WO2023074363A1 - Pluripotent stem cell-derived cerebrovascular endothelial cells and production method thereof - Google Patents
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Definitions
- the brain has a blood-brain barrier (BBB).
- BBB blood-brain barrier
- the BBB severely restricts the penetration of substances such as drugs into brain cells (Non-Patent Document 1). Due to the restriction of substance penetration by the BBB, the contribution of drugs to many central nervous system diseases such as Alzheimer's disease is extremely low, and sufficient treatment satisfaction has not been obtained.
- P-glycoprotein a drug efflux transporter
- MDR/TAP subfamily of phosphorylated ABC transporters with a molecular weight of about 180,000. family B Member 1) or MDR1 (Multiple drug resistance 1).
- Drug efflux by P-gp encoded by the MDR1 gene is involved in drug resistance and has an important function.
- P-gp is expressed on the blood-side cell membrane and has been shown to strictly restrict the membrane permeability of substances between brain tissue and blood.
- pluripotent stem cell-derived cerebrovascular endothelial cells that have been produced so far cannot be said to have sufficient expression of P-gp.
- Patent No. 6478464 Patent Publication No. 2015-159785
- the present invention provides pluripotent stem cell-derived cerebral vascular endothelial cells that accurately reflect the transport function of the human BBB and have a higher level of drug efflux than conventional pluripotent stem cell-derived cerebral vascular endothelial cells.
- An object of the present invention is to provide cerebral vascular endothelial cells having a port function and a method for producing the same.
- the present inventors found that, after undergoing the step of introducing the MDR1 gene that encodes P-gp into pluripotent stem cells, a higher level of drug efflux transduction was achieved by inducing differentiation into cerebral vascular endothelial cells.
- the inventors have found that it is possible to provide cerebrovascular endothelial cells having a port function, and completed the present invention.
- 6. The pluripotent stem cell-derived cerebrovascular endothelial cell according to 4 or 5 above, which has a P-gp function equal to or greater than that retained by primary cerebral vascular endothelial cells. 7. 6. A method for analyzing the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to 4 or 5 above. 8. 6. A method for evaluating pharmacokinetics at the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to 4 or 5 above.
- the pluripotent stem cells introduced with the P-gp-encoding MDR1 gene used to generate the cerebral vascular endothelial cells of the present invention retained the pluripotent potential inherent in pluripotent stem cells. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention highly expressed P-gp, had a high barrier function, and retained an excellent drug efflux transporter function.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have an excellent drug efflux transporter function that has not been achieved with immortalized cerebrovascular endothelial cell lines established from human cerebral vascular endothelial cells.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention accurately reflect the transport function of the human BBB, and it is possible to obtain cerebral endothelial cells with the same properties as primary cerebral vascular endothelial cells. I was able to overcome the challenges that were difficult.
- FIG. 2 shows the structure of a lentiviral vector plasmid for introducing the MDR1 gene encoding P-gp into pluripotent stem cells.
- FIG. 2 shows the results of confirming the characteristics of the established MDR1 gene-introduced iPS cells (MDR1-iPS cells).
- Figure 2 (A) shows the results of confirming that Venus is expressed in the obtained cells
- Figure 2 (B) shows MDR1-iPS cells, MCS-iPS cells, and non-transfected cells. Morphology for iPS cells.
- Example 1 shows the results of confirming the expression of P-gp and undifferentiated markers (Nanog, Oct3/4) in MDR1-iPS cells and MCS-iPS cells established in Example 1.
- FIG. Figure 3(A) shows that P-gp is highly expressed in MDR1-iPS cells compared to MCS-iPS cells, and Figure 3(B) shows an undifferentiated marker (Nanog and Oct3/4) are equivalent to those in MCS-iPS cells.
- (Experimental example 1) 4 shows the results of observing the morphology of MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells produced in Example 2.
- FIG. 1 shows that observing the morphology of MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells produced in Example 2.
- Example 2-1 2 shows the results of confirming the expression of vascular endothelial cell markers (PECAM1, VE-cadherin) in the MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2.
- Example 2-1) 4 shows the results of barrier function evaluation of MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2.
- FIG. FIG. 6(A) shows the results of the intermembrane electrical resistance value
- FIG. 6(B) shows the evaluation of substance permeability using NaF.
- Example 2-2 shows the results of confirming the expression of tight junction-related markers (Claudin-5, Occludin and ZO-1) in MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells prepared in Example 2.
- (Experimental example 2-2) 2 shows the functional evaluation results of P-gp expressed in MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells prepared in Example 2.
- Example 2-3 2 shows the results of evaluating the uptake of Rhodamine-123 in the MDR1-iPS cell-derived cerebral vascular endothelial cells and the MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2.
- FIG. (Experimental example 2-3) 4 shows the results of permeability evaluation using quinidine for the MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2.
- the present invention relates to a pluripotent stem cell-derived cerebrovascular endothelial cell and a method for producing the same, which is a cerebral vascular endothelial cell that can accurately reflect the transport function of the human BBB, and which is a drug efflux transporter, P-gp.
- An object of the present invention is to provide cerebral vascular endothelial cells with excellent functions.
- Cerebrovascular endothelial cells generally refer to vascular endothelial cells that have brain-specific properties and/or functions.
- vascular endothelial cells that have not acquired tissue or organ specificity are simply referred to as “vascular endothelial cells” to distinguish them from “cerebral vascular endothelial cells”.
- the "pluripotent stem cell-derived cerebrovascular endothelial cells” of the present invention are, for example, “primary cerebral vascular endothelial cells” obtained from non-human animals such as humans or mammals, and " used in distinction from “immortalized cerebrovascular endothelial cell line”.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention are characterized by high functions of the drug efflux transporter P-gp.
- Pluripotent stem cell-derived cerebrovascular endothelial cells having such properties are characterized by first undergoing a step of overexpressing MDR1 (P-gp) in pluripotent stem cells, and then inducing differentiation into cerebrovascular endothelial cells.
- Pluripotent stem cells overexpressing MDR1 (P-gp) may be treated to induce differentiation into cerebral vascular endothelial cells.
- vascular endothelial cells that have not acquired organ-specificity may be collected and then induced to differentiate into cerebral vascular endothelial cells.
- pluripotent stem cells are stem cells that can differentiate into all types of cells that make up the body, iPS cells (induced pluripotent stem cells) and ES cells (Embryonic Stem Cells) etc. are exemplified. Pluripotent stem cells in the present invention are particularly preferably human-derived cells, particularly human iPS cells. "Pluripotent stem cell” as used herein means a cell that has both self-renewal and pluripotency. “Pluripotency” is used interchangeably with “pluripotency” and refers to the property possessed by cells, and refers to the ability to differentiate into cells belonging to various tissues and organs.
- iPS cells induce the reprogramming of differentiated cells without using eggs, embryos, or ES cells, and have the same pluripotency and proliferative potential as ES cells. It refers to induced pluripotent stem cells, and was created in 2006 for the first time in the world from mouse fibroblasts. Furthermore, the human iPS cells were successfully established by transfecting human-derived fibroblasts with OCT3/4, SOX2, KLF4, and C-MYC, the human homologous genes of the four genes used to establish mouse iPS cells. reported (Cell 131: 861-872, 2007).
- the iPS cells used in the present invention may be iPS cells produced by a method known per se as described above, or iPS cells produced by a new method that will be developed in the future.
- a method for culturing pluripotent stem cells such as iPS cells is not particularly limited, and a method known per se can be used.
- a medium capable of maintaining the undifferentiated state and pluripotency of iPS cells or ES cells or a medium suitable for inducing differentiation a medium known per se or a new medium to be developed in the future can be used.
- EC medium such as DMEM and/or DMEM Ham's F-12 (DMEM/F12), Human Endothelial-SFM, mTeSR1 (modified Tenneille Serum Replacer 1) medium that does not require feeder cells, etc. are used as needed. can do.
- bFGF basic fibroblast growth factor
- FGF2 basic fibroblast growth factor
- KOSR knock- outserum replacement
- LIF leukemia inhibitory factor
- bFGF basic fibroblast growth factor
- CSTI-7 primate ES cell proliferation medium
- subculturing may be performed as appropriate. For example, when the cells become confluent or subconfluent, a part of the cells can be collected and transferred to another culture container to continue the culture.
- the number of cells at passage is not particularly limited, but is, for example, about 1 ⁇ 10 5 to 1 ⁇ 10 6 /cm 2 , preferably about 1 ⁇ 10 4 to 3 ⁇ 10 4 /cm 2 . can be done.
- pre-treat the cells with a ROCK inhibitor Rho-associated coiled-coil forming kinase/Rho-associated kinase
- Y-27632 ROCK inhibitor
- Cells can also be cultured on a culture surface coated with a basement membrane component, an adhesion molecule, or the like for the purpose of improving cell viability and growth rate, promoting differentiation induction, cell selection, and the like.
- materials/components used to coat culture surfaces include matrigel, collagen I, collagen IV, fibronectin, laminin, gelatin, polylysine, vitronectin, and the like.
- MDR1 gene encoding P-gp into pluripotent stem cells Introduction of the MDR1 gene into pluripotent stem cells can be performed using a viral vector such as a lentiviral vector or an adenoviral vector carrying a target gene.
- a target gene can be effectively introduced into pluripotent stem cells by using a lentiviral vector or an adenoviral vector.
- Promoters that can be used include, for example, EF, RSV, pCMV, CA ( ⁇ -actin promoter/CMV enhancer) and the like.
- the drug efflux transporter P-gp is also called ABCB1 or MDR1.
- P-gp specifically refers to the protein and MDR1 gene refers to the gene encoding P-gp.
- the MDR1 gene is indicated by GenBank Access No. AF016535, and the MDR1 gene to be introduced into pluripotent stem cells is at least the sequence of the protein coding region (coding sequence: CDS) among the nucleotide sequences specified by GenBank Access No. AF016535. including.
- Pluripotent stem cells into which the MDR1 gene has been introduced are characterized by high expression of P-gp.
- the MDR1 gene-introduced pluripotent stem cells express at least 100 times more P-gp, and the expression level is 100 to 2000 times, preferably 150 to 1000 times, as compared to pluripotent stem cells that have not been introduced with the MDR1 gene. about two times, most preferably about 900 times.
- the MDR1 gene-introduced pluripotent stem cells of the present invention are cells that maintain both the inherent properties of pluripotent stem cells, that is, self-renewal ability and pluripotency, in addition to highly expressing P-gp.
- Pluripotency refers to the ability to differentiate into cells belonging to various tissues and organs, and retains markers such as Nanog and Oct3/4 like normal pluripotent stem cells.
- the method for producing cerebral vascular endothelial cells from MDR1 gene-introduced pluripotent stem cells can apply a method for producing cerebral vascular endothelial cells from pluripotent stem cells known per se. , 4 can be referred to.
- vascular endothelial cells that have not acquired tissue- or organ-specificity can be first collected from MDR1 gene-introduced pluripotent stem cells, and then induced to differentiate into cerebral vascular endothelial cells.
- vascular endothelial cells which are CD34-expressing cells, can be first collected and then induced to differentiate into cerebral vascular endothelial cells.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have good adhesiveness and the ability to regulate substance transport, similar to the cerebral vascular endothelial cells that constitute the BBB in vivo. Adhesiveness and ability to regulate substance transport can be confirmed by a technique known per se, and can be confirmed, for example, by the method described in Experimental Examples described later. The ability to regulate substance transport and adhesiveness characteristic of cerebrovascular endothelial cells can be confirmed by measuring electrical resistance and analyzing the expression of tight junction-related genes.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have particularly high P-gp function. Drug efflux by P-gp is involved in drug resistance and has an important function.
- P-gp is expressed on the blood side cell membrane and strictly restricts the membrane permeability of substances between brain tissue and blood. Also in the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention, P-gp is preferably expressed on the blood-side cell membrane.
- Examples of tight junction-related genes include Claudin-5, Occludin, and ZO-1.
- the ability to regulate substance transport can be confirmed by substance permeability analysis using labeled dextran, the expression of transporters that are specifically expressed in cerebrovascular endothelial cells, and the substance transport functions of these transporters. can.
- Examples of the transporter include P-gp, BCRP (cancer resistance protein), Glut1 (glucose transporter type 1), MRP4 (multidrug resistance protein 4), MRP1 (multidrug resistance protein 1), and the like.
- the pluripotent stem cell-derived cerebral vascular endothelial cells of the present invention contain BCRP, Glut1, MRP4, MRP1, etc. to the same extent as cerebral vascular endothelial cells produced by differentiation induction from pluripotent stem cells into which the MDR1 gene has not been introduced. It is sufficient if it is expressed.
- Vascular endothelial cell markers include CD34, CD31, von Willebrand Factor (vWF), and VE-cadherin.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention are induced to differentiate from pluripotent stem cells into which the MDR1 (P-gp) gene has not been introduced, regardless of whether the step of collecting CD34-expressing cells is included. It is sufficient that CD31, vWF, VE-cadherin, etc. are expressed at the same level as the cerebral vascular endothelial cells produced by the method described above.
- Humoral factors that can be used in the present invention to induce differentiation into brain vascular endothelial cells include, for example, bone morphogenetic protein 4 (BMP4), activin A, mesoderm-inducing factors such as FGF2, and vascular endothelial cell growth factors (vascular endothelial growth factor: VEGF), platelet growth factors such as thrombopoietin (TPO), stem cell factor (SCF), FMS-like tyrosinekinase 3 Ligand (Flt3L), Examples include hematopoietic factors such as interleukin-6 (IL-6)/soluble IL-6 receptor complex (IL-6/sIL-6R) and Notch ligand (hereinafter referred to as Notch ligand).
- BMP4 bone morphogenetic protein 4
- activin A mesoderm-inducing factors
- FGF2 vascular endothelial cell growth factor
- VEGF vascular endothelial growth factor
- platelet growth factors such as
- a single humoral factor may be used, or a plurality thereof may be used at the same time.
- other cells pericytes, astrocytes, etc. may be used in combination.
- the pluripotent stem cell-derived cerebral endothelial cells of the present invention increased the expression of P-gp to the same level as primary cultured human cerebral endothelial cells.
- the pluripotent stem cell-derived cerebrovascular endothelial cells express at least 100 times more P-gp than pluripotent stem cell-derived cerebral endothelial cells into which the MDR1 gene has not been introduced.
- the expression level is 100- to 2000-fold, preferably about 150- to 1000-fold, most preferably about 900-fold.
- the expression of transporter proteins other than P-gp in the pluripotent stem cell-derived cerebrovascular endothelial cells is comparable to that of pluripotent stem cell-derived cerebral endothelial cells into which the MDR1 gene has not been introduced.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention can be used for analysis of the blood-brain barrier. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention can also be used to evaluate pharmacokinetics at the blood-brain barrier.
- the present invention also extends to a method for analyzing the blood-brain barrier using pluripotent stem cell-derived cerebrovascular endothelial cells and a method for evaluating pharmacokinetics in the blood-brain barrier.
- Example 1 Establishment of MDR1-introduced pluripotent stem cells This example describes the establishment of MDR1-iPS cells in which the MDR1 gene was incorporated into human iPS cells (iMR90-4 cells: purchased from WiCell).
- MDR1-iPS cells Human iPS cells (iMR90-4 cells, purchased from WiCell) were placed on Matrigel basement membrane matrix (Growth Factor Reduced Matrigel (R) , manufactured by Corning) and mTeSR1. It was cultured using -cGMP (modified Tenneille Serum Replacer 1-cGMP) medium. The virus solution prepared above was allowed to act for 24 hours and cultured for 3 to 4 days.
- iPS cells Human iPS cells (iMR90-4 cells, purchased from WiCell) were placed on Matrigel basement membrane matrix (Growth Factor Reduced Matrigel (R) , manufactured by Corning) and mTeSR1. It was cultured using -cGMP (modified Tenneille Serum Replacer 1-cGMP) medium. The virus solution prepared above was allowed to act for 24 hours and cultured for 3 to 4 days.
- -cGMP modified Tenneille Serum Replacer 1-cGMP
- MDR1-expressing cells MDR1-expressing cells
- MCS-iPS cells MCS gene-introduced iPS cells
- Expression of Venus was confirmed again in the obtained cells (Fig. 2A).
- MDR1-iPS cells, MCS-iPS cells and non-transfected iPS cells were each cultured in mTeSR1-cGMP medium. No morphological difference was observed between MDR1-iPS cells, MCS-iPS cells, and iPS cells not transfected (Fig. 2B).
- Example 1 Evaluation of MDR1-iPS cells and MCS-iPS cells
- mRNA of P-gp and undifferentiated markers Nag, Oct3/4
- the expression level was measured by quantitative PCR as follows.
