Method for purifying neural crest cells or corneal epithelial cells

Abstract

The present invention provides a method for purifying neural crest cells or corneal epithelial cells.

Description

Technical Field

[0001] The present invention relates to a method for purifying neural crest cells and the like, and more particularly to a method for purifying neural crest cells using laminin 211 and the like. In another aspect, the present invention relates to a method for purifying corneal epithelial cells using laminin 332 and the like.

Background Art

[0002] In recent years, with the establishment of iPS cells, the development of new treatment methods for diseases involving cell transplantation has been making great progress. As one of the cells that can be suitably used for cell transplantation therapy, neural crest cells having broad multipotency and thus also referred to as the "fourth germ layer" have attracted attention.

[0003] A plurality of methods for preparing neural crest cells have been reported. For example, Non-Patent Document 1 discloses a method of inducing the differentiation of iPS cells in the presence of a TGF-β signal inhibitor, a Wnt signal activator, etc., preparing a cell population containing neural crest cells, and recovering only neural crest cells from the cell population using a cell sorter and expanding and culturing them. Non-Patent Document 2 also discloses that by inducing the differentiation of ES cells in the presence of FGF2 and a TGFβ signal inhibitor, etc., preparing a cell population containing neural crest cells, and further culturing the cell population in a gelatin-containing medium in the presence of FGF2 and a TGF-β inhibitor for a long time, relatively highly pure neural crest cells can be prepared.

[0004] In the field of cell transplantation therapy, corneal epithelial cells are also one of the cells attracting attention. Corneal epithelial cells constitute the surface of the cornea and function as a barrier to protect the cornea from the outside world and to take in oxygen into the cornea. Most disorders of corneal epithelial cells lead to a decrease in visual acuity and significantly reduce the QOL of patients. Therefore, there is a high need to establish a method for efficiently producing transplantable corneal epithelial cells for treating such disorders.

[0005] As a method for preparing corneal epithelial cells, for example, a method for inducing differentiation of corneal epithelial cells by culturing pluripotent stem cells in a serum-free medium without BMP for a certain period in the presence of stromal cells or amniotic membrane-derived factors has been reported (Patent Document 1).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Using the methods disclosed in Non-Patent Documents 1 and 2, neural crest cells can be prepared. However, there are still problems with these methods. For example, in the method disclosed in Non-Patent Document 1, a step of collecting neural crest cells using a cell sorter is essential, but the use of a cell sorter has various problems such as the yield, purity, viability, and functional decline of the sorted target cells, as well as the costs associated with purchasing and maintaining the cell sorter. In addition, in the method of Non-Patent Document 2, although it does not involve the use of a cell sorter, continuous passage is required to obtain highly pure neural crest cells, and since its preparation requires a relatively long period, there is a problem with efficiency.

[0009] Moreover, corneal epithelial cells can be prepared by using the method disclosed in Patent Document 1. However, similar to the case of the above-mentioned neural crest cells, in order to obtain highly pure corneal epithelial cells using this method, a step of sorting corneal epithelial cells from a cell population containing induced corneal epithelial cells using a cell sorter or the like is required, and there are problems with efficiency.

Means for Solving the Problems

[0010] As a result of intensive studies on the above problems, the present inventors have found that by expanding and culturing a cell population containing neural crest cells using laminin 211 as a substrate, neural crest cells can be purified without sorting the neural crest cells using a cell sorter or the like. The present inventors have also found that using this method, neural crest cells can be purified in an extremely short period compared to conventional purification methods. Furthermore, the present inventors have found that by expanding and culturing a cell population containing corneal epithelial cells exceeding a certain ratio using laminin 332 as a substrate, corneal epithelial cells can be purified without sorting the corneal epithelial cells using a cell sorter or the like, and based on such findings, further research has been conducted to complete the present invention. That is, the present invention is as follows.

[0011] [1] A method for purifying neural crest cells, comprising the following steps: Step 1) A step of obtaining a cell population containing neural crest cells, and Step 2) A step of expanding and culturing the cell population obtained in Step 1 using laminin 211 as a substrate. [2] The method according to [1], wherein the cell population containing neural crest cells obtained in Step 1 is subjected to Step 2 without being subjected to a sorting treatment of neural crest cells. [3] The method according to [1] or [2], wherein the culture period in Step 2 is 1 to 21 days. [4] The method according to any one of [1] to [3], wherein the cell population containing neural crest cells is derived from pluripotent stem cells. [5] The method according to [4], wherein the pluripotent stem cells are iPS cells. [6] A method for producing purified neural crest cells, comprising the following steps: Step 1) obtaining a cell population containing neural crest cells, and Step 2) expanding and culturing the cell population obtained in Step 1 using laminin 211 as a substrate. [7] The method according to [6], characterized in that the cell population containing neural crest cells obtained in Step 1 is subjected to Step 2 without being subjected to a sorting process for neural crest cells. [8] The method according to [6] or [7], wherein the culture period of Step 2 is 1 to 21 days. [9] The method according to any one of [6] to [8], wherein the cell population containing neural crest cells is derived from pluripotent stem cells.

[10] The method according to [9], wherein the pluripotent stem cells are iPS cells.

[11] A method for purifying corneal epithelial cells, comprising the following steps: Step 1) obtaining a cell population in which the proportion of corneal epithelial cells is 25% or more, and Step 2) expanding and culturing the cell population obtained in Step 1 using laminin 332 as a substrate.

[12] The method according to

[11] , characterized in that the cell population containing corneal epithelial cells obtained in Step 1 is subjected to Step 2 without being subjected to a sorting process for corneal epithelial cells.

