JP2017023019A - Method for inducing differentiation from pluripotent stem cells to mesodermal progenitor cells and blood vessel progenitor cells - Google Patents
Method for inducing differentiation from pluripotent stem cells to mesodermal progenitor cells and blood vessel progenitor cells Download PDFInfo
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Abstract
Description
本発明は、多能性幹細胞からの胚葉前駆細胞および血液血管前駆細胞の製造方法に関する。 The present invention relates to a method for producing germ layer progenitor cells and blood vascular progenitor cells from pluripotent stem cells.
胚性幹細胞（ES細胞）や体細胞へ未分化細胞特異的遺伝子を導入することで得られる
人工多能性幹細胞（iPS細胞）など多能性を有する細胞がこれまでに報告されている（特
許文献１または２）。そこで、これらの多能性幹細胞から分化誘導された細胞を移植する再生医療やin vitroでの病態モデルの作製に注目されている。
胚性幹細胞から内皮細胞や血球系細胞を作製するためには、発生を模して、中胚葉前駆細胞を作製し、各細胞へと分化誘導する多段階工程が試みられている（非特許文献１、２または３）。また、この工程において、異種細胞や生物由来原料を用いることでは安定的な収量が得られないことから、限定された成分のみを用いて多能性幹細胞から中胚葉前駆細胞、さらには、各分化細胞へと誘導する方法が提示されている（特許文献３、非特許文献４）。
Multipotent cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) obtained by introducing undifferentiated cell-specific genes into somatic cells have been reported (patents) Literature 1 or 2). Therefore, attention has been focused on regenerative medicine in which cells induced to differentiate from these pluripotent stem cells are transplanted and in vitro pathological model creation.
In order to produce endothelial cells and blood cells from embryonic stem cells, a multi-step process has been attempted in which mesoderm progenitor cells are produced by mimicking development and induced to differentiate into each cell (non-patent literature). 1, 2, or 3). In addition, in this process, since stable yields cannot be obtained by using heterogeneous cells or biological materials, pluripotent stem cells, mesoderm progenitor cells, and each differentiation using only limited components. Methods for inducing cells have been proposed (Patent Document 3, Non-Patent Document 4).
本発明の目的は、多能性幹細胞から効率よく中胚葉前駆細胞および血液血管前駆細胞を製造することである。したがって、本発明の課題は、ヒト多能性幹細胞、特にヒト人工多能性幹細胞を中胚葉前駆細胞および血液血管前駆細胞へ効率よく分化誘導する方法を提供することである。 An object of the present invention is to efficiently produce mesoderm progenitor cells and blood vascular progenitor cells from pluripotent stem cells. Accordingly, an object of the present invention is to provide a method for efficiently inducing differentiation of human pluripotent stem cells, particularly human induced pluripotent stem cells, into mesoderm progenitor cells and blood vascular progenitor cells.
本発明者らは、上記の課題を解決すべく、多能性幹細胞から中胚葉前駆細胞へと誘導する工程において、ラミニン５１１をコートした培養容器を用いてＢＭＰ、ＧＳＫ３β阻害剤およびＶＥＧＦを含む培養液中で培養することで、短期間で高分化能を有する中胚葉前駆細胞を製造することに成功した。さらに、得られた中胚葉前駆細胞を、ＶＥＧＦ、ＳＣＦ、ｂＦＧＦおよびＴＧＦβ阻害剤を含む培養液中で接着培養することで、高分化能な血液血管前駆細胞を製造することに成功し、本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventors have induced a culture containing BMP, GSK3β inhibitor and VEGF using a culture container coated with laminin 511 in the step of inducing pluripotent stem cells into mesoderm progenitor cells. By culturing in the liquid, we succeeded in producing mesoderm progenitor cells having high differentiation ability in a short period of time. Furthermore, the obtained mesoderm progenitor cells were successfully cultured in a culture solution containing VEGF, SCF, bFGF and TGFβ inhibitor to successfully produce highly differentiated blood vascular progenitor cells. It came to complete.
すなわち、本発明は以下の通りである。
［１］多能性幹細胞から中胚葉前駆細胞を製造する方法であって、
多能性幹細胞を、ラミニン（ＬＭ）５１１またはその断片でコーティングされた培養器材上にて、ＢＭＰ（Bone Morphogenetic Protein）、ＶＥＧＦ（Vascular Endothelial Gro
wth Factor）およびＧＳＫ（Glycogen Synthase Kinase）３β阻害剤を含む培養液中で接着培養する工程を含む方法。
［２］前記ラミニン５１１の断片がラミニン５１１Ｅ８である、［１］に記載の方法。
［３］前記ＧＳＫ３β阻害剤がＣＨＩＲ９９０２１である、［１］または［２］に記載の方法。
［４］前記工程が2日間以上行われる、［１］〜［３］のいずれか１項に記載の方法。
［５］中胚葉前駆細胞から血液血管前駆細胞を製造する方法であって、
中胚葉前駆細胞をＶＥＧＦ、ＳＣＦ（Stem Cell Factor）、ｂＦＧＦ（basic Fibroblast
Growth Factor）およびＴＧＦ（Transforming Growth Factor）β阻害剤を含む培養液中で接着培養する工程を含む方法。
［６］前記培養液がＴＧＦβを含有しない培養液である、［５］に記載の方法。
［７］前記中胚葉前駆細胞が、［１］〜［４］のいずれか１項に記載の方法で製造された細胞である、［５］または［６］に記載の方法。
［８］前記ＴＧＦβ阻害剤がＳＢ４３１５４２である、［５］〜［７］のいずれか１項に記載の方法。
［９］前記工程が2日間以上行われる、［５］〜［８］のいずれか１項に記載の方法。
［１０］次の工程を含む多能性幹細胞から、血液血管前駆細胞を製造する方法であって、（１）多能性幹細胞を、ラミニン５１１またはその断片でコーティングされた培養器材上にて、ＢＭＰ、ＶＥＧＦおよびＧＳＫ３β阻害剤を含む培養液中で接着培養する工程、および
（２）前記工程（１）で得られた細胞をＶＥＧＦ、ＳＣＦ、ｂＦＧＦおよびＴＧＦβ阻害剤を含む培養液中で接着培養する工程。
［１１］前記ラミニン５１１の断片がラミニン５１１Ｅ８である、［１０］に記載の方法。
［１２］前記ＧＳＫ３β阻害剤がＣＨＩＲ９９０２１である、［１０］または［１１］に記載の方法。
［１３］前記工程（２）で用いる培養液がＴＧＦβを含有しない培養液である、［１０］〜［１２］のいずれか１項に記載の方法。
［１４］前記ＴＧＦβ阻害剤がＳＢ４３１５４２である、［１０］〜［１３］のいずれか１項に記載の方法。
［１５］前記工程（１）が2日間以上行われる、［１０］〜［１４］のいずれか１項に記
載の方法。
［１６］前記工程（２）が2日間以上行われる、［１０］〜［１５］のいずれか１項に記
載の方法。
That is, the present invention is as follows.
[1] A method for producing mesoderm progenitor cells from pluripotent stem cells,
Pluripotent stem cells are cultured on culture equipment coated with laminin (LM) 511 or a fragment thereof, and BMP (Bone Morphogenetic Protein) and VEGF (Vascular Endothelial Gro
a method comprising adhesion culture in a culture solution containing a wth factor) and a GSK (Glycogen Synthase Kinase) 3β inhibitor.
[2] The method according to [1], wherein the laminin 511 fragment is laminin 511E8.
[3] The method according to [1] or [2], wherein the GSK3β inhibitor is CHIR99021.
[4] The method according to any one of [1] to [3], wherein the step is performed for 2 days or more.
[5] A method for producing blood vascular progenitor cells from mesoderm progenitor cells,
Mesodermal progenitor cells are VEGF, SCF (Stem Cell Factor), bFGF (basic Fibroblast)
A method comprising a step of adhesion culture in a culture solution containing a growth factor) and a TGF (Transforming Growth Factor) β inhibitor.
[6] The method according to [5], wherein the culture solution is a culture solution not containing TGFβ.
