JP7452799B2 - Method for producing hepatocytes - Google Patents

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JP7452799B2
JP7452799B2 JP2020563365A JP2020563365A JP7452799B2 JP 7452799 B2 JP7452799 B2 JP 7452799B2 JP 2020563365 A JP2020563365 A JP 2020563365A JP 2020563365 A JP2020563365 A JP 2020563365A JP 7452799 B2 JP7452799 B2 JP 7452799B2
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健二 長船
勝太郎 安田
伸二 上本
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Description

本発明は細胞関連技術に関し、より詳しくは機能的に成熟した哺乳動物肝細胞の製造方法に関する。 The present invention relates to cell-related technology, and more particularly to a method for producing functionally mature mammalian hepatocytes.

現在、人工多能性幹(iPS)細胞や胚性幹(ES)細胞から分化誘導される肝細胞や、線維芽細胞など肝細胞以外の細胞種に転写因子の組み合わせを導入することによって作製される誘導肝細胞を用いた、肝細胞移植療法(肝臓再生医療)、肝臓の再構築、肝疾患モデル作製、創薬、薬剤肝毒性評価系の構築などが期待されている。しかし、現在までのところ、機能的に未熟な胎児期の肝細胞に相当する肝細胞の作製にとどまり、成体の体内に存在する肝細胞に近い機能を有する肝細胞を作製することは可能となっていない。 Currently, hepatocytes that are induced to differentiate from induced pluripotent stem (iPS) cells or embryonic stem (ES) cells, and cell types other than hepatocytes such as fibroblasts, are produced by introducing a combination of transcription factors. The use of induced hepatocytes is expected to be used for hepatocyte transplantation therapy (liver regenerative medicine), liver reconstruction, liver disease model creation, drug discovery, and the construction of drug hepatotoxicity evaluation systems. However, to date, we have only been able to create hepatocytes that correspond to functionally immature fetal hepatocytes, and it has not been possible to create hepatocytes that have functions similar to the hepatocytes that exist in the adult body. Not yet.

非特許文献1には、マウス胎仔および成体線維芽細胞にHnf4aと、Foxa1、Foxa2またはFoxa3の遺伝子を導入することで誘導肝細胞を作製したことが報告されている。
非特許文献2には、マウス成体線維芽細胞にGata4、Hnf1aおよびFoxa3の遺伝子を導入することで誘導肝細胞を作製したことが報告されている。
非特許文献3には、マウス胎仔線維芽細胞にHnf1bおよびFoxa3の遺伝子を導入することで誘導肝細胞を作製したことが報告されている。
非特許文献4には、ヒト線維芽細胞にFOXA3、HNF1AおよびHNF4Aの遺伝子を導入することで誘導肝細胞を作製したことが報告されている。
非特許文献5には、ヒト線維芽細胞にHNF1A、HNF4A、HNF6、ATF5、PROX1およびCEBPAの遺伝子を導入することで誘導肝細胞を作製したことが報告されている。
しかしながら、これまでに報告されている誘導肝細胞は肝マーカーの発現量が少なく、機能的に未熟で、上記のような再生医療等の目的に使用できるものではなかった。
Non-Patent Document 1 reports that induced hepatocytes were produced by introducing Hnf4a and Foxa1, Foxa2, or Foxa3 genes into mouse embryos and adult fibroblasts.
Non-Patent Document 2 reports that induced hepatocytes were produced by introducing Gata4, Hnf1a, and Foxa3 genes into mouse adult fibroblasts.
Non-Patent Document 3 reports that induced hepatocytes were produced by introducing Hnf1b and Foxa3 genes into mouse fetal fibroblasts.
Non-Patent Document 4 reports that induced hepatocytes were produced by introducing the genes of FOXA3, HNF1A, and HNF4A into human fibroblasts.
Non-Patent Document 5 reports that induced hepatocytes were produced by introducing the genes of HNF1A, HNF4A, HNF6, ATF5, PROX1, and CEBPA into human fibroblasts.
However, the induced hepatocytes that have been reported so far express low levels of liver markers and are functionally immature, so they cannot be used for purposes such as regenerative medicine as described above.

Sekiya S. et al., Nature 475(7356), 390-393 (2011)Sekiya S. et al., Nature 475(7356), 390-393 (2011) Huang P. et al., Nature 475(7356), 386-389 (2011)Huang P. et al., Nature 475(7356), 386-389 (2011) Yu B. et al., Cell Stem Cell 13(3) 328-340, (2013)Yu B. et al., Cell Stem Cell 13(3) 328-340, (2013) Huang P. et al., Cell Stem Cell 14(3), 370-384 (2014)Huang P. et al., Cell Stem Cell 14(3), 370-384 (2014) Du Y. et al., Cell Stem Cell 14(3), 394-403 (2014):Du Y. et al., Cell Stem Cell 14(3), 394-403 (2014):

本発明は機能的に成熟した肝細胞を効率よく製造する方法を提供することを課題とする。 An object of the present invention is to provide a method for efficiently producing functionally mature hepatocytes.

上記課題を解決するために、発明者らは、マウス成体肝細胞に発現する遺伝子の発現プロファイルを探索し、iPS干渉法(Hikichi T. 2013 Apr 16;110(16):6412-7)を用いて成熟肝細胞を誘導する転写因子の組み合わせの絞り込みを行った。その結果、Hnf4a(Hepatocyte nuclear factor 4 alpha)、Foxa3(Forkhead Box A3)、Cebpa(CCAAT/enhancer-binding protein alpha)、Cebpd(CCAAT/enhancer-binding protein delta)、Hnf6(Hepatocyte nuclear factor 6)およびOnecut2(One cut domain family member 2)の6種類の転写因子の組み合わせ(6因子)の遺伝子導入により、マウスおよびヒトの線維芽細胞から最も機能的に成熟した肝細胞を作製することに成功した。また、前述の6因子に加えMycを導入すること、およびTGF-β受容体阻害剤を用いて培養することにより、機能的成熟度をさらに上げることができることを見出した。以上の発見に基づき、本発明を完成させた。 In order to solve the above problems, the inventors searched the expression profile of genes expressed in mouse adult hepatocytes and used iPS interference method (Hikichi T. 2013 Apr 16;110(16):6412-7). We narrowed down the combination of transcription factors that induce mature hepatocytes. As a result, Hnf4a (Hepatocyte nuclear factor 4 alpha), Foxa3 (Forkhead Box A3), Cebpa (CCAAT/enhancer-binding protein alpha), Cebpd (CCAAT/enhancer-binding protein delta), Hnf6 (Hepatocyte nuclear factor 6), and Onecut2 By introducing a combination of six types of transcription factors (one cut domain family member 2) (six factors), we succeeded in producing the most functionally mature hepatocytes from mouse and human fibroblasts. We also found that the degree of functional maturity could be further increased by introducing Myc in addition to the six factors mentioned above and by culturing with a TGF-β receptor inhibitor. Based on the above findings, the present invention was completed.

したがって、本発明の要旨は以下のとおりである。
[1]Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2が導入された体細胞を用意する工程、および当該体細胞を培養して肝細胞へと誘導する工程、を含む、肝細胞の製造方法。
[2]体細胞が線維芽細胞である、[1]に記載の肝細胞の製造方法。
[3]体細胞がヒトまたはマウス由来である、[1]または[2]に記載の肝細胞の製造方法。
[4]Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2がヒトまたはマウス由来である、[1]~[3]のいずれかに記載の肝細胞の製造方法。
[5]Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2がレトロウイルスベクターを用いて導入された、[1]~[4]のいずれかに記載の肝細胞の製造方法。
[6]前記体細胞は、さらにMycが導入されている、[1]~[5]のいずれかに記載の肝細胞の製造方法。
[7]前記培養工程が20日以上行われる、[1]~[6]のいずれかに記載の肝細胞の製造方法。
[8]前記培養工程において培地はTGFβ(Transforming growth factor beta)受容体阻害剤を含む、[1]~[7]のいずれかに記載の肝細胞の製造方法。
[9]TGFβ受容体阻害剤はA83-01である、請求項8に記載の肝細胞の製造方法。
[10] [1]~[9]のいずれかに記載の方法によって製造された肝細胞。
[11] [10]に記載の肝細胞を含む、再生医療用細胞製剤。
[12]下記工程を(i)-(iii)の順番で含む、被検物質の毒性評価方法;
(i)[1]~[9]のいずれかに記載の方法によって製造された肝細胞に被検物質を接触させる工程、
(ii)前記肝細胞の生存率または培養上清中の肝障害マーカー量を測定する工程、および
(iii)前記被検物質と接触させた肝細胞の生存率が、被検物質と接触させなかった肝細胞(対照細胞)の生存率よりも低値、または、前記被検物質と接触させた肝細胞の培養上清中の肝障害マーカー量が、対照細胞の培養上清中の肝障害マーカー量よりも高値である被検物質を、肝毒性を有すると評価する工程。
Therefore, the gist of the present invention is as follows.
[1] A method for producing hepatocytes, comprising the steps of preparing somatic cells into which Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 have been introduced, and culturing the somatic cells to induce them into hepatocytes.
[2] The method for producing hepatocytes according to [1], wherein the somatic cells are fibroblasts.
[3] The method for producing hepatocytes according to [1] or [2], wherein the somatic cells are derived from humans or mice.
[4] The method for producing hepatocytes according to any one of [1] to [3], wherein Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 are derived from humans or mice.
[5] The method for producing hepatocytes according to any one of [1] to [4], wherein Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 are introduced using a retroviral vector.
[6] The method for producing hepatocytes according to any one of [1] to [5], wherein the somatic cells are further introduced with Myc.
[7] The method for producing hepatocytes according to any one of [1] to [6], wherein the culturing step is performed for 20 days or more.
[8] The method for producing hepatocytes according to any one of [1] to [7], wherein in the culturing step, the medium contains a TGFβ (Transforming growth factor beta) receptor inhibitor.
[9] The method for producing hepatocytes according to claim 8, wherein the TGFβ receptor inhibitor is A83-01.
[10] Hepatocytes produced by the method according to any one of [1] to [9].
[11] A cell preparation for regenerative medicine, comprising the hepatocytes described in [10].
[12] A method for evaluating the toxicity of a test substance, comprising the following steps in the order of (i) to (iii);
(i) A step of bringing a test substance into contact with hepatocytes produced by the method according to any one of [1] to [9],
(ii) measuring the survival rate of the hepatocytes or the amount of liver damage marker in the culture supernatant, and (iii) the survival rate of the hepatocytes that have been brought into contact with the test substance is not determined when the hepatocytes have not come into contact with the test substance. The survival rate of hepatocytes (control cells) is lower than the survival rate of hepatocytes (control cells), or the amount of liver damage marker in the culture supernatant of hepatocytes contacted with the test substance is lower than that of the liver damage marker in the culture supernatant of control cells. A process of evaluating a test substance whose value is higher than the amount as having hepatotoxicity.