- Total RNA extracted from each cell was treated with RNase (New England Biolabs) and reverse-transcribed using SuperScript IV VILO Master Mix (ThermoFisher Scientific) to synthesize cDNA.
- RNase New England Biolabs
- SuperScript IV VILO Master Mix ThermoFisher Scientific
- mRNA expression levels of MDR1 (P-gp), Nanog and Oct3/4 genes were quantified using the StepOnePlus real-time PCR system (ThermoFisher Scientific).
- GAPDH was used as an internal standard gene.
- ⁇ MDR1 gene amplification primer Forward CCATGCTCAGACAGGATGTG (sequence number 1) Reverse ACAGCAAGCCTGGAACCTAT (SEQ ID NO: 2) ⁇ Nanog gene amplification primer Forward AGAAGGCCTCAGCACCTAC (SEQ ID NO: 3) Reverse GGCCTGATTGTTCCAGGATT (SEQ ID NO: 4) ⁇ Oct3/4 gene amplification primers Forward CTTGAATCCCGAATGGAAAGGG (SEQ ID NO: 5) Reverse GTGTATATCCCAGGGTGATCCTC (SEQ ID NO: 6) ⁇ Primer for GAPDH gene amplification Forward GGTGGTCTCCTCTGACTTCAACGT (sequence number 7) Reverse GTGGTCGTTGAGGGCAATG (SEQ ID NO: 8)
- the MDR1-iPS cells and MCS-iPS cells established in Example 1 were treated with a separation/dispersion reagent (ESGRO Complete Accutase, manufactured by Merck-Millipor) to obtain single cells.
- ESGRO Complete Accutase manufactured by Merck-Millipor
- mTeSR1-cGMP medium containing Y-27632 manufactured by Wako
- ROCK Raster-binding kinase
- each iPS cell adjusted to 0.8-1.2 ⁇ 10 5 cells/well
- Growth Factor Reduced Matrigel (R ) were plated on 6-well plates coated with 24 hours after seeding, the medium was replaced with mTeSR1-cGMP medium and cultured for 2 days.
- Unconditioned Medium DMEM/F-12, 20% KOSR (KnockOut TM Serum Replacement, ThermoFisher Scientific), 1% Non-essential amino acid (Nacalai), 0.5% GlutaMAX (TM) (ThermoFisher Scientific), 0.0007% 2- mercaptoethanol
- serum-free medium for endothelial cell culture ESFM, human endotherial SFM, ThermoFisher Scientific
- 1% human platelet-derived serum Sigma
- 10 ⁇ M all-trans-retinoic acid Wako
- 20 ng/mL human bFGF Keratayama chemical
- Example 2-1 Morphological evaluation of iPS cell-derived cerebral endothelial cells and evaluation of vascular endothelial cell markers
- MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cells prepared in Example 2 were used.
- the derived brain endothelial cells were evaluated for morphology and endothelial cell markers.
- Vascular endothelial cell marker mRNA expression of vascular endothelial cell markers (PECAM1, VE-cadherin) for MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed by quantitative PCR method. bottom. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed in the same manner as in Experimental Example 1.
- vascular endothelial cell markers PECAM1, VE-cadherin
- Example 2-2 Evaluation of barrier function of iPS cell-derived cerebral vascular endothelial cells
- the barrier function of MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2 was evaluated. evaluated for functionality.
- Transmembrane electrical resistance value Transmembrane electrical resistance value (TEER) was measured for MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells.
- the intermembrane electrical resistance value was measured using Millicell ERS-2 (manufactured by Merck-Millipore). The measurement method was performed according to the method described in the instruction manual for Millicell ERS-2.
- TEER was calculated according to the following formula.
- MDR1-iPS cell-derived cerebral vascular endothelial cells and hCMEC/D3 cells human-derived immortalized cerebral capillary endothelial cell line widely used as cerebral intravascular cell line
- isolated human cerebral capillaries primary culture
- P-gp (MDR1) mRNA in MDR1-iPS cell-derived cerebrovascular endothelial cells was measured in hCMEC/D3 cells and reported in human isolated brain capillaries (primary culture) (ref). was almost the same. It was confirmed that introduction of the MDR1 gene increased the expression of P-gp to the same level as that of primary cultured human cerebral endothelial cells.
- Rhodamine-123 was measured using a fluorescence intensity meter GENIOS (Spectra FLUOR Plus, manufactured by TECAN) (excitation wavelength: 485 nm, fluorescence wavelength: 530 nm). Rhodamine-123 concentration was calculated by preparing a calibration curve by serially diluting the Rhodamine-123 solution. The amount of Rhodamine-123 detected was normalized using cell number.
- the apparent permeability coefficient (Papp) of quinidine was calculated from the following formula.
- dQ/dt, D0 and S denote the transport rate of quinidine, the initial concentration on the donor side and the surface area of the transwell, respectively.
- the Efflux ratio was calculated from Papp in the A-to-B and B-to-A directions.
- Quinidine was quantified using Nexera-XR HPLC system and LC-MS/MS connected to QTRAP4500. The electrospray ionization method was used for the mass spectrometer, and the measurement was performed in the positive mode. The m/z values of quinidine and dantrolene were monitored at 325.1/307.1 and 325.1/114.0, respectively.
- a Synergi Hydro-RP column (2.0 ⁇ 50 mm, 25 ⁇ m, Phenomenex) was used for chromatographic separation, and gradient analysis with 10 mM ammonium formate (0.2% formic acid) and methanol was performed at a flow rate of 0.4 mL/min. Data analysis was performed with Analyst 1.6.1 software.
- the MDR1 gene-introduced pluripotent stem cells used to generate the cerebral vascular endothelial cells of the present invention highly expressed P-gp and retained the pluripotent potential inherent in pluripotent stem cells. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention highly expressed P-gp, had a high barrier function, and retained an excellent function as a drug efflux transporter.
- the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have an excellent drug efflux transporter function that has not been achieved with immortalized cerebrovascular endothelial cell lines established from human cerebral vascular endothelial cells.
- the pluripotent stem cell-derived cerebral vascular endothelial cells of the present invention accurately reflect the transport function of the human BBB, and primary cerebral vascular endothelial cells can be obtained with similar properties. I was able to overcome the challenges that were difficult. That is, according to the cerebral vascular endothelial cells obtained by the present invention, it is possible to easily obtain homogeneous cerebral vascular endothelial cells that have a function that accurately reflects the transport function of the human BBB. It is possible to uniformly evaluate disease therapeutic drugs, etc., and is useful for human clinical trials and the like.
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Abstract
Provided are pluripotent stem cell-derived cerebrovascular endothelial cells capable of accurately reflecting the transport function of human BBB and a production method of the same. The cerebrovascular endothelial cells are obtained by introducing MDR1 gene encoding P-gp into pluripotent stem cells and then inducing the differentiation into the cerebrovascular endothelial cells. The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention express P-gp at a high level, have a high barrier function, and retain an excellent function as a drug efflux transporter. The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have an excellent drug efflux transporter function that has never been achieved by immortalized cerebrovascular endothelial cell lines established from human cerebrovascular endothelial cells. Moreover, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention accurately reflect the transport function of human BBB and thus make it possible to overcome the problem of the difficulty of obtaining cerebrovascular endothelial cells having comparable properties occurring in primary cerebrovascular endothelial cells.
Description
本発明は、ヒトの血液脳関門（blood brain barrier：BBB）の輸送機能を精度よく反映する多能性幹細胞由来脳血管内皮細胞に関し、さらにその製造方法に関する。
The present invention relates to pluripotent stem cell-derived cerebrovascular endothelial cells that accurately reflect the transport function of the human blood brain barrier (BBB), and also to a method for producing the same.
本出願は、参照によりここに援用されるところの日本出願特願2021-178573号優先権を請求する。
This application claims priority from Japanese Patent Application No. 2021-178573, which is incorporated herein by reference.
脳には血液脳関門（BBB）が存在している。その中心構成成分である脳毛細血管内皮細胞は強固なタイトジャンクションを形成し、さらに特異的トランスポーターを発現している。BBBにより、薬剤等の物質の脳細胞への透過は厳しく制限されている（非特許文献１）。BBBによる物質透過の制限のため、アルツハイマー等の中枢神経疾患の多くでは薬剤の貢献度は著しく低く、十分な治療満足度が得られていない。
The brain has a blood-brain barrier (BBB). The brain capillary endothelial cells, which are the central component, form strong tight junctions and express specific transporters. The BBB severely restricts the penetration of substances such as drugs into brain cells (Non-Patent Document 1). Due to the restriction of substance penetration by the BBB, the contribution of drugs to many central nervous system diseases such as Alzheimer's disease is extremely low, and sufficient treatment satisfaction has not been obtained.
薬物排出トランスポーターであるP糖タンパク質（P-glycoprotein：P-gp）は、分子量約18万のリン酸化ABC輸送体のMDR/TAPサブファミリーに属する分子であり、ABCB1（ATP-binding Cassette Sub-family B Member 1）又はMDR1（Multiple drug resistance 1）とも呼ばれる。MDR1遺伝子にコードされたP-gpによる薬物の排出は薬剤耐性等に関与しており、重要な機能を有する。特に脳毛細血管内皮細胞（BCEC）においてP-gpは血液側細胞膜に発現し、脳組織と血液との間の物質の膜透過性を厳密に制限していることが明らかにされている。
P-glycoprotein (P-gp), a drug efflux transporter, is a molecule belonging to the MDR/TAP subfamily of phosphorylated ABC transporters with a molecular weight of about 180,000. family B Member 1) or MDR1 (Multiple drug resistance 1). Drug efflux by P-gp encoded by the MDR1 gene is involved in drug resistance and has an important function. Especially in brain capillary endothelial cells (BCEC), P-gp is expressed on the blood-side cell membrane and has been shown to strictly restrict the membrane permeability of substances between brain tissue and blood.
従来より、BBBの機能解明ならびに創薬研究への応用を目指して、ラット等の実験動物由来の血管内皮細胞を用いて、in vitro BBBモデルが構築されてきた。しかし、実験動物を用いたモデルではヒトの薬物の輸送機能を精度よく反映できないため、中枢神経疾患治療薬のヒト臨床試験における成功率が依然として低い。ヒトの生体内のBBB機能を反映し得るヒト脳血管内皮細胞を用いたin vitro BBBモデルの開発が望まれるが、ヒト毛細脳血管内皮細胞の大量入手は困難であるため創薬研究への応用が制限されているのが現状である。ヒト脳血管内皮細胞として不死化細胞株が樹立されているが、初代脳血管内皮細胞に比べて、種々のタイトジャンクション形成タンパク質の発現が低いことが問題視されている（非特許文献２）。
Conventionally, an in vitro BBB model has been constructed using vascular endothelial cells derived from experimental animals such as rats, with the aim of elucidating the functions of the BBB and applying it to drug discovery research. However, the success rate in human clinical trials of therapeutic drugs for central nervous system diseases is still low because models using experimental animals cannot accurately reflect the drug transport function of humans. Development of an in vitro BBB model using human cerebrovascular endothelial cells, which can reflect the BBB function in the human body, is desired. is currently limited. Immortalized cell lines have been established as human cerebral vascular endothelial cells, but the low expression of various tight junction-forming proteins compared to primary cerebral vascular endothelial cells is regarded as a problem (Non-Patent Document 2).
多能性幹細胞から脳血管内皮細胞を製造する方法が試みられてきた。iPS細胞（人工多能性幹細胞：induced pluripotent stem cell）をWnt7Wnt/β-カテニンシグナル経路を刺激し、アストロサイトと共培養することでiPS細胞由来脳微小血管内皮細胞（BMECs）を作製することが報告されている（非特許文献３）。iPS細胞由来BMECsをレチノイン酸で処理したところ、BBBの機能が増強したことが報告されている（非特許文献４）。更にヒト多能性幹細胞から分化誘導されたCD34発現細胞を培養し、前記培養した細胞を脳由来細胞を培養した培地に接触させて培養する脳血管内皮細胞への分化誘導方法についても開示がある（特許文献１）。
Attempts have been made to produce cerebrovascular endothelial cells from pluripotent stem cells. It is possible to generate iPS cell-derived brain microvascular endothelial cells (BMECs) by stimulating the Wnt7Wnt/β-catenin signaling pathway in iPS cells (induced pluripotent stem cells) and co-culturing them with astrocytes. reported (Non-Patent Document 3). It has been reported that treatment of iPS cell-derived BMECs with retinoic acid enhanced the function of the BBB (Non-Patent Document 4). Furthermore, there is also disclosed a method of inducing differentiation into cerebrovascular endothelial cells by culturing CD34-expressing cells induced to differentiate from human pluripotent stem cells, and culturing the cultured cells by contacting them with a medium in which brain-derived cells were cultured. (Patent document 1).
しかしながら、これまでに作製されてきた多能性幹細胞由来脳血管内皮細胞は、P-gpの発現は十分とはいえない。
However, the pluripotent stem cell-derived cerebrovascular endothelial cells that have been produced so far cannot be said to have sufficient expression of P-gp.
本発明は、多能性幹細胞由来脳血管内皮細胞であって、ヒトのBBBの輸送機能を精度よく反映し、従来の多能性幹細胞由来脳血管内皮細胞と比較して、より高い薬物排出トランスポート機能を有する脳血管内皮細胞及びその製造方法を提供することを課題とする。
The present invention provides pluripotent stem cell-derived cerebral vascular endothelial cells that accurately reflect the transport function of the human BBB and have a higher level of drug efflux than conventional pluripotent stem cell-derived cerebral vascular endothelial cells. An object of the present invention is to provide cerebral vascular endothelial cells having a port function and a method for producing the same.
本発明者らは、鋭意検討を重ねた結果、多能性幹細胞にP-gpをコードするMDR1遺伝子を導入する工程を経た後、脳血管内皮細胞へと分化誘導することでより高い薬物排出トランスポート機能を有する脳血管内皮細胞を提供しうることを見出し、本発明を完成した。
As a result of intensive studies, the present inventors found that, after undergoing the step of introducing the MDR1 gene that encodes P-gp into pluripotent stem cells, a higher level of drug efflux transduction was achieved by inducing differentiation into cerebral vascular endothelial cells. The inventors have found that it is possible to provide cerebrovascular endothelial cells having a port function, and completed the present invention.
すなわち本発明は、以下よりなる。
１．以下の工程を含む多能性幹細胞から脳血管内皮細胞を製造する方法：
１）多能性幹細胞にP-gpをコードするMDR1遺伝子を導入する工程；及び
２）前記工程１）の遺伝子導入多能性幹細胞を、脳由来細胞に分化誘導する工程。
２．前記１）のP-gpをコードするMDR1遺伝子を導入した多能性幹細胞が、未分化性を維持しながら、MDR1遺伝子を導入していない多能性幹細胞に比べてP-gpの発現量が100～2000倍に高発現した多能性幹細胞である、前項１に記載の脳血管内皮細胞を製造する方法。
３．多能性幹細胞が、iPS細胞である、前項１に記載の脳血管内皮細胞を製造する方法。
４．MDR1遺伝子を導入した多能性幹細胞由来の脳血管内皮細胞であって、MDR1遺伝子を導入していない多能性幹細胞由来の脳血管内皮細胞に比べてP-gpの発現量が100～2000倍に高発現していることを特徴とする、多能性幹細胞由来脳血管内皮細胞。
５．前項１に記載の製造方法により製造された多能性幹細胞由来脳血管内皮細胞。
６．初代脳血管内皮細胞が保持するP-gp機能と同等若しくはそれ以上のP-gp機能を担持することを特徴とする前項４又は５に記載の多能性幹細胞由来脳血管内皮細胞。
７．前項４又は５に記載の多能性幹細胞由来脳血管内皮細胞を用いる、血液脳関門の解析方法。
８．前項４又は５に記載の多能性幹細胞由来脳血管内皮細胞を用いる、血液脳関門における薬物動態の評価方法。 That is, the present invention consists of the following.
1. A method for producing cerebrovascular endothelial cells from pluripotent stem cells comprising the steps of:
1) a step of introducing the MDR1 gene encoding P-gp into the pluripotent stem cells; and 2) a step of inducing differentiation of the gene-introduced pluripotent stem cells of step 1) into brain-derived cells.
2. The pluripotent stem cells introduced with the MDR1 gene encoding the P-gp of 1) maintain undifferentiated state, while the expression level of P-gp is higher than that of the pluripotent stem cells without the introduction of the MDR1 gene. The method for producing the cerebrovascular endothelial cells according to the precedingitem 1, which are pluripotent stem cells expressed 100 to 2000 times higher.