[7] The method according to [5] or [6], wherein the mesoderm progenitor cell is a cell produced by the method according to any one of [1] to [4].
[8] The method according to any one of [5] to [7], wherein the TGFβ inhibitor is SB431542.
[9] The method according to any one of [5] to [8], wherein the step is performed for 2 days or more.
[10] A method for producing blood vascular progenitor cells from pluripotent stem cells comprising the following steps, comprising: (1) pluripotent stem cells on a culture device coated with laminin 511 or a fragment thereof; A step of adhesion culture in a culture solution containing BMP, VEGF and GSK3β inhibitor; and (2) adhesion culture of the cells obtained in the step (1) in a culture solution containing VEGF, SCF, bFGF and a TGFβ inhibitor. Process.
[11] The method according to [10], wherein the laminin 511 fragment is laminin 511E8.
[12] The method according to [10] or [11], wherein the GSK3β inhibitor is CHIR99021.
[13] The method according to any one of [10] to [12], wherein the culture solution used in the step (2) is a culture solution not containing TGFβ.
[14] The method according to any one of [10] to [13], wherein the TGFβ inhibitor is SB431542.
[15] The method according to any one of [10] to [14], wherein the step (1) is performed for 2 days or more.
[16] The method according to any one of [10] to [15], wherein the step (2) is performed for 2 days or more.
本発明によれば、中胚葉前駆細胞および血液血管前駆細胞を得ることができる。 According to the present invention, mesodermal progenitor cells and blood vascular progenitor cells can be obtained.
本発明を以下で詳細に説明する。 The present invention is described in detail below.
＜多能性幹細胞＞
本発明で使用可能な多能性幹細胞は、生体に存在するすべての細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞であり、それには、特に限定されないが、例えば胚性幹(ES)細胞、核移植により得られるクローン胚由来の胚性幹(ntES)細胞、精子幹細胞（「GS細胞」）、胚性生殖細胞（「EG細胞」）、人工多能性幹(iPS)細胞、培養線
維芽細胞や骨髄幹細胞由来の多能性細胞（Muse細胞）などが含まれる。好ましい多能性幹細胞は、ES細胞、ntES細胞、およびiPS細胞である。
<Pluripotent stem cells>
The pluripotent stem cell that can be used in the present invention is a stem cell that has pluripotency that can be differentiated into all cells existing in a living body and also has proliferative ability, and is not particularly limited. For example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic germ cells (“EG cells”), artificial pluripotency Examples include sex stem (iPS) cells, cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.
(A) 胚性幹細胞
ES細胞は、ヒトやマウスなどの哺乳動物の初期胚（例えば胚盤胞）の内部細胞塊から樹立された、多能性と自己複製による増殖能を有する幹細胞である。
(A) Embryonic stem cells
ES cells are stem cells established from the inner cell mass of early embryos of mammals such as humans and mice (for example, blastocysts) and having the pluripotency and the ability to proliferate by self-replication.
ES細胞は、受精卵の8細胞期、桑実胚後の胚である胚盤胞の内部細胞塊に由来する胚由
来の幹細胞であり、成体を構成するあらゆる細胞に分化する能力、いわゆる分化多能性と、自己複製による増殖能とを有している。ES細胞は、マウスで1981年に発見され(M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156)、その後、ヒト、サルなどの霊長類でもES細胞株が樹立された (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848;J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165)。
ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (JA Thomson et al. al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod ., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).
ES細胞は、対象動物の受精卵の胚盤胞から内部細胞塊を取出し、内部細胞塊を線維芽細胞のフィーダー上で培養することによって樹立することができる。また、継代培養による細胞の維持は、白血病抑制因子(leukemia inhibitory factor (LIF))、塩基性線維芽細胞成長因子(basic fibroblast growth factor (bFGF))などの物質を添加した培養液を用い
て行うことができる。ヒトおよびサルのES細胞の樹立と維持の方法については、例えばUSP5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92:7844-7848；Thomson JA, et al. (1998), Science. 282:1145-1147； H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932； M. Ueno et al. (2006), Proc. Natl.
Acad. Sci. USA, 103:9554-9559； H. Suemori et al. (2001), Dev. Dyn., 222:273-279;H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585；Klimanskaya I, et al. (2006), Nature. 444:481-485などに記載されている。
ES cells can be established by removing an inner cell mass from a blastocyst of a fertilized egg of a subject animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). It can be carried out. For methods of establishing and maintaining human and monkey ES cells, see, for example, USP 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. US A. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc Natl.
Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci USA, 99: 1580-1585; Klimanskaya I, et al. (2006), Nature. 444: 481-485.
ES細胞作製のための培養液として、例えば0.1mM 2-メルカプトエタノール、0.1mM 非必須アミノ酸、2mM L-グルタミン酸、20% KSRおよび4ng/ml bFGFを補充したDMEM/F-12培養
液を使用し、37℃、2% CO2/98% 空気の湿潤雰囲気下でヒトES細胞を維持することができ
る(O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224)。また、ES細胞は、3〜4日おきに継代する必要があり、このとき、継代は、例えば1mM CaCl2および20% KSRを含
有するPBS中の0.25% トリプシンおよび0.1mg/mlコラゲナーゼIVを用いて行うことができ
る。
For example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF is used as the culture medium for ES cell production. Human ES cells can be maintained in a humid atmosphere of 37 ° C., 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
ES細胞の選択は、一般に、アルカリホスファターゼ、Oct-3/4、Nanogなどの遺伝子マーカーの発現を指標にしてReal-Time PCR法で行うことができる。特に、ヒトES細胞の選択
では、OCT-3/4、NANOG、ECADなどの遺伝子マーカーの発現を指標とすることができる(E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452)。
In general, ES cells can be selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index. In particular, in the selection of human ES cells, expression of gene markers such as OCT-3 / 4, NANOG, ECAD and the like can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443 -452).
ヒトＥＳ細胞株は、例えばWA01(H1)およびWA09(H9)は、WiCell Reserch Instituteから
、KhES-1、KhES-2およびKhES-3は、京都大学再生医科学研究所(京都、日本)から入手可能である。
Human ES cell lines are obtained from, for example, WA01 (H1) and WA09 (H9) from the WiCell Research Institute, and KhES-1, KhES-2, and KhES-3 from Kyoto University Institute for Regenerative Medicine (Kyoto, Japan). Is possible.
(B) 精子幹細胞
精子幹細胞は、精巣由来の多能性幹細胞であり、精子形成のための起源となる細胞である。この細胞は、ES細胞と同様に、種々の系列の細胞に分化誘導可能であり、例えばマウス胚盤胞に移植するとキメラマウスを作出できるなどの性質をもつ(M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012)。神経膠細胞系由来神経栄養因子(glial cell line-derived neurotrophic factor (GDNF))を含む培養液で自己複製可能であるし、またES細胞と同様の培養条件下で
継代を繰り返すことによって、精子幹細胞を得ることができる(竹林正則ら(2008),実験医学,26巻,5号(増刊),41〜46頁,羊土社(東京、日本))。
(B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is capable of self-replication in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and by repeating subculture under the same culture conditions as ES cells, Stem cells can be obtained (Masatake Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 41-46, Yodosha (Tokyo, Japan)).
(C) 胚性生殖細胞
胚性生殖細胞は、胎生期の始原生殖細胞から樹立される、ES細胞と同様な多能性をもつ細胞であり、LIF、bFGF、幹細胞因子(stem cell factor)などの物質の存在下で始原生殖
細胞を培養することによって樹立しうる(Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551)。
(C) Embryonic germ cells Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
(D) 人工多能性幹細胞
人工多能性幹(iPS)細胞は、ある特定の再プログラミング因子を、DNA又はタンパク質の形態で体細胞に導入することによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能、を有する体細胞由来の人工の幹細胞である（K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M.
