JP7072756B2 - Method for inducing differentiation of pluripotent stem cells into mesoderm progenitor cells and blood vascular progenitor cells - Google Patents

Description

The present invention relates to a method for producing germ layer progenitor cells and blood vessel progenitor cells from pluripotent stem cells.

Pluripotent cells such as embryonic stem cells (ES cells) and artificial pluripotent stem cells (iPS cells) obtained by introducing undifferentiated cell-specific genes into somatic cells have been reported so far (patented patents). Document 1 or 2). Therefore, attention is being paid to regenerative medicine in which cells induced to differentiate from these pluripotent stem cells are transplanted and to create an in vitro pathological model.
In order to produce endothelial cells and blood cell lineage cells from embryonic stem cells, a multi-step process of producing mesoderm progenitor cells by simulating development and inducing differentiation into each cell has been attempted (Non-Patent Documents). 1, 2 or 3). In addition, since stable yields cannot be obtained by using heterologous cells or biological raw materials in this step, pluripotent stem cells, mesoderm progenitor cells, and further differentiation using only limited components. A method for inducing cells has been presented (Patent Document 3, Non-Patent Document 4).

USP 5,843,780 WO 2007/069666 WO 2011/115308

Lu SJ, et al, Nat Methods. 4: 501-509, 2007 Nishikawa SI, et al, Development. 125: 1747-1757, 1998 Sumi T, et al, Development. 135: 2969-2979, 2008 Niwa A, et al, PLoS One.6: e22261, 2011

An object of the present invention is to efficiently produce mesoderm progenitor cells and blood vascular progenitor cells from pluripotent stem cells. Therefore, 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.

In order to solve the above-mentioned problems, the present inventors use a culture vessel coated with laminin 511 in a step of inducing pluripotent stem cells to mesoderm progenitor cells and culture them containing BMP, GSK3β inhibitor and VEGF. By culturing in liquid, we succeeded in producing mesoderm progenitor cells with high differentiation potential in a short period of time. Furthermore, by adhering and culturing the obtained mesoderm progenitor cells in a culture medium containing VEGF, SCF, bFGF and TGFβ inhibitors, we succeeded in producing highly differentiateable blood vascular progenitor cells, and the present invention. Has been completed.

That is, the present invention is as follows.
[1] A method for producing mesoderm progenitor cells from pluripotent stem cells.
BMP (Bone Morphogenetic Protein), VEGF (Vascular Endothelial Gro) on pluripotent stem cells coated with laminin (LM) 511 or a fragment thereof.
A method comprising a step of adhesive culture in a culture medium containing a wth factor) and a GSK (Glycogen Synthase Kinase) 3β inhibitor.
[2] The method according to [1], wherein the fragment of laminin 511 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 two days or more.
[5] A method for producing blood vessel progenitor cells from mesoderm progenitor cells.
A method comprising adhering and culturing mesoderm progenitor cells in a culture medium containing VEGF, SCF (Stem Cell Factor), bFGF (basic Fibroblast Growth Factor) and TGF (Transforming Growth Factor) β inhibitor.
[6] The method according to [5], wherein the culture solution is a culture solution containing no 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 two days or more.
[10] A method for producing blood vascular precursor cells from pluripotent stem cells including the following steps, wherein (1) the pluripotent stem cells are coated with laminin 511 or a fragment thereof on a culture device. Adhesive culture in a culture medium containing BMP, VEGF and GSK3β inhibitors, and (2) Adhesion culture of cells obtained in step (1) above in a culture medium containing VEGF, SCF, bFGF and TGFβ inhibitors. Process to do.
[11] The method according to [10], wherein the fragment of laminin 511 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 containing no 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 two days or more.
[16] The method according to any one of [10] to [15], wherein the step (2) is performed for two days or more.

According to the present invention, mesoderm progenitor cells and blood vessel progenitor cells can be obtained.

FIG. 1 shows a microscopic image of blood vessel progenitor cells derived from ES cells on the 10th day of culture (photograph). FIG. 2 shows the results of FACS of the cell group on the 10th day of culture of blood vessel progenitor cells derived from ES cells. The horizontal axis of FIG. 2A shows the fluorescence intensity of the CD34 antibody, and the vertical axis shows the fluorescence intensity of the KDR antibody. The horizontal axis of FIG. 2B shows the fluorescence intensity of the CD34 antibody (left figure and the center figure) or the CD43 antibody (right figure), and the vertical axis shows the fluorescence intensity of the CD43 antibody (left figure) or the CD45 antibody (center figure and the right figure). Shows fluorescence intensity. FIG. 3 shows a microscopic image (A) and a Meigimza-stained image (B) of cells on the 10th day after dissociation of cells derived from iPS cells and isolation of CD34-positive cells (photograph). .. The right figure of FIG. 3B shows a partially enlarged view of the left figure.

The present invention will be described in detail below.

<Pluripotent stem cells>
The pluripotent stem cell that can be used in the present invention is a stem cell that has pluripotency capable of differentiating into all cells existing in a living body and also has proliferative ability, and is not particularly limited thereto. For example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells (“GS cells”), embryonic germ cells (“EG cells”), artificial pluripotency Includes sex stem (iPS) cells, cultured fibroblasts and pluripotent cells (Muse cells) derived from bone marrow stem cells. Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

(A) Embryonic stem cells
ES cells are pluripotent and self-replicating stem cells established from the inner cell mass of early embryos (eg, blastocysts) of mammals such as humans and mice.

ES cells are embryo-derived stem cells derived from the internal cell mass of the scutellum vesicle, which is the embryo after the mulberry embryo, at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called polymorphism. It has the ability and the 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 ES cell lines were subsequently established in primates such as humans and monkeys (JA Thomson et. 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 cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on the feeder of fibroblasts. For cell maintenance by subculture, use a culture medium 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 ES cells in humans and monkeys, 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, etc.

As the culture medium for ES cell production, 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 was used. , 37 ° C, 2% CO ₂ / 98% Human ES cells can be maintained in a moist atmosphere of air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). Also, ES cells need to be passaged every 3-4 days, where passage is 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing, for example, 1 mM CaCl ₂ and 20% KSR. Can be done using.

Selection of ES cells can generally be performed by the Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, and Nanog as an index. In particular, the selection of human ES cells can be indexed by the expression of genetic markers such as OCT-3 / 4, NANOG, ECAD (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).

Human ES cell lines, for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is possible.

(B) Sperm stem cells Sperm stem cells are pluripotent stem cells derived from the testis and are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various lineages of cells, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It is self-renewable in a culture medium containing a glial cell line-derived neurotrophic factor (GDNF), and sperm by repeating passage under the same culture conditions as ES cells. Stem cells can be obtained (Masanori Takebayashi et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

(C) Embryonic germ cells Embryonic germ cells are cells with pluripotency similar to ES cells, which are established from primordial germ cells in the embryonic period, such as LIF, bFGF, and stem cell factor. It can be established by culturing primordial germ cells in the presence of the substance of (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550. -551).

(D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are similar to ES cells, which can be produced by introducing certain reprogramming factors into somatic cells in the form of DNA or proteins. Artificial stem cells derived from somatic cells with equivalent properties, such as pluripotency and self-renewal proliferation (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.
Et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666). The reprogramming factor may be a gene specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiated ES cells, or a gene product thereof, and is not particularly limited, but is, for example, 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 / It is a combination of 4 and KLF4.

These factors may be introduced into somatic cells in the form of proteins, eg, by techniques such as lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA, eg, viruses, plasmids. , Vectors such as artificial chromosomes, lipofection, liposomes, microinjection and other techniques can be introduced into somatic cells. As virus vectors, retrovirus vector, lentiviral vector (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector and the like. Further, the artificial chromosome vector includes, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the nuclear reprogramming substance can be expressed, and further, if necessary, a drug resistance gene (drug resistance gene). For example, canamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, selection marker sequence such as diphtheriatoxin gene, reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. Can include. In addition, in the above vector, after introduction into somatic cells, LoxP sequences are added before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to the promoter. You may have.

In addition to the above factors, for example, histone deacetylase (HDAC) inhibitors [eg, valproic acid (VPA) (Nat. Biotechnol., 26 (7): 795), to increase induction efficiency during reprogramming. -797 (2008)), small molecule inhibitors such as tricostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDACs (eg, HDAC1 siRNA Smartpool®, HuSH 29mer shRNA Constructs against HDAC1 (eg, HDAC1 siRNA 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 Agents [eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA against G9a and shRNA (eg, G9a siRNA (human) (Santa Cruz Biotechnology), etc.), 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 for 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, miRNAs such as PloS Biology, 6 (10), 2237-2247 (2008)), miR-291-3p, miR-294, miR-295 ( RL Judson
et al., Nat. Biotech., 27: 459-461) (2009) etc. can be used.

Culture media for iPS cell induction include, for example, (1) DMEM, DMEM / F12 or DME medium containing 10 to 15% FBS (in addition to these media, LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, β-mercaptoethanol and the like can be appropriately contained.), (2) ES cell culture medium containing bFGF or SCF, for example, mouse ES cell culture medium (eg, TX-WES medium, thrombo). X) or primate ES cell culture medium (eg, primate (human & monkey) ES cell medium, reprocell, Kyoto, Japan), etc. are included.

Examples of culture methods include contacting somatic cells with reprogramming factors (DNA or protein) on DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO ₂ at 37 ° C. for approximately 4 After culturing for ~ 7 days, the cells are re-sown on feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.) and bFGF-containing primate ES cells approximately 10 days after contact between somatic cells and reprogramming factors. It can be cultured in culture medium to give rise to iPS-like colonies about 30-about 45 days or more after contact.

Alternatively, as an alternative culture method, DMEM medium containing 10% FBS (for example, LIF) 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. can be appropriately contained.)
ES-like colonies can be formed after about 30 days or more. Desirably, instead of feeder cells, the reprogrammed somatic cells 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.

In addition to this, a method of culturing using a medium containing no serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci US A. 106: 15720-15725). Furthermore, in order to increase the 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).

During the above culture, the fresh culture solution and the culture solution are exchanged once a day from the second day after the start of the culture. 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 culture dish.

iPS cells can be selected according to the shape of the formed colonies. On the other hand, when a drug resistance gene expressed in conjunction with a gene expressed when somatic cells are reprogrammed (for example, Oct3 / 4, Nanog) is introduced as a marker gene, a culture medium containing the corresponding drug (selection). Established iPS cells can be selected by culturing in a culture medium). In addition, iPS cells are selected by observing with a fluorescence microscope when the marker gene is a fluorescent protein gene, by adding a luminescent substrate when it is a luciferase gene, and by adding a chromogenic substrate when it is a luciferase gene. can do.

As used herein, the term "somatic cell" refers to any animal cell (preferably a mammalian cell, including human) except germline cells such as eggs, egg mother cells, ES cells or totipotent cells. say. Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passed cells, and established cells are all included. Specifically, somatic cells are, for example, (1).
Tissue stem cells (somatic stem cells) such as nerve 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, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, renal cells, fat cells, and other differentiated cells are exemplified. ..

When iPS cells are used as a material for transplant cells, it is desirable to use somatic cells having the same or substantially the same HLA genotype of the transplanted individual from the viewpoint of not causing rejection. Here, "substantially the same" means that the transplanted cells have the same HLA genotype to the extent that the immune response can be suppressed by the immunosuppressant, and for example, HLA-A and HLA-B. It is a somatic cell having an HLA type in which 3 loci of HLA-DR or 4 loci with HLA-C are matched.

(E) ES cells derived from cloned embryos obtained by nuclear transplantation
nt ES cells are ES cells derived from cloned embryos produced by nuclear transplantation technology and have almost the same characteristics as ES cells derived from fertilized eggs (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, ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nuclei of unfertilized eggs with the nuclei of somatic cells are nt ES (nuclear transfer ES) cells. For the production of nt ES cells, a combination of nuclear transplantation 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 (Special Edition), pp. 47-52). In nuclear transplantation, somatic cell nuclei can be injected into unenucleated unfertilized eggs of mammals and cultured for several hours to initialize them.

<Mesoderm progenitor cells>
In the present invention, the mesoderm is a body cavity and the mesothelium, muscle, skeleton, skin dermal, connective tissue, heart / blood vessel (including vascular epithelium), blood (including blood cells), and blood (including blood cells) that support the mesoderm during development. It includes the germ layer composed of cells capable of forming lymphatic vessels, spleen, kidney and urinary tract, and gonads (testis, uterus, gonad epithelium). In the present invention, the mesoderm progenitor cell is a cell expressing at least one marker gene selected from, for example, a marker gene consisting of T (synonymous with Brachyury), KDR, FOXF1, FLK1, BMP4, MOX1 and SDF1. The mesoderm progenitor cells are not distinguished from the mesoderm progenitor cells, and those having weak expression of the above marker gene may be referred to as mesoderm progenitor cells. The mesoderm 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.

<Blood vessel progenitor cells>
In the present invention, a blood vessel precursor cell is a cell composed of cells having an ability to be induced into a blood lineage cell or a vascular endothelial cell, and is also referred to as a hemangioblast or hemangioblast. In the present invention, blood vessel progenitor cells are exemplified as cells expressing CD34 and KDR. The blood vessel 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.

<Manufacturing process of mesoderm progenitor cells>
The present invention comprises pluripotent stem cells being adherently cultured in a culture medium containing BMP, GSK3β inhibitor and VEGF on a culture device coated with laminin 511 or a fragment thereof. A method for producing germ layer progenitor cells is provided.

In the present invention, adhesion culture means culturing in a scaffold-dependent manner using a culture vessel having a surface treatment suitable for cell adhesion or a culture vessel coated with an extracellular matrix.

In the present invention, laminin 511 (laminin composed of α5 chain, β1 chain, and 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, is a heterotrimer composed of three subunit chains of α chain, β chain, and γ chain, and is a huge sugar having a molecular weight of 800,000 Da. It is a protein. A heterotrimeric molecule in which three subunit chains are associated on the C-terminal side to form a coiled coil structure and stabilized by a disulfide bond. Therefore, laminin 511 means laminin in which the α chain is α5, the β chain is β1, and the γ chain is γ1. Further, laminin may be a mutant, and is not particularly limited as long as it is a mutant having integrin-binding activity. Laminin is preferably of human origin.

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) may be used. Laminin 51
1E8 is available, for example, from Nippi.

The coating treatment can be performed by putting a solution containing laminin 511 or a fragment thereof in a culture vessel and then appropriately removing the solution.

The BMP used in the process for producing mesoderm progenitor cells is at least one BMP selected from the group consisting of BMP2, BMP4 and BMP7, and is preferably BMP4. BMP is preferably of human origin.

In the present invention, when BMP4 is used in the process of producing mesoderm progenitor cells, the concentration of BMP4 in the culture medium is not particularly limited, but is 5 ng / ml to 200 ng / ml and 10 ng / ml to 100 ng / ml. , 20 ng / ml to 80 ng / ml are exemplified. Preferably, it is 80 ng / ml.

In the present invention, the GSK3β inhibitor refers to the kinase activity of the GSK-3β protein (eg, β).
It is defined as a substance that inhibits the ability to phosphorylate catenin), and many are already known. For example, BIO (also known as GSK-3β inhibitor IX; 6-bromoinzylvin 3'- Oxime), maleimide derivative SB216763 (3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indole-3-yl) -1H-pyrrole-2,5-dione), phenylαbromomethyl GSK-3β inhibitor VII (4-dibromoacetophenone), which is a ketone compound, and L803-mts (also known as GSK-3β peptide inhibitor), which is a cell membrane-penetrating phosphorylated peptide; Myr-N-GKEAPPAPPQpSP-NH ₂ (SEQ ID NO: 1)) and CHIR99021 with high selectivity (6- [2- [4- (2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino] ethylamino] pyridine-3-carbonitrile). These compounds are commercially available from, for example, Calbiochem, Biomol, etc. and can be easily used, but they may be obtained from other sources or may be prepared by themselves. The GSK-3β inhibitor used in the present invention is preferably CHI.
It can be R99021.

The concentrations of CHIR99021 in the culture medium of the present invention are, for example, 1nM, 10nM, 50nM, 100nM, 500nM, 750nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM.
, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM or concentrations between them, but not limited to these. It is preferably 2 μM.

The concentration of VEGF in the culture medium in the process of producing 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 of human origin.

The culture medium used in the process for producing mesoderm progenitor cells can be prepared by using the medium used for culturing animal cells as the basal medium and appropriately adding BMP, GSK3β inhibitor and VEGF. The basal medium includes, for example, Glassgow'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), Stempr
Includes o34SFM medium (Life Technologies) and mixed media thereof. The basal medium may be, for example, albumin, transferrin, Knockout Serum, as required.
Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol It 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. , Buffers, inorganic salts, etc. may also contain one or more substances. The preferred basal medium is mTesR1 medium or Essential 8.

Regarding the culture conditions in the process of producing mesoderm _progenitor cells, the culture temperature is not particularly limited, but is about 30 to 40 ° _C , preferably about 37 ° C. The concentration is preferably about 2-5%. The culture period is 2 days or more, preferably 2 to 3 days.

<Manufacturing process of blood vessel progenitor cells>
The present invention provides a method for producing blood vascular progenitor cells from mesoderm progenitor cells, which comprises the step of adhering and culturing mesoderm progenitor cells in a culture medium containing VEGF, SCF, bFGF and TGFβ inhibitors.

In the process of producing blood vessel precursor cells, the coating agent of the culture vessel used for adhesive culture is not particularly limited, and laminin or a fragment thereof, Matrixel, collagen, and fibronectin are exemplified, but the above-mentioned mesoderm precursor cells are simply exemplified. Laminin 511 or a fragment thereof can be used as in the manufacturing process of. When the step for producing blood vessel progenitor cells is carried out following the above-mentioned step for producing mesoderm progenitor cells, it can be carried out by appropriately exchanging the culture medium.

The concentration of VEGF in the culture medium in the process of producing blood vessel 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.

The concentration of SCF in the culture medium in the process of producing blood vessel 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 50 ng / ml. The SCF is preferably of human origin.

The concentration of bFGF in the culture medium in the process of producing blood vessel 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 50 ng / ml. The bFGF is preferably derived from humans.

In the present invention, the TGFβ inhibitor is defined as a substance that inhibits the binding of TGFβ to its receptor, or a substance that inhibits the downstream signal after TGFβ binds to the receptor, and the downstream signal is TGFβ type II. Phosphorylation of TGFβI type receptor by the receptor, phosphorylation of Smad (R-Smad) by this TGFβI type receptor, etc. are exemplified. Many TGFβ inhibitors are already known, for example, SB431542, SB202190 (RK 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). These compounds are commercially available, for example, from STEM GENT and the like and can be easily used, but they may be obtained from other sources or may be prepared by themselves. The TGFβ inhibitor used in the present invention may preferably be SB431542.

The concentrations of SB431542 in the culture medium of the blood vessel progenitor cell manufacturing process are, for example, 1nM, 10nM, 50nM, 100nM, 500nM, 750nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9
Concentrations such as, but not limited to, μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, or between them. It is preferably 2 μM.

The culture medium used in the process for producing blood vascular progenitor cells can be prepared by using the medium used for culturing animal cells as the basal medium and appropriately adding VEGF, SCF, bFGF and TGFβ inhibitors. The basal medium includes, