Method for purifying retinal pigment epithelial cells

Abstract

Provided are: a method for producing retinal pigment epithelial cells, which improves the efficiency of differentiation induction in which pluripotent stem cells are differentiated into retinal pigment epithelial cells, and which can provide high-purity retinal pigment epithelial cells in a short period of time by means of a simple and convenient operation; the present invention relates to a method for culturing retinal pigment epithelial cells which can stably grow and culture cells, a method for evaluating toxicity / drug efficacy using retinal pigment epithelial cells useful in transplantation therapy, and a therapeutic drug for retinal diseases. The present invention relates to a method for producing retinal pigment epithelial cells comprising a step for adherent culture of human pluripotent stem cells by using a coated laminin-E8 fragment, a method for culturing retinal pigment epithelial cells comprising a step for adherent culture of retinal pigment epithelial cells by using a coated laminin-E8 fragment, and a method for culturing retinal pigment epithelial cells comprising a step for adherent culture of human pluripotent stem cells by using a coated laminin-E8 fragment. A method for evaluating the toxicity or efficacy of the retinal pigment epithelial cells produced or cultured by the method; and a therapeutic agent for retinal diseases comprising retinal pigment epithelial cells.

Description

[0001] This application is a divisional application of an invention patent application filed on October 9, 2014, with application number 201480055673.5 and invention name “Method for purifying retinal pigment epithelial cells”. Technical Field

[0002] The present invention relates to a method for efficiently producing retinal pigment epithelial (RPE) cells from human pluripotent stem cells, a method for stably culturing retinal pigment epithelial cells, and the like. Background Art

[0003] As a method for producing retinal pigment epithelial cells from pluripotent stem cells, the following methods are known: a method called SFEB method comprising culturing ES cells as suspended aggregates in a serum-free medium (Patent Document 1, etc.), a method comprising inducing 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. (Non-Patent Document 1, etc.). However, due to low differentiation induction efficiency, these methods require a combination of multiple steps of adhesion culture and suspension culture to obtain a highly concentrated cell population of retinal pigment epithelial cells, and have the following problems, such as the need for a purification step with a high workload and a long time, including selectively picking colonies of pigment cells under an optical microscope. In addition, these methods have the problem of easy cell loss during subculture. Therefore, a method that can stably obtain high-purity retinal pigment epithelial cells by a simple and easy method has been required.

[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 describes that laminin-511E8 maintains binding activity to α6β1 integrin, which is at the same level as the binding activity of full-length laminin-511, and Patent Document 2 describes that by using the laminin-511E8, pluripotent stem cells can be stably immobilized in a culture dish, with the result that the cells can be maintained in culture 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 the differentiation induction efficiency of retinal pigment epithelial cells differentiated into by pluripotent stem cells adheres to laminin-111 and matrigel and increases significantly. 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 One object of the present invention is to provide a method for producing retinal pigment epithelial cells that improves the differentiation induction efficiency of pluripotent stem cells into retinal pigment epithelial cells and can provide high-purity retinal pigment epithelial cells in a short time through simple and convenient operations, a method for culturing retinal pigment epithelial cells that can stably grow and culture cells, a method for evaluating the toxicity / efficacy of retinal pigment epithelial cells that can be used for transplantation therapy, and a therapeutic drug for retinal diseases.

[0008] Solutions to technical problems The present inventors have conducted in-depth research to try to achieve the above-mentioned purpose, and found that when human pluripotent stem cells are cultured on a culture substrate coated with laminin-E8, the inoculated pluripotent stem cells quickly adhere to the culture substrate, a large number of pigment cells are produced from an early stage, the yield of retinal pigment epithelial cells can be significantly improved, the cells are not easily damaged during the culture medium exchange, the purification step can be simplified, and a high-purity cell population can be obtained in a short time. As a result, the operability and economic efficiency problems in inducing pluripotent stem cells to differentiate into retinal pigment epithelial cells can be significantly improved, and the target retinal pigment epithelial cells can be stably and efficiently produced, which leads to the completion of the present invention.

