JP6296399B2 - Methods for inducing differentiation of induced pluripotent stem cells into intestinal epithelial cells - Google Patents

Description

  The present invention relates to a method for inducing differentiation of induced pluripotent stem (iPS) into intestinal epithelial cells and use thereof. This application claims the priority based on the Japan patent application 2013-36434 for which it applied on February 26, 2013, The whole content of the said patent application is used by reference.

  Since there are many drug metabolizing enzymes and drug transporters in the small intestine, like the liver, it is very important as an organ involved in the first-pass effect of drugs. Therefore, evaluating membrane permeability and metabolism of pharmaceuticals in the small intestine from the early stage of pharmaceutical development is necessary for the development of pharmaceuticals with excellent pharmacokinetic properties. At present, human colon cancer-derived Caco-2 cells are frequently used as a model system for the small intestine. However, the expression pattern of drug transporters in Caco-2 cells is different from that in the human small intestine. In addition, since Caco-2 cells show almost no drug-metabolizing enzyme expression or enzyme induction, it is difficult to accurately evaluate the pharmacokinetics in the small intestine. Therefore, in order to comprehensively evaluate drug metabolism and membrane permeability in the small intestine, it is desirable to use primary small intestinal epithelial cells, but it is difficult to obtain primary small intestinal epithelial cells.

  By the way, human induced pluripotent stem (iPS) cells were established in 2007 by Yamanaka et al. This human iPS cell is a cell having pluripotency and almost unlimited proliferation ability similar to the human embryonic stem (ES) cell established by Thomson et al. In 1998. Human iPS cells have fewer ethical problems than human ES cells and are expected to be a stable cell source for drug development.

  In addition, 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 intestinal cells 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).

JP 2008-206510 A Special table 2012-511935 gazette

Ueda T et al., Biochem Biophys Res Commun. 2010 Jan 1; 391 (1): 38-42. McCracken KW et al., Nat Protoc. 2011 Nov 10; 6 (12): 1920-8 Spence JR, Nature. 2011 Feb 3; 470 (7332): 105-109.

  Currently, there are few reports on the differentiation of iPS cells into the intestinal tract. As far as the present inventors know, a report (Non-patent Document 1) 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. There are only reports (Non-Patent Documents 2 and 3). Further, 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. Furthermore, the differentiation induction method induces differentiation using a large amount of extremely expensive growth factors and cytokines and is not suitable for practical use.

  For practical application, it is desired to use a low-molecular compound that is inexpensive and easy to handle as a differentiation-inducing factor. In order to meet such a demand, 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.

  In view of the above problems, 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 TGFβ 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. Furthermore, 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 TGFβ 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.

  Intestinal epithelial cell-like cells that were successfully produced expressed various marker molecules specific to the intestinal epithelium and had a peptide transport function. In addition, induction of drug metabolizing enzyme (CYP3A4) expression via vitamin D receptor was also observed. Thus, it was confirmed that the cell which showed the characteristic very similar to an intestinal epithelial cell was acquired successfully.

  As a result of further investigation, the effectiveness of MEK1 inhibitor, DNA methylation inhibitor and TGFβ receptor inhibitor as inducers and the usefulness of this method were further supported.

The present invention shown below is mainly based on the above results and considerations by the present inventors.
[1] 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 TGFβ 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 TGFβ receptor inhibitor, and EGF.
[3] The method according to [1] or [2], wherein the culture period in step (3) is 7 to 30 days.
[4] The MEK1 inhibitor is PD98059, the DNA methylation inhibitor is 5-aza-2′-deoxycytidine, and the TGFβ 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 the step (1).
[6] The method according to any one of [1] to [5], wherein FGF2 is used as a differentiation inducing factor in the step (2).
[7] The method according to any one of [1] to [6], wherein 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) A step of culturing in the presence of EGF and one or more compounds selected from the group consisting of MEK1 inhibitor, DNA methylation inhibitor and TGFβ receptor inhibitor, following step (3-1) .
[8] The method according to [7], wherein the culture period of step (3-1) is 3 days to 14 days, and the culture period of step (3-2) is 3 days to 21 days.
[9] The method according to [7] or [8], wherein the GSK inhibitor is GSK3iXV and the BMP inhibitor is dorsomorphin.
[10] The method according to any one of [1] to [9], wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
[11] An intestinal epithelial cell-like cell obtained by the method according to any one of [1] to [10].
[12] A method for evaluating the pharmacokinetics of a test substance using the intestinal epithelial cell-like cells according to [11].
[13] 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].