本発明により機能的に成熟した哺乳動物肝細胞を効率よく製造することができる。
本発明の方法により得られる哺乳動物成熟肝細胞を用いることにより、難治性肝疾患に対する肝細胞移植療法(肝臓再生医療)、肝疾患モデル作製、肝細胞を用いた創薬、薬剤肝毒性評価等をこれまでにない高い精度で実施可能となる。また、本発明の方法により得られる肝細胞は、肝細胞の分化および成熟化機構の解明の目的にも使用できる。
According to the present invention, functionally mature mammalian hepatocytes can be efficiently produced.
By using mature mammalian hepatocytes obtained by the method of the present invention, hepatocyte transplantation therapy (liver regenerative medicine) for intractable liver diseases, liver disease model production, drug discovery using hepatocytes, drug hepatotoxicity evaluation, etc. can be performed with unprecedented precision. Furthermore, hepatocytes obtained by the method of the present invention can also be used for the purpose of elucidating the differentiation and maturation mechanism of hepatocytes.

2因子(Hnf4a+Foxa3)または6因子(Hnf4a+Foxa3+Cebpa+Cebpd+Hnf6+Onecut2)を導入して誘導された肝細胞における各遺伝子の発現量を示すグラフ。Graph showing the expression level of each gene in hepatocytes induced by introducing two factors (Hnf4a + Foxa3) or six factors (Hnf4a + Foxa3 + Cebpa + Cebpd + Hnf6 + Onecut2). 6因子を導入して誘導された肝細胞をA83-01のあるなしで培養した時のVimentin、Cola1(コラーゲン1a1)、Afp(Alpha-fetoprotein)の発現量を示すグラフ。Graph showing the expression levels of Vimentin, Cola1 (collagen 1a1), and Afp (Alpha-fetoprotein) when hepatocytes induced by introducing 6 factors were cultured with or without A83-01. 6因子を導入して誘導された肝細胞をA83-01のあるなしで培養した時のアルブミンの分泌量を示すグラフ。Graph showing the amount of albumin secreted when hepatocytes induced by introducing 6 factors were cultured with or without A83-01. 6因子およびMycの強制発現により得られた誘導肝細胞の免疫染色結果を示す図(写真)。略称は次の通り:Alb, Albumin. E-cad, E-cadherin. Ho, Hoechst 33342。Figure (photograph) showing the results of immunostaining of induced hepatocytes obtained by forced expression of factor 6 and Myc. The abbreviations are: Alb, Albumin. E-cad, E-cadherin. Ho, Hoechst 33342. 培地中への24時間あたりのAlbumin分泌量をELISAにより測定した結果を示す図。Albumin分泌量は、免疫染色でAlbumin陽性となる細胞数で補正した。N=3で行い、平均値±標準偏差で示した。**P < 0.01(one-way ANOVA with Tukey’s correction)。A diagram showing the results of measuring the amount of albumin secreted into the medium per 24 hours by ELISA. The albumin secretion amount was corrected by the number of albumin-positive cells by immunostaining. The test was conducted with N=3, and the results are shown as the mean value ± standard deviation. **P < 0.01 (one-way ANOVA with Tukey’s correction). 各細胞においてCyp3a11の酵素活性を比較した結果を示す図。N=3で行い、平均値±標準偏差で示した。*P < 0.05, **P < 0.01(one-way ANOVA with Tukey’s correction)A diagram showing the results of comparing the enzyme activity of Cyp3a11 in each cell. The test was conducted with N=3, and the results are shown as the mean value ± standard deviation. *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s correction) 6因子+Mycを導入して誘導された肝細胞において、各薬剤で刺激した時の、CYP遺伝子の発現変動を示すグラフ。矢印は発現量が顕著に増加した遺伝子を指す。Graph showing changes in expression of CYP genes when hepatocytes induced by introducing factor 6 + Myc are stimulated with various drugs. Arrows point to genes whose expression levels have significantly increased. 2~6因子を導入して誘導された肝細胞におけるCps1(Carbamoylphosphate synthase I)遺伝子の発現量(n = 3, mean ± SD)を示すグラフ。βアクチン(Actb)で標準化したのち、2因子(Hnf4a+Foxa3)を導入して誘導された肝細胞における値を1として表した。Graph showing the expression level (n = 3, mean ± SD) of the Cps1 (Carbamoylphosphate synthase I) gene in hepatocytes induced by introducing 2 to 6 factors. After normalization with β-actin (Actb), the value in hepatocytes induced by introducing two factors (Hnf4a + Foxa3) was expressed as 1. 6因子およびMycをヒト細胞に導入することにより得られた誘導肝細胞の免疫染色結果を示す図(写真)。Figure (photograph) showing the results of immunostaining of induced hepatocytes obtained by introducing Factor 6 and Myc into human cells.

本発明の肝細胞の製造方法は、Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2が導入された体細胞を用意する工程、および当該体細胞を培養して肝細胞へと誘導する工程、を含む。 The method for producing hepatocytes of the present invention includes the steps of preparing somatic cells into which Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 have been introduced, and culturing the somatic cells to induce them into hepatocytes. .

<6因子導入体細胞を用意する工程>
ここで、用意するとは、体細胞に6因子を導入して新たに用意する場合に限られず、6因子の一部が既に導入された体細胞に残りの因子すべてを導入する場合や、予め6因子が導入された体細胞を調達してくる場合も含む。
<Step of preparing 6-factor-introduced somatic cells>
Here, "preparing" is not limited to the case where six factors are newly prepared by introducing them into somatic cells, but also when all the remaining factors are introduced into somatic cells into which some of the six factors have already been introduced, or when six factors are introduced in advance into somatic cells. This also includes cases in which somatic cells into which factors have been introduced are procured.

<体細胞>
本発明において、体細胞は肝細胞以外の体細胞であれば特に限定されないが、例えば線維芽細胞、上皮細胞(皮膚表皮細胞、口腔粘膜上皮細胞、気道粘膜上皮細胞、腸管粘膜上皮細胞など)、表皮細胞、歯肉細胞(歯肉線維芽細胞、歯肉上皮細胞)、歯髄細胞、白色脂肪細胞、 皮下脂肪、内臓脂肪、筋肉、血液細胞などが挙げられる。また、間葉系幹細胞(Mesenchymal stem cell: MSC)、腸幹細胞、皮膚幹細胞、毛包幹細胞、色素細胞幹細胞などの体性幹細胞から分化誘導し、あるいは脱分化させ、あるいはリプログラミングさせて作製した体細胞も挙げられる。
<Somatic cells>
In the present invention, somatic cells are not particularly limited as long as they are somatic cells other than hepatocytes, such as fibroblasts, epithelial cells (skin epidermal cells, oral mucosal epithelial cells, respiratory tract mucosal epithelial cells, intestinal mucosal epithelial cells, etc.), Examples include epidermal cells, gingival cells (gingival fibroblasts, gingival epithelial cells), dental pulp cells, white adipocytes, subcutaneous fat, visceral fat, muscle, and blood cells. In addition, bodies created by inducing differentiation, dedifferentiation, or reprogramming from somatic stem cells such as mesenchymal stem cells (MSCs), intestinal stem cells, skin stem cells, hair follicle stem cells, and pigment cell stem cells. Also included are cells.

体細胞の由来は、成体であっても小児であっても胎児であってもよい。体細胞は、ヒト、マウス、ラット、ブタ等のヒト以外の哺乳動物から単離されたものであっても、その継代培養細胞であってもよい。線維芽細胞としては、例えば、胎児線維芽細胞、尾先端部由来線維芽細胞、心臓線維芽細胞、包皮線維芽細胞、皮膚線維芽細胞、肺線維芽細胞等を用いることができる。 The somatic cells may be derived from adults, children, or fetuses. The somatic cells may be those isolated from non-human mammals such as humans, mice, rats, pigs, etc., or may be subcultured cells thereof. As the fibroblasts, for example, fetal fibroblasts, tail tip-derived fibroblasts, cardiac fibroblasts, foreskin fibroblasts, skin fibroblasts, lung fibroblasts, etc. can be used.

また、得られる肝細胞を移植用細胞の材料として用いる場合、拒絶反応が起こらないという観点から、当該肝細胞の製造に用いる体細胞は、移植先の個体のHLA遺伝子型が同一または実質的に同一である体細胞であることが望ましい。ここで、「実質的に同一」とは、移植した細胞に対して免疫抑制剤により免疫反応が抑制できる程度にHLA遺伝子型が一致していることであり、例えば、移植先の個体とHLA-A、HLA-BおよびHLA-DRの3遺伝子座またはHLA-Cを加えた4遺伝子座が一致するHLA型を有する体細胞である。 In addition, when the obtained hepatocytes are used as material for transplantation cells, from the viewpoint of preventing rejection, the somatic cells used for the production of the hepatocytes must have the same or substantially the same HLA genotype as the recipient individual. Preferably, the somatic cells are identical. Here, "substantially the same" means that the HLA genotype matches the transplanted cells to the extent that the immune response can be suppressed by immunosuppressants. A, somatic cells with HLA types that match the three loci of HLA-B and HLA-DR or the four loci including HLA-C.

<6因子>
6因子として、Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2を用いる。
<6 factors>
Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 are used as the 6 factors.