3. 2. The method for producing cerebrovascular endothelial cells according to the precedingitem 1, wherein the pluripotent stem cells are iPS cells.
4. Pluripotent stem cell-derived cerebrovascular endothelial cells transfected with the MDR1 gene showed 100 to 2000-fold expression of P-gp compared to pluripotent stem cell-derived cerebral endothelial cells not transfected with the MDR1 gene. A pluripotent stem cell-derived cerebrovascular endothelial cell characterized by high expression in
5. A pluripotent stem cell-derived cerebrovascular endothelial cell produced by the production method according to the precedingitem 1.
6. 6. The pluripotent stem cell-derived cerebrovascular endothelial cell according to 4 or 5 above, which has a P-gp function equal to or greater than that retained by primary cerebral vascular endothelial cells.
7. 6. A method for analyzing the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to 4 or 5 above.
8. 6. A method for evaluating pharmacokinetics at the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to 4 or 5 above.
１．以下の工程を含む多能性幹細胞から脳血管内皮細胞を製造する方法：
１）多能性幹細胞にP-gpをコードするMDR1遺伝子を導入する工程；及び
２）前記工程１）の遺伝子導入多能性幹細胞を、脳由来細胞に分化誘導する工程。
２．前記１）のP-gpをコードするMDR1遺伝子を導入した多能性幹細胞が、未分化性を維持しながら、MDR1遺伝子を導入していない多能性幹細胞に比べてP-gpの発現量が100～2000倍に高発現した多能性幹細胞である、前項１に記載の脳血管内皮細胞を製造する方法。
３．多能性幹細胞が、iPS細胞である、前項１に記載の脳血管内皮細胞を製造する方法。
４．MDR1遺伝子を導入した多能性幹細胞由来の脳血管内皮細胞であって、MDR1遺伝子を導入していない多能性幹細胞由来の脳血管内皮細胞に比べてP-gpの発現量が100～2000倍に高発現していることを特徴とする、多能性幹細胞由来脳血管内皮細胞。
５．前項１に記載の製造方法により製造された多能性幹細胞由来脳血管内皮細胞。
６．初代脳血管内皮細胞が保持するP-gp機能と同等若しくはそれ以上のP-gp機能を担持することを特徴とする前項４又は５に記載の多能性幹細胞由来脳血管内皮細胞。
７．前項４又は５に記載の多能性幹細胞由来脳血管内皮細胞を用いる、血液脳関門の解析方法。
８．前項４又は５に記載の多能性幹細胞由来脳血管内皮細胞を用いる、血液脳関門における薬物動態の評価方法。 That is, the present invention consists of the following.
1. A method for producing cerebrovascular endothelial cells from pluripotent stem cells comprising the steps of:
1) a step of introducing the MDR1 gene encoding P-gp into the pluripotent stem cells; and 2) a step of inducing differentiation of the gene-introduced pluripotent stem cells of step 1) into brain-derived cells.
2. The pluripotent stem cells introduced with the MDR1 gene encoding the P-gp of 1) maintain undifferentiated state, while the expression level of P-gp is higher than that of the pluripotent stem cells without the introduction of the MDR1 gene. The method for producing the cerebrovascular endothelial cells according to the preceding
3. 2. The method for producing cerebrovascular endothelial cells according to the preceding
4. Pluripotent stem cell-derived cerebrovascular endothelial cells transfected with the MDR1 gene showed 100 to 2000-fold expression of P-gp compared to pluripotent stem cell-derived cerebral endothelial cells not transfected with the MDR1 gene. A pluripotent stem cell-derived cerebrovascular endothelial cell characterized by high expression in
5. A pluripotent stem cell-derived cerebrovascular endothelial cell produced by the production method according to the preceding
6. 6. The pluripotent stem cell-derived cerebrovascular endothelial cell according to 4 or 5 above, which has a P-gp function equal to or greater than that retained by primary cerebral vascular endothelial cells.
7. 6. A method for analyzing the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to 4 or 5 above.
8. 6. A method for evaluating pharmacokinetics at the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to 4 or 5 above.
本発明の脳血管内皮細胞の作製に使用したP-gpをコードするMDR1遺伝子を導入した多能性幹細胞は、多能性幹細胞が本来有している多分化能を保持していた。さらに、本発明の多能性幹細胞由来脳血管内皮細胞はP-gpを高発現しており、高いバリア機能を有し、かつ優れた薬物排出トランスポーター機能を保持していた。本発明の多能性幹細胞由来脳血管内皮細胞は、ヒト脳血管内皮細胞から樹立された不死化脳血管内皮細胞株では達成されなかった優れた薬物排出トランスポーター機能を有する。さらに本発明の多能性幹細胞由来脳血管内皮細胞は、ヒトのBBBの輸送機能を精度よく反映しており、初代脳血管内皮細胞では、同等の性質を有する脳血管内皮細胞を入手することが困難であったという課題を克服することができた。
The pluripotent stem cells introduced with the P-gp-encoding MDR1 gene used to generate the cerebral vascular endothelial cells of the present invention retained the pluripotent potential inherent in pluripotent stem cells. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention highly expressed P-gp, had a high barrier function, and retained an excellent drug efflux transporter function. The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have an excellent drug efflux transporter function that has not been achieved with immortalized cerebrovascular endothelial cell lines established from human cerebral vascular endothelial cells. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention accurately reflect the transport function of the human BBB, and it is possible to obtain cerebral endothelial cells with the same properties as primary cerebral vascular endothelial cells. I was able to overcome the challenges that were difficult.
本発明は、多能性幹細胞由来の脳血管内皮細胞及びその製造方法に関し、ヒトのBBBの輸送機能を精度よく反映し得る脳血管内皮細胞であって、薬物排出トランスポーターであるP-gpの機能が優れた脳血管内皮細胞を提供することを課題とする。脳血管内皮細胞は一般的に、脳特異的な性質及び／又は機能を有している血管内皮細胞をいう。
The present invention relates to a pluripotent stem cell-derived cerebrovascular endothelial cell and a method for producing the same, which is a cerebral vascular endothelial cell that can accurately reflect the transport function of the human BBB, and which is a drug efflux transporter, P-gp. An object of the present invention is to provide cerebral vascular endothelial cells with excellent functions. Cerebrovascular endothelial cells generally refer to vascular endothelial cells that have brain-specific properties and/or functions.
本明細書においては、組織又は臓器特異性を獲得していない血管内皮細胞を、単に「血管内皮細胞」と称し、「脳血管内皮細胞」とは区別することとする。さらに、本発明の「多能性幹細胞由来脳血管内皮細胞」は、例えばヒト又は哺乳動物等の非ヒト動物から得られた「初代脳血管内皮細胞」やヒト脳血管内皮細胞から樹立された「不死化脳血管内皮細胞株」とは区別して使用される。
In the present specification, vascular endothelial cells that have not acquired tissue or organ specificity are simply referred to as "vascular endothelial cells" to distinguish them from "cerebral vascular endothelial cells". Furthermore, the "pluripotent stem cell-derived cerebrovascular endothelial cells" of the present invention are, for example, "primary cerebral vascular endothelial cells" obtained from non-human animals such as humans or mammals, and " used in distinction from "immortalized cerebrovascular endothelial cell line".
本発明の多能性幹細胞由来脳血管内皮細胞は、薬物排出トランスポーターであるP-gpの機能が高いことを特徴とする。係る性質を有する多能性幹細胞由来脳血管内皮細胞は、まず多能性幹細胞にMDR1（P-gp）を過剰発現させる工程を経た後、脳血管内皮細胞へと分化誘導することを特徴とする。MDR1（P-gp）を過剰発現させた多能性幹細胞を、脳血管内皮細胞へと分化誘導処理しても良いし、MDR1（P-gp）を過剰発現させた多能性幹細胞からまず組織又は臓器特異性を獲得していない血管内皮細胞を収集し、その後脳血管内皮細胞へと分化誘導しても良い。
The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention are characterized by high functions of the drug efflux transporter P-gp. Pluripotent stem cell-derived cerebrovascular endothelial cells having such properties are characterized by first undergoing a step of overexpressing MDR1 (P-gp) in pluripotent stem cells, and then inducing differentiation into cerebrovascular endothelial cells. . Pluripotent stem cells overexpressing MDR1 (P-gp) may be treated to induce differentiation into cerebral vascular endothelial cells. Alternatively, vascular endothelial cells that have not acquired organ-specificity may be collected and then induced to differentiate into cerebral vascular endothelial cells.
（多能性幹細胞）
本発明において、「多能性幹細胞」とは身体を構成する全ての種類の細胞に分化出来る幹細胞であり、iPS細胞（人工多能性幹細胞：induced pluripotent stem cell）やES細胞（Embryonic Stem Cell）等が例示される。本発明における多能性幹細胞は特に好適には、ヒト由来細胞であり特にヒトiPS細胞が挙げられる。「多能性幹細胞」は、本明細書中で用いられる場合、自己複製能と多分化能を共に有する細胞を意味する。「多能性（pluripotency）」とは、「多分化能」と互換可能に使用され、細胞の有する性質をいい、様々な組織や器官に属する細胞に分化し得る能力をいう。 (pluripotent stem cells)
In the present invention, "pluripotent stem cells" are stem cells that can differentiate into all types of cells that make up the body, iPS cells (induced pluripotent stem cells) and ES cells (Embryonic Stem Cells) etc. are exemplified. Pluripotent stem cells in the present invention are particularly preferably human-derived cells, particularly human iPS cells. "Pluripotent stem cell" as used herein means a cell that has both self-renewal and pluripotency. "Pluripotency" is used interchangeably with "pluripotency" and refers to the property possessed by cells, and refers to the ability to differentiate into cells belonging to various tissues and organs.
本発明において、「多能性幹細胞」とは身体を構成する全ての種類の細胞に分化出来る幹細胞であり、iPS細胞（人工多能性幹細胞：induced pluripotent stem cell）やES細胞（Embryonic Stem Cell）等が例示される。本発明における多能性幹細胞は特に好適には、ヒト由来細胞であり特にヒトiPS細胞が挙げられる。「多能性幹細胞」は、本明細書中で用いられる場合、自己複製能と多分化能を共に有する細胞を意味する。「多能性（pluripotency）」とは、「多分化能」と互換可能に使用され、細胞の有する性質をいい、様々な組織や器官に属する細胞に分化し得る能力をいう。 (pluripotent stem cells)
In the present invention, "pluripotent stem cells" are stem cells that can differentiate into all types of cells that make up the body, iPS cells (induced pluripotent stem cells) and ES cells (Embryonic Stem Cells) etc. are exemplified. Pluripotent stem cells in the present invention are particularly preferably human-derived cells, particularly human iPS cells. "Pluripotent stem cell" as used herein means a cell that has both self-renewal and pluripotency. "Pluripotency" is used interchangeably with "pluripotency" and refers to the property possessed by cells, and refers to the ability to differentiate into cells belonging to various tissues and organs.
iPS細胞とは、体細胞へ数種類の遺伝子を導入することにより、卵子、胚やES細胞を利用せずに分化細胞の初期化を誘導し、ES細胞と同様な多能性や増殖能を有する誘導多能性幹細胞をいい、2006年にマウスの線維芽細胞から世界で初めて作られた。さらに、マウスiPS細胞の樹立に用いた４遺伝子のヒト相同遺伝子であるOCT3/4、SOX2、KLF4、C-MYCを、ヒト由来線維芽細胞に導入してヒトiPS細胞の樹立に成功したことが報告されている(Cell 131: 861-872, 2007)。本発明で使用されるiPS細胞は、上記のような自体公知の方法により作製されたiPS細胞、又は今後開発される新たな方法により作製されるiPS細胞であってもよい。
By introducing several types of genes into somatic cells, iPS cells induce the reprogramming of differentiated cells without using eggs, embryos, or ES cells, and have the same pluripotency and proliferative potential as ES cells. It refers to induced pluripotent stem cells, and was created in 2006 for the first time in the world from mouse fibroblasts. Furthermore, the human iPS cells were successfully established by transfecting human-derived fibroblasts with OCT3/4, SOX2, KLF4, and C-MYC, the human homologous genes of the four genes used to establish mouse iPS cells. reported (Cell 131: 861-872, 2007). The iPS cells used in the present invention may be iPS cells produced by a method known per se as described above, or iPS cells produced by a new method that will be developed in the future.
iPS細胞等の多能性幹細胞の培養方法は特に限定されず、自体公知の方法によることができる。iPS細胞又はES細胞等の未分化性及び多能性を維持可能な培地や分化誘導に適した培地として、自体公知の培地、又は今後開発される新たな培地を用いることができる。具体的には、DMEM及び／又はDMEM Ham's F-12（DMEM/F12）、Human Endothelial-SFM等のEC培地、フィーダー細胞不要なmTeSR1（modified Tenneille Serum Replacer 1）培地等を適宜必要に応じて使用することができる。これらの培地に、必要に応じて、血清又は血清代替物、塩基性線維芽細胞成長因子（basic fibroblast growth factor：bFGF（FGF2とも称する））、レチノイン酸及びTGF-β阻害剤、KOSR（knock-out serum replacement）、LIF（白血病阻止因子）やbFGF（塩基性線維芽細胞増殖因子）などを添加することができる。また市販の霊長類ES細胞用培地、霊長類ES細胞増殖用基礎培地hESF-GRO、霊長類ES細胞分化誘導用基礎培地hESF-DIF、霊長類ES細胞増殖用培地CSTI-7等を用いることができる。培地には、iPS細胞等の多能性幹細胞の培養に適する自体公知の添加物、例えば、N2サプリメント、B27サプリメント、インシュリン、bFGF、アクチビン(activin) A、ヘパリン、ROCKインヒビターやGSK-3インヒビターなどの各種インヒビター等から選択される１種又は複数種の添加物を適当な濃度で添加することができる。培地及びその添加物は、使用する細胞、分化状態等により適宜選択し、使用することができる。例えば、Tiss. Cult. Res. Commun., 27: 139-147 (2008) に記載の方法によることができる。
A method for culturing pluripotent stem cells such as iPS cells is not particularly limited, and a method known per se can be used. As a medium capable of maintaining the undifferentiated state and pluripotency of iPS cells or ES cells or a medium suitable for inducing differentiation, a medium known per se or a new medium to be developed in the future can be used. Specifically, EC medium such as DMEM and/or DMEM Ham's F-12 (DMEM/F12), Human Endothelial-SFM, mTeSR1 (modified Tenneille Serum Replacer 1) medium that does not require feeder cells, etc. are used as needed. can do. In these media, if necessary, serum or serum replacement, basic fibroblast growth factor (bFGF (also referred to as FGF2)), retinoic acid and TGF-β inhibitor, KOSR (knock- outserum replacement), LIF (leukemia inhibitory factor), bFGF (basic fibroblast growth factor), etc. can be added. In addition, commercially available primate ES cell medium, primate ES cell proliferation basal medium hESF-GRO, primate ES cell differentiation induction basal medium hESF-DIF, primate ES cell proliferation medium CSTI-7, etc. can be used. can. The medium contains known additives suitable for culturing pluripotent stem cells such as iPS cells, such as N2 supplement, B27 supplement, insulin, bFGF, activin A, heparin, ROCK inhibitor and GSK-3 inhibitor. can be added in an appropriate concentration of one or more additives selected from various inhibitors of The medium and its additives can be appropriately selected and used depending on the cells to be used, the state of differentiation, and the like. For example, the method described in Tiss. Cult. Res. Commun., 27: 139-147 (2008) can be used.
培養工程において、適宜継代培養を行ってもよい。例えばコンフルエント又はサブコンフルエントになった際に細胞の一部を採取して別の培養容器に移し、培養を継続することができる。継代時の細胞数としては特に限定されないが、例えば約1×105個～1×106個/cm2、好ましくは約1×104個～3×104個/cm2とすることができる。培地交換や継代培養などに伴う、細胞の回収の際には、細胞死を抑制するためにY-27632等のROCK阻害剤（Rho-associated coiled-coil forming kinase/Rho結合キナーゼ）で予め細胞を処理しておいてもよい。
In the culture step, subculturing may be performed as appropriate. For example, when the cells become confluent or subconfluent, a part of the cells can be collected and transferred to another culture container to continue the culture. The number of cells at passage is not particularly limited, but is, for example, about 1×10 5 to 1×10 6 /cm 2 , preferably about 1×10 4 to 3×10 4 /cm 2 . can be done. When harvesting cells associated with medium exchange or subculture, pre-treat the cells with a ROCK inhibitor (Rho-associated coiled-coil forming kinase/Rho-associated kinase) such as Y-27632 to suppress cell death. may be processed.