Effect of FGF2, FGF4 and Wnt3a on differentiation from human iPS cells to intestinal stem cells. Each data was expressed as mean ± S.D. (N = 3). The addition amount of FGF2 and FGF4 is 250 ng / mL, and the addition amount of Wnt3a is 50 ng / mL. Effect of serum concentration on differentiation of human iPS cells into intestinal epithelial cells. Each data was expressed as mean ± S.D. (N = 3). N.D .: Cannot be detected. Expression of CDX2, DPP4, SLC46A1 / PCFT in differentiated intestinal epithelial cell-like cells. Each data was expressed as mean ± S.D. (N = 3). Differentiation schedule from human iPS cells to intestinal epithelial cells and morphological changes of the cells during the differentiation process. Immunofluorescence staining of sucrase-isomaltase in differentiated intestinal epithelial cell-like cells. (A) Immunofluorescence staining of sucrase-isomaltase (red). (B) Nuclear staining with DAPI (blue). (C) An image that combines (A) and (B). Scale bar is 50 μm. Uptake of β-Ala-Lys-AMCA in differentiated intestinal epithelial cell-like cells. (A) Intracellular uptake of β-Ala-Lys-AMCA (blue). (B) Immunofluorescence staining of sucrase-isomaltase (red). (C) An image that combines (A) and (B). Scale bar is 50 μm. Effect of GSK3 and BMP inhibitors on differentiation into intestinal epithelial cells. Each data was expressed as mean ± S.D. (N = 3). GSK3i is GSK3iXV and BMPi is Dorsomorphin. Effect of PD98059 and 5-aza-2'-deoxycytidine on differentiation into intestinal epithelial cells. Each data was expressed as mean ± S.D. (N = 3). N.D .: Cannot be detected. 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Effect of A-83-01 on differentiation into intestinal epithelial cells. Each data was expressed as mean ± S.D. (N = 3). Induction effect of 1α, 25-dihydroxyvitamin D _{3 on} CYP3A4 expression in intestinal epithelial cell-like cells differentiated with A-83-01. Each data was expressed as mean ± SD (n = 3). VD3 is l [alpha], a 25-dihydroxyvitamin D _3. Effect of low molecular weight compounds on differentiation into intestinal epithelial cells. Each data was expressed as mean ± SD (n = 3). GSK3i is GSK3iXV, BMPi is dorsomorphin, 5-aza-2′-dC is 5-aza-2′-deoxycytidine, VD3 is 1α, 25-dihydroxyvitamin D ₃ , and RIF is rifampicin. Effect of each compound on differentiation into intestinal epithelial cells. Intestinal specific homeobox (ISX) (top) and villin 1 (bottom) were used as marker genes. Each data was expressed as mean ± S.D. (N = 3). In the case of long-term addition, the compound was added from day 8 to day 26 (18 days) after the start of differentiation. GSK3i is GSK3iXV, BMPi is dorsomorphin, and 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Effect of each compound on differentiation into intestinal epithelial cells. Sucrase-isomaltase (upper) and SLC15A1 / PEPT1 (lower) were used as marker genes. Each data was expressed as mean ± S.D. (N = 3). In the case of long-term addition, the compound was added from day 8 to day 26 (18 days) after the start of differentiation. GSK3i is GSK3iXV, BMPi is dorsomorphin, and 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Effect of each compound on differentiation into intestinal epithelial cells. ABCB1 / MDR1 (top) and ABCG2 / BCRP (bottom) were used as marker genes. Each data was expressed as mean ± S.D. (N = 3). In the case of long-term addition, the compound was added from day 8 to day 26 (18 days) after the start of differentiation. GSK3i is GSK3iXV, BMPi is dorsomorphin, and 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Immunofluorescence staining of sucrase-isomaltase in intestinal epithelial cell-like cells differentiated with low molecular weight compounds. 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Scale bar is 500μm. Effect of each compound on differentiation into intestinal epithelial cells. CYP1A1 / 2 (top) and CYP2C9 (bottom) were used as marker genes. Each data was expressed as mean ± S.D. (N = 3). In the case of long-term addition, the compound was added from day 8 to day 26 (18 days) after the start of differentiation. GSK3i is GSK3iXV, BMPi is dorsomorphin, and 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Effect of each compound on differentiation into intestinal epithelial cells. CYP2C19 (top) and CYP2D6 (bottom) were used as marker genes. Each data was expressed as mean ± S.D. (N = 3). In the case of long-term addition, the compound was added from day 8 to day 26 (18 days) after the start of differentiation. GSK3i is GSK3iXV, BMPi is dorsomorphin, and 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Effect of each compound on differentiation into intestinal epithelial cells. Uridine diphosphate-glucuronyltransferase (upper) and sulfotransferase (lower) were used as marker genes. Each data was expressed as mean ± S.D. (N = 3). In the case of long-term addition, the compound was added from day 8 to day 26 (18 days) after the start of differentiation. GSK3i is GSK3iXV, BMPi is dorsomorphin, and 5-aza-2'-dC is 5-aza-2'-deoxycytidine. Inducing effect of 1α, 25-dihydroxyvitamin D _{3 on} CYP3A4 expression in intestinal epithelial cell-like cells differentiated under each condition. Each data was expressed as mean ± SD (n = 3). GSK3i is GSK3iXV, BMPi is dorsomorphin, 5-aza-2'-dC is 5-aza-2'-deoxycytidine, VD3 is l [alpha], a 25-dihydroxyvitamin D _3. Uptake of β-Ala-Lys-AMCA in intestinal epithelial cell-like cells differentiated with low molecular weight compounds. Between control (when the temperature condition during uptake is 37 ° C) (left), when an inhibitor (ibuprofen) is added (center), and when the temperature condition during uptake is 4 ° C (right) , Β-Ala-Lys-AMCA intracellular uptake (blue) was compared. Scale bar is 500μm. Differentiation induction by various compounds. The effects of TGF-β inhibitors and MEK inhibitors on differentiation into intestinal epithelial cells were verified using various compounds. The average value was expressed as ± S.D. (N = 3). Differentiation induction by various compounds. The effects of DNA methylation inhibitors on differentiation into intestinal epithelial cells were verified using various compounds. The average value was expressed as ± S.D. (N = 3). 5-aza-2'-dC is 5-aza-2'-deoxycytidine and 5-azaC is 5-azacytidine. Expression of LGR5 and sucrase-isomaltase in intestinal epithelial cell-like cells differentiated with a more effective compound combination. Each data was expressed as mean ± S.D. (N = 3). 5-aza-2'-dC is 5-aza-2'-deoxycytidine. SLC15A1 / PEPT1 expression (upper) in intestinal epithelial cell-like cells differentiated by a more effective combination of compounds, and the induction effect of 1α, 25-dihydroxyvitamin D _{3 on} CYP3A4 expression. Each data was expressed as mean ± SD (n = 3). 5-aza-2'-dC is 5-aza-2'-deoxycytidine and VD3 is 1α, 25-dihydroxyvitamin D3.

