JPWO2019208736A1 - Method for inducing differentiation of pluripotent stem cells into brain capillary endothelial cells - Google Patents

Abstract

It is an object of the present invention to provide a method for inducing differentiation of cerebral vascular endothelial cells capable of forming tight junctions that are strong and can be maintained for a long period of time from pluripotent stem cells, and their uses. (1) A step of culturing pluripotent stem cells in the absence of FGF2 to reduce undifferentiation, and (2) a step of differentiating the cells obtained in step (1) into cerebral capillary endothelial cells. Then, the pluripotent stem cells are induced to differentiate into brain capillary endothelial cells by a step including culturing in the presence of FGF2, retinoic acid and TGF-β inhibitor.

Description

The present invention relates to a method for inducing differentiation of pluripotent stem cells into brain microvascular endothelial cells (BMECs) and their uses / applications. This application claims priority based on Japanese Patent Application No. 2018-0876770 filed on April 27, 2018, and the entire contents of the patent application are incorporated by reference.

The blood-brain barrier (BBB) has a very important biological barrier function that regulates molecular transport between the blood and the brain. BBB is composed of cerebral capillary endothelial cells (BMEC) and is characterized by strong tight junctions and the expression of various efflux transporters.

In new drug development, knowing BBB permeability is essential for predicting the efficacy or safety of drugs, and it is desirable to use normal human cerebrovascular tissue for that purpose, but normal human cerebrovascular tissue is difficult to obtain. is there. Therefore, tests using experimental animals or tumor cells have been conducted, but there is a problem that these have different functionality as compared with human tissues. BBB in vitro reconstruction models using monkey-derived cells or rat-derived cells are commercially available (monkey-type BBB kit ^TM , rat-type BBB kit ^TM (Pharmacocell Co., Ltd.)).

In recent years, embryonic stem cells (ES cells) and induced pluripotent stem cells (induced), which have pluripotency similar to ES cells and almost infinite proliferative potential, and are expected to be used for drug discovery research. A BBB model using pluripotent stem cells (iPS cells) is being developed (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1).

U.S. Patent Application Publication No. 2017/0283772 A1 Japanese Unexamined Patent Publication No. 2015-159785

Ethan S. Lippmann et al. Sci Reports. 2014; 4: 4160

As mentioned above, there are attempts to build a BBB model using ES cells and iPS cells. Various improvements have been made to obtain a BBB model with a somewhat strong tight junction. However, the inability to maintain the barrier function for a long period of time is a particular problem. In addition to low expression of vascular endothelial cell markers, transendothelial electrical resistance (TEER), which is an index of tight junctions, fluctuates greatly, and improvement in reproducibility is also desired. Furthermore, freeze-thaw of differentiated cells has not been fully investigated.

Therefore, the present invention is a method for inducing differentiation of cerebral vascular endothelial cells (BMEC) capable of forming tight junctions that are strong and can be maintained for a long period of time from pluripotent stem cells in order to enable the construction of a highly practical BBB model. And to provide its use.

In view of the above problems, studies were conducted with the aim of enhancing the function of BMEC tight junctions by using low-molecular-weight compounds that are inexpensive and have little difference between lots, and to construct a BBB model that can maintain that function for a long period of time. Specifically, we focused on TGF-β inhibitors and investigated the effects or effects of using TGF-β inhibitors in inducing differentiation into BMEC. As a result, when differentiation was induced using a medium to which a TGF-β inhibitor was added, the characteristics as vascular endothelial cells became remarkable. Furthermore, it was found that the TEER value was dramatically improved and a stronger tight junction could be formed. Moreover, the TEER value remained at a high level, and it was possible to maintain the tight junction function for a long period of time. Furthermore, the functions of drug transporters P-gp and BCRP were also confirmed.

As described above, the present inventors have found a low molecular weight compound effective for improving the tight junction function, and succeeded in constructing a highly practical BBB model. The following inventions are based on the results.
[1] A method for inducing differentiation of pluripotent stem cells into cerebral capillary endothelial cells, which comprises the following steps (1) and (2):
(1) A step of culturing pluripotent stem cells in the absence of FGF2 to reduce undifferentiated state;
(2) A step of differentiating the cells obtained in step (1) into brain capillary endothelial cells, which comprises culturing in the presence of FGF2, retinoic acid and a TGF-β inhibitor.
[2] The method according to [1], wherein a medium containing serum or serum substitute, non-essential amino acid, L-glutamic acid and 2-mercaptoethanol is used for culturing in step (1).
[3] The method according to [1] or [2], wherein a medium containing serum or serum substitute, FGF2, retinoic acid and a TGF-β inhibitor is used for culturing in step (2).
[4] The method according to any one of [1] to [3], wherein the culture period of the step (1) is 3 to 9 days.
[5] The method according to any one of [1] to [4], wherein the culture period of the step (2) is 2 to 10 days.
[6] The method according to any one of [1] to [5], wherein the transendothelial electrical resistance value of the cell layer formed by the step (2) is 1000 Ω × cm ^{2 or more.}
[7] The method according to any one of [1] to [6], wherein the cell layer formed by the step (2) ^{maintains a transendothelial electrical resistance value exceeding 1000 Ω × cm 2 for 5 days or more.}
[8] Step (2) is performed in the presence of (2-1) FGF2 and retinoic acid and after the culture of (2-2) FGF2, retinoic acid, and TGF-β inhibitor. The method according to any one of [1] to [7], which comprises culturing in the presence.
[9] A medium containing serum or serum substitute, FGF2, and retinoic acid was used for the culture in step (2-1), and serum or serum substitute, FGF2, and retinoic acid were used for the culture in step (2-2). The method according to [8], which uses a medium containing an acid and a TGF-β inhibitor.
[10] The method according to [8] or [9], wherein the culture period of step (2-1) is 1 to 5 days, and the culture period of step (2-2) is 1 to 4 days. ..
[11] Step (2) is performed in the presence of (2-1) FGF2 and retinoic acid and after the culturing of (2-2) FGF2, retinoic acid, and TGF-β inhibitor. The method according to any one of [1] to [7], which comprises culturing in the presence and culturing in the presence of (2-3) TGF-β inhibitor performed after the culturing.
[12] A medium containing serum or serum substitute, FGF2, and retinoic acid was used for culturing in step (2-1), and serum or serum substitute, FGF2, retinoin was used for culturing in step (2-2). [11], wherein a medium containing an acid and a TGF-β inhibitor is used, and a serum or a serum substitute and a medium containing a TGF-β inhibitor are used for culturing in step (2-3). Method.
[13] The culture period of step (2-1) is 1 to 5 days, the culture period of step (2-2) is 1 to 4 days, and the culture period of step (2-3) is 1. The method according to [11] or [12], which is 1 to 4 days.
[14] One or more TGF-β inhibitors selected from the group consisting of A-83-01, SB-431542, RepSox, SB-505124, SB-525334, LY-2157299, LY-364947, SD208 and D4476. The method according to any one of [1] to [13], which is a compound.
[15] The method according to any one of [1] to [14], wherein the pluripotent stem cell is an induced pluripotent stem cell.
[16] The method according to [15], wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
[17] A cell layer obtained by the method according to any one of [1] to [16].
[18] A method for evaluating the blood-brain barrier permeability of a test substance using the cell layer according to [17].
[19] The method according to [18], which comprises the following steps (i) to (iii):
(I) Step of preparing the cell layer according to [17];
(Ii) A step of bringing the test substance into contact with the cell layer;
(Iii) A step of quantifying a test substance that has permeated the cell layer and evaluating the permeability of the test substance.
[20] A method for evaluating the effect of a test substance on the blood-brain barrier barrier function using the cell layer according to [17].