ら,Nat. Biotechnol. 26:101-106 (2008)；国際公開WO 2007/069666）。再プログラム化
因子は、ES細胞に特異的に発現している遺伝子またはES細胞の未分化維持に重要な役割を果たす遺伝子もしくはその遺伝子産物であれば良く、特に限定されないが、例えばOCT3/4、SOX2及びKLF4； OCT3/4、KLF4及びC-MYC； OCT3/4、SOX2、KLF4及びC-MYC； OCT3/4及
びSOX2； OCT3/4、SOX2及びNANOG； OCT3/4、SOX2及びLIN28； OCT3/4及びKLF4などの組
み合わせである。
(D) Artificial pluripotent stem cellsInduced pluripotent stem (iPS) cells are almost the same as ES cells that can be created by introducing certain reprogramming factors into somatic cells in the form of DNA or protein. Artificial stem cells derived from somatic cells with equivalent properties, such as pluripotency and proliferation by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M.
Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666). The reprogramming factor is not particularly limited as long as it is a gene specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof, such as OCT3 / 4, SOX2 and KLF4; OCT3 / 4, KLF4 and C-MYC; OCT3 / 4, SOX2, KLF4 and C-MYC; OCT3 / 4 and SOX2; OCT3 / 4, SOX2 and NANOG; OCT3 / 4, SOX2 and LIN28; OCT3 / Combination of 4 and KLF4.
これらの因子は、タンパク質の形態で、例えばリポフェクション、細胞膜透過性ペプチドとの結合、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してもよいし、あるいは、DNAの形態で、例えば、ウイルス、プラスミド、人工染色体などのベクター
、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。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（以上、Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007）、アデノウイルスベクター(Science, 322, 945-949, 2008)、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(Science, 322:949-953, 2008)。ベクターには、核初期化物質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、緑色蛍光タンパク質(GFP)、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。また、上記ベクターには、体細胞への導入後、再プログラミング化因子をコードする遺伝子もしくはプロモーターとそれに結合する再
プログラミング化因子をコードする遺伝子を共に切除するために、それらの前後にLoxP配列を有してもよい。
These factors may be introduced into somatic cells in the form of proteins, for example, by lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA, for example, viruses, plasmids. It can be introduced into a somatic cell by a technique such as a vector such as an artificial chromosome, lipofection, liposome, or microinjection. Examples of viral vectors include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors and the like. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed. Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can be included. In addition, after introduction into a somatic cell, the above vector contains a LoxP sequence before and after the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. You may have.
再プログラム化に際して、誘導効率を高めるために、上記の因子の他に、例えば、ヒストンデアセチラーゼ（HDAC）阻害剤［例えば、バルプロ酸 (VPA)(Nat. Biotechnol., 26(7): 795-797 (2008))、トリコスタチンA、酪酸ナトリウム、MC 1293、M344等の低分子阻
害剤、HDACに対するsiRNAおよびshRNA（例、HDAC1 siRNA Smartpool (登録商標：Millipore)、HuSH 29mer shRNA Constructs against HDAC1 (OriGene)等）等の核酸性発現阻害剤など］、DNAメチルトランスフェラーゼ阻害剤（例えば5’-azacytidine）（Nat. Biotechnol., 26(7): 795-797 (2008))、G9aヒストンメチルトランスフェラーゼ阻害剤［例えば
、BIX-01294 (Cell Stem Cell, 2: 525-528 (2008))等の低分子阻害剤、G9aに対するsiRNAおよびshRNA（例、G9a siRNA(human) (Santa Cruz Biotechnology)等）等の核酸性発現
阻害剤など］、L-channel calcium agonist (例えばBayk8644) (Cell Stem Cell, 3, 568-574 (2008))、p53阻害剤（例えばp53に対するsiRNAおよびshRNA (Cell Stem Cell, 3, 475-479 (2008))、UTF1(Cell Stem Cell, 3, 475-479 (2008))、Wnt Signaling（例えばsoluble Wnt3a）（Cell Stem Cell, 3, 132-135 (2008)）、2i/LIF (2iはmitogen-activated protein kinase signallingおよびglycogen synthase kinase-3の阻害剤、PloS Biology, 6(10), 2237-2247 (2008))、miR-291-3p、miR-294、miR-295などのmiRNA(R.L. Judson
et al., Nat. Biotech., 27:459-461) (2009)等を使用することができる。
In order to increase induction efficiency upon reprogramming, in addition to the above factors, for example, a histone deacetylase (HDAC) inhibitor [for example, valproic acid (VPA) (Nat. Biotechnol., 26 (7): 795 -797 (2008)), small molecule inhibitors such as trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg, HDAC1 siRNA Smartpool (registered trademark: Millipore), HuSH 29mer shRNA Constructs against HDAC1 ( Nucleic acid expression inhibitors such as OriGene)], DNA methyltransferase inhibitors (eg 5'-azacytidine) (Nat. Biotechnol., 26 (7): 795-797 (2008)), G9a histone methyltransferase inhibition Such as small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology), etc.) Nucleic acid expression inhibitors, etc.], L-channel calcium agonist (eg Bayk8644) (Cell Stem Cell, 3, 568 -574 (2008)), p53 inhibitors (eg siRNA and shRNA against p53 (Cell Stem Cell, 3, 475-479 (2008)), UTF1 (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling (Eg soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 2i / LIF (2i is an inhibitor of mitogen-activated protein kinase signaling and glycogen synthase kinase-3, PloS Biology, 6 (10), 2237-2247 (2008)), miR-291-3p, miR-294, miR-295 and other miRNAs (RL Judson
et al., Nat. Biotech., 27: 459-461) (2009) and the like can be used.
iPS細胞誘導のための培養培地としては、例えば(1) 10〜15％FBSを含有するDMEM、DMEM/F12又はDME培地（これらの培地にはさらに、LIF、penicillin/streptomycin、puromycin、L-グルタミン、非必須アミノ酸類、β-メルカプトエタノールなどを適宜含むことがで
きる。）、(2) bFGF又はSCFを含有するES細胞培養用培地、例えばマウスES細胞培養用培
地(例えばTX-WES培地、トロンボＸ社)又は霊長類ES細胞培養用培地（例えば霊長類（ヒト＆サル）ES細胞用培地、リプロセル、京都、日本）、などが含まれる。
Examples of the culture medium for iPS cell induction include (1) DMEM, DMEM / F12 or DME medium containing 10 to 15% FBS (these media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , (2) ES cell culture medium containing bFGF or SCF, for example, mouse ES cell culture medium (for example, TX-WES medium, thrombos. X) or primate ES cell culture medium (eg, primate (human & monkey) ES cell culture medium, Reprocell, Kyoto, Japan).
培養法の例としては、たとえば、37℃、5％CO2存在下にて、10％FBS含有DMEM又はDMEM/F12培地上で体細胞と再プログラム化因子(DNA又はタンパク質)を接触させ約4〜7日間培養し、その後、細胞をフィーダー細胞(たとえば、マイトマイシンC処理STO細胞、SNL細胞等)上にまきなおし、体細胞と再プログラム化因子の接触から約10日後からbFGF含有霊長類ES細胞培養用培地で培養し、該接触から約30〜約45日又はそれ以上ののちにiPS様コロニーを生じさせることができる。 As an example of the culture method, for example, a somatic cell and a reprogramming factor (DNA or protein) are brought into contact with each other on a DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO 2 at 37 ° C. Incubate for ~ 7 days, then re-spread cells onto feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and bFGF-containing primate ES cells approximately 10 days after contact of somatic cells with reprogramming factors Culturing in a culture medium can generate iPS-like colonies about 30 to about 45 days or more after the contact.
あるいは、その代替培養法として、37℃、5% CO2存在下にて、フィーダー細胞(たとえ
ば、マイトマイシンC処理STO細胞、SNL細胞等)上で10％FBS含有DMEM培地（これにはさら
に、LIF、ペニシリン/ストレプトマイシン、ピューロマイシン、L-グルタミン、非必須アミノ酸類、β-メルカプトエタノールなどを適宜含むことができる。）で培養し、約25〜
約30日又はそれ以上ののちにES様コロニーを生じさせることができる。望ましくは、フィーダー細胞の代わりに、初期化される体細胞そのものを用いる（Takahashi K, et al. (2009), PLoS One. 4:e8067またはWO2010/137746）、もしくは細胞外基質（例えば、Laminin-5（WO2009/123349）およびマトリゲル（BD社））を用いる方法が例示される。
Alternatively, as an alternative culture method, DMEM medium containing 10% FBS on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) in the presence of 5% CO 2 at 37 ° C. , Penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.).