  The present invention relates to a method for inducing differentiation of an induced pluripotent stem cell (iPS cell) into an intestinal epithelial cell lineage (hereinafter also referred to as “differentiation induction method of the present invention”). According to the present invention, cells exhibiting characteristics similar to those of intestinal epithelial cells constituting intestinal tissue of a living body, that is, intestinal epithelial cell-like cells can be obtained.

  “Artificial pluripotent stem cells (iPS cells)” are cells having pluripotency (multipotency) and proliferative ability, which are 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.

  The most basic method for producing iPS cells 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). 3 factors except c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), Oct3 / 4 and Klf4 (Kim JB, et al: Nature 454 (7204) , 646-650, 2008), or the establishment of iPS cells by introducing only Oct3 / 4 (Kim JB, et al: Cell 136 (3), 411-419, 2009) has been reported. In addition, a method for introducing a protein, which is an expression product of a gene, into a cell (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al : Cell Stem Cell 4, 472-476, 2009). On the other hand, by using the inhibitor BIX-01294 for histone methyltransferase G9a, the histone deacetylase inhibitor valproic acid (VPA) or BayK8644, production efficiency can be improved and factors to be introduced can be reduced. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J , et al: PLoS. Biol. 6 (10), e 253, 2008). Studies on gene transfer methods are also underway, and in addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903 ), 945-949, 2008), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vector (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766- 770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009), or A technique using an episomal vector (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) has been developed.

  Cells that have undergone transformation (reprogramming) into iPS cells are expressed pluripotent stem cell markers (undifferentiation 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 the RIKEN BioResource Center.

  As used herein, “inducing differentiation” refers to acting to differentiate along a specific cell lineage. In the present invention, 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)). Hereinafter, details of each process will be described.

<Process (1) Differentiation into endoderm-like cells>
In this step, iPS cells are cultured and differentiated into endoderm-like cells. In other words, 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. For example, the cells are cultured in a medium supplemented with activin A according to a conventional method. In this case, 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. From the viewpoint of cell growth rate and maintenance, it is preferable to add serum or serum replacement (Knockout serum replacement (KSR), etc.) to the medium. 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 / β-catenin signaling pathway (for example, hexachlorophene, quercetin, Wnt3a which is a Wnt ligand) may be added to the medium to promote differentiation into endoderm-like cells.

  In a preferred embodiment, 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. In 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 (culture period) of the step (1) is, for example, 1 day to 10 days, preferably 2 days to 7 days. When adopting a two-stage culture as the step (1), the first stage culture period is, for example, 1 day to 7 days, preferably 2 days to 5 days, and the second stage culture period is, for example, 1 day to 6 days. Days, preferably 1 to 4 days.

<Step (2) Differentiation into intestinal stem cell-like cells>
In this step, the endoderm-like cells obtained in step (1) are cultured and differentiated into intestinal stem cell-like cells. In other words, 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. Preferably, the culture is performed in the presence of FGF2 (fibroblast growth factor 2) based on the experimental results shown in the Examples described later. Preferably, human FGF2 (for example, human recombinant FGF2) is used.

  Typically, the cell population obtained through the step (1) or a part thereof is subjected to the step (2) without sorting. On the other hand, after selecting endoderm-like cells from the cell population obtained through step (1), step (2) may be carried out. For example, the endoderm-like cells may be selected with a flow cytometer (cell sorter) using a cell surface marker as an index.

  “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 (culture period) of the step (2) 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.

  Differentiation into intestinal stem cell-like cells can be determined or evaluated using, for example, the expression of an intestinal stem cell marker as an index. Examples of 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>
In this step, one or more compounds selected from the group consisting of MEK1 inhibitor, DNA methylation inhibitor and TGFβ receptor inhibitor (hereinafter also referred to as “first inducer”) and EGF (hereinafter referred to as “second” In the presence of an inducer), the intestinal stem cell-like cells obtained in step (2) are differentiated into intestinal epithelial cell-like cells. Typically, the cell population obtained through the step (2) or a part thereof is subjected to the step (3) without sorting. On the other hand, after intestinal stem cell-like cells are selected from the cell population obtained through the step (2), 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.