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

[0010] [1] A method for producing retinal pigment epithelial cells, comprising the step of allowing human pluripotent stem cells to adhere and culture on a culture substrate coated with a laminin-E8 fragment.

[0011] [2] The method of [1], wherein the laminin-E8 fragment is a laminin-511E8 fragment.

[0012] [3] The method of [1] or [2], wherein the adhesion culture step is performed in the presence of a differentiation inducing factor.

[0013] [4] The method of any one of [1] to [3], wherein the human pluripotent stem cells are human iPS cells.

[0014] [5] Retinal pigment epithelial cells produced by allowing human pluripotent stem cells to adhere to a culture substrate coated with laminin-E8 fragments.

[0015] [6] Retinal pigment epithelial cell sheets produced by allowing human pluripotent stem cells to adhere to a culture substrate coated with laminin-E8 fragments.

[0016] [7] A method for expanding retinal pigment epithelial cells, comprising the step of allowing the retinal pigment epithelial cells to adhere and culture on a culture substrate coated with a laminin-E8 fragment.

[0017] [8] A method for purifying retinal pigment epithelial cells, comprising the step of allowing the retinal pigment epithelial cells to adhere and culture on a culture substrate coated with a laminin-E8 fragment.

[0018] [9] A therapeutic agent for retinal diseases, comprising retinal pigment epithelial cells produced by the method of any one of [1] to [4], or retinal pigment epithelial cells cultured by the method of [7].

[0019] Effects of the Invention According to the present invention, retinal pigment epithelial cells can be produced from pluripotent stem cells with high yield. In addition, differentiation induction efficiency is improved, and the cell colony comprising high-concentration retinal pigment epithelial cells can be obtained, and highly purified retinal pigment epithelial cells can be produced by simple and easy purification operation. In addition, according to the present invention, because retinal pigment epithelial cells can stably adhere to, the cell is not easy to lose during culture medium exchange, and can be stably passed down to posterity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram showing the structure of laminin-E8.

[0021] Figure 2 Shown are the expressions of RPE-related genes in RPE cells induced from human iPS cells (201B7).

[0022] Figure 3 The cell adhesion states on days 1, 2, and 3 after the start of differentiation induction of iPS cells in Comparative Example 3 are shown, wherein the iPS cells were seeded on a matrix coated with laminin-511 E8, Matrigel, or full-length laminin-511.

[0023] Figure 4 Shown are cell populations on days 15 and 40 after starting differentiation induction of iPS cells plated on a substrate coated with laminin-511 E8, Matrigel, or laminin-511 full length in Comparative Example 3. Specific implementation plan

[0024] 1. Production of retinal pigment epithelial cells The present invention is a method for producing retinal pigment epithelial cells, the method comprising the step of adhesion culture of human pluripotent stem cells using a culture substrate coated with a laminin-E8 fragment (hereinafter referred to as "the production method of the present invention").

[0025] 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.

[0026] 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.

[0027] "Retinal pigment epithelial cells" in the present invention refer to epithelial cells that constitute 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.). Progenitor cells of retinal pigment epithelial cells refer to cells that are directed and induced to differentiate into retinal cells, and whether they are progenitor cells can be confirmed by the expression of cell markers (MITF, Pax6, Rx, Crx, 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.

[0028] 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 number of each of the α chain (α1-α5), the β chain (β1-β4), and the γ chain (γ1-γ3) is combined to determine the name of the laminin. For example, the laminin composed of the combination of α1 chain, β1 chain, and γ1 chain is named laminin 111, the laminin composed of the combination of α5 chain, β1 chain, and γ1 chain is named laminin-511, and the laminin composed of the 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.

[0029] 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, there is also a fragment corresponding to the E8 fragment of mouse laminin-111 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.

[0030] 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 functional aspects of (1) and the structural aspects of (2) are described in more detail below.

[0031] (1) Function of laminin-E8 Examples of laminin-E8 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 / 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. Examples of integrins expressed on the surface of human pluripotent stem cells include α6β1 integrin and the like, and examples of integrins expressed on the surface of human retinal pigment epithelial cells include α6β1 integrin, α3β1 integrin, α7β1 integrin and the like.

