JP6265385B2 - Cell amplification using amino acid preparations - Google Patents

Cell amplification using amino acid preparations Download PDF

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JP6265385B2
JP6265385B2 JP2015518230A JP2015518230A JP6265385B2 JP 6265385 B2 JP6265385 B2 JP 6265385B2 JP 2015518230 A JP2015518230 A JP 2015518230A JP 2015518230 A JP2015518230 A JP 2015518230A JP 6265385 B2 JP6265385 B2 JP 6265385B2
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hepatocytes
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valine
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JPWO2014188994A1 (en
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英樹 谷口
英樹 谷口
貴則 武部
貴則 武部
康晴 上野
康晴 上野
博之 小池
博之 小池
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Yokohama City University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids

Description

本発明は、アミノ酸製剤による細胞増幅法に関し、より詳細には、アミノ酸製剤を用いて細胞を培養することにより、細胞を増幅する方法に関する。   The present invention relates to a cell amplification method using an amino acid preparation, and more particularly to a method for amplifying a cell by culturing the cell using the amino acid preparation.

近年、様々な機能細胞へ分化する能力を有するiPS細胞などの多能性幹細胞を分化誘導することにより、創薬スクリーニングや再生医療に有益なヒト機能細胞を創出する方法が注目されている。   In recent years, attention has been focused on methods for creating human functional cells useful for drug discovery screening and regenerative medicine by inducing differentiation of pluripotent stem cells such as iPS cells having the ability to differentiate into various functional cells.

一般的には、多能性幹細胞の培養系にサイトカインを始めとしたタンパク質製剤などを添加することで、幹細胞の維持増幅、及び各種機能細胞への分化誘導が試みられている(特許文献１〜３、非特許文献１)。   In general, by adding a protein preparation such as a cytokine to a culture system of pluripotent stem cells, maintenance and amplification of stem cells and induction of differentiation into various functional cells have been attempted (Patent Documents 1 to 3). 3. Non-patent document 1).

しかし、上記のような従来の培養法においては、目的とする臓器細胞を分化誘導する上で必要となるタンパク質製剤が極めて高額であることが重大な課題となっている。中でも肝臓などのように、再生医療・産業応用に超大量の細胞（1011cells）が必要な臓器の場合、細胞の製造に莫大なコストを要することとなり、多能性幹細胞を利用した応用を阻む最大の障壁と考えられている。However, in the conventional culture methods as described above, it is a serious problem that the protein preparation required for inducing differentiation of the target organ cells is extremely expensive. In particular, in the case of organs that require an extremely large number of cells (10 11 cells) for regenerative medicine and industrial applications, such as the liver, the production of cells requires enormous costs, and applications using pluripotent stem cells are required. It is considered the greatest barrier to block.

したがって、ES細胞あるいはiPS細胞などの多能性幹細胞を用いた再生医療・産業応用の実現化を目指すためには、いかに効率よくかつ安価に未分化な臓器細胞から機能細胞を大量に精製するか、という点が重大な課題と考えられている。   Therefore, in order to realize regenerative medicine / industrial application using pluripotent stem cells such as ES cells or iPS cells, how efficiently and inexpensively purify functional cells in large quantities from undifferentiated organ cells This is considered a serious issue.

特開２０１１−２０６０６４JP2011-206064 特開２０１２−２２８２６３JP2012-228263A 特開２０１０−７５１９９JP 2010-75199 A

Hannan N.R., et al., Nat. Protoc., 8(2), 430-437 (2013)Hannan N.R., et al., Nat. Protoc., 8 (2), 430-437 (2013)

本発明は、効率よく安価に未分化臓器細胞から機能細胞を創出する方法を提供することを目的とする。   An object of the present invention is to provide a method for efficiently and inexpensively creating functional cells from undifferentiated organ cells.

本発明者らは、大量創出を試みる臓器細胞の元となる、分化中間段階に存在する未分化な細胞と、それ以外の分化段階に存在する細胞との代謝特性の違いに着目した。   The present inventors paid attention to the difference in metabolic characteristics between undifferentiated cells existing in an intermediate differentiation stage and cells existing in other differentiation stages, which are the source of organ cells that attempt mass creation.

まず、異なる分化段階の細胞における代謝特性の差異を検出することを目的として、胎生初期から成体に至るまでの肝臓を対象として、メタボローム解析・トランスクリプトーム解析を行った。その結果、未分化な細胞が高頻度に存在する胎生初期（E9.5〜11.5）の肝臓において特異的に分岐鎖アミノ酸の分解酵素であるアミノ基転移酵素(branched-chain aminotransferase: BCAT1)が高発現しており、分岐鎖アミノ酸の分解が亢進していることを見出した。   First, metabolome analysis and transcriptome analysis were performed on the liver from the early embryonic stage to adulthood in order to detect the difference in metabolic characteristics in cells at different stages of differentiation. As a result, in the early embryonic liver (E9.5 to 11.5) in which undifferentiated cells are frequently present, the branched-chain aminotransferase (BCAT1), which specifically degrades branched-chain amino acids, is high. It was expressed, and it was found that the degradation of branched chain amino acids was enhanced.

この結果から、細胞増殖が活発な未分化な細胞において、分岐鎖アミノ酸の代謝要求度が著しく高いものと仮説を立て、未分化細胞への分岐鎖アミノ酸添加による影響の評価を行った。BCAT1が機能する分岐鎖アミノ酸(L-バリン、L-ロイシン、L-イソロイシン) 、ならびに比較対象や従来の培養系に頻繁に利用されているとして市販のアミノ酸製剤カクテルが細胞へ与える影響について、胎仔肝臓細胞を用いて検討した。   From these results, it was hypothesized that the metabolic demand of branched chain amino acids was remarkably high in undifferentiated cells with active cell proliferation, and the effects of adding branched chain amino acids to undifferentiated cells were evaluated. Regarding the effect of branched-chain amino acids (L-valine, L-leucine, L-isoleucine) on which BCAT1 functions, and commercially available amino acid cocktails that are frequently used in comparison and conventional culture systems, on the fetus Examination was performed using liver cells.

その結果、胎生初期(E11.5)の肝臓由来未分化細胞を用いた場合、アミノ酸非添加群、市販の必須アミノ酸製剤添加群、および市販の非必須アミノ酸製剤添加群と比較して、分岐鎖アミノ酸を添加した群において高い増殖性を示し、肝細胞特異的マーカーであるアルブミンを強く発現する細胞から構成されるコロニーが高頻度に出現することを見出した。さらに、遺伝子発現解析より、分岐鎖アミノ酸添加群ではアルブミンの発現が上昇することを確認した。このような分岐鎖アミノ酸による効果は、胎生中期(E15.5)の肝臓細胞を用いた解析では確認されなかったことから、胎生初期に存在する未分化な肝臓細胞でのみ特異的に分岐鎖アミノ酸添加による効果が現れることを発見した。   As a result, when using undifferentiated liver-derived cells in the early embryonic period (E11.5), the branched chain compared to the amino acid non-addition group, the commercially available essential amino acid preparation added group, and the commercially available non-essential amino acid preparation added group It was found that colonies composed of cells exhibiting high proliferation in the group to which amino acids were added and composed of cells that strongly express albumin, a hepatocyte-specific marker, frequently appear. Furthermore, it was confirmed from the gene expression analysis that albumin expression was increased in the branched chain amino acid addition group. The effect of such branched chain amino acids was not confirmed by analysis using liver cells in the mid-embryonic stage (E15.5). It was discovered that the effect of addition appears.

以上の結果から、未分化な肝臓細胞に対して分岐鎖アミノ酸を添加することで、増殖性が亢進し、アルブミンを発現する肝細胞が高頻度に出現することが見いだされた。したがって、分岐鎖アミノ酸は、未分化な臓器細胞の、一過性初期増殖(transit amplification)の誘導を介して、機能細胞への分化を誘導する作用を有しているものと考えられる。   From the above results, it was found that by adding a branched chain amino acid to undifferentiated liver cells, the proliferation was enhanced and hepatocytes expressing albumin appeared frequently. Therefore, it is considered that the branched chain amino acid has an action of inducing differentiation of undifferentiated organ cells into functional cells through induction of transient amplification (transit amplification).

本発明の要旨は以下の通りである。
（１）1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することを含む、未分化肝細胞の増幅方法。
（２）1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することにより、未分化肝細胞が増幅し、かつ肝細胞への分化が誘導される（１）記載の方法。
（３）分岐鎖アミノ酸が、バリン、ロイシン、イソロイシン及びそれらの組み合わせからなる群より選択される（１）又は（２）記載の方法。
（４）1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することを含む、肝細胞への分化誘導方法。
（５）1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することにより、未分化肝細胞を増幅し、かつ肝細胞への分化を誘導することを含む、肝細胞の調製方法。
（６）1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することを含む、肝細胞への分化が可能な細胞の選別法。
（７）1 mM以上の濃度の分岐鎖アミノ酸を含む、培地。
（８）未分化肝細胞を増幅するために用いられる（７）記載の培地。
（９）未分化肝細胞を肝細胞へ分化誘導するために用いられる（７）記載の培地。
（１０）分岐鎖アミノ酸を含む、未分化肝細胞増幅促進剤。
（１１）分岐鎖アミノ酸を含む、肝細胞への分化誘導剤。The gist of the present invention is as follows.
(1) A method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid having a concentration of 1 mM or more.
(2) The culture of undifferentiated hepatocytes in the presence of a branched-chain amino acid at a concentration of 1 mM or more amplifies undifferentiated hepatocytes and induces differentiation into hepatocytes (1) Method.
(3) The method according to (1) or (2), wherein the branched chain amino acid is selected from the group consisting of valine, leucine, isoleucine and combinations thereof.
(4) A method for inducing differentiation into hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.
(5) Hepatocytes comprising amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. Preparation method.
(6) A method for selecting cells capable of differentiating into hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched-chain amino acid at a concentration of 1 mM or more.
(7) A medium containing a branched chain amino acid at a concentration of 1 mM or more.
(8) The medium according to (7), which is used for amplifying undifferentiated hepatocytes.
(9) The medium according to (7), which is used for inducing differentiation of undifferentiated hepatocytes into hepatocytes.
(10) An undifferentiated hepatocyte amplification promoter containing a branched chain amino acid.
(11) An agent for inducing differentiation into hepatocytes, comprising a branched chain amino acid.

本発明により提供される培養方法および培地によって、ES/iPS細胞に由来する組織幹細胞などの未分化な臓器細胞から安価にかつ効率的に機能細胞を創出することが可能となる。これにより創薬スクリーニングに必要なヒト肝細胞の大量創出に有用な細胞操作技術となることが期待される。   By the culture method and medium provided by the present invention, it becomes possible to efficiently and efficiently create functional cells from undifferentiated organ cells such as tissue stem cells derived from ES / iPS cells. This is expected to become a cell manipulation technique useful for creating a large amount of human hepatocytes necessary for drug discovery screening.

