Coating agent for inducing differentiation of pluripotent stem cells into brain capillary endothelial-like cells and its use

Abstract

Provided is a technique that enables stable supply of brain capillary endothelium-like cells. This coating agent for inducing differentiation of pluripotent stem cells into brain capillary endothelium-like cells includes at least one component of a laminin 221 fragment and N-terminal vitronectin.

Description

Technical Field

[0001] The present disclosure relates to a coating agent for inducing differentiation of pluripotent stem cells into brain microvascular endothelial-like cells. This application is based on Japanese Application No. 2020-021031 filed on February 10, 2020, the content of which is incorporated herein by reference.

Background Art

[0002] Brain microvascular endothelial cells (BMECs), which are one of the constituent cells of the blood-brain barrier (BBB), inhibit the non-specific invasion of substances into the brain parenchyma by strong cell-cell adhesion and the expression of efflux transporters. In drug discovery, this strong barrier function inhibits the transfer of drug candidate drugs to the brain parenchyma (nerve) side, and thus drug development may be aborted. For this reason, a screening model capable of evaluating pharmacokinetics in the human BBB is desired, and differentiation induction of pluripotent stem cells into brain microvascular endothelial-like cells (iBMECs) has been carried out (for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In Patent Document 1, when inducing the differentiation of human induced pluripotent stem cells (iPS cells) into iBMECs, culture is performed using a culture dish coated with Matrigel. However, Matrigel has the property of solidifying when the temperature rises, and since coating requires complicated operations, it has been difficult to maintain reproducibility. In addition, since Matrigel is produced from mouse granulomas, the differentiation efficiency of iBMECs varies depending on the manufacturing company and production lot of Matrigel. For this reason, the inventors of the present invention have conducted intensive research and have invented a technique that enables the stable supply of iBMECs.

Means for Solving the Problems

[0005] The present invention has been made to solve the above-described problems and can be realized in the following forms.

[0006] (1) According to one form of the present invention, a coating agent for inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells is provided. The coating agent of this form contains at least one of a laminin 221 fragment and N-terminal vitronectin. According to the coating agent of this form, it is possible to suppress a decrease in reproducibility when inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells, and it is also possible to suppress fluctuations in differentiation efficiency, so that brain capillary endothelial-like cells can be stably supplied.

[0007] (2) In the coating agent of the above form, the component may be contained in the coating agent at 0.01 nmol / L or more and 10 mmol / L or less. According to the coating agent of this form, it is possible to further suppress a decrease in differentiation efficiency.

[0008] (3) According to another form of the present invention, a differentiation induction kit is provided. The differentiation induction kit of this form includes the above coating agent and cells. According to the differentiation induction kit of this form, it is possible to easily induce the differentiation of pluripotent stem cells into brain capillary endothelial-like cells.

[0009] (4) The present invention can also be realized by using it as a coating agent for inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells, which contains at least one of the components of laminin 221 fragment and N-terminal vitronectin.

[0010] (5) According to another aspect of the present invention, a culture dish including a coating layer coated with a coating agent for inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells is provided. In the culture dish of this aspect, the coating agent contains at least one of the components of laminin 221 fragment and N-terminal vitronectin. According to the culture dish of this aspect, it is possible to suppress a decrease in reproducibility when inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells, and also to suppress fluctuations in differentiation efficiency, so that brain capillary endothelial-like cells can be stably supplied.

[0011] (6) In the culture dish of the above aspect, the component may be contained in an amount of 1 ng or more and 1 mg or less per 1 cm 2 of the coated area of the coating layer. According to the coating agent of this aspect, it is possible to further suppress a decrease in differentiation efficiency.

[0012] (7) According to another aspect of the present invention, a method for producing brain capillary endothelial-like cells by inducing the differentiation of pluripotent stem cells is provided. The method for producing brain capillary endothelial-like cells of this aspect includes a step of culturing the pluripotent stem cells using at least one of the components of laminin 221 fragment and N-terminal vitronectin. According to the method for producing brain capillary endothelial-like cells of this aspect, it is possible to suppress a decrease in reproducibility when inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells, and also to suppress fluctuations in differentiation efficiency, so that brain capillary endothelial-like cells can be stably supplied.

[0013] (8) In the method for producing brain capillary endothelial-like cells of the above aspect, the pluripotent stem cells may be induced pluripotent stem cells.

[0014] (9) In the method for producing brain capillary endothelial-like cells of the above-described form, the induced pluripotent stem cells may be human induced pluripotent stem cells.

[0015] (10) According to another aspect of the present invention, there is provided a method for evaluating the blood-brain barrier permeability of a test substance using a cell layer of brain capillary endothelial-like cells obtained by the method for producing brain capillary endothelial-like cells of the above-described form.

[0016] (11) The method for evaluating the blood-brain barrier permeability of a test substance of the above-described form may include the following steps (i) to (iii). (i) A step of preparing the cell layer; (ii) A step of bringing the test substance into contact with the cell layer; (iii) A step of evaluating the permeability of the test substance by quantifying the test substance that has permeated through the cell layer.

[0017] (12) According to another aspect of the present invention, there is provided a method for evaluating the effect of a test substance on the blood-brain barrier function using a cell layer of brain capillary endothelial-like cells obtained by the method for producing brain capillary endothelial-like cells of the above-described form.

[0018] (13) According to another aspect of the present invention, there is provided a method for inducing differentiation of pluripotent stem cells into brain capillary endothelial-like cells. The method for inducing differentiation of pluripotent stem cells of this form into brain capillary endothelial-like cells includes a step of culturing the pluripotent stem cells using at least one component of a laminin 221 fragment and an N-terminal vitronectin. According to the method for inducing differentiation of pluripotent stem cells of this form into brain capillary endothelial-like cells, it is possible to suppress a decrease in reproducibility when inducing differentiation of pluripotent stem cells into brain capillary endothelial-like cells, and it is also possible to suppress fluctuations in differentiation efficiency, so that brain capillary endothelial-like cells can be stably supplied.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] The disclosure of this specification relates to a coating agent for inducing the differentiation of pluripotent stem cells into brain capillary endothelial-like cells. Brain capillary endothelial cells (hereinafter also referred to as "BMECs") are the main cells that constitute the blood-brain barrier (hereinafter also referred to as "BBB"). The BBB has a structure in which pericytes and astrocytes cover the periphery of BMECs. According to the present disclosure, cells similar to BMECs, that is, brain capillary endothelial-like cells (hereinafter also referred to as "iBMECs") that exhibit the characteristics of BMECs, can be obtained. The iBMECs obtained by the method of the present disclosure are useful for constructing a BBB model and are used, for example, in the evaluation of the brain permeability (blood-brain barrier permeability) of a test substance (typically a drug).