TEER value of cells obtained by inducing differentiation using a TGF-β inhibitor. The TEER value measured on the 10th day after the start of differentiation is shown. Mean ± SD (n = 3); ^*** P <0.001 vs control. Cell permeability obtained by inducing differentiation with a TGF-β inhibitor. The membrane permeability coefficient of Lucifer Yellow measured on the 10th day after the start of differentiation is shown. Mean ± SD (n = 3); ^* P <0.05 vs control. Immunofluorescent staining of cells obtained by inducing differentiation with a TGF-β inhibitor. Occludin / Claudin-5 / ZO-1, tight junction related proteins. DAPI, nuclear staining. Scale bar, 500 μm. Time-dependent changes in TEER values in cells obtained by inducing differentiation with a TGF-β inhibitor. The TEER values measured from the 9th day after the start of differentiation are shown. Mean ± SD (n = 3); ^*** P <0.001 vs control. Cell permeability obtained by inducing differentiation with a TGF-β inhibitor. The permeability coefficient of Lucifer Yellow measured from the 9th day to the 13th day after the start of differentiation is shown. Mean ± SD (n = 3); ^* P <0.05, ^** P <0.01, ^*** P <0.001 vs control. Transport experiments in cells obtained by inducing differentiation using a TGF-β inhibitor. The amount of rhodamine 123 (P-gp transport activity) accumulated per cellular protein measured on the 10th day after the start of differentiation was shown. Mean ± SD (n = 3); ^*** P <0.001 vs CsA-. Transport experiments in cells obtained by inducing differentiation using a TGF-β inhibitor. The accumulated amount of Hoechst 33342 (BCRP transport activity) per cell protein amount measured on the 10th day after the start of differentiation was shown. Mean ± SD (n = 3) ^*** P <0.001 vs Ko143-. Immunofluorescent staining of cells obtained by inducing differentiation with a TGF-β inhibitor. VE-cadherin, an intercellular adhesion molecule. DAPI, nuclear staining. Scale bar, 100 μm. Mean ± SD (n = 3) ^*** P <0.001 vs control. An uptake test of acetylated LDL in cells obtained by inducing differentiation using a TGF-β inhibitor. The number of acetylated LDL-positive cells measured on the 10th day after the start of differentiation was shown. DAPI, nuclear staining. Scale bar, 100 μm. Mean ± SD (n = 3) ^*** P <0.001 vs control. Angiogenic ability test in cells obtained by inducing differentiation with a TGF-β inhibitor. From the 8th day after the start of differentiation, cells were cultured on Matrigel under the condition of addition of VEGF, which is an angiogenesis-promoting factor, and on the 10th day, the formation of vascular-like structures was observed by staining with calcein-AM. Scale bar, 500 μm. Immunofluorescent staining of cells obtained by inducing differentiation with a TGF-β inhibitor. BCRP / P-gp / GLUT1, drug transporter. DAPI, nuclear staining. Scale bar, 100 μm. Mean ± SD (n = 3) ^*** P <0.05 vs control. Immunofluorescent staining of cells obtained by inducing differentiation using a TGF-β inhibitor (cell cross-sectional view). BCRP / P-gp / GLUT1, drug transporter. DAPI, nuclear staining. Upper side, apical; lower side, basal. TEER value of cells obtained by freezing and thawing on the 8th day after the start of differentiation and inducing differentiation using a TGF-β inhibitor. The TEER value measured on the 10th day after the start of differentiation is shown. Mean ± SD (n = 3); ^** P <0.01 vs non-freeze thaw. Time-dependent changes in TEER values in cells obtained by freezing and thawing on the 8th day after the start of differentiation and inducing differentiation using a TGF-β inhibitor. The TEER values measured from the 10th day to the 19th day after the start of differentiation are shown. Mean ± S.D. (n = 3). Immunofluorescent staining of cells obtained by freezing and thawing on the 8th day after the start of differentiation and inducing differentiation with a TGF-β inhibitor. Occludin / ZO-1, tight junction related protein. DAPI, nuclear staining. Scale bar, 50 μm. Immunofluorescent staining of cells obtained by freezing and thawing on the 8th day after the start of differentiation and inducing differentiation with a TGF-β inhibitor. BCRP / P-gp, drug transporter. Mean ± S.D. (n = 3). Gene expression of cells obtained by freezing and thawing on the 8th day after the start of differentiation and inducing differentiation using a TGF-β inhibitor. MDR1 (P-gp) / BCRP / GLUT1, drug transporter; Occludin / ZO-1, tight junction-related protein; VE-cadherin, cell-cell adhesion molecule. Mean ± SD (n = 3); ^* P <0.05 vs non-freeze thawing.

The present invention relates to a method for inducing differentiation of pluripotent stem cells into cerebral capillary endothelial cells (hereinafter, also referred to as "method for inducing differentiation of the present invention"). Cerebral capillary endothelial cells (hereinafter sometimes referred to as "BMEC") are the major cells that make up the blood-brain barrier. The blood-brain barrier consists of a structure in which pericytes and astrocytes cover the BMEC. According to the present invention, cells similar to BMEC, which are the main cells constituting the blood-brain barrier of a living body, that is, cells exhibiting BMEC characteristics (hereinafter, also referred to as "BMEC-like cells") can be obtained. The BMEC-like cells obtained by the method of the present invention are useful for constructing a blood-brain barrier model, and are used, for example, for evaluating the translocation (cerebral vascular barrier permeability) of a test substance (typically a drug) into the brain. To.