  The presence of one or more compounds selected from the group consisting of MEK1 inhibitor, DNA methylation inhibitor and TGFβ receptor inhibitor (first inducer) and EGF (second inducer) is the first inducer And the second inducing factor 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. Similarly, examples of DNA methylation inhibitors include 5-aza-2'-deoxycytidine, 5-azacytidine, RG108, and zebralin. As for the TGFβ receptor inhibitor, it is preferable to consider that A-83-01 used in the examples described later exhibits inhibitory activity on TGF-β receptors ALK4, ALK5, and ALK7. What shows inhibitory activity with respect to one or more of ALK4, ALK5, and ALK7 may be used. For example, A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox satisfy the conditions.

  An example of the concentration of MEK1 inhibitor added (in the case of PD98059) is 4 μM to 100 μM, preferably 10 to 40 μM. Similarly, an example of the addition concentration of the methylation inhibitor (in the case of 5-aza-2′-deoxycytidine) is 1 μM to 25 μM, preferably 2.5 μM to 10 μM, and the addition concentration of the TGFβ receptor inhibitor An example (in the case of A-83-01) is 0.1 μM to 2.5 μM, preferably 0.2 μM to 1 μM.

  In a preferred embodiment, two or more of MEK1 inhibitor, DNA methylation inhibitor, and TGFβ receptor inhibitor are used in combination as the first inducer. 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. Most preferably, all (ie, three types) of first inducers are used in combination. As shown in the examples described later, 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 TGFβ receptor inhibitor) It was confirmed that differentiation was further promoted when 83-01 was used in combination.

  The period (culture period) of the step (3) 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.

  Differentiation into intestinal epithelial cell-like cells 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. Examples of 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 . Among them, sucrase-isomaltase having high specificity for the intestinal epithelium, CYP3A4, which is a major drug-metabolizing enzyme in the small intestine, and SLC15A1 / PEPT1 involved in peptide absorption in the small intestine are particularly effective markers.

  If you want to obtain 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. The cultured cell population may be selected and sorted as an index.

  In a preferred embodiment, 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 TGFβ receptor inhibitors The step (3-2) of culturing is performed. Therefore, in this embodiment, the culture in the presence of a GSK inhibitor and / or a 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.

  When performing step (3-1) and step (3-2) 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.

In each step (steps (1), (2), (3), (3-1), (3-2)) constituting the present invention, subculture may be performed midway. For example, when the cells become confluent or subconfluent, a part of the cells is collected and transferred to another culture vessel, and the culture is continued. In order to promote differentiation, it is preferable to set the cell density low. For example, the cells may be seeded at a cell density of about 1 × 10 ⁴ cells / cm ² to 1 × 10 ⁶ cells / cm ² .

  When collecting cells due to medium exchange or subculture, in order to suppress cell death, cells are pre-treated with a ROCK inhibitor (Rho-associated coiled-coil forming kinase / Rho-binding kinase) such as Y-27632. Should be processed.

Other culture conditions (culture temperature etc.) in each step (steps (1), (2), (3), (3-1), (3-2)) constituting the present invention are animal cell cultures. In this case, the conditions generally adopted may be used. That is, for example, it may be cultured in an environment of 37 ° C. and 5% CO ₂ . In addition, as a basic medium, Iskov modified Dulbecco medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, Gibco, etc.), Dulbecco modified Eagle medium (D-MEM) (Nacalai Tesque, Sigma, Gibco, etc.), Glasgow basic medium (Gibco, etc.), RPMI1640 medium, etc. can be used. Two or more basic media may be used in combination. In steps (2), (3), (3-1), and (3-2), a basic medium (for example, a mixed medium of D-MEM and Ham F12 medium, D-MEM) suitable for epithelial cell culture is used. It is preferable to use it. Examples of 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. Typically, cells are cultured two-dimensionally using a culture dish or the like. According to the method of the present invention, intestinal epithelial cell-like cells can be obtained from iPS cells by two-dimensional culture. However, 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. In other words, the intestinal epithelial cell-like cell of the present invention can be used for evaluating the pharmacokinetics of a compound.

  Specifically, the intestinal epithelial cell-like cell of the present invention can be used to test the absorbability or membrane permeability of a 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. In the method, (i) 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; (Iii) 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).

  In step (i), intestinal epithelial cell-like cells are typically cultured on a semipermeable membrane (porous membrane) to form a cell layer. Specifically, for example, by using a culture vessel equipped with a culture insert (for example, Transwell (registered trademark) provided by Corning), seeding and culturing cells in the culture insert, intestinal epithelial cells A cell layer composed of like-like cells is obtained.

  “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.

  As the test substance, organic compounds or inorganic compounds having various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) Existing components or candidate components such as pharmaceuticals and nutritional foods are also preferable test substances. Extracts, cell extracts, culture supernatants, etc. 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 arbitrarily set. 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.