[13] In the expansion culture of Step 2, the coating amount of laminin 332 used as a substrate is 0.01 μg / cm 2 or more and less than 0.5 μg / cm 2 The method according to

[11] or

[12] .

[14] The method according to any one of

[11] to

[13] , wherein the cell population containing corneal epithelial cells is derived from pluripotent stem cells.

[15] The method according to

[14] , wherein the pluripotent stem cells are iPS cells.

[16] A method for producing purified corneal epithelial cells, comprising the following steps: Step 1) obtaining a cell population in which the proportion of corneal epithelial cells is 25% or more, and Step 2) expanding and culturing the cell population obtained in Step 1 using laminin 332 as a substrate.

[17] The method according to

[16] , characterized in that the cell population containing corneal epithelial cells obtained in Step 1 is subjected to Step 2 without being subjected to the sorting process of corneal epithelial cells.

[18] In the expansion culture of Step 2, the coating amount of laminin 332 used as a scaffold is 0.01 μg / cm 2 or more and less than 0.5 μg / cm 2 The method according to

[16] or

[17] .

[19] The method according to any one of

[16] to

[18] , wherein the cell population containing corneal epithelial cells is derived from pluripotent stem cells.

[20] The method according to

[19] , wherein the pluripotent stem cells are iPS cells.

Advantages of the Invention

[0012] According to the present invention, neural crest cells can be purified from a cell population containing neural crest cells very simply and in a short period of time. Further, according to the present invention, highly pure neural crest cells can be prepared very simply and in a short period of time. Furthermore, according to the present invention, corneal epithelial cells can be purified from a cell population containing corneal epithelial cells very simply. Also, according to the present invention, highly pure corneal epithelial cells can be prepared very simply.

Embodiments for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail.

[0014] 1. Method for purifying neural crest cells The present invention provides a method for purifying neural crest cells (hereinafter sometimes referred to as "Purification Method 1 of the Present Invention") including the following steps: Step 1) A step of preparing a cell population containing neural crest cells, and Step 2) A step of expanding and culturing the cell population obtained in Step 1 using laminin 211 as a scaffold.

[0015] "Neural crest cells (also referred to as "NCC")" refer to cells that are epithelialized from the neural crest, a structure that is temporarily formed between the epidermal ectoderm and the neural plate during the early development of vertebrates, and are induced to various sites within the embryo after the epithelial-to-mesenchymal transition. The term "neural crest cells" in this specification includes not only cells collected from a living body, but also neural crest cells derived from pluripotent stem cells and cells passaged therefrom. In the purification method of the present invention, the origin of neural crest cells is not particularly limited and may be from any vertebrate, but neural crest cells derived from mammals are preferred. Such mammals include, but are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans.

[0016] As a method for preparing a cell population containing neural crest cells in Step 1 of Purification Method 1 of the present invention, when the cell population is a cell population containing neural crest cells derived from a living body, it can be prepared by collecting a cell population from tissues derived from the neural crest in a living body (for example, bone marrow, dorsal root ganglia of the spinal cord, heart, cornea, iris, dental pulp, and olfactory mucosa, etc.). In the case of a cell population containing neural crest cells derived from pluripotent stem cells, as described above, a plurality of preparation methods are known. As an example, a method of culturing pluripotent stem cells in a culture solution containing a TGFβ inhibitor and a GSK-3β inhibitor is exemplified. By inducing differentiation of iPS cells with a culture solution containing a TGFβ inhibitor and a GSK-3β inhibitor, the iPS cells differentiate into colonies (self-formed ectodermal autonomous multi-zone: SEAM) having multi-banded regions composed of various cell lineages of the eye. Neural crest cells may be included in SEAM.

[0017] Whether or not the thus obtained cell population contains neural crest cells may be confirmed by a method known per se for the expression of one or more of neural crest cell-specific marker genes such as TFAP2a, SOX9, SOX10, TWIST1, PAX3, etc. Further, a protein present on the cell surface of neural crest cells such as CD271 protein (also referred to as "p75(NTR)") can also be used as a neural crest cell-specific marker. The cell population containing neural crest cells used in the purification method 1 of the present invention is preferably derived from pluripotent stem cells, and more preferably derived from iPS cells.

[0018] In the present invention, the pluripotent stem cells are stem cells that have pluripotency to differentiate into many cells existing in a living body and also have a proliferative ability, and any cells induced into the intermediate mesoderm cells used in the present invention are included. The pluripotent stem cells are not particularly limited, and examples thereof include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, pluripotent cells (Muse cells) derived from cultured fibroblasts and bone marrow stem cells, and the like. Preferred pluripotent stem cells are iPS cells from the viewpoint that they can be obtained without destroying embryos, eggs, etc. in the manufacturing process, and more preferably human iPS cells.

[0019] Methods for producing iPS cells are known in the art and can be produced by introducing reprogramming factors into any somatic cell. Here, reprogramming factors include, for example, genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, etc. These reprogramming factors can be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al.The combinations described in (2008), Cell Stem Cell, 3, 568-574; Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479; Marson A, (2008), Cell Stem Cell, 3, 132-135; Feng B, et al. (2009), Nat. Cell Biol. 11:197-203; R.L. Judson et al., (2009), Nat. Biotechnol., 27:459-461; Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917; Kim JB, et al. (2009), Nature. 461:649-643; Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503; Heng JC, et al. (2010), Cell Stem Cell. 6:167-74; Han J, et al. (2010), Nature. 463:1096-100; Mali P, et al. (2010), Stem Cells. 28:713-720; Maekawa M, et al. (2011), Nature. 474:225-9 are exemplified.