[0032] The laminin-E8 in the present invention shows a binding specificity for integrins, at least equivalent to the binding specificity for each laminin. It is preferred to use a laminin-E8 that shows a particularly strong affinity for integrins. "Laminin-E8 that shows a particularly strong affinity for integrins" is a laminin fragment that shows a significantly low dissociation constant, as measured by known methods, for example, by the dissociation constants measured by the method shown in Table 1 of The Journal of Biological Chemistry (2009) 284, pp.7820-7831.

[0033] As laminin-E8 in the present invention, laminin-E8 having strong cell adhesion activity is preferably used. "Laminin-E8 having strong cell adhesion activity" is laminin-E8 that shows significantly strong adhesion activity in a cell adhesion test using a measurement method by a known method and shows not less than 400 cells / mm at a coating concentration of laminin-E8 not more than 10 nM. 2 When performing a cell adhesion assay as described in The Journal of Biological Chemistry (2007) 282, pp. 11144-11154, for example.

[0034] In the present invention, as laminin-E8, laminin-E8 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 their progenitor cells in the late stage of differentiation induction is preferably used. From such an aspect, in particular, laminin-511 E8 is highly preferred among laminin-E8 because it has binding specificity to α3β1 integrin and α7β1 integrin in addition to α6β1 integrin, and can promote the improvement of differentiation induction efficiency of pluripotent stem cells into retinal pigment epithelial cells, or the stabilization of maintenance culture of retinal pigment epithelial cells by improving adhesion activity to retinal pigment epithelial cells.

[0035] (2) Structure of laminin-E8 The laminin-E8 in the present invention is 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 1The 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.

[0036] As described above, the laminin-E8 in the present invention may be an enzyme-treated product obtained by treating natural laminin with elastase, or a recombinant produced by gene recombination. When the laminin-E8 of the present invention is a recombinant, a tag may be bonded to the N-terminus for purification purposes, 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 laminin-E8 is not particularly limited, as long as the binding activity of the corresponding full-length (native) laminin to integrin is maintained and the cell adhesion is not impaired.

[0037] In the laminin-E8 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.

[0038] 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.

[0039] 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.).

[0040] 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.

[0041] The "culture substrate" used in the present invention can be produced by coating the surface of the culture vessel with the laminin-E8 of the present invention. As used herein, the surface of the "coated" culture vessel means that laminin-E8 is adsorbed on the surface of the culture vessel by some interactions between laminin-E8 and the culture vessel surface, wherein the orientation of the laminin-E8 does not cause special problems for obtaining 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, dishes (also referred to as culture dishes), covered culture dishes and plates (microtiter plates, microplates, deep well plates, etc. of 6 holes, 24 holes, 48 ​​holes, 96 holes, 384 holes, 9600 holes, etc.), bottles, slides, tubes, cell factories, roller bottles, rotating bottles, hollow fibers, microcarriers, beads, etc. The culture substrate in the present invention can be applied with appropriate surface treatment as long as the cell adhesion of laminin-E8 is not impaired.

[0042] "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-E8 and do not float in the culture medium even when the culture vessel is gently shaken during culture. Since the laminin-E8 used in the present invention can show very excellent cell adhesion, all cells after cell seeding are preferably uniformly dispersed by a method including rapidly shaking the culture vessel, etc. The target cells may be suspended cultured in a culture vessel containing laminin-E8 before or after adhesion culture, as long as the purpose of the present invention can be achieved.

[0043] The substratum is composed of a basal medium, serum and / or serum replacement, and other components. As a basal medium, one or more synthetic culture media that are commonly used to culture mammalian cells can be used in combination, and for example, commercially available products such as DMEM, GMEM, etc. can be obtained.

[0044] As the serum, serum derived from mammals such as cattle, humans, 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., and serum substitutes N2, B27, etc. for neural cell culture can be obtained. In the present invention, serum substitutes are preferred, and KSR is particularly preferred from the aspect of quality management of target cells.