さらに、多能性幹細胞を臨床応用するためには大量の細胞を得ることに加え、腫瘍形成や異所性組織形成の原因となる組織幹細胞以前の多能性幹細胞や他臓器へ分化した細胞を除去することが必要不可欠となる。本発明によって、より未分化な細胞や目的としない臓器細胞が生存不可能な代謝環境を構築することにより、目的とする未分化な臓器細胞のみを安価に大量に精製することが可能となるものと期待される。従来技術のように、遺伝子改変やFACSなど複雑な技術を用いる必要性がないことから、極めて優位性が高い安価かつ単純な方法となる。   Furthermore, in order to clinically apply pluripotent stem cells, in addition to obtaining a large amount of cells, pluripotent stem cells before tissue stem cells that cause tumor formation and ectopic tissue formation and cells that have differentiated into other organs It is essential to remove it. By constructing a metabolic environment in which more undifferentiated cells and unintended organ cells cannot survive according to the present invention, only target undifferentiated organ cells can be purified in large quantities at low cost. It is expected. Since there is no need to use complicated techniques such as genetic modification and FACS as in the prior art, it is an inexpensive and simple method with extremely high advantages.

本発明により、従来用いられてきたタンパク質製剤よりも安価なアミノ酸製剤を用いて、効率的に細胞を増幅・選別することができる培養方法が提供される。   INDUSTRIAL APPLICABILITY The present invention provides a culture method that can efficiently amplify and sort cells using an amino acid preparation that is less expensive than conventionally used protein preparations.

本明細書は、本願の優先権の基礎である日本国特許出願、特願2013-106289の明細書および／または図面に記載される内容を包含する。   This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2013-106289, which is the basis of the priority of the present application.

肝発生初期において特徴的な活性を示す代謝経路の抽出。(A)トランスクリプトーム解析による異なる分化段階の肝臓におけるアミノ酸代謝関連遺伝子発現量の比較。トランスクリプトーム解析より算出した、胎生9.5、10.5、11.5、13.5、15.5、17.5、19.5日目マウス胎仔、出生0、3日目乳仔および8週齢成体マウスの肝臓細胞中の各アミノ酸代謝経路に関与する遺伝子群の発現量をヒートマップで図示したもの。ヒートマップの色は平均値を黒として、赤: 2倍以上の増加、緑: 2倍以上の減少、を示している。複数のマーカーの中でもBcat1が顕著に肝発生初期特異的に発現することを示す。(B) 定量RT-PCRを用いたマウス分岐差アミノ酸代謝酵素(Bcat1)の発現確認。(A)の結果と同様に、肝発生の初期でのみBcat1の発現が亢進していることが確認される。(C)免疫組織化学染色による発生初期マウス胎児肝臓(胎生11.5日目)におけるBcat1の発現確認。Ck8/18で染色される胎児肝臓において、Bcat1の発現が確認される。赤: Bcat1、緑: Ck8/18(上皮細胞マーカー)、青: DAPI。He: 心臓、 Li: 肝臓。Extraction of metabolic pathways that exhibit characteristic activities in the early stages of liver development. (A) Comparison of expression levels of genes related to amino acid metabolism in livers at different stages of differentiation by transcriptome analysis. Each amino acid metabolism pathway in the liver cells of embryonic day 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, 19.5 day mouse fetus, birth day 0, day 3 infant and 8 week old adult mouse calculated from transcriptome analysis The expression level of the gene group involved in is shown in the heat map. The color of the heat map indicates that the average value is black, red: an increase of 2 times or more, and green: a decrease of 2 times or more. It shows that Bcat1 is remarkably expressed in the early stage of liver development among several markers. (B) Confirmation of expression of mouse branching difference amino acid metabolizing enzyme (Bcat1) using quantitative RT-PCR. Similar to the results of (A), it is confirmed that the expression of Bcat1 is increased only in the early stage of liver development. (C) Confirmation of Bcat1 expression in early embryonic mouse fetal liver (embryonic day 11.5) by immunohistochemical staining. Bcat1 expression is confirmed in fetal liver stained with Ck8 / 18. Red: Bcat1, Green: Ck8 / 18 (epithelial cell marker), Blue: DAPI. He: Heart, Li: Liver. メタボローム解析による異なる分化段階の肝臓におけるアミノ酸量の比較。(A)分岐差アミノ酸代謝経路図。Bcatは分岐差アミノ酸(BCAA)の代謝の初期段階を担っており、分岐差アミノ酸代謝物は最終的にクエン酸回路へと取り込まれ、エネルギー産生に用いられる。(B)メタボローム解析より算出した、胎生11.5、19.5日目マウス胎仔および8週齢成体マウスの肝組織中の各アミノ酸濃度の比較。Leu: ロイシン, Val: バリン, Ile: イソロイシン, Arg: アルギニン, Asn: アスパラギン, Asp: アスパラギン酸, Cys: システイン, Glu: グルタミン酸, Phe: フェニルアラニン, Pro: プロリン, Ser: セリン, Thr: トレオニン。縦軸の単位はnmol/g。分岐差アミノ酸(Leu, Val, Ile)は胎生11.5日目において肝臓中の含有量が低いことが確認される。Comparison of amino acid content in livers at different stages of differentiation by metabolomic analysis. (A) Branching difference amino acid metabolic pathway diagram. Bcat is responsible for the initial stage of metabolism of branched-chain amino acids (BCAA), and branched-chain amino acid metabolites are finally taken into the citric acid cycle and used for energy production. (B) Comparison of amino acid concentrations in liver tissues of embryonic day 11.5 and 19.5 day mouse fetuses and 8-week-old adult mice, calculated from metabolomic analysis. Leu: leucine, Val: valine, Ile: isoleucine, Arg: arginine, Asn: asparagine, Asp: aspartic acid, Cys: cysteine, Glu: glutamic acid, Phe: phenylalanine, Pro: proline, Ser: serine, Thr: threonine. The unit of the vertical axis is nmol / g. It is confirmed that the content of divergent amino acids (Leu, Val, Ile) is low in the liver on embryonic day 11.5. 妊娠マウスへ分岐差アミノ酸非含有飼料を与えたときの胎児肝形成に与える影響評価。(A)妊娠8.5日目から分岐差アミノ酸非含有飼料を与えた、胎生13.5日目マウスおよび肝臓像。(B)同マウス胎児重量、(C)肝重量、(D)胎児重量に占める肝臓重量の割合。分岐差アミノ酸非含有飼料を与えたマウスでは肝臓の形成が阻害される。Evaluation of the effect on fetal liver formation when a diet containing no branched-chain amino acids is given to pregnant mice. (A) Embryonic day 13.5 mice and liver images fed with a diet containing no branching difference amino acids from the 8.5th day of pregnancy. (B) Mouse fetal weight, (C) Liver weight, (D) Liver weight ratio to fetal weight. Liver formation is inhibited in mice fed a diet containing no branching difference amino acids. 妊娠マウスへバリン非含有飼料を与えた時の胎児肝前駆細胞に与える影響評価。(A)妊娠8.5日目からバリン非含有飼料を与えた、胎生13.5日目マウス肝臓細胞のフローサイトメトリー解析。血球細胞マーカーとしてCD45/TER119を、肝前駆細胞マーカーとしてDlk1を用いた。(B) (A)に基づくDlk1陽性細胞の割合。バリン非含有飼料を与えられたマウス胎児は肝臓中に含まれる肝前駆細胞の割合も減少する。Evaluation of the effect on fetal liver progenitor cells when a valine-free diet was given to pregnant mice. (A) Flow cytometric analysis of embryonic day 13.5 mouse liver cells fed a valine-free diet from day 8.5 of gestation. CD45 / TER119 was used as a blood cell marker, and Dlk1 was used as a hepatic progenitor cell marker. (B) Percentage of Dlk1-positive cells based on (A). Mouse fetuses fed a valine-free diet also have a reduced proportion of hepatic progenitor cells in the liver. 分岐鎖アミノ酸の中でもL-バリンを豊富に含む培養条件下で発生初期のマウス肝臓細胞の増殖が亢進する。(A)基礎組成の培地へ分岐鎖アミノ酸(L-バリン、L-イソロイシン, L-ロイシン)を0.4 mM〜4 mMの範囲の濃度で添加し、胎生11日目マウス胎仔肝臓由来細胞を6日間培養した後の90個以上で構成されるコロニー数の変化。Contはアミノ酸非添加群を示し、Contの値に対する増加率を%で示している。(B)基礎組成の培地へ分岐鎖アミノ酸(L-バリン、L-イソロイシン, L-ロイシン)および非必須アミノ酸を4 mMの濃度で添加し、胎生11.5、13.5、15.5日目マウス胎仔肝臓由来細胞を6日間培養した後の90個以上で構成されるコロニー数の変化。左は非添加群(control)およびL-バリン添加時の培養6日目のコロニーの写真。スケールバーは200μm。右は各アミノ酸添加し、マウス胎仔肝臓由来細胞を6日間培養した後の90個以上で構成されるコロニー数の変化。BCAAはL-バリン、L-イソロイシン、L-ロイシンを各4 mM添加したもの、NEAAは非必須アミノ酸添加群を示し、Contの値に対する増加率を%で示している。上段から胎生11.5日目、胎生13.5日目、胎生15.5日目のデータ。エラーバーは標準誤差。有意差検定はMann-Whitney’s U test。Nは3-12。Proliferation of mouse liver cells in the early stages of development is enhanced under culture conditions rich in L-valine among branched chain amino acids. (A) A branched chain amino acid (L-valine, L-isoleucine, L-leucine) is added to a medium having a basic composition at a concentration ranging from 0.4 mM to 4 mM, and embryonic day 11 mouse fetal liver-derived cells are added for 6 days. Change in the number of colonies composed of 90 or more after culturing. Cont indicates a group not added with amino acid, and the rate of increase relative to the value of Cont is indicated in%. (B) Branched-chain amino acids (L-valine, L-isoleucine, L-leucine) and non-essential amino acids were added to the basic composition medium at a concentration of 4 mM, and embryonic day 11.5, 13.5, 15.5 mouse embryonic liver-derived cells Change in the number of colonies composed of 90 or more after 6 days of culture. The left is a photograph of a colony on the 6th day of culture when the control group and L-valine were added. The scale bar is 200 μm. On the right is the change in the number of colonies composed of 90 or more after adding each amino acid and culturing mouse fetal liver-derived cells for 6 days. BCAA represents L-valine, L-isoleucine and L-leucine added at 4 mM each, NEAA represents a non-essential amino acid addition group, and the percentage increase relative to the value of Cont is indicated in%. From the top row, data for embryonic day 11.5, embryonic day 13.5, and embryonic day 15.5. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 3-12. L-バリンを豊富に含む培養条件下ではマウス肝幹・前駆細胞の肝細胞への分化が促進される。(A)分岐鎖アミノ酸を4 mM添加し6日間培養した後のコロニーの免疫染色像。赤: アルブミン(ALB)、緑: サイトケラチン(Ck)7、青: DAPI。上から非添加群、L-バリン、L-イソロイシン、L-ロイシン添加群。(B)免疫染色像から算出した、分岐鎖アミノ酸添加時のコロニー中の各系譜細胞の存在率の比較。円グラフ内の黒部分がALB陽性かつCk7陰性細胞、灰部分がALB陰性かつCk7陽性細胞、白部分がALB/Ck7共陽性細胞の割合。上から非添加群、L-バリン、L-イソロイシン、L-ロイシンの結果である。 (C)基礎培地へアミノ酸を添加し培養6日後の細胞を対象としたALBおよびCk7の遺伝子発現量比較。非添加群の値を1としたときの相対値で示している。エラーバーは標準誤差。有意差検定はMann-Whitney’s U test。Nは4-8。Under culture conditions rich in L-valine, differentiation of mouse hepatic stem / progenitor cells into hepatocytes is promoted. (A) An immunostained image of a colony after adding 6 mM of a branched chain amino acid and culturing for 6 days. Red: Albumin (ALB), Green: Cytokeratin (Ck) 7, Blue: DAPI. From the top, no addition group, L-valine, L-isoleucine, L-leucine addition group. (B) Comparison of the abundance of each lineage cell in the colony when branched-chain amino acids were added, calculated from immunostained images. The black part in the pie chart is the ratio of ALB positive and Ck7 negative cells, the gray part is ALB negative and Ck7 positive cells, and the white part is the ratio of ALB / Ck7 co-positive cells. From the top, the results for the non-added group, L-valine, L-isoleucine and L-leucine are shown. (C) Comparison of gene expression levels of ALB and Ck7 in cells after 6 days in culture after adding amino acids to the basal medium. The value is shown as a relative value when the value of the non-addition group is 1. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 4-8. 分岐鎖アミノ酸を豊富に含む培養条件下では未分化なヒト肝臓細胞の増殖および肝細胞への分化が促進される。(A)本実験で用いた分化誘導プロトコルの概略図。各分化段階の細胞として、培養系への添加因子を変更するタイムポイント毎に、Stage1、2、３，４と定義した。(B) 各分化段階におけるヒトiPS由来肝臓細胞およびヒト肝臓細胞を対象としたヒトBCAT1遺伝子の発現量比較。iPS細胞の分化過程においては、Stage2におけるBCAT1の発現が最も高いことが確認された。(C)分岐鎖アミノ酸添加時のヒトiPS由来肝内胚葉細胞の増殖性の変化。(D)分岐鎖アミノ酸添加時のヒトiPS由来肝内胚葉細胞の分化の変化。エラーバーは標準誤差。有意差検定はMann-Whitney’s U test。Nは4-8。Under culture conditions rich in branched chain amino acids, proliferation of undifferentiated human liver cells and differentiation into hepatocytes are promoted. (A) Schematic diagram of the differentiation induction protocol used in this experiment. As cells at each differentiation stage, Stages 1, 2, 3, and 4 were defined for each time point at which the factors added to the culture system were changed. (B) Comparison of human BCAT1 gene expression levels in human iPS-derived liver cells and human liver cells in each differentiation stage. In the differentiation process of iPS cells, it was confirmed that the expression of BCAT1 in Stage2 was the highest. (C) Proliferative change of human iPS-derived hepatic endoderm cells upon addition of branched chain amino acids. (D) Change in differentiation of human iPS-derived hepatic endoderm cells upon addition of branched chain amino acids. Error bars are standard errors. Significant difference test is Mann-Whitney ’s U test. N is 4-8. ヒトiPS細胞由来肝芽創出過程における分岐差アミノ酸添加効果の検証。(A)実験プロトコルの概略。ヒトiPS細胞由来肝臓細胞(Stage2)、ヒト臍帯静脈内皮細胞(HUVEC)および間葉系幹細胞(MSC)を共培養することで肝芽を創出した。その後肝芽を分岐鎖アミノ酸過剰の培地で培養した。(B)2週間培養後の肝芽の形態。(C)肝芽の平均半径および増殖の高い肝芽の割合。Verification of the effect of addition of branching amino acids in the process of creating human iPS cell-derived liver buds. (A) Outline of experimental protocol. Liver buds were created by co-culturing human iPS cell-derived liver cells (Stage2), human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC). Thereafter, the liver buds were cultured in a medium containing excess branched-chain amino acids. (B) Hepatic bud morphology after 2 weeks of culture. (C) Average radius of liver buds and percentage of hepatic buds with high proliferation. 分岐鎖アミノ酸の濃度を変化させたときのマウス肝臓細胞の細胞増殖亢進効果の検討。(A)基礎組成の培地へ分岐鎖アミノ酸(L-バリン、L-イソロイシン, L-ロイシン)を0.4 mM〜20 mMの範囲の濃度で添加し、胎生11日目マウス胎仔肝臓由来細胞を6日間培養した後の90個以上で構成されるコロニー数の変化。横軸はそれぞれの分岐鎖アミノ酸添加濃度(mM)を示し、非添加群(0 mM)の値に対する増加率を%で示している。エラーバーは標準誤差。Nは3-12。Examination of the effect of increasing cell proliferation of mouse liver cells when the concentration of branched chain amino acids is changed. (A) A branched chain amino acid (L-valine, L-isoleucine, L-leucine) is added to a medium having a basic composition at a concentration ranging from 0.4 mM to 20 mM, and embryonic day 11 mouse fetal liver-derived cells are added for 6 days. Change in the number of colonies composed of 90 or more after culturing. The horizontal axis shows the concentration of each branched chain amino acid added (mM), and the percentage increase relative to the value of the non-added group (0 mM) is shown in%. Error bars are standard errors. N is 3-12.