"Pluripotent stem cells" are the ability to differentiate into all the cells that make up the living body (pluripotency) and the ability to produce daughter cells that have the same differentiation ability as self through cell division (self-renewal ability). ) And a cell. Differentiation pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing for the presence or absence of teratomy including each of the three germ layers (ectoderm, mesoderm, endoderm).

Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), etc., but cells having both differentiation pluripotency and self-renewal ability As long as it is, it is not limited to this. Preferably, ES cells or iPS cells are used. More preferably, iPS cells are used. Pluripotent stem cells are preferably mammalian cells (eg, primates such as humans and chimpanzees, rodents such as mice and rats), particularly preferably human cells. Therefore, in the most preferred embodiment of the present invention, human iPS cells are used as pluripotent stem cells.

ES cells can be established, for example, by culturing an early embryo before implantation, an inner cell mass constituting the early embryo, a single blastomere, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, JA et al., Science, 282, 1145-1147 (1998)). As early embryos, early embryos prepared by nuclear transfer of somatic cell nuclei may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256). (1998)), Akira Iriya 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). Participatory embryos may be used as the initial locus (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 papers, Strelchenko N., et al. Reprod Biomed Online. 9 for the production of ES cells. : 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, PM et Al. Methods in Enzymology, Vol.365, 2003 etc. will be helpful. The fused ES cells obtained by cell fusion of ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

Some ES cells are available from storage institutions or are commercially available. For example, human ES cells can be obtained from the Institute for Frontier Medical Sciences, Kyoto University (for example, KhES-1, KhES-2 and KhES-3), WiCell Research Institute, ESI BIO and the like.

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)).

"Induced pluripotent stem cells (iPS cells)" are cells having pluripotency (pluripotency) and proliferative ability, which are produced by reprogramming somatic cells by introducing a reprogramming factor or the like. Induced pluripotent stem cells exhibit properties similar to ES cells. The somatic cells used for producing iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. Further, the origin thereof is not particularly limited, but somatic cells of mammals (for example, primates such as humans and chimpanzees, rodents such as mice and rats), particularly preferably human somatic cells are used. iPS cells can be produced by various methods reported so far. In addition, it is naturally assumed that the iPS cell production method to be developed in the future will be applied.

The most basic method for producing iPS cells is to introduce four transcription factors, Oct3 / 4, Sox2, Klf4 and c-Myc, 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). Human iPS cells have been reported to be established by the introduction of four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). Three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), two factors Oct3 / 4 and Klf4 (Kim JB, et al: Nature 454 (7204)) , 646-650, 2008), or the establishment of iPS cells by introduction of Oct3 / 4 alone (Kim JB, et al: Cell 136 (3), 411-419, 2009) has also been reported. In addition, a method for introducing a protein, which is an 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, by using the inhibitor BIX-01294 for histone methyltransferase G9a, the histone deacetylase inhibitor valproic acid (VPA), BayK8644, etc., it is possible to improve the production efficiency and reduce the factors to be introduced. (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). In addition to retroviruses, gene transfer methods have also been studied, including lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007) and adenoviruses (Stadtfeld M, et al: Science 322 (5903)). ), 945-949, 2008), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vector (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 Technologies using episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) for gene transfer have been developed.

Cells that have undergone transformation into iPS cells, that is, reprogramming, express pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1 and Cript. Etc. can be selected as an index. The selected cells are collected as iPS cells.

iPS cells can also be provided by, for example, the National University Corporation Kyoto University or the National Research and Development Corporation RIKEN BioResource Center.

As used herein, "inducing differentiation" means acting to differentiate along a specific cell lineage. In the present invention, pluripotent stem cells are induced to differentiate into brain capillary endothelial cells (BMEC). The method for inducing differentiation of the present invention is roughly divided into two steps, that is, (1) a step of culturing pluripotent stem cells in the absence of FGF2 to reduce undifferentiation (step (1)) and a step (1). ) Is a step of differentiating the cells obtained in () into brain capillary endothelial cells (BMEC), which includes culturing in the presence of FGF2, retinoic acid and TGF-β inhibitor (step (2)). Including. The details of each step will be described below.

<Step (1) Decrease in undifferentiated pluripotent stem cells>
In this step, the undifferentiated state of pluripotent stem cells is reduced by culturing in the absence of FGF2. By going through this step, more reliable differentiation induction to BMEC, improvement of differentiation induction efficiency, etc. can be expected. "Undifferentiated" is a term that can be exchanged for pluripotency, and loss of pluripotency also corresponds to "decrease in undifferentiated".

"In the absence of FGF2" means using a medium containing no FGF2. Therefore, in step (1), culturing is performed using a medium that does not contain FGF2 (in other words, a medium that does not contain FGF2). From the viewpoint of cell proliferation rate and maintenance, it is preferable to add serum or serum replacement (Knockout serum replacement (KSR), etc.) to the medium. The serum is not limited to fetal bovine serum, and human serum, sheep serum and the like can also be used. The amount of serum or serum substitute added is, for example, 0.1% (v / v) to 30% (v / v).

Other components that can be added to the medium include non-essential amino acids, L-glutamic acid, and 2-mercaptoethanol. The amount of these components added is not particularly limited, but for example, the amount of non-essential amino acids added is 0.08% (v / v) to 8% (v / v), and the amount of L-glutamic acid added is 0.1% (v / v). ) ~ 10% (v / v), the amount of 2-mercaptoethanol added is 0.01 mM ~ 1 mM. In a preferred embodiment, the culture of step (1) is carried out using a medium containing serum or serum substitute, non-essential amino acids, L-glutamic acid and 2-mercaptoethanol.

The period (culture period) of the step (1) is, for example, 3 to 9 days, preferably 4 to 7 days. If the culture period is too short, the expected effect (decrease in undifferentiated state) cannot be sufficiently obtained. On the other hand, if the culture period is too long, it causes a decrease in maturation and unintended differentiation (differentiation into another cell).

The decrease in undifferentiated state should be confirmed or evaluated by using the expression level of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4 and Esg-1 as an index. Can be done.