[0045] 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%.

[0046] 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 part or all of the differentiation induction step. 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.

[0047] In addition to the above components, the culture medium may contain other components commonly used for culturing mammalian cells.

[0048] 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.

[0049] 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 into retinal pigment epithelial cells. Examples of retinal pigment epithelial cell differentiation inducing factors include Nodal signal inhibitors, Wnt signal inhibitors, Sonic hedgehog signal inhibitors, and activin signal promoters, etc.

[0050] Nodal signal inhibitors are not particularly limited, as long as they 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.

[0051] 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.

[0052] Examples of the activin signaling promoter include proteins belonging to the activin family, activin receptors, activin receptor agonists, and the like.

[0053] 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.

[0054] In the production of the present invention, it is preferred to use a combination of a Nodal signaling inhibitor (eg, SB431542) and a Wnt signaling inhibitor (eg, CKI-7) as a differentiation inducing factor.

[0055] According to the culture induced pluripotent stem cell of said method, it is differentiated into retinal pigment epithelial cell, thereby retinal pigment epithelial cell can generate at the 25th-45th day of inoculation pluripotent stem cell usually.The generation of retinal pigment epithelial cell can be confirmed according to said method.When the generation of retinal pigment epithelial cell is confirmed, this culture medium is exchanged with the culture medium for maintaining retinal pigment epithelial cell, and, for example, described cell preferably further cultivates 5-10 days, simultaneously with the total amount of the frequency exchange culture medium of being no more than 3 days once.As a result, the cell colony and the polygonal flat cell colony of the melanin dye deposition that adhere to basal layer can be observed more clearly.

[0056] 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.

[0057] 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. In the present invention, serum substitutes are preferred, and B27 is particularly preferred from the aspect of quality management of target cells.

[0058] Examples of other components include L-glutamine, sodium penicillin, streptomycin sulfate and the like.

[0059] The production method of the present invention may further include the step of purifying retinal pigment epithelial cells by concentration operation. Because the production method of the present invention significantly improves the differentiation induction efficiency, high-purity retinal pigment epithelial cells can be obtained in a short production cycle by simple and easy operation. The method for concentrating retinal pigment epithelial cells is not particularly limited, as long as it is a method commonly known as concentrated cells, and for example, such as filtration, centrifugation, perfusion separation, flow cytometry separation, separation by the capture of the carrier of immobilized antibodies, etc. can be used. Preferably, a method such as filtering using a nylon net, etc. can be utilized.

[0060] According to production method of the present invention, human pluripotent stem cells can be rapidly adhered to culture vessel via laminin-E8 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. Therefore, cell colonies with high concentration of retinal pigment epithelial cells can be obtained in a large number. In addition, cell purification can be carried out by simple and easy operation, the necessary time of purification step can be shortened, and retinal pigment epithelial cells can be produced 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 in 3 below, as cell colony used as a cell transplantation therapeutic drug for the treatment of retinal diseases, the sheet of retinal pigment epithelial cells is useful.

[0061] 2. Method for proliferation of retinal pigment epithelial cells The present invention also relates to a method for expanding retinal pigment epithelial cells, the method comprising the step of adhesion culture of retinal pigment epithelial cells using a culture substrate coated with a laminin-E8 fragment (hereinafter referred to as "the expansion method of the present invention"). According to the expansion method of the present invention, retinal pigment epithelial cells can be easily cultured by conventional methods.

[0062] As the retinal pigment epithelial cell in the amplification method of the present invention, the retinal pigment epithelial cell derived from a mammal can be used. The example of mammal includes mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cattle, horse, goat, monkey and people. When producing retinal pigment epithelial cells for transplanting into the purpose of people, preferably use human retinal pigment epithelial cells etc.