以下、本発明を詳細に説明する。   Hereinafter, the present invention will be described in detail.

本発明は、1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することを含む、未分化肝細胞の増幅方法を提供する。   The present invention provides a method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more.

本発明の方法において、分岐鎖アミノ酸の濃度は、1 mM以上であり、好ましくは、 1 〜 20 mMであり、より好ましくは、 2 〜 10 mMである。   In the method of the present invention, the concentration of the branched chain amino acid is 1 mM or more, preferably 1 to 20 mM, and more preferably 2 to 10 mM.

分岐鎖アミノ酸としては、バリン、ロイシン、イソロイシン並びにこれらの誘導体などのアミノ酸(Dおよび/またはL-アミノ酸の両方)、及びそれらの組み合わせを例示することができ、好適にはバリンである。   Examples of branched chain amino acids include amino acids (both D and / or L-amino acids) such as valine, leucine, isoleucine and derivatives thereof, and combinations thereof, and valine is preferred.

未分化肝細胞の「未分化」とは、分化が完全に終わっていない状態をいい、未分化肝細胞とは、肝細胞に分化可能なあらゆる細胞を含む概念である。未分化肝細胞としては、iPS細胞やES細胞などの多能性幹細胞や生体組織に由来する未分化な臓器（例えば、肝臓）細胞などを例示することができる。未分化肝細胞は、BCAT1の発現が亢進されるステージにあるとよい。未分化肝臓細胞はコーティングされた細胞培養容器において接着培養するとよい。細胞培養容器のコーティング剤は、マトリゲル、ラミニン、コラーゲン、ゼラチン、フィブロネクチンならびに細胞外マトリックスなどである。培養は34℃〜38℃、好ましくは37℃の温度で行い、ＣＯ２濃度は２％〜１０％が好ましく、５％が最も好ましい。“Undifferentiated” of undifferentiated hepatocytes refers to a state in which differentiation is not completely completed, and undifferentiated hepatocytes is a concept including all cells that can differentiate into hepatocytes. Examples of undifferentiated hepatocytes include pluripotent stem cells such as iPS cells and ES cells, and undifferentiated organ (eg, liver) cells derived from living tissue. The undifferentiated hepatocytes are preferably in a stage where the expression of BCAT1 is enhanced. Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of the coating agent for the cell culture container include matrigel, laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO 2 concentration is preferably 2% to 10%, and most preferably 5%.

1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することにより、未分化肝細胞が増幅し、かつ肝細胞への分化が誘導される。従って、本発明は、1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することを含む、肝細胞への分化誘導方法も提供する。未分化肝臓細胞はコーティングされた細胞培養容器において接着培養するとよい。細胞培養容器のコーティング剤は、マトリゲル、ラミニン、コラーゲン、ゼラチン、フィブロネクチンならびに細胞外マトリックスなどである。培養は34℃〜38℃、好ましくは37℃の温度で行い、ＣＯ２濃度は２％〜１０％が好ましく、５％が最も好ましい。By culturing undifferentiated hepatocytes in the presence of a branched chain amino acid having a concentration of 1 mM or more, undifferentiated hepatocytes are amplified and differentiation into hepatocytes is induced. Therefore, the present invention also provides a method for inducing differentiation into hepatocytes, including culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of the coating agent for the cell culture container include matrigel, laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO 2 concentration is preferably 2% to 10%, and most preferably 5%.

本発明の方法により、未分化肝細胞を増幅し、かつ肝細胞への分化を誘導することは、肝細胞の調製に利用することができる。従って、本発明は、1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することにより、未分化肝細胞を増幅し、かつ肝細胞への分化を誘導することを含む、肝細胞の調製方法も提供するものである。   Amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by the method of the present invention can be used for the preparation of hepatocytes. Therefore, the present invention includes amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. A method for preparing hepatocytes is also provided.