Hnf4aはHepatocyte nuclear factor 4 alphaと呼ばれ、糖代謝や脂質代謝、薬物代謝など、多くの肝細胞機能に関わる遺伝子の発現を調節することが知られている。ヒトHNF4Aタンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_849180(配列番号1)に登録されており、マウスHNF4aタンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_032287(配列番号2)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトHNF4aタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_178849に登録されており、マウスHNF4aタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_008261に登録されている。 Hnf4a is called Hepatocyte nuclear factor 4 alpha, and is known to regulate the expression of genes involved in many liver cell functions, such as glucose metabolism, lipid metabolism, and drug metabolism. The amino acid sequence of human HNF4A protein is registered, for example, in GenBank Accession No. NP_849180 (SEQ ID NO: 1), and the amino acid sequence of mouse HNF4a protein is registered, for example, in GenBank Accession No. NP_032287 (SEQ ID NO: 2). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human HNF4a protein is registered, for example, in GenBank Accession No. NM_178849, and the base sequence encoding mouse HNF4a protein is registered, for example, in GenBank Accession No. NM_008261.

Foxa3はForkhead box A3と呼ばれ、肝細胞においては糖新生に関わる遺伝子などの発現を調節することが知られている。ヒトFOXA3タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_004488(配列番号3)に登録されており、マウスFoxa3タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_032286(配列番号4)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトFOXA3タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_004497に登録されており、マウスFoxa3タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_008260に登録されている。 Foxa3 is called Forkhead box A3 and is known to regulate the expression of genes involved in gluconeogenesis in hepatocytes. The amino acid sequence of human FOXA3 protein is registered, for example, in GenBank Accession No. NP_004488 (SEQ ID NO: 3), and the amino acid sequence of mouse Foxa3 protein is registered, for example, in GenBank Accession No. NP_032286 (SEQ ID NO: 4). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human FOXA3 protein is registered, for example, in GenBank Accession No. NM_004497, and the base sequence encoding mouse Foxa3 protein is registered, for example, in GenBank Accession No. NM_008260.

CebpaはCCAAT/enhancer-binding protein alphaと呼ばれ、肝細胞分化や肝細胞における糖・脂質代謝を調節することが知られている。ヒトCEBPA タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_004355(配列番号5)に登録されており、マウスCebpa タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_031704(配列番号6)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトCEBPAタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_004364に登録されており、マウスCebpa タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_007678に登録されている。 Cebpa is called CCAAT/enhancer-binding protein alpha and is known to regulate hepatocyte differentiation and sugar and lipid metabolism in hepatocytes. The amino acid sequence of human CEBPA protein is registered, for example, in GenBank Accession No. NP_004355 (SEQ ID NO: 5), and the amino acid sequence of mouse Cebpa protein is registered, for example, in GenBank Accession No. NP_031704 (SEQ ID NO: 6). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human CEBPA protein is registered, for example, in GenBank Accession No. NM_004364, and the base sequence encoding mouse Cebpa protein is registered, for example, in GenBank Accession No. NM_007678.

CebpdはCCAAT/enhancer-binding protein deltaと呼ばれ、肝細胞においてはIL-6によるシグナルの下流に位置し、急性期反応に関わることが知られている。ヒトCEBPDタンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_005186(配列番号7)に登録されており、マウスCebpdタンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_031705(配列番号8)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトCEBPDタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_005195に登録されており、マウスCebpdタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_007679に登録されている。 Cebpd is called CCAAT/enhancer-binding protein delta, and is known to be located downstream of IL-6 signals in hepatocytes and involved in acute phase reactions. The amino acid sequence of human CEBPD protein is registered, for example, in GenBank Accession No. NP_005186 (SEQ ID NO: 7), and the amino acid sequence of mouse Cebpd protein is registered, for example, in GenBank Accession No. NP_031705 (SEQ ID NO: 8). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human CEBPD protein is registered, for example, in GenBank Accession No. NM_005195, and the base sequence encoding mouse Cebpd protein is registered, for example, in GenBank Accession No. NM_007679.

Hnf6はHepatocyte nuclear factor 6と呼ばれ、肝細胞分化や肝細胞における糖・脂質代謝を調節することが知られている。ヒトHNF6タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_004489(配列番号9)に登録されており、マウスHnf6タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_032288(配列番号10)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトHNF6タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_004498に登録されており、マウスHnf6タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_008262に登録されている。 Hnf6 is called Hepatocyte nuclear factor 6 and is known to regulate hepatocyte differentiation and sugar and lipid metabolism in hepatocytes. The amino acid sequence of human HNF6 protein is registered, for example, in GenBank Accession No. NP_004489 (SEQ ID NO: 9), and the amino acid sequence of mouse Hnf6 protein is registered, for example, in GenBank Accession No. NP_032288 (SEQ ID NO: 10). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human HNF6 protein is registered, for example, in GenBank Accession No. NM_004498, and the base sequence encoding mouse Hnf6 protein is registered, for example, in GenBank Accession No. NM_008262.

Onecut2はOnecut domain family member 2と呼ばれ、Foxa2遺伝子の発現を誘導するなど、肝細胞の分化や代謝に関与することが知られている。ヒトONECUT2タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_004843(配列番号11)に登録されており、マウスOnecut2タンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_919244(配列番号12)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトONECUT2タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_004852に登録されており、マウスOnecut2タンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_194268に登録されている。 Onecut2 is called Onecut domain family member 2, and is known to be involved in hepatocyte differentiation and metabolism, including inducing the expression of the Foxa2 gene. The amino acid sequence of human ONECUT2 protein is registered, for example, in GenBank Accession No. NP_004843 (SEQ ID NO: 11), and the amino acid sequence of mouse Onecut2 protein is registered, for example, in GenBank Accession No. NP_919244 (SEQ ID NO: 12). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human ONECUT2 protein is registered, for example, in GenBank Accession No. NM_004852, and the base sequence encoding mouse Onecut2 protein is registered, for example, in GenBank Accession No. NM_194268.

さらに、Mycが導入されることが好ましい。ヒトMYCタンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_002458(配列番号13)に登録されており、マウスMycタンパク質のアミノ酸配列は例えば、GenBank Accession No. NP_034979(配列番号14)に登録されている。ただし、これら特定のアミノ酸配列を有するタンパク質には限定されず、転写因子としての機能を有する限り、これらの配列と90%以上、好ましくは95%以上、より好ましくは98%以上の同一性を有するアミノ酸配列を有するタンパク質でもよい。ヒトMYCタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_002467に登録されており、マウスMycタンパク質をコードする塩基配列は例えば、GenBank Accession No. NM_010849に登録されている。 Furthermore, it is preferable that Myc is introduced. The amino acid sequence of human MYC protein is registered, for example, in GenBank Accession No. NP_002458 (SEQ ID NO: 13), and the amino acid sequence of mouse Myc protein is registered, for example, in GenBank Accession No. NP_034979 (SEQ ID NO: 14). However, it is not limited to proteins having these specific amino acid sequences, and as long as it has a function as a transcription factor, it has an identity of 90% or more, preferably 95% or more, more preferably 98% or more with these sequences. It may be a protein having an amino acid sequence. The base sequence encoding human MYC protein is registered, for example, in GenBank Accession No. NM_002467, and the base sequence encoding mouse Myc protein is registered, for example, in GenBank Accession No. NM_010849.

上記6因子およびMycは同時に導入されてもよいし、個別因子または複数因子が順次導入されてもよい。 The above six factors and Myc may be introduced simultaneously, or individual factors or multiple factors may be introduced sequentially.

これらの転写因子の導入により、これらの転写因子の発現量(遺伝子レベルまたはタンパク質レベル)は、導入前の細胞における発現量の5倍以上に維持されることが好ましく、10倍以上に維持されることがより好ましい。前記発現量が前記レベルに維持される期間としては、例えば、少なくとも7日間、好ましくは10日間、さらに好ましくは14日間、特に好ましくは肝細胞への分化が終了するまでの期間である。これにより、成熟肝細胞への高効率での分化誘導が可能になる。肝細胞への分化が終了して実験に供されるまで持続することがさらに好ましい。これにより、成熟肝細胞への高効率な分化誘導と、得られた成熟肝細胞機能の機能が良好に維持される場合がある。 By introducing these transcription factors, the expression level (gene level or protein level) of these transcription factors is preferably maintained at 5 times or more, and 10 times or more, as compared to the expression level in the cell before introduction. It is more preferable. The period during which the expression level is maintained at the above level is, for example, at least 7 days, preferably 10 days, more preferably 14 days, particularly preferably until the differentiation into hepatocytes is completed. This makes it possible to induce differentiation into mature hepatocytes with high efficiency. It is more preferable that the differentiation into hepatocytes is continued until the differentiation into hepatocytes is completed and the cells are used for experiments. This may lead to highly efficient induction of differentiation into mature hepatocytes and good maintenance of the functions of the obtained mature hepatocytes.

本発明において、上記の転写因子は遺伝子レベルで体細胞に導入されてもよいし、タンパク質レベルで体細胞に導入されてもよい。なお、内因性で発現している転写因子であれば内因性転写因子の発現を誘導してもよい。すなわち、転写因子の導入は内因性転写因子の誘導を含む。 In the present invention, the above transcription factors may be introduced into somatic cells at the gene level or at the protein level. Note that expression of an endogenous transcription factor may be induced as long as it is an endogenously expressed transcription factor. That is, introduction of transcription factors includes induction of endogenous transcription factors.

<導入方法>
遺伝子(DNA)の形態で導入する場合、例えば、ウイルス、プラスミド、人工染色体などのベクターをリポフェクション、リポソーム、マイクロインジェクションなどの手法によって体細胞内に導入することができる。導入は一過的導入でもよいし、安定的導入でもよい。
ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、センダイウイルスベクターなどが例示される。また、人工染色体ベクターとしては、例えばヒト人工染色体(HAC)、酵母人工染色体(YAC)、細菌人工染色体(BAC、PAC)などが含まれる。プラスミドとしては、哺乳動物細胞用プラスミドを使用しうる。ベクターには、導入遺伝子が発現可能なように、プロモーター、エンハンサー、リボゾーム結合配列、ターミネーター、ポリアデニル化サイトなどの制御配列を含むことができるし、さらに、必要に応じて、薬剤耐性遺伝子(例えばカナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子など)、チミジンキナーゼ遺伝子、ジフテリアトキシン遺伝子などの選択マーカー配列、蛍光タンパク質、βグルクロニダーゼ(GUS)、FLAGなどのレポーター遺伝子配列などを含むことができる。プロモーターとして、SV40プロモーター、 LTRプロモーター、CMV (cytomegalovirus)プロモーター、RSV (Rous sarcoma virus)プロモーター、MoMuLV (Moloney mouse leukemia virus) LTR、HSV-TK (herpes simplex virus thymidine kinase)プロモーター、EF-αプロモーター、CAGプロモーターおよびTREプロモーター(tetO 配列が7回連続したTet応答配列をもつCMV 最小プロモーター)が例示される。
<Introduction method>
When introducing a gene (DNA) in the form of a gene, for example, vectors such as viruses, plasmids, and artificial chromosomes can be introduced into somatic cells by techniques such as lipofection, liposomes, and microinjection. Introduction may be temporary or stable.
Examples of viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors. Furthermore, examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), and the like. As the plasmid, a plasmid for mammalian cells can be used. The vector can contain control sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc., so that the transgene can be expressed, and may also contain drug resistance genes (e.g. kanamycin), if necessary. resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), selection marker sequences such as thymidine kinase gene and diphtheria toxin gene, reporter gene sequences such as fluorescent protein, β-glucuronidase (GUS), and FLAG. As promoters, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF-α promoter, CAG Examples include the promoter and the TRE promoter (CMV minimal promoter with a Tet response element consisting of seven consecutive tetO sequences).

RNAの形態で導入する場合、例えばエレクトロポレーション、リポフェクション、マイクロインジェクションなどの手法によって多能性幹細胞内に導入してもよい。 When introduced in the form of RNA, it may be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, and microinjection.

タンパク質の形態で導入する場合、例えばリポフェクション、細胞膜透過性ペプチド(例えば、HIV由来のTATおよびポリアルギニン)との融合、マイクロインジェクションなどの手法によって多能性幹細胞内に導入してもよい。
6因子の発現は、少なくとも7日間、好ましくは10日間、さらに好ましくは14日間、特に好ましくは肝細胞への分化が終了するまでの期間、持続することが望ましい。これにより、成熟肝細胞への高効率での分化誘導が可能になる。さらに、分化が終了して実験に供されるまで持続してもよい。これにより、得られた成熟肝細胞の機能が良好に維持される場合がある。
When introduced in the form of a protein, it may be introduced into pluripotent stem cells by techniques such as lipofection, fusion with cell membrane permeable peptides (eg, HIV-derived TAT and polyarginine), and microinjection.
It is desirable that the expression of factor 6 continues for at least 7 days, preferably 10 days, more preferably 14 days, particularly preferably until the completion of differentiation into hepatocytes. This makes it possible to induce differentiation into mature hepatocytes with high efficiency. Furthermore, it may continue until differentiation is completed and used for experiments. Thereby, the function of the obtained mature hepatocytes may be maintained well.

<培養工程>
転写因子が導入された細胞を培養する工程に使用する培養液は、特に限定されないが、動物細胞の培養に用いられる培地を基礎培地とし、適切な成分を添加して調製することができる。基礎培地には、例えばIscove's Modified Dulbecco's Medium(IMDM)培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium(ライフテクノロジーズ)およびこれらの混合培地などが包含される。HCM BulletKit Medium、William’s E Medium (Thermo Fisher Scientific), Leibovitz’s L-15 Medium (Thermo Fisher Scientific)などの肝細胞用培地も好ましく用いることができる。培地には、血清が含有されていてもよいし、あるいは無血清を使用してもよい。必要に応じて、基礎培地は、例えば、アルブミン、インスリン、トランスフェリン、セレン、脂肪酸、微量元素、2-メルカプトエタノール、チオールグリセロール、脂質、アミノ酸、L-グルタミン、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類、副腎皮質ホルモン(デキサメタゾンなどのホルモン、サイトカインなどの物質も含有し得る。
サイトカインとしては、肝細胞増殖因子(HGF)やオンコスタチンM(OSM)などが挙げられ、これらは、例えば、1~100ng/mLの濃度で添加される。
<Culture process>
The culture solution used in the step of culturing cells into which transcription factors have been introduced is not particularly limited, but can be prepared by using a medium used for culturing animal cells as a basal medium and adding appropriate components. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, and Fischer's medium. , Neurobasal Medium (Life Technologies) and mixed media thereof. Hepatocyte media such as HCM BulletKit Medium, William's E Medium (Thermo Fisher Scientific), and Leibovitz's L-15 Medium (Thermo Fisher Scientific) can also be preferably used. The medium may contain serum or may be serum-free. If necessary, the basal medium contains, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiol glycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low It may also contain substances such as molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, hormones such as corticosteroids (dexamethasone), cytokines.
Examples of cytokines include hepatocyte growth factor (HGF) and oncostatin M (OSM), which are added at a concentration of, for example, 1 to 100 ng/mL.

<TGFβ受容体阻害剤>
培地にはTGFβ受容体阻害剤を添加することが好ましい。TGFβ受容体阻害剤とは、TGFβの受容体への結合からSMADへと続くシグナル伝達を阻害する物質であり、受容体であるALKファミリーへの結合を阻害する物質、またはALKファミリーによるSMADのリン酸化を阻害する物質が挙げられ、例えば、Lefty-1(NCBI Accession No.として、マウス:NM_010094、ヒト:NM_020997が例示される)、SB431542、SB202190(以上、R.K.Lindemann et al., Mol. Cancer, 2003, 2:20)、SB505124 (GlaxoSmithKline)、 NPC30345、SD093、SD908、SD208 (Scios)、LY2109761、LY364947、 LY580276 (Lilly Research Laboratories)、A83-01(WO 2009146408) およびこれらの誘導体などが例示される。本発明で使用されるTGFβ受容体阻害剤は、好ましくは、A83-01であり得る。
<TGFβ receptor inhibitor>
It is preferable to add a TGFβ receptor inhibitor to the medium. A TGFβ receptor inhibitor is a substance that inhibits the signal transmission from the binding of TGFβ to its receptor to SMAD. Substances that inhibit oxidation include, for example, Lefty-1 (NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (RKLindemann et al., Mol. Cancer, 2003). , 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009146408), and derivatives thereof. The TGFβ receptor inhibitor used in the present invention may preferably be A83-01.

培養液中におけるA83-01の濃度は、ALK5を阻害する濃度であれば特に限定されないが、例えば、500nMから5μMである。 The concentration of A83-01 in the culture solution is not particularly limited as long as it inhibits ALK5, and is, for example, 500 nM to 5 μM.

培養は接着培養でも浮遊培養でもよく、用いる体細胞の種類によって適宜選択できる。浮遊培養によって行われる場合、細胞を培養容器へ非接着の状態で凝集体(スフェアとも言う)を形成させて培養することが望ましく、このような培養は、特に限定はされないが、細胞との接着性を向上させる目的で人工的に処理(例えば、細胞外マトリックス等によるコーティング処理)されていない培養容器、若しくは、人工的に接着を抑制する処理(例えば、ポリヒドロキシエチルメタクリル酸(poly-HEMA)、非イオン性の界面活性ポリオール(Pluronic F-127等)またはリン脂質類似構造物(例えば、2-メタクリロイルオキシエチルホスホリルコリンを構成単位とする水溶性ポリマー(Lipidure))によるコーティング処理した培養容器を使用することによって行うことができる。 The culture may be either adherent culture or suspension culture, which can be selected as appropriate depending on the type of somatic cells used. When performing suspension culture, it is desirable to culture cells in a culture container in a non-adherent state by forming aggregates (also called spheres). Culture vessels that have not been artificially treated to improve adhesion (e.g., coating with extracellular matrix, etc.), or artificially treated to suppress adhesion (e.g., polyhydroxyethyl methacrylic acid (poly-HEMA)) , use a culture vessel coated with a nonionic surfactant polyol (such as Pluronic F-127) or a phospholipid-like structure (for example, a water-soluble polymer with 2-methacryloyloxyethylphosphorylcholine as a constituent unit (Lipidure)). This can be done by

接着培養によって培養が行われる場合、細胞外基質をコーティング処理された培養容器を用いて培養することによって行うことができる。コーティング処理は、細胞外基質を含有する溶液を培養容器に入れた後、当該溶液を適宜除くことによって行い得る。ここで、細胞外基質とは、細胞の外に存在する超分子構造体であり、天然由来であっても、人工物(組換え体)であってもよい。例えば、ポリリジン、ポリオルニチン、コラーゲン、プロテオグリカン、フィブロネクチン、ヒアルロン酸、テネイシン、エンタクチン、エラスチン、フィブリリン、ラミニンといった物質およびこれらの断片が挙げられる。これらの細胞外基質は、組み合わせて用いられてもよく、例えば、BD Matrigel(商標)などの細胞からの調製物であってもよい。 When culturing is carried out by adhesive culture, it can be carried out by culturing using a culture vessel coated with extracellular matrix. The coating treatment can be performed by putting a solution containing an extracellular matrix into a culture container and then removing the solution as appropriate. Here, the extracellular matrix is a supramolecular structure that exists outside of cells, and may be of natural origin or artificial (recombinant). Examples include substances such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, and fragments thereof. These extracellular matrices may be used in combination, for example in preparations from cells such as BD Matrigel™.

培養条件は、特に限定されないが、例えば、約37℃~約42℃程度、約37~約39℃程度が好ましい。CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2~5%である。また、培養期間については、当業者であれば細胞数などをモニターしながら、適宜決定することが可能である。日数は特に限定されないが、例えば、10日間以上、12日以上、14日以上、16日以上、18日以上、20日以上、25日以上である。 Culture conditions are not particularly limited, but are preferably about 37°C to about 42°C, about 37°C to about 39°C, for example. Cultivation is carried out in an atmosphere of CO 2 -containing air, the CO 2 concentration being preferably about 2-5%. Furthermore, a person skilled in the art can appropriately determine the culture period while monitoring the number of cells and the like. The number of days is not particularly limited, but is, for example, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 25 days or more.

<肝細胞>
肝細胞が得られたことは、アルブミンなどの発現を測定することにより評価できる。
肝細胞は、他の細胞種が含まれる細胞集団として得られてもよく、純化されてもよい。本発明の方法で得られる細胞集団は、例えば、3%以上、5%以上、10%以上もしくは20%以上の肝細胞を含むが、好ましくは、50%以上、60%以上、70%以上または80%以上の肝細胞を含む。なお、肝細胞を純化する方法する方法として、アシアログライコプロテイン(ASGP)受容体に対する抗体などを用いて染色し、染色された細胞をフローサイトメーター(FACS)や磁気細胞分離装置(MACS)を用いて濃縮する方法が例示される。
<Hepatocytes>
Obtaining hepatocytes can be evaluated by measuring the expression of albumin and the like.
Hepatocytes may be obtained as a cell population containing other cell types, or may be purified. The cell population obtained by the method of the present invention contains, for example, 3% or more, 5% or more, 10% or more, or 20% or more of hepatocytes, but preferably 50% or more, 60% or more, 70% or more, or Contains over 80% hepatocytes. In addition, as a method to purify hepatocytes, they are stained using an antibody against the asialoglycoprotein (ASGP) receptor, and the stained cells are collected using a flow cytometer (FACS) or magnetic cell sorter (MACS). An example is a method of concentrating.

本発明で得られた肝細胞は上記6因子、好ましくはさらにMycが導入されたことにより、成体の肝細胞としての機能を有する。例えば、肝細胞はアルブミンの分泌に加え、シトクロムP450活性、薬物代謝、グリコーゲンの蓄積、低比重リポタンパク質(LDL)の取り込み、アンモニア代謝と尿素合成などの肝細胞の機能の1つ以上を有する。 The hepatocytes obtained according to the present invention have the functions of adult hepatocytes due to the introduction of the above six factors, preferably Myc. For example, in addition to secreting albumin, hepatocytes have one or more of the functions of hepatocytes, such as cytochrome P450 activity, drug metabolism, glycogen accumulation, low-density lipoprotein (LDL) uptake, ammonia metabolism and urea synthesis.

<細胞製剤>
本発明では、上述した方法により得られた肝細胞を含む、肝疾患治療等に使用される再生医療用細胞製剤を提供する。
患者への細胞製剤の投与方法としては、例えば、得られた肝細胞をシート化して、患者の肝臓に貼付する方法、得られた肝細胞を生理食塩水等に懸濁させ、患者の肝臓に直接移植する方法、マトリゲル等から構成されたスキャフォールド上で三次元培養し、得られた肝細胞塊を移植する方法などが挙げられる。
<Cell preparations>
The present invention provides a cell preparation for regenerative medicine, which contains hepatocytes obtained by the method described above and is used for treating liver diseases.
Methods for administering cell preparations to patients include, for example, forming the obtained hepatocytes into a sheet and pasting it on the patient's liver, suspending the obtained hepatocytes in physiological saline, etc., and injecting the obtained hepatocytes into the patient's liver. Examples include a method of direct transplantation, a method of three-dimensional culture on a scaffold made of matrigel, etc., and a method of transplanting the obtained hepatocyte mass.

本発明において、細胞製剤に含まれる肝細胞の細胞数は、移植片が投与後に生着できれば特に限定されなく、患部の大きさや体躯の大きさに合わせて適宜増減して調製すればよい。 In the present invention, the number of hepatocytes contained in the cell preparation is not particularly limited as long as the graft can survive after administration, and may be adjusted as appropriate depending on the size of the affected area and body size.

<肝細胞を用いた薬物評価>
本発明の方法で調製した肝細胞を用いて被検物質の代謝についてin vitroで試験することができる。
当該被検物質の代謝試験は、例えば、(i)本発明の方法で得られた肝細胞に被検物質を接触させる工程、および(ii)被検物質の代謝産物を検出する工程を含むことができる。
工程(i)での「接触」とは、本発明によって製造された肝細胞を培養する培地に被検物質を添加することによって行われ得る。被検化合物の添加のタイミングは特に限定されない。従って、被検物質を含まない培地で培養を開始した後、ある時点で被検物質を添加することにしても、予め被検物質を含む培地で培養を開始することにしてもよい。
<Drug evaluation using hepatocytes>
Metabolism of a test substance can be tested in vitro using hepatocytes prepared by the method of the present invention.
The metabolism test of the test substance may include, for example, (i) a step of bringing the test substance into contact with hepatocytes obtained by the method of the present invention, and (ii) a step of detecting metabolites of the test substance. Can be done.
"Contact" in step (i) may be performed by adding a test substance to a medium in which hepatocytes produced according to the present invention are cultured. The timing of addition of the test compound is not particularly limited. Therefore, the test substance may be added at a certain point after culturing is started in a medium that does not contain the test substance, or the culture may be started in a medium containing the test substance in advance.

被検物質には、例えば、医薬品や栄養食品等の既存成分或いは候補成分などが包含される。被検物質は天然物由来であっても、或いは合成によるものであってもよい。後者の場合には例えばコンビナトリアル合成の手法を利用して効率的なアッセイ系を構築することができる。 The test substance includes, for example, existing components or candidate components of pharmaceuticals, nutritional foods, and the like. The test substance may be derived from natural products or may be synthetic. In the latter case, an efficient assay system can be constructed using, for example, combinatorial synthesis techniques.

被検物質を接触させる期間は任意に設定可能である。接触期間は例えば10分間~3日間、好ましくは1時間~1日間である。接触を複数回に分けて行うことにしてもよい。 The period of contact with the test substance can be set arbitrarily. The contact period is, for example, 10 minutes to 3 days, preferably 1 hour to 1 day. The contact may be performed in multiple steps.

被検物質の代謝とは、エステルなどの加水分解、酸化反応(特にシトクロムP450による酸化)、還元反応などによる被検物質の化学修飾が例示され、このような代謝の有無は、例えば、代謝産物の検出によって行うことができる。好ましくは、工程(i)の後、得られた培養液をサンプルとして、予想される代謝産物を定性的又は定量的に測定する。測定方法は被検物質並びにその予測される代謝産物に応じて適切なものを選択すればよいが、例えば、質量分析、液体クロマトグラフィー、免疫学的手法(例えば蛍光免疫測定法(FIA法)、酵素免疫測定法(EIA法))等を採用可能である。 Metabolism of a test substance is exemplified by chemical modification of the test substance by hydrolysis of esters, oxidation reactions (especially oxidation by cytochrome P450), reduction reactions, etc. This can be done by detecting Preferably, after step (i), the resulting culture solution is used as a sample to qualitatively or quantitatively measure expected metabolites. Appropriate measurement methods may be selected depending on the test substance and its predicted metabolites; examples include mass spectrometry, liquid chromatography, immunological methods (e.g., fluorescence immunoassay (FIA method), Enzyme immunoassay (EIA method) etc. can be adopted.

被検物質の代謝産物が検出されたとき、「被検物質が代謝された」と判定する。また、代謝産物の量に応じて被検物質の代謝量を評価することができる。 When a metabolite of the test substance is detected, it is determined that "the test substance has been metabolized." Furthermore, the amount of metabolism of the test substance can be evaluated according to the amount of metabolites.

また、本発明で得られた肝細胞を用いて薬物代謝酵素(例えば、シトクロム、UGT)の発現誘導を確認することができる。薬物代謝酵素の発現はmRNAレベル又はタンパク質レベルで評価することができる。例えば、薬物代謝酵素のmRNAレベルに上昇を認めたとき、「被検物質は薬物相互作用を起こす疑いがある」と判定することができる。 Furthermore, the induction of expression of drug-metabolizing enzymes (eg, cytochromes, UGT) can be confirmed using the hepatocytes obtained according to the present invention. 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 a drug-metabolizing enzyme is observed, it can be determined that "the test substance is suspected of causing a drug interaction."

一方、本発明の方法で調製した肝細胞を用いて被検物質の毒性を試験することもできる。当該方法は、(i)本発明の方法で得られた肝細胞に被検物質を接触させる工程と、(ii)工程(i)後の肝細胞の状態を調べる工程と、(iii)(ii)の結果に基づいて被検物質の毒性を評価する工程、とを含むことができる。工程(i)は、前記被検物質の代謝と同様である。 On the other hand, the toxicity of a test substance can also be tested using hepatocytes prepared by the method of the present invention. The method includes (i) a step of bringing a test substance into contact with the hepatocytes obtained by the method of the present invention, (ii) a step of examining the state of the hepatocytes after step (i), and (iii) (iii) ) of evaluating the toxicity of the test substance based on the results of the step. Step (i) is the same as the metabolism of the test substance.

工程(ii)では、被検物質を接触させた後の肝細胞の状態を調べ、工程(iii)において被検物質の毒性を評価する。肝細胞の状態は、生存率の測定、細胞形態の観察、培養液(培養上清)中の肝障害マーカー(GOT、GPT等)の測定などによって把握することができる。例えば、被検物質の接触によって生存率の低下を認めたとき、「被検物質は肝毒性を有する」と判定することができる。また、被検物質の接触によって細胞形態の異常を認めたときや、培養液中の肝障害マーカーの量が上昇したときも同様に、「被検物質は肝毒性を有する」と判定することができる。生存率の低下の程度や肝障害マーカーの量に応じて定量的な判定を行ってもよい。なお、前記「生存率の低下」は、目安として、被検物質と接触させなかった肝細胞(対照細胞)の生存率に対し、90%以下、80%以下、70%以下、60%以下、50%以下の生存率であってもよい。また、前記「肝障害マーカーの量が上昇」は、目安として、対照細胞の生存率に対し、110%以上、120%以上、130%以下、140%以上、または150%以上の前記マーカー量であってもよい。
また、工程(ii)において被検物質のTC50を解析し、工程(iii)において、当該TC50値が特定の値以下の被検物質に対して「肝毒性を有する」と判定してもよい。被検物質の肝細胞に対するTC50を調べることで、人体への被検物質の適正投与量の予測に貢献することができる。
In step (ii), the state of the hepatocytes after contact with the test substance is examined, and in step (iii), the toxicity of the test substance is evaluated. The state of hepatocytes can be ascertained by measuring survival rate, observing cell morphology, and measuring liver damage markers (GOT, GPT, etc.) in the culture medium (culture supernatant). For example, when a decrease in survival rate is observed due to contact with a test substance, it can be determined that "the test substance has hepatotoxicity." Similarly, when an abnormality in cell morphology is observed due to contact with the test substance, or when the amount of liver damage markers in the culture solution increases, it is also possible to determine that the test substance is hepatotoxic. can. Quantitative determination may be made depending on the degree of decrease in survival rate and the amount of liver damage marker. The above-mentioned "decreased survival rate" is, as a guide, 90% or less, 80% or less, 70% or less, 60% or less, with respect to the survival rate of hepatocytes that were not contacted with the test substance (control cells). The survival rate may be less than 50%. In addition, the above-mentioned "increase in the amount of liver damage marker" means, as a guideline, when the amount of the marker is 110% or more, 120% or more, 130% or less, 140% or more, or 150% or more of the survival rate of control cells. There may be.
Alternatively, the TC50 of the test substance may be analyzed in step (ii), and the test substance whose TC50 value is below a specific value may be determined to be "hepatotoxic" in step (iii). . Examining the TC 50 of a test substance to hepatocytes can contribute to predicting the appropriate dose of the test substance to the human body.