  In the step (iii), the test substance that has permeated the cell layer is quantified. For example, when a culture vessel equipped with a culture insert such as Transwell (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. Based on the quantification result (the amount of the test substance that has permeated the cell layer) and the amount of the test substance used (typically, the amount added to the medium), the absorbability or membrane permeability of the test substance is judged and evaluated. .

  As another aspect (second aspect), the present invention also provides a method for evaluating metabolism or absorption of a test substance. In 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. .

  The step (I), that is, the contact between the intestinal epithelial cell-like cell and the test substance can be carried out in the same manner as in the above step (ii).

  After step (I), the metabolism or absorption of the test substance is measured and evaluated (step (II)). Immediately after step (I), that is, after contact with the test substance, metabolism or absorption may be 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.

  Typically, when a metabolite of a test substance is detected, it is determined or evaluated that “the test substance has been metabolized”. Further, 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).

  It is also possible to measure the metabolism of a test substance using the expression of drug metabolizing enzymes (cytochrome (particularly CYP3A4), UGT (particularly UGT1A8, UGT1A10), SULT1A3, etc.) in intestinal epithelial cell-like cells as an index. 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”. As in the case of determining a metabolite as an index, quantitative determination / evaluation may be performed based on the expression level of a drug metabolizing enzyme.

  In order to evaluate the absorption of the test substance, for example, the remaining amount of the test substance in the culture solution is measured. Usually, 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. Typically, when a decrease in the content of the test substance in the culture medium is observed, it is determined and evaluated that “the test substance has been absorbed”. Further, 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.

  Metabolism measurement / evaluation and absorption measurement / evaluation 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. For example, 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.

  Dimethyl sulfoxide (DMSO) and 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. In addition, 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.

<Examination of conditions for inducing differentiation of intestinal epithelial cells 1>
Aiming at the construction of a model system of the small intestine useful for pharmacokinetic studies, conditions for inducing differentiation of intestinal epithelial cells from human iPS cells were examined. In the following study, we focused on the possibility of low molecular weight compounds as differentiation-inducing factors, and aimed to produce cells that can be used in evaluation systems for pharmacokinetic tests by a simpler method.

1. 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.

(2) Medium
For the culture of MEF, Dulbecco containing 10% fetal bovine serum (FBS), 2 mmol / L L-glutamine (L-Glu), 1% non-essential amino acid (NEAA), 100 units / mL penicillin G, 100 μg / mL streptomycin Modified Eagle medium (DMEM) was used. 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) was used as the MEF stripping solution, and Cell Banker 1 was used as the MEF stock solution. 20% knockout serum replacement (KSR), 0.8% NEAA, 2 mmol / L L-Glu, 0.1 mmol / L 2-mercaptoethanol (2-MeE), 5 ng / mL fibroblasts for maintenance culture of human iPS cells DMEM Ham's F-12 (DMEM / F12) containing cell growth factor (FGF) 2 was used. Dulbecco's phosphate buffered saline (PBS) containing 1 mg / mL collagenase IV, 0.25% trypsin, 20% KSR, 1 mmol / L calcium chloride was used as the human iPS cell detachment solution. As a preservation solution for human iPS cells, a cryopreservation solution for primate ES / iPS cells was used.

(3) Culture of human iPS cells 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.

(4) Differentiation of human iPS cells into intestinal epithelial cells Differentiation of human iPS cells into intestinal epithelial cells started when human iPS cells accounted for approximately 70% of the undifferentiated colony in the culture dish. did. 2% FBS, 100 ng / mL activin A for 2 days in Roswell Park Memorial Institute (RPMI) + glutamax medium containing 0.5% FBS, 100 ng / mL activin A, 100 units / mL penicillin G, 100 μg / mL streptomycin It was differentiated into endoderm by culturing in RPMI + glutamax medium containing 100 units / mL penicillin G and 100 μg / mL streptomycin for 1 day. Thereafter, 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. After this treatment, Y-27632 (Rho binding kinase inhibitor) was added to 10 μmol / L, and cells treated at 37 ° C in a CO ₂ incubator for 60 minutes under 5% CO ₂ /95% air conditions 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. Then 2% FBS, 2 mmol / L L-Glu, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 100 units / mL penicillin G, 100 μg / mL streptomycin, 20 ng / mL epidermal growth factor (EGF ), 1 day in DMEM / F12 containing 10 μmol / L Y-27632, 2% FBS, 2 mmol / L L-Glu, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 100 units / mL penicillin G It was differentiated into intestinal epithelial cells by culturing in DMEM / F12 containing 100 μg / mL streptomycin and 20 ng / mL epidermal growth factor (EGF) for 18 days. 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 μg / mL streptomycin, 20 ng / Add 1α, 25-dihydroxyvitamin D ₃ (VD3) to 10 nmol / L or rifampicin 40 μmol / L to DMEM / F12 containing mL epidermal growth factor (EGF) and incubate for 48 hours before recovery. I went there.

In order to evaluate the effect of low molecular weight compounds on differentiation induction, culture was also performed under the following conditions (a) to (c).
(A) GSK3iXV (125 nM) and dorsomorphin (1 μM) 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 μM), 5-aza-2′-deoxycytidine (5 μM), and A-83-01 (0.5 μM) were added to the culture for 18 days from the start of differentiation (other culture conditions) Is as above).
(C) GSK3iXV (125 nM) and dorsomorphin (1 μM) were added to the culture solution for 6 days from the 8th day after the start of differentiation, and PD98059 (20 μM), 5-aza-2′-deoxycytidine (5 μM), A- 83-01 (0.5 μM) was added for 12 days from the 14th day after the start of differentiation (other culture conditions were as described above).