[0020] Somatic cells include, but are not limited to, somatic cells of a fetus (fetal), somatic cells of a newborn (neonatal), and somatic cells of a mature healthy or diseased individual, and also include any of primary cultured cells, subcultured cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, etc., (2) tissue progenitor cells, (3) blood cells (peripheral blood cells, cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells, and adipose cells, and other differentiated cells.

[0021] The mammal from which somatic cells are collected is not particularly limited, but is preferably a human.

[0022] In a cell population containing neural crest cells, the proportion of neural crest cells can vary greatly depending on the collected tissue and differentiation induction conditions. In one aspect of the purification method 1 of the present invention, the proportion of neural crest cells in a cell population containing neural crest cells can be, for example, 1 to 95%, 1 to 90%, 1 to 85%, 1 to 80%, 1 to 75%, 1 to 70%, 1 to 65%, 1 to 60%, 1 to 55%, 1 to 50%, 1 to 45%, 1 to 40%, 1 to 35%, 1 to 30%, 1 to 25%, 1 to 20%, 1 to 15%, or 1 to 10%, but is not limited thereto. In another aspect, the proportion of neural crest cells in a cell population containing neural crest cells can be, for example, 25 to 95%, 25 to 90%, 25 to 85%, 25 to 80%, 25 to 75%, 25 to 70%, 25 to 65%, 25 to 60%, 25 to 55%, 25 to 50%, 25 to 45%, 25 to 40%, or 25 to 35%, but is not limited thereto. In another aspect, the proportion of neural crest cells in a cell population containing neural crest cells can be, for example, 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 50 to 75%, 50 to 70%, 50 to 65%, or 50 to 60%, but is not limited thereto. In another aspect, the proportion of neural crest cells in a cell population containing neural crest cells can be, for example, 75 to 95%, 75 to 90%, or 75 to 85%, but is not limited thereto.

[0023] Here, "purification" in the purification method 1 of the present invention means that as a result of subjecting the cell population containing neural crest cells prepared in step 1 to step 2, the proportion of neural crest cells in the cell population increases compared to the proportion at the time of preparation in step 1. In one aspect, the proportion of neural crest cells in the cell population purified by the purification method 1 of the present invention is 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100%, but is not limited thereto.

[0024] The cell population containing neural crest cells prepared in Step 1 of the purification method 1 of the present invention may, if necessary, be subjected to mechanical dispersion treatment, dispersion treatment with enzymes such as collagenase and trypsin, and / or dispersion treatment with a chelating agent such as EDTA, etc., to obtain a single-cell state. In addition, when the cell population is made into single cells, a ROCK inhibitor may be added for the purpose of suppressing cell death. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK). For example, Y-27632, Fasudil / HA1077, H-1152, Wf-536, and their derivatives, etc. can be mentioned. In addition, other known low-molecular compounds can also be used as the ROCK inhibitor (for example, refer to US Patent Application Publication Nos. 2005 / 0209261, 2005 / 0192304, 2004 / 0014755, 2004 / 0002508, 2004 / 0002507, 2003 / 0125344, 2003 / 0087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, 2004 / 039796).

[0025] The cell population prepared in Step 1 may be subjected to Step 2 after being subjected to the sorting treatment of neural crest cells, but preferably, it is subjected to Step 2 without being subjected to the sorting treatment of neural crest cells. In addition, examples of the sorting treatment of neural crest cells include sorting by a cell sorter using a fluorescently labeled CD271-specific antibody, sorting by magnetic beads conjugated with a CD271-specific antibody, and sorting by an affinity column immobilized with a CD271-specific antibody, etc., but are not limited thereto.

[0026] In Step 2 of the purification method 1 of the present invention, the cell population obtained in Step 1 is subjected to expansion culture. In addition, in this specification, "expansion culture" is a concept including culture for maintaining and / or proliferating desired cells, and preferably, it can be culture for proliferating desired cells.

[0027] In the purification method 1 of the present invention, since the purpose is to purify neural crest cells, as culture conditions, conditions suitable for the maintenance and / or proliferation of neural crest cells can be used.

[0028] The culture conditions for expanding neural crest cells are not particularly limited as long as the neural crest cells can be expanded in culture, and culture conditions known per se can be used. As an example, a method of culturing in a culture solution containing a TGFβ inhibitor, EGF (epidermal growth factor), and FGF2 (fibroblast growth factor 2) is exemplified.

[0029] The medium used for the expansion culture of neural crest cells can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM medium, Medium199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen), RPMI-base medium, StemFit (registered trademark) AK03N medium, and mixed media thereof. In this step, preferably, StemFit (registered trademark) AK03N medium is used. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol (2ME), thioglycerol, etc., or may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0030] In the present invention, the TGFβ inhibitor is a substance that inhibits the signal transduction from the binding of TGFβ to its receptor to SMAD, and is not particularly limited as long as it is a substance that inhibits the binding to the receptor ALK family or a substance that inhibits the phosphorylation of SMAD by the ALK family. In the present invention, examples of the TGFβ inhibitor include Lefty-1 (exemplified by mouse: NM_010094 and human: NM_020997 as NCBI Accession No.), SB431542, SB202190 (R.K. Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009 / 146408), and derivatives thereof. The TGFβ inhibitor used for the expansion culture of neural crest cells can preferably be SB431542.

[0031] The concentration of the TGFβ inhibitor such as SB431542 in the culture medium is not particularly limited as long as it is a concentration that inhibits ALK5, but is preferably 1 nM to 50 μM, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, but is not limited thereto. More preferably, it is 10 μM.