本発明の多能性幹細胞を培養する工程において、浮遊条件ではなく接着させて培養することが好ましい。培養温度などの培養条件は、動物細胞の培養において一般に採用されている条件とすればよい。培養温度は約30～40℃、好ましくは37±0.5℃である。培養時のCO2濃度は約1～10％、好ましくは約5％が適当である。培養担体としては培養皿などを用いて二次元的に細胞を培養することができるし、ゲル状の培養基材や３次元培養プレートなどを用いた３次元培養を行うこともできる。細胞の生存率や増殖率の向上、分化誘導の促進、細胞のセレクション等のため、基底膜成分や接着分子などでコートした培養面上で細胞を培養することもできる。培養面のコートに用いられる材料／成分の例として、マトリゲル、コラーゲンI、コラーゲンIV、フィブロネクチン（Fibronectin）、ラミニン、ゼラチン、ポリリジン、ビトロネクチン等が挙げられる。
In the step of culturing the pluripotent stem cells of the present invention, it is preferable to culture them in an adherent state rather than in suspension. Cultivation conditions such as culture temperature may be conditions generally employed in culturing animal cells. The culture temperature is about 30-40°C, preferably 37±0.5°C. A CO 2 concentration of about 1 to 10%, preferably about 5% is appropriate during culture. Cells can be cultured two-dimensionally using a culture dish or the like as a culture carrier, and three-dimensional culture can also be performed using a gel-like culture substrate, a three-dimensional culture plate, or the like. Cells can also be cultured on a culture surface coated with a basement membrane component, an adhesion molecule, or the like for the purpose of improving cell viability and growth rate, promoting differentiation induction, cell selection, and the like. Examples of materials/components used to coat culture surfaces include matrigel, collagen I, collagen IV, fibronectin, laminin, gelatin, polylysine, vitronectin, and the like.
（多能性幹細胞へのP-gpをコードするMDR1遺伝子の導入）
多能性幹細胞へのMDR1遺伝子の導入は、レンチウイルスベクターやアデノウイルスベクター等のウイルスベクターに目的遺伝子を搭載したベクターを用いて導入することができる。レンチウイルスベクター又はアデノウイルスベクターを用いることで、効果的に目的遺伝子を多能性幹細胞へ導入することができる。使用可能なプロモーターとしては、例えばEF、RSV、pCMV、CA（β-actin promoter/CMV enhancer）等が挙げられる。背景技術の欄にも示したが、薬物排出トランスポーターであるP-gpは、ABCB1又はMDR1とも呼ばれる。本明細書において、P-gpは特にタンパク質を意図し、MDR1遺伝子はP-gpをコードする遺伝子を意図する。 (Introduction of MDR1 gene encoding P-gp into pluripotent stem cells)
Introduction of the MDR1 gene into pluripotent stem cells can be performed using a viral vector such as a lentiviral vector or an adenoviral vector carrying a target gene. A target gene can be effectively introduced into pluripotent stem cells by using a lentiviral vector or an adenoviral vector. Promoters that can be used include, for example, EF, RSV, pCMV, CA (β-actin promoter/CMV enhancer) and the like. As shown in the background art section, the drug efflux transporter P-gp is also called ABCB1 or MDR1. As used herein, P-gp specifically refers to the protein and MDR1 gene refers to the gene encoding P-gp.
多能性幹細胞へのMDR1遺伝子の導入は、レンチウイルスベクターやアデノウイルスベクター等のウイルスベクターに目的遺伝子を搭載したベクターを用いて導入することができる。レンチウイルスベクター又はアデノウイルスベクターを用いることで、効果的に目的遺伝子を多能性幹細胞へ導入することができる。使用可能なプロモーターとしては、例えばEF、RSV、pCMV、CA（β-actin promoter/CMV enhancer）等が挙げられる。背景技術の欄にも示したが、薬物排出トランスポーターであるP-gpは、ABCB1又はMDR1とも呼ばれる。本明細書において、P-gpは特にタンパク質を意図し、MDR1遺伝子はP-gpをコードする遺伝子を意図する。 (Introduction of MDR1 gene encoding P-gp into pluripotent stem cells)
Introduction of the MDR1 gene into pluripotent stem cells can be performed using a viral vector such as a lentiviral vector or an adenoviral vector carrying a target gene. A target gene can be effectively introduced into pluripotent stem cells by using a lentiviral vector or an adenoviral vector. Promoters that can be used include, for example, EF, RSV, pCMV, CA (β-actin promoter/CMV enhancer) and the like. As shown in the background art section, the drug efflux transporter P-gp is also called ABCB1 or MDR1. As used herein, P-gp specifically refers to the protein and MDR1 gene refers to the gene encoding P-gp.
MDR1遺伝子は、GenBank Access No. AF016535で示され、多能性幹細胞へ導入するMDR1遺伝子は、GenBank Access No. AF016535で特定される塩基配列のうち、少なくともタンパク質コード領域の配列（coding sequence：CDS）を含む。
The MDR1 gene is indicated by GenBank Access No. AF016535, and the MDR1 gene to be introduced into pluripotent stem cells is at least the sequence of the protein coding region (coding sequence: CDS) among the nucleotide sequences specified by GenBank Access No. AF016535. including.
上記によりMDR1遺伝子を導入した多能性幹細胞（MDR1遺伝子導入多能性幹細胞）は、P-gpを高発現することを特徴する。当該MDR1遺伝子導入多能性幹細胞は、MDR1遺伝子導入していない多能性幹細胞に比べて少なくとも100倍以上のP-gpを発現しており、発現量は100～2000倍、好ましくは150～1000倍程度であり、最も好ましくは約900倍である。本発明のMDR1遺伝子導入多能性幹細胞は、P-gpを高発現する他は多能性幹細胞が本来有する性質、即ち自己複製能と多分化能を共に維持した細胞である。多分化能とは様々な組織や器官に属する細胞に分化し得る能力をいい、通常の多能性幹細胞と同様にNanogやOct3/4等のマーカーを保持している。
Pluripotent stem cells into which the MDR1 gene has been introduced (MDR1 gene-introduced pluripotent stem cells) are characterized by high expression of P-gp. The MDR1 gene-introduced pluripotent stem cells express at least 100 times more P-gp, and the expression level is 100 to 2000 times, preferably 150 to 1000 times, as compared to pluripotent stem cells that have not been introduced with the MDR1 gene. about two times, most preferably about 900 times. The MDR1 gene-introduced pluripotent stem cells of the present invention are cells that maintain both the inherent properties of pluripotent stem cells, that is, self-renewal ability and pluripotency, in addition to highly expressing P-gp. Pluripotency refers to the ability to differentiate into cells belonging to various tissues and organs, and retains markers such as Nanog and Oct3/4 like normal pluripotent stem cells.
（脳血管内皮細胞への分化誘導）
本発明において、MDR1遺伝子導入多能性幹細胞から脳血管内皮細胞を作製する方法は、自体公知の多能性幹細胞から脳血管内皮細胞を作製する方法を適用することができ、例えば非特許文献３、４に示す方法を参酌することができる。また、MDR1遺伝子導入多能性幹細胞から、まず組織又は臓器特異性を獲得していない血管内皮細胞を収集し、その後脳血管内皮細胞へと分化誘導することもできる。例えば特許文献１の記載を参酌し、まずCD34発現細胞である血管内皮細胞を収集し、その後脳血管内皮細胞へと分化誘導して作製することができる。 (Induction of differentiation into cerebrovascular endothelial cells)
In the present invention, the method for producing cerebral vascular endothelial cells from MDR1 gene-introduced pluripotent stem cells can apply a method for producing cerebral vascular endothelial cells from pluripotent stem cells known per se. , 4 can be referred to. Alternatively, vascular endothelial cells that have not acquired tissue- or organ-specificity can be first collected from MDR1 gene-introduced pluripotent stem cells, and then induced to differentiate into cerebral vascular endothelial cells. For example, referring to the description ofPatent Document 1, vascular endothelial cells, which are CD34-expressing cells, can be first collected and then induced to differentiate into cerebral vascular endothelial cells.
本発明において、MDR1遺伝子導入多能性幹細胞から脳血管内皮細胞を作製する方法は、自体公知の多能性幹細胞から脳血管内皮細胞を作製する方法を適用することができ、例えば非特許文献３、４に示す方法を参酌することができる。また、MDR1遺伝子導入多能性幹細胞から、まず組織又は臓器特異性を獲得していない血管内皮細胞を収集し、その後脳血管内皮細胞へと分化誘導することもできる。例えば特許文献１の記載を参酌し、まずCD34発現細胞である血管内皮細胞を収集し、その後脳血管内皮細胞へと分化誘導して作製することができる。 (Induction of differentiation into cerebrovascular endothelial cells)
In the present invention, the method for producing cerebral vascular endothelial cells from MDR1 gene-introduced pluripotent stem cells can apply a method for producing cerebral vascular endothelial cells from pluripotent stem cells known per se. , 4 can be referred to. Alternatively, vascular endothelial cells that have not acquired tissue- or organ-specificity can be first collected from MDR1 gene-introduced pluripotent stem cells, and then induced to differentiate into cerebral vascular endothelial cells. For example, referring to the description of
本発明の多能性幹細胞由来脳血管内皮細胞は、生体内のBBBを構成する脳血管内皮細胞と同様に、良好な接着性と物質輸送の調節能を有する。接着性と物質輸送の調節能は、自体公知の手法により確認することができるが、例えば後述する実験例に記載の方法により確認することができる。脳血管内皮細胞に特徴的な物質輸送の調節能、接着性は、電気抵抗を測定及びタイトジャンクション関連遺伝子の発現を解析することにより確認することができる。本発明の多能性幹細胞由来脳血管内皮細胞は、特に高いP-gp機能を有する。P-gpによる薬物の排出は薬剤耐性等に関与しており、重要な機能を有する。特に脳毛細血管内皮細胞においP-gpは血液側細胞膜に発現し、脳組織と血液との間の物質の膜透過性を厳密に制限していることが明らかにされている。本発明の多能性幹細胞由来脳血管内皮細胞においても、P-gpは血液側細胞膜に発現しているのが好適である。
The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have good adhesiveness and the ability to regulate substance transport, similar to the cerebral vascular endothelial cells that constitute the BBB in vivo. Adhesiveness and ability to regulate substance transport can be confirmed by a technique known per se, and can be confirmed, for example, by the method described in Experimental Examples described later. The ability to regulate substance transport and adhesiveness characteristic of cerebrovascular endothelial cells can be confirmed by measuring electrical resistance and analyzing the expression of tight junction-related genes. The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have particularly high P-gp function. Drug efflux by P-gp is involved in drug resistance and has an important function. Especially in brain capillary endothelial cells, it has been revealed that P-gp is expressed on the blood side cell membrane and strictly restricts the membrane permeability of substances between brain tissue and blood. Also in the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention, P-gp is preferably expressed on the blood-side cell membrane.
タイトジャンクション関連遺伝子としては、Claudin-5、Occludin、ZO-1が例示される。物質輸送の調節能は、標識されたデキストランを用いた物質透過性解析及び脳血管内皮細胞に特異的に発現するトランスポーターの発現、当該トランスポーターの物質輸送機能を解析することにより確認することができる。当該トランスポーターとしては、例えばP-gp、BCRP（cancer resistance protein）、Glut1（glucose transporter type 1）、MRP4（multidrug resistance protein 4）、MRP1（multidrug resistance protein 1）等が例示される。本発明の多能性幹細胞由来脳血管内皮細胞は、MDR1遺伝子を導入していない多能性幹細胞から分化誘導して作製された脳血管内皮細胞と同程度のBCRP、Glut1、MRP4、MRP1等が発現していればよい。
Examples of tight junction-related genes include Claudin-5, Occludin, and ZO-1. The ability to regulate substance transport can be confirmed by substance permeability analysis using labeled dextran, the expression of transporters that are specifically expressed in cerebrovascular endothelial cells, and the substance transport functions of these transporters. can. Examples of the transporter include P-gp, BCRP (cancer resistance protein), Glut1 (glucose transporter type 1), MRP4 (multidrug resistance protein 4), MRP1 (multidrug resistance protein 1), and the like. The pluripotent stem cell-derived cerebral vascular endothelial cells of the present invention contain BCRP, Glut1, MRP4, MRP1, etc. to the same extent as cerebral vascular endothelial cells produced by differentiation induction from pluripotent stem cells into which the MDR1 gene has not been introduced. It is sufficient if it is expressed.
本発明において多能性幹細胞から脳血管内皮細胞への分化誘導前に血管内皮細胞を収集する工程を含むことができる。血管内皮細胞取得するために、CD34発現細胞を収集することができる。CD34発現細胞は、多能性幹細胞から分化誘導されたものであればいかなるものであってもよい。多能性幹細胞を中胚葉誘導因子や造血因子等の液性因子を含む培地で培養することにより、中胚葉細胞に分化誘導される。中胚葉細胞は、心筋細胞、血管内皮細胞、血管平滑筋細胞、血液細胞などに分化する細胞である。多能性幹細胞又は前記分化誘導処理した中胚葉細胞を液性因子を含む培地で培養し、培養後の細胞集団からCD34発現細胞を単離してもよい。CD34発現細胞を単離して培養することで血管内皮細胞へ効率的に分化誘導することができる。中胚葉細胞又は血管内皮細胞へ分化誘導するための培養工程では、BMP4、アクチビンA、及びVEGF等の液性因子を培養液に含めることができる。また上記液性因子は、ヒト由来であることが好ましい。
The present invention can include a step of collecting vascular endothelial cells before inducing differentiation from pluripotent stem cells to cerebral vascular endothelial cells. CD34-expressing cells can be harvested to obtain vascular endothelial cells. Any CD34-expressing cell may be used as long as it is induced to differentiate from pluripotent stem cells. Differentiation into mesodermal cells is induced by culturing pluripotent stem cells in a medium containing humoral factors such as mesoderm-inducing factors and hematopoietic factors. Mesoderm cells are cells that differentiate into cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, blood cells, and the like. The pluripotent stem cells or mesodermal cells subjected to differentiation induction treatment may be cultured in a medium containing humoral factors, and CD34-expressing cells may be isolated from the cultured cell population. By isolating and culturing CD34-expressing cells, differentiation into vascular endothelial cells can be efficiently induced. In the culture step for inducing differentiation into mesoderm cells or vascular endothelial cells, humoral factors such as BMP4, activin A, and VEGF can be included in the culture solution. Moreover, the humoral factor is preferably derived from humans.
血管内皮細胞マーカーとしては、CD34、CD31、von Willebrand Factor（vWF）、VE-cadherinが挙げられる。本発明の多能性幹細胞由来脳血管内皮細胞は、CD34発現細胞を収集する工程を含むか否かに係わらず、MDR1（P-gp）遺伝子を導入していない多能性幹細胞から分化誘導して作製された脳血管内皮細胞と同程度のCD31、vWF、VE-cadherin等が発現していればよい。
Vascular endothelial cell markers include CD34, CD31, von Willebrand Factor (vWF), and VE-cadherin. The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention are induced to differentiate from pluripotent stem cells into which the MDR1 (P-gp) gene has not been introduced, regardless of whether the step of collecting CD34-expressing cells is included. It is sufficient that CD31, vWF, VE-cadherin, etc. are expressed at the same level as the cerebral vascular endothelial cells produced by the method described above.
本発明において脳血管内皮細胞への分化誘導に使用可能な液性因子として、例えば骨形成タンパク質4（bone morphogenetic protein4：BMP4）、アクチビンA、及びFGF2などの中胚葉誘導因子、血管内皮細胞増殖因子（vascular endothelial growth factor：VEGF）、トロンボポイエチン（thrombopoietin：TPO）などの血小板増殖因子、幹細胞因子（stem cell factor：SCF）、 FMS様チロシンキナーゼ3リガンド（FMS-like tyrosinekinase 3 Ligand：Flt3L）、インターロイキン-6（IL-6）／可溶性IL-6受容体複合体（IL-6/sIL-6R）、及びノッチリガンド（以下、Notchリガンドと称する）などの造血因子等が挙げられる。液性因子は、単独で使用してもよいし、複数を同時に使用してもよい。本発明の脳血管内皮細胞で構成された細胞層に加え、他の細胞（ペリサイト、アストロサイト等）を併用してもよい。
Humoral factors that can be used in the present invention to induce differentiation into brain vascular endothelial cells include, for example, bone morphogenetic protein 4 (BMP4), activin A, mesoderm-inducing factors such as FGF2, and vascular endothelial cell growth factors (vascular endothelial growth factor: VEGF), platelet growth factors such as thrombopoietin (TPO), stem cell factor (SCF), FMS-like tyrosinekinase 3 Ligand (Flt3L), Examples include hematopoietic factors such as interleukin-6 (IL-6)/soluble IL-6 receptor complex (IL-6/sIL-6R) and Notch ligand (hereinafter referred to as Notch ligand). A single humoral factor may be used, or a plurality thereof may be used at the same time. In addition to the cell layer composed of the cerebrovascular endothelial cells of the present invention, other cells (pericytes, astrocytes, etc.) may be used in combination.