[0021] The term "pluripotent stem cell" refers to a cell that has both the ability to differentiate into all cells that make up a living body (pluripotency) and the ability to produce daughter cells with the same differentiation ability as itself through cell division (self-renewal ability). Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing for the formation of teratomas containing cells of each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0022] Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), etc., but are not limited thereto as long as they are cells having both pluripotency and self-renewal ability. Preferably, ES cells or iPS cells are used. More preferably, iPS cells are used. Pluripotent stem cells are preferably cells of mammals (for example, primates such as humans and chimpanzees, rodents such as mice and rats, etc.), particularly preferably human cells. Therefore, in the most preferred embodiment of the present disclosure, human iPS cells are used as pluripotent stem cells.

[0023] ES cells can be established, for example, by culturing pre-implantation early embryos, inner cell masses constituting the early embryos, single blastomeres, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, J. A. et al., Science, 282, 1145-1147 (1998)). As the early embryo, an early embryo produced by nuclear transfer of a somatic cell nucleus may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Akira Iritani et al. (Protein, Nucleic Acid, Enzyme, 44, 892 (1999)), Baguisi et al. (Nature Biotechnology, 17, 456 (1999)), Wakayama et al. (Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000), Tachibana et al. (Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013) in press)). As the early embryo, a parthenogenetic embryo may be used (Kim et al. (Science, 315, 482-486 (2007)), Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al. (Cloning Stem Cells, 9, 432-449 (2007)), Revazova et al. (Cloning Stem Cells, 10, 11-24 (2008)).In addition to the above-mentioned papers, for the generation of ES cells, Strelchenko N., et al. Reprod Biomed Online. 9: 623-629, 2004; Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al Stem Cells 24: 2669-2676, 2006; Wassarman, P.M. et al. Methods in Enzymology, Vol.365, 2003, etc. are also reference-worthy. It should be noted that the fused ES cells obtained by cell fusion of ES cells and somatic cells are also included in the embryonic stem cells in the present disclosure.

[0024] Some ES cells are available from preservation institutions or are commercially available. For example, for human ES cells, they can be obtained from the Institute for Frontier Medical Sciences, Kyoto University (such as KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc. In addition, ES cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, SCF, etc. (Matsui et al., Cell, 70, 841-847 (1992), Shamblott et al., Proc. Natl. Acad. Sci. USA, 95 (23), 13726-13731 (1998), Turnpenny et al., Stem Cells, 21(5), 598-609,(2003)).

[0025] iPS cells are cells with pluripotency (multipotency) and proliferation ability, which are produced by reprogramming somatic cells through the introduction of reprogramming factors, etc. iPS cells exhibit properties similar to those of ES cells. The somatic cells used for the production of iPS cells are not particularly limited and may be differentiated somatic cells or undifferentiated stem cells. Also, their origin is not particularly limited, but preferably somatic cells of mammals (for example, primates such as humans and chimpanzees, rodents such as mice and rats) are used, and particularly preferably human somatic cells. iPS cells can be produced by various methods reported so far. It is also naturally assumed that iPS cell production methods to be developed in the future will be applied.

[0026] The most basic method for producing iPS cells is a method of introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, which are transcription factors, into cells using a virus (Takahashi K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007). There are reports of the establishment of human iPS cells by introducing four factors, Oct4, Sox2, Lin28, and Nanog (Yu J, et al: Science 318(5858), 1917-1920, 2007). The establishment of iPS cells by introducing three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), two factors, Oct3 / 4 and Klf4 (Kim J B, et al: Nature 454 (7204), 646-650, 2008), or only Oct3 / 4 (Kim J B, et al: Cell 136 (3), 411-419, 2009) has also been reported. In addition, a method of introducing a protein, which is the expression product of a gene, into cells (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al: Cell Stem Cell 4, 472-476, 2009) has also been reported. On the other hand, there are reports that it is possible to improve the production efficiency and reduce the factors to be introduced by using an inhibitor BIX-01294 against histone methyltransferase G9a, a histone deacetylase inhibitor valproic acid (VPA), or BayK8644, etc. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J, et al: PLoS. Biol. 6 (10), e 253, 2008).Regarding gene transfer methods, studies are also underway. In addition to retroviruses, lentiviruses (Yu J, et al: Science 318(5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903), 945-949, 2008), plasmids (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been used for gene transfer and related technologies have been developed.

[0027] Cells in which transformation, i.e., reprogramming (reinitialization) into iPS cells has occurred can be selected using as indicators the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript. The selected cells are recovered as iPS cells.

[0028] iPS cells can also be obtained, for example, from the National University Corporation Kyoto University or the RIKEN BioResource Center, National Institute of Advanced Industrial Science and Technology.

[0029] As used herein, "inducing differentiation" means acting to cause differentiation along a specific cell lineage. In the present disclosure, when inducing the differentiation of pluripotent stem cells into brain microvascular endothelial-like cells (iBMECs), a coating agent containing at least one of laminin 221 fragment (hereinafter also referred to as "LN221F") and N-terminal vitronectin (hereinafter also referred to as "VTN-N") is used. Details of the coating agent are described below.

[0030] <Coating agent> Generally, for the purpose of improving cell viability and proliferation rate, promoting differentiation induction, cell selection, etc., cells may be cultured on a culture surface coated with basement membrane components, adhesion molecules, etc. Matrigel, which has been conventionally used as a coating agent, has laminin 111 as a main component and contains collagen type IV, heparan sulfate proteoglycan, entactin / nidogen, and various growth factors.

[0031] Laminin is a heterotrimeric molecule composed of three subunit chains, an α chain, a β chain, and a γ chain. Five types of α chains (α1-α5), three types of β chains (β1-β3), and three types of γ chains (γ1-γ3) are known. Known reference sequence RNAs for laminin are NM_000426, NM_001079823 (Subunit alpha2), NM_002291 (Subunit beta1), NM_002292 (Subunit beta2), NM_000228 (Subunit beta3), NM_001318046, NM_001318047, NM_001318048, NM_007356 (Subunit beta4), NM_002293 (Subunit gamma 1), NM_005562, NM_018891 (Subunit gamma 2).