<Step (2) Differentiation into brain capillary endothelial cells (BMEC)>
In this step, pluripotent stem cells whose undifferentiated state has been reduced by step (1) are cultured and differentiated into BMEC. In other words, the cells obtained in step (1) are cultured under conditions that induce differentiation into BMEC. In the present invention, culturing is carried out in the presence of FGF2, retinoic acid and a TGF-β inhibitor to induce differentiation into BMEC.

"In the presence of FGF2, retinoic acid and TGF-β inhibitor" is synonymous with the condition that FGF2, retinoic acid and TGF-β inhibitor are added to the medium. Therefore, in order to carry out culturing in the presence of FGF2, retinoic acid and a TGF-β inhibitor, a medium to which at least these three components have been added may be used. As FGF2, human FGF2 (for example, human recombinant FGF2) is preferably used. An example of the concentration of FGF2 added is 1 ng / mL to 500 ng / mL. Similarly, an example of the concentration of retinoic acid added is 0.1 μM to 100 μM. As TGF-β inhibitors, A-83-01 (selective inhibitor of TGF-βI type receptors ALK4, ALK5, ALK7) and SB-431542 (selective of TGF-βI type receptors ALK4, ALK5, ALK7) Inhibitor), RepSox (selective inhibitor of TGF-βI type receptor ALK5), SB-505124 (selective inhibitor of TGF-βI type receptor ALK4, ALK5, ALK7), SB525334 (selective inhibitor of TGF-βI type receptor ALK5), SB525334 (TGF-βI type receptor) ALK5 selective inhibitor), LY2157299 (TGF-β receptor inhibitor), LY364947 (TGF-βI type receptor selective ATP competition inhibitor), SD208 (TGF-βI type receptor inhibitor), D4476 ( Casein kinase 1 and TGF-βI type receptor ALK5 selective inhibitor) can be used, and an example of the addition concentration of TGF-β receptor inhibitor is 0.1 μM to 10 μM (A-83-01). Case), 0.1 μM to 10 μM (for SB-431542), 0.1 μM to 10 μM (for RepSox). Regarding the concentration of addition when a compound different from the exemplified compound is used, those skilled in the art can consider the difference between the characteristics of the compound used and the characteristics of the exemplified compound (particularly the difference in activity). It can be set according to the concentration range. Further, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to an example described later.

From the viewpoint of cell proliferation rate and maintenance, it is preferable to add serum or serum replacement (Knockout serum replacement (KSR), etc.) to the medium. The serum is not limited to fetal bovine serum, and human serum, sheep serum and the like can also be used. Also, plasma-derived serum (PDS) may be used instead of normal serum. The amount of serum or serum substitute added is, for example, 0.1% (v / v) to 10% (v / v).

In a preferred embodiment, the culture of step (2) is carried out using a medium containing serum or serum substitute, FGF2, retinoic acid and a TGF-β inhibitor.

The period (culture period) of the step (2) is, for example, 2 to 10 days, preferably 3 to 7 days. If the culture period is too short, the expected effect (induction of differentiation into BMEC) cannot be sufficiently obtained. On the other hand, if the culture period is too long, unintended differentiation (differentiation into another cell) may occur.

Step (2) may be composed of two-step culture. Specifically, for example, the culture in the presence of step (2-1) FGF2 and retinoic acid, and the step (2-2) FGF2, retinoic acid, and TGF-β inhibitor performed after the culture. Culturing in the presence is performed, and the cells obtained in step (1) are induced to differentiate into BMEC. According to this aspect, the cell adhesion rate at the time of passage is improved. Typically, a medium containing serum or serum substitute, FGF2, and retinoic acid is used for culturing in step (2-1), and serum or serum substitute is used for culturing in step (2-2). , FGF2, retinoic acid, and TGF-β inhibitor. The concentration of each component contained in the medium is as described above. The culture period of step (2-1) is, for example, 1 to 5 days, preferably 2 to 3 days, and the culture period of step (2-2) is, for example, 1 to 4 days, preferably 1 to 2 days. It's a day.

In another aspect, step (2) comprises a three-step culture. Specifically, for example, a culture in the presence of step (2-1) FGF2 and retinoic acid, and a step (2-2) FGF2, retinoic acid, and TGF-β inhibitor performed after the culture. And the cells obtained in step (1) are induced to differentiate into BMEC by culturing in the presence of TGF-β inhibitor, which is performed after the culturing in step (2-3). To do. This aspect is advantageous in increasing the TEER value. Typically, a medium containing serum or serum substitute, FGF2, and retinoic acid is used for culturing in step (2-1), and serum or serum substitute is used for culturing in step (2-2). , FGF2, retinoic acid, and TGF-β inhibitor are used, and for the culture in step (2-3), a medium containing serum or serum substitute and TGF-β inhibitor is used. The concentration of each component contained in the medium is as described above. The culture period of step (2-1) is, for example, 1 to 5 days, preferably 2 to 3 days, and the culture period of step (2-2) is, for example, 1 to 4 days, preferably 1 to 2 days. It is a day, and the culture period of the step (2-3) is, for example, 1 to 4 days, preferably 1 to 2 days.

Differentiation into BMEC, in other words, the acquisition of BMEC-like cells, for example, expression of vascular endothelial markers (eg VE-cadherin), transporters important or characteristic of BMEC (eg BCRP, P- The expression of gp, GLUT1), the expression of tight junction markers important or characteristic for BMEC (for example, Claudin5, Occludin, ZO-1), etc. can be used as an index for judgment or evaluation.

In each step (step (1), step (2), step (2-1), step (2-2), step (2-3)) that can constitute the present invention, subculture is performed in the middle. May be good. For example, when the cells become confluent or subconfluent, a part of the cells is collected and transferred to another culture vessel, and the culture is continued.

At the time of cell recovery due to medium exchange or subculture, cells are previously treated with a ROCK inhibitor (Rho-associated coiled-coil forming kinase / Rho-binding kinase) such as Y-27632 to suppress cell death. May be processed.

Other culture conditions (culture temperature, etc.) in each step constituting the present invention may be conditions generally adopted in the culture of animal cells. That is, for example, the cells may be cultured in an environment of 37 ° C. and 5% CO _2. Further, in step (1), it is preferable to use a basal medium suitable for culturing animal cells, such as DMEM Ham's F-12 (DMEM / F12). In step (2) (including the case of performing two-step or three-step culture), it is preferable to use a basal medium suitable for culturing endothelial cells such as Human Endothelial-SFM. Typically, cells are two-dimensionally cultured using a culture dish or the like. However, 3D culture may be carried out using a gel-like culture substrate, a 3D culture plate, or the like.