本発明の多能性幹細胞由来脳血管内皮細胞は、 P-gpの発現が初代培養ヒト脳血管内皮細胞と同程度にまで上昇したことが確認された。当該多能性幹細胞由来脳血管内皮細胞は、MDR1遺伝子を導入していない多能性幹細胞由来脳血管内皮細胞に比べて、少なくとも100倍以上のP-gpを発現しており、P-gpの発現量は100～2000倍、好ましくは150～1000倍程度であり、最も好ましくは約900倍である。当該多能性幹細胞由来脳血管内皮細胞は、P-gp以外のトランスポータータンパク質の発現はMDR1遺伝子導入していない多能性幹細胞由来脳血管内皮細胞と同程度である。
It was confirmed that the pluripotent stem cell-derived cerebral endothelial cells of the present invention increased the expression of P-gp to the same level as primary cultured human cerebral endothelial cells. The pluripotent stem cell-derived cerebrovascular endothelial cells express at least 100 times more P-gp than pluripotent stem cell-derived cerebral endothelial cells into which the MDR1 gene has not been introduced. The expression level is 100- to 2000-fold, preferably about 150- to 1000-fold, most preferably about 900-fold. The expression of transporter proteins other than P-gp in the pluripotent stem cell-derived cerebrovascular endothelial cells is comparable to that of pluripotent stem cell-derived cerebral endothelial cells into which the MDR1 gene has not been introduced.
上記により、本発明の多能性幹細胞由来脳血管内皮細胞は血液脳関門の解析に使用することができる。さらに、本発明の多能性幹細胞由来脳血管内皮細胞は、血液脳関門における薬物動態の評価にも使用することができる。本発明は、多能性幹細胞由来脳血管内皮細胞を用いる血液脳関門の解析方法や血液脳関門における薬物動態の評価方法にも及ぶ。
According to the above, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention can be used for analysis of the blood-brain barrier. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention can also be used to evaluate pharmacokinetics at the blood-brain barrier. The present invention also extends to a method for analyzing the blood-brain barrier using pluripotent stem cell-derived cerebrovascular endothelial cells and a method for evaluating pharmacokinetics in the blood-brain barrier.
本発明の理解を深めるために、実施例及び実験例を示して本発明を具体的に説明するが、本発明はこれらに限定されるものではないことは、いうまでもない。
In order to deepen the understanding of the present invention, the present invention will be specifically described by showing Examples and Experimental Examples, but it goes without saying that the present invention is not limited to these.
（実施例１）MDR1導入多能性幹細胞の樹立
本実施例では、ヒトiPS細胞（iMR90-4細胞：WiCellより購入）にMDR1遺伝子を組み込んだMDR1-iPS細胞の樹立について説明する。 (Example 1) Establishment of MDR1-introduced pluripotent stem cells This example describes the establishment of MDR1-iPS cells in which the MDR1 gene was incorporated into human iPS cells (iMR90-4 cells: purchased from WiCell).
本実施例では、ヒトiPS細胞（iMR90-4細胞：WiCellより購入）にMDR1遺伝子を組み込んだMDR1-iPS細胞の樹立について説明する。 (Example 1) Establishment of MDR1-introduced pluripotent stem cells This example describes the establishment of MDR1-iPS cells in which the MDR1 gene was incorporated into human iPS cells (iMR90-4 cells: purchased from WiCell).
（１）クローニング及びレンチウイルスベクタープラスミドの作製
MDR1遺伝子に関し、GenBank Access No. AF016535で特定される塩基配列のうち、CDS部分をレンチウイルスベクタープラスミドに導入した。レンチウイルスベクタープラスミドとしてCSII-EF-MCS-IRES-Venus（理研より入手）を使用した。上記単離したMDR1遺伝子をCSII-EF-MCS-IRES-VenusのNotI/BamHIサイトに導入し、EFプロモーター下でのMDR1遺伝子及びVenus（蛍光タンパク質）を発現するレンチウイルスベクター（CSII-EF-MDR1-IRES-Venus）を作製した。コントロールベクタープラスミドはCSII-EF-MCS-IRES-Venusとした（図１）。 (1) Cloning and Preparation of Lentiviral Vector Plasmid Regarding the MDR1 gene, the CDS portion of the nucleotide sequence specified by GenBank Access No. AF016535 was introduced into a lentiviral vector plasmid. CSII-EF-MCS-IRES-Venus (obtained from RIKEN) was used as a lentiviral vector plasmid. The MDR1 gene isolated above is introduced into the NotI/BamHI site of CSII-EF-MCS-IRES-Venus, and a lentiviral vector expressing the MDR1 gene and Venus (fluorescent protein) under the EF promoter (CSII-EF-MDR1 -IRES-Venus). The control vector plasmid was CSII-EF-MCS-IRES-Venus (Fig. 1).
MDR1遺伝子に関し、GenBank Access No. AF016535で特定される塩基配列のうち、CDS部分をレンチウイルスベクタープラスミドに導入した。レンチウイルスベクタープラスミドとしてCSII-EF-MCS-IRES-Venus（理研より入手）を使用した。上記単離したMDR1遺伝子をCSII-EF-MCS-IRES-VenusのNotI/BamHIサイトに導入し、EFプロモーター下でのMDR1遺伝子及びVenus（蛍光タンパク質）を発現するレンチウイルスベクター（CSII-EF-MDR1-IRES-Venus）を作製した。コントロールベクタープラスミドはCSII-EF-MCS-IRES-Venusとした（図１）。 (1) Cloning and Preparation of Lentiviral Vector Plasmid Regarding the MDR1 gene, the CDS portion of the nucleotide sequence specified by GenBank Access No. AF016535 was introduced into a lentiviral vector plasmid. CSII-EF-MCS-IRES-Venus (obtained from RIKEN) was used as a lentiviral vector plasmid. The MDR1 gene isolated above is introduced into the NotI/BamHI site of CSII-EF-MCS-IRES-Venus, and a lentiviral vector expressing the MDR1 gene and Venus (fluorescent protein) under the EF promoter (CSII-EF-MDR1 -IRES-Venus). The control vector plasmid was CSII-EF-MCS-IRES-Venus (Fig. 1).
（２）レンチウイルス溶液の調製
上記により各遺伝子を搭載したレンチウイルスベクター（CSII-EF-MDR1-IRES-Venus又はCSII-EF-MCS-IRES-Venus）と、pCMV-VSVG-RSV-REV及びpCAG-HIVgpをヒト培養細胞HEK293T細胞にLipofectamine(R) 2000（ThermoFisher Scientific）を用いて常法に従いトランスフェクトした。翌日、フォルスコリン（Forskolin）含有培地で培地交換を行い更に48時間培養した。その後培養上清を回収し、50,000×gで2時間遠心処理し、濃縮したウイルス溶液を調製した。 (2) Preparation of lentiviral solution Lentiviral vector (CSII-EF-MDR1-IRES-Venus or CSII-EF-MCS-IRES-Venus) loaded with each gene as described above, pCMV-VSVG-RSV-REV and pCAG -HIVgp was transfected into cultured human HEK293T cells using Lipofectamine (R) 2000 (ThermoFisher Scientific) according to a standard method. The next day, the medium was replaced with a medium containing Forskolin, and the cells were further cultured for 48 hours. After that, the culture supernatant was collected and centrifuged at 50,000×g for 2 hours to prepare a concentrated virus solution.
上記により各遺伝子を搭載したレンチウイルスベクター（CSII-EF-MDR1-IRES-Venus又はCSII-EF-MCS-IRES-Venus）と、pCMV-VSVG-RSV-REV及びpCAG-HIVgpをヒト培養細胞HEK293T細胞にLipofectamine(R) 2000（ThermoFisher Scientific）を用いて常法に従いトランスフェクトした。翌日、フォルスコリン（Forskolin）含有培地で培地交換を行い更に48時間培養した。その後培養上清を回収し、50,000×gで2時間遠心処理し、濃縮したウイルス溶液を調製した。 (2) Preparation of lentiviral solution Lentiviral vector (CSII-EF-MDR1-IRES-Venus or CSII-EF-MCS-IRES-Venus) loaded with each gene as described above, pCMV-VSVG-RSV-REV and pCAG -HIVgp was transfected into cultured human HEK293T cells using Lipofectamine (R) 2000 (ThermoFisher Scientific) according to a standard method. The next day, the medium was replaced with a medium containing Forskolin, and the cells were further cultured for 48 hours. After that, the culture supernatant was collected and centrifuged at 50,000×g for 2 hours to prepare a concentrated virus solution.
（３）MDR1遺伝子導入iPS細胞（MDR1-iPS細胞）の樹立
ヒトiPS細胞（iMR90-4細胞、WiCellより購入）をマトリゲル基底膜マトリックス（Growth Factor Reduced Matrigel(R) 、Corning社製）上でmTeSR1-cGMP（modified Tenneille Serum Replacer 1-cGMP）培地を用いて培養した。上記調製したウイルス溶液を24時間作用させ3日～4日間培養を行った。その後セルソーター（SH800、SONY社製）を用いてVenus陽性細胞（MDR1発現細胞）のみを単離し、再度マトリゲル基底膜マトリックス上に細胞を播種後、MDR1-iPS細胞株のモノクローン化を行った、コントロールとしてMCS遺伝子導入iPS細胞（MCS-iPS細胞）を同手法で樹立し、モノクローン化を行った。得られた細胞において、Venusが発現していることを再度確認した（図２Ａ）。MDR1-iPS細胞、MCS-iPS細胞及び遺伝子導入していないiPS細胞は各々mTeSR1-cGMP培地で培養した。MDR1-iPS細胞、MCS-iPS細胞及び、遺伝子導入していないiPS細胞は形態的な違いは観察されなかった（図２Ｂ）。 (3) Establishment of MDR1 gene-introduced iPS cells (MDR1-iPS cells) Human iPS cells (iMR90-4 cells, purchased from WiCell) were placed on Matrigel basement membrane matrix (Growth Factor Reduced Matrigel (R) , manufactured by Corning) and mTeSR1. It was cultured using -cGMP (modified Tenneille Serum Replacer 1-cGMP) medium. The virus solution prepared above was allowed to act for 24 hours and cultured for 3 to 4 days. After that, using a cell sorter (SH800, manufactured by SONY), only Venus-positive cells (MDR1-expressing cells) were isolated, and after seeding the cells again on Matrigel basement membrane matrix, the MDR1-iPS cell line was monocloned. As a control, MCS gene-introduced iPS cells (MCS-iPS cells) were established by the same method and monocloned. Expression of Venus was confirmed again in the obtained cells (Fig. 2A). MDR1-iPS cells, MCS-iPS cells and non-transfected iPS cells were each cultured in mTeSR1-cGMP medium. No morphological difference was observed between MDR1-iPS cells, MCS-iPS cells, and iPS cells not transfected (Fig. 2B).
ヒトiPS細胞（iMR90-4細胞、WiCellより購入）をマトリゲル基底膜マトリックス（Growth Factor Reduced Matrigel(R) 、Corning社製）上でmTeSR1-cGMP（modified Tenneille Serum Replacer 1-cGMP）培地を用いて培養した。上記調製したウイルス溶液を24時間作用させ3日～4日間培養を行った。その後セルソーター（SH800、SONY社製）を用いてVenus陽性細胞（MDR1発現細胞）のみを単離し、再度マトリゲル基底膜マトリックス上に細胞を播種後、MDR1-iPS細胞株のモノクローン化を行った、コントロールとしてMCS遺伝子導入iPS細胞（MCS-iPS細胞）を同手法で樹立し、モノクローン化を行った。得られた細胞において、Venusが発現していることを再度確認した（図２Ａ）。MDR1-iPS細胞、MCS-iPS細胞及び遺伝子導入していないiPS細胞は各々mTeSR1-cGMP培地で培養した。MDR1-iPS細胞、MCS-iPS細胞及び、遺伝子導入していないiPS細胞は形態的な違いは観察されなかった（図２Ｂ）。 (3) Establishment of MDR1 gene-introduced iPS cells (MDR1-iPS cells) Human iPS cells (iMR90-4 cells, purchased from WiCell) were placed on Matrigel basement membrane matrix (Growth Factor Reduced Matrigel (R) , manufactured by Corning) and mTeSR1. It was cultured using -cGMP (modified Tenneille Serum Replacer 1-cGMP) medium. The virus solution prepared above was allowed to act for 24 hours and cultured for 3 to 4 days. After that, using a cell sorter (SH800, manufactured by SONY), only Venus-positive cells (MDR1-expressing cells) were isolated, and after seeding the cells again on Matrigel basement membrane matrix, the MDR1-iPS cell line was monocloned. As a control, MCS gene-introduced iPS cells (MCS-iPS cells) were established by the same method and monocloned. Expression of Venus was confirmed again in the obtained cells (Fig. 2A). MDR1-iPS cells, MCS-iPS cells and non-transfected iPS cells were each cultured in mTeSR1-cGMP medium. No morphological difference was observed between MDR1-iPS cells, MCS-iPS cells, and iPS cells not transfected (Fig. 2B).
（実験例１）MDR1-iPS細胞及びMCS-iPS細胞の評価
実施例１で樹立したMDR1-iPS細胞及びMCS-iPS細胞について、P-gp及び未分化マーカー（Nanog、Oct3/4）のmRNAを各々定量的PCR法にて確認し、未分化状態を評価した。定量的PCRによる発現量の測定は以下の手順で行った。 (Experimental Example 1) Evaluation of MDR1-iPS cells and MCS-iPS cells For the MDR1-iPS cells and MCS-iPS cells established in Example 1, mRNA of P-gp and undifferentiated markers (Nanog, Oct3/4) Each was confirmed by a quantitative PCR method, and the undifferentiated state was evaluated. The expression level was measured by quantitative PCR as follows.
実施例１で樹立したMDR1-iPS細胞及びMCS-iPS細胞について、P-gp及び未分化マーカー（Nanog、Oct3/4）のmRNAを各々定量的PCR法にて確認し、未分化状態を評価した。定量的PCRによる発現量の測定は以下の手順で行った。 (Experimental Example 1) Evaluation of MDR1-iPS cells and MCS-iPS cells For the MDR1-iPS cells and MCS-iPS cells established in Example 1, mRNA of P-gp and undifferentiated markers (Nanog, Oct3/4) Each was confirmed by a quantitative PCR method, and the undifferentiated state was evaluated. The expression level was measured by quantitative PCR as follows.
フィブロネクチン及びIV型コラーゲン上で培養したMDR1-iPS細胞及びMCS-iPS細胞、並びに遺伝子導入していないiPS細胞について、RNAiso Plus（TaKaRa）を用いてTotal RNAを抽出した。各細胞から抽出したTotal RNAをRNase（New England Biolabs）で処理し、SuperScript IV VILO Master Mix（ThermoFisher Scientific）を用いて逆転写処理し、cDNAを合成した。Fast S(R) Green Master Mix（ThermoFisher Scientific）にて増幅し、StepOnePlusリアルタイムPCRシステム（ThermoFisher Scientific）でMDR1（P-gp）、Nanog及びOct3/4各遺伝子のmRNA発現量を定量した。内部標準遺伝子はGAPDHを用いた。
Total RNA was extracted using RNAiso Plus (TaKaRa) from MDR1-iPS cells and MCS-iPS cells cultured on fibronectin and type IV collagen, and iPS cells not transfected with a gene. Total RNA extracted from each cell was treated with RNase (New England Biolabs) and reverse-transcribed using SuperScript IV VILO Master Mix (ThermoFisher Scientific) to synthesize cDNA. After amplification with Fast S (R) Green Master Mix (ThermoFisher Scientific), mRNA expression levels of MDR1 (P-gp), Nanog and Oct3/4 genes were quantified using the StepOnePlus real-time PCR system (ThermoFisher Scientific). GAPDH was used as an internal standard gene.
