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WO2014132933A1 - Méthode d'induction d'une différenciation de cellules souches pluripotentes induites en cellules épithéliales intestinales - Google Patents

Méthode d'induction d'une différenciation de cellules souches pluripotentes induites en cellules épithéliales intestinales Download PDF

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WO2014132933A1
WO2014132933A1 PCT/JP2014/054379 JP2014054379W WO2014132933A1 WO 2014132933 A1 WO2014132933 A1 WO 2014132933A1 JP 2014054379 W JP2014054379 W JP 2014054379W WO 2014132933 A1 WO2014132933 A1 WO 2014132933A1
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cell
inhibitor
intestinal epithelial
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岳洋 岩尾
民秀 松永
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Nagoya City University
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for inducing differentiation of induced pluripotent stem cells (iPS) into intestinal epithelial cells and uses thereof.
  • iPS induced pluripotent stem cells
  • iPS induced pluripotent stem
  • Patent Document 1 In order to provide intestinal epithelial cells used for drug absorption tests and the like, a method for selectively obtaining intestinal stem / progenitor cells from cells derived from the intestinal tract has been reported (Patent Document 1). In addition, a method for producing or maintaining pluripotent cells using an ALK5 inhibitor has been proposed (Patent Document 2).
  • Non-patent Document 1 a report that an embryoid body was formed from mouse iPS cells and an intestine-like tissue was prepared, and an intestine-like tissue was prepared from human iPS cells by three-dimensional culture.
  • Non-Patent Documents 2 and 3 reports that the differentiation induction methods in these reports are complicated, and the differentiation efficiency is not sufficient, and pharmacokinetic analysis has not been performed in detail.
  • the differentiation induction method induces differentiation using a large amount of extremely expensive growth factors and cytokines and is not suitable for practical use.
  • an object of the present invention is to provide a novel method and its use for efficiently inducing differentiation of iPS cells into intestinal epithelial cells.
  • the present inventors have the same multipotency and almost unlimited proliferation ability as human ES cells, and are expected to be used for drug discovery research from human iPS cells to intestinal epithelial cell-like cells. I decided to try to make. At that time, we focused on the possibility of low molecular weight compounds as differentiation-inducing factors, and aimed to create cells that can be used in evaluation systems for pharmacokinetic tests by a simpler method. As a result of detailed investigation, MEK1 inhibitor, DNA methylation inhibitor and TGF ⁇ receptor inhibition were used as inducers when inducing intestinal stem cell-like cells obtained through endoderm-like cells into intestinal epithelial cell-like cells The agent was found to be effective.
  • differentiation induction efficiency increased when cultured in the presence of GSK inhibitor and BMP inhibitor at the initial stage of inducing differentiation of intestinal stem cell-like cells into intestinal epithelial cell-like cells. Furthermore, when a MEK1 inhibitor, DNA methylation inhibitor and TGF ⁇ receptor inhibitor were used in combination, differentiation induction into the intestinal epithelium was further promoted. On the other hand, it has been found that FGF2 is particularly preferable as an inducing factor in inducing differentiation of endoderm-like cells into intestinal stem cell-like cells.
  • the intestinal epithelial cell-like cells that were successfully produced expressed various marker molecules specific to the intestinal epithelium and had a peptide transport function.
  • induction of drug metabolizing enzyme (CYP3A4) expression via vitamin D receptor was also observed.
  • CYP3A4 drug metabolizing enzyme
  • a method for inducing differentiation of induced pluripotent stem cells into intestinal epithelial cells comprising the following steps (1) to (3): (1) a step of differentiating induced pluripotent stem cells into endoderm-like cells; (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (3) Differentiating intestinal stem cell-like cells obtained in step (2) into intestinal epithelial cell-like cells, selected from the group consisting of MEK1 inhibitors, DNA methylation inhibitors and TGF ⁇ receptor inhibitors Comprising culturing in the presence of one or more compounds and EGF.
  • [2] The method according to [1], wherein the culture in the step (3) is performed in the presence of a MEK1 inhibitor, a DNA methylation inhibitor, a TGF ⁇ receptor inhibitor, and EGF.
  • [3] The method according to [1] or [2], wherein the culture period in the step (3) is 7 days to 30 days.
  • the MEK1 inhibitor is PD98059
  • the DNA methylation inhibitor is 5-aza-2′-deoxycytidine
  • the TGF ⁇ receptor inhibitor is A-83-01.
  • [1] to [3] The method as described in any one of.
  • [5] The method according to any one of [1] to [4], wherein activin A is used as the differentiation-inducing factor in step (1).
  • step (3) comprises the following steps (3-1) and (3-2): (3-1) A step of culturing the intestinal stem cell-like cell obtained in step (2) in the presence of a GSK inhibitor and / or a BMP inhibitor and EGF; (3-2) Step (3-1) followed by culturing in the presence of EGF and one or more compounds selected from the group consisting of MEK1 inhibitor, DNA methylation inhibitor and TGF ⁇ receptor inhibitor .
  • the method according to [12], comprising the following steps (i) to (iii): (I) A step of preparing a cell layer composed of the intestinal epithelial cell-like cells according to [11]; (Ii) contacting the test substance with the cell layer; (Iii) A step of quantifying the test substance that has permeated through the cell layer and evaluating the absorbability or membrane permeability of the test substance. [14] The method according to [12], comprising the following steps (I) and (II): (I) A step of bringing a test substance into contact with the intestinal epithelial cell-like cell according to [11]; (II) A step of measuring / evaluating the metabolism or absorption of the test substance. [15] A cell preparation comprising the intestinal epithelial cell-like cell according to [11].
  • GSK3i is GSK3iXV
  • BMPi is dorsomorphin
  • 5-aza-2′-dC is 5-aza-2′-deoxycytidine
  • VD3 is 1 ⁇ , 25-dihydroxyvitamin D 3
  • GSK3i is GSK3iXV
  • BMPi is dorsomorphin
  • GSK3i is GSK3iXV
  • BMPi is dorsomorphin
  • 5-aza-2'-dC is 5-aza-2'-deoxycytidine.
  • ABCB1 / MDR1 top
  • ABCG2 / BCRP bottom
  • GSK3i GSK3iXV
  • BMPi dorsomorphin
  • 5-aza-2'-dC 5-aza-2'-deoxycytidine.
  • 5-aza-2'-dC is 5-aza-2'-deoxycytidine.
  • GSK3i GSK3iXV
  • BMPi dorsomorphin
  • GSK3i is GSK3iXV
  • BMPi is dorsomorphin
  • 5-aza-2'-dC is 5-aza-2'-deoxycytidine.
  • GSK3i is GSK3iXV
  • BMPi is dorsomorphin
  • 5-aza-2'-dC is 5-aza-2'-deoxycytidine
  • VD3 is l [alpha], a 25-dihydroxyvitamin D 3.
  • SLC15A1 / PEPT1 expression (upper) in intestinal epithelial cell-like cells differentiated by a more effective combination of compounds, and the induction effect of 1 ⁇ , 25-dihydroxyvitamin D 3 on CYP3A4 expression.
  • 5-aza-2'-dC is 5-aza-2'-deoxycytidine and VD3 is 1 ⁇ , 25-dihydroxyvitamin D3.
  • the present invention relates to a method for inducing differentiation of induced pluripotent stem cells (iPS cells) into the intestinal epithelial cell lineage (hereinafter also referred to as “differentiation induction method of the present invention”).
  • iPS cells induced pluripotent stem cells
  • intestinal epithelial cell lineage hereinafter also referred to as “differentiation induction method of the present invention.
  • iPS cells are cells having pluripotency (pluripotency) and proliferative ability produced by reprogramming somatic cells by introduction of reprogramming factors. Artificial pluripotent stem cells exhibit properties similar to ES cells. Somatic cells used for the production of iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. Although the origin thereof is not particularly limited, preferably, somatic cells of mammals (eg, primates such as humans and chimpanzees, rodents such as mice and rats), particularly preferably human somatic cells are used. iPS cells can be prepared by various methods reported so far. In addition, it is naturally assumed that an iPS cell production method developed in the future will be applied.
  • mammals eg, primates such as humans and chimpanzees, rodents such as mice and rats
  • iPS cell production The most basic method of iPS cell production is to introduce four factors, transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007).
  • Human iPS cells have been reported to be established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318 (5858), 1917-1920, 2007).
  • Three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol.
  • lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903 ), 945-949, 2008), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766- 770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or Techniques using episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been developed.
  • pluripotent stem cell markers such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript Etc. can be selected as an index.
  • the selected cells are collected as iPS cells.
  • IPS cells can also be provided from, for example, Kyoto University or RIKEN BioResource Center.
  • inducing differentiation means acting to differentiate along a specific cell lineage.
  • iPS cells are induced to differentiate into intestinal epithelial cells.
  • the differentiation induction method of the present invention is roughly divided into three stages of induction processes, that is, a process of differentiating iPS cells into endoderm-like cells (step (1)), and the obtained endoderm-like cells into intestinal stem cell-like cells. And the step of differentiating (step (2)) and the step of differentiating the obtained intestinal stem cell-like cells into intestinal epithelial cell-like cells (step (3)).
  • iPS cells are cultured and differentiated into endoderm-like cells.
  • iPS cells are cultured under conditions that induce differentiation into endoderm-like cells.
  • the culture conditions are not particularly limited as long as iPS cells differentiate into endoderm-like cells.
  • the cells are cultured in a medium supplemented with activin A according to a conventional method.
  • the concentration of activin A in the medium is, for example, 10 ng / ml to 200 ng / ml, preferably 20 ng / ml to 150 ng / ml.
  • serum or serum replacement Knockout serum replacement (KSR), etc.
  • Serum is not limited to fetal bovine serum, and human serum, sheep serum, and the like can also be used.
  • the amount of serum or serum replacement added is, for example, 0.1% (v / v) to 10% (v / v).
  • An inhibitor of the Wnt / ⁇ -catenin signaling pathway eg, hexachlorophene, quercetin, Wnt3a, which is a Wnt ligand
  • Wnt3a which is a Wnt ligand
  • two-stage culture is performed as step (1).
  • the first stage culture is performed in a medium supplemented with a relatively low concentration of serum (eg, 0.1% (v / v) to 1% (v / v)), and the second stage culture is the first stage culture.
  • a medium with a higher serum concentration for example, the serum concentration is 1% (v / v) to 10% (v / v)).
  • Adopting the two-stage culture in this way is preferable in that the growth of undifferentiated cells is suppressed by the first-stage culture and the differentiated cells are grown in the subsequent second stage.
  • the period of step (1) is, for example, 1 day to 10 days, preferably 2 days to 7 days.
  • the first stage culture period is, for example, 1 to 7 days, preferably 2 to 5 days
  • the second stage culture period is, for example, 1 to 6 days. Days, preferably 1 to 4 days.
  • the endoderm-like cells obtained in step (1) are cultured and differentiated into intestinal stem cell-like cells.
  • the endoderm cells are cultured under conditions that induce differentiation into intestinal stem cell-like cells.
  • the culture conditions are not particularly limited as long as the endoderm-like cells differentiate into intestinal stem cell-like cells.
  • the culture is performed in the presence of FGF2 (fibroblast growth factor 2) based on the experimental results shown in the Examples described later.
  • FGF2 fibroblast growth factor 2
  • human FGF2 for example, human recombinant FGF2
  • step (2) may be carried out.
  • the endoderm-like cells may be selected with a flow cytometer (cell sorter) using a cell surface marker as an index.
  • FGF2 In the presence of FGF2 is synonymous with the condition in which FGF2 is added to the medium. Therefore, in order to perform culture in the presence of FGF2, a medium supplemented with FGF2 may be used.
  • An example of the concentration of FGF2 added is 100 ng / mL to 500 ng / mL.
  • the period of step (2) (culture period) is, for example, 2 days to 10 days, preferably 3 days to 7 days. If the culture period is too short, the expected effects (increased differentiation efficiency, promotion of acquisition of functions as intestinal stem cells) cannot be sufficiently obtained. On the other hand, if the culture period is too long, the differentiation efficiency is lowered.
  • intestinal stem cell-like cells Differentiation into intestinal stem cell-like cells can be determined or evaluated using, for example, the expression of intestinal stem cell marker as an index.
  • intestinal stem cell markers are G protein-coupled receptor 5 (LGR5) and ephrin B2 receptor (EphB2) containing leucine-rich repeats.
  • Step (3) Differentiation into intestinal epithelial cell-like cells>
  • the intestinal stem cell-like cells obtained in step (2) are differentiated into intestinal epithelial cell-like cells.
  • the cell population obtained through the step (2) or a part thereof is subjected to the step (3) without sorting.
  • the step (3) may be performed. Selection of intestinal stem cell-like cells may be performed, for example, with a flow cytometer (cell sorter) using a cell surface marker as an index.
  • first inducer DNA methylation inhibitor and TGF ⁇ receptor inhibitor
  • second inducer EGF
  • the first inducer and the second inducer are synonymous with the conditions added to the medium. Therefore, in order to perform culture in the presence of the first inducer and the second inducer, a medium to which the first inducer and the second inducer are added may be used.
  • Examples of MEK1 inhibitors include PD98059, PD184352, PD184161, PD0325901, U0126, MEK inhibitor I, MEK inhibitor II, MEK1 / 2 inhibitor II, and SL327.
  • examples of DNA methylation inhibitors include 5-aza-2'-deoxycytidine, 5-azacytidine, RG108, and zebralin.
  • A-83-01 used in the examples described later exhibits inhibitory activity on TGF- ⁇ receptors ALK4, ALK5, and ALK7. What shows inhibitory activity with respect to one or more of ALK4, ALK5, and ALK7 may be used.