[0032] In addition, the concentration of EGF in the medium is preferably 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but is not limited thereto. More preferably, it is 20 ng / ml.

[0033] In addition, the concentration of FGF2 in the medium is preferably 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but is not limited thereto. More preferably, it is 20 ng / ml.

[0034] In addition, as long as the expansion culture of neural crest cells can be achieved, components other than the above can also be added to the medium.

[0035] The expansion culture of neural crest cells in Step 2 may be either adherent culture or suspension culture, but is preferably performed by adherent culture.

[0036] In the purification method 1 of the present invention, laminin 211 is used to achieve the purification of neural crest cells.

[0037] Laminin is a glycoprotein that is a major constituent molecule of the basement membrane. Laminin is known to be involved in various cellular functions such as cell adhesion, cell proliferation, metastasis, and differentiation. Laminin is composed of a heterotrimer having one each of the α, β, and γ subunit chains. Currently, five types of α subunit chains (α1, α2, α3, α4, α5), three types of β subunit chains (β1, β2, β3), and three types of γ subunit chains (γ1, γ2, γ3) are known to exist. Depending on the combination of these subunit chains, the existence of 15 types of laminin isoforms has currently been confirmed in humans. Laminin 211 used in the purification method of the present invention is a laminin composed of the subunit chains of the α2 chain, β1 chain, and γ1 chain. It is preferable that the origin of laminin coincides with the organism from which the neural crest cells are derived (for example, when using human-derived neural crest cells, it is preferable to use human-derived laminin 211). Incidentally, although it is known that the laminin E8 fragment composed only of the integrin-binding site has stronger cell adhesion activity than full-length laminin (see Miyazaki T. et al., Nat Commun. 2012;3:1236), laminin 211 used in the purification method of the present invention is the full-length laminin 211 protein, not a fragment. Laminin 211 may be prepared using a gene recombination technique known per se, or a commercially available one may be used.

[0038] In Step 2, when expanding the cell population in suspension culture, laminin 211 may be added to the medium so that the floating cells can use it as a scaffold. Suspension culture can be carried out by a method known per se. As an example, there is a method of expanding the cell population in suspension culture in a medium supplemented with laminin 211 while stirring the medium using a spinner flask or the like. Alternatively, the cell population can also be expanded in suspension culture by using a polysaccharide (for example, methylcellulose, xanthan gum, gellan gum, etc.) having an effect of suspending cells by adding it to the medium in combination with laminin 211. The concentration of laminin added and the like may be appropriately set in consideration of various conditions such as the seeding density of the cell population and the concentration of the polysaccharide used in combination. In the present specification, suspension culture means a culture method in which cells are cultured without adhering to the surface of the culture vessel. The suspension culture may or may not involve physical stirring. Also, the cells to be cultured may be uniformly dispersed or non-uniformly dispersed in the medium.

[0039] In Step 2, when expanding the cell population in adherent culture, laminin 211 is coated on the surface of the culture vessel. The coating amount of laminin 211 is not particularly limited as long as the desired effect of the present invention is obtained, and a generally recommended coating amount may be used. As an example, the coating amount of laminin 211 is 0.1 ng / cm 2 ~1000 ng / cm 2 , preferably 0.5 ng / cm 2 ~500 ng / cm 2 , more preferably 1 ng / cm 2 ~250 ng / cm 2 , even more preferably 2 ng / cm 2 ~100 ng / cm 2 However, it is not limited thereto.

[0040] In addition, although the culture period in Step 2 can vary depending on culture conditions, culture methods, the proportion of neural crest cells in the cell population, etc., purification of neural crest cells can be achieved in a relatively short period. As an example of the culture period, it can be 1 to 21 days, 1 to 20 days, 1 to 19 days, 1 to 18 days, 1 to 17 days, 1 to 16 days, 1 to 15 days, 1 to 14 days, 1 to 13 days, 1 to 12 days, 1 to 11 days, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, or 1 to 3 days, but is not limited thereto.

[0041] The culture temperature in Step 2 is not particularly limited as long as neural crest cells can be cultured, but is 30 to 40°C, preferably about 37°C. Also, the CO2 concentration during culture in Step 2 is not particularly limited as long as neural crest cells can be cultured, but is 2 to 5%, preferably about 5%.

[0042] 2. Method for producing neural crest cells The present invention also provides a method for producing purified neural crest cells (hereinafter sometimes referred to as "Production Method 1 of the present invention") including the following steps: Step 1) A step of preparing a cell population containing neural crest cells, and Step 2) A step of expanding and culturing the cell population obtained in Step 1 using laminin 211 as a scaffold.

[0043] Various conditions in the production method of the present invention are the same as those in Purification Method 1 of the present invention.

[0044] 3. Method for purifying corneal epithelial cells The present invention provides a method for purifying corneal epithelial cells (hereinafter sometimes referred to as "Purification Method 2 of the present invention") including the following steps: Step 1) A step of obtaining a cell population in which the proportion of corneal epithelial cells is 25% or more, and Step 2) A step of expanding and culturing the cell population obtained in Step 1 using laminin 332 as a scaffold.

[0045] "Corneal epithelial cells (also referred to as "CEC")" are the outermost cells of the cornea that constitute the corneal epithelial layer. Corneal epithelial cells are derived from the epidermal ectoderm. The term "corneal epithelial cells" in this specification includes not only cells collected from a living body, but also corneal epithelial cells derived from pluripotent stem cells and cells obtained by subculturing them. In the purification method 2 of the present invention, the origin of corneal epithelial cells is not particularly limited and may be from any vertebrate, but corneal epithelial cells derived from mammals are preferred. Such mammals include, but are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. In addition, the term "corneal epithelial cells" in this specification is a concept that may include corneal epithelial stem cells and / or corneal epithelial progenitor cells.

[0046] As a method for preparing a cell population containing corneal epithelial cells in step 1 of the purification method 2 of the present invention, when the cell population is a cell population containing corneal epithelial cells derived from a living body, it can be prepared by collecting the cell population from the corneal epithelial layer. In the case of a cell population containing corneal epithelial cells derived from pluripotent stem cells, as described above, a known preparation method can be adopted. As an example, for example, the method shown in the examples of the present application, or a method of culturing pluripotent stem cells in a serum-free medium containing no BMP for a certain period in the presence of stromal cells or amnion-derived factors, can be exemplified. By these methods, pluripotent stem cells (e.g., iPS cells) differentiate into SEAM. SEAM may contain corneal epithelial cells.

[0047] Whether the thus obtained cell population contains corneal epithelial cells can be confirmed by a method known per se for detecting the expression of one or more corneal epithelial cell-specific marker genes such as PAX-6, Cytokeratin 12, or Cytokeratin 3. The cell population containing corneal epithelial cells used in the purification method 2 of the present invention is preferably derived from pluripotent stem cells, and more preferably derived from iPS cells. The "pluripotent stem cells" in the present invention are as described above.

[0048] In a cell population containing corneal epithelial cells, the proportion of corneal epithelial cells can vary significantly depending on the tissue collected and the differentiation induction conditions. In one aspect of the purification method 2 of the present invention, the proportion of corneal epithelial cells in a cell population containing corneal epithelial cells can be, for example, 1 to 95%, 1 to 90%, 1 to 85%, 1 to 80%, 1 to 75%, 1 to 70%, 1 to 65%, 1 to 60%, 1 to 55%, 1 to 50%, 1 to 45%, 1 to 40%, 1 to 35%, 1 to 30%, 1 to 25%, 1 to 20%, 1 to 15%, or 1 to 10%, but is not limited thereto. In another aspect, the proportion of corneal epithelial cells in a cell population containing corneal epithelial cells can be, for example, 25 to 95%, 25 to 90%, 25 to 85%, 25 to 80%, 25 to 75%, 25 to 70%, 25 to 65%, 25 to 60%, 25 to 55%, 25 to 50%, 25 to 45%, 25 to 40%, or 25 to 35%, but is not limited thereto. In another aspect, the proportion of corneal epithelial cells in a cell population containing corneal epithelial cells can be, for example, 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 50 to 75%, 50 to 70%, 50 to 65%, or 50 to 60%, but is not limited thereto. In another aspect, the proportion of corneal epithelial cells in a cell population containing corneal epithelial cells can be, for example, 75 to 95%, 75 to 90%, or 75 to 85%, but is not limited thereto.

[0049] For a cell population in which the proportion of corneal epithelial cells is less than 25%, the proportion of corneal epithelial cells can be increased to 25% or more by performing sorting treatment of corneal epithelial cells. The sorting treatment of corneal epithelial cells can be carried out by a method known per se. As an example, a method of identifying a marker specifically expressed on the surface of corneal epithelial cells and selecting cells expressing the marker can be mentioned, but is not limited thereto.

[0050] In the purification method 2 of the present invention, corneal epithelial cells can be purified by expanding and culturing a cell population in which the proportion of corneal epithelial cells is 25% or more, using laminin 332 as a scaffold. The proportion of corneal epithelial cells in the cell population is usually 25% or more, preferably 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, but is not limited thereto.

[0051] "Purification" in the purification method 2 of the present invention means that as a result of subjecting the cell population containing corneal epithelial cells prepared in step 1 to step 2, the proportion of corneal epithelial cells in the cell population increases compared to the proportion at the time of preparation in step 1. In one aspect, the proportion of corneal epithelial cells in the cell population purified by the purification method 2 of the present invention is 90 - 100%, 91 - 100%, 92 - 100%, 93 - 100%, 94 - 100%, 95 - 100%, 96 - 100%, 97 - 100%, 98 - 100%, 99 - 100%, or 100%, but is not limited thereto.

[0052] The cell population containing corneal epithelial cells prepared in step 1 of the purification method 2 of the present invention may be subjected to mechanical dispersion treatment, dispersion treatment with enzymes such as collagenase and trypsin, and / or dispersion treatment with a chelating agent such as EDTA, etc., to obtain a single-cell state. In addition, when the cell population is made into single cells, a ROCK inhibitor may be added for the purpose of suppressing cell death. The ROCK inhibitor is as described above.

[0053] The cell population prepared in step 1 may be subjected to corneal epithelial cell sorting treatment and then to step 2, but preferably, it is subjected to step 2 without being subjected to corneal epithelial cell sorting treatment.

[0054] In step 2 of the purification method 2 of the present invention, the cell population obtained in step 1 is subjected to expansion culture. In addition, the meaning of "expansion culture" in this specification is as described above.

[0055] In the purification method 2 of the present invention, since the purpose is to purify corneal epithelial cells, as culture conditions, conditions suitable for the maintenance and / or proliferation of corneal epithelial cells can be used.

[0056] The culture conditions for the expansion culture of corneal epithelial cells are not particularly limited as long as the corneal epithelial cells can be expanded, and culture conditions known per se can be used. As an example, a method of culturing in a culture solution containing KGF (Keratinocyte growth factor) and a Rho kinase inhibitor is exemplified.