[0032] Laminin 221 is a laminin molecule composed of subunit chains of α2 chain, β2 chain, and γ1 chain and is known to be abundant in the heart.

[0033] As used herein, the "laminin 221 fragment (LN221F)" means a fragment (E8 fragment) corresponding to the integrin-binding site of laminin 221. In addition, the "laminin 221 fragment (LN221F)" in this specification is assumed to include proteins highly homologous to LN221F. As used herein, the "protein highly homologous to LN221F" refers to a protein whose amino acid sequence contains an amino acid sequence that is 80% or more identical to the amino acid sequence of LN221F. That is, the "protein highly homologous to LN221F" includes proteins with longer amino acid sequences than LN221F. From the perspective of enhancing the barrier function, the homology of the protein highly homologous to LN221F is preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. Generally, the molecular weight of the laminin 221 fragment (LN221F) is about 150 kDa to about 170 kDa. In this specification, it is estimated that the molecular weight of the laminin 221 fragment (LN221F) is 150 kDa.

[0034] As used herein, "N-terminal vitronectin (VTN-N)" refers to a protein in which the N-terminal of vitronectin is deleted and which contains an RGD sequence. In addition, "N-terminal vitronectin (VTN-N)" as used herein is assumed to include proteins highly homologous to VTN-N. As used herein, a "protein highly homologous to VTN-N" refers to a protein that contains an amino acid sequence that is 70% or more identical to the amino acid sequence of VTN-N in the amino acid sequence constituting such protein, or a protein that contains an RGD sequence in the amino acid sequence. "Proteins highly homologous to VTN-N" include proteins having an amino acid sequence longer than that of VTN-N. From the viewpoint of enhancing the barrier function, the homology of a protein highly homologous to VTN-N is preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. As the Reference Sequence RNA of vitronectin, NM_000638 is known, and as the Reference Sequence protein of vitronectin, N NP_000629 is known. In addition, in this specification, it is presumed that the molecular weight of N-terminal vitronectin (VTN-N) is 47.6 kDa.

[0035] The coating agent of the present disclosure may contain only one of LN221F and VTN-N, or may contain both LN221F and VTN-N. That is, the coating agent of the present disclosure contains at least one of the components of LN221F and VTN-N. The coating agent of the present disclosure preferably contains LN221F. In addition, the coating agent of the present disclosure may exclude Matrigel. The above components may be contained in the coating agent at 0.01 nmol / L or more and may be contained at 10 mmol / L or less. The content of the above components is preferably 0.1 nmol / L or more, more preferably 1 nmol / L or more, in the coating agent. The content of the above components is preferably 1 mmol / L or less, more preferably 0.1 mmol / L or less, in the coating agent. When the above components are contained in the coating agent at 0.01 nmol / L or more, the differentiation efficiency can be improved. In addition, when the above components are contained in the coating agent at 1 mmol / L or less, an increase in the cost required for differentiation induction can be suppressed.

[0036] By diluting the coating agent containing the above components with a medium or the like, pouring it into a culture dish, a Transwell insert, etc., and allowing it to stand, a culture dish or the like with a coated layer can be obtained. Hereinafter, forming a coated layer is also referred to as "coating". The above components may be contained at 1 ng or more and may be contained at 1 mg or less per 1 cm of the area of the culture dish, that is, the coated area of the coated layer. 2 The content of the above components is preferably 10 ng or more, more preferably 0.1 μg or more, per 1 cm of the coated area. Also, the content of the above components is preferably 0.1 mg or less, more preferably 10 μg or less, per 1 cm of the coated area. When the above components are contained at 1 ng or more per 1 cm of the coated area of the coated layer, a decrease in the differentiation efficiency can be more suppressed. In addition, when the above components are contained at 1 mg or less per 1 cm of the coated area of the coated layer, an increase in the cost required for differentiation induction can be suppressed. 2 2 2 2 ​​​​

[0037] <Differentiation induction kit> The above coating agent may be provided in the form of a kit combined with cells for manufacturing iBMECs. That is, according to another aspect of the present disclosure, a differentiation induction kit comprising the above coating agent and cells is provided. As the cells in this kit, pluripotent stem cells or intermediate cells in the differentiation process of pluripotent stem cells as described above can be used. Such pluripotent stem cells are preferably ES cells or iPS cells, and more preferably iPS cells. Further, as such pluripotent stem cells, cells of mammals (for example, primates such as humans and chimpanzees, rodents such as mice and rats, etc.) are preferably used, and human cells are more preferably used. As the intermediate cells in the differentiation process, vascular endothelial progenitor cells are preferably used. That is, the cells in this kit are particularly preferably vascular endothelial progenitor cells differentiated from human iPS cells. Further, the state of the cells in this kit may be, for example, a state stored at 37°C or the like, a cryopreserved state, or the like. The cells in this kit are preferably cryopreserved cells from the viewpoints of transportation and handling. The user of the kit can easily induce the differentiation of cells into iBMECs by coating the coating agent on a culture dish or the like and then seeding and culturing the cells, or by seeding the coating agent and the cells on a culture dish or the like and culturing the cells. During the culture, other materials may be used in combination with the coating agent and the cells contained in the kit, and subculture may be appropriately performed. Note that, as the culture dish, for example, any form of culture dish such as a petri dish or a cell culture plate having a plurality of wells can be used.

[0038] Since LN221F and VTN-N do not have the property of solidifying due to temperature rise like Matrigel, they are easier to handle compared to Matrigel, and the coating procedure is simple, so it is possible to suppress a decrease in reproducibility in differentiation induction. Also, since LN221F and VTN-N are composed of a single component and have little lot-to-lot variation, it is possible to suppress fluctuations in the differentiation efficiency of iBMECs due to differences in manufacturing companies or manufacturing lots. Therefore, according to the present disclosure, it becomes possible to stably supply iBMECs.

[0039] <Method for producing iBMECs by inducing differentiation of pluripotent stem cells> The method for producing iBMECs by inducing differentiation of pluripotent stem cells in the present disclosure includes a step of culturing pluripotent stem cells using at least one of the components LN221F and VTN-N. This step may be a step of seeding pluripotent stem cells on a culture dish or the like coated with at least one of the components LN221F and VTN-N at the time of subculture and culturing using a medium.