  • A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox satisfy the condition.
  • concentration of MEK1 inhibitor added is 4 ⁇ M to 100 ⁇ M, preferably 10 to 40 ⁇ M.
  • an example of the addition concentration of a methylation inhibitor in the case of 5-aza-2′-deoxycytidine is 1 ⁇ M to 25 ⁇ M, preferably 2.5 ⁇ M to 10 ⁇ M
  • the addition concentration of the TGF ⁇ receptor inhibitor is 0.1 ⁇ M to 2.5 ⁇ M, preferably 0.2 ⁇ M to 1 ⁇ M.
  • two or more of MEK1 inhibitor, DNA methylation inhibitor, and TGF ⁇ receptor inhibitor are used in combination as the first inducer.
  • first inducers By using two or more different first inducers in combination, an additive or synergistic effect can be obtained, and differentiation into intestinal epithelium can be promoted.
  • all (ie, three types) of first inducers are used in combination.
  • three types of first inducers in the examples, PD98059 as a MEK1 inhibitor, 5-aza-2'-deoxycytidine as a DNA methylation inhibitor, and A- as a TGF ⁇ receptor inhibitor
  • the period of step (3) (culture period) is, for example, 7 days to 30 days, preferably 10 days to 20 days. If the culture period is too short, the expected effects (increased differentiation efficiency, promotion of acquisition of functions as intestinal epithelial cells) cannot be sufficiently obtained. On the other hand, if the culture period is too long, the differentiation efficiency is lowered.
  • intestinal epithelial cell marker expression can be determined or evaluated using, for example, intestinal epithelial cell marker expression, peptide uptake, or induction of drug metabolizing enzyme expression via vitamin D receptor as an index.
  • intestinal epithelial cell markers include ATP-binding cassette transporter B1 / multidrug resistance protein 1 (ABCB1 / MDR1), caudal homeobox transcription factor 2 (CDX2), cytochrome P450-3A4 (CYP3A4), dipeptidyl peptidase 4 (DPP4), SLC (solute carrier) family member 15A1 / peptide transporter 1 (SLC15A1 / PEPT1), SLC (solute carrier) family member 46A1 / proton conjugated folate transporter (SLC46A1 / PCFT), sucrase-isomaltase .
  • ABSB1 / MDR1 multidrug resistance protein 1
  • CDX2 caudal homeobox transcription factor 2
  • CYP3A4 cytochro
  • sucrase-isomaltase having high specificity for the intestinal epithelium, CYP3A4, which is a main drug-metabolizing enzyme in the small intestine, and SLC15A1 / PEPT1 involved in the absorption of peptides in the small intestine are particularly effective markers.
  • a cell population consisting only of the target cells (intestinal epithelial cell-like cells) or a cell population that contains the target cells in a high ratio (high purity)
  • select a cell surface marker that is characteristic for the target cells may be selected and sorted as an index.
  • the step (3) is a two-stage culture, that is, the step of culturing the intestinal stem cell-like cells obtained in the step (2) in the presence of a GSK inhibitor and / or a BMP inhibitor and EGF (3- 1) followed by the presence of one or more compounds (first inducer) and EGF (second inducer) selected from the group consisting of MEK1 inhibitors, DNA methylation inhibitors and TGF ⁇ receptor inhibitors
  • the step (3-2) of culturing is performed. Therefore, in this embodiment, the culture in the presence of the GSK inhibitor and / or the BMP inhibitor is performed before the culture using the first inducer and the second inducer.
  • the main purpose of culturing in the presence of GSK inhibitors and / or BMP inhibitors is to promote intestinal stem cell proliferation.
  • step (3-1) and step (3-2) are performed as step (3)
  • the culture period of step (3-1) is, for example, 3 days to 14 days, preferably 4 days to 10 days.
  • the culture period of (3-2) is, for example, 3 days to 21 days, preferably 5 days to 15 days.
  • step (1), (2), (3), (3-1), (3-2)) constituting the present invention subculture may be performed midway.
  • the cells may be seeded at a cell density of about 1 ⁇ 10 4 cells / cm 2 to 1 ⁇ 10 6 cells / cm 2 .
  • ROCK inhibitor Rho-associated coiled-coil forming kinase / Rho-binding kinase
  • IMDM Iskov modified Dulbecco medium
  • HamF12 Ham F12 medium
  • D-MEM Dulbecco modified Eagle medium
  • Gibco, etc. Glasgow basic medium
  • a basic medium for example, a mixed medium of D-MEM and Ham F12 medium, D-MEM
  • D-MEM Ham F12 medium
  • components that can be added to the medium include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium.