(5) Total ribonucleic acid (RNA) extraction Total 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.

(6) Reverse Transcription Reaction For the synthesis of complementary DNA (cDNA), PrimeScript (registered trademark) RT reagent Kit (Perfect Real Time) (Takara Bio Inc.) was used. The operation followed the attached manual.

(7) Real-time reverse transcription polymerase chain reaction (Real-Time RT-PCR)
Real-Time RT-PCR was performed using cDNA as a template using SYBR (registered trademark) Premix Ex Taq II (Perfect Real Time) (Takara Bio Inc.). The operation followed the attached manual. The measurement result was corrected using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control.

(8) 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 (registered trademark) 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 μg / mL 4 ′, 6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature in the dark.

(9) β-Ala-Lys-AMCA uptake experiment Cells used in the uptake experiment were cultured on a cover glass. After culturing, the cells were incubated with DMEM / F12 containing 25 μM β-Ala-Lys-AMCA for 4 hours at 37 ° C. in a CO ₂ incubator under 5% CO ₂ /95% air. 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. Thereafter, when immunostaining was performed, membrane permeation treatment was performed for 5 minutes at room temperature using 0.1% Triton X, and blocking treatment was performed for 20 minutes at room temperature using 2% skim milk. Furthermore, 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 (registered trademark) 568 goat anti-rabbit IgG antibody (Invitrogen; 1: 500) for 60 minutes at room temperature in the dark.

The characteristics of the marker gene used in this study are shown below.
ABCB1 / MDR1 (ATP-binding cassette transporter B1 / multidrug resistance protein 1): P glycoprotein that functions as an efflux transporter.
CDX2 (caudal homeobox transcription factor 2): A transcription factor involved in intestinal differentiation.
CYP3A4 (cytochrome P450 3A4): a major drug-metabolizing enzyme in the small intestine.
DPP4 (dipeptidyl peptidase 4): A serine protease that is also expressed in the intestinal epithelium.
LGR5 (G protein-coupled receptor including leucine-rich repeat): a marker for intestinal stem cells.
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.

2. Results (1) Examination of differentiation conditions into intestinal epithelial cells Various conditions (differentiation-inducing factors, etc.) were examined in inducing differentiation of human iPS cells into intestinal epithelial cells. First, when the effect of FGF2, FGF4, and Wnt3a on differentiation into intestinal stem cells was examined, LGR, which is a marker for intestinal stem cells, was higher in the FGF2-treated group than in the FGF4-treated group. Moreover, the expression was not changed by Wnt3a treatment (FIG. 1).

  Next, the serum concentration for differentiation into intestinal epithelial cells was examined. As a result, by setting the FBS concentration in the medium to 2%, 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).

  In cells differentiated into intestinal epithelial cells (ie, intestinal epithelial cell-like cells), CDX2, which is a transcription factor involved in intestinal differentiation, and DPP4 and SLC46A1, which are abundant in intestinal epithelium, are almost the same as human small intestine or Further expression was observed (FIG. 3). A dome-like structure was also observed in the differentiated cells (FIG. 4). Furthermore, when the protein expression of sucrase-isomaltase was confirmed by immunofluorescence staining, sufficient expression was observed (FIG. 5). In this way, human iPS cells were successfully induced to differentiate into intestinal epithelial cells by treatment with FGF2 and two-dimensional culture employing 2% FBS concentration.

(2) Uptake of β-Ala-Lys-AMCA in differentiated intestinal epithelial cell-like cells A peptide transporter is expressed in the intestinal epithelium, which is involved in absorption of the peptide from the intestinal tract. Since intestinal epithelial cell-like cells obtained by differentiation of human iPS cells expressed the peptide transporter SLC15A1 / PEPT1, we decided to confirm its function. As a result, uptake of β-Ala-Lys-AMCA, a peptide transporter substrate, was observed in intestinal epithelial cell-like cells (FIG. 6A). Furthermore, expression of sucrase-isomaltase, a marker specific to intestinal epithelial cells, was also observed in cells in which β-Ala-Lys-AMCA was taken up (FIGS. 6B and 6C). Thus, it was shown that human iPS cells could be induced to differentiate into intestinal epithelial cell-like cells having a peptide transport function.

(3) Effect of low molecular weight compounds on differentiation into intestinal epithelial cells It is known that activation of Wnt signals and suppression of BMP signals are involved in the proliferation of intestinal stem cells. Therefore, we investigated the effects of GSK inhibitors and BMP inhibitors on differentiation into intestinal stem cells using GSK3iXV, which activates Wnt signals by inhibiting GSK, and dorsomorphin, a BMP inhibitor. As a result, the expression of LGR5 and EphB2, which are markers of intestinal stem cells, was increased by treatment with these two low molecular compounds from day 8 to day 14 after the start of differentiation (FIG. 7). From this, it was suggested that it can differentiate efficiently into an intestinal stem cell by controlling a signal related to differentiation of the intestinal tract.