各遺伝子及び内部標準遺伝子増幅用のプライマーは以下を用いた。
・MDR1遺伝子増幅用プライマー
Forward CCATGCTCAGACAGGATGTG（配列番号１）
Reverse ACAGCAAGCCTGGAACCTAT（配列番号２）
・Nanog遺伝子増幅用プライマー
Forward AGAAGGCCTCAGCACCTAC（配列番号３）
Reverse GGCCTGATTGTTCCAGGATT（配列番号４）
・Oct3/4遺伝子増幅用プライマー
Forward CTTGAATCCCGAATGGAAAGGG（配列番号５）
Reverse GTGTATATCCCAGGGTGATCCTC（配列番号６）
・GAPDH遺伝子増幅用プライマー
Forward GGTGGTCTCCTCTGACTTCAACGT（配列番号７）
Reverse GTGGTCGTTGAGGGCAATG（配列番号８） The following primers were used for amplification of each gene and internal standard gene.
・MDR1 gene amplification primer
Forward CCATGCTCAGACAGGATGTG (sequence number 1)
Reverse ACAGCAAGCCTGGAACCTAT (SEQ ID NO: 2)
・Nanog gene amplification primer
Forward AGAAGGCCTCAGCACCTAC (SEQ ID NO: 3)
Reverse GGCCTGATTGTTCCAGGATT (SEQ ID NO: 4)
・Oct3/4 gene amplification primers
Forward CTTGAATCCCGAATGGAAAGGG (SEQ ID NO: 5)
Reverse GTGTATATCCCAGGGTGATCCTC (SEQ ID NO: 6)
・Primer for GAPDH gene amplification
Forward GGTGGTCTCCTCTGACTTCAACGT (sequence number 7)
Reverse GTGGTCGTTGAGGGCAATG (SEQ ID NO: 8)
・MDR1遺伝子増幅用プライマー
Forward CCATGCTCAGACAGGATGTG（配列番号１）
Reverse ACAGCAAGCCTGGAACCTAT（配列番号２）
・Nanog遺伝子増幅用プライマー
Forward AGAAGGCCTCAGCACCTAC（配列番号３）
Reverse GGCCTGATTGTTCCAGGATT（配列番号４）
・Oct3/4遺伝子増幅用プライマー
Forward CTTGAATCCCGAATGGAAAGGG（配列番号５）
Reverse GTGTATATCCCAGGGTGATCCTC（配列番号６）
・GAPDH遺伝子増幅用プライマー
Forward GGTGGTCTCCTCTGACTTCAACGT（配列番号７）
Reverse GTGGTCGTTGAGGGCAATG（配列番号８） The following primers were used for amplification of each gene and internal standard gene.
・MDR1 gene amplification primer
Forward CCATGCTCAGACAGGATGTG (sequence number 1)
Reverse ACAGCAAGCCTGGAACCTAT (SEQ ID NO: 2)
・Nanog gene amplification primer
Forward AGAAGGCCTCAGCACCTAC (SEQ ID NO: 3)
Reverse GGCCTGATTGTTCCAGGATT (SEQ ID NO: 4)
・Oct3/4 gene amplification primers
Forward CTTGAATCCCGAATGGAAAGGG (SEQ ID NO: 5)
Reverse GTGTATATCCCAGGGTGATCCTC (SEQ ID NO: 6)
・Primer for GAPDH gene amplification
Forward GGTGGTCTCCTCTGACTTCAACGT (sequence number 7)
Reverse GTGGTCGTTGAGGGCAATG (SEQ ID NO: 8)
上記の結果、MCS-iPS細胞と比較してMDR1-iPS細胞ではP-gpが高発現していることが確認された（図３Ａ）。さらに未分化マーカーであるNanog及びOct3/4の発現について解析したところ、MDR1-iPS細胞及びMCS-iPS細胞のいずれも未分化マーカーの発現は維持されていた（図３Ｂ）。以上の結果より、作製したMDR1-iPS細胞は未分化性を維持しながら、P-gpを高発現することが示された。
The above results confirmed that P-gp was highly expressed in MDR1-iPS cells compared to MCS-iPS cells (Fig. 3A). Furthermore, when the expression of undifferentiation markers Nanog and Oct3/4 was analyzed, the expression of undifferentiation markers was maintained in both MDR1-iPS cells and MCS-iPS cells (Fig. 3B). The above results showed that the prepared MDR1-iPS cells highly expressed P-gp while maintaining undifferentiation.
（実施例２）多能性幹細胞から脳血管内皮細胞への分化誘導
本実施例では、実施例１で樹立したMDR1-iPS細胞又はMCS-iPS細胞から脳血管内皮細胞への分化誘導について説明する。ヒトiPS細胞から脳血管内皮細胞への分化誘導は、Lippmannらの報告（非特許文献３及び４）に基づいて行った。 (Example 2) Differentiation induction from pluripotent stem cells to cerebral vascular endothelial cells In this example, induction of differentiation from MDR1-iPS cells or MCS-iPS cells established in Example 1 to cerebral vascular endothelial cells will be described. . Human iPS cells were induced to differentiate into cerebrovascular endothelial cells based on reports by Lippmann et al. (Non-Patent Documents 3 and 4).
本実施例では、実施例１で樹立したMDR1-iPS細胞又はMCS-iPS細胞から脳血管内皮細胞への分化誘導について説明する。ヒトiPS細胞から脳血管内皮細胞への分化誘導は、Lippmannらの報告（非特許文献３及び４）に基づいて行った。 (Example 2) Differentiation induction from pluripotent stem cells to cerebral vascular endothelial cells In this example, induction of differentiation from MDR1-iPS cells or MCS-iPS cells established in Example 1 to cerebral vascular endothelial cells will be described. . Human iPS cells were induced to differentiate into cerebrovascular endothelial cells based on reports by Lippmann et al. (Non-Patent Documents 3 and 4).
実施例１で樹立したMDR1-iPS細胞及MCS-iPS細胞について、分離・分散用試薬（ESGRO Complete Accutase、Merck-Millipor社製）で処理し、各々単細胞とした。その後ROCK（Rho結合キナーゼ）阻害剤であるY-27632（Wako社製）含有mTeSR1-cGMP培地を用い、0.8～1.2×105cells/wellに調整した各iPS細胞を、Growth Factor Reduced Matrigel(R)をコートした6ウェルプレート上に播種した。播種24時間後に、mTeSR1-cGMP培地を用いて培地交換を行い、2日間培養した。次にUnconditioned Medium（DMEM/F-12、20％KOSR（KnockOutTM Serum Replacement、ThermoFisher Scientific）、1％Non-essential amino acid（ナカライ）、0.5％GlutaMAX(TM)（ThermoFisher Scientific）、0.0007％2-メルカプトエタノール）を用いて6日間培養した。その後、内皮細胞培養用無血清培地（ESFM、human endotherial SFM、ThermoFisher Scientific）、1％ human platelet-derived serum（Sigma）に10μM all-trans-レチノイン酸（Wako）及び20 ng/mL human bFGF（片山化学）を添加した培地で2日間培養した。分離・分散用試薬で処理した各細胞をフィブロネクチン及びIV型コラーゲン（Pharmco-Cell）コートした24ウェルインサートに3.3×105cells/insert又はフィブロネクチン及びIV型コラーゲン（Pharmco-Cell）コートした24ウェルプレートに2.5×105cells/wellで培養し、さらに2日間培養し、MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞を各々作製した。
The MDR1-iPS cells and MCS-iPS cells established in Example 1 were treated with a separation/dispersion reagent (ESGRO Complete Accutase, manufactured by Merck-Millipor) to obtain single cells. After that, using mTeSR1-cGMP medium containing Y-27632 (manufactured by Wako), a ROCK (Rho-binding kinase) inhibitor, each iPS cell adjusted to 0.8-1.2 × 10 5 cells/well, Growth Factor Reduced Matrigel (R ) were plated on 6-well plates coated with 24 hours after seeding, the medium was replaced with mTeSR1-cGMP medium and cultured for 2 days. Unconditioned Medium (DMEM/F-12, 20% KOSR (KnockOut TM Serum Replacement, ThermoFisher Scientific), 1% Non-essential amino acid (Nacalai), 0.5% GlutaMAX (TM) (ThermoFisher Scientific), 0.0007% 2- mercaptoethanol) and cultured for 6 days. After that, serum-free medium for endothelial cell culture (ESFM, human endotherial SFM, ThermoFisher Scientific), 1% human platelet-derived serum (Sigma), 10 μM all-trans-retinoic acid (Wako) and 20 ng/mL human bFGF (Katayama chemical) was added for 2 days. 3.3×10 5 cells/insert of each cell treated with a reagent for separation/dispersion into a 24-well insert coated with fibronectin and type IV collagen (Pharmco-Cell) or a 24-well plate coated with fibronectin and type IV collagen (Pharmco-Cell) After culturing at 2.5×10 5 cells/well for 2 days, MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells were prepared.
（実験例２－１）iPS細胞由来脳血管内皮細胞の形態学的及び血管内皮細胞マーカーの評価
本実験例では、実施例２で作製したMDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について、形態及び血管内皮細胞マーカーについて評価した。 (Experimental Example 2-1) Morphological evaluation of iPS cell-derived cerebral endothelial cells and evaluation of vascular endothelial cell markers In this experimental example, the MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cells prepared in Example 2 were used. The derived brain endothelial cells were evaluated for morphology and endothelial cell markers.
本実験例では、実施例２で作製したMDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について、形態及び血管内皮細胞マーカーについて評価した。 (Experimental Example 2-1) Morphological evaluation of iPS cell-derived cerebral endothelial cells and evaluation of vascular endothelial cell markers In this experimental example, the MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cells prepared in Example 2 were used. The derived brain endothelial cells were evaluated for morphology and endothelial cell markers.
（１）形態
実施例２で作製したMDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について各々形態的な違いは観察されなかった（図４）。 (1) Morphology No morphological difference was observed between the MDR1-iPS cell-derived cerebral endothelial cells and the MCS-iPS cell-derived cerebral endothelial cells produced in Example 2 (Fig. 4).
実施例２で作製したMDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について各々形態的な違いは観察されなかった（図４）。 (1) Morphology No morphological difference was observed between the MDR1-iPS cell-derived cerebral endothelial cells and the MCS-iPS cell-derived cerebral endothelial cells produced in Example 2 (Fig. 4).
（２）血管内皮細胞マーカー
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について血管内皮細胞マーカー（PECAM1、VE-cadherin）のmRNA発現を各々定量的PCR法にて確認した。内部標準遺伝子としてGAPDHを用いた。定量的PCRによるmRNA発現量の測定は、実験例１と同手法により行った。 (2) Vascular endothelial cell marker mRNA expression of vascular endothelial cell markers (PECAM1, VE-cadherin) for MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed by quantitative PCR method. bottom. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed in the same manner as in Experimental Example 1.
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について血管内皮細胞マーカー（PECAM1、VE-cadherin）のmRNA発現を各々定量的PCR法にて確認した。内部標準遺伝子としてGAPDHを用いた。定量的PCRによるmRNA発現量の測定は、実験例１と同手法により行った。 (2) Vascular endothelial cell marker mRNA expression of vascular endothelial cell markers (PECAM1, VE-cadherin) for MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed by quantitative PCR method. bottom. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed in the same manner as in Experimental Example 1.
各遺伝子増幅用のプライマーは以下を用いた。
・PECAM1遺伝子増幅用プライマー
Forward GAGTATTACTGCACAGCCTTCA（配列番号９）
Reverse AACCACTGCAATAAGTCCTTTC（配列番号１０）
・VE-cadherin遺伝子増幅用プライマー
Forward TCACGATAACACGGCCAACA（配列番号１１）
Reverse TGGCATCCCATTGTCTGAGA（配列番号１２） The following primers were used for each gene amplification.
・PECAM1 gene amplification primer
Forward GAGTATTACTGCACAGCCTTCA (sequence number 9)
Reverse AACCACTGCAATAAGTCCTTTC (SEQ ID NO: 10)
・Primer for VE-cadherin gene amplification
Forward TCACGATAACACGGCCAACA (SEQ ID NO: 11)
Reverse TGGCATCCCATTGTCTGAGA (SEQ ID NO: 12)
・PECAM1遺伝子増幅用プライマー
Forward GAGTATTACTGCACAGCCTTCA（配列番号９）
Reverse AACCACTGCAATAAGTCCTTTC（配列番号１０）
・VE-cadherin遺伝子増幅用プライマー
Forward TCACGATAACACGGCCAACA（配列番号１１）
Reverse TGGCATCCCATTGTCTGAGA（配列番号１２） The following primers were used for each gene amplification.
・PECAM1 gene amplification primer
Forward GAGTATTACTGCACAGCCTTCA (sequence number 9)
Reverse AACCACTGCAATAAGTCCTTTC (SEQ ID NO: 10)
・Primer for VE-cadherin gene amplification
Forward TCACGATAACACGGCCAACA (SEQ ID NO: 11)
Reverse TGGCATCCCATTGTCTGAGA (SEQ ID NO: 12)
上記の結果、MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について血管内皮細胞マーカー（PECAM1、VE-cadherin）の発現に関して有意な差は観察されなかった（図５）。
As a result, no significant difference in the expression of vascular endothelial cell markers (PECAM1, VE-cadherin) was observed between MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells (Fig. 5). .
（実験例２－２）iPS細胞由来脳血管内皮細胞のバリア機能評価
本実験例では、実施例２で作製したMDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のバリア機能について評価した。 (Experimental Example 2-2) Evaluation of barrier function of iPS cell-derived cerebral vascular endothelial cells In this experimental example, the barrier function of MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2 was evaluated. evaluated for functionality.
本実験例では、実施例２で作製したMDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のバリア機能について評価した。 (Experimental Example 2-2) Evaluation of barrier function of iPS cell-derived cerebral vascular endothelial cells In this experimental example, the barrier function of MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells prepared in Example 2 was evaluated. evaluated for functionality.
（１）膜間電気抵抗値
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について膜間電気抵抗値（TEER）を測定した。膜間電気抵抗値はMillicell ERS-2（Merk-Millipore社製）を用いて測定した。測定方法は、Millicell ERS-2の取扱い説明書に記載の方法に従って行った。TEERは以下の計算式に従い算出した。
(1) Transmembrane electrical resistance value Transmembrane electrical resistance value (TEER) was measured for MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells. The intermembrane electrical resistance value was measured using Millicell ERS-2 (manufactured by Merck-Millipore). The measurement method was performed according to the method described in the instruction manual for Millicell ERS-2. TEER was calculated according to the following formula.
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について膜間電気抵抗値（TEER）を測定した。膜間電気抵抗値はMillicell ERS-2（Merk-Millipore社製）を用いて測定した。測定方法は、Millicell ERS-2の取扱い説明書に記載の方法に従って行った。TEERは以下の計算式に従い算出した。
膜間電気抵抗値は、MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞共に高値を示した（図６Ａ）。
Both the MDR1-iPS cell-derived cerebral vascular endothelial cells and the MCS-iPS cell-derived cerebral vascular endothelial cells showed high transmembrane electrical resistance values (Fig. 6A).
（２）NaF（sodium fluorescein）を用いた物質透過性の評価
NaFを用いた物質透過性評価を行った。HEPES含有Dulbecco's Phosphate Buffered Saline（DPBS）でNaFを10μg/mLに調製した。インサート内の培地を10μg/mlのNaF溶液に置換し、プレート側の培地をNaF不含のDPBSにて置換した。30分培養後、プレート側の培地を攪拌した後に回収し、インサートからプレート側へ移行したNaF量を蛍光強度計GENIOS （Spectra FLUOR Plus（TECAN製）を用いて測定した（励起波長：485 nm、蛍光波長：535 nm）。NaF溶液の段階希釈により検量線を作成して濃度を算出した。そして、得られた濃度を用いて透過係数（apparent permeability coefficient：Papp）（10-6cm/s）を以下の計算式に従い算出した。MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞共に低値を示し、有意な差は観察されなかった（図６Ｂ）。
(2) Evaluation of material permeability using NaF (sodium fluorescein) Material permeability evaluation using NaF was performed. NaF was adjusted to 10 μg/mL with Dulbecco's Phosphate Buffered Saline (DPBS) containing HEPES. The medium in the insert was replaced with a 10 μg/ml NaF solution, and the medium on the plate side was replaced with NaF-free DPBS. After culturing for 30 minutes, the medium on the plate side was stirred and then collected, and the amount of NaF transferred from the insert to the plate side was measured using a fluorescence intensity meter GENIOS (Spectra FLUOR Plus (manufactured by TECAN) (excitation wavelength: 485 nm, Fluorescence wavelength: 535 nm).The concentration was calculated by preparing a calibration curve by serially diluting the NaF solution.Then, the obtained concentration was used to calculate the apparent permeability coefficient (Papp) (10 -6 cm/s). was calculated according to the following formula: Both MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells showed low values, and no significant difference was observed (Fig. 6B).