In order to improve the survival rate and proliferation rate of cells, promote differentiation induction, select cells, etc., it is advisable to culture cells on a culture surface coated with basement membrane components or adhesion molecules. Examples of materials / components used for coating the culture surface include matrigel, collagen I, collagen IV, fibronectin, laminin, gelatin, polylysine, and vitronectin. In a preferred embodiment, step (2) is divided into two stages (step (2-1) and step (2-2)) or three-step culture (step (2-1), step (2-2) and step (2). -3)) In the latter stage of culturing, that is, in the case of two-stage culturing, step (2-2), and in the case of three-stage culturing, step (2-2) and (2-3) culturing. Use a culture surface coated with fibronectin and collagen IV. According to this feature, a cell selection effect can be expected. In the case of this aspect, for the culture in the first stage of the step (2), for example, a matrigel-coated culture surface may be used in order to promote differentiation induction.

Differentiation into BMEC is induced by step (2), and cells (BMEC-like cells) showing characteristics similar to BMEC, which are the main cells constituting the blood-brain barrier of a living body, are obtained. If the culture of step (2) is continued, or if the culture is continued under the culture conditions suitable for the maintenance and proliferation of BMEC, a monolayer (cell layer) of BMEC-like cells is formed. According to the method of the present invention, a cell layer having an excellent barrier function can be obtained. The barrier function of the cell layer obtained by the method of the present invention can be characterized by strong tight junctions and maintenance of tight junctions over a long period of time. According to the present invention, it is possible to form a cell layer having a transendothelium electrical resistance value of 1000 Ω × cm ^{2 or more, which is an index of tight junction.} TEER values of cell layer formed by the method of the present invention (the maximum value) is typically a ^{2500Ω × cm 2 ~3500Ω × cm 2} . In addition, the cell layer formed by the method of the present invention ^{has a TEER value of more than 1000 Ω × cm 2} for 5 days or more (for example, 5 to 7 days), preferably 6 days or more (for example, 6 to 8 days), and further. It is preferably maintained for 7 days or longer (for example, 7 to 9 days), and even more preferably for 8 days or longer (for example, 8 days). If the TEER value exceeds 2000 Ω × cm ² , maintain it for 2 days or more (for example, 2 days, 3 days), preferably 4 days or more (for example, 4 days).

The barrier function of the cell layer obtained by the method of the present invention can be further characterized by having the functions of drug transporters P-gp and BCRP.

As described above, it is possible to form a cell layer composed of BMEC-like cells by the step (2). In one aspect, when the culture of step (2) is composed from the beginning or from the middle (for example, when step (2) is composed of step (2-1) and step (2-2), step (2-2) is performed. When the step (2) is composed of the step (2-1), the step (2-2) and the step (2-3), the step (2-2) and the step (2-3), or the step (2-3) 2-3) will be performed on a semi-permeable membrane (porous membrane), and a cell layer of BMEC-like cells will be formed on the semi-permeable membrane. This aspect is particularly effective when the cell layer formed by the method of the present invention is used for various assays. For example, using a culture vessel equipped with a culture insert (having a culture surface made of a semi-permeable membrane) (for example, Transwell® provided by Corning Inc.), cells are cultured in the insert to obtain a cell layer. To form.

The second aspect of the present invention relates to the use of cells (BMEC-like cells) prepared by the method for inducing differentiation of the present invention. As described above, according to the method for inducing differentiation of the present invention, it is possible to obtain a cell layer composed of BMEC-like cells. The cell layer can be used for the BBB model. For example, an assay using the cell layer is useful for evaluating the BBB permeability of a test substance such as a drug or a drug candidate. Therefore, a method for evaluating the BBB permeability of a test substance using the cell layer obtained by the method for inducing differentiation of the present invention (hereinafter, referred to as "the BBB permeability evaluation method of the present invention") is provided. In the BBB permeability evaluation method of the present invention, for example, the following steps (i) to (iii) are performed.
(I) Step of preparing the cell layer obtained by the method for inducing differentiation of the present invention (ii) Step of contacting the test substance with the cell layer (iii) Quantifying the test substance that has permeated the cell layer and subjecting it Step to evaluate the permeability of the test substance

In step (i), the cell layer obtained by the method for inducing differentiation of the present invention is prepared. It is possible to carry out step (i) as part of step (2) of the differentiation induction method of the present invention. That is, in one aspect, the differentiation induction method of the present invention and the BBB permeability evaluation method of the present invention are carried out as a series of assays, and the culture of step (2) is carried out from the beginning or from the middle of the semi-permeable membrane (porous). It is performed on a membrane) to form a cell layer composed of BMEC-like cells on a semi-permeable membrane. In another embodiment, the BMEC-like cells obtained by the method for inducing differentiation of the present invention are cultured on a semi-permeable membrane to form a cell layer. For example, using a culture vessel equipped with a culture insert, cells are seeded and cultured in the culture insert to obtain a cell layer composed of BMEC-like cells.

In addition to the cell layer composed of BMEC-like cells, other cells (pericytes, astrocytes, etc.) may be used in combination. For example, by adopting a culture vessel equipped with a culture insert as described above, the cell layer of BMEC-like cells is formed in the culture insert (the cell layer is formed on the upper surface of the bottom of the culture insert), and the bottom of the culture insert is formed. Perisite can be cultivated while adhering to the back surface (perisite adhesive co-culture system), perisite can be cultivated in the compartment between the culture insert and the well (perisite non-adhesive co-culture system), or the bottom of the culture insert. Perisite is cultivated while adhering to the back surface, and astrosite is cultivated in the section between the culture insert and the well (perisite adherent / non-adhesive astrosite co-culture system), or adhered to the bottom back surface of the culture insert. It is possible to cultivate astrosites in (astrosite adhesion co-culture system).

The "contact" in step (ii) is typically performed by adding the test substance to the medium. The timing of addition of the test substance is not particularly limited. Therefore, after culturing in a medium containing no test substance, the test substance may be added at a certain point in time, or the culture may be started in a medium containing the test substance in advance.