  • BSA bovine serum albumin
  • NEAA non-essential amino acids
  • cells are cultured two-dimensionally using a culture dish or the like.
  • intestinal epithelial cell-like cells can be obtained from iPS cells by two-dimensional culture.
  • three-dimensional culture using a gel culture substrate or a three-dimensional culture plate may be performed.
  • the second aspect of the present invention relates to the use of intestinal epithelial cell-like cells prepared by the differentiation induction method of the present invention.
  • Various assays are provided as a first use.
  • the intestinal epithelial cell-like cell of the present invention can be used in a model system of the small intestine, and is useful for evaluating pharmacokinetics (absorption, metabolism, etc.) in the small intestine.
  • the intestinal epithelial cell-like cell of the present invention can be used for evaluating the pharmacokinetics of a compound.
  • the intestinal epithelial cell-like cell of the present invention can be used to test the absorbability or membrane permeability of the test substance. That is, the present invention provides, as one of the uses of intestinal epithelial cell-like cells, a method (first aspect) for evaluating the absorbability or membrane permeability of a test substance.
  • a method for evaluating the absorbability or membrane permeability of a test substance.
  • a step of preparing a cell layer composed of intestinal epithelial cell-like cells obtained by the differentiation induction method of the present invention and (ii) a step of bringing a test substance into contact with the cell layer;
  • a step of quantifying the test substance that has permeated the cell layer and evaluating the absorbability or membrane permeability of the test substance is performed.
  • the absorbability of the test substance can also be evaluated by the method described later (second aspect).
  • intestinal epithelial cell-like cells are typically cultured on a semipermeable membrane (porous membrane) to form a cell layer.
  • a semipermeable membrane for example, Transwell (registered trademark) provided by Corning
  • a culture insert for example, Transwell (registered trademark) provided by Corning
  • “Contact” in step (ii) is typically performed by adding a test substance to the medium.
  • the timing of adding the test substance is not particularly limited. Therefore, after culturing is started in a medium not containing the test substance, the test substance may be added at a certain point in time, or the culture may be started in advance in a medium containing the test substance.
  • organic compounds or inorganic compounds having various molecular sizes can be used as the test substance.
  • organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.)
  • test substances may be used as test substances, and by adding two or more kinds of test substances at the same time, the interaction between the test substances, synergism, etc. will be investigated.
  • the test substance may be derived from a natural product or may be synthetic, in which case, for example, a combinatorial synthesis technique is used. It is possible to construct a rate assay systems.
  • the period for contacting the test substance can be set arbitrarily.
  • the contact period is, for example, 10 minutes to 3 days, preferably 1 hour to 1 day.
  • the contact may be performed in a plurality of times.
  • the test substance that has permeated the cell layer is quantified.
  • a culture vessel equipped with a culture insert such as Transwell (registered trademark) is used, a test substance leaked from the culture insert, that is, a test substance moved into the lower container through the cell layer Is quantified by a measuring method such as mass spectrometry, liquid chromatography, immunological technique (for example, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method)) or the like according to the test substance.
  • FIA method fluorescence immunoassay
  • EIA method enzyme immunoassay
  • the present invention also provides a method for evaluating metabolism or absorption of a test substance as another aspect (second aspect).
  • this method (I) a step of contacting a test substance with the intestinal epithelial cell-like cell obtained by the differentiation induction method of the present invention, and (II) a step of measuring and evaluating the metabolism or absorption of the test substance. .
  • Step (I) that is, the contact between the intestinal epithelial cell-like cells and the test substance can be carried out in the same manner as in the above step (ii).
  • step (II) the metabolism or absorption of the test substance is measured and evaluated (step (II)).
  • step (I) Immediately after step (I), that is, after contact with the test substance, metabolism or absorption is measured and evaluated without a substantial time interval, or for a certain time (for example, 10 minutes to 5 hours) After elapse of time, metabolism or absorption may be measured and evaluated. Metabolism can be measured, for example, by detecting a metabolite. In this case, the expected metabolite is usually measured qualitatively or quantitatively using the culture solution after step (I) as a sample. An appropriate measurement method may be selected according to the metabolite. For example, mass spectrometry, liquid chromatography, immunological method (eg, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method)) ) Etc. can be adopted.
  • FFA method fluorescence immunoassay
  • EIA method enzyme immunoassay
  • the metabolic amount of the test substance can be evaluated according to the amount of the metabolite.
  • the metabolic efficiency of the test substance may be calculated based on the detection result of the metabolite and the amount of the test substance used (typically, the amount added to the medium).