後述の実施例に示すように、マウスの場合、胎生初期（E11.5）の肝臓由来未分化細胞に分岐鎖アミノ酸を添加すると、肝細胞特異的マーカーであるアルブミンを強く発現する細胞から構成されるコロニーが高頻度に出現した。よって、本発明の方法において、1 mM以上の濃度の分岐鎖アミノ酸の存在下で培養する未分化肝細胞は、マウスで言うと、E11.5の時期に対応する分化段階のレベルの細胞であることが好ましい。この分化段階は、肝臓の幹・前駆細胞が大量に存在するステージである。細胞がそのような時期にあることは、肝臓の分化マーカー（例えば、アルブミン(ALB)、α-フェトプロテイン(AFP)、トランスサイレチン(TTR)、forkhead box protein A2(FOXA2)、Hepatocyte nuclear factor 4 alpha(HNF4A)、レチノール結合タンパク質4(RBP4)、シトクロムP450(CYP)3A4、CYP3A7、グルコース-6-ホスファターゼ(G6PC)、GATA binding protein 4/6(GATA4/6)、アシアロ糖タンパク質レセプター1(ASGR1)、アンジオテンシノーゲン(AGT)、トランスフェリン(TRF)、アポリポプロテインA-1(APOA1)、フィブリノゲンα鎖(FGA)、ホモゲンチジン酸(HGD)、Carbamoyl-Phosphate Synthase 1（CPS1）など）の発現を指標として、推測することができる。具体的には、GATA4/6、HNF4A、HHEX1、AFPなどのマーカーの発現レベルがES細胞やiPS細胞と比較して増加していれば、マウスE11.5の時期に対応する分化段階のレベルの細胞であると推測することができる。また、ALB、RBP4,ASGR1、AGT、TRF、FGA、APOA1、HGD、CPS1などのマーカーの発現レベルが他の分化段階の細胞と比較して減少していれば、マウスE11.5の時期に対応する分化段階のレベルの細胞であると推測することができる。また、一方で、BCAT1の発現を指標にして、推測することも可能である。具体的には、BCAT1の発現レベルがES細胞やiPS細胞と比較して増加していれば、マウスE11.5の時期に対応する分化段階のレベルの細胞であると推測することができる。   As shown in the Examples below, in the case of mice, when branched chain amino acids are added to liver-derived undifferentiated cells in the early embryonic period (E11.5), they are composed of cells that strongly express albumin, a hepatocyte-specific marker. Appeared frequently. Therefore, in the method of the present invention, undifferentiated hepatocytes cultured in the presence of a branched chain amino acid at a concentration of 1 mM or more are cells at a differentiation stage level corresponding to the period of E11.5 in mice. It is preferable. This differentiation stage is a stage in which a large amount of liver stem / progenitor cells are present. The presence of cells in such a period means that liver differentiation markers (eg, albumin (ALB), α-fetoprotein (AFP), transthyretin (TTR), forkhead box protein A2 (FOXA2), Hepatocyte nuclear factor 4 alpha (HNF4A), retinol binding protein 4 (RBP4), cytochrome P450 (CYP) 3A4, CYP3A7, glucose-6-phosphatase (G6PC), GATA binding protein 4/6 (GATA4 / 6), asialoglycoprotein receptor 1 (ASGR1) , Angiotensinogen (AGT), transferrin (TRF), apolipoprotein A-1 (APOA1), fibrinogen alpha chain (FGA), homogentisic acid (HGD), Carbamoyl-Phosphate Synthase 1 (CPS1) Can be guessed. Specifically, if the expression level of markers such as GATA4 / 6, HNF4A, HHEX1, and AFP is increased compared to ES cells and iPS cells, the level of differentiation stage corresponding to the time of mouse E11.5 It can be assumed that it is a cell. Also, if the expression level of markers such as ALB, RBP4, ASGR1, AGT, TRF, FGA, APOA1, HGD, CPS1 is reduced compared to cells in other differentiation stages, it corresponds to the time of mouse E11.5 It can be assumed that the cells are at the level of differentiation stage. On the other hand, it is also possible to estimate using the expression of BCAT1 as an index. Specifically, if the expression level of BCAT1 is increased as compared with ES cells or iPS cells, it can be assumed that the cells are at the level of differentiation corresponding to the time of mouse E11.5.

また、後述の実施例で示すように、マウス胎児由来の未分化肝臓細胞を培養後、分化状態を検討するため免疫染色を行ったところ、分岐鎖アミノ酸添加群は非添加群と比較して、アルブミン陽性の肝細胞が増加し、サイトケラチン7陽性の胆管上皮細胞の出現が低下していた。また、遺伝子発現解析からも、分岐鎖アミノ酸（特にL-バリン）添加時に、サイトケラチン7遺伝子の発現減少およびアルブミン遺伝子の発現増加が確認された。従って、分岐鎖アミノ酸添加により、胆管上皮細胞の出現を抑制し、肝細胞を効率的に創出することが可能と考えられる。従って、本発明は、1 mM以上の濃度の分岐鎖アミノ酸の存在下で、未分化肝細胞を培養することを含む、肝細胞への分化が可能な細胞の選別法も提供する。未分化肝臓細胞はコーティングされた細胞培養容器において接着培養するとよい。細胞培養容器のコーティング剤は、ラミニン、コラーゲン、ゼラチン、フィブロネクチンならびに細胞外マトリックスなどである。培養は34℃〜38℃、好ましくは37℃の温度で行い、ＣＯ２濃度は２％〜１０％が好ましく、５％が最も好ましい。In addition, as shown in Examples below, after culturing undifferentiated liver cells derived from mouse fetuses, immunostaining was performed in order to examine the differentiation state, the branched chain amino acid added group was compared with the non-added group, Albumin-positive hepatocytes increased, and the appearance of cytokeratin 7-positive bile duct epithelial cells decreased. Moreover, gene expression analysis also confirmed that cytokeratin 7 gene expression decreased and albumin gene expression increased when branched chain amino acid (particularly L-valine) was added. Therefore, it is considered that the addition of branched chain amino acids can suppress the appearance of bile duct epithelial cells and efficiently create hepatocytes. Therefore, the present invention also provides a method for selecting cells capable of differentiating into hepatocytes, including culturing undifferentiated hepatocytes in the presence of a branched chain amino acid at a concentration of 1 mM or more. Undifferentiated liver cells may be adherently cultured in a coated cell culture vessel. Examples of coating agents for cell culture containers include laminin, collagen, gelatin, fibronectin and extracellular matrix. Culturing is performed at a temperature of 34 ° C. to 38 ° C., preferably 37 ° C., and the CO 2 concentration is preferably 2% to 10%, and most preferably 5%.

さらに、本発明は、1 mM以上の濃度の分岐鎖アミノ酸を含む、培地を提供する。   Furthermore, the present invention provides a medium containing a branched chain amino acid at a concentration of 1 mM or more.

本発明の培地は、未分化肝細胞を増幅するために用いることができる。また、本発明の培地は、未分化肝細胞を肝細胞へ分化誘導するために用いることができる。さらに、本発明の培地は、肝細胞への分化が可能な細胞を選別するために用いることもできる。   The medium of the present invention can be used to amplify undifferentiated hepatocytes. The medium of the present invention can be used for inducing differentiation of undifferentiated hepatocytes into hepatocytes. Furthermore, the culture medium of the present invention can also be used to select cells that can differentiate into hepatocytes.

本発明の培地には、分岐鎖アミノ酸以外のアミノ酸(グルタミン、アラニン、アルギニン、アスパラギン、システイン、グルタミン酸、グリシン、ヒスチジン、リシン、メチオニン、フェニルアラニン、プロリン、セリン、トレオニン、トリプトファンおよびチロシン、並びに、これらの誘導体などのアミノ酸(Dおよび/またはL-アミノ酸の両方))、フェノールレッド、ピルビン酸塩、HEPES、微量金属、リン酸塩、酢酸塩、ビタミン類、アスコルビン酸、ニコチンアミド、2-メルカプトエタノール、デキサメタゾン、インスリン、上皮成長因子(EGF)、肝細胞増殖因子(HGF)、アクチビンA、塩基性繊維芽細胞増殖因子(bFGF)、骨形成タンパク質(BMP)4、オンコスタチンM、ヒドロコルチゾン、ヘパリン、血管内皮細胞成長因子(VEGF)、インシュリン様成長因子(R3-IGF)-1、ウシ脳抽出物(BBE)、ウシ胎児血清(FBS)、トランスフェリン、ウシ血清アルブミン(BSA)、血清代替物(N2、B27サプリメントなど)、緩衝剤、抗生物質(ゲンタマイシン、ペニシリン、ストレプトマイシン、アンホテリシン-B等)等の他の成分を添加してもよい。   The medium of the present invention contains amino acids other than branched chain amino acids (glutamine, alanine, arginine, asparagine, cysteine, glutamic acid, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine, and these Amino acids such as derivatives (both D and / or L-amino acids)), phenol red, pyruvate, HEPES, trace metals, phosphates, acetates, vitamins, ascorbic acid, nicotinamide, 2-mercaptoethanol, Dexamethasone, insulin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), activin A, basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP) 4, oncostatin M, hydrocortisone, heparin, blood vessel Endothelial cell growth factor (VEGF), insulin-like growth factor (R3- IGF) -1, bovine brain extract (BBE), fetal bovine serum (FBS), transferrin, bovine serum albumin (BSA), serum replacement (N2, B27 supplement etc.), buffer, antibiotics (gentamicin, penicillin, Other components such as streptomycin, amphotericin-B and the like may be added.

本発明の培地の溶媒としては、水、血清、または、pH緩衝溶液などを用いることができる。あるいはまた、市販の培地（例えば、ダルベッコ改変イーグル培地(DMEM)、E-MEM、IMDM、乳糖含有glucose-free DMEM、ハム(Ham)F12、RPMI-1640、ウイリアムズE、など、及びそれらの混和物）に分岐鎖アミノ酸を添加して、1 mM以上の濃度の分岐鎖アミノ酸を含むように調整してもよい。   As the medium of the medium of the present invention, water, serum, pH buffer solution or the like can be used. Alternatively, commercially available media (eg Dulbecco's Modified Eagle Medium (DMEM), E-MEM, IMDM, lactose-containing glucose-free DMEM, Ham F12, RPMI-1640, Williams E, etc., and mixtures thereof A branched chain amino acid may be added to) so as to contain a branched chain amino acid having a concentration of 1 mM or more.

未分化肝細胞については、上述した。   The undifferentiated hepatocytes have been described above.

本発明において、分岐鎖アミノ酸は、未分化肝細胞の増幅を促進することできる。従って、本発明は、分岐鎖アミノ酸を含む、未分化肝細胞増幅促進剤を提供する。   In the present invention, branched chain amino acids can promote amplification of undifferentiated hepatocytes. Therefore, the present invention provides an undifferentiated hepatocyte amplification promoter comprising a branched chain amino acid.

また、分岐鎖アミノ酸は未分化肝細胞から肝細胞への分化を誘導することができる。従って、本発明は、分岐鎖アミノ酸を含む、肝細胞への分化誘導剤を提供する。   In addition, branched chain amino acids can induce differentiation from undifferentiated hepatocytes to hepatocytes. Therefore, the present invention provides an agent for inducing differentiation into hepatocytes containing a branched chain amino acid.

本発明は、アミノ酸製剤（分岐鎖アミノ酸）を添加するという安価かつ単純な方法を用いることにより、ヒト臓器細胞の工業的製造に向けて劇的なコストダウンが可能な基盤的培養技術となる。本発明者らが過去に開発した技術（「組織・臓器の作製方法」WO2013/047639）と連動することにより、再生医療や産業応用上極めて有益な細胞操作技術となる。例えば、本発明によりヒトiPS細胞由来ヒト肝細胞を効率的に増幅することで、創薬開発において必要なヒト成熟肝細胞を大量に安価に製造することが可能となる。また、個人毎に樹立された複数のiPS細胞株を用いることで、人種・性別・個体差などスペックの明らかなヒト成熟肝細胞を、安定的かつ安価に大量供給することが可能となる。これにより、創薬開発における課題であった個人における反応性の相違を検出する革新的なスクリーニング技術となることが期待される。   The present invention provides a basic culture technique capable of drastically reducing costs for industrial production of human organ cells by using an inexpensive and simple method of adding an amino acid preparation (branched chain amino acid). By cooperating with a technique developed in the past by the present inventors (“Tissue / Organ Production Method” WO2013 / 047639), it becomes a cell manipulation technique that is extremely useful for regenerative medicine and industrial applications. For example, by efficiently amplifying human iPS cell-derived human hepatocytes according to the present invention, it becomes possible to produce a large amount of mature human hepatocytes necessary for drug development at low cost. In addition, by using a plurality of iPS cell lines established for each individual, it becomes possible to stably supply a large amount of mature human hepatocytes having clear specifications such as race, sex, and individual differences. This is expected to provide an innovative screening technique for detecting differences in individual responsiveness, which was a challenge in drug development.

