of claims 6 to 8, wherein the cell detachment solution comprises trypsin.

10. The method according to any one of claims 6 to 9, wherein the pore size of the filter is 20 to 70 μm.

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