ES-like colonies can be generated after about 30 days or more. Desirably, instead of feeder cells, somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.
この他にも、血清を含有しない培地を用いて培養する方法も例示される（Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106:15720-15725）。さらに、樹立効率を上げる
ため、低酸素条件（0.1％以上、15％以下の酸素濃度）によりiPS細胞を樹立しても良い（Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241またはWO2010/013845）。
In addition, a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106: 15720-15725). Furthermore, in order to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
上記培養の間には、培養開始2日目以降から毎日1回新鮮な培養液と培養液交換を行う。また、核初期化に使用する体細胞の細胞数は、限定されないが、培養ディッシュ100cm2あ
たり約5×103〜約5×106細胞の範囲である。
During the culture, the culture medium is exchanged with a fresh culture medium once a day from the second day onward. The number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 × 10 3 to about 5 × 10 6 cells per 100 cm 2 of culture dish.
iPS細胞は、形成したコロニーの形状により選択することが可能である。一方、体細胞
が初期化された場合に発現する遺伝子（例えば、Oct3/4、Nanog）と連動して発現する薬
剤耐性遺伝子をマーカー遺伝子として導入した場合は、対応する薬剤を含む培養液（選択培養液）で培養を行うことにより樹立したiPS細胞を選択することができる。また、マー
カー遺伝子が蛍光タンパク質遺伝子の場合は蛍光顕微鏡で観察することによって、発光酵素遺伝子の場合は発光基質を加えることによって、また発色酵素遺伝子の場合は発色基質を加えることによって、iPS細胞を選択することができる。
iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture solution containing the corresponding drug (selection The established iPS cells can be selected by culturing with the culture medium. In addition, if the marker gene is a fluorescent protein gene, iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.
本明細書中で使用する「体細胞」なる用語は、卵子、卵母細胞、ES細胞などの生殖系列細胞または分化全能性細胞を除くあらゆる動物細胞（好ましくは、ヒトを含む哺乳動物細胞）をいう。体細胞には、非限定的に、胎児(仔)の体細胞、新生児(仔)の体細胞、および成熟した健全なもしくは疾患性の体細胞のいずれも包含されるし、また、初代培養細胞、継代細胞、および株化細胞のいずれも包含される。具体的には、体細胞は、例えば（1）
神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）、（2）組織前駆細胞、（3）リンパ球、上皮細胞、内皮細胞、筋肉細胞、線維芽細胞（皮膚細胞等）、毛細胞、肝細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞(膵外分泌細胞等)、脳細胞、肺細胞、腎細胞および脂肪細胞等の分化した細胞などが例示される。
As used herein, the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell. Say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines. Specifically, somatic cells are, for example, (1)
Tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin Cells), hair cells, hepatocytes, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells, etc.), differentiated cells such as brain cells, lung cells, kidney cells and adipocytes, etc. .
また、iPS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、移植先の個体のHLA遺伝子型が同一もしくは実質的に同一である体細胞を用いる
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、HLA-A、HLA-BおよびHLA-DRの3遺伝子座あるいは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。
When iPS cells are used as a material for transplantation cells, it is desirable to use somatic cells having the same or substantially the same HLA genotype of the transplant destination individual from the viewpoint of preventing rejection. Here, “substantially the same” means that the HLA genotype matches the transplanted cells to such an extent that an immune response can be suppressed by an immunosuppressive agent. For example, HLA-A, HLA-B And somatic cells having an HLA type in which 3 loci of HLA-DR or 4 loci plus HLA-C are matched.
(E) 核移植により得られたクローン胚由来のES細胞
nt ES細胞は、核移植技術によって作製されたクローン胚由来のES細胞であり、受精卵
由来のES細胞とほぼ同じ特性を有している（T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502）。すなわち、未受精卵の核を体細胞の核と置換することによって得られたクローン胚由来の胚盤胞の内部細胞塊から樹立されたES細胞がnt ES(nuclear transfer ES)細胞である。nt ES細胞の作製のためには、核移植技術（J.B. Cibelli et al. (1998), Nature Biotechnol., 16:642-646）とES細胞作製技術(上記)との組み合
わせが利用される(若山清香ら(2008),実験医学,26巻,5号(増刊), 47〜52頁)。核移植においては、哺乳動物の除核した未受精卵に、体細胞の核を注入し、数時間培養することで初期化することができる。
(E) Cloned embryo-derived ES cells obtained by nuclear transfer
The nt ES cell is a cloned embryo-derived ES cell produced by a nuclear transfer technique and has almost the same characteristics as an ES cell derived from a fertilized egg (T. Wakayama et al. (2001), Science, 292). : 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell. For the production of nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52). Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.
＜中胚葉前駆細胞＞
本発明において、中胚葉とは、発生の過程で体腔及びそれを裏打ちする中皮、筋肉、骨格、皮膚真皮、結合組織、心臓・血管（血管内皮も含む)、血液（血液細胞も含む）、リ
ンパ管や脾臓、腎臓及び尿管、性腺（精巣、子宮、性腺上皮）をつくる能力を有した細胞から構成される胚葉を包含する。本発明において、中胚葉前駆細胞は、例えば、Ｔ（Ｂｒａｃｈｙｕｒｙと同義）、ＫＤＲ、ＦＯＸＦ１、ＦＬＫ１、ＢＭＰ４、ＭＯＸ１及びＳＤＦ１から成るマーカー遺伝子から選択される少なくとも一つのマーカー遺伝子が発現する細胞である。中胚葉前駆細胞には、中胚葉細胞と区別されるものではなく、上記マーカー遺伝子の発現が弱いものを中胚葉前駆細胞と称してもよい。本発明において製造される、中胚葉前駆細胞は他の細胞種が含まれる細胞集団として製造されてもよく、例えば、５０％以上、６０％以上、７０％以上、８０％以上または９０％以上の中胚葉前駆細胞が含ま
れる細胞集団である。
<Mesodermal progenitor cells>
In the present invention, the mesoderm refers to the body cavity and the mesothelium, muscle, skeleton, skin dermis, connective tissue, heart / blood vessels (including vascular endothelium), blood (including blood cells), It includes the germ layers composed of cells that have the ability to produce lymphatic vessels, spleen, kidney and ureter, and gonadal (testis, uterus, gonadal epithelium). In the present invention, a mesoderm progenitor cell is a cell that expresses at least one marker gene selected from marker genes consisting of T (synonymous with Brachyury), KDR, FOXF1, FLK1, BMP4, MOX1, and SDF1, for example. Mesodermal progenitor cells are not distinguished from mesoderm cells, and cells with weak expression of the marker gene may be referred to as mesoderm progenitor cells. Mesodermal progenitor cells produced in the present invention may be produced as a cell population containing other cell types, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. A cell population containing mesoderm progenitor cells.
＜血液血管前駆細胞＞
本発明において、血液血管前駆細胞とは、血液系細胞または血管内皮細胞へ誘導される能力を有した細胞から構成される細胞であり、血管芽細胞またはヘマンジオブラストとも称される。本発明において、血液血管前駆細胞は、ＣＤ３４及びＫＤＲを発現する細胞として例示される。本発明において製造される、血液血管前駆細胞は他の細胞種が含まれる細胞集団として製造されてもよく、例えば、５０％以上、６０％以上、７０％以上、８０％以上または９０％以上の血液血管前駆細胞が含まれる細胞集団である。
<Blood blood vessel progenitor cells>
In the present invention, blood vascular progenitor cells are cells composed of cells capable of being induced into blood cells or vascular endothelial cells, and are also referred to as hemangioblasts or hemangioblasts. In the present invention, blood vascular progenitor cells are exemplified as cells that express CD34 and KDR. The blood vascular progenitor cells produced in the present invention may be produced as a cell population containing other cell types, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. A cell population containing blood vessel progenitor cells.