以下、実施例により本発明を更に詳細に説明する。本実施例において、特に断りがない限り、アミノ酸は、L-アミノ酸である。
〔実施例１〕
結果
メタボローム・トランスクリプトーム解析による異なる分化段階の肝臓における代謝特性の比較
異なる分化段階の細胞における代謝関連遺伝子の発現の差異を検出するために、胎生初期から成体に至るまでのマウス肝臓細胞を対象として、トランスクリプトーム解析を行った。各代謝経路における代表的な分子の発現を比較した結果、未分化な肝幹・前駆細胞が高頻度に存在する発生初期 (胎生9.5〜11.5 日目)の肝臓では、それ以降の発生段階の肝臓に比べて、脂質代謝、ビリルビン代謝、尿素回路を始めとする成体肝臓での主要な代謝に関わる多くの遺伝子発現が低いことが明らかとなった(データは示さず)。一方でアミノ酸代謝関連遺伝子の一部は肝発生初期でのみ高い発現を示し、中でも特に分岐鎖アミノ酸代謝遺伝子Branched-chain aminotransferase 1(Bcat1)の発現亢進が顕著であることが確認された(図 1A)。この結果について、定量PCRにより解析を行った場合も同様に肝発生初期特異的な発現亢進が認められた(図1B)。さらに、発生初期(胎生11.5日目)の肝臓を対象に免疫組織化学的解析を行った所、肝臓中で高いBcat1タンパク質の発現が確認された。Hereinafter, the present invention will be described in more detail with reference to examples. In this example, unless otherwise specified, the amino acid is an L-amino acid.
[Example 1]
result
Comparison of metabolic characteristics in livers of different differentiation stages by metabolomic and transcriptome analysis To detect differences in the expression of metabolism-related genes in cells of different differentiation stages, we used mouse liver cells from early embryonic stages to adulthood. Transcriptome analysis was performed. As a result of comparing the expression of representative molecules in each metabolic pathway, the liver in the early developmental stage (embryonic day 9.5 to 11.5) in which undifferentiated hepatic stem / progenitor cells are frequently present is found in the liver in the later developmental stages. In contrast, the expression of many genes involved in major metabolism in the adult liver including lipid metabolism, bilirubin metabolism, and urea cycle was found to be lower (data not shown). On the other hand, some of the genes related to amino acid metabolism showed high expression only in the early stage of liver development, and in particular, it was confirmed that the expression of branched-chain amino acid metabolism gene Branched-chain aminotransferase 1 (Bcat1) was particularly remarkable (Figure 1A). ). Similarly, when the results were analyzed by quantitative PCR, specific expression enhancement in the early stage of liver development was observed (FIG. 1B). Furthermore, when immunohistochemical analysis was performed on the liver in the early stage of development (embryonic day 11.5), high Bcat1 protein expression was confirmed in the liver.

そこで、アミノ酸代謝関連遺伝子の機能を確認するため、メタボローム解析を用いて肝発生過程における各代謝産物量を比較した。図2Aに示す様にBcat1は分岐鎖アミノ酸(L-バリン、L-ロイシン、L-イソロイシン)の代謝初期反応を担う酵素である。メタボローム解析の結果、アミノ酸の中でも分岐鎖アミノ酸(L-バリン、L-ロイシン、L-イソロイシン)濃度が肝発生初期(胎生11日目)に低いことが明らかとなった(図 2)。以上の結果から、肝発生初期に存在する未分化な肝臓細胞において、分岐鎖アミノ酸の代謝活性が高いものと仮説を立て、分岐鎖アミノ酸の機能的検証を行った。   In order to confirm the functions of amino acid metabolism-related genes, metabolome analysis was used to compare the amount of each metabolite in the liver development process. As shown in FIG. 2A, Bcat1 is an enzyme responsible for the initial metabolic reaction of branched chain amino acids (L-valine, L-leucine, L-isoleucine). As a result of metabolomic analysis, it was found that the concentration of branched chain amino acids (L-valine, L-leucine, L-isoleucine) among amino acids was low in the early stage of liver development (embryonic day 11) (Fig. 2). Based on the above results, we hypothesized that the metabolic activity of branched-chain amino acids is high in undifferentiated liver cells existing in the early stages of liver development, and functionally verified the branched-chain amino acids.

母体のL-バリン摂取量の減少は胎児肝幹/前駆細胞の頻度を減少させる
発生初期の胎児肝臓形成に与える分岐鎖アミノ酸の効果を検証するため、肝発生が開始する妊娠8.5日目の母体マウスへ分岐鎖アミノ酸非含有飼料を与え、胎生13.5日目の胎児を解析した。その結果、分岐鎖アミノ酸非含有飼料を与えた群ではコントロール群と比較して、肝重量が大きく減少することが確認された(図３)。この結果から発生初期の肝形成過程に分岐鎖アミノ酸が重要な役割を担っていることが示唆される。同様に、L-バリン非含有飼料を与えたマウス胎児においても肝臓の形成阻害が確認された(データは示さず)。 Decreased maternal L-valine intake reduces fetal liver stem / progenitor cell frequency. To verify the effects of branched-chain amino acids on early fetal liver formation, maternal day 8.5 gestation when liver development begins Mice were fed a diet containing no branched-chain amino acids, and the embryos on embryonic day 13.5 were analyzed. As a result, it was confirmed that the liver weight was significantly reduced in the group fed with the diet not containing branched chain amino acids compared to the control group (FIG. 3). This result suggests that branched-chain amino acids play an important role in the early hepatogenesis process. Similarly, inhibition of liver formation was also confirmed in mouse fetuses fed with L-valine-free diet (data not shown).

次に、マウス胎児肝幹/前駆細胞に与える影響について評価するため、妊娠8.5日目の母体マウスへL-バリン非含有飼料を与え、胎生13.5日目胎児肝臓中に含まれる肝幹/前駆細胞を解析するため、Dlk1を指標にフローサイトメトリー解析を実施した。その結果、コントロール群に比べて、L-バリン非含有飼料を与えた群ではDlk1陽性細胞の頻度が大きく減少することが確認された(図４)。これらの結果から、L-バリンの欠損が、肝幹/前駆細胞の頻度を減少させ、肝発生初期過程を阻害することが示唆された。   Next, in order to evaluate the effects on mouse fetal liver stem / progenitor cells, the L-valine-free diet was given to the 8.5th day of pregnancy, and the liver stem / progenitor cells contained in the embryonic day 13.5 fetal liver Therefore, flow cytometry analysis was performed using Dlk1 as an index. As a result, it was confirmed that the frequency of Dlk1-positive cells was greatly reduced in the group fed with L-valine-free feed compared to the control group (FIG. 4). These results suggest that L-valine deficiency decreases the frequency of hepatic stem / progenitor cells and inhibits the early process of liver development.

L-バリンを豊富に含む培養条件ではマウス肝幹/前駆細胞の増殖を亢進させる
BCAT1が代謝反応を担っているL-ロイシン、L-イソロイシン、L-バリン、ならびに従来の培養系に頻繁に利用されているものとして市販のアミノ酸製剤カクテルが細胞増殖へ与える影響について、マウス胎仔肝臓細胞を用いて検討した。まず、胎生初期 (E11.5) の肝臓由来未分化細胞を用いて、各分岐鎖アミノ酸添加による影響について評価した。分岐鎖アミノ酸添加濃度を変更したときの増殖促進効果を検討したところ、アミノ酸添加濃度が0.4あるいは0.8 mM 時には90個以上の細胞から構成される増殖性の高いコロニーの出現頻度がコントロールと比べて同等であったのに対し、L-バリン4 mM添加時には31%増加することが明らかとなった (図 5A)。そこで、胎生初期の肝臓由来未分化細胞へ各分岐鎖アミノ酸を4 mM添加した時の影響をアミノ酸非添加群、市販の必須アミノ酸製剤添加群、および市販の非必須アミノ酸製剤添加群と比較した。その結果、非添加群と比較して、4 mMのL-バリンを添加した群においてのみ高い増殖性を示すコロニーの頻度が増加することが確認された(図 5B)。このとき、他の分岐鎖アミノ酸(L-イソロイシン、L-ロイシン)および、市販の非必須、必須アミノ酸製剤を4 mM添加した群では高い増殖性を示すコロニーの出現頻度に差は見られなかった。 Increased proliferation of mouse hepatic stem / progenitor cells in culture conditions rich in L-valine
Regarding the effect of L-leucine, L-isoleucine, L-valine, which are responsible for metabolic reactions of BCAT1, and commercially available amino acid cocktails that are frequently used in conventional culture systems, on the cell growth, mouse fetal liver It examined using the cell. First, the effects of each branched chain amino acid addition were evaluated using undifferentiated liver-derived cells in the early embryonic period (E11.5). When the growth promotion effect when changing the concentration of branched chain amino acids was examined, when the amino acid addition concentration was 0.4 or 0.8 mM, the appearance frequency of highly proliferative colonies composed of 90 or more cells was equivalent to that of the control. On the other hand, when L-valine 4 mM was added, it was found to increase 31% (FIG. 5A). Therefore, the effect of adding 4 mM of each branched chain amino acid to liver-derived undifferentiated cells in the early embryonic period was compared with the amino acid non-added group, the commercially available essential amino acid preparation added group, and the commercially available non-essential amino acid preparation added group. As a result, it was confirmed that the frequency of colonies exhibiting high proliferative activity increased only in the group to which 4 mM L-valine was added compared to the non-added group (FIG. 5B). At this time, there was no difference in the appearance frequency of colonies showing high growth in the group to which 4 mM of other branched chain amino acids (L-isoleucine, L-leucine) and commercially available non-essential and essential amino acid preparations were added. .