NaFを用いた物質透過性評価を行った。HEPES含有Dulbecco's Phosphate Buffered Saline（DPBS）でNaFを10μg/mLに調製した。インサート内の培地を10μg/mlのNaF溶液に置換し、プレート側の培地をNaF不含のDPBSにて置換した。30分培養後、プレート側の培地を攪拌した後に回収し、インサートからプレート側へ移行したNaF量を蛍光強度計GENIOS （Spectra FLUOR Plus（TECAN製）を用いて測定した（励起波長：485 nm、蛍光波長：535 nm）。NaF溶液の段階希釈により検量線を作成して濃度を算出した。そして、得られた濃度を用いて透過係数（apparent permeability coefficient：Papp）（10-6cm/s）を以下の計算式に従い算出した。MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞共に低値を示し、有意な差は観察されなかった（図６Ｂ）。
（３）タイトジャンクション関連遺伝子
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のタイトジャンクション関連遺伝子であるClaudin-5、Occludin及びZO-1についてmRNA発現を各々定量的PCR法にて確認した。内部標準遺伝子としてGAPDHを用いた。定量的PCRによるmRNA発現量の測定は、実験例２－１と同手法により行った。MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞共に有意な差は観察されなかった（図７）。 (3) Tight-junction-associated genes Claudin-5, Occludin, and ZO-1, which are tight-junction-associated genes in MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells, were analyzed for mRNA expression by quantitative PCR. Confirmed by law. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed by the same method as in Experimental Example 2-1. No significant difference was observed between MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells (Fig. 7).
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のタイトジャンクション関連遺伝子であるClaudin-5、Occludin及びZO-1についてmRNA発現を各々定量的PCR法にて確認した。内部標準遺伝子としてGAPDHを用いた。定量的PCRによるmRNA発現量の測定は、実験例２－１と同手法により行った。MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞共に有意な差は観察されなかった（図７）。 (3) Tight-junction-associated genes Claudin-5, Occludin, and ZO-1, which are tight-junction-associated genes in MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells, were analyzed for mRNA expression by quantitative PCR. Confirmed by law. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed by the same method as in Experimental Example 2-1. No significant difference was observed between MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells (Fig. 7).
各遺伝子増幅用のプライマーは以下を用いた。
・Claudin-5遺伝子増幅用プライマー
Forward GTTCGTTGCGCTCTTCGTGA（配列番号１３）
Reverse GCTCGTACTTCTGCGACACG（配列番号１４）
・Occludin遺伝子増幅用プライマー
Forward CAGCAGCGGTGGTAACTTTG（配列番号１５）
Reverse TCCCTGATCCAGTCCTCCTC（配列番号１６）
・ZO-1遺伝子増幅用プライマー
Forward TGATCATTCCAGGCACTCG（配列番号１７）
Reverse CTCTTCATCTCTACTCCGGAGACT（配列番号１８） The following primers were used for each gene amplification.
・Claudin-5 gene amplification primer
Forward GTTCGTTGCGCTCTTCGTGA (sequence number 13)
Reverse GCTCGTACTTCTGCGACACG (SEQ ID NO: 14)
・Occludin gene amplification primer
Forward CAGCAGCGGTGGTAACTTTG (SEQ ID NO: 15)
Reverse TCCCTGATCCAGTCCTCCTC (SEQ ID NO: 16)
・ZO-1 gene amplification primer
Forward TGATCATTCCAGGCACTCG (sequence number 17)
Reverse CTCTTCATCTCTACTCCGGAGACT (SEQ ID NO: 18)
・Claudin-5遺伝子増幅用プライマー
Forward GTTCGTTGCGCTCTTCGTGA（配列番号１３）
Reverse GCTCGTACTTCTGCGACACG（配列番号１４）
・Occludin遺伝子増幅用プライマー
Forward CAGCAGCGGTGGTAACTTTG（配列番号１５）
Reverse TCCCTGATCCAGTCCTCCTC（配列番号１６）
・ZO-1遺伝子増幅用プライマー
Forward TGATCATTCCAGGCACTCG（配列番号１７）
Reverse CTCTTCATCTCTACTCCGGAGACT（配列番号１８） The following primers were used for each gene amplification.
・Claudin-5 gene amplification primer
Forward GTTCGTTGCGCTCTTCGTGA (sequence number 13)
Reverse GCTCGTACTTCTGCGACACG (SEQ ID NO: 14)
・Occludin gene amplification primer
Forward CAGCAGCGGTGGTAACTTTG (SEQ ID NO: 15)
Reverse TCCCTGATCCAGTCCTCCTC (SEQ ID NO: 16)
・ZO-1 gene amplification primer
Forward TGATCATTCCAGGCACTCG (sequence number 17)
Reverse CTCTTCATCTCTACTCCGGAGACT (SEQ ID NO: 18)
以上の結果、MDR1-iPS細胞由来脳血管内皮細胞についてiPS細胞由来脳血管内皮細胞の高いバリア能が維持されており、MDR1（P-gp）の過剰発現は脳血管内皮細胞のバリア機能に影響されないことが示された。
As a result, the high barrier ability of iPS cell-derived cerebral vascular endothelial cells was maintained for MDR1-iPS cell-derived cerebral vascular endothelial cells, and overexpression of MDR1 (P-gp) affected the barrier function of cerebral vascular endothelial cells. was shown not to.
（実験例２－３）iPS細胞由来脳血管内皮細胞のP-gp機能評価
iPS細胞由来脳血管内皮細胞に発現するP-gpについて解析を行った。 (Experimental Example 2-3) Evaluation of P-gp function in iPS cell-derived cerebral vascular endothelial cells P-gp expressed in iPS cell-derived cerebral vascular endothelial cells was analyzed.
iPS細胞由来脳血管内皮細胞に発現するP-gpについて解析を行った。 (Experimental Example 2-3) Evaluation of P-gp function in iPS cell-derived cerebral vascular endothelial cells P-gp expressed in iPS cell-derived cerebral vascular endothelial cells was analyzed.
（１）MDR1（P-gp）のmRNAの発現
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のMDR1遺伝子の発現を定量的PCR法にて確認した。定量的PCRによる測定は、実験例２－１と同手法により行った。MDR1-iPS細胞由来脳血管内皮細胞でのMDR1はMCS-iPS細胞由来脳血管内皮細胞に比べて約900倍発現量が増加した。 (1) Expression of MDR1 (P-gp) mRNA The expression of the MDR1 gene in MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed by quantitative PCR. Measurement by quantitative PCR was performed by the same method as in Experimental Example 2-1. The expression level of MDR1 in MDR1-iPS cell-derived cerebral vascular endothelial cells increased approximately 900-fold compared to that in MCS-iPS cell-derived cerebral vascular endothelial cells.
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のMDR1遺伝子の発現を定量的PCR法にて確認した。定量的PCRによる測定は、実験例２－１と同手法により行った。MDR1-iPS細胞由来脳血管内皮細胞でのMDR1はMCS-iPS細胞由来脳血管内皮細胞に比べて約900倍発現量が増加した。 (1) Expression of MDR1 (P-gp) mRNA The expression of the MDR1 gene in MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed by quantitative PCR. Measurement by quantitative PCR was performed by the same method as in Experimental Example 2-1. The expression level of MDR1 in MDR1-iPS cell-derived cerebral vascular endothelial cells increased approximately 900-fold compared to that in MCS-iPS cell-derived cerebral vascular endothelial cells.
また、MDR1-iPS細胞由来脳血管内皮細胞とhCMEC/D3細胞（脳血管内細胞株として汎用されているヒト由来不死化脳毛細血管内皮細胞株）やヒト単離脳毛細血管（初代培養）について、各種トランスポーター発現量を比較した結果を図８に示す。その結果、MDR1-iPS細胞由来脳血管内皮細胞のP-gp（MDR1）のmRNAの発現量はhCMEC/D3細胞の実測値やヒト単離脳毛細血管（初代培養）での報告値（ref）とほぼ同程度であった。MDR1遺伝子を導入することで P-gpの発現が初代培養ヒト脳血管内皮細胞と同程度にまで上昇したことが確認された。
In addition, about MDR1-iPS cell-derived cerebral vascular endothelial cells and hCMEC/D3 cells (human-derived immortalized cerebral capillary endothelial cell line widely used as cerebral intravascular cell line) and isolated human cerebral capillaries (primary culture) , and the results of comparing the expression levels of various transporters are shown in FIG. As a result, the expression level of P-gp (MDR1) mRNA in MDR1-iPS cell-derived cerebrovascular endothelial cells was measured in hCMEC/D3 cells and reported in human isolated brain capillaries (primary culture) (ref). was almost the same. It was confirmed that introduction of the MDR1 gene increased the expression of P-gp to the same level as that of primary cultured human cerebral endothelial cells.
（２）P-gp機能評価
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について、排出トランスポーターであるP-gpの機能を、P-gpの基質であるRhodamine-123（Sigma社製）を用いて取り込み実験を行い、解析した。24ウェルプレートに播種した細胞をトランスポート緩衝液（HBSS、10 mM HEPES、10 mM sodium pyrubate、pH7.4）を用いて洗浄後、トランスポート緩衝液、又はMDR1阻害剤として10μM シクロスポリンA（Cyclosporine A）を含むランスポート緩衝液で1時間37℃プレインキュベートした。その後トランスポート緩衝液を除去し、新たに10μM Rhodamine-123を含むトランスポート緩衝液を添加した。細胞は、RIPA緩衝液（25 mM トリス-HCl、pH 7.6、150 mM NaCl、1% NP-40, 1% デオキシコール酸ナトリウム、0.1%ドデシル硫酸ナトリウム（SDS）、ThermoFisher Scientific）で回収し、Rhodamine-123を蛍光強度計GENIOS （Spectra FLUOR Plus、TECAN製）を用いて測定した（励起波長：485 nm、蛍光波長：530 nm）。Rhodamine-123溶液の段階希釈により検量線を作成してRhodamine-123濃度を算出した。検出されたRhodamine-123量は、細胞数を用いて標準化した。 (2) P-gp function evaluation Regarding MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells, the function of P-gp, an efflux transporter, An uptake experiment was performed using 123 (manufactured by Sigma) and analyzed. After washing the cells seeded in a 24-well plate with transport buffer (HBSS, 10 mM HEPES, 10 mM sodium pyrubate, pH 7.4), transport buffer or 10 μM Cyclosporine A (Cyclosporine A ) in transport buffer for 1 hour at 37°C. After that, the transport buffer was removed and a new transport buffer containing 10 μM Rhodamine-123 was added. Cells were harvested in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), ThermoFisher Scientific) and washed with Rhodamine. -123 was measured using a fluorescence intensity meter GENIOS (Spectra FLUOR Plus, manufactured by TECAN) (excitation wavelength: 485 nm, fluorescence wavelength: 530 nm). Rhodamine-123 concentration was calculated by preparing a calibration curve by serially diluting the Rhodamine-123 solution. The amount of Rhodamine-123 detected was normalized using cell number.
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞について、排出トランスポーターであるP-gpの機能を、P-gpの基質であるRhodamine-123（Sigma社製）を用いて取り込み実験を行い、解析した。24ウェルプレートに播種した細胞をトランスポート緩衝液（HBSS、10 mM HEPES、10 mM sodium pyrubate、pH7.4）を用いて洗浄後、トランスポート緩衝液、又はMDR1阻害剤として10μM シクロスポリンA（Cyclosporine A）を含むランスポート緩衝液で1時間37℃プレインキュベートした。その後トランスポート緩衝液を除去し、新たに10μM Rhodamine-123を含むトランスポート緩衝液を添加した。細胞は、RIPA緩衝液（25 mM トリス-HCl、pH 7.6、150 mM NaCl、1% NP-40, 1% デオキシコール酸ナトリウム、0.1%ドデシル硫酸ナトリウム（SDS）、ThermoFisher Scientific）で回収し、Rhodamine-123を蛍光強度計GENIOS （Spectra FLUOR Plus、TECAN製）を用いて測定した（励起波長：485 nm、蛍光波長：530 nm）。Rhodamine-123溶液の段階希釈により検量線を作成してRhodamine-123濃度を算出した。検出されたRhodamine-123量は、細胞数を用いて標準化した。 (2) P-gp function evaluation Regarding MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells, the function of P-gp, an efflux transporter, An uptake experiment was performed using 123 (manufactured by Sigma) and analyzed. After washing the cells seeded in a 24-well plate with transport buffer (HBSS, 10 mM HEPES, 10 mM sodium pyrubate, pH 7.4), transport buffer or 10 μM Cyclosporine A (Cyclosporine A ) in transport buffer for 1 hour at 37°C. After that, the transport buffer was removed and a new transport buffer containing 10 μM Rhodamine-123 was added. Cells were harvested in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), ThermoFisher Scientific) and washed with Rhodamine. -123 was measured using a fluorescence intensity meter GENIOS (Spectra FLUOR Plus, manufactured by TECAN) (excitation wavelength: 485 nm, fluorescence wavelength: 530 nm). Rhodamine-123 concentration was calculated by preparing a calibration curve by serially diluting the Rhodamine-123 solution. The amount of Rhodamine-123 detected was normalized using cell number.
その結果、MDR1-iPS細胞由来脳血管内皮細胞ではMDR1遺伝子を導入することにより、Rhodamine-123の排出が促進されるため、MCS-iPS細胞由来脳血管内皮細胞に比べてRhodamine-123の取り込みが減少した。MDR1阻害剤であるシクロスポリンA存在下ではMDR1遺伝子導入の有無にかかわらずRhodamine-123の取り込みが増加した（図９）。これにより導入した MDR1遺伝子は機能的なP-gpを発現することが確認された。
As a result, introduction of the MDR1 gene in MDR1-iPS cell-derived cerebrovascular endothelial cells promoted the excretion of Rhodamine-123. Diminished. In the presence of MDR1 inhibitor cyclosporin A, Rhodamine-123 uptake increased regardless of the presence or absence of MDR1 gene transfer (Fig. 9). This confirmed that the introduced MDR1 gene expressed functional P-gp.
（３）薬物透過性試験
排出トランスポーターであるP-gpの機能を、P-gpの基質であるキニジン（quinidine）を用いて透過性実験を行った。キニジンを含むアッセイ緩衝液（HBSS、10 mM HEPES、10 mM sodium pyrubate、pH7.4）をドナー側に添加し、37℃にてアッセイを開始した。キニジンの血液側（luminal）から脳側（abluminal）（A-to-B）及び脳側から血液側（B-to-A）への輸送を評価するとともに、MDR1阻害剤として10μMシクロスポリンA及び10μM PSC833を使用した輸送能評価を行った。阻害剤をドナー側及びレシーバー側に添加し、30分間、37℃でプレインキュベートした後、透過実験を行った。 (3) Drug Permeability Test The function of P-gp, an efflux transporter, was investigated using quinidine, which is a substrate of P-gp. An assay buffer containing quinidine (HBSS, 10 mM HEPES, 10 mM sodium pyrubate, pH 7.4) was added to the donor side to initiate the assay at 37°C. The transport of quinidine from the blood side (luminal) to the brain side (A-to-B) and from the brain side to the blood side (B-to-A) was evaluated, and 10 μM cyclosporine A and 10 μM cyclosporin A as MDR1 inhibitors were evaluated. Transportability evaluation using PSC833 was performed. Inhibitors were added to the donor and receiver sides and pre-incubated for 30 min at 37° C. prior to permeation experiments.
排出トランスポーターであるP-gpの機能を、P-gpの基質であるキニジン（quinidine）を用いて透過性実験を行った。キニジンを含むアッセイ緩衝液（HBSS、10 mM HEPES、10 mM sodium pyrubate、pH7.4）をドナー側に添加し、37℃にてアッセイを開始した。キニジンの血液側（luminal）から脳側（abluminal）（A-to-B）及び脳側から血液側（B-to-A）への輸送を評価するとともに、MDR1阻害剤として10μMシクロスポリンA及び10μM PSC833を使用した輸送能評価を行った。阻害剤をドナー側及びレシーバー側に添加し、30分間、37℃でプレインキュベートした後、透過実験を行った。 (3) Drug Permeability Test The function of P-gp, an efflux transporter, was investigated using quinidine, which is a substrate of P-gp. An assay buffer containing quinidine (HBSS, 10 mM HEPES, 10 mM sodium pyrubate, pH 7.4) was added to the donor side to initiate the assay at 37°C. The transport of quinidine from the blood side (luminal) to the brain side (A-to-B) and from the brain side to the blood side (B-to-A) was evaluated, and 10 μM cyclosporine A and 10 μM cyclosporin A as MDR1 inhibitors were evaluated. Transportability evaluation using PSC833 was performed. Inhibitors were added to the donor and receiver sides and pre-incubated for 30 min at 37° C. prior to permeation experiments.