＜中胚葉前駆細胞の製造工程＞
本発明は、多能性幹細胞を、ラミニン５１１またはその断片でコーティングされた培養器材上にて、ＢＭＰ、ＧＳＫ３β阻害剤およびＶＥＧＦを含む培養液中で接着培養する工程を含む多能性幹細胞から中胚葉前駆細胞を製造する方法を提供する。
<Process for producing mesoderm progenitor cells>
The present invention relates to pluripotent stem cells comprising a step of adhesion-culturing pluripotent stem cells on a culture device coated with laminin 511 or a fragment thereof in a culture medium containing BMP, a GSK3β inhibitor and VEGF. A method for producing germ layer progenitor cells is provided.
本発明において、接着培養とは、細胞接着に適した表面加工をした培養容器または細胞外基質でコーティング処理された培養容器を用いて足場依存的に培養することを意味する。 In the present invention, adhesion culture means culturing in a scaffold-dependent manner using a culture vessel that has been surface-treated suitable for cell adhesion or a culture vessel that has been coated with an extracellular matrix.
本発明において、細胞外基質として、ラミニン５１１（α５鎖、β１鎖、γ１鎖からなるラミニン）またはその断片が好適に用いられる。本発明においてラミニンとは、基底膜の主要な細胞接着分子であり、α鎖、β鎖、及びγ鎖の３本のサブユニット鎖からなるヘテロ３量体で、分子量８０万Ｄａの巨大な糖タンパク質である。３本のサブユニット鎖がＣ末端側で会合してコイルドコイル構造を作りジスルフィド結合によって安定化したヘテロ３量体分子を言う。従って、ラミニン５１１とは、α鎖がα５であり、β鎖がβ１であり、ならびにγ鎖がγ１であるラミニンを意味する。さらに、ラミニンは、変異体であってもよく、インテグリン結合活性を有している変異体であれば、特に限定されない。ラミニンはヒト由来のものが好ましい。 In the present invention, laminin 511 (laminin composed of α5 chain, β1 chain, γ1 chain) or a fragment thereof is preferably used as the extracellular matrix. In the present invention, laminin is a major cell adhesion molecule of the basement membrane, a heterotrimer composed of three subunit chains of α chain, β chain, and γ chain, and a huge sugar having a molecular weight of 800,000 Da. It is a protein. A heterotrimeric molecule in which three subunit chains associate at the C-terminal to form a coiled-coil structure and is stabilized by disulfide bonds. Thus, laminin 511 means laminin in which the α chain is α5, the β chain is β1, and the γ chain is γ1. Furthermore, laminin may be a mutant and is not particularly limited as long as it is a mutant having integrin binding activity. Laminin is preferably derived from human.
本発明において、ラミニン断片は、エラスターゼにて消化して得られる断片であるＥ８フラグメント（ラミニン５１１のＥ８フラグメント（ラミニン５１１Ｅ８ともいう））（Ido H, et al, J Biol Chem. 2007, 282, 11144-11154）であってもよい。ラミニン５１
１Ｅ８は、例えば、ニッピ社から入手可能である。
In the present invention, the laminin fragment is an E8 fragment (E8 fragment of laminin 511 (also referred to as laminin 511E8)) (Ido H, et al, J Biol Chem. 2007, 282, 11144) which is a fragment obtained by digestion with elastase. -11154). Laminin 51
1E8 is available from Nippi, for example.
コーティング処理は、ラミニン５１１またはその断片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。 The coating treatment can be performed by appropriately removing the solution after putting a solution containing laminin 511 or a fragment thereof in a culture vessel.
中胚葉前駆細胞の製造工程で用いるＢＭＰは、ＢＭＰ２、ＢＭＰ４およびＢＭＰ７から成る群より選択される少なくとも一つのＢＭＰであり、好ましくは、ＢＭＰ４である。ＢＭＰはヒト由来のものが好ましい。 The BMP used in the production process of mesoderm progenitor cells is at least one BMP selected from the group consisting of BMP2, BMP4 and BMP7, preferably BMP4. BMP is preferably derived from human.
本発明において、中胚葉前駆細胞の製造工程にＢＭＰ４を用いる場合、培養液中におけるＢＭＰ４の濃度は、特に限定されないが、5 ng/mlから200 ng/ml、10 ng/mlから100 ng/ml、20 ng/mlから80 ng/mlが例示される。好ましくは、80 ng/mlである。 In the present invention, when BMP4 is used in the production process of mesoderm progenitor cells, the concentration of BMP4 in the culture solution is not particularly limited, but is 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml. 20 ng / ml to 80 ng / ml. Preferably, it is 80 ng / ml.
本発明において、GSK3β阻害剤とは、GSK-3βタンパク質のキナーゼ活性（例えば、β
カテニンに対するリン酸化能）を阻害する物質として定義され、既に多数のものが知られているが、例えば、インジルビン誘導体であるBIO（別名、GSK-3β阻害剤IX；6-ブロモインジルビン3'-オキシム）、マレイミド誘導体であるSB216763（3-(2,4-ジクロロフェニル)-4-(1-メチル-1H-インドール-3-イル)-1H-ピロール-2,5-ジオン）、フェニルαブロモメ
チルケトン化合物であるGSK-3β阻害剤VII（4-ジブロモアセトフェノン）、細胞膜透過型のリン酸化ペプチドであるL803-mts（別名、GSK-3βペプチド阻害剤；Myr-N-GKEAPPAPPQpSP-NH2（配列番号１））および高い選択性を有するCHIR99021(6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile)が挙げられる。これらの化合物は、例えばCalbiochem社やBiomol社等から市販
されており容易に利用することが可能であるが、他の入手先から入手してもよく、あるいはまた自ら作製してもよい。本発明で使用されるGSK-3β阻害剤は、好ましくは、ＣＨＩ
Ｒ９９０２１であり得る。
In the present invention, GSK3β inhibitor refers to kinase activity of GSK-3β protein (for example, β
Many substances are defined as substances that inhibit catenin phosphorylation ability. For example, BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3′-) is an indirubin derivative. Oxime), maleimide derivative SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), phenyl α bromomethyl GSK-3β inhibitor VII (4-dibromoacetophenone), a ketone compound, L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQpSP-NH 2) (SEQ ID NO: 1)) and CHIR99021 (6- [2- [4- (2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino] ethylamino] with high selectivity pyridine-3-carbonitrile). These compounds are commercially available from, for example, Calbiochem and Biomol, and can be easily used, but may be obtained from other sources or may be prepared by themselves. The GSK-3β inhibitor used in the present invention is preferably CHI.
May be R99021.
本発明の培養液中におけるＣＨＩＲ９９０２１の濃度は、例えば、1nM、10nM、50nM、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM、10μM
、15μM、20μM、25μM、30μM、40μM、50μMまたはこれらの間の濃度であるがこれらに限定されない。好ましくは、2μMである。
The concentration of CHIR99021 in the culture medium of the present invention is, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM.
, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM or a concentration therebetween, but is not limited thereto. Preferably, it is 2 μM.
中胚葉前駆細胞の製造工程の培養液中におけるVEGFの濃度は、特に限定されないが、5 ng/mlから200 ng/ml、10 ng/mlから100 ng/ml、または20 ng/mlから80 ng/mlが例示され
る。好ましくは、80 ng/mlである。VEGFはヒト由来のものが好ましい。
The concentration of VEGF in the culture medium of the production process of mesoderm progenitor cells is not particularly limited, but is 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, or 20 ng / ml to 80 ng / ml is exemplified. Preferably, it is 80 ng / ml. VEGF is preferably derived from human.