Typically, a drug or a drug candidate substance is used as the test substance. However, the test substance is not particularly limited, and organic compounds or inorganic compounds having various molecular sizes can be used as the test substance. Examples of organic compounds are nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycoglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1,) B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) can be exemplified. Plant extracts, cell extracts, culture supernatants, etc. may be used as test substances. 2 By adding more than one kind of test substance at the same time, the interaction, synergistic action, etc. between the test substances may be investigated. The test substance may be derived from a natural product or synthetic. In the latter case, for example, a method of combinatorial synthesis can be used to construct an efficient assay system.

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

In the step (iii), the test substance that has permeated the cell layer is quantified. For example, when a culture vessel equipped with a culture insert such as Transwell® is used, the test substance that has permeated the culture insert, that is, the upper container (culture insert) or the lower container via the cell layer. Mass analysis, liquid chromatography, immunological method (for example, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method)) for the test substance transferred into the (well) Quantify by the measuring method such as. The membrane permeability of the test substance is evaluated based on the quantification result (the amount of the test substance that has permeated the cell layer) and the amount of the test substance used (typically the amount added to the medium). In addition to membrane permeability, evaluate the absorption by the cell layer (absorption), the effect on the cell layer (for example, the effect on the barrier function), the expression or the effect on the function of the transporter (for example, BCRP or P-gp), etc. You may decide. Since absorbability is usually in a two-sided relationship with permeability, it can be evaluated in the same manner as in the case of permeability. The effect on the barrier function can be evaluated by measuring the TEER value, permeation test using a non-absorbable marker, and the like. The effect on the expression of the transporter can be evaluated by an immunological method, Western blotting, flow cytometry, etc., and the effect on the function can be evaluated by, for example, an activity test using a substrate.

As can be seen from the above explanation, the effect of the test substance on the BBB function (for example, improvement, decrease, or breakdown of BBB function) can also be evaluated by using the cell layer composed of BMEC-like cells. Therefore, the present invention is applied to an evaluation method targeting or targeting the BBB function, that is, the BBB function of the test substance, as another use of the cell layer composed of BMEC-like cells obtained by the method for inducing differentiation of the present invention. It also provides a way to assess the impact of. The evaluation method is useful as a means for searching for, for example, a substance that strengthens (improves) the barrier function, a substance that protects the barrier function, a substance that regulates the barrier function, and the like. It can also be used to evaluate toxicity to BBB. In the evaluation method, similarly to the above-mentioned BBB permeability evaluation method, a step of preparing the cell layer and a step of bringing the test substance into contact with the cell layer are performed, and then the influence on the barrier function of the cell layer is evaluated. The method for evaluating the effect on the barrier function is as described above.

The following studies were conducted with the aim of enhancing the function of BMEC tight junctions by using low-molecular-weight compounds that are inexpensive and have little difference between lots, and to construct a BBB model that can maintain that function for a long period of time.

1. 1. Method (1) Cell Human iPS cell 610B1 strain (physical chemistry) established by introducing 5 human factors (Oct3 / 4, Sox2, Klf4, L-Myc, Lin28) into umbilical cord blood using an episomal vector (pCXLE). The experiment was conducted using (purchased from the laboratory). Mouse embryonic fibroblasts (MEF) were used as feeder cells.

(2) Medium
Dalbecco containing 10% fetal bovine serum (FBS), 2 mmol / L L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), 100 units / mL penicillin G, 100 μg / mL streptomycin for MEF culture Modified Eagle's medium (DMEM) was used. 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) was used as the MEF stripping solution, and Cerbunker 1 was used as the MEF preservative solution. 20% knockout serum substitute (KSR), 0.8% NEAA, 2 mmol / L L-Glu, 0.1 mmol / L 2-mercaptoethanol (2-MeE), 5 ng / mL fibroblasts for maintenance culture of human iPS cells DMEM Ham's F-12 (DMEM / F12) containing cell growth factor (FGF) 2 was used. Dulbeccoline buffered saline (PBS) containing 1 mg / mL collagenase IV, 0.25% trypsin, 20% KSR, and 1 mmol / L calcium chloride was used as the exfoliation solution for human iPS cells. Cell Reservoir One (Nacalai Tesque) was used as the preservation solution for human iPS cells.

(3) Culture of human iPS cells Human iPS cells are ^{seeded on MEF (6 × 10 5} cells / 100 mm dish) _{treated with mitomycin C and placed in a CO 2} incubator _{under 5% CO 2} / 95% air conditions. The cells were cultured at 37 ° C. Passage of human iPS cells was performed at a split ratio of 1: 2 to 1: 3 after culturing for 3 to 5 days. Human iPS cells were changed medium 48 hours after thawing and daily thereafter.

(4) Differentiation of human iPS cells into brain capillary endothelial cells (BMEC) Differentiation of human iPS cells into BMEC was performed by matrigel (growth factor removal) diluted 30-fold with a medium for human iPS cells at the time of passage. The cells were seeded on a coated culture dish and cultured in StemSure® hPSC medium containing 35 ng / mL FGF2, and started with the proportion of undifferentiated colonies reaching about 70%. After culturing for 6 days in DMEM / F12 containing 20% KSR, 0.8% NEAA, 2 mmol / L L-Glu, 0.1 mmol / L 2-MeE, 1% plasma-derived serum (PDS), 10 nM retinoic acid (RA) ), Human Endothelial-SFM containing 25 ng / mL FGF2 was cultured for 2 days. It was then stripped with actase and seeded in Transwell inserts or culture dishes pre-coated with 400 μg / mL fibronectin and 100 μg / mL collagen type IV. Differentiation was induced to BMEC by culturing in Human Endothelial-SFM containing 1% PDS, 10 nM RA, 25 ng / mL FGF2 for 1 day and in Human Endothelial-SFM containing 1% PDS for 1 day. In addition, 1 nM A-83-01, 1 nM SB-431542, and 1 nM RepSox were added from the 8th to the 10th day after the start of differentiation, and the effect on differentiation into BMEC was examined. When the evaluation was performed after the 11th day, the medium was changed with Human Endothelial-SFM containing 1% PDS on the 10th day, and the medium was not changed until the evaluation day thereafter.

(5) Immunofluorescent staining After the induction of differentiation, the obtained cells (BMEC-like cells) were fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton-X for 25 minutes. Then, it was blocked with 5% donkey serum for 20 minutes, and the primary antibody was reacted at 4 ° C. overnight. Then, the cells were washed, the secondary antibody was reacted at room temperature for 1 hour, and nuclear staining was performed using 4', 6-diamidino-2-phenylindole (DAPI). Fluorescence was observed using an Operetta microscope. Regarding P-gp / BCRP / GLUT1, after the differentiation induction was completed, the obtained cells (BMEC-like cells) were washed 3 times with PBS containing 0.1% BSA, fixed with 4% paraformaldehyde, and contained again with 0.1% BSA. After washing with PBS, permeation treatment was performed with PBS containing 0.1% Triton-X for 5 minutes. Subsequently, after washing with PBS containing 0.1% BSA, the primary antibody was reacted at 4 ° C. overnight. Subsequent operations were the same as above.