次に、より発生の進行したE13.5およびE15.5の肝臓由来細胞に対して同様にアミノ酸添加による細胞の増殖性に与える影響を評価した。その結果、E11.5の肝臓由来細胞と異なり、増殖性の高いコロニーの出現頻度はいずれの分岐鎖アミノ酸溶液を加えた場合もコントロールと比べて同等あるいは減少していた。以上の結果から、L-バリンによる増殖促進効果は、胎生中期(E13.5〜)以降の肝臓細胞を用いた解析では確認されなかったことから、胎生初期に存在する未分化な肝臓細胞でのみ特異的にL-バリンの添加による増殖促進効果が現れることが示された。   Next, the effect of addition of amino acids on the proliferation of cells was similarly evaluated for E13.5 and E15.5 liver-derived cells, which were more advanced. As a result, unlike the E11.5 liver-derived cells, the appearance frequency of highly proliferative colonies was the same or decreased compared to the control when any branched-chain amino acid solution was added. Based on the above results, the growth-promoting effect of L-valine was not confirmed by analysis using liver cells after the middle embryonic period (E13.5 ~), so only in undifferentiated liver cells existing in the early embryonic period. It was shown that the growth promoting effect by adding L-valine specifically appears.

Ｌ-バリンを豊富に含む培養条件ではマウス肝幹/前駆細胞の肝細胞への分化を亢進させる
次に我々は、L-ロイシン、L-イソロイシン、L-バリンが細胞分化へ与える影響について検討した。胎生初期 (E11.5) の肝臓由来未分化細胞を用いて、各分岐鎖アミノ酸添加による影響についてアミノ酸非添加群と比較した。分岐鎖アミノ酸4 mM添加した培地で6日間培養後、免疫染色により肝幹・前駆細胞の分化状態を評価した結果、L-バリン添加時に肝細胞特異的マーカーであるアルブミンのみを強く発現する細胞が多数存在するコロニーの頻度が23±15%増加することを見出した(図 6AB)。さらに、培養後のコロニーに対して遺伝子発現解析を行ったところ、L-バリン添加群では胆管上皮特異的マーカーであるサイトケラチン(Ck7)の発現量増加がみられないのに対して、アルブミンの発現量は60±18%増加することを確認した(図 6C)。一方、イソロイシン添加時にはアルブミンの発現は39%減少し、ロイシンの添加時にはアルブミンおよびCk7の双方の発現量がそれぞれ50%,51%ずつ増加していた(図 6C)。以上の結果から、胎生初期のマウス肝臓細胞は、L-バリンが培地内に豊富に存在する場合に、肝細胞への分化を亢進することが明らかとなった。 L-valine-rich culture conditions enhance the differentiation of mouse hepatic stem / progenitor cells into hepatocytes. Next, we examined the effects of L-leucine, L-isoleucine, and L-valine on cell differentiation. . The effect of each branched-chain amino acid addition was compared with the non-amino acid addition group using liver-derived undifferentiated cells in the early embryonic period (E11.5). After culturing for 6 days in a medium supplemented with 4 mM branched-chain amino acid, the differentiation state of hepatic stem / progenitor cells was evaluated by immunostaining. As a result, when L-valine was added, cells that strongly express only albumin, a hepatocyte-specific marker, were found. It was found that the frequency of colonies present in large numbers increased by 23 ± 15% (FIG. 6AB). Furthermore, when gene expression analysis was performed on colonies after culturing, no increase in the expression level of cytokeratin (Ck7), a bile duct epithelium-specific marker, was observed in the L-valine addition group, whereas albumin It was confirmed that the expression level increased by 60 ± 18% (FIG. 6C). On the other hand, when isoleucine was added, albumin expression was reduced by 39%, and when leucine was added, the expression levels of both albumin and Ck7 were increased by 50% and 51%, respectively (FIG. 6C). From the above results, it was clarified that mouse embryonic cells in the early embryonic stage are enhanced to differentiate into hepatocytes when L-valine is abundant in the medium.

Ｌ-バリンを豊富に含む培養条件ではヒト肝臓細胞の増殖および肝細胞への分化を亢進する
次に我々は、発生初期のマウス肝臓細胞でみられた、Ｌ-バリンによる増殖・分化促進効果がヒト未分化肝臓細胞へも適応されるか検証した。まず、ヒトiPS細胞由来肝臓細胞を用いて各分化段階におけるBCAT1の発現を比較したところ、肝発生初期の分化段階に相当する細胞(図7AのStage2)、より分化の進行した段階の細胞では低いことが明らかとなった(図 7A)。そこで、次に、最もBCAT1の発現亢進が見られたStage2のiPS細胞由来肝臓細胞を用いて各分岐鎖アミノ酸を添加し、増殖に与える影響を検討した。その結果、分岐鎖アミノ酸非添加群と比較して、L-バリン4 mM添加時に、培養6日目の細胞数が11.3(±3.1)%増加することを見いだした。このとき、市販の非必須、必須アミノ酸製剤を4 mM添加した群では培養後の細胞数に差は見られなかった。以上の結果より、未分化な肝臓細胞が高頻度に存在する分化段階の細胞へ分岐鎖アミノ酸を添加することで細胞増殖が亢進することが明らかになった(図 7B)。次に、分岐鎖アミノ酸がiPS細胞由来肝臓細胞の分化へ与える影響について検討した。分岐鎖アミノ酸4 mM添加した培地で8日間培養後の細胞に対して遺伝子発現解析を行ったところ、L-バリン添加群では、肝細胞の分化マーカーであるアルブミンおよびRBP4の発現量がそれぞれ、52±28%、58±2%増加することを確認した(図 7C)。一方、イソロイシン添加時にはこれらマーカーの発現が大きく減少することが確認された。以上の結果から、iPS細胞由来肝臓細胞は、L-バリンが培地内に豊富に存在する場合に、増殖を亢進し、肝細胞への分化を亢進することが明らかとなった。 Culture conditions rich in L-valine enhance human liver cell proliferation and differentiation into hepatocytes. Next, we observed the effects of L-valine on the proliferation and differentiation promoting effects observed in early mouse liver cells. It was verified whether it was also applicable to human undifferentiated liver cells. First, when comparing the expression of BCAT1 in each differentiation stage using human iPS cell-derived liver cells, the cell corresponding to the differentiation stage in the early stage of liver development (Stage 2 in FIG. 7A) is low in the cells in the stage of more advanced differentiation. It became clear (Fig. 7A). Then, each branched-chain amino acid was added using Stage 2 iPS cell-derived liver cells, which showed the highest expression of BCAT1, and the effect on proliferation was examined. As a result, it was found that the number of cells on the 6th day of culture increased by 11.3 (± 3.1)% when 4 mM L-valine was added compared to the group without branched chain amino acid. At this time, there was no difference in the number of cells after culture in the group to which 4 mM of a commercially available non-essential and essential amino acid preparation was added. From the above results, it has been clarified that cell proliferation is enhanced by adding branched chain amino acids to cells in a differentiation stage where undifferentiated liver cells are frequently present (FIG. 7B). Next, the effect of branched chain amino acids on the differentiation of iPS cell-derived liver cells was examined. Gene expression analysis was performed on cells cultured for 8 days in a medium supplemented with 4 mM branched-chain amino acids. In the L-valine addition group, the expression levels of albumin and RBP4, which are differentiation markers for hepatocytes, were 52 and 52 respectively. Increases of ± 28% and 58 ± 2% were confirmed (FIG. 7C). On the other hand, it was confirmed that the expression of these markers greatly decreased when isoleucine was added. From the above results, it has been clarified that iPS cell-derived hepatocytes enhance proliferation and differentiation into hepatocytes when L-valine is abundant in the medium.

L-バリンの添加によりヒトiPS細胞由来肝芽中の細胞増殖が亢進する
肝前駆細胞に相当する分化段階(Stage2)のヒトiPS細胞由来肝臓細胞、ヒト臍帯静脈内皮細胞および間葉系幹細胞の共培養により、ヒトiPS細胞由来肝芽を創出した(図8A)。創出された肝芽をさらに、特定の分岐鎖アミノ酸を多量に含む培地を用いて培養し、その大きさを比較した結果、L-バリンを多量に含む培地で培養した肝芽は通常組成の培地で培養した場合よりも大きな肝芽が創出されることが確認された(図8BC)。以上の結果から、L-バリンを多量に含む培地は、ヒトiPS細胞由来肝前駆細胞に対して、二次元的な培養系のみではなく、立体組織中の細胞においても効果があることが確認された。Addition of L-valine enhances cell proliferation in human iPS cell-derived hepatoblasts Differentiated (Stage2) human iPS cell-derived hepatocytes, human umbilical vein endothelial cells, and mesenchymal stem cells By culture, human iPS cell-derived hepatoblasts were created (FIG. 8A). The resulting hepatic bud was further cultured in a medium containing a large amount of a specific branched chain amino acid, and the size was compared. As a result, the hepatic bud cultured in a medium containing a large amount of L-valine was a medium of normal composition. It was confirmed that larger liver buds were created than when cultured in (FIG. 8BC). From the above results, it was confirmed that a medium containing a large amount of L-valine is effective not only in a two-dimensional culture system but also in cells in three-dimensional tissues against human iPS cell-derived hepatic progenitor cells. It was.

本発明により、特定の分化段階の細胞をアミノ酸添加により増幅することが可能となった。   According to the present invention, it is possible to amplify cells at a specific differentiation stage by adding amino acids.

当分化段階の細胞をバリン添加条件下で継代培養することにより、未添加条件に比べて1.5倍程度増幅が可能である。未分化肝臓細胞は以前の特許（WO2009/139419）により、性質を維持したまま継代培養を繰り返し、分化誘導により最終的に機能細胞の創出に利用することができる。   By subculturing cells at this differentiation stage under valine-added conditions, amplification is possible about 1.5 times as compared with unadded conditions. Undifferentiated liver cells can be repeatedly used for the creation of functional cells by induction of differentiation according to a previous patent (WO2009 / 139419) by repeating subculture while maintaining the properties.

したがって、本発明により、前記未分化細胞において１．５倍の増殖亢進を誘導できるということは、その後指数関数的（１．５のべき乗）に拡大培養することが可能であり、大量の機能細胞を得るためにきわめて有益な技術となる。   Therefore, according to the present invention, the fact that 1.5-fold increase in proliferation can be induced in the undifferentiated cells can be expanded thereafter exponentially (power of 1.5), and a large amount of functional cells It becomes a very useful technique to obtain.

実験材料及び方法
実験動物
本実験では、日本エス・エル・シーより購入した野生型マウス(C57BL6/J)を用いた。全ての動物実験は、公立大学法人横浜市立大学福浦キャンパス動物実験指針に基づき、当該委員会の承認のもとに施行した。Experimental materials and methods
Experimental animals In this experiment, wild type mice (C57BL6 / J) purchased from Japan SLC were used. All animal experiments were conducted with the approval of the committee based on the guidelines for animal experiments at Yokohama City University Fukuura Campus.