[0057] The medium used for the expansion culture of corneal epithelial cells can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM medium, Medium199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen), RPMI-base medium, StemFit (registered trademark) AK03N medium, and mixed media thereof. In this step, preferably, StemFit (registered trademark) AK03N medium is used. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursor, trace elements, 2-mercaptoethanol (2ME), thioglycerol, etc., or may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0058] The concentration of KGF in the culture medium is not particularly limited as long as it enables the proliferation of corneal epithelial cells, but is preferably 0.1 to 200 ng / mL. For example, it can be 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, but is not limited thereto. More preferably, it is 20 ng / mL.

[0059] Also, as long as the expansion culture of corneal epithelial cells can be achieved, components other than the above can be added to the medium.

[0060] The expansion culture of corneal epithelial cells in Step 2 may be either adherent culture or suspension culture, but is preferably performed by adherent culture.

[0061] In the purification method 2 of the present invention, in order to achieve the purification of corneal epithelial cells, laminin 332 is used as a substrate material.

[0062] The laminin 332 used in the purification method 2 of the present invention is the full-length laminin 332 protein, not a fragment. Laminin 332 may be prepared using a gene recombination technique known per se, or a commercially available product may be used.

[0063] In Step 2, when the cell population is expanded in suspension culture, laminin 332 may be added to the medium so that the suspended cells can use it as a substrate. The method and conditions of suspension culture are as described above.

[0064] In Step 2, when expanding the cell population by adherent culture, laminin 332 is coated on the surface of the culture vessel. The coating amount of laminin 332 is not particularly limited as long as the desired effect of the present invention can be obtained, and the usually recommended coating amount may be used. As an example, the coating amount of laminin 332 is 0.01 μg / cm 2 or more and 0.5 μg / cm 2 less, preferably 0.05 μg / cm 2 or more and 0.4 μg / cm 2 less, more preferably 0.05 μg / cm 2 or more and 0.3 μg / cm 2 less, even more preferably 0.05 μg / cm 2 or more and 0.25 μg / cm 2 less, but not limited thereto.

[0065] Also, the culture period in Step 2 can vary depending on culture conditions, culture methods, the proportion of corneal epithelial cells contained in the cell population, etc., and can be appropriately set. As an example of the culture period, it is 1 to 50 days, 1 to 40 days, 1 to 30 days, 1 to 20 days, 1 to 17 days, 1 to 16 days, 1 to 15 days, 1 to 14 days, 1 to 13 days, 1 to 12 days, 1 to 11 days, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, or 1 to 3 days, but not limited thereto.

[0066] The culture temperature in Step 2 is not particularly limited as long as corneal epithelial cells can be cultured, but it is 30 to 40 °C, preferably about 37 °C. Also, the CO2 concentration during culture in Step 2 is not particularly limited as long as corneal epithelial cells can be cultured, but it is 2 to 5%, preferably about 5%.

[0067] 4. Method for producing corneal epithelial cells The present invention also provides a method for producing purified corneal epithelial cells (hereinafter sometimes referred to as "Production Method 2 of the present invention") including the following steps: Step 1) obtaining a cell population in which the proportion of corneal epithelial cells is 25% or more, and Step 2) expanding and culturing the cell population obtained in Step 1 using laminin 332 as a scaffold.

[0068] Various conditions in Production Method 2 of the present invention are the same as those in Purification Method 2 of the present invention.

[0069] The present invention will be described in more detail in the following examples, but the present invention is not limited by these examples.

Example

[0070] [Test Example 1] Evaluation of the scaffolding function of laminin 211 on iPS cell culture (Materials and Methods) The iPS cell line 201B7 (iPS portal) was used. The cells were seeded at 2300 cells / well in a 24-well plate coated with laminin 511-E8 fragment (iMatrix511, Nippi: 0.26 - 6.58 μg / cm 2 ), or laminin 211 (Biolamina: 0.05 - 6.58 μg / cm 2 ), and cultured in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) medium at 37°C under 5% CO2 for 5 days to evaluate cell adhesion and colony formation. Also, cell adhesion was evaluated by observing under a microscope at the 5th day of culture. Moreover, cell proliferation was evaluated by determining colony formation at each time point by observing under a microscope at the 0th day and 5th day of culture and comparing them. The results are shown in Table 1.

[0071]

Table 1

[0072] As shown in Table 1, when the laminin 511-E8 fragment was used as a substrate, good cell adhesion and colony formation were shown regardless of the coating amount. On the other hand, when laminin 211 was used as a substrate, no cell adhesion or colony formation was observed regardless of the coating amount, similar to the negative control (non-coat).

[0073] [Example 1] Preparation of neural crest cells from iPS cells (Materials and Methods) 1. Induction of differentiation from iPS cells into neural crest cells The iPS cell line 201B7 (iPS portal) was used. iPS cells were seeded at 6500 cells / well on 6-well plates coated with the laminin 511-E8 fragment (iMatrix511, Nippi) and cultured in StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.) medium at 37°C under 5% CO2 for 5 days. Subsequently, the medium was changed to StemFit AK03N (solution A + solution B) supplemented with SB431542 (Stemgent, Inc., 10 μM) and CHIR99021 (Wako, 0.3 μM (condition 1) or 0.9 μM (condition 2)), and differentiation induction into neural crest cells was carried out at 37°C under 5% CO2 for 14 days. The ratio of neural crest cells at the end of induction was calculated by determining the ratio of cells with high expression of the CD271 protein using FACS. In addition, the gene expression status of neural crest cell markers was examined by RT-PCR. The primers used for RT-PCR are shown below.