以下、実施例を挙げて本発明を具体的に説明するが、本発明の態様は以下には限定されない。 EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the embodiments of the present invention are not limited to the following.

[実施例1]
以下の手順でレトロウイルスベクターを使用して線維芽細胞に遺伝子導入を行うことにより誘導肝細胞(iHeps)を作製し、得られたiHepsを用いて肝細胞マーカーの発現などを解析した。
[Example 1]
Induced hepatocytes (iHeps) were created by introducing genes into fibroblasts using a retroviral vector according to the following procedure, and the expression of hepatocyte markers was analyzed using the obtained iHeps.

ウイルスベクター作製
マウスのHnf4a遺伝子、Foxa3遺伝子、Cebpa遺伝子、Cebpd遺伝子、Hnf6遺伝子およびOnecut2遺伝子をそれぞれpMXsプラスミドに組み込み、各遺伝子導入用のレトロウイルスベクターをそれぞれ作製した。また、マウスのMyc遺伝子導入用のレトロウイルスベクターも同様に作製した。なお、pMXsは東京大学の北村俊雄教授より譲り受けた(Exp Hematol. 2003 Nov;31(11):1007-14.)。
Viral vector production The mouse Hnf4a gene, Foxa3 gene, Cebpa gene, Cebpd gene, Hnf6 gene, and Onecut2 gene were each incorporated into the pMXs plasmid, and retroviral vectors for each gene introduction were prepared. In addition, a retroviral vector for introducing the Myc gene into mice was similarly created. Note that pMXs was a gift from Professor Toshio Kitamura of the University of Tokyo (Exp Hematol. 2003 Nov;31(11):1007-14.).

ウイルス上清準備
1. Plat-E (Cell Biolabs, Inc., Cat# RV-101) を、抗生物質を含まない10% FBS/DMEM (FBS (Thermo Fisher Scientific), DMEM (Nacalai tesque, Cat# 08459-64)) に懸濁し、ゼラチンコートした6-well plate (Greiner) に播種 (5.0 × 105 cells/1.5 ml/well)し、37℃ 5% CO2 で一晩インキュベートした。
Viral supernatant preparation
1. Plat-E (Cell Biolabs, Inc., Cat# RV-101) in 10% FBS/DMEM without antibiotics (FBS (Thermo Fisher Scientific), DMEM (Nacalai tesque, Cat# 08459-64)) The cells were suspended in a gelatin-coated 6-well plate (Greiner) and seeded (5.0 × 10 5 cells/1.5 ml/well), and incubated overnight at 37°C and 5% CO 2 .

2. Plat-Eに対し、各転写因子のplasmidのlipofectionを行った。
37℃のOpti-MEM (Thermo Fisher Scientific, Cat# 31985062) 50 μlにFuGENE 6 (Promega, Cat# E2692) 4.5 μlを加え、vortex後に5分間室温静置した。そこに各転写因子のレトロウイルスベクター (pMXs) 500 ng/μlを加え、20分間室温静置し、その後、1で用意したPlat-Eの各wellに加え、37℃ 5% CO2 で一晩インキュベートした。
2. Lipofection of plasmid of each transcription factor was performed on Plat-E.
4.5 μl of FuGENE 6 (Promega, Cat# E2692) was added to 50 μl of Opti-MEM (Thermo Fisher Scientific, Cat# 31985062) at 37°C, and after vortexing, the mixture was allowed to stand at room temperature for 5 minutes. Add 500 ng/μl of retroviral vectors (pMXs) for each transcription factor, leave to stand at room temperature for 20 minutes, then add to each well of Plat-E prepared in step 1 and incubate overnight at 37°C with 5% CO 2. Incubated.

3. 抗生物質を含まない10%FBS/DMEM 1.6 ml/wellで培地交換し、37℃ 5% CO2で一晩インキュベートし、それをウイルス上清として用いた。 3. The medium was replaced with 1.6 ml/well of 10% FBS/DMEM without antibiotics, incubated overnight at 37°C and 5% CO 2 , and used as the virus supernatant.

MEFs (mouse embryonic fibroblasts)準備
E13.5 妊娠マウス (C57BL/6J) の胎仔より得た。継代は行わず、Cell Reservoir One (Nacalai tesque, Cat# 07485-44)を細胞保存液として用いて、-80℃凍結保存した。
MEFs (mouse embryonic fibroblasts) preparation
Obtained from the fetus of an E13.5 pregnant mouse (C57BL/6J). Cells were not passaged and stored frozen at -80°C using Cell Reservoir One (Nacalai tesque, Cat# 07485-44) as a cell storage solution.

iHep induction
1. Viral transduction前日 (day-1)、凍結保存していたMEFsを起眠し、Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat# 354230) でコーティングした12-well plate (Greiner) 上に播種 (5.0 × 104 cells/well)した。
iHep induction
1. The day before viral transduction (day-1), the frozen MEFs were awakened and placed on a 12-well plate (Greiner) coated with Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat# 354230). It was seeded (5.0 × 10 4 cells/well).

2. Viral transduction当日(day0)、Plat-Eの培養上清をウイルス上清として回収し、filtering (Millex-HP Syringe Filter Unit 0.45 μm, Merck Millipore, Cat# SLHP033RS)した。 2. On the day of viral transduction (day 0), the culture supernatant of Plat-E was collected as a virus supernatant and filtered (Millex-HP Syringe Filter Unit 0.45 μm, Merck Millipore, Cat# SLHP033RS).

3. 各転写因子のウイルス上清を各100 μl(c-Mycは12.5 μl), 10% FBS/DMEM 87.5 μl, Polybrene (nacalai tesque, Cat# 12996-81) 0.7 μlを加え、そのうち500 μlを、前日MEFsを播種した12-well plateの1wellあたりに加えた。 3. Add 100 μl of virus supernatant for each transcription factor (12.5 μl for c-Myc), 87.5 μl of 10% FBS/DMEM, and 0.7 μl of Polybrene (nacalai tesque, Cat# 12996-81), of which 500 μl , was added to each well of a 12-well plate in which MEFs had been seeded the previous day.

4. 37℃, 5% CO2 で一晩インキュベートし、ウイルス感染を行った。 4. Virus infection was performed by incubating overnight at 37°C, 5% CO2 .

5. 翌日 (day1) 培地交換 (10% FBS/DMEM)した。 5. The next day (day1), the medium was replaced (10% FBS/DMEM).

6. Day3にも培地交換 (10% FBS/DMEM)した。 6. The medium was replaced (10% FBS/DMEM) on Day 3 as well.

7. Day4でMatrigelでコートされた12-well plateに1:10の割合でreplateした。培地は10% FBS/DMEM/F12, GlutaMAXをベースにしたHepatic specification培地 (DMEM/F12, GlutaMAX (Thermo Fisher Scientific, Cat# 10565042) 360 ml, FBS 40 ml, Nicotinamide (STEMCELL Technologies, Cat# 07154) 10mM, ITS-X (Thermo Fisher Scientific, Cat# 51500056) 400μl, Dexamethasone (Merck, Cat# D4902-25MG) 0.1μM, 2-Mercaptoethanol (Thermo Fisher Scientific, Cat# 21985023) 50 μM, Penicillin-Streptomycin (Thermo Fisher Scientific, Cat# 15140122) 2 ml)とし、Day6に培地交換を行った。 7. On Day 4, replate was applied to a 12-well plate coated with Matrigel at a ratio of 1:10. Medium: 10% FBS/DMEM/F12, GlutaMAX-based Hepatic specification medium (DMEM/F12, GlutaMAX (Thermo Fisher Scientific, Cat# 10565042) 360 ml, FBS 40 ml, Nicotinamide (STEMCELL Technologies, Cat# 07154) 10mM , ITS-X (Thermo Fisher Scientific, Cat# 51500056) 400μl, Dexamethasone (Merck, Cat# D4902-25MG) 0.1μM, 2-Mercaptoethanol (Thermo Fisher Scientific, Cat# 21985023) 50 μM, Penicillin-Streptomycin (Thermo Fisher Scientific , Cat# 15140122) 2 ml), and the medium was replaced on Day 6.

8. Day7から、hepatic maturation培地(HCM BulletKit Medium (Lonza, Cat# CC-3198), HGF (Peprotech, Cat# 100-39) 20 ng/ml, OSM (Peprotech, Cat# 300-10) 20 ng/ml, A83-01 (Merck, Cat# 616454-2MGCN) 1 μM)に変更して、2日に一度培地交換した。
Day25前後で遺伝子発現解析等の実験に供した。
8. From Day7, add hepatic maturation medium (HCM BulletKit Medium (Lonza, Cat# CC-3198), HGF (Peprotech, Cat# 100-39) 20 ng/ml, OSM (Peprotech, Cat# 300-10) 20 ng/ ml, A83-01 (Merck, Cat# 616454-2MGCN) 1 μM), and the medium was replaced once every two days.
It was subjected to experiments such as gene expression analysis around Day 25.