[0040] As the medium used during differentiation induction, for example, a basal medium such as DMEM Ham's F-12 (DMEM / F12) or Human Endothelial-SFM may be employed. It is preferable to add serum or a serum substitute (such as Knockout serum replacement (KSR)) to the medium from the viewpoints of cell growth rate and maintenance. The serum is not limited to fetal bovine serum, and human serum, sheep serum, etc. can also be used. Also, instead of normal serum, plasma-derived serum (PDS) may be used. The addition amount of the serum or serum substitute is, for example, 0.1% (v / v) to 10% (v / v). It is preferable to add fibroblast growth factor (FGF) 2 such as human FGF2 (for example, human recombinant FGF2) to the medium, and the addition concentration of FGF2 may be, for example, 1 ng / mL to 500 ng / mL.

[0041] The culture period for differentiation induction may be, for example, 6 to 30 days, preferably 8 to 12 days. By setting the culture period as such, it is possible to suppress the deterioration of the differentiation induction efficiency into iBMECs and also to suppress the occurrence of unintended differentiation (differentiation into other cells).

[0042] During the differentiation induction, subculture may be performed. For example, when the cells become confluent or sub-confluent, a part of the cells may be collected and transferred to another culture vessel to continue the culture. When collecting cells accompanied by medium replacement or subculture, etc., the cells may be pretreated with a ROCK inhibitor such as Y-27632 (Rho-associated coiled-coil forming kinase / Rho-binding kinase) in order to suppress cell death. Also, in order to enhance the cell selection effect, during the differentiation induction, the cells may be seeded on a culture surface coated with fibronectin and collagen IV.

[0043] Other culture conditions (such as culture temperature) may be the conditions generally adopted in the culture of animal cells. For example, it may be cultured in an environment of 37°C and 5% CO2. Also, not limited to the method of culturing cells two-dimensionally using a culture dish, etc., three-dimensional culture may be carried out using a three-dimensional culture plate, etc. containing a coating layer coated with the above coating agent. Also, not limited to the mode in which the above coating agent is pre-coated on a culture instrument such as a culture dish, etc., a mode in which the use of the above coating agent and the seeding of cells are performed simultaneously may also be possible.

[0044] By the above-described differentiation induction method, iBMECs are produced from pluripotent stem cells. By continuously culturing under culture conditions suitable for the maintenance and proliferation of iBMECs, a monolayer (cell layer) of iBMECs is formed. According to the method of the present disclosure, a cell layer having excellent barrier function can be obtained. The barrier function of the cell layer obtained by the method of the present disclosure can be characterized by strong tight junctions and the maintenance of tight junctions over a long period of time.

[0045] Here, generally, in the in vivo of rats, the transendothelial electrical resistance (TEER value), which is an index of tight junctions in BMECs, is 1000 - 2000 Ω×cm 2 or so, and as a model of the human BBB, it is also desirable to have a TEER value of 1000 Ω×cm 2 or more.

[0046] According to the method for producing iBMECs of this form, a cell layer with a TEER value of 1000 Ω×cm 2 or more can be formed, and a TEER value exceeding 1000 Ω×cm 2 can be maintained for a long period, such as 9 days or more.

[0047] The barrier function of the cell layer obtained by the method of the present disclosure can be further characterized by having the functions of drug transporters (such as BCRP, P-gp, GLUT1) that are important or characteristic in BMECs. Also, the functions of iBMECs can be determined or evaluated by using, as an index, the expression of tight junction markers (such as Claudin5, Occludin, ZO-1) that are important or characteristic in BMECs.

[0048] In the later stage of differentiation induction, by performing the culture on a semipermeable membrane (porous membrane), a cell layer of iBMECs can be formed on the semipermeable membrane. This mode is particularly effective when using the cell layer of iBMECs obtained by the production method of the present disclosure for various assays. For example, a culture container equipped with a culture insert (having a culture surface made of a semipermeable membrane) (such as Transwell (registered trademark) provided by Corning) can be used, and cell culture can be performed in the insert to form a cell layer.

[0049] Other embodiments of the present disclosure relate to the use of iBMECs produced by inducing differentiation by the above method. As described above, according to the present disclosure, it is possible to obtain a cell layer composed of iBMECs, and the cell layer can be used for a BBB model. For example, an assay using the cell layer is useful for evaluating the BBB permeability of a test substance such as a pharmaceutical or a pharmaceutical candidate. Therefore, provided is a method for evaluating the BBB permeability of a test substance (hereinafter, also referred to as "the BBB permeability evaluation method of the present disclosure") using the cell layer of iBMECs obtained by the production method of the present disclosure. In the BBB permeability evaluation method of the present disclosure, the following steps (i) to (iii) are performed. (i) A step of preparing a cell layer of iBMECs obtained by the production method of the present disclosure (ii) A step of bringing a test substance into contact with the cell layer (iii) A step of evaluating the permeability of the test substance by quantifying the test substance that has permeated the cell layer

[0050] In step (i), a cell layer of iBMECs obtained by the production method of the present disclosure is prepared. In addition to the cell layer of iBMECs, other cells (pericytes, astrocytes, etc.) may be used in combination. For example, a culture vessel equipped with a culture insert is adopted to form a cell layer of iBMECs in the culture insert (a cell layer is formed on the upper surface of the bottom of the culture insert), and pericytes are cultured in a state of adhering to the bottom back surface of the culture insert (pericyte adhesion co-culture system), pericytes are cultured in the compartment between the culture insert and the well (pericyte non-adhesion co-culture system), pericytes are cultured in a state of adhering to the bottom back surface of the culture insert and astrocytes are cultured in the compartment between the culture insert and the well (pericyte adhesion / astrocyte non-adhesion co-culture system), or astrocytes are cultured in a state of adhering to the bottom back surface of the culture insert (astrocyte adhesion co-culture system).

[0051] In step (ii), the "contact" is typically carried out by adding the test substance to the culture medium. The timing of adding the test substance is not particularly limited. Therefore, after starting the culture in a medium without the test substance, the test substance may be added at a certain point, or the culture may be started in a medium containing the test substance in advance.

[0052] Typically, a pharmaceutical or a substance as a pharmaceutical candidate is used as the test substance. However, the test substance is not particularly limited, and organic compounds or inorganic compounds with various molecular sizes can be used as the test substance. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.)). Plant extracts, cell extracts, culture supernatants, etc. may be used as the test substance. By adding two or more test substances simultaneously, the interaction, synergistic effect, etc. between the test substances may be investigated. The test substance may be derived from natural products or may be synthetic. In the latter case, for example, an efficient assay system can be constructed using combinatorial synthesis techniques.