マウス胎仔肝臓細胞の分取
野生型妊娠マウスに麻酔を行った後、腹腔内から胎仔を取り出し双眼実体顕微鏡下で胎仔肝臓を採取した。採取した胎仔肝臓を、0.2 ％ トリプシン、5 % 牛胎児血清(FBS: ICN) を含むDMEM / F12 (Invitrogen)内に浸し、氷上で30分間インキュベートした後、37 ℃で15分間振盪させた。その後穏やかなピペッティング操作により細胞を分散させた。遠心処理後、5%FBSを含むDMEM/F12にて2回洗浄した。得られた胎仔細胞はナイロンメッシュ(径40μm)に通過させて単一細胞のみを回収し、以後の実験に用いた。 Preparation of mouse fetal liver cells After anesthetizing a wild-type pregnant mouse, the fetus was taken out from the abdominal cavity and the fetal liver was collected under a binocular stereomicroscope. The collected fetal liver was immersed in DMEM / F12 (Invitrogen) containing 0.2% trypsin and 5% fetal bovine serum (FBS: ICN), incubated on ice for 30 minutes, and then shaken at 37 ° C. for 15 minutes. Thereafter, the cells were dispersed by gentle pipetting. After centrifugation, the plate was washed twice with DMEM / F12 containing 5% FBS. The obtained fetal cells were passed through a nylon mesh (diameter 40 μm) to collect only single cells and used for the subsequent experiments.

肝臓細胞の培養
単離した胎仔肝臓細胞をLaminin(BD)でコートした6 well プレート(BD)上に1000 cells / cm2の条件で播種し、培養を行った。細胞培養には独自に調製した培地を用いた。培養用培地は10 % FBS、 1 μg / mL Insulin (Wako)、1 x 10-7 M Dexamethasone (Sigma)、10 mmol / L nicotinamide (Sigma)、 2 mmol / L L-glutamine (Invitrogen)、50 mmol / L 2-mercaptoethanol (Invitrogen)、5 mmol / L HEPES (Dojindo) 2.6 x 10-4 mol / L L-Ascorbic acid 2-Phosphate (Sigma)、1 x penicillin/streptomycin (Invitrogen)を含むDMEM/F12培地を基礎組成とし、そこへ分岐鎖アミノ酸溶液(イソロイシン: Ile、ロイシン: Leu、バリン: Val)あるいは、市販の必須アミノ酸溶液(GIBCO)、非必須アミノ酸溶液(GIBCO)を添加することで各種アミノ酸の培養に与える影響を評価した。なお、コントロールには等量の溶媒(PBS)を添加した。細胞播種後、細胞接着が確認された時点で培地を交換し、交換前の培地へさらに20 ng/ ml Epidermal growth factor (Sigma)、 25 ng / mL Hepatocyte growth factor (クリングルファーマ)を加えた培地で培養を続けた。培養は37 ℃、5 % CO2気相下で行った。 Culture of liver cells Isolated fetal liver cells were seeded on a 6-well plate (BD) coated with Laminin (BD) at 1000 cells / cm 2 and cultured. A uniquely prepared medium was used for cell culture. Culture medium is 10% FBS, 1 μg / mL Insulin (Wako), 1 x 10 -7 M Dexamethasone (Sigma), 10 mmol / L nicotinamide (Sigma), 2 mmol / L L-glutamine (Invitrogen), 50 mmol / L 2-mercaptoethanol (Invitrogen), 5 mmol / L HEPES (Dojindo) 2.6 x 10 -4 mol / L L-Ascorbic acid 2-Phosphate (Sigma), DMEM / F12 medium containing 1 x penicillin / streptomycin (Invitrogen) Is added to the branched chain amino acid solution (isoleucine: Ile, leucine: Leu, valine: Val), or commercially available essential amino acid solution (GIBCO), non-essential amino acid solution (GIBCO). The influence on culture was evaluated. An equal amount of solvent (PBS) was added to the control. After cell seeding, when the cell adhesion is confirmed, the medium is changed, and the medium before replacement is further added with 20 ng / ml Epidermal growth factor (Sigma) and 25 ng / mL Hepatocyte growth factor (Kringle Pharma). The culture was continued. Incubation was performed at 37 ° C. in a 5% CO 2 gas phase.

免疫染色
培地を除去した後、PBSで洗浄した。培養細胞の固定は1:1混合のアセトン/メタノールを加え-30 ℃で10分間、あるいは4 % PFA中で室温10分間反応させることで行った。固定済みの細胞サンプルは0.05 % Tweeen20を含んだPBSで5分間1回洗浄し、二次抗体作製時の免疫動物血清(Goat serum) (Sigma)を10 %含むPBS溶液中で、室温2時間インキュベートした。その後4 ℃で一晩、一次抗体反応を行った。一次抗体反応後、0.05 % Tween20を含んだPBSで5分間3回洗浄し、その後、遮光下において二次抗体を1時間、室温処理した。処理後、0.05 % Tween20を含んだPBSで3回洗浄し、傾向保護材(Vector)で封入し、正立型蛍光顕微鏡(Zeiss Axio システム)下で観察した。使用した一次抗体は、マウス抗Ck7抗体 (Dako) (1:100)、ウサギ抗Albumin抗体(Biogenesis) (1:500)、ウサギ抗AFP抗体 (MP bio) (1:200)を使用した。二次抗体は、Alexa488標識ヤギ抗マウスIgG1抗体 (Molecular Probe) (1:500) 、Aelxa555(Molecular Probe)標識ヤギ抗ウサギIgG抗体 (Molecular Probe) (1:500)である。After removing the immunostaining medium, it was washed with PBS. The cultured cells were fixed by adding 1: 1 acetone / methanol and reacting at −30 ° C. for 10 minutes or in 4% PFA for 10 minutes at room temperature. Fixed cell samples are washed once with PBS containing 0.05% Tweeen20 for 5 minutes and incubated in PBS solution containing 10% immunized animal serum (Goat serum) (Sigma) for 2 hours at room temperature. did. Thereafter, the primary antibody reaction was performed overnight at 4 ° C. After the primary antibody reaction, the plate was washed 3 times for 5 minutes with PBS containing 0.05% Tween 20, and then the secondary antibody was treated at room temperature for 1 hour under light shielding. After the treatment, the plate was washed three times with PBS containing 0.05% Tween 20, sealed with a trend protector (Vector), and observed under an upright fluorescence microscope (Zeiss Axio system). The primary antibodies used were mouse anti-Ck7 antibody (Dako) (1: 100), rabbit anti-Albumin antibody (Biogenesis) (1: 500), and rabbit anti-AFP antibody (MP bio) (1: 200). Secondary antibodies are Alexa488-labeled goat anti-mouse IgG1 antibody (Molecular Probe) (1: 500) and Aelxa555 (Molecular Probe) -labeled goat anti-rabbit IgG antibody (Molecular Probe) (1: 500).

Total RNAの調製
各発生段階における胎仔・乳児野生型マウスから得た非血球画分（CD45−Ter119−）、あるいは、経門脈的脱血操作を施した8週齢マウス肝臓からtotalRNAを回収し、定量PCR法を用いて目的遺伝子の発現を検討した。 Total RNA non blood cell fraction obtained from fetal, and infant wild-type mice at each developmental stage preparation of (CD45 - Ter119 -), or the totalRNA recovered from 8-week-old mouse liver which has been subjected to through portal vein manner blood removal operation Then, the expression of the target gene was examined using a quantitative PCR method.

非血球細胞の単離は次のように行った。野生型マウスの肝臓から、分取した肝臓細胞を、Biotin標識抗マウスTER119抗体 (PharMingen)、Biotin標識抗マウスCD45抗体(PharMingen)を加え、氷上にて20分間反応させた。3 % FBSを含むPBS溶液を加えて遠心操作を実施し、洗浄を行った。その後、IMag (BD)を氷上にて30分間反応させた。細胞懸濁液を2mlのEDTA 入りPBSで懸濁し、磁石によりCD45・Ter119陽性細胞を除去し、非血球細胞とした。   Non-hemocyte cells were isolated as follows. Biotin-labeled anti-mouse TER119 antibody (PharMingen) and Biotin-labeled anti-mouse CD45 antibody (PharMingen) were added to the liver cells collected from the wild-type mouse liver, and reacted on ice for 20 minutes. A PBS solution containing 3% FBS was added to perform centrifugation and washing. Thereafter, IMag (BD) was reacted on ice for 30 minutes. The cell suspension was suspended in 2 ml of PBS containing EDTA, and CD45 / Ter119 positive cells were removed with a magnet to obtain non-hemocyte cells.

TotalRNAの抽出はTRIzol (Invitrogen)を用いた。なお、成体肝臓からのRNA抽出に際しては、液体窒素中で組織片を物理的に破損させた後、TRIzolを加えた。TRIzolを用いたtotalRNA抽出はWAKO社のマニュアルに従って行った。なお、RNA 溶液に混入しているゲノムDNAはDeoxyribonuclease I, Amplification Grade（Invitrogen）により分解させた。その後、SuperScriptTM III Reverse Transcriptase（Invitrogen）を用いてrandomプライマー存在下で逆転写反応を行い、cDNA 溶液を得た。これらの操作方法はKit付属のマニュアルに従った。Total RNA was extracted using TRIzol (Invitrogen). When RNA was extracted from the adult liver, TRIzol was added after the tissue pieces were physically damaged in liquid nitrogen. Total RNA extraction using TRIzol was performed according to the WAKO manual. Genomic DNA mixed in the RNA solution was degraded by Deoxyribonuclease I, Amplification Grade (Invitrogen). Thereafter, a reverse transcription reaction was performed in the presence of a random primer using SuperScript ™ III Reverse Transcriptase (Invitrogen) to obtain a cDNA solution. These operation methods followed the manual attached to Kit.

定量PCR
cDNA溶液をテンプレートとして定量を行った。定量解析は比較CT法を用いた。マウス細胞の解析では、それぞれのサンプルのGAPDH遺伝子の測定値によって、反応に用いたcDNA量の補正を行うことで相対定量を施行した。ヒト細胞の解析では、18Sの発現量により補正を行った。 Quantitative PCR
Quantification was performed using the cDNA solution as a template. The quantitative analysis used the comparative CT method. In the analysis of mouse cells, relative quantification was performed by correcting the amount of cDNA used in the reaction based on the measured value of the GAPDH gene in each sample. In the analysis of human cells, correction was performed based on the expression level of 18S.

トランスクリプトーム解析
total RNA溶液をテンプレートとして、Agilent マイクロアレイ解析を行った。アレイデータはGrobal normalization法を用いて正規化し、検体間の遺伝子発現の差異を検討した。得られたデータからヒートマップを作成し、各発生段階における遺伝子発現量の比較を行った。各代謝経路に関わる遺伝子の抽出は、Gene Ontology分類を元に行った。 Transcriptome analysis
Agilent microarray analysis was performed using the total RNA solution as a template. The array data was normalized using the Grobal normalization method, and the difference in gene expression between specimens was examined. A heat map was created from the obtained data, and the gene expression level at each developmental stage was compared. Extraction of genes related to each metabolic pathway was performed based on Gene Ontology classification.