遺伝子発現はRT-qPCRで解析した。
Albuminの分泌量の定量はELISAキット(Mouse Albumin ELISA Kit (BETHYL Laboratories)を使用した。
Cyp3a11の活性測定においては、基質となるMidazolamを培地に加え、Cyp3a11による水酸化により産生されたHydroxy midazolamの24時間あたりの培地中への分泌量を、Mass spectrometryで測定した。
Gene expression was analyzed by RT-qPCR.
The amount of secreted albumin was quantified using an ELISA kit (Mouse Albumin ELISA Kit (BETHYL Laboratories)).
To measure the activity of Cyp3a11, midazolam as a substrate was added to the medium, and the amount of Hydroxy midazolam produced by hydroxylation by Cyp3a11 secreted into the medium per 24 hours was measured by mass spectrometry.

<結果>
発明者らは、マウス成体肝細胞に発現する遺伝子の発現プロファイルを探索し、iPS干渉法を用いて成熟肝細胞を誘導する転写因子の組み合わせの絞り込みを行った。その結果、Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6, Onecut2の6因子の組み合わせが成熟肝細胞の誘導に最適であることを見出した。
図1に、6因子の優位性を示すため、2因子(Hnf4a, Foxa3)のみを導入して誘導した場合とで、肝細胞マーカー遺伝子の発現を比較した。その結果、6因子の導入は、2因子に比べ、顕著に肝細胞マーカー遺伝子を増加させた。Cdx2は中腸・後腸マーカー遺伝子(腸管マーカー遺伝子 肝臓は後方前腸由来)であり、既報(Morris et al Cell 2014 Aug 14;158(4):889-902)で2因子から作製したiHepsはCdx2 positive細胞であることが示されたが、6因子を用いることで、Cdx2の発現は著明に低下した。また、Afpは肝芽細胞マーカーであるが、6因子を用いることで、Afpの発現も低下したことから、より成熟した肝細胞に近いiHepsが得られたことが示された。
<Results>
The inventors searched for the expression profile of genes expressed in adult mouse hepatocytes, and used iPS interference to narrow down the combination of transcription factors that induce mature hepatocytes. As a result, we found that a combination of six factors, Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6, and Onecut2, was optimal for inducing mature hepatocytes.
In FIG. 1, in order to show the superiority of the six factors, the expression of hepatocyte marker genes was compared between the case where only two factors (Hnf4a and Foxa3) were introduced and induced. As a result, introduction of 6 factors significantly increased hepatocyte marker genes compared to 2 factors. Cdx2 is a midgut/hindgut marker gene (intestinal marker gene; the liver originates from the posterior foregut), and as previously reported (Morris et al Cell 2014 Aug 14;158(4):889-902), iHeps produced from two factors Although these cells were shown to be Cdx2 positive cells, Cdx2 expression was markedly reduced by using 6 factors. In addition, Afp is a hepatoblast marker, and by using the 6 factors, Afp expression was also reduced, indicating that iHeps that are more similar to mature hepatocytes were obtained.

次に、6因子を導入したのちの培養条件を最適化するため、肝細胞を効率的に誘導できる培地成分の検討を行ったところ、TGFβ受容体阻害剤であるA83-01の添加が有効であることを見出した。hepatic maturation培地にA83-01を添加した場合と添加しない場合で培養を行って得られた肝細胞におけるマーカーの発現を調べた結果を図2、3に示す。その結果、A83-01の添加はアルブミン分泌を増加させた。
一方、A83-01はVimentin、Col1a1、Afpなどの脱分化因子の発現を低下させ、肝細胞の分化効率を上げることが分かった。
Next, in order to optimize the culture conditions after introducing the six factors, we investigated medium components that can efficiently induce hepatocytes, and found that the addition of A83-01, a TGFβ receptor inhibitor, was effective. I discovered something. Figures 2 and 3 show the results of examining the expression of markers in hepatocytes obtained by culturing with and without the addition of A83-01 to the hepatic maturation medium. As a result, addition of A83-01 increased albumin secretion.
On the other hand, A83-01 was found to decrease the expression of dedifferentiation factors such as Vimentin, Col1a1, and Afp, and increase the efficiency of hepatocyte differentiation.

以下、6因子とともにMyc遺伝子を導入して誘導し、hepatic maturation培地にA83-01を添加して培養して得られた肝細胞を用いて実験を行った。
図4には、6因子およびMycの強制発現により得られたiHeps (6TF-Myc iHeps) の免疫染色結果を示す。Albumin陽性の上皮様細胞(6TF-Myc iHeps)が敷石状に拡がっていることがわかる。
Hereinafter, experiments were conducted using hepatocytes obtained by introducing and inducing the Myc gene together with 6 factors and culturing with A83-01 added to a hepatic maturation medium.
Figure 4 shows the results of immunostaining of iHeps (6TF-Myc iHeps) obtained by forced expression of 6 factors and Myc. It can be seen that albumin-positive epithelial-like cells (6TF-Myc iHeps) are spread like cobblestones.

図5では、6TF-Myc iHepsを用い、培地中への24時間あたりのAlbumin分泌量をELISAにより測定した。なお、Albumin分泌量は、免疫染色でAlbumin陽性となる細胞数で補正して示した。その結果、6因子にMycを組み合わせた場合、得られる誘導肝細胞におけるアルブミン分泌量は6因子の場合に比べても顕著に多く、初代培養肝細胞に匹敵するほどであった。 In FIG. 5, the amount of albumin secreted into the medium per 24 hours was measured by ELISA using 6TF-Myc iHeps. Note that the amount of albumin secreted was corrected by the number of cells that were positive for albumin in immunostaining. As a result, when 6 factors were combined with Myc, the amount of albumin secreted by the resulting induced hepatocytes was significantly higher than in the case of 6 factors, and was comparable to that of primary cultured hepatocytes.

図6では、肝細胞による薬物代謝において、主要な役割を果たすCytochrome P450 (CYP)のうち、代表的なCyp3a11の酵素活性を、各誘導肝細胞又は初代培養肝細胞で比較した。Hydroxy midazolamの分泌量は、免疫染色でAlbumin陽性となる細胞数で補正した。代謝産物であるHydroxy midazolamの産生量が多いほど、酵素活性が高いことを意味し、より成熟化した肝細胞機能を有することを示す。図6によれば、6因子にMycを組み合わせた場合、得られる誘導肝細胞における酵素活性は6因子の場合に比べても顕著に高く、初代培養肝細胞に匹敵するほどであった。 In FIG. 6, the enzyme activity of Cyp3a11, a representative enzyme among Cytochrome P450 (CYP) that plays a major role in drug metabolism by hepatocytes, was compared in each induced hepatocyte or primary cultured hepatocyte. The amount of hydroxy midazolam secreted was corrected by the number of albumin-positive cells by immunostaining. The higher the production amount of the metabolite Hydroxy midazolam, the higher the enzyme activity, which indicates that the liver cells have more mature functions. According to FIG. 6, when 6 factors were combined with Myc, the enzyme activity in the resulting induced hepatocytes was significantly higher than in the case of 6 factors, and comparable to primary cultured hepatocytes.

図7では、6TF-Myc iHepsを用い、種々の薬剤に対するCYP遺伝子の発現誘導を調べた。その結果、RifampicinではCyp3a11が誘導され、OmeprazoleではCyp1a2が誘導され、TCP0B0PではCyp2b10が誘導され、得られた誘導肝細胞は、薬剤に対応したCYPを活性化し、正常肝細胞の応答プロファイルを有していることが分かった。 In FIG. 7, 6TF-Myc iHeps was used to investigate the induction of CYP gene expression in response to various drugs. As a result, Rifampicin induced Cyp3a11, Omeprazole induced Cyp1a2, and TCP0B0P induced Cyp2b10, and the resulting induced hepatocytes activated CYP corresponding to the drug and had the response profile of normal hepatocytes. I found out that

[実施例2]
6因子の必要性の検討
以下の手順で6因子のうちの2~6因子を導入し、肝細胞分化を誘導した。
iHep induction
1. Viral transduction前日 (day-1)、凍結保存していたMEFsを起眠し、Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat# 354230) でコーティングした12-well plate (Greiner) 上に播種 (5.0 × 104 cells/well)。
2. Viral transduction当日(day0)、Plat-Eの培養上清をウイルス上清として回収し、filtering (Millex-HP Syringe Filter Unit 0.45 μm, Merck Millipore, Cat# SLHP033RS)を行った。
3. 各転写因子のウイルス上清を各100 μl, Polybrene (nacalai tesque, Cat# 12996-81) 0.6 μl を加え、そのうち500 μlを、前日MEFsを播種した12-well plateの1wellあたりに加えた。
4. 37℃, 5% CO2 で一晩インキュベートし、ウイルス完成を行った。
5. 翌日 (day1) 培地交換 (10% FBS/DMEM)した。
6. Day3にも培地交換 (10% FBS/DMEM)した。
7. Day4で10% FBS/DMEM/F12, GlutaMAXをベースにしたHepatic specification培地 (DMEM/F12, GlutaMAX (Thermo Fisher Scientific, Cat# 10565042) 360 ml, FBS 40 ml, Nicotinamide (STEMCELL Technologies, Cat# 07154) 10mM, ITS-X (Thermo Fisher Scientific, Cat# 51500056) 400 μl, Dexamethasone (Merck, Cat# D4902-25MG) 0.1 μM, 2-Mercaptoethanol (Thermo Fisher Scientific, Cat# 21985023) 50 μM, Penicillin-Streptomycin (Thermo Fisher Scientific, Cat# 15140122) 2 ml)に培地交換した。さらに2日に一度培地交換した。
8. Day11から、hepatic maturation培地(HCM BulletKit Medium (Lonza, Cat# CC-3198), HGF (Peprotech, Cat# 100-39) 20 ng/ml, OSM (Peprotech, Cat# 300-10) 20 ng/ml)に変更して、2日に一度培地交換した。Day19前後で実験に供した。
[Example 2]
Consideration of the necessity of 6 factors
Two to six of the six factors were introduced according to the following procedure to induce hepatocyte differentiation.
iHep induction
1. The day before viral transduction (day-1), the frozen MEFs were awakened and placed on a 12-well plate (Greiner) coated with Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat# 354230). Seeding (5.0 × 10 4 cells/well).
2. On the day of viral transduction (day 0), the culture supernatant of Plat-E was collected as a virus supernatant and filtered (Millex-HP Syringe Filter Unit 0.45 μm, Merck Millipore, Cat# SLHP033RS).
3. Add 100 μl of virus supernatant of each transcription factor and 0.6 μl of Polybrene (Nacalai Tesque, Cat# 12996-81), of which 500 μl was added per well of the 12-well plate that had been seeded with MEFs the previous day. .
4. Virus completion was performed by incubating overnight at 37°C and 5% CO2 .
5. The next day (day1), the medium was replaced (10% FBS/DMEM).
6. The medium was replaced (10% FBS/DMEM) on Day 3 as well.
7. On Day 4, add 10% FBS/DMEM/F12, GlutaMAX based Hepatic specification medium (DMEM/F12, GlutaMAX (Thermo Fisher Scientific, Cat# 10565042) 360 ml, FBS 40 ml, Nicotinamide (STEMCELL Technologies, Cat# 07154) ) 10mM, ITS-X (Thermo Fisher Scientific, Cat# 51500056) 400 μl, Dexamethasone (Merck, Cat# D4902-25MG) 0.1 μM, 2-Mercaptoethanol (Thermo Fisher Scientific, Cat# 21985023) 50 μM, Penicillin-Streptomycin ( The medium was replaced with 2 ml (Thermo Fisher Scientific, Cat# 15140122). Furthermore, the medium was replaced once every two days.
8. From Day 11, add hepatic maturation medium (HCM BulletKit Medium (Lonza, Cat# CC-3198), HGF (Peprotech, Cat# 100-39) 20 ng/ml, OSM (Peprotech, Cat# 300-10) 20 ng/ ml), and the medium was replaced once every two days. It was used for experiments around Day 19.