中胚葉前駆細胞の製造工程で用いる培養液は、動物細胞の培養に用いられる培地を基礎培地として、適宜ＢＭＰ、ＧＳＫ３β阻害剤およびＶＥＧＦを添加することによって調製することができる。基礎培地としては、例えば、Glasgow's Minimal Essential Medium(GMEM)培地、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）、ｍＴｅｓＲ１培地（ライフテクノロジーズ）、Essential 8（ライフテクノロジーズ）、Ｓｔｅｍｐｒ
ｏ３４ＳＦＭ培地（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。基礎培地には、必要に応じて、例えば、アルブミン、トランスフェリン、Knockout Serum
Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2サプリメント（Invitrogen）、B27サプリメント（Invitrogen）、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、3'-チオールグリセロールなどの1つ以上の血清代替物を含んで
もよいし、脂質、アミノ酸、L-グルタミン、Glutamax（Invitrogen）、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、無機塩類などの1つ以上の物質も含有し得る。好ましい基礎培地は、ｍＴｅｓＲ１培地またはEssential 8である。
The culture solution used in the production process of mesoderm progenitor cells can be prepared by appropriately adding BMP, a GSK3β inhibitor and VEGF using a medium used for culturing animal cells as a basal medium. As the basal medium, for example, Glasgow's Minimal Essential Medium (GMEM) 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, Fischer's Medium, Neurobasal Medium (Life Technologies), mTesR1 Medium (Life Technologies), Essential 8 (Life Technologies), Temprr
o34SFM medium (Life Technologies) and mixed media thereof are included. Basal media include, for example, albumin, transferrin, Knockout Serum as needed.
Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol, 3'-thiolglycerol May contain one or more serum substitutes such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate It may also contain one or more substances such as buffering agents, inorganic salts. A preferred basal medium is mTesR1 medium or Essential 8.
中胚葉前駆細胞の製造工程の培養条件について、培養温度は、特に限定されないが、約30〜40℃、好ましくは約37℃であり、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。培養期間は、2日以上であり、2日〜3日間が好ましい。 Regarding the culture conditions of the production process of mesoderm progenitor cells, the culture temperature is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO 2 -containing air, and CO 2 The concentration is preferably about 2-5%. The culture period is 2 days or more, and preferably 2 days to 3 days.
＜血液血管前駆細胞の製造工程＞
本発明は、中胚葉前駆細胞をＶＥＧＦ、ＳＣＦ、ｂＦＧＦおよびＴＧＦβ阻害剤を含む培養液中で接着培養する工程を含む中胚葉前駆細胞から血液血管前駆細胞を製造する方法を提供する。
<Manufacturing process of blood vascular progenitor cells>
The present invention provides a method for producing blood vascular progenitor cells from mesoderm progenitor cells, comprising the step of adhesion culture of mesoderm progenitor cells in a culture medium containing VEGF, SCF, bFGF and a TGFβ inhibitor.
血液血管前駆細胞の製造工程では、接着培養に用いる培養容器のコーティング剤は特に制限されず、ラミニンまたはその断片、Ｍａｔｒｉｇｅｌ、コラーゲン、およびフィブロネクチンが例示されるが、簡便には前述した中胚葉前駆細胞の製造工程と同じくラミニン５１１またはその断片を用いることができる。前述した中胚葉前駆細胞の製造工程に引き続き、血液血管前駆細胞の製造工程を行う場合、適宜培養液を交換することで実施し得る。 In the production process of blood vascular progenitor cells, the coating agent for the culture vessel used for adhesion culture is not particularly limited, and examples thereof include laminin or a fragment thereof, Matrigel, collagen, and fibronectin. Laminin 511 or a fragment thereof can be used in the same manner as in the manufacturing process. Subsequent to the process for producing mesoderm progenitor cells described above, the process for producing blood vascular progenitor cells can be carried out by appropriately replacing the culture medium.
血液血管前駆細胞の製造工程の培養液中におけるVEGFの濃度は、特に限定されないが、5 ng/mlから200 ng/ml、10 ng/mlから100 ng/ml、または20 ng/mlから80 ng/mlが例示さ
れる。好ましくは、80 ng/mlである。
The concentration of VEGF in the culture medium of the blood vessel progenitor cell production process is not particularly limited, but is 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, or 20 ng / ml to 80 ng / ml is exemplified. Preferably, it is 80 ng / ml.
血液血管前駆細胞の製造工程の培養液中におけるＳＣＦの濃度は、特に限定されないが、5 ng/mlから200 ng/ml、10 ng/mlから100 ng/ml、または20 ng/mlから80 ng/mlが例示
される。好ましくは、50 ng/mlである。ＳＣＦはヒト由来のものが好ましい。
The concentration of SCF in the culture medium of the blood vessel progenitor cell production process is not particularly limited, but is 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, or 20 ng / ml to 80 ng. / ml is exemplified. Preferably, it is 50 ng / ml. SCF is preferably derived from human.
血液血管前駆細胞の製造工程の培養液中におけるｂＦＧＦの濃度は、特に限定されないが、5 ng/mlから200 ng/ml、10 ng/mlから100 ng/ml、または20 ng/mlから80 ng/mlが例
示される。好ましくは、50 ng/mlである。ｂＦＧＦはヒト由来のものが好ましい。
The concentration of bFGF in the culture medium of the blood vessel progenitor cell production process is not particularly limited, but is 5 ng / ml to 200 ng / ml, 10 ng / ml to 100 ng / ml, or 20 ng / ml to 80 ng. / ml is exemplified. Preferably, it is 50 ng / ml. bFGF is preferably derived from human.
本発明において、ＴＧＦβ阻害剤とは、ＴＧＦβとその受容体の結合を阻害する物質、またはTGFβが受容体に結合後の下流シグナルを阻害する物質として定義され、、当該下
流シグナルとは、TGFβII型受容体によるTGFβI型受容体をリン酸化、このTGFβI型受容
体によるSmad(R-Smad)のリン酸化などが例示される。ＴＧＦβ阻害剤として既に多数のものが知られているが、例えば、SB431542、SB202190(R.K.Lindemann et al., Mol. Cancer
2:20 (2003))、SB505124 (GlaxoSmithKline)、NPC30345 、SD093、SD908、SD208 (Scios)、LY2109761、LY364947、LY580276 (Lilly Research Laboratories)、A-83-01 (WO 2009146408)などが挙げられる。これらの化合物は、例えばSTEMGENT社等から市販されており
容易に利用することが可能であるが、他の入手先から入手してもよく、あるいはまた自ら作製してもよい。本発明で使用されるＴＧＦβ阻害剤は、好ましくは、SB431542であり得る。
In the present invention, a TGFβ inhibitor is defined as a substance that inhibits the binding of TGFβ and its receptor, or a substance that inhibits a downstream signal after TGFβ binds to the receptor, and the downstream signal is TGFβ type II Examples include phosphorylation of TGFβ type I receptor by the receptor, phosphorylation of Smad (R-Smad) by this TGFβ type I receptor, and the like. Many TGFβ inhibitors are already known. For example, SB431542, SB202190 (RKLindemann et al., Mol. Cancer
2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009146408), and the like. These compounds are commercially available from, for example, STEMGENT and can be easily used, but may be obtained from other sources or may be prepared by themselves. The TGFβ inhibitor used in the present invention may preferably be SB431542.
血液血管前駆細胞の製造工程の培養液中におけるSB431542の濃度は、例えば、1nM、10nM、50nM、100nM、500nM、750nM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9
μM、10μM、15μM、20μM、25μM、30μM、40μM、50μMまたはこれらの間の濃度であるがこれらに限定されない。好ましくは、2μMである。
The concentration of SB431542 in the culture medium of the blood vessel progenitor cell production process is, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9
The concentration is, but not limited to, μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM. Preferably, it is 2 μM.