[0063] The retinal pigment epithelial cells in the amplification method of the present invention refer to epithelial cells constituting the retinal pigment epithelium, and their progenitor cells. Examples thereof include retinal pigment epithelial cells derived from living organisms; retinal pigment epithelial cells induced to differentiate from undifferentiated cells (pluripotent stem cells, precursors of retinal pigment epithelial cells, etc.); retinal pigment epithelial cells transdifferentiated from precursors of cells other than differentiated cells or retinal pigment epithelial cells, preferably retinal pigment epithelial cells induced to differentiate from undifferentiated cells, and more preferably retinal pigment epithelial cells induced to differentiate from pluripotent stem cells, which are obtained by the above-mentioned production method of the present invention.

[0064] The concentration of retinal pigment epithelial cells used in the expansion method of the present invention is not particularly limited as long as uniform adhesion culture of retinal pigment epithelial cells can be performed. For example, when a 10 cm dish is used, it is 1×10 5 -1×10 8cells, preferably 2×10 6 -5×10 7 cells, more preferably 5×10 5 -1×10 7 cells.

[0065] The laminin-E8 fragment in amplification method of the present invention is similar to above-mentioned, and the coating method of culture matrix is ​​also similar.Therefore, by cultivating the retinal pigment epithelial cell obtained by described production method of the present invention in the same culture vessel of coating laminin-E8 fragment, the retinal pigment epithelial cell obtained can directly be amplified in situ, and can obtain in a large number.In addition, according to amplification method of the present invention, the cell that may not be induced to differentiate in the retinal pigment epithelial cell of differentiation induction can be reduced relatively.Therefore, amplification method of the present invention also can be used as the purification method of retinal pigment epithelial cell.

[0066] Regarding laminin-E8 used in the expansion method of the present invention, laminin-511 E8 is particularly preferred among laminin-E8 because it has binding specificity to α3β1 integrin and α7β1 integrin in addition to α6β1 integrin, and can promote stabilization and cell proliferation of maintenance culture of retinal pigment epithelial cells by enhancing adhesion activity to retinal pigment epithelial cells.

[0067] According to the expansion method of the present invention, since the retinal pigment epithelial cells are quickly fixed to the culture vessel via laminin-E8, which has excellent cell adhesion, cell loss during the culture medium exchange process can be suppressed, and the 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 group can also be used directly or after being separated and recovered from the culture matrix, as a therapeutic drug for retinal diseases, as shown below.

[0068] 3. Retinal Pigment Epithelial Cells The retinal pigment epithelial cells obtained by the production method or amplification method of the present invention are cultured on laminin-E8, particularly laminin-511 E8. Therefore, they can be easily separated and recovered from the culture matrix as cells alone or as sheets, and have excellent properties.

[0069] 4. Drugs for the treatment of retinal diseases The retinal pigment epithelial cells produced by the production method of the present invention and the retinal pigment epithelial cells amplified by the amplification 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 with other diseases, such as diabetes.

[0070] 5. Toxicity and efficacy evaluation of drugs The retinal pigment epithelial cells produced by the 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 stimulation, toxicity tests such as apoptosis evaluation, 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 through various gene markers, ELISA of cytokines, etc.), phototoxicity toxicity tests, 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, cells can be provided by inoculating a plate, a cell suspension, a sheet thereof, or a compact thereof. They can be used as an extrapolation test for human and animal tests.

[0071] The disclosures of any publications cited in this specification, including patents and patent applications, are hereby incorporated by reference in their entirety to the same extent as if fully disclosed herein.

[0072] The present invention is explained in more detail below by referring to Examples, which should not be construed as limitative.

[0073] EXAMPLES 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).

[0074] 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.

[0075] The above-mentioned differentiation induction was performed according to the following schedule, and the differentiation induction in the examples and comparative examples in this specification followed this schedule. 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).

[0076] 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 colonies.At the 47th day, reclaim the cell colony containing pigment cell.

[0077] 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.

[0078] Research Example 2 Production of RPE cells (other iPS cells) Pigment cells were obtained by a method similar to that of 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).

[0079] As a result, like Example 1, the generation rate of pigment cells was significantly increased and the differentiation induction efficiency was significantly improved compared with the seeded iPS cells.