メタボローム解析
各発生期におけるマウス肝臓を採取し、ヒューマン・メタボローム・テクノロジーズ(HMT)社にてメタボローム解析を行った。マウス肝臓試料と内部標準物質50μMを含んだメタノール溶液を破砕用チューブに入れ、冷却下にて卓上型破砕機(bms)を用いて破砕した。これにクロロホルムおよびMilli-Q水を加えて撹拌し、遠心分離を行った。遠心分離後、水層を限外濾過チューブに移し取った。これを遠心し、限外濾過処理を行った。濾液を乾固させ、再びMilli-Q水に溶解して測定に供した。 Metabolome analysis Mouse liver at each developmental stage was collected and metabolome analysis was performed by Human Metabolome Technologies (HMT). A methanol solution containing a mouse liver sample and an internal standard substance of 50 μM was placed in a crushing tube, and crushed using a desktop crusher (bms) under cooling. Chloroform and Milli-Q water were added to this and stirred, followed by centrifugation. After centrifugation, the aqueous layer was transferred to an ultrafiltration tube. This was centrifuged and subjected to ultrafiltration treatment. The filtrate was dried and dissolved in Milli-Q water again for measurement.

測定は、キャピラリー電気泳動-飛行時間型質量分析計のカチオンモード、アニオンモードによる測定を実施した。本試験ではHMT代謝物質データベースに登録された物質を対象として解析を行った。   The measurement was carried out in a cation mode and an anion mode of a capillary electrophoresis-time-of-flight mass spectrometer. In this study, analysis was performed on substances registered in the HMT metabolite database.

検出されたピークは、自動積分ソフトウェア(MasterHands ver.2.9.0.9)を用いて自動抽出し、ピーク情報として質量電荷費、泳動時間とピーク面積値を得た。得られたピーク面積値は相対面積値に変換した。また、これらのデータにはNa+やK+などのアダクトイオンおよび、脱水、脱アンモニウムなどのフラグメントイオンが含まれているので、これらの分子量関連イオンを削除した。精査したピークについて各試料間のピークの照合・整列化を行った。   The detected peaks were automatically extracted using automatic integration software (MasterHands ver.2.9.0.9), and mass charge cost, migration time and peak area values were obtained as peak information. The obtained peak area value was converted into a relative area value. These data included adduct ions such as Na + and K + and fragment ions such as dehydration and deammonium, so these molecular weight related ions were deleted. For the examined peaks, the peaks between each sample were collated and aligned.

フローサイトメトリー解析
胎生13.5日目胎児より分取した肝臓細胞へ抗Dlk1ラット抗体(LSBio)、PE標識抗CD45抗体(PharMingen)およびPE標識抗TER119抗体(PharMingen)を加え、氷上にて30分間反応させた。3%FBSを含むPBS溶液を加えて遠心操作を実施し、洗浄を行った。その後、2次抗体としてAlexa647標識抗ラット抗体を加え、20分間反応させた。再び洗浄操作を行った後、FACSAriaにより細胞の蛍光強度を測定した。Flow cytometry analysis Add anti-Dlk1 rat antibody (LSBio), PE-labeled anti-CD45 antibody (PharMingen) and PE-labeled anti-TER119 antibody (PharMingen) to liver cells collected from fetal day 13.5 fetuses, and react on ice for 30 minutes I let you. A PBS solution containing 3% FBS was added to perform centrifugation and washing. Thereafter, Alexa647-labeled anti-rat antibody was added as a secondary antibody and allowed to react for 20 minutes. After washing again, the fluorescence intensity of the cells was measured by FACSAria.

ヒトiPS細胞由来肝芽の創出
肝前駆細胞に相当する分化段階のヒトiPS細胞由来肝臓細胞（東京大学中内教授より供与して頂いたiPS細胞から当研究室で分化誘導した肝臓細胞）、ヒト臍帯静脈内皮細胞（ロンザ）および間葉系幹細胞（ロンザ）の三種類を共培養することで肝芽の創出を行った。細胞をそれぞれ10：7：2の比率で混合し、マイクロウェルプレートで培養することで創出された肝芽は、その後特定のアミノ酸が豊富に含まれる、あるいはアミノ酸が少量しか含まれない培地(RPMIとEGMを1:1で混合したもの)で2週間培養し、そのサイズを計測した。肝芽のサイズの計測はIN Cell Analyzer 2000を用いて、肝芽の呈する蛍光を基に算出した。 Creation of human iPS cell-derived hepatic buds Differentiated human iPS cell-derived liver cells corresponding to hepatic progenitor cells (liver cells differentiated in this laboratory from iPS cells donated by Professor Nakauchi of the University of Tokyo), human Liver buds were created by co-culturing three types of umbilical vein endothelial cells (Lonza) and mesenchymal stem cells (Lonza). Liver buds created by mixing cells at a ratio of 10: 7: 2 and culturing them in a microwell plate are then a medium rich in specific amino acids or a small amount of amino acids (RPMI). And EGM mixed 1: 1) for 2 weeks, and the size was measured. The size of the liver bud was measured using IN Cell Analyzer 2000 based on the fluorescence exhibited by the liver bud.

〔実施例２〕
細胞増殖促進に有効なL-バリンの添加濃度を明らかにするため、さまざまな濃度でL-バリンを添加し、胎生11.5日目のマウス胎仔肝臓細胞への細胞増殖促進効果を検討した。その結果、L-バリンを4mM添加した時に細胞増殖促進効果が最も高く、0.8-20mMの濃度範囲のときに増殖を促進する傾向が観察された(図9)。この結果から、L-バリンの添加は0.8-20mMの範囲で細胞増殖亢進に有効であることが示唆された。[Example 2]
In order to clarify the effective concentration of L-valine for promoting cell growth, L-valine was added at various concentrations, and the effect of promoting cell growth on embryonic day 11.5 mouse fetal liver cells was examined. As a result, the cell growth promoting effect was the highest when 4 mM of L-valine was added, and the tendency to promote the growth was observed when the concentration range was 0.8-20 mM (FIG. 9). From these results, it was suggested that the addition of L-valine is effective in enhancing cell proliferation in the range of 0.8-20 mM.

実験材料及び方法
実験は「実施例１」のマウス胎仔肝臓細胞の分取、肝臓細胞の培養と同様に行った。Experimental materials and methods Experiments were performed in the same manner as in Example 1, the collection of mouse fetal liver cells and the culture of liver cells .

本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。   All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

本発明は、細胞培養技術や細胞操作技術として、再生医療や医薬品のスクリーニングなどに利用することができる。本発明は、臓器細胞の増幅用培地製剤や、臓器細胞から分化誘導した肝細胞の選別用培地製剤などの開発につながる。   The present invention can be used as a cell culture technique or a cell manipulation technique for regenerative medicine or drug screening. The present invention leads to the development of a medium preparation for amplifying organ cells, a medium preparation for sorting hepatocytes induced to differentiate from organ cells, and the like.

Claims (8)

１．０〜２０ｍＭの濃度のバリンの存在下で、未分化肝細胞を培養することを含む、未分化肝細胞の増幅方法であって、未分化肝細胞がマウス胎児のＥ１１．５の時期に対応する分化段階のレベルの細胞である前記方法。A method for amplifying undifferentiated hepatocytes, comprising culturing undifferentiated hepatocytes in the presence of valine at a concentration of 1.0 to 20 mM, wherein the undifferentiated hepatocytes are in the period of E11.5 of a mouse fetus. Said method wherein the cells are at the level of the corresponding differentiation stage. １．０〜２０ｍＭの濃度のバリンの存在下で、未分化肝細胞を培養することにより、未分化肝細胞を増幅し、かつ肝細胞への分化を誘導することを含む、肝細胞の調製方法であって、未分化肝細胞がマウス胎児のＥ１１．５の時期に対応する分化段階のレベルの細胞である前記方法。A method for preparing hepatocytes, comprising amplifying undifferentiated hepatocytes and inducing differentiation into hepatocytes by culturing undifferentiated hepatocytes in the presence of valine at a concentration of 1.0 to 20 mM. Wherein the undifferentiated hepatocytes are cells at a differentiation stage level corresponding to the E11.5 period of the mouse fetus. 未分化肝細胞において、ＧＡＴＡ ｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ ４／６、Ｈｅｐａｔｏｃｙｔｅ ｎｕｃｌｅａｒ ｆａｃｔｏｒ ４ ａｌｐｈａ、ＨＨＥＸ１及びα−フェトプロテインからなる群より選択される少なくとも１つのマーカーの発現レベルがＥＳ細胞及び／又はｉＰＳ細胞と比較して増加している請求項１２又は１３に記載の方法。In undifferentiated hepatocytes, the expression level of at least one marker selected from the group consisting of GATA binding protein 4/6, Hepatocyte nuclear factor 4 alpha, HHEX1 and α-fetoprotein is compared with ES cells and / or iPS cells. 14. A method according to claim 12 or 13, wherein the method is increasing. 未分化肝細胞において、アルブミン、レチノール結合タンパク質４、アシアロ糖タンパク質レセプター１、アンジオテンシノーゲン、トランスフェリン、フィブリノゲンα鎖、アポリポプロテインＡ−１、ホモゲンチジン酸及びＣａｒｂａｍｏｙｌ−Ｐｈｏｓｐｈａｔｅ Ｓｙｎｔｈａｓｅ １からなる群より選択される少なくとも１つのマーカーの発現レベルが他の分化段階の細胞と比較して減少している請求項１２又は１３に記載の方法。In undifferentiated hepatocytes, selected from the group consisting of albumin, retinol binding protein 4, asialoglycoprotein receptor 1, angiotensinogen, transferrin, fibrinogen α chain, apolipoprotein A-1, homogentisic acid and Carbamoyl-Phosphate Synthase 1 14. The method according to claim 12 or 13, wherein the expression level of at least one marker is reduced compared to cells of other differentiation stages. 未分化肝細胞において、ＢＣＡＴ１の発現レベルがＥＳ細胞及び／又はｉＰＳ細胞と比較して増加している請求項１２又は１３に記載の方法。The method according to claim 12 or 13, wherein the expression level of BCAT1 is increased in undifferentiated hepatocytes compared to ES cells and / or iPS cells. １．０〜２０ｍＭの濃度のバリンの存在下で、未分化肝細胞を培養することにより、胆管上皮細胞の出現を抑制し、肝細胞を創出することを含む、肝細胞への分化が可能な細胞の選別法。By culturing undifferentiated hepatocytes in the presence of valine at a concentration of 1.0 to 20 mM, it is possible to differentiate into hepatocytes, including inhibiting the appearance of bile duct epithelial cells and creating hepatocytes. Cell sorting method. バリンの濃度が２〜１０ｍＭである請求項１２〜１７のいずれかに記載の方法。The method according to any one of claims 12 to 17, wherein the concentration of valine is 2 to 10 mM. バリンの濃度が４ｍＭである請求項１８記載の方法。The method according to claim 18, wherein the concentration of valine is 4 mM.
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