[0074] Marker gene Primer ID Taqman Cat.No. TFAP2a Hs00271528_CE A15629 SOX9 Hs01001343_g1 4331182 TWIST1 Hs01675818_s1 4331182 (Using β-actin as a reference gene (Hs01101944_s1, 4331182))

[0075] 2. Selective proliferation of neural crest cells The cell population (SEAM) containing neural crest cells prepared in 1. above was made into single cells using TrypLE Select (Thermo Fisher). The single cell population was seeded onto a 6-well plate coated with various scaffolds and cultured under culture conditions suitable for the expansion culture of neural crest cells. More specifically, the cells were cultured for 9 - 10 days at 37 °C under 5% CO2 in a medium prepared by adding SB431542 (10 μM), Epithelium growth factor (Sigma Chemical, 20 ng / mL), and StemFit AK03N Solution C (Ajinomoto Co., Inc., 0.08%) to StemFit AK03N (Solution A + Solution B). After culturing for 9 - 10 days, when the confluency reached approximately 90%, the ratio of neural crest cells in the cell population cultured on each scaffold and the expression status of neural crest cell gene markers were determined by the same method as in 1. above.

[0076] The following scaffolds were used. (1) Laminin 511-E8 fragment (Nippi, 31.3 ng / cm 2 and 312.5 ng / cm 2 ) (2) Laminin 211 (Biolamina, 6.3 ng / cm 2 and 62.5 ng / cm 2 ) (3) Vitronectin (Life Technologies, 31.3 ng / cm 2 and 312.5 ng / cm 2 ) (4) Fibronectin (Sigma Chemical, 1562.5 ng / cm 2 and 3125.0 ng / cm 2 ) In addition, each of the scaffolds (1) - (3) was coated on the surface of the 6-well plate by directly suspending it in the culture medium. The scaffold (4) was dissolved in 1 mL of PBS(-), added to the wells, and left standing at room temperature for 1 hour to coat the surface of the 6-well plate.

[0077] The results are shown in Tables 2 and 3.

[0078]

Table 2

[0079]

Table 3

[0080] As shown in Table 2, when a cell population containing neural crest cells differentiated from iPS cells was expanded using laminin 211 as a substrate, regardless of the proportion of neural crest cells in the cell population to be subjected to the expansion culture and the coating amount of laminin 211 on the surface of the culture vessel, the proportion of neural crest cells in the cell population could be significantly increased.

[0081] [Example 2] Cell proliferation test The purified neural crest cells obtained by expanding the cell population under differentiation induction condition 2 in Example 1 using each substrate material for 9 days were collected, re-seeded on a 6-well plate coated with each substrate material, and further cultured for 5 days under the culture conditions used in the purification step of Example 1. At the time point 5 days after the culture, the cells were made into single cells using TrypLE Select, and the cell number was measured. The results are shown in Table 4.

[0082]

Table 4

[0083] As shown in Table 4, the cell proliferation activity of the neural crest cells purified using laminin 211 as a substrate was good. Considering that, as shown in Table 2 above, the neural crest cells purified using laminin 211 as a substrate have a very high purity, it is suggested that expansion culture using laminin 211 is extremely preferable in the production of neural crest cells with high purity.

[0084] [Test Example 2] Evaluation of the scaffolding function of laminin 332 on iPS cell culture (Materials and Methods) The iPS cell line 201B7 (iPS portal) was seeded at 2500 cells / well onto 12-well plates coated with Laminin511-E8 fragment (iMatrix511, Nippi: 0.05 - 5.00 μg / cm 2 ) or Laminin332 (Biolamina: 0.05 - 5.00 μg / cm 2 ), and cultured for 5 days to evaluate cell adhesion and proliferation. The results of cell adhesion from the day after seeding to day 5 of culture and cell proliferation on day 5 of culture are shown in Table 5.

[0085]

Table 5

[0086] As shown in Table 5, the iPS cells adhered and proliferated at any coating amount with the Laminin511-E8 fragment. On the other hand, with Laminin332, the iPS cells adhered and proliferated at a coating amount of 0.50 μg / cm 2 or more.

[0087] [Example 3] Preparation of corneal epithelial cells from iPS cells (Materials and Methods) 1. Induction of corneal epithelial cells from iPS cells The iPS cell line used was 201B7 (iPS portal). The Laminin511-E8 fragment (iMatrix511, Nippi: 0.25, 0.50, or 5.00 μg / cm 2 ) or Laminin332 (Biolamina: 0.5 or 5.00 μg / cm 2) A 12-well plate coated with [coating material] was seeded with iPS cells at a density of 2500 cells / well and cultured in StemFit (registered trademark) AK03N medium at 37°C under 5% CO2 for 5 days. Subsequently, the medium was switched to StemFit AK03N (solution A + solution B) supplemented with SB431542 (Stemgent, Inc., 10 μM) and CHIR99021 (Wako, 0.6 μM), and corneal epithelial cell differentiation was induced at 37°C under 5% CO2 for 15 days. The differentiation induction rate of corneal epithelial cells was evaluated by FACS for the proportion of cells expressing the corneal epithelial cell markers PAX-6 and Cytokeratin12 protein. The proportion of corneal epithelial cells obtained at the end of the induction is shown in Table 6.