(6) Measurement of transendothelial electrical resistance (TEER) value After the differentiation induction was completed, measurement was performed using Millicell ERS-2 according to the attached manual.

(7) Permeation test of lucifer yellow After completion of differentiation, HBSS (Hanks buffer salt solution) containing lucifer yellow is added to the apical membrane side of the cell culture insert, incubated at 37 ° C, and sampled over time from the basement membrane side. did. HBSS is 137 mmol / L sodium chloride, 5.4 mmol / L potassium chloride, 0.81 mmol / L magnesium sulfate, 0.44 mmol / L potassium dihydrogen phosphate, 0.34 mmol / L disodium hydrogen phosphate, 1.3 mmol / L calcium chloride , 4.2 mmol / L sodium hydrogen carbonate, 5.6 mmol / L D-glucose, pH 7.4 containing 10 mmol / L HEPES was used. The apparent film permeability coefficient of Lucifer Yellow (excitation wavelength 428 nm, fluorescence wavelength 540 nm) was calculated from the fluorescence intensity measured using a fluorescence plate reader.

Accumulation test method: After completion of differentiation induction, HBSS containing 10 μM rhodamine 123 or 20 μM Hoechst 33342 was added, incubated at 37 ° C., and 60 minutes later, cells were lysed with 5% Triton X-100. 10 μM CsA and 20 μM Ko 143 were used as inhibitors, respectively. Rhodamine 123 (excitation wavelength 485 nm, fluorescence wavelength 528 nm) and hexist 33342 (excitation wavelength 355 nm, fluorescence wavelength 460 nm) contained in the cell lysate were measured with a Synergy HTX multimode plate reader. The ^{amount of cellular protein was measured using the Pierce TM} BCA Protein Assay Kit, and the fluorescence intensity was corrected by the amount of protein.

(8) Acetylated LDL uptake test The cells after completion of differentiation were treated with 10 μg / mL Dil-labeled acetylated LDL (Ac-LDL, Alfa Aesar) for 5 hours and treated with 10 μg / mL Hoechist 33342 for 30 minutes. After washing with the medium, the observation was performed with the Operetta High-Content Imaging System.

(9) Angioplasty test On the 8th day after the start of differentiation, cells were seeded on Matrigel (BD Bioscience) and treated with Human Endothelial-SFM containing 1% PDS, 40 ng / mL VEGF, and 1 nM A-83-01 20. -Cultured for 24 hours. Then, it was treated with Calcein-AM for 30 minutes and observed with a fluorescence microscope.

(10) Freezing and thawing On the 8th day after the start of differentiation, cells were detached with actase and stored at -80 ° C for 60-90 minutes with TC-protector (KAC). The cells were then thawed and seeded in a Transwell insert or culture dish pre-coated with 400 μg / mL fibronectin and 100 μg / mL collagen type IV. Differentiation was induced to BMEC by culturing in Human Endothelial-SFM containing 1% PDS, 10 nM RA, 25 ng / mL FGF2 for 1 day and in Human Endothelial-SFM containing 1% PDS for 1 day. In addition, 1 nM A-83-01 was added from cell thawing to the 10th day, and the effect on differentiation into BMEC was examined.

(11) Gene analysis After completion of differentiation, use the Agencourt RNAdvance Tissue Kit (Beckman Coulter) to purify mRNA according to the attached manual, and then perform qPCR using KAPA SYBR FAST qPCR Kit Master mix (2 ×) ABI Prism (Kapa Biosystems). I did.

2. Results / Discussion (1) Induction of differentiation into BMEC using TGF-β inhibitor The effect of the TGF-β inhibitor added when inducing differentiation of human iPS cells into BMEC was investigated. As a result, in the group to which A-83-01 (1 nM), SB-431542 (1 nM), and RepSox (1 nM) were added, the TEER value increased about 2.5 times as compared with the control group (Fig. 1). ).

When a permeation test was performed using Lucifer Yellow, a substance transported by the paracellular pathway, the permeability coefficient was lower in the group to which A-83-01, SB-431542, and RepSox were added than in the control group. (Fig. 2).

In addition, as a result of immunofluorescence staining, the expression level of the vascular endothelial cell marker VE-cadherin (Fig. 8) increased in the A-83-01-added group, and the tight junction-related proteins Occludin and Claudin 5, ZO-1 It was found that the localization of (Fig. 3) to the cell membrane was stable (Fig. 3). Furthermore, the expression of pharmacokinetic-related proteins P-gp, GLUT1 and BCRP was confirmed (Fig. 11).

From the above, it was suggested that the addition of a TGF-β inhibitor stabilizes the membrane localization of tight junction-related proteins and forms a strong tight junction.

(2) Changes in TEER value over time
Regarding BMEC-like cells obtained by inducing differentiation by adding A-83-01 (1 nM), we investigated how TEER changes over time in each of the three different cell lines. As a result, regardless of which cell line was used to induce differentiation, the group to which A-83-01 was added ^{maintained a high TEER value (1000 Ω × cm 2} or more) compared to the control group (Fig. 4). ..

In addition, in a transmission test using Lucifer Yellow, we investigated how the permeability coefficient changes over time. As a result, in the group to which A-83-01 was added, the permeability coefficient was maintained at a low value as compared with the control group (Fig. 5).

From the above, it was suggested that long-term maintenance of strong tight junctions is possible by inducing differentiation into BMEC using a TGF-β inhibitor.

(3) Transport experiment of Rhodamine 123 and Hexto 33342
The activities of drug transporters P-gp and BCRP were evaluated for BMEC-like cells obtained by inducing differentiation by adding A-83-01 (1 nM). In the obtained cells, it was confirmed that each transporter was expressed on the apical side by immunofluorescent staining (Fig. 12). From the results of the transport amount of rhodamine 123 and the suppression of the transport amount by the inhibitor (CsA) (Fig. 6), the activity of P-gp could be confirmed by using the accumulation test method. On the other hand, from the results of the transport volume of Hoechst 33342 and the transport volume suppression by the inhibitor (Ko 143) (Fig. 7), the activity of BCRP could be confirmed in the accumulation test method as well as P-gp.