[0053] The period for contacting the test substance can be arbitrarily set. The contact period is, for example, 10 minutes to 3 days, preferably 1 hour to 1 day.

[0054] In step (iii), the test substance that has passed through the cell layer is quantified. For example, when using a culture vessel equipped with a culture insert such as Transwell (registered trademark), the test substance that has passed through the culture insert, that is, the test substance that has moved into the upper vessel (culture insert) or the lower vessel (well) through the cell layer, is quantified by a measurement method corresponding to the test substance. Examples of the measurement method include measurement methods such as mass spectrometry, liquid chromatography, immunological techniques (e.g., Fluorescent Immunoassay (FIA method), Enzyme Immunoassay (EIA method)). Based on the quantification result (the amount of the test substance that has passed through the cell layer) and the amount of the test substance used (typically the amount added to the medium), the membrane permeability of the test substance is evaluated. In addition to membrane permeability, absorption by the cell layer (absorbability), the effect on the cell layer (e.g., the effect on the barrier function), the effect on the expression or function of transporters (e.g., BCRP, P-gp), etc. may also be evaluated. Usually, since absorbability is the reverse of permeability, it can be evaluated by the same method as in the case of permeability. The effect on the barrier function can be evaluated by measuring the TEER value, a permeation test using a non-absorbable marker, etc. Also, the effect on the expression of transporters can be evaluated by immunological techniques, Western blotting, flow cytometry, etc., and the effect on the same function can be evaluated by, for example, an activity test using a substrate.

[0055] As can be seen from the above description, by using the cell layer of iBMECs, the effect of a test substance on the BBB function (for example, improvement, decrease, or breakdown of the BBB function) can also be evaluated. Therefore, the present disclosure also provides, as another use of the cell layer of iBMECs produced by the production method of the present disclosure, an evaluation method targeting or directed to the BBB function, that is, a method for evaluating the effect of a test substance on the BBB function. The evaluation method is useful, for example, as a means for searching for substances that enhance (improve) the barrier function, substances that protect the barrier function, substances that regulate the barrier function, and the like. It can also be used for evaluating the toxicity to the BBB. In this evaluation method, similar to the above-described BBB permeability evaluation method, a step of preparing a cell layer and a step of bringing a test substance into contact with the cell layer are performed, and then the effect on the barrier function of the cell layer is evaluated. The method for evaluating the effect on the barrier function is as described above.

Example

[0056] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. In the following description, unless otherwise specified, “%” indicates volume / volume %, and “w / v%” indicates weight / volume %. The materials used in the examples were obtained from the companies shown in Table 1, respectively.

[0057]

Table 1

[0058] 1. Materials and methods (1) Medium For the culture of mouse embryonic fibroblasts (MEF) as feeder cells, Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mmol / L L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), and 1×penicillin-streptomycin was used. For the detachment solution of MEF, 0.05 w / v% trypsin-ethylenediaminetetraacetic acid (EDTA) was used, and for the preservation solution of MEF, Cell Banker 1 was used. For the maintenance culture of human iPS cells, DMEM / F12 containing 20% KnockOut Serum Replacement (KSR), 0.8% NEAA, 2 mmol / L L-Glu, 0.1 mmol / L 2-mercaptoethanol (2-MeE), and 5 ng / mL fibroblast growth factor (FGF) 2 was used. For the detachment solution of human iPS cells, Dulbecco's phosphate-buffered saline (PBS) containing 1 mg / mL collagenase IV, 0.25 w / v% trypsin, 20% KSR, and 1 mmol / L calcium chloride was used. For the preservation solution of human iPS cells, Cell Reservoir One was used.

[0059] (2) Culture of human iPS cells As human iPS cells, the 610B1 strain and 648A1 strain (obtained from the RIKEN Institute respectively) were used. The human iPS cells were seeded on mitomycin C-treated MEF (6×10 5 cells / 100 mm dish) and cultured at 37°C in a CO2 incubator under the condition of 5% CO2 / 95% air. The passage of human iPS cells was performed at a split ratio of 1:2 to 1:4 after culturing for 3 to 5 days.

[0060] (3) Coating agent Matrigel GFR (Growth Factor Reduced), Fibronectin (hereinafter also referred to as "FBN"), N-terminal Vitronectin (hereinafter also referred to as "VTN-N"), Laminin 221 fragment (hereinafter also referred to as "LN221F"), Laminin 411 fragment (hereinafter also referred to as "LN411F"), and Laminin 511 fragment (hereinafter also referred to as "LN511F") were each used as a coating agent at the start of differentiation induction. Also, a combination of Fibronectin and Collagen type IV was used as a coating agent during subculture in the middle of differentiation induction. In the following description, Matrigel GFR is also simply referred to as "Matrigel". The concentration of the coating agent containing the above LN221F was 175 μg / 0.35 mL, and the concentration of the coating agent containing the above VTN-N was 0.5 mg / mL.

[0061] (4) Formation of the coat layer Using each of the above coating agents, a coat layer was formed. For Matrigel, it was diluted 30-fold with the maintenance medium for human iPS cells on ice, poured into a well plate, and allowed to stand at 37°C for 30 minutes or more to form a coat layer. For FBN, VTN-N, LN221F, LN411F, and LN511F, they were diluted with PBS to a concentration of 1 μg / cm 2 and then poured into a well plate and allowed to stand at 37°C for 1 to 2 hours to form a coat layer respectively. Also, for the combination of Fibronectin and Collagen type IV, they were diluted with PBS to concentrations of 100 μg / mL and 400 μg / mL respectively, poured into a Transwell insert, and allowed to stand at 37°C for 2 hours or more to form a coat layer.

[0062] (5) Induction of differentiation of human iPS cells into iBMECs Figure 1 is an explanatory diagram showing the differentiation protocol of human iPS cells into iBMECs. As shown in Figure 1, when inducing the differentiation of human iPS cells into iBMECs, Matrigel and a single basement membrane component as described above were used as coating agents from day 0 to day 8 of iBMECs differentiation. The differentiation induction was started at the time of subculture by seeding on a culture dish coated with each of the above coating agents and culturing in StemSure hPSC medium containing 35 ng / mL FGF2 until the proportion occupied by undifferentiated colonies reached about 60 - 70% (day 0 of differentiation). After culturing for 6 days using a DMEM / F12 based medium supplemented with 20% KSR, 0.8% NEAA, 2 mmol / L L-Glu, and 0.1 mmol / L 2-MeE (day 6 of differentiation), 10 μM all-trans retinoic acid (RA) and 20 ng / mL FGF2 were added to a HE-SFM based medium supplemented with 1% platelet-poor plasma derived serum (PDS) and 1× penicillin-streptomycin, and cultured for 2 days. Then (day 8 of differentiation), the cells were detached with Accutase and seeded at 3×10 5 cells / well on a Transwell insert or culture dish coated with Fibronectin and Collagen type IV. The cells were cultured for 1 day in a HE-SFM based medium containing 10 μM RA and 20 ng / mL FGF2, and then (day 9 of differentiation), differentiated into iBMECs by culturing in a HE-SFM based medium (without addition of RA and FGF2). Medium change was not performed after day 9 of differentiation.