  • drug metabolizing enzymes cytochrome (particularly CYP3A4), UGT (particularly UGT1A8, UGT1A10), SULT1A3, etc.
  • the expression of drug metabolizing enzymes can be assessed at the mRNA level or protein level. For example, when an increase in the mRNA level of the drug-metabolizing enzyme is observed, it can be determined that “the test substance has been metabolized”. Similarly, when an increase in the activity of the drug-metabolizing enzyme is observed, it can be determined that “the test substance has been metabolized”.
  • quantitative determination / evaluation may be performed based on the expression level of a drug metabolizing enzyme.
  • the remaining amount of the test substance in the culture solution is measured.
  • the test substance is quantified using the culture solution after step (I) as a sample.
  • An appropriate measuring method may be selected according to the test substance. For example, mass spectrometry, liquid chromatography, immunological techniques (for example, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method)) and the like can be employed.
  • FFA method fluorescence immunoassay
  • EIA method enzyme immunoassay
  • the amount of absorption or absorption efficiency of the test substance can be determined and evaluated according to the degree of decrease.
  • the absorption can also be evaluated by measuring the amount of the test substance taken up into the cells.
  • the measurement / evaluation of metabolism and the measurement / evaluation of absorption may be performed simultaneously or in parallel.
  • a cell preparation containing intestinal epithelial cell-like cells is provided as a second use of the intestinal epithelial cell-like cells prepared by the differentiation induction method of the present invention.
  • the cell preparation of the present invention can be applied to the treatment of various intestinal diseases. In particular, it is expected to be used as a material for regeneration / reconstruction of intestinal epithelial tissue that has been impaired (including dysfunction). That is, contribution to regenerative medicine can be expected.
  • the cell preparation of the present invention can be prepared, for example, by suspending intestinal epithelial cell-like cells obtained by the method of the present invention in a physiological saline, a buffer solution (for example, a phosphate buffer solution) or the like.
  • 1 ⁇ 10 5 to 1 ⁇ 10 10 cells may be contained as a single dose so that a therapeutically effective amount of cells can be administered.
  • the content of the cells can be appropriately adjusted in consideration of the purpose of use, the target disease, the sex of the application target (recipient), age, weight, the state of the affected area, the state of the cells, and the like.
  • DMSO Dimethyl sulfoxide
  • serum albumin for the purpose of cell protection, antibiotics for the purpose of blocking bacterial contamination, and various components (vitamins for the purpose of cell activation, proliferation or differentiation induction, etc. , Cytokines, growth factors, steroids, etc.) may be included in the cell preparation of the present invention.
  • other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc. You may make it contain in the cell formulation of this invention.
  • Method (1) Cells Human iPS cells (iPS-51: Windy) are expressed in human fetal lung fibroblasts MRC-5, octamer binding protein 3/4 (OCT3 / 4), sex determining region Y-box 2 (SOX2), After introducing kruppel-like factor 4 (KLF4), v-myc myelocytomatosis viral oncogene homolog (avian) (c-MYC) using a pantropic retrovirus vector, human ES cell-like colonies are cloned, Granted by Dr. Akihiro Umezawa, National Center for Child Health and Development. Mouse fetal fibroblasts (MEF) were used as feeder cells.
  • KLF4 kruppel-like factor 4
  • c-MYC v-myc myelocytomatosis viral oncogene homolog
  • 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 cultured in a CO 2 incubator under 5% CO 2 /95% air conditions. Incubated at 0 ° 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.
  • the cells were cultured in DMEM / F12 containing 2% FBS, 1% glutamax and 250 ng / mL FGF2 for 4 days to differentiate into intestinal stem cells.
  • Y-27632 Ras binding kinase inhibitor
  • the cells were peeled off with actase and seeded in a 24-well plate for cell culture coated with Matrigel from which growth factors had been removed, which had been diluted 30-fold with a medium for human iPS cells.
  • Drug inducer treatment of drug metabolizing enzymes is 2% FBS, 2 mmol / L L-Glu, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 100 units / mL penicillin G, 100 ⁇ g / mL streptomycin, 20 ng / Add 1 ⁇ , 25-dihydroxyvitamin D 3 (VD3) to 10 nmol / L or rifampicin 40 ⁇ mol / L to DMEM / F12 containing mL epidermal growth factor (EGF) and incubate for 48 hours before recovery. I went there.
  • culture was also performed under the following conditions (a) to (c).
  • (A) GSK3iXV (125 nM) and dorsomorphin (1 ⁇ M) were added to the culture solution for 6 days from the 8th day after the start of differentiation (other culture conditions are as described above).