[0088]

Table 6

[0089] 2. Selective proliferation of corneal epithelial cells The cell population (SEAM) containing corneal epithelial cells prepared in 1. above was made into single cells using TrypLE Select (Thermo Fisher). The single-cell population was seeded onto a 6-well plate coated with Laminin332 at a concentration of 0.06 μg / cm 2 and seeded at a density of 2×10 5 cells / well and cultured under culture conditions suitable for the expansion culture of corneal epithelial cells. More specifically, it was cultured in a medium prepared by adding Keratinocyte growth factor (Peprotech, 20 ng / mL) and Y-27632 (Wako, 10 μM) to StemFit AK03N (solution A + solution B) at 37°C under 5% CO2. The proportion of corneal epithelial cells at the time of the first passage of purified culture was determined by evaluating the proportion of cells expressing the corneal epithelial cell markers PAX-6 and Cytokeratin12 protein by FACS in the same manner as in 1. above. The results are shown in Table 7.

[0090]

Table 7

[0091] At the time of the first passage of the purified culture, when the ratio of PAX-6 and Cytokeratin12 positive cells (i.e., CEC) at the end of induction was less than 25%, the cells did not grow sufficiently. However, when the ratio of PAX-6 and Cytokeratin12 positive cells at the end of induction was 25% or more, the ratio of PAX-6 and Cytokeratin12 positive cells improved under all conditions.

Industrial Applicability

[0092] According to the present invention, neural crest cells can be purified from a cell population containing neural crest cells simply and in a very short period of time. Further, according to the present invention, highly pure neural crest cells can be produced simply and in a very short period of time. Furthermore, according to the present invention, corneal epithelial cells can be purified from a cell population containing corneal epithelial cells simply. Also, according to the present invention, highly pure corneal epithelial cells can be produced simply. Therefore, the present invention is extremely useful, for example, in the field of regenerative medicine.

[0093] This application is based on Japanese Patent Application No. 2019-091988 (filing date: May 15, 2019) and Japanese Patent Application No. 2019-186280 (filing date: October 9, 2019), the contents of which are incorporated herein in their entirety.

Claims

1. A method for purifying neural crest cells, comprising the following steps: A step of expanding and culturing a cell population containing neural crest cells using full-length laminin 211 as a substrate, wherein the culture medium used in the step contains a TGFβ inhibitor and an Epithelium Growth Factor.

2. The method according to claim 1, wherein the cell population containing the neural crest cells is subjected to the above step without being subjected to a sorting process of neural crest cells.

3. The method according to claim 1 or 2, wherein the culture period of the above step is 1 to 21 days.

4. The method according to any one of claims 1 to 3, wherein the cell population containing neural crest cells is derived from pluripotent stem cells.

5. The method according to claim 4, wherein the pluripotent stem cells are iPS cells.

6. A method for producing purified neural crest cells, comprising the following steps: A step of expanding and culturing a cell population containing neural crest cells using full-length laminin 211 as a substrate, wherein the culture medium used in the step contains a TGFβ inhibitor and an Epithelium Growth Factor.

7. The method according to claim 6, wherein the cell population containing the neural crest cells is subjected to the above step without being subjected to a sorting process of neural crest cells.

8. The method according to claim 6 or 7, wherein the culture period of the above step is 1 to 21 days.

9. The method according to any one of claims 6 to 8, wherein the cell population containing neural crest cells is derived from pluripotent stem cells.

10. The method according to claim 9, wherein the pluripotent stem cells are iPS cells.

11. A method for purifying corneal epithelial cells, comprising the following steps: A step of expanding and culturing a cell population in which the proportion of corneal epithelial cells is 25% or more using full-length laminin 332 as a substrate (however, the method excludes embodiments including a step of subjecting a cell population in which the proportion of the corneal epithelial cells is 25% or more to magnetic cell separation to obtain CD200-negative / SSEA-4-positive cells), wherein the culture medium used in the step contains a Keratinocyte Growth Factor.

12. The method according to claim 11, wherein the cell population in which the proportion of the corneal epithelial cells is 25% or more is subjected to the above step without being subjected to a sorting process of corneal epithelial cells.

13. In the scale-up culture of the above process, the coating amount of full-length laminin 332 used as a scaffold is 0.01 μg / cm 2 or more and less than 0.5 μg / cm 2 The method according to claim 11 or 12. **Claim 14**: The method according to any one of claims 11 to 13, wherein the cell population in which the proportion of the corneal epithelial cells is 25% or more is derived from pluripotent stem cells. **Claim 15** **Claim 16**: The method according to claim 14, wherein the pluripotent stem cells are iPS cells. **Claim 16** **Claim 17**: A method for producing purified corneal epithelial cells, comprising the following steps: A step of expanding and culturing a cell population in which the proportion of corneal epithelial cells is 25% or more, using full-length laminin 332 as a scaffold (However, this method excludes embodiments that include a step of subjecting a cell population in which the proportion of the corneal epithelial cells is 25% or more to magnetic cell separation to obtain CD200-negative / SSEA-4-positive cells), wherein the medium used in the above step contains Keratinocyte Growth Factor. **Claim 18**: The method according to claim 16, wherein the cell population in which the proportion of the corneal epithelial cells is 25% or more is subjected to the above step without being subjected to sorting treatment of corneal epithelial cells.

18. In the scale-up culture of the above process, the coating amount of full-length laminin 332 used as a scaffold is 0.01 μg / cm 2 or more and less than 0.5 μg / cm 2 The method according to claim 16 or 17. **Claim 19**: The method according to any one of claims 16 to 18, wherein the cell population in which the proportion of the corneal epithelial cells is 25% or more is derived from pluripotent stem cells. **Claim 20** **Claim 21**: The method according to claim 19, wherein the pluripotent stem cells are iPS cells.

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