  Next, regarding the differentiation from intestinal stem cells into intestinal epithelial cells, studies using low molecular weight compounds were performed. Activation of the MEK pathway inhibits differentiation into intestinal epithelial cells, and it has been reported that DNA methylation is involved in CYP expression (Lemleux E, et al: Am. J Physiol. Gastrointest. Liver Phsiol. 301 (4), G719-G730, 2011; Habano W, et al: BMC Cancer 11, 81, 2011). Therefore, when inducing differentiation from intestinal stem cells to intestinal epithelial cells, treatment with PD98059, a MEK1 inhibitor, and 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, sucrase-isomaltase, LGR5, Increased expression levels of SLC15A1 / PEPT1 and CYP3A4 were observed (FIG. 8).

In addition, when treated with A-83-01, an inhibitor of TGF-β receptors ALK4, ALK5, and ALK7, a dramatic increase in the expression levels of sucrase-isomaltase, SLC15A1 / PEPT1, and ABCB1 / MDR1 was observed. (FIG. 9). On the other hand, the expression level of LGR5 decreased (FIG. 9). Moreover, regarding CYP3A4, an increase in the expression level was observed by treatment with A-83-01 (FIG. 10). Furthermore, when treated with 1α, 25-dihydroxyvitamin D ₃ which is a ligand of vitamin D receptor for 48 hours, the expression of CYP3A4 increased about 30-fold and enzyme induction was also observed (FIG. 10). A-83-01 has an inhibitory effect on TGF-β-induced epithelial-mesenchymal transition. The above results suggest that this inhibitory action is effective for differentiation into intestinal epithelial cells.

As described above, GSK3iXV and dorsomorphin were found to have an effect of promoting differentiation into intestinal stem cells. On the other hand, 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 (upper figure 11). In addition, the combined use of these compounds increased the expression of CYP3A4 by about 20 times, and the treatment with 1α, 25-dihydroxyvitamin D ₃ further induced the expression by about 10 times (lower part of FIG. 11). On the other hand, no induction of expression was observed with rifampicin. This result shows that intestinal epithelial cell-like cells have functions characteristic of intestinal epithelial cells.

  From the above results, it was revealed that these low molecular weight compounds are useful for differentiation from human iPS cells to intestinal epithelial cells and can promote differentiation more effectively when used in combination. When PD98059, 5-aza-2′-deoxycytidine and A-83-01 were used in combination, the expression levels of sucrase-isomaltase and CYP3A4 were higher than when they were used alone (FIG. 11). ) Regardless of the use of GSK3iXV and dorsomorphin, it can be said that combining these three low molecular weight compounds is effective.

3. Summary As described above, we succeeded in producing functional intestinal epithelial cell-like cells (with peptide transport function and drug-metabolizing enzyme-inducing ability) from human iPS cells by a simple method, MEK1 inhibitor, DNA methyl Inhibitor, TGF-β receptor inhibitor, GSK inhibitor and BMP inhibitor (especially MEK1 inhibitor, DNA methylation inhibitor and TGF-β receptor inhibitor in combination, MEK1 inhibitor, DNA methylation) Inhibitors, TGF-β receptor inhibitors, combined use of GSK inhibitors and BMP inhibitors) were found to be useful as inducers for inducing differentiation of human iPS cells into intestinal epithelial cells. Establishment of a simple and inexpensive differentiation induction method using low molecular weight compounds is considered to be a great achievement.

<Examination of conditions for inducing differentiation of intestinal epithelial cells 2>
Further studies were conducted regarding the effectiveness of MEK1 inhibitors, DNA methylation inhibitors and TGFβ receptor inhibitors as inducers. Specifically, the addition timing and combination of these compounds were examined in detail, and a group of compounds having the same action was used to examine the relationship between the action of the compound and the induction effect.

1. Method The experimental method and the experimental conditions were the same as those described in <Examination of conditions for inducing differentiation of intestinal epithelial cells 1> except for the following points (A) to (C).
(A) As the culture conditions for differentiating human iPS cells into intestinal epithelial cells (corresponding to 1. (4) of <Study 1 for inducing differentiation of intestinal epithelial cells> corresponds), the following (d) to ( i) was set and compared.
(D) GSK3iXV (125 nM), dorsomorphin (1 μM) for 6 days from the 8th day after initiation of differentiation, then PD98059 (20 μM), 5-aza-2′-deoxycytidine (5 μM), and A-83-01 (0.5 μM) or SB431542 (10 μM) was added to the culture for 12 days from the 14th day after the start of differentiation.
(E) PD98059 (20 μM), 5-aza-2′-deoxycytidine (5 μM), and A-83-01 (0.5 μM) or SB431542 (10 μM) are added to the culture for 12 days from the 14th day after the start of differentiation. did.
(F) PD98059 (20 μM), 5-aza-2′-deoxycytidine (5 μM), and A-83-01 (0.5 μM) or SB431542 (10 μM) 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 μM), PD0325901 (0.1 μM), PD184161 (1 μM), PD184352 (0.1 μM), SL327 (1 μM) or U0126 (10 μM) is used as a compound having the same action. used. The other conditions were the same as (f).
(H) Instead of 5-aza-2′-deoxycytidine (5 μM), 5-azacytidine (10 μM), RG108 (10 μM) or zebraline (10 μM) was used as a compound having the same action. The other conditions were the same as (f).
(I) Instead of A-83-01 (0.5 μM), D4476 (10 μM), GW788388 (10 μM), LY364947 (5 μM), RepSox (10 μM) or SB431542 (10 μM) was used as a compound exhibiting the same action. . The other conditions were the same as (f).