血液血管前駆細胞の製造工程で用いる培養液は、動物細胞の培養に用いられる培地を基礎培地として、適宜ＶＥＧＦ、ＳＣＦ、ｂＦＧＦおよびＴＧＦβ阻害剤を添加することによって調製することができる。基礎培地としては、例えば、Glasgow's Minimal Essential Medium(GMEM)培地、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）、ｍ
ＴｅｓＲ１培地（ライフテクノロジーズ）、Essential 8（ライフテクノロジーズ）、Essential 6（ライフテクノロジーズ）、Ｓｔｅｍｐｒｏ３４ＳＦＭ培地（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。基礎培地には、必要に応じて、例えば、アルブミン、トランスフェリン、Knockout Serum Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2サプリメント（Invitrogen）、B27サプリメント（Invitrogen）、脂
肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、3'-チオー
ルグリセロールなどの1つ以上の血清代替物を含んでもよいし、脂質、アミノ酸、L-グル
タミン、Glutamax（Invitrogen）、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、無機塩類などの1つ以上の物質も含有し得
る。好ましい基礎培地は、ＴＧＦβを含有しない基礎培地であり、このような培地として例えば、Essential 6が挙げられる。
The culture solution used in the blood vessel progenitor cell production process can be prepared by appropriately adding VEGF, SCF, bFGF, and TGFβ inhibitor using a medium used for animal cell culture as a basal medium. As the basal medium, for example, Glasgow's Minimal Essential Medium (GMEM) 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, Fischer's Medium, Neurobasal Medium (Life Technologies), m
Examples include TesR1 medium (Life Technologies), Essential 8 (Life Technologies), Essential 6 (Life Technologies), Tempro34 SFM medium (Life Technologies), and mixed media thereof. For basal media, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin May include one or more serum substitutes such as collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, It may also contain one or more substances such as vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like. A preferred basal medium is a basal medium not containing TGFβ. Examples of such a medium include Essential 6.
血液血管前駆細胞の製造工程の培養条件について、培養温度は、特に限定されないが、約30〜40℃、好ましくは約37℃であり、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。培養期間は、2日以上であり、2日〜3日間が好ましい。
Regarding the culture conditions of the blood vessel progenitor cell production process, the culture temperature is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO 2 -containing air, and CO 2 The concentration is preferably about 2-5%. The culture period is 2 days or more, and preferably 2 days to 3 days.
＜キット＞
本発明での他の実施態様において、多能性幹細胞から中胚葉前駆細胞を作製するキットおよび/または中胚葉前駆細胞から血液血管前駆細胞を作製するキットが含まれる。当該
キットには、上述した中胚葉前駆細胞を作製する各工程に使用する培養液、添加剤または培養容器等が含まれる。本キットには、さらに製造工程の手順を記載した書面や説明書を含んでもよい。
<Kit>
In other embodiments of the invention, kits for producing mesoderm progenitor cells from pluripotent stem cells and / or kits for producing blood vascular progenitor cells from mesoderm progenitor cells are included. The kit includes a culture solution, an additive, a culture container, or the like used for each step of producing the above-described mesoderm progenitor cells. The kit may further include a document or instructions describing the procedure of the manufacturing process.
以下に実施例を挙げて本発明をより具体的に説明するが、本発明がこれらに限定されないことは言うまでもない。 Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.
細胞および培養
ヒトＥＳ細胞（ＫｈＥＳ１）は、京都大学再生医科学研究所より受領し、従来の方法で培養した（Suemori H, et al. Biochem Biophys Res Commun. 345:926-32, 2006）。ヒトiPS細胞は、臍帯血単核球に４因子をエピソーマルベクター（Okita K, et al, Nat Methods. 8, 409-412, 2011）により導入して作製した（CB-iPSCと言う）。
Cells and cultured human ES cells (KhES1) were received from the Institute of Regenerative Medicine, Kyoto University and cultured by conventional methods (Suemori H, et al. Biochem Biophys Res Commun. 345: 926-32, 2006). Human iPS cells were prepared by introducing 4 factors into cord blood mononuclear cells using an episomal vector (Okita K, et al, Nat Methods. 8, 409-412, 2011) (referred to as CB-iPSC).
ヒトＥＳ細胞およびヒトiPS細胞の維持培養はＳＮＬフィーダー上で、５ｍｇ／ｍＬのbFGF (WAKO)を添加したＥＳ培地(ReproCELL)を用いて行った。また、継代はＣＴＫ溶液（
０．２５％トリプシン(Life technologies)、０．１％コラゲナーゼＩＶ(Life technologies),、２０％ＫＳＲ、及び１ｍＭＣａＣl２）によって約３０秒間室温で処理して細胞を解離させ、既存の方法（Suemori, H. et al. Biochemical and Biophysical Research Communications 345, 926932 (2006)）により、ＳＮＬ細胞を除去した。
Maintenance culture of human ES cells and human iPS cells was performed on an SNL feeder using ES medium (ReproCELL) supplemented with 5 mg / mL bFGF (WAKO). In addition, the passage is CTK solution (
Cells were dissociated by treatment with 0.25% trypsin (Life technologies), 0.1% collagenase IV (Life technologies), 20% KSR, and 1 mM CaCl 2 ) for about 30 seconds at room temperature, and the existing method (Suemori, H. et al. Biochemical and Biophysical Research Communications 345, 926932 (2006)), SNL cells were removed.
多能性幹細胞から中胚葉前駆細胞への分化誘導
ヒトＥＳ細胞をLM511 (Biolamina)でコーティングしたプレートに5 colonies/cm2の密
度で播種し、mTeSR1で培養した。コロニーの直径が750μmになるまで増殖させた後、2 μM CHIR99021 (Wako), 80 ng/mL BMP4 (R&D Systems（RSD）), 80 ng/mL VEGF (RSD)を含
むEssential 8 (Life technologies)培地に交換し、2日培養し、KDR陽性の中胚葉前駆細
胞を得た。
Differentiation-induced human ES cells from pluripotent stem cells to mesoderm progenitor cells were seeded at a density of 5 colonies / cm 2 on a plate coated with LM511 (Biolamina) and cultured in mTeSR1. Essential 8 (Life technologies) medium containing 2 μM CHIR99021 (Wako), 80 ng / mL BMP4 (R & D Systems (RSD)), 80 ng / mL VEGF (RSD) after growing to a colony diameter of 750 μm And cultured for 2 days to obtain KDR-positive mesoderm progenitor cells.
中胚葉前駆細胞から血液血管前駆細胞への分化誘導
上記の方法で得られた中胚葉前駆細胞の培地を吸引除去し、2 μM SB431542 (STEMGENT), 80 ng/mL VEGF (RSD), 50 ng/mL bFGF (WAKO)および50 ng/mL SCF (RSD)を含むEssential 6 (Life technologies)培地（TGFβの除去）に交換し、2日培養し、ＣＤ３４陽性の
血液血管前駆細胞を得た。
Induction of differentiation from mesoderm progenitor cells to blood vascular progenitor cells Aspirate the medium of mesoderm progenitor cells obtained by the above method, 2 μM SB431542 (STEMGENT), 80 ng / mL VEGF (RSD), 50 ng / The medium was replaced with an Essential 6 (Life technologies) medium (removal of TGFβ) containing mL bFGF (WAKO) and 50 ng / mL SCF (RSD) and cultured for 2 days to obtain CD34-positive blood vessel progenitor cells.
血液血管前駆細胞の評価
上記の方法で得られた血液血管前駆細胞の培地を吸引除去し、20ng/mL VEGF, 50ng/mL SCF, 50ng/mL IL（Interleukin）3 (RSD), 50ng/mL FL（Flt-3 Ligand Protein）(RSD), 50ng/mL IL6 (RSD), 10U/mL EPO（Erythropoietin）(Merk)およびITSX（Insulin-transferrin-sodium selenite-X：Life technologies）を含むStemline II (SIGMA)培地に交換し、2日培養した。
Evaluation of blood vascular progenitor cells The blood vascular progenitor cell culture medium obtained by the above method is removed by suction, and 20 ng / mL VEGF, 50 ng / mL SCF, 50 ng / mL IL (Interleukin) 3 (RSD), 50 ng / mL FL Stemline II (SIGMA) including (Flt-3 Ligand Protein) (RSD), 50ng / mL IL6 (RSD), 10U / mL EPO (Erythropoietin) (Merk) and ITSX (Insulin-transferrin-sodium selenite-X: Life technologies) ) The medium was changed and cultured for 2 days.
