WO2019208736A1 - Procédé d'induction de différenciation de cellules souches pluripotentes en cellules endothéliales microvasculaires cérébrales - Google Patents

Procédé d'induction de différenciation de cellules souches pluripotentes en cellules endothéliales microvasculaires cérébrales Download PDF

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WO2019208736A1
WO2019208736A1 PCT/JP2019/017799 JP2019017799W WO2019208736A1 WO 2019208736 A1 WO2019208736 A1 WO 2019208736A1 JP 2019017799 W JP2019017799 W JP 2019017799W WO 2019208736 A1 WO2019208736 A1 WO 2019208736A1
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cells
culture
fgf2
tgf
inhibitor
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PCT/JP2019/017799
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English (en)
Japanese (ja)
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民秀 松永
真大 坡下
美紗季 山下
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公立大学法人名古屋市立大学
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Priority to JP2020515585A priority Critical patent/JP7251812B2/ja
Publication of WO2019208736A1 publication Critical patent/WO2019208736A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • BBB blood-brain barrier
  • BMEC brain capillary endothelial cells
  • the culture in the step (2) is performed using a medium containing serum or a serum substitute, FGF2, retinoic acid, and a TGF- ⁇ inhibitor.
  • the step (2) comprises three stages of culture. Specifically, for example, step (2-1) culturing in the presence of FGF2 and retinoic acid, and step (2-2) FGF2, retinoic acid, and TGF- ⁇ inhibitor performed after the culturing. And in the presence of a TGF- ⁇ inhibitor, the cells obtained in the step (1) are induced to differentiate into BMEC. To do.
  • This embodiment is advantageous for increasing the TEER value.
  • a culture medium containing serum or serum substitute, FGF2, and retinoic acid is used for the culture in step (2-1), and serum or serum substitute is used for the culture in step (2-2).
  • the culture surface coated with matrigel may be used for the early culture in the step (2) in order to promote differentiation induction.
  • the test substance that has permeated the cell layer is quantified.
  • a culture vessel equipped with a culture insert such as Transwell (registered trademark)
  • a test substance that has passed through the culture insert that is, an upper vessel (culture insert) or a lower vessel through a cell layer
  • the test substance that has moved into the (well) is subjected to mass spectrometry, liquid chromatography, immunological technique (for example, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method))
  • Quantify using a measurement method such as The membrane permeability of the test substance is evaluated based on the quantification result (the amount of the test substance that has passed through the cell layer) and the amount of the test substance used (typically, the amount added to the medium).
  • BBB BBB permeability evaluation method
  • a step of preparing a cell layer and a step of bringing a test substance into contact with the cell layer are performed, and then the influence of the cell layer on the barrier function is evaluated.
  • the method for evaluating the influence on the barrier function is as described above.
  • Method 1 Cells Human iPS cell 610B1 strain (RIKEN) established by introducing human 5 factors (Oct3 / 4, Sox2, Klf4, L-Myc, Lin28) into cord blood using an episomal vector (pCXLE) The experiment was conducted using the (purchased from the laboratory). Mouse fetal fibroblasts (MEF) were used as feeder cells.
  • RIKEN Human iPS cell 610B1 strain
  • pCXLE episomal vector
  • FBS fetal bovine serum
  • L-Glu 2 mmol / L L-glutamine
  • NEAA non-essential amino acid
  • 100 units / mL penicillin G 100 ⁇ g / mL Dulbecco's modified Eagle medium (DMEM) containing streptomycin was used.
  • EDTA trypsin-ethylenediaminetetraacetic acid
  • Cell Banker 1 was used as the MEF stock solution.
  • Human iPS cells were seeded on MEF (6 ⁇ 10 5 cells / 100 mm dish) treated with mitomycin C, and in a CO 2 incubator under 5% CO 2 /95% air conditions. Cultured at 37 ° C. Human iPS cells were subcultured at a split ratio of 1: 2 to 1: 3 after 3-5 days of culture. For human iPS cells, the medium was changed 48 hours after thawing and thereafter daily.
  • BMEC brain capillary endothelial cells
  • the obtained cells were washed 3 times with PBS containing 0.1% BSA, fixed with 4% paraformaldehyde, and again containing 0.1% BSA. After washing with PBS, permeation treatment was performed for 5 minutes with PBS containing 0.1% Triton-X. Subsequently, after washing with PBS containing 0.1% BSA, the primary antibody was reacted at 4 ° C. overnight. Subsequent operations were the same as described above.
  • TEER transendothelial electrical resistance
  • HBSS is 137 mmol / L sodium chloride, 5.4 mmol / L potassium chloride, 0.81 mmol / L magnesium sulfate, 0.44 mmol / L potassium dihydrogen phosphate, 0.34 mmol / L disodium hydrogen phosphate, 1.3 mmol / L calcium chloride 4.2 mmol / L sodium hydrogen carbonate, 5.6 mmol / L D-glucose, and pH 7.4 containing 10 mmol / L HEPES were used.
  • the apparent membrane transmission coefficient of Lucifer Yellow (excitation wavelength: 428 nm, fluorescence wavelength: 540 nm) was calculated from the fluorescence intensity measured using a fluorescence plate reader.
  • Acetylated LDL uptake test As a result of evaluating the acetylated LDL uptake ability of BMEC-like cells obtained by inducing differentiation by adding A-83-01 (1 nM), characteristics of vascular endothelial cells Uptake of acetylated LDL, which is one of these, was confirmed (FIG. 9). In the A-83-01-treated group, uptake of acetylated LDL was confirmed in more cells.
  • angiogenesis which is one of the characteristics of vascular endothelial cells, was confirmed (FIG. 10).
  • A-83-01 treatment group a blood vessel-like structure was confirmed with a smaller number of cells.
  • Freezing and thawing Freezing and thawing treatment can affect the structure and function of cells. Freeze-thaw treatment was performed in the middle of differentiation induction, and the effect was examined. As a result, TEER decreased by freezing and thawing in the control group, but TEER did not change in the A-83-01 addition group (FIG. 13). Further, when TEER was measured over time for the A-83-01 addition group, the freeze-thaw group showed the same TEER as the non-freeze-thaw group (FIG. 14).
  • the freeze-thaw group maintained a gene expression level equal to or higher than that of the non-freeze-thaw group (FIG. 17).
  • cells that enable the construction of a BBB model with a high barrier function can be induced to differentiate from pluripotent stem cells.
  • the BBB model constructed using the present invention is excellent in practicality, and can be used, for example, in an evaluation system for efficacy / safety of pharmaceuticals.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention aborde le problème consistant à fournir : un procédé pour induire la différenciation de cellules souches pluripotentes en cellules endothéliales cérébrovasculaires pouvant former des jonctions serrées qui sont stables et qui peuvent être maintenues pendant une longue période de temps ; et une utilisation dudit procédé. Dans la présente invention, la différenciation de cellules souches pluripotentes en cellules endothéliales microvasculaires cérébrales est induite au moyen : d'une étape (1) consistant à cultiver des cellules souches pluripotentes en l'absence de FGF2 et à réduire la dédifférenciation ; et une étape (2) consistant à différencier les cellules obtenues à l'étape (1) en cellules endothéliales microvasculaires cérébrales, ladite étape comprenant la culture en présence de FGF2, d'acide rétinoïque et d'un inhibiteur de TGF-β.
PCT/JP2019/017799 2018-04-27 2019-04-25 Procédé d'induction de différenciation de cellules souches pluripotentes en cellules endothéliales microvasculaires cérébrales WO2019208736A1 (fr)

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JP2020515585A JP7251812B2 (ja) 2018-04-27 2019-04-25 多能性幹細胞から脳毛細血管内皮細胞への分化誘導方法

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JP2018-087670 2018-04-27
JP2018087670 2018-04-27

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Cited By (2)

* Cited by examiner, † Cited by third party
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WO2021161991A1 (fr) * 2020-02-10 2021-08-19 公立大学法人名古屋市立大学 Agent de revêtement pour induire la différenciation des cellules souches pluripotentes en cellules de type endothélium capillaire cérébral et son utilisation
WO2022244841A1 (fr) * 2021-05-20 2022-11-24 公立大学法人名古屋市立大学 Procédé de production de cellules de type endothélial capillaire cérébral et son utilisation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112640887B (zh) * 2020-12-25 2022-05-13 武汉睿健医药科技有限公司 一种神经干细胞冻存液及其应用

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US20120015395A1 (en) * 2010-06-17 2012-01-19 Shusta Eric V Human Blood-Brain Barrier Endothelial Cells Derived From Pluripotent Stem Cells and Blood-Brain Barrier Model Thereof
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US20140127800A1 (en) * 2012-11-08 2014-05-08 Wisconsin Alumni Research Foundation Retinoic Acid Enhanced Human Stem Cell Derived Blood Brain Barrier Model
US20170283772A1 (en) * 2016-04-05 2017-10-05 Wisconsin Alumni Research Foundation Methods for differentiation of human pluripotent stem cells to brain microvascular endothelial cells
WO2019058140A1 (fr) * 2017-09-25 2019-03-28 Oxford University Innovation Ltd. Génération de cellules endothéliales microvasculaires cérébrales à partir de cellules souches pluripotentes pour modéliser la barrière hémato-encéphalique humaine et l'unité neurovasculaire

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US20140127800A1 (en) * 2012-11-08 2014-05-08 Wisconsin Alumni Research Foundation Retinoic Acid Enhanced Human Stem Cell Derived Blood Brain Barrier Model
US20170283772A1 (en) * 2016-04-05 2017-10-05 Wisconsin Alumni Research Foundation Methods for differentiation of human pluripotent stem cells to brain microvascular endothelial cells
WO2019058140A1 (fr) * 2017-09-25 2019-03-28 Oxford University Innovation Ltd. Génération de cellules endothéliales microvasculaires cérébrales à partir de cellules souches pluripotentes pour modéliser la barrière hémato-encéphalique humaine et l'unité neurovasculaire

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KATT, MORIAH E. ET AL.: "Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype", PLOS ONE, vol. 11, no. 4, 12 April 2016 (2016-04-12), pages 1 - 18, XP055648234, DOI: 10.1371/journal.pone.0152105 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021161991A1 (fr) * 2020-02-10 2021-08-19 公立大学法人名古屋市立大学 Agent de revêtement pour induire la différenciation des cellules souches pluripotentes en cellules de type endothélium capillaire cérébral et son utilisation
CN115175986A (zh) * 2020-02-10 2022-10-11 公立大学法人名古屋市立大学 用于诱导多能干细胞向脑毛细血管内皮样细胞分化的涂布剂及其利用
WO2022244841A1 (fr) * 2021-05-20 2022-11-24 公立大学法人名古屋市立大学 Procédé de production de cellules de type endothélial capillaire cérébral et son utilisation

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