[0080] Example 3 Expansion of RPE cells derived from iPS cells The cell colony that comprises pigment cell at the 47th day, it experiences adhesion culture in the culture dish of embodiment 1 and embodiment 2, handles with 0.01% trypsin-0.53 mM EDTA, and cell aggregate is separated from culture dish.Then, intercellular adhesion is separated by gentle suction.Protease liquid in cell mixture and its residual impurity are removed together with centrifugal supernatant, then, unnecessary cell is separated by the filtration separation method through cell strainer (DB Falcon Cell Strainer 40 μm Nylon), and reclaims the cell colony (48 days) that contains RPE cell.

[0081] The obtained cells were divided into 9x10 6 10 cells / 9 cm dish were seeded in the RPE maintenance medium described in Example 1, in 5 dishes coated with laminin-511 E8 as in Example 1, and static culture was performed until about day 50, when adhesion of retinal pigment epithelial cell colonies was confirmed.

[0082] 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, then filtered and separated using a filter, inoculated 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 Example 1 and Example 2. The purity (n = 4) of the obtained cell colony was 96.4%, 100%, 98.6% or 99.6%.

[0083] As an example, purity calculation data for a dish having a purity of 98.6% is shown.

[0084] For purity, immunostaining is performed for Pax6 and Bestrophin, and when any one is dyed, cell is considered to be RPE cell.When fluorescence is not observed, the presence or absence of melanin in the test cell is checked, and the cell is judged to be RPE cell (because some pigments suppress fluorescence observation, so even when fluorescence is not observed, cell is also judged to be RPE cell in the presence of pigment) based on the confirmation of pigment.Determine purity by including the method that each is added as positive cell.

[0085] This example provides similar results even when a different line of iPS cells (201B7) is used.

[0086] Comparative Example 1: A differentiation induction step was performed by a method similar to that of Example 1, except that in the differentiation induction step of 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).

[0087] 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.

[0088] Comparative Example 2: Differentiation induction was performed by a method similar to that of Example 1, except that in the differentiation induction step of 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.

[0089] 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 Example 1, and the differentiation induction efficiency was also significantly lower.

[0090] (Evaluation 1) Expression of RPE cell markers: According to the method described in Journal of Cell Science 2009 Sep 1 1223169-79, the pigment cells obtained in Example 1-3 were subjected to RT-PCR analysis using primers with the following sequences. As a result, it was found that RPE cell-specific genes (RPE65, CRALBP, MERTK, BEST1) were expressed, similar to commercially available human RPE cell lines, thereby confirming RPE cells.

[0091] 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.

[0092] (Evaluation 2) Cytokine secretion ability: The pigment cells obtained in Examples 1-3 were tested for PEDF production 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).

[0093] Table 2: PEDF secretion

[0094] This example provides similar results even when a different line of iPS cells (201B7) is used.

[0095] (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), the pigment cells obtained in embodiment 1-3 are analyzed for phagocytic ability. As a result, it is confirmed that the cell has the phagocytic ability of the same level as commercially available human RPE cell lines. This embodiment provides similar results, even when using the iPS cells (201B7) of different lines. In addition, even when according to the method described in The Lancet 2012379 713-720, using the iPS cells (201B7) of different lines and pHrodGreen E. coli BioParticles (registered trademark) conjugate (Molecular Probes, P35366) for phagocytosis, similar results are also obtained.

[0096] Comparative Example 3 Differentiation induction was performed by a method similar to that in Example 1 and using iPS cells (201B7, provided by Kyoto University) derived from human skin (fibroblasts) in the same manner as in Example 2 and a culture dish coated with any of the following 1) to 3). Two tests were performed, the test including simultaneously performing 3 matrices to induce differentiation, and one supplementary test for each matrix.

[0097] 1) Laminin-511 E8 (molecular weight 150,000) (iMatrix511: manufactured by Nippi, NIP-8920-02) was used for the analysis of the fibroblasts at 0.5 μg / cm 2 Package.

[0098] 2) Recombinant human laminin-511 full length (molecular weight 776,000) (sold by VERITAS Corporation (produced by BioLamina) / BLA-LN511) was used at 2.59 μg / cm 2 Used for wrapping.