[0063] (6) Measurement of transendothelial electrical resistance (TEER) value The TEER values of iBMECs derived from the 610B1 strain (day 10 of differentiation) induced to differentiate on Matrigel or FBN, VTN-N, LN221F, LN411F, LN511F were measured. The number of samples was 3, and based on the TEER values of iBMECs induced to differentiate on Matrigel (hereinafter also referred to as "Matrigel-iBMECs"), the relative TEER values of iBMECs induced to differentiate on FBN, VTN-N, LN221F, LN411F, LN511F were determined. Also, for each of the 610B1 strain and 648A1 strain, the TEER values between Matrigel-iBMECs and iBMECs induced to differentiate on LN221F (hereinafter also referred to as "LN221F-iBMECs") were measured from day 1 after seeding (day 9 of differentiation) to day 10 after seeding (day 18 of differentiation). The number of samples was 6, and the time-course change of the TEER value was determined. The TEER value was measured using a Millicell ERS-2 (chopstick type) according to the attached manual. The liquid volume of the medium was 300 μL on the apical side and 800 μL on the basal side.

[0064] (7) Permeability test of fluorescein isothiocyanate-dextran 4 kDa (FD4) and lucifer yellow (LY) The permeability tests of FD4 and LY were performed on Matrigel-iBMECs and LN221F-iBMECs for each of the 610B1 strain and 648A1 strain. The number of samples was 6. On day 10 of differentiation, the medium was replaced with transport buffer (HBSS containing 10 mM HEPES solution) and cultured at 37°C for 20 minutes. A transport buffer containing 1 mg / mL FD4 or 300 μM LY was added to the apical side. After incubating at 37°C for 60 minutes, 100 μL of the solution was collected from the basolateral side. The liquid volume of the transport buffer was 300 μL on the apical side and 800 μL on the basal side. The fluorescence intensity of FD4 or LY was measured using a Synergy HTX multimode plate reader and analyzed with Gen5 data analysis software.

[0065] (8) RNA extraction Extraction was performed according to the attached manual of the Agencourt (registered trademark) RNAdvance Tissue Kit.

[0066] (9) Reverse transcription reaction Synthesis of complementary DNA (cDNA) was carried out using ReverTra Ace (registered trademark) qPCR RT Master Mix according to the attached manual.

[0067] (10) RT-qPCR method Analysis of the gene expression levels of Matrigel-iBMECs and LN221F-iBMECs (day 10 of differentiation) derived from the 610B1 strain, and human-derived primary BMECs (hereinafter also referred to as "hBMECs") was performed by RT-qPCR. The number of samples of Matrigel-iBMECs and LN221F-iBMECs was 3, and the number of samples of hBMECs was 1. RT-qPCR was performed using the KAPA SYBR Fast qPCR Kit with cDNA as a template according to the attached manual. The primer sequences used in RT-qPCR are shown in Table 2. The obtained results were corrected using HPRT1 as an endogenous control. Based on the mRNA expression level in Matrigel-iBMECs, the relative expression levels of mRNA in LN221F-iBMECs and hBMECs were determined. The sequence of the forward primer for CDH5 is shown as SEQ ID NO: 1, the sequence of the reverse primer for CDH5 is shown as SEQ ID NO: 2, the sequence of the forward primer for MDR1 is shown as SEQ ID NO: 3, the sequence of the reverse primer for MDR1 is shown as SEQ ID NO: 4, the sequence of the forward primer for BCRP is shown as SEQ ID NO: 5, the sequence of the reverse primer for BCRP is shown as SEQ ID NO: 6, the sequence of the forward primer for GLUT1 is shown as SEQ ID NO: 7, the sequence of the reverse primer for GLUT1 is shown as SEQ ID NO: 8, the sequence of the forward primer for Occludin is shown as SEQ ID NO: 9, the sequence of the reverse primer for Occludin is shown as SEQ ID NO: 10, the sequence of the forward primer for ZO-1 is shown as SEQ ID NO: 11, the sequence of the reverse primer for ZO-1 is shown as SEQ ID NO: 12, the sequence of the forward primer for LAT1 is shown as SEQ ID NO: 13, the sequence of the reverse primer for LAT1 is shown as SEQ ID NO: 14, the sequence of the forward primer for HPRT1 is shown as SEQ ID NO: 15, and the sequence of the reverse primer for HPRT1 is shown as SEQ ID NO: 16, as the primers used in RT-qPCR.

[0068]

Table 2

[0069] (11) Immunostaining method To analyze the protein expression levels and localization of BMEC markers in Matrigel-iBMECs and LN221F-iBMECs (day 10 of differentiation) derived from the 610B1 strain, analysis by immunostaining was performed. The antibodies used in the immunostaining are shown in Table 3. For Zonula occludens-1 (ZO-1) and occludin, cells on a 96-well plate were fixed in 4 w / v% paraformaldehyde at room temperature for 15 minutes, washed twice with PBS containing glycine, and then permeabilized with PBS containing 0.1 w / v% Triton X-100 at room temperature for 25 minutes. After blocking with 5% donkey serum at room temperature for 20 minutes, the primary antibody was reacted at room temperature for 2 hours. After washing three times with PBS, the secondary antibody was reacted at room temperature at a 200-fold dilution for 60 minutes. At this time, 1 μg / mL DAPI, a nuclear staining reagent, was also reacted simultaneously. Then, it was washed three times with PBS and analyzed using an Opera high-content imaging system. For Cadherin 5 (CDH5), claudin 5, P-glycoprotein (P-gp), and breast cancer resistant protein (BCRP), cells on a 96-well plate were washed three times with D-PBS (-) containing 0.1 w / v% BSA, fixed in 4 w / v% paraformaldehyde for 15 minutes, washed three times again with PBS containing 0.1 w / v% BSA, and then permeabilized with PBS containing 0.1 w / v% Triton X-100 for 5 minutes. Subsequently, after washing three times with PBS containing 0.1 w / v% BSA, the primary antibody was reacted at 4°C overnight. Then, it was washed three times with PBS containing 0.1 w / v% BSA, and the secondary antibodies (Anti-Rabbit, Anti-Mouse) were reacted at room temperature at a 200-fold dilution for 60 minutes. Then, it was washed three times with PBS containing 0.1 w / v% BSA, and 1 μg / mL DAPI was reacted for 5 minutes. Then, it was reacted in 4 w / v% paraformaldehyde for 5 minutes. Subsequently, it was washed three times with PBS and analyzed using an Opera high-content imaging system.