  • (B) PD98059 (20 ⁇ M), 5-aza-2′-deoxycytidine (5 ⁇ M), and A-83-01 (0.5 ⁇ M) were added to the culture for 18 days from the start of differentiation (other culture conditions) Is as above).
  • RNA extraction Total ribonucleic acid (RNA) extraction
  • RNA was extracted according to the attached manual of RNeasy (registered trademark) Mini Kit (Qiagen) after completion of induction of differentiation of human iPS cells.
  • Real-Time RT-PCR Real-time reverse transcription polymerase chain reaction
  • SYBR registered trademark
  • Premix Ex Taq II Perfect Real Time
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • Sucrase-isomaltase immunofluorescent staining Cells used for immunostaining were cultured on a cover glass. After culturing, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, 0.1% Triton X was used for membrane permeation for 5 minutes at room temperature, and 2% skim milk was used for 20 minutes at room temperature. Blocking treatment was performed. Thereafter, the primary antibody was a rabbit anti-human sucrase-isomaltase antibody (Sigma; 1: 200) for 60 minutes at room temperature, and the secondary antibody was an Alexa Fluor® 568 goat anti-rabbit IgG antibody (Invitrogen; 1: 500) for 60 minutes at room temperature in the dark. Nuclear staining was performed by treating 1 ⁇ g / mL 4 ′, 6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature in the dark.
  • DAPI 6-diamidino-2-phenylindole
  • the primary antibody was a rabbit anti-human sucrase-isomaltase antibody (Sigma; 1: 200) for 60 minutes at room temperature
  • the secondary antibody was an Alexa Fluor® 568 goat anti-rabbit IgG antibody (Invitrogen; 1: 500) for 60 minutes at room temperature in the dark.
  • ABCB1 / MDR1 ATP-binding cassette transporter B1 / multidrug resistance protein 1
  • CDX2 cartilage growth factor 2
  • CYP3A4 cytochrome P450 3A4
  • DPP4 dipeptidyl peptidase 4
  • LGR5 G protein-coupled receptor including leucine-rich repeat
  • SLC15A1 / PEPT1 SLC (solute carrier) family member 15A1 / peptide transporter 1: expressed on the apical membrane side of the small intestine.
  • SLC46A1 / PCFT SLC (solute carrier) family member 46A1 / proton conjugated folate transporter: expressed in the small intestine.
  • Sucrase-isomaltase A disaccharide-degrading enzyme present in the intestinal epithelium and a marker specific to the intestinal epithelium.
  • the serum concentration for differentiation into intestinal epithelial cells was examined.
  • the intestinal epithelium-specific marker sucrase-isomaltase, the peptide transporter expressed in the intestinal epithelium, SLC15A1 / PEPT1, and the main intestinal metabolism Increased expression of the enzyme CYP3A4 was observed (FIG. 2).
  • sucrase-isomaltase a marker specific to intestinal epithelial cells
  • ⁇ -Ala-Lys-AMCA was taken up
  • GSK3iXV and dorsomorphin were found to have an effect of promoting differentiation into intestinal stem cells.
  • PD98059, 5-aza-2′-deoxycytidine and A-83-01 were found to have an effect of promoting differentiation into intestinal epithelial cells. Therefore, in order to find conditions for inducing differentiation of iPS cells into intestinal epithelial cells more efficiently, the combined effect of these compounds was examined. As a result, after treatment with GSK3iXV and dorsomorphin, strong expression of sucrase-isomaltase was observed when treated with PD98059, 5-aza-2′-deoxycytidine and A-83-01 (FIG. 11 upper).
  • PD98059 (20 ⁇ M), 5-aza-2′-deoxycytidine (5 ⁇ M), and A-83-01 (0.5 ⁇ M) or SB431542 (10 ⁇ M) were added to the culture for 18 days from the 8th day after the start of differentiation. did.
  • G instead of PD98059, MEK inhibitor II (10 ⁇ M), PD0325901 (0.1 ⁇ M), PD184161 (1 ⁇ M), PD184352 (0.1 ⁇ M), SL327 (1 ⁇ M) or U0126 (10 ⁇ M) is used as a compound having the same action. used. The other conditions were the same as (f).
  • Intestinal specific homeobox (ISX): A transcription factor expressed in the intestinal epithelium.
  • Villin 1 a component of microvilli of small intestinal epithelial cells, expressed on the brush border of absorbing epithelial cells.
  • ABCG2 / BCRP ATP binding cassette transporter G2 / breast cancer resistance protein, efflux transporter.
  • CYP1A1 / 2, CYP2C9, CYP2C19, CYP2D6 These are enzymes involved in drug metabolism (phase 1 reaction).
  • Uridine diphosphate-glucuronyltransferase A drug metabolizing enzyme that performs glucuronidation reaction.