From the above, it was shown that it is possible to confirm the transporter activity even in the cells to which the TGF-β inhibitor was added.

(4) Acetylated LDL uptake test
As a result of evaluating the uptake ability of acetylated LDL in BMEC-like cells obtained by inducing differentiation by adding A-83-01 (1 nM), acetylated LDL, which is one of the characteristics of vascular endothelial cells, was evaluated. Was confirmed (Fig. 9). Uptake of acetylated LDL was confirmed in more cells in the A-83-01 treatment group.

(5) Angioplasty test As a result of evaluating the angioplasty ability using cells on the 8th day after the start of differentiation, angioplasty, which is one of the characteristics of vascular endothelial cells, was confirmed (Fig. 10). In the A-83-01 treatment group, a vascular-like structure was confirmed with a smaller number of cells.

(6) Freeze-thaw Freeze-thaw treatment can affect the structure and function of cells. Freezing and thawing treatment was performed during the induction of differentiation, and its effect was investigated. As a result, TEER decreased by freezing and thawing in the control group, but TEER did not change in the A-83-01-added group (Fig. 13). Furthermore, when the TEER of the A-83-01-added group was measured over time, the freeze-thaw group showed the same TEER as the non-freeze-thaw group (Fig. 14).

On the other hand, as a result of immunostaining of tight junction proteins, continuous tight junctions were maintained even after freezing and thawing in the A-83-01-added group (Fig. 15).

In addition, as a result of immunostaining the transporter and measuring the fluorescence intensity, the same protein expression level was confirmed even after freezing and thawing in both the control group and the A-83-01-added group (Fig. 16).

Furthermore, as a result of gene expression analysis of the A-83-01-added group, the freeze-thaw group maintained the gene expression level equal to or higher than that of the non-freeze-thaw group (Fig. 17).

4. Summary Based on the above results, in this study, we succeeded in finding a new low-molecular-weight compound that stabilizes the localization of tight junction-related proteins and improves their functions in the induction of differentiation from human iPS cells to BMEC. In addition, it was clarified that the BMEC-like cells obtained by inducing differentiation using this low molecular weight compound maintained a strong tight junction for a long period of time. Furthermore, it was shown that this low molecular weight compound also has an effect of reducing cell damage during freezing and thawing.

According to the present invention, it is possible to induce differentiation of cells (BMEC-like cells) that enable the construction of a BBB model with a high barrier function from pluripotent stem cells. The BBB model constructed by utilizing the present invention is excellent in practicality and can be used, for example, in an evaluation system for efficacy / safety of pharmaceutical products.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (20)

A method for inducing differentiation of pluripotent stem cells into cerebral capillary endothelial cells, which comprises the following steps (1) and (2):
(1) A step of culturing pluripotent stem cells in the absence of FGF2 to reduce undifferentiated state;
(2) A step of differentiating the cells obtained in step (1) into brain capillary endothelial cells, which comprises culturing in the presence of FGF2, retinoic acid and a TGF-β inhibitor. The method of claim 1, wherein a medium containing serum or serum substitute, non-essential amino acids, L-glutamic acid and 2-mercaptoethanol is used for culturing in step (1). The method according to claim 1 or 2, wherein a medium containing serum or a serum substitute, FGF2, retinoic acid and a TGF-β inhibitor is used for culturing in step (2). The method according to any one of claims 1 to 3, wherein the culture period of the step (1) is 3 to 9 days. The method according to any one of claims 1 to 4, wherein the culture period of the step (2) is 2 to 10 days. The method according to any one of claims 1 to 5, wherein the transendothelial electrical resistance value of the cell layer formed by the step (2) is 1000 Ω × cm ^{2 or more.} The method according to any one of claims 1 to 6, wherein the cell layer formed by the step (2) ^{maintains a transendothelial electrical resistance value exceeding 1000 Ω × cm 2 for 5 days or more.} Step (2) is performed in the presence of (2-1) FGF2 and retinoic acid and after the culture, in the presence of (2-2) FGF2, retinoic acid, and TGF-β inhibitor. The method according to any one of claims 1 to 7, which comprises culturing. A medium containing serum or serum substitute, FGF2, and retinoic acid was used for the culture of step (2-1), and serum or serum substitute, FGF2, retinoic acid, and TGF were used for the culture of step (2-2). The method according to claim 8, wherein a medium containing a -β inhibitor is used. The method according to claim 8 or 9, wherein the culturing period of the step (2-1) is 1 to 5 days, and the culturing period of the step (2-2) is 1 to 4 days. Step (2) is performed in the presence of (2-1) FGF2 and retinoic acid and after the culture, in the presence of (2-2) FGF2, retinoic acid, and TGF-β inhibitor. The method according to any one of claims 1 to 7, which comprises culturing in the presence of (2-3) TGF-β inhibitor, which is carried out after the culturing. A medium containing serum or serum substitute, FGF2, and retinoic acid was used for the culture of step (2-1), and serum or serum substitute, FGF2, retinoic acid, and retinoic acid were used for the culture of step (2-2). The method according to claim 11, wherein a medium containing a TGF-β inhibitor is used, and a medium containing serum or a serum substitute and a TGF-β inhibitor is used for culturing in step (2-3). The culture period of step (2-1) is 1 to 5 days, the culture period of step (2-2) is 1 to 4 days, and the culture period of step (2-3) is 1 to 4 days. The method of claim 11 or 12, which is a day. The TGF-β inhibitor is one or more compounds selected from the group consisting of A-83-01, SB-431542, RepSox, SB-505124, SB-525334, LY-2157299, LY-364947, SD208 and D4476. , The method according to any one of claims 1 to 13. The method according to any one of claims 1 to 14, wherein the pluripotent stem cell is an induced pluripotent stem cell. The method of claim 15, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell. The cell layer obtained by the method according to any one of claims 1 to 16. A method for evaluating the blood-brain barrier permeability of a test substance using the cell layer according to claim 17. The method according to claim 18, which comprises the following steps (i) to (iii):
(I) The step of preparing the cell layer according to claim 17;
(Ii) A step of bringing the test substance into contact with the cell layer;
(Iii) A step of quantifying a test substance that has permeated the cell layer and evaluating the permeability of the test substance. A method for evaluating the effect of a test substance on the blood-brain barrier barrier function using the cell layer according to claim 17.

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