[0070]

Table 3

[0071] (12) Functional analysis of P-gp and BCRP For Matrigel-iBMECs and LN221F-iBMECs (on the 10th day of differentiation) derived from the 610B1 strain, a substrate uptake test was performed for functional analysis of P-gp and BCRP as efflux transporters. Rhodamine 123 and Hoechst 33342 were used as substrates. The medium was removed, and the cells on the 96-well plate were pre-incubated at 37 °C for 15 minutes in transport buffer. The number of samples was set to 6. In the presence or absence of 10 μM cyclosporine A (CsA), an inhibitor of P-gp, or 20 μM Ko 143, an inhibitor of BCRP, the cells were incubated at 37 °C for 60 minutes in transport buffer containing 10 μM rhodamine 123 or 10 μM Hoechst 33342. Then, the cells were washed three times with PBS and lysed with PBS containing 5 w / v% Triton X-100, and the fluorescence intensities of rhodamine 123 and Hoechst 33342 were measured with a Synergy HTX multimode plate reader and analyzed with Gen5 data analysis software.

[0072] 2. Results and discussion (1) Relative TEER values based on Matrigel-iBMECs Figure 2 is an explanatory diagram showing the relative TEER values of iBMECs induced to differentiate on each coat layer. In Figure 2, the vertical axis indicates the relative TEER value when the TEER value of Matrigel-iBMECs is set to 1.0, and the horizontal axis indicates the types of iBMECs. From the results of Figure 2, it was found that iBMECs induced to differentiate on LN221F and VTN-N showed higher TEER values than iBMECs induced to differentiate on Matrigel. In particular, LN221F-iBMECs showed TEER values approximately twice as high as those of Matrigel-iBMECs. Therefore, it can be said that iBMECs induced to differentiate on LN221F or VTN-N have a higher barrier function than iBMECs induced to differentiate on Matrigel.

[0073] (2)Temporal change of TEER value Figure 3 is an explanatory diagram showing the temporal change of TEER values in Matrigel-iBMECs and LN221F-iBMECs derived from the 610B1 strain. Figure 4 is an explanatory diagram showing the temporal change of TEER values in Matrigel-iBMECs and LN221F-iBMECs derived from the 648A1 strain. In Figures 3 and 4, the vertical axis indicates the TEER value (Ω×cm 2 ), and the horizontal axis indicates the number of days elapsed after seeding. From the results shown in Figures 3 and 4, it was found that LN221F-iBMECs showed higher TEER values over a longer period than Matrigel-iBMECs regardless of whether the 610B1 strain or the 648A1 strain was used as human iPS cells. That is, it can be said that iBMECs induced to differentiate on LN221F have a higher barrier function over a longer period than iBMECs induced to differentiate on Matrigel. Also, regardless of whether the 610B1 strain or the 648A1 strain was used as human iPS cells, LN221F-iBMECs showed TEER values of 1000 Ω×cm 2 or higher over a long period, and it was found to be useful as a model of the human BBB.

[0074] (3)Permeability test FIG. 5 is an explanatory diagram showing the results of the FD4 permeability test in Matrigel-iBMECs and LN221F-iBMECs. FIG. 6 is an explanatory diagram showing the results of the LY permeability test in Matrigel-iBMECs and LN221F-iBMECs. In FIGS. 5 and 6, the vertical axis represents the permeability coefficient (10 -6 cm / sec), and the horizontal axis represents the type of iBMECs. From the results shown in FIGS. 5 and 6, regardless of whether the 610B1 strain or the 648A1 strain was used as the human iPS cells, and regardless of whether FD4 or LY, which is an index of paracellular pathway permeability, was used as the permeation target, LN221F-iBMECs showed a lower permeability coefficient than Matrigel-iBMECs. It was found that they showed a high TEER value over a long period. That is, it can be said that iBMECs induced to differentiate on LN221F have a higher barrier function than iBMECs induced to differentiate on Matrigel.

[0075] From the results of the TEER value measurement and the permeability test, it was suggested that LN221F-iBMECs have a higher barrier function compared to Matrigel-iBMECs. Therefore, it was found that LN221F-iBMECs are useful as a model of the human BBB.

[0076] (4) Analysis of gene expression levels by RT-qPCR FIG. 7 is an explanatory diagram showing the analysis results of the expression levels of each gene in Matrigel-iBMECs, LN221F-iBMECs, and hBMECs. In FIG. 7, the vertical axis represents the relative mRNA expression level when the mRNA expression level of each gene in Matrigel-iBMECs is set to 1.0, and the horizontal axis represents the type of iBMECs or BMECs. From the results shown in FIG. 7, regardless of which of CDH5, MDR1, BCRP, ZO-1, Occludin, GLUT1, and LAT1 was used as the BMEC marker, LN221F-iBMECs showed almost the same expression level as Matrigel-iBMECs.

[0077] (5) Confirmation test of protein expression by immunostaining Figure 8 is an explanatory diagram showing the results of protein expression analysis by immunostaining in Matrigel-iBMECs and LN221F-iBMECs. In Figure 8, a 50-μm scale bar is shown together with the image obtained by immunofluorescence staining. From the results shown in Figure 8, it was found that LN221F-iBMECs exhibited almost the same protein expression level and localization as Matrigel-iBMECs regardless of which of CDH5, P-gp, BCRP, ZO-1, Occludin, and Claudin 5 was used as the BMEC marker.

[0078] From the results of the confirmation tests of gene expression and protein expression, it was found that LN221F-iBMECs have properties as BMECs similar to those of Matrigel-iBMECs. Therefore, it was found that LN221F-iBMECs are useful as a model of the human BBB.