<結果>
図8に示すように、6因子のうち1つでも欠けると、肝細胞へのconversion 効率が大幅に落ちることが確認できた。なお、本実施例ではMyc遺伝子は導入しておらず、Myc遺伝子はなくとも6因子で肝細胞分化が効率よく実施できることが示された。
<Results>
As shown in Figure 8, it was confirmed that if even one of the six factors is missing, the conversion efficiency into hepatocytes is significantly reduced. Note that in this example, the Myc gene was not introduced, and it was shown that hepatocyte differentiation can be efficiently performed with at least six factors without the Myc gene.

[実施例3]
次に、ヒト体細胞を用いて以下の手順で肝細胞分化を誘導した。
iHep induction
1. Viral transduction前日 (day-1)、凍結保存していたHDF-Slc7a1(Yamakawa, Stem Cells 2016; gift from Okita lab.)を起眠し、Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat# 354230) でコーティングした12-well plate (Greiner) 上に播種 (5.0 × 104 cells/well)した。
2. Viral transduction当日(day0)、Plat-Eの培養上清をウイルス上清として回収し、filtering (Millex-HP Syringe Filter Unit 0.45 μm, Merck Millipore, Cat# SLHP033RS)した。
3. 各転写因子のウイルス上清を各100 μl, c-Myc 12.5 μl, 10% FBS/DMEM 87.5 μl, Polybrene (nacalai tesque, Cat# 12996-81) 0.7 μl を加え、そのうち500 μlを、前日HDF-Slc7a1を播種した12-well plateの1wellあたりに加えた。
4. 37℃, 5% CO2 で一晩インキュベートし、ウイルス完成を行った。
5. 翌日 (day1) 培地交換 (10% FBS/DMEM)した。
6. Day3にも培地交換 (10% FBS/DMEM)した。
7. Day4に、Matrigelでコーティングした12-well plate上に1:10の割合で再播種した。培地は10% FBS/DMEM/F12, GlutaMAXをベースにしたHepatic specification培地 (DMEM/F12, GlutaMAX (Thermo Fisher Scientific, Cat# 10565042) 360 ml, FBS 40 ml, Nicotinamide (STEMCELL Technologies, Cat# 07154) 10mM, ITS-X (Thermo Fisher Scientific, Cat# 51500056) 400 μl, Dexamethasone (Merck, Cat# D4902-25MG) 0.1 μM, 2-Mercaptoethanol (Thermo Fisher Scientific, Cat# 21985023) 50 μM, Penicillin-Streptomycin (Thermo Fisher Scientific, Cat# 15140122) 2 ml)に培地交換した。
8. Day6から、hepatic maturation培地(HCM BulletKit Medium (Lonza, Cat# CC-3198), HGF (Peprotech, Cat# 100-39) 20 ng/ml, OSM (Peprotech, Cat# 300-10) 20 ng/ml)に変更して、2日に一度培地交換した。Day20前後で実験に供した。
[Example 3]
Next, hepatocyte differentiation was induced using human somatic cells according to the following procedure.
iHep induction
1. The day before Viral transduction (day-1), we woke up the frozen HDF-Slc7a1 (Yamakawa, Stem Cells 2016; gift from Okita lab.) and treated it with Matrigel (BD Matrigel Matrix Growth Factor Reduced, BD Biosciences, Cat). The cells were seeded (5.0 × 10 4 cells/well) on a 12-well plate (Greiner) coated with # 354230).
2. On the day of viral transduction (day 0), the culture supernatant of Plat-E was collected as a virus supernatant and filtered (Millex-HP Syringe Filter Unit 0.45 μm, Merck Millipore, Cat# SLHP033RS).
3. Add 100 μl of virus supernatant for each transcription factor, 12.5 μl of c-Myc, 87.5 μl of 10% FBS/DMEM, and 0.7 μl of Polybrene (nacalai tesque, Cat# 12996-81), of which 500 μl was added from the previous day. It was added to each well of a 12-well plate in which HDF-Slc7a1 was seeded.
4. Virus completion was performed by incubating overnight at 37°C and 5% CO2 .
5. The next day (day1), the medium was replaced (10% FBS/DMEM).
6. The medium was replaced (10% FBS/DMEM) on Day 3 as well.
7. On Day 4, the cells were reseeded at a ratio of 1:10 onto a 12-well plate coated with Matrigel. Medium: 10% FBS/DMEM/F12, GlutaMAX-based Hepatic specification medium (DMEM/F12, GlutaMAX (Thermo Fisher Scientific, Cat# 10565042) 360 ml, FBS 40 ml, Nicotinamide (STEMCELL Technologies, Cat# 07154) 10mM , ITS-X (Thermo Fisher Scientific, Cat# 51500056) 400 μl, Dexamethasone (Merck, Cat# D4902-25MG) 0.1 μM, 2-Mercaptoethanol (Thermo Fisher Scientific, Cat# 21985023) 50 μM, Penicillin-Streptomycin (Thermo Fisher The medium was replaced with 2 ml (Scientific, Cat# 15140122).
8. From Day 6, add hepatic maturation medium (HCM BulletKit Medium (Lonza, Cat# CC-3198), HGF (Peprotech, Cat# 100-39) 20 ng/ml, OSM (Peprotech, Cat# 300-10) 20 ng/ml. ml), and the medium was replaced once every two days. It was used for experiments around Day 20.

<結果>
図9に示すように、ヒト細胞を用いても同様にアルブミン陽性の肝細胞を誘導できることが分かった。
<Results>
As shown in FIG. 9, it was found that albumin-positive hepatocytes could be similarly induced using human cells.

Claims (8)

Hnf4a(Hepatocyte nuclear factor 4 alpha)、Foxa3(Forkhead Box A3)、Cebpa(CCAAT/enhancer-binding protein alpha)、Cebpd(CCAAT/enhancer-binding protein delta)、Hnf6(Hepatocyte nuclear factor 6)およびOnecut2(Onecut domain family member 2)が導入された線維芽細胞を用意する工程、および
当該線維芽細胞を培養して肝細胞へと誘導する工程、を含む、肝細胞の製造方法。
Hnf4a (Hepatocyte nuclear factor 4 alpha), Foxa3 (Forkhead Box A3), Cebpa (CCAAT/enhancer-binding protein alpha), Cebpd (CCAAT/enhancer-binding protein delta), Hnf6 (Hepatocyte nuclear factor 6) and Onecut2 (Onecut domain A method for producing hepatocytes , comprising the steps of preparing fibroblasts into which family member 2) has been introduced, and culturing the fibroblasts to induce them into hepatocytes.
線維芽細胞がヒトまたはマウス由来である、請求項に記載の肝細胞の製造方法。 The method for producing hepatocytes according to claim 1 , wherein the fibroblasts are derived from humans or mice. Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2がヒトまたはマウス由来である、請求
項1または2に記載の肝細胞の製造方法。
The method for producing hepatocytes according to claim 1 or 2 , wherein Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 are derived from human or mouse.
Hnf4a、Foxa3、Cebpa、Cebpd、Hnf6およびOnecut2がレトロウイルスベクターを用いて導
入された、請求項1~のいずれか一項に記載の肝細胞の製造方法。
The method for producing hepatocytes according to any one of claims 1 to 3 , wherein Hnf4a, Foxa3, Cebpa, Cebpd, Hnf6 and Onecut2 are introduced using a retroviral vector.
前記線維芽細胞は、さらにMycが導入されている、請求項1~のいずれか一項に記載の
肝細胞の製造方法。
The method for producing hepatocytes according to any one of claims 1 to 4 , wherein the fibroblasts are further introduced with Myc.
前記培養工程が20日以上行われる、請求項1~のいずれか一項に記載の肝細胞の製造方法。 The method for producing hepatocytes according to any one of claims 1 to 5 , wherein the culturing step is performed for 20 days or more. 前記培養工程において培地はTGFβ(Transforming growth factor beta)受容体阻害剤を含む、請求項1~のいずれか一項に記載の肝細胞の製造方法。 The method for producing hepatocytes according to any one of claims 1 to 6 , wherein in the culturing step, the medium contains a TGFβ (Transforming growth factor beta) receptor inhibitor. TGFβ受容体阻害剤はA83-01である、請求項に記載の肝細胞の製造方法。 The method for producing hepatocytes according to claim 7 , wherein the TGFβ receptor inhibitor is A83-01.
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