さらに、培地を吸引除去し、50ng/mL SCF, 50ng/mL IL6 (RSD), 10U/mL EPO (Merk)お
よびITSXを含むStemline II (SIGMA)培地に交換し、4日培養した。培養２日目に同じ培地へ交換を行った。
Furthermore, the medium was removed by aspiration and replaced with Stemline II (SIGMA) medium containing 50 ng / mL SCF, 50 ng / mL IL6 (RSD), 10 U / mL EPO (Merk) and ITSX, and cultured for 4 days. On the second day of culture, the same medium was changed.
その結果、接着した細胞は、内皮細胞様に分化していることが確認された（図１）。さらに、得られた細胞を回収し、FACS解析を行ったところ、KDR+CD34+を示す内皮細胞への
分化（図２A）およびCD34+CD43+細胞、CD34+CD45+細胞またはCD43+CD45+細胞を示す造血
細胞への分化（図２B）が得られることが確認された。
As a result, it was confirmed that the adhered cells were differentiated into endothelial cells (FIG. 1). Furthermore, the obtained cells were collected and subjected to FACS analysis. As a result, differentiation into endothelial cells showing KDR + CD34 + (FIG. 2A) and CD34 + CD43 + cells, CD34 + CD45 + cells, or CD43 + CD45 + cells. It was confirmed that differentiation into hematopoietic cells (FIG. 2B) was obtained.
この結果から、上述の方法で多能性幹細胞から得られた細胞は、内皮細胞および造血細胞といった中胚葉系細胞への誘導能を有する中胚葉前駆細胞であることが確認され、さらに、当該中胚葉前駆細胞から得られた細胞は、内皮細胞および造血細胞への誘導能を有する血液血管前駆細胞であることが確認された。 From this result, it was confirmed that the cells obtained from the pluripotent stem cells by the above-described method are mesoderm progenitor cells having the ability to induce mesodermal cells such as endothelial cells and hematopoietic cells, and Cells obtained from germinal progenitor cells were confirmed to be blood vessel progenitor cells having the ability to induce endothelial cells and hematopoietic cells.
同様に、CB-iPSCを用いて上記の方法で中胚葉前駆細胞を経て血液血管前駆細胞集団を
作製した。この血液血管前駆細胞集団からCD34陽性細胞をMACS（磁気細胞分離装置）（ミルテニー社）を用いて単離し、Matrigel (BD Biosciences)上に播種し、上記と同様の方
法にて分化誘導行ったところ、接着した細胞は、内皮細胞様に分化していることが確認され（図３A）、浮遊した細胞をメイギムザ染色したところ、血球系細胞が存在しているこ
とが確認された（図３B）。
Similarly, a blood vessel progenitor cell population was prepared via mesoderm progenitor cells by the above method using CB-iPSC. CD34 positive cells were isolated from this blood vascular progenitor cell population using MACS (Magnetic Cell Separation Device) (Milteny), seeded on Matrigel (BD Biosciences), and differentiated by the same method as above. The adherent cells were confirmed to have differentiated into endothelial cells (FIG. 3A), and the floating cells were stained with Meigemza to confirm the presence of blood cells (FIG. 3B).
この結果から、上記の方法で分化誘導した４日目細胞中のCD34陽性分画が、血液血管前駆細胞であることが示された。 From this result, it was shown that the CD34 positive fraction in the day 4 cells induced to differentiate by the above method is blood vessel progenitor cells.
中胚葉前駆細胞への分化誘導において、LM511をLM511E8（iMatrix-511、ニッピ）に置
き換えたところ、同様の結果が得られた。
When LM511 was replaced with LM511E8 (iMatrix-511, Nippi) in the induction of differentiation into mesoderm progenitor cells, similar results were obtained.
本発明により、ＥＳ細胞やｉＰＳ細胞などの多能性幹細胞から、血液血管前駆細胞を作製することが可能になる。血液血管前駆細胞は、血液系細胞または血管内皮細胞を誘導することが可能であり、当該細胞は、血液製剤の製造または血管再生医療の分野で使用することができる。 According to the present invention, blood vessel progenitor cells can be prepared from pluripotent stem cells such as ES cells and iPS cells. Blood vascular progenitor cells can induce blood lineage cells or vascular endothelial cells, and the cells can be used in the field of blood product production or vascular regenerative medicine.
Claims (16)
多能性幹細胞を、ラミニン５１１またはその断片でコーティングされた培養器材上にて、ＢＭＰ、ＶＥＧＦおよびＧＳＫ３β阻害剤を含む培養液中で接着培養する工程を含む方法。 A method for producing mesoderm progenitor cells from pluripotent stem cells,
A method comprising the step of adhesion-culturing pluripotent stem cells in a culture medium containing BMP, VEGF, and a GSK3β inhibitor on a culture device coated with laminin 511 or a fragment thereof.
中胚葉前駆細胞をＶＥＧＦ、ＳＣＦ、ｂＦＧＦおよびＴＧＦβ阻害剤を含む培養液中で接着培養する工程を含む方法。 A method for producing blood vascular progenitor cells from mesoderm progenitor cells,
A method comprising the step of adhesion-culturing mesoderm progenitor cells in a culture medium containing VEGF, SCF, bFGF and a TGFβ inhibitor.
（１）多能性幹細胞を、ラミニン５１１またはその断片でコーティングされた培養器材上にて、ＢＭＰ、ＶＥＧＦおよびＧＳＫ３β阻害剤を含む培養液中で接着培養する工程、および
（２）前記工程（１）で得られた細胞をＶＥＧＦ、ＳＣＦ、ｂＦＧＦおよびＴＧＦβ阻害剤を含む培養液中で接着培養する工程。 A method for producing blood vascular progenitor cells from pluripotent stem cells comprising the following steps:
(1) Adhesive culture of pluripotent stem cells on a culture device coated with laminin 511 or a fragment thereof in a culture solution containing BMP, VEGF, and a GSK3β inhibitor, and (2) the step (1) The step of adhesion culture of the cells obtained in (1) in a culture medium containing VEGF, SCF, bFGF and a TGFβ inhibitor.
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| CN107937338A (en) * | 2017-11-30 | 2018-04-20 | 中山大学 | A kind of mesodermal lineage mescenchymal stem cell from multipotential stem cell and preparation method thereof |
| WO2020090903A1 (en) * | 2018-10-31 | 2020-05-07 | 国立大学法人京都大学 | Method for producing pluripotent stem cell having released differentiation resistance to mesendoderm |
| WO2020218579A1 (en) * | 2019-04-26 | 2020-10-29 | 国立大学法人京都大学 | Method for producing pluripotent stem cells conditioned for induction of differentiation |
| WO2021085462A1 (en) * | 2019-10-29 | 2021-05-06 | 国立大学法人京都大学 | Method for producing hemogenic endothelial cell and/or hematopoietic progenitor cell from pluripotent stem cell |
| CN113046318A (en) * | 2021-04-13 | 2021-06-29 | 中国科学院深圳先进技术研究院 | Culture medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells |
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| CN107937338A (en) * | 2017-11-30 | 2018-04-20 | 中山大学 | A kind of mesodermal lineage mescenchymal stem cell from multipotential stem cell and preparation method thereof |
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| US20220010283A1 (en) * | 2018-10-31 | 2022-01-13 | Kyoto University | Method for producing pluripotent stem cell having released differentiation resistance to mesendoderm |
| JP7437766B2 (en) | 2018-10-31 | 2024-02-26 | 国立大学法人京都大学 | Method for producing pluripotent stem cells that are free from mesendoderm differentiation resistance |
| WO2020218579A1 (en) * | 2019-04-26 | 2020-10-29 | 国立大学法人京都大学 | Method for producing pluripotent stem cells conditioned for induction of differentiation |
| WO2021085462A1 (en) * | 2019-10-29 | 2021-05-06 | 国立大学法人京都大学 | Method for producing hemogenic endothelial cell and/or hematopoietic progenitor cell from pluripotent stem cell |
| CN113046318A (en) * | 2021-04-13 | 2021-06-29 | 中国科学院深圳先进技术研究院 | Culture medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells |
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