[0079] (6) Substrate uptake test Figure 9 is an explanatory diagram showing the results of functional analysis of P-gp in Matrigel-iBMECs and LN221F-iBMECs. Figure 10 is an explanatory diagram showing the results of functional analysis of BCRP in Matrigel-iBMECs and LN221F-iBMECs. In Figures 9 and 10, the vertical axis indicates the relative substrate accumulation amount when the substrate accumulation amount in the absence of an inhibitor for the efflux transporter is set to 1.0, and the horizontal axis indicates the presence or absence of an inhibitor for the efflux transporter. From the results shown in Figures 9 and 10, it was confirmed that, similar to Matrigel-iBMECs, the substrate accumulation amount in LN221F-iBMECs increased in the presence of an inhibitor for the efflux transporter compared to that in the absence of such an inhibitor. For this reason, it was found that LN221F-iBMECs have the functions of P-gp and BCRP similar to those of Matrigel-iBMECs.

[0080] 3. Summary From the above results, it was found that by using LN221F or VTN-N as a coating agent for inducing the differentiation of iBMECs, iBMECs with a higher barrier function can be produced compared to the case of using Matrigel.

Industrial Applicability

[0081] According to the present disclosure, it becomes possible to stably induce the differentiation of iBMECs with a high barrier function from pluripotent stem cells, which enables the construction of a BBB model. Therefore, iBMECs can be stably supplied, contributing to the establishment of a supply system of iBMECs for pharmaceutical companies and research institutions. In addition, the BBB model constructed using the present disclosure can be used, for example, in an evaluation system for the efficacy / safety of pharmaceuticals.

[0082] This invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in this invention within the scope that those skilled in the art can easily conceive without departing from the description of the claims. The contents of the papers, published patent gazettes, and patent gazettes explicitly stated in this specification shall be cited by incorporating all of their contents.

Sequence Listing Free-Text

[0083] SEQ ID NO: 1: Description of artificial sequence: CDH5 forward primer SEQ ID NO: 2: Description of artificial sequence: CDH5 reverse primer SEQ ID NO: 3: Description of artificial sequence: MDR1 forward primer SEQ ID NO: 4: Description of artificial sequence: MDR1 reverse primer SEQ ID NO: 5: Description of artificial sequence: BCRP forward primer SEQ ID NO: 6: Description of artificial sequence: BCRP reverse primer SEQ ID NO: 7: Description of artificial sequence: GLUT1 forward primer SEQ ID NO: 8: Description of artificial sequence: GLUT1 reverse primer SEQ ID NO: 9: Description of artificial sequence: Occludin forward primer Sequence number 10: Description of artificial sequence: Occludin reverse primer Sequence number 11: Description of artificial sequence: ZO-1 forward primer Sequence number 12: Description of artificial sequence: ZO-1 reverse primer Sequence number 13: Description of artificial sequence: LAT1 forward primer Sequence number 14: Description of artificial sequence: LAT1 reverse primer Sequence number 15: Description of artificial sequence: HPRT1 forward primer Sequence number 16: Description of artificial sequence: HPRT1 reverse primer

Claims

1. A coating agent for inducing differentiation from pluripotent stem cells into brain capillary endothelial-like cells, comprising a component of a laminin 221 fragment, wherein the laminin 221 fragment means a fragment corresponding to the integrin-binding site in laminin 221 (E8 fragment), and the amino acid sequence constituting the protein contains an amino acid sequence that is 95% or more identical to the amino acid sequence of the laminin 221 fragment, the coating agent.

2. The coating agent according to Claim 1, wherein the component is contained in the coating agent at 0.01 nmol / L or more and 10 mmol / L or less, the coating agent.

3. A differentiation induction kit comprising the coating agent according to Claim 1 or Claim 2 and cells. The differentiation induction kit.

4. A coating agent for inducing differentiation from pluripotent stem cells into brain capillary endothelial-like cells, comprising a component of a laminin 221 fragment, wherein the laminin 221 fragment means a fragment corresponding to the integrin-binding site in laminin 221 (E8 fragment), and the amino acid sequence constituting the protein contains an amino acid sequence that is 95% or more identical to the amino acid sequence of the laminin 221 fragment, wherein, per 1 cm2 of the coated area of the coated layer coated with the coating agent, the component is contained at 1 ng or more, and it is used as the coating agent at the start of differentiation induction.

5. A culture dish comprising a coated layer coated with a coating agent for inducing differentiation from pluripotent stem cells into brain capillary endothelial-like cells, wherein the coating agent comprises a component of a laminin 221 fragment, wherein the laminin 221 fragment means a fragment corresponding to the integrin-binding site in laminin 221 (E8 fragment), and the amino acid sequence constituting the protein contains an amino acid sequence that is 95% or more identical to the amino acid sequence of the laminin 221 fragment, wherein the component is contained at 1 ng or more per 1 cm2 of the coated area of the coated layer, the culture dish.

6. The culture dish according to Claim 5, The above component is contained at 1 ng or more and 1 mg or less per 1 cm ２ of the coating area of the coating layer, the culture dish.

7. A method for producing brain capillary endothelial-like cells by inducing differentiation of pluripotent stem cells, comprising the step of culturing the pluripotent stem cells using a component of a laminin 221 fragment at the start of differentiation induction, wherein the component is contained at 1 ng or more per 1 cm2 of the area of the culture dish. The laminin-221 fragment means a fragment (E8 fragment) corresponding to the integrin-binding site of laminin-221, and includes an amino acid sequence that is 95% or more identical to the amino acid sequence of the laminin-221 fragment in the amino acid sequence constituting the protein. Method for producing brain capillary endothelial-like cells.

8. In the method for producing brain capillary endothelial-like cells according to Claim 7, the pluripotent stem cells are induced pluripotent stem cells. Method for producing brain capillary endothelial-like cells.

9. In the method for producing brain capillary endothelial-like cells according to Claim 8, the induced pluripotent stem cells are human induced pluripotent stem cells. Method for producing brain capillary endothelial-like cells.

10. A method for inducing differentiation of pluripotent stem cells into brain capillary endothelial-like cells, comprising a step of culturing the pluripotent stem cells using a component of the laminin-221 fragment at the start of differentiation induction, wherein the component is contained at 1 ng or more per 1 cm2 of the area of the culture dish, the laminin-221 fragment means a fragment (E8 fragment) corresponding to the integrin-binding site of laminin-221, and includes an amino acid sequence that is 95% or more identical to the amino acid sequence of the laminin-221 fragment in the amino acid sequence constituting the protein. Method.

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