キニジンの見かけの透過係数（Papp）は以下の公式より算出した。
The apparent permeability coefficient (Papp) of quinidine was calculated from the following formula.
上記においてdQ/dt、D0及びSは、それぞれキニジンの輸送速度、ドナー側の初期濃度及びトランスウェルの表面積を示す。Efflux ratioは、A-to-B及びB-to-A方向のPappから算出した。
またキニジンはNexera-XR HPLCシステム及びQTRAP4500を接続したLC-MS/MSを用いて定量した。質量分析計にはエレクトロスプレーイオン化法を用い、ポジティブモードによる測定を行った。キニジン及びダントロレンのm/zは、それぞれお325.1/307.1、325.1/114.0の値をモニターした。クロマトグラフィーの分離にはSynergi Hydro-RPカラム（2.0×50 mm、25μm、Phenomenex）を用い、10 mMギ酸アンモニウム（0.2％ギ酸）及びメタノールによるグラジエント分析を0.4 mL/minの流速で行った。データの解析はAnalyst 1.6.1 ソフトウェアにて行った。
In the above, dQ/dt, D0 and S denote the transport rate of quinidine, the initial concentration on the donor side and the surface area of the transwell, respectively. The Efflux ratio was calculated from Papp in the A-to-B and B-to-A directions.
Quinidine was quantified using Nexera-XR HPLC system and LC-MS/MS connected to QTRAP4500. The electrospray ionization method was used for the mass spectrometer, and the measurement was performed in the positive mode. The m/z values of quinidine and dantrolene were monitored at 325.1/307.1 and 325.1/114.0, respectively. A Synergi Hydro-RP column (2.0×50 mm, 25 μm, Phenomenex) was used for chromatographic separation, and gradient analysis with 10 mM ammonium formate (0.2% formic acid) and methanol was performed at a flow rate of 0.4 mL/min. Data analysis was performed with Analyst 1.6.1 software.
上記の結果、MDR1-iPS細胞由来脳血管内皮細胞ではキニジンは血液側から脳側（A-to-B）方向に比べ脳側から血液側（B-to-A）方向へ多く輸送され（図１０）、Efflux ratio（B-to-A/A-to-B）は2.06であった。又、シクロスポリンAやPSC833などのMDR1阻害剤存在下ではEfflux ratioは1.0程度まで低下することが示された（表１）。
As a result, more quinidine was transported from the brain side to the blood side (B-to-A) than from the blood side to the brain side (A-to-B) in MDR1-iPS cell-derived cerebrovascular endothelial cells (Fig. 10), Efflux ratio (B-to-A/A-to-B) was 2.06. In addition, it was shown that the Efflux ratio decreased to about 1.0 in the presence of MDR1 inhibitors such as cyclosporin A and PSC833 (Table 1).
以上の結果よりMDR1-iPS細胞由来脳血管内皮細胞では機能的なP-gpが発現していることが示された。
The above results indicated that functional P-gp was expressed in MDR1-iPS cell-derived cerebrovascular endothelial cells.
（４）その他のトランスポーター
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のトランスポーター関連遺伝子の発現を確認した。物質の脳内輸送を厳しく制御しているため、脳血管内皮細胞には様々なトランスポーターが発現している。そこで、MDR1（P-gp）以外のトランスポーター関連遺伝子として、BCRP、Glut1及びMRP1についてmRNA発現を各々定量的PCR法にて確認した。内部標準遺伝子としてGAPDHを用いた。定量的PCRによるmRNA発現量の測定は、実験例２－１と同手法により行った。MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞の間で有意な差は観察されなかった（図１１）。 (4) Other transporters The expression of transporter-related genes in MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed. Various transporters are expressed in cerebral vascular endothelial cells to strictly control intracerebral transport of substances. Therefore, mRNA expression of BCRP, Glut1 and MRP1 as transporter-related genes other than MDR1 (P-gp) was confirmed by quantitative PCR. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed by the same method as in Experimental Example 2-1. No significant difference was observed between MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells (Fig. 11).
MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のトランスポーター関連遺伝子の発現を確認した。物質の脳内輸送を厳しく制御しているため、脳血管内皮細胞には様々なトランスポーターが発現している。そこで、MDR1（P-gp）以外のトランスポーター関連遺伝子として、BCRP、Glut1及びMRP1についてmRNA発現を各々定量的PCR法にて確認した。内部標準遺伝子としてGAPDHを用いた。定量的PCRによるmRNA発現量の測定は、実験例２－１と同手法により行った。MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞の間で有意な差は観察されなかった（図１１）。 (4) Other transporters The expression of transporter-related genes in MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells was confirmed. Various transporters are expressed in cerebral vascular endothelial cells to strictly control intracerebral transport of substances. Therefore, mRNA expression of BCRP, Glut1 and MRP1 as transporter-related genes other than MDR1 (P-gp) was confirmed by quantitative PCR. GAPDH was used as an internal standard gene. Measurement of the mRNA expression level by quantitative PCR was performed by the same method as in Experimental Example 2-1. No significant difference was observed between MDR1-iPS cell-derived cerebral endothelial cells and MCS-iPS cell-derived cerebral endothelial cells (Fig. 11).
・BCRP遺伝子増幅用プライマー
Forward CTCTTCGGCTTGCAACAACT（配列番号１９）
Reverse TTCTCCTCCAGACACACCAC（配列番号２０）
・Glut1遺伝子増幅用プライマー
Forward GCTATGGGGAGAGCATCCTG（配列番号２１）
Reverse AAGGCCAGCAGGTTCATCAT（配列番号２２）
・MRP1遺伝子増幅用プライマー
Forward CAAGGTGGATGCGAATGAGG（配列番号２３）
Reverse TGAGGAAGTAGGGCCCAAAG（配列番号２４） ・BCRP gene amplification primer
Forward CTCTTCGGCTTGCAACAACT (SEQ ID NO: 19)
Reverse TTCTCCTCCAGACACACCAC (SEQ ID NO: 20)
・Glut1 gene amplification primer
Forward GCTATGGGGAGAGCATCCTG (SEQ ID NO: 21)
Reverse AAGGCCAGGTTCATCAT (SEQ ID NO: 22)
・MRP1 gene amplification primer
Forward CAAGGTGGATGCGAATGAGG (SEQ ID NO: 23)
Reverse TGAGGAAGTAGGGCCCAAAG (SEQ ID NO: 24)
Forward CTCTTCGGCTTGCAACAACT（配列番号１９）
Reverse TTCTCCTCCAGACACACCAC（配列番号２０）
・Glut1遺伝子増幅用プライマー
Forward GCTATGGGGAGAGCATCCTG（配列番号２１）
Reverse AAGGCCAGCAGGTTCATCAT（配列番号２２）
・MRP1遺伝子増幅用プライマー
Forward CAAGGTGGATGCGAATGAGG（配列番号２３）
Reverse TGAGGAAGTAGGGCCCAAAG（配列番号２４） ・BCRP gene amplification primer
Forward CTCTTCGGCTTGCAACAACT (SEQ ID NO: 19)
Reverse TTCTCCTCCAGACACACCAC (SEQ ID NO: 20)
・Glut1 gene amplification primer
Forward GCTATGGGGAGAGCATCCTG (SEQ ID NO: 21)
Reverse AAGGCCAGGTTCATCAT (SEQ ID NO: 22)
・MRP1 gene amplification primer
Forward CAAGGTGGATGCGAATGAGG (SEQ ID NO: 23)
Reverse TGAGGAAGTAGGGCCCAAAG (SEQ ID NO: 24)
トランスポーター関連遺伝子のうちBCRPの基質であるダントロレン（Dantrolene）を用いて、上記（３）のキニジンの薬物動態評価法と同手法により透過性試験を行った。その結果、MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞ではいずれもBCRPは血液側から脳側（A-to-B）方向に比べ脳側から血液側（B-to-A）方向へ多く輸送されたが両細胞の間で有意な差は観察されなかった（図１２）。以上より、MDR1-iPS細胞由来脳血管内皮細胞及びMCS-iPS細胞由来脳血管内皮細胞のいずれについてもBCRPは同程度機能することが確認された。
Among the transporter-related genes, a permeability test was conducted using the same method as the pharmacokinetic evaluation method for quinidine in (3) above, using Dantrolene, which is a substrate of BCRP. As a result, in both MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells, BCRP was higher in the direction from the blood side to the brain side (A-to-B) than in the direction from the brain side to the blood side (B- To-A) was highly transported, but no significant difference was observed between the two cells (Fig. 12). From the above, it was confirmed that BCRP functions to the same extent in both MDR1-iPS cell-derived cerebral vascular endothelial cells and MCS-iPS cell-derived cerebral vascular endothelial cells.
上記結果より、P-gpを過剰発現させた多能性幹細胞由来脳血管内皮細胞であっても、MDR1を除く他のトランスポーターの発現や機能に影響を与えないことが示された。
From the above results, it was shown that even pluripotent stem cell-derived cerebrovascular endothelial cells overexpressing P-gp do not affect the expression or function of other transporters other than MDR1.
本発明の脳血管内皮細胞の作製に使用したMDR1遺伝子を導入した多能性幹細胞は、P-gpを高発現し、多能性幹細胞が本来有している多分化能を保持していた。さらに、本発明の多能性幹細胞由来脳血管内皮細胞は、P-gpを高発現しており、高いバリア機能を有し、かつ薬物排出トランスポーターとして優れた機能を保持していた。本発明の多能性幹細胞由来脳血管内皮細胞は、ヒト脳血管内皮細胞から樹立された不死化脳血管内皮細胞株では達成されなかった優れた薬物排出トランスポーター機能を有する。
The MDR1 gene-introduced pluripotent stem cells used to generate the cerebral vascular endothelial cells of the present invention highly expressed P-gp and retained the pluripotent potential inherent in pluripotent stem cells. Furthermore, the pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention highly expressed P-gp, had a high barrier function, and retained an excellent function as a drug efflux transporter. The pluripotent stem cell-derived cerebrovascular endothelial cells of the present invention have an excellent drug efflux transporter function that has not been achieved with immortalized cerebrovascular endothelial cell lines established from human cerebral vascular endothelial cells.
本発明の多能性幹細胞由来脳血管内皮細胞は、ヒトのBBBの輸送機能を精度よく反映しており、さらに初代脳血管内皮細胞では、同等の性質を有する脳血管内皮細胞を入手することが困難であったという課題を克服することができた。すなわち本発明により得られた脳血管内皮細胞によれば、ヒトのBBBの輸送機能を精度よく反映する機能を有し、均質な脳血管内皮細胞を容易に入手することが可能となり、例えば中枢神経疾患治療薬等の評価を均質に行うことができ、ヒト臨床試験等に利用可能であり、有用である。
The pluripotent stem cell-derived cerebral vascular endothelial cells of the present invention accurately reflect the transport function of the human BBB, and primary cerebral vascular endothelial cells can be obtained with similar properties. I was able to overcome the challenges that were difficult. That is, according to the cerebral vascular endothelial cells obtained by the present invention, it is possible to easily obtain homogeneous cerebral vascular endothelial cells that have a function that accurately reflects the transport function of the human BBB. It is possible to uniformly evaluate disease therapeutic drugs, etc., and is useful for human clinical trials and the like.
Claims (8)
- 以下の工程を含む多能性幹細胞から脳血管内皮細胞を製造する方法：
１）多能性幹細胞にP-gpをコードするMDR1遺伝子を導入する工程；及び
２）前記工程１）の遺伝子導入多能性幹細胞を、脳由来細胞に分化誘導する工程。 A method for producing cerebrovascular endothelial cells from pluripotent stem cells comprising the steps of:
1) a step of introducing the MDR1 gene encoding P-gp into the pluripotent stem cells; and 2) a step of inducing differentiation of the gene-introduced pluripotent stem cells of step 1) into brain-derived cells. - 前記１）のP-gpをコードするMDR1遺伝子を導入した多能性幹細胞が、未分化性を維持しながら、MDR1遺伝子を導入していない多能性幹細胞に比べてP-gpの発現量が100～2000倍に高発現した多能性幹細胞である、請求項１に記載の脳血管内皮細胞を製造する方法。 The pluripotent stem cells introduced with the MDR1 gene encoding the P-gp of 1) maintain undifferentiated state, while the expression level of P-gp is higher than that of the pluripotent stem cells without the introduction of the MDR1 gene. The method for producing cerebrovascular endothelial cells according to claim 1, wherein the pluripotent stem cells are highly expressed 100- to 2000-fold.
- 多能性幹細胞が、iPS細胞である、請求項１に記載の脳血管内皮細胞を製造する方法。 The method for producing cerebrovascular endothelial cells according to claim 1, wherein the pluripotent stem cells are iPS cells.
- MDR1遺伝子を導入した多能性幹細胞由来の脳血管内皮細胞であって、MDR1遺伝子を導入していない多能性幹細胞由来の脳血管内皮細胞に比べてP-gpの発現量が100～2000倍に高発現していることを特徴とする、多能性幹細胞由来脳血管内皮細胞。 Pluripotent stem cell-derived cerebrovascular endothelial cells transfected with the MDR1 gene showed 100 to 2000-fold expression of P-gp compared to pluripotent stem cell-derived cerebral endothelial cells not transfected with the MDR1 gene. A pluripotent stem cell-derived cerebrovascular endothelial cell characterized by high expression in
- 請求項１に記載の製造方法により製造された多能性幹細胞由来脳血管内皮細胞。 A pluripotent stem cell-derived cerebrovascular endothelial cell produced by the production method according to claim 1 .
- 初代脳血管内皮細胞が保持するP-gp機能と同等若しくはそれ以上のP-gp機能を担持することを特徴とする請求項４又は５に記載の多能性幹細胞由来脳血管内皮細胞。 6. The pluripotent stem cell-derived cerebrovascular endothelial cell according to claim 4 or 5, which has a P-gp function equal to or higher than that retained by primary cerebral vascular endothelial cells.
- 請求項４又は５に記載の多能性幹細胞由来脳血管内皮細胞を用いる、血液脳関門の解析方法。 A method for analyzing the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to claim 4 or 5.
- 請求項４又は５に記載の多能性幹細胞由来脳血管内皮細胞を用いる、血液脳関門における薬物動態の評価方法。
A method for evaluating pharmacokinetics at the blood-brain barrier, using the pluripotent stem cell-derived cerebrovascular endothelial cells according to claim 4 or 5.
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| JP2015159785A (en) * | 2014-02-28 | 2015-09-07 | 国立研究開発法人医薬基盤・健康・栄養研究所 | Method of producing cerebrovascular endothelial cell from pluripotent stem cell |
| US20190048316A1 (en) * | 2016-02-16 | 2019-02-14 | President And Fellows Of Harvard College | Improved blood-brain barrier endothelial cells derived from pluripotent stem cells for blood-brain barrier models |
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| JP2015159785A (en) * | 2014-02-28 | 2015-09-07 | 国立研究開発法人医薬基盤・健康・栄養研究所 | Method of producing cerebrovascular endothelial cell from pluripotent stem cell |
| US20190048316A1 (en) * | 2016-02-16 | 2019-02-14 | President And Fellows Of Harvard College | Improved blood-brain barrier endothelial cells derived from pluripotent stem cells for blood-brain barrier models |
Non-Patent Citations (2)
| Title |
|---|
| SAKO, DAIKI ET AL.: "28C-pm05S Evaluation of transporter function in P-gp expressing human iPS cell-derived brain microvascular endothelial cells", 142ND ANNUAL MEETING OF THE PHARMACEUTICAL SOCIETY OF JAPAN; NAGOYA, JAPAN [VIRTUAL MEETING]; MARCH 25-28, 2022, vol. 142, 4 March 2022 (2022-03-04) - 28 March 2022 (2022-03-28), XP009546684 * |
| TOMOKO YAMAGUCHI, BIMAO JIANG NISHIJIMA, HONGYAN ZHANG, YOSHIHARU DEGUCHI, KENJI KAWABATA: "28Q-pm089 Development of blood-brain barrier model using MDR1-overexpressed iPS cells", 140TH ANNUAL MEETING OF THE PHARMACEUTICAL SOCIETY OF JAPAN; KYOTO, JAPAN; MARCH 25-28, 2020, vol. 140, 1 January 2020 (2020-01-01) - 28 March 2020 (2020-03-28), XP009546683 * |
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