(B) In addition to those listed above, the following marker genes were also used as indicators for inducing differentiation into intestinal epithelial cells.
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) β-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 μM β-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. Thereafter, when immunostaining was performed, membrane permeation treatment was performed for 5 minutes at room temperature using 0.1% Triton X, and blocking treatment was performed for 20 minutes at room temperature using 2% skim milk. Furthermore, 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-mouse IgG antibody (Invitrogen; 1: 500) for 60 minutes at room temperature in the dark.

2. Result It is an intestinal marker gene by using GSK3i XV, Dorsomorphin, PD98059, 5-aza2'-deoxycytidine, A-83-01 or SB431542 during differentiation from human iPS cells to intestinal epithelial cells Expression of SLC15A1, ABCB1 / MDR1, and ABCG2 / BCRP, which are drug transporters expressed in the intestinal specific homeobox (ISX), villin 1, sucrase-isomaltase, and intestinal tract, increased (FIGS. 12 to 14). With respect to sucrase-isomaltase, an intestinal specific marker, differentiation with PD98059, 5-aza-2'-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), and intestinal epithelial cells It was also shown to have a function as (FIGS. 16 to 18).

  For 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. Thus, when differentiated intestinal epithelial cell-like cells were treated with 1α, 25-dihydroxyvitamin D3, the expression of CYP3A4 was strongly induced compared to the control. In addition to gene expression, metabolic activity was increased by differentiation using low molecular weight compounds. Induction of activity by 1α, 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. Furthermore, 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.

  In order to investigate whether the above-mentioned differentiation-inducing effect is unique to each low molecular weight compound used or due to the action of these low molecular weight compounds, PD98059, 5-aza 2'-deoxycytidine, A-83- The effect of a compound group (TGF-β inhibitor, MEK inhibitor, DNA methylation inhibitor) having the same action as 01 was examined. As a result, there was a tendency that differentiation into intestinal epithelial cells was promoted by using a TGF-β inhibitor, a MEK inhibitor, and a DNA methylation inhibitor (FIGS. 21 and 22). In addition, by combining GW788388, MEK inhibitor II and zebralin, which are considered to be more effective among these compounds, increased expression of sucrase-isomaltase and CYP3A4 genes, and 1α, 25-dihydroxyvitamin D3 The same effects as when differentiation was performed using PD98059, 5-aza-2′-deoxycytidine and A-83-01, such as induction of CYP3A4 expression, were observed (FIGS. 23 and 24).

  The above results confirmed that GSK3β inhibitor, BMP inhibitor, TGF-β 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.

3. Summary As described above, 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.

  According to the present invention, functional 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. In addition, 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.

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

Claims (14)

A method for inducing differentiation of an induced pluripotent stem cell into an intestinal epithelial cell, 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) A step of differentiating the intestinal stem cell-like cells obtained in step (2) into intestinal epithelial cell-like cells in the presence of a MEK1 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and EGF Including culturing in   The method according to claim 1, wherein the culture period in step (3) is 7 days to 30 days.   The method according to claim 1 or 3, wherein the MEK1 inhibitor is PD98059, the DNA methylation inhibitor is 5-aza-2'-deoxycytidine, and the TGFβ receptor inhibitor is A-83-01.   The method according to any one of claims 1, 3, and 4, wherein activin A is used as a differentiation-inducing factor in the step (1).   The method according to any one of claims 1 and 3 to 5, wherein FGF2 is used as a differentiation inducing factor in the step (2). The method according to any one of claims 1 and 3 to 6, wherein 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) A step of culturing in the presence of EGF and one or more compounds selected from the group consisting of a MEK1 inhibitor, a DNA methylation inhibitor and a TGFβ receptor inhibitor following the step (3-1). .   The method according to claim 7, wherein the culture period in the step (3-1) is 3 days to 14 days, and the culture period in the step (3-2) is 3 days to 21 days.   The method according to claim 7 or 8, wherein the GSK inhibitor is GSK3iXV and the BMP inhibitor is dorsomorphin.   The method according to any one of claims 1 and 3 to 9, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.   Intestinal epithelial cell-like cells obtained by the method according to any one of claims 1, 3 to 10.   A method for evaluating the pharmacokinetics of a test substance using the intestinal epithelial cell-like cell according to claim 11. The method according to claim 12, comprising the following steps (i) to (iii):
(I) a step of preparing a cell layer composed of intestinal epithelial cell-like cells according to claim 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. The method of claim 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 claim 11;
(II) A step of measuring / evaluating the metabolism or absorption of the test substance.   A cell preparation comprising the intestinal epithelial cell-like cell according to claim 11.

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