[0099] 3) Matrigel (manufactured by BD, 354230 (total protein concentration: 9-12 mg / ml)) (basal lamina matrix, a soluble basal lamina preparation derived from mouse Engelbreth-Holm-Swarm (EHS) sarcoma, containing major molecules of the basal lamina, type IV collagen, entactin, perlecan, laminin-111) was dissolved overnight at 4°C and stirred at 10 mL / 57 cm in a cold state. 2 (0.175 ml / cm 2 ) is used to coat culture vessels.

[0100] The cell adhesion status on day 1, day 2, and day 3 from the start of differentiation induction is shown in Figure 3 . In the case of the matrix coated with laminin-511 E8, from the second day of seeding, the cells grew while uniformly adhering to the entire surface of the culture dish and quickly entered the differentiation induction process. In the case of the matrix coated with matrigel (laminin-111), the cells adhered inconsistently, had partial invasion of the gel, and ultimately failed to form a complete surface layer. In the case of the matrix coated with laminin full length, the adhesion was weak, and the cells partially formed colonies on the second day of seeding. It took 4 - 5 days for the cells to grow and spread over the entire surface of the dish. As a result, the differentiation induction process became uneven, leaving a portion where the differentiation induction did not proceed.

[0101] The status of the cell population at day 15 from differentiation induction and before the cell filtration separation step (approximately 40 days) is shown in Figure 4 . In the case of laminin-511 E8 coated matrix, cells were scattered on the whole surface for about 15 days, and the accumulation of stratified cells thickened, and melanin color development was observed. About 40 days, cells were evenly stratified and thick, and no basal rupture or delamination was found. Clear cell aggregates containing melanin were found on the top of the multilayer. In the case of matrigel coated matrix, no uniform layer of adherent cells was found for about 15 days, and cells were sucked into the inside of the gel. No clear cell membrane with a single layer was formed on the basal side for about 40 days, and no melanin color development was observed. In the case of laminin full-length coated matrix, adherent cells were observed on the whole surface for about 15 days, but differentiation induction was not carried out in many places, and sometimes rupture or delamination of the adhesion surface layer was found in other places. In addition, cells with poor stratification were found on the top, and the color development of melanin color was very weak. Stratified cells gradually fell off from about 20 days, and the cell membrane on the basal side was significantly ruptured for about 40 days.

[0102] According to above-mentioned, in the situation of matrigel coated matrix, observe growth failure and differentiation induction failure, and obviously show the fragility that adhesion cell number reduces and stratified cell number, the efficiency of the RPE cell outward appearance shown by melanin color development, and its differentiation induction rate speed are all significantly low, and the productive rate of the RPE cell finally obtained in the situation of laminin full-length coated matrix is ​​low.Therefore, clarified that in the present invention, the culture matrix coated with laminin-E8 segment can produce retinal pigment epithelial cell very effectively.

[0103] Industrial Applicability According to the production method of the present invention, retinal pigment epithelial cells can be effectively produced by using a simple and easy method of a culture matrix coated with laminin-E8 fragment. The production method of the present invention is excellent in differentiation induction efficiency, and retinal pigment epithelial cells can be purified by simple and easy operation, and retinal pigment epithelial cells can be produced with high yield by suppressing 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 normal and disease model cells.

[0104] 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 producing retinal pigment epithelial cells, comprising the step of differentiating adherently cultured human pluripotent stem cells on a culture substrate coated with a laminin-511E8 fragment.

2. The method according to claim 1, wherein the step of differentiating is performed in the presence of differentiation inducing factors.

3. The method according to claim 1 or 2, wherein the human pluripotent stem cells are human iPS cells.

4. A method for expanding retinal pigment epithelial cells, comprising the step of allowing the retinal pigment epithelial cells to adhere and culture on a culture substrate coated with a laminin-511E8 fragment.

5. A method for purifying retinal pigment epithelial cells, comprising the step of allowing the retinal pigment epithelial cells to adhere and culture on a culture substrate coated with a laminin-511E8 fragment.

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