  • Sulfyltransferase A drug-metabolizing enzyme that performs a sulfate conjugation reaction.
  • (C) ⁇ -Ala-Lys-AMCA uptake experiment was performed as follows. First, the cells used for the uptake experiment were cultured on a cover glass. After the culture, the cells were incubated with DMEM / F12 containing 25 ⁇ M ⁇ -Ala-Lys-AMCA for 4 hours at 37 ° C. or 4 ° C. in a CO 2 incubator under 5% CO 2 /95% air. For inhibition experiments, ibuprofen was added to the uptake solution to a concentration of 10 mM. After incubation, uptake was stopped by washing the cells with ice-cold PBS. Immediately thereafter, fixation was performed using 4% paraformaldehyde at room temperature for 30 minutes.
  • the primary antibody was a rabbit anti-human sucrase-isomaltase antibody (Sigma; 1: 200) for 60 minutes at room temperature
  • the secondary antibody was an Alexa Fluor® 568 goat anti-mouse IgG antibody (Invitrogen; 1: 500) for 60 minutes at room temperature in the dark.
  • sucrase-isomaltase an intestinal specific marker, differentiation with PD98059, 5-aza2'-deoxycytidine, A-83-01 not only increased gene expression but also immunofluorescent staining positive cells The ratio of increased also (FIG. 15).
  • Intestinal epithelial cell-like cells differentiated with low molecular weight compounds show drug-metabolizing enzyme activities (cytochrome P450 (CYP), uridine diphosphate-glucuronosyltransferase and sulphate transferase activities). It has also been shown to have a function as (FIGS. 16 to 18).
  • CYP3A4 which is a major metabolic enzyme in the intestinal tract
  • gene expression was remarkably increased by using these low molecular compounds (FIG. 19).
  • Intestinal CYP3A4 is known to be induced through vitamin D receptors.
  • the expression of CYP3A4 was strongly induced compared to the control.
  • metabolic activity was increased by differentiation using low molecular weight compounds.
  • Induction of activity by 1 ⁇ , 25-dihydroxyvitamin D3 was also observed in intestinal epithelial cell-like cells differentiated with low molecular weight compounds, suggesting that it has a characteristic function in the intestine regarding drug metabolism.
  • dipeptide uptake was also observed in differentiated intestinal epithelial-like cells. This uptake was suppressed by adding ibuprofen, an inhibitor of the peptide transporter (SLC15A1 / PEPT1), and setting the uptake temperature to 4 ° C. (FIG. 20). This suggested that it has a transport function via a peptide transporter.
  • GSK3 ⁇ inhibitor, BMP inhibitor, TGF- ⁇ inhibitor, MEK inhibitor and DNA methylation inhibitor promote the differentiation of human iPS cells into intestinal epithelial cells.
  • the obtained cells were confirmed to be cells having a pharmacokinetic function characteristic of intestinal epithelial cells.
  • intestinal epithelial cell-like cells having a peptide transport function, drug metabolizing enzyme activity and induction ability were successfully produced from human iPS cells by a simple method. The details of the conditions required for the inducer were also clarified.
  • Intestinal epithelial cell-like cells can be easily and efficiently prepared from iPS cells.
  • Intestinal epithelial cell-like cells are useful as a model system for the small intestine, and can be used for evaluation of absorption, metabolism, and membrane permeability. Also, it is expected to be used as an active ingredient of cell preparations for treating various intestinal diseases or as a material for regenerative medicine.

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Abstract

L'objectif de la présente invention est de fournir une nouvelle méthode d'induction efficace de la différentiation de cellules souches pluripotentes induites en cellules épithéliales intestinales, et son utilisation. La différenciation de cellules souches pluripotentes induites en cellules épithéliales intestinales est induite par : une étape dans laquelle des cellules souches pluripotentes induites sont différenciées en cellules de type endoderme ; une étape dans laquelle les cellules de type endoderme obtenues à l'étape précédente sont différenciées en cellules de type cellule souche intestinale ; et une étape dans laquelle les cellules de type cellule souche intestinale obtenues à l'étape précédente sont différenciées en cellules de type cellule épithéliale intestinale, ladite étape incluant la culture en présence d'EGF et d'au moins un composé sélectionné dans le groupe constitué par des inhibiteurs de MEK1, des inhibiteurs de méthylation d'ADN, et des inhibiteurs du récepteur TFGβ.
PCT/JP2014/054379 2013-02-26 2014-02-24 Méthode d'induction d'une différenciation de cellules souches pluripotentes induites en cellules épithéliales intestinales Ceased WO2014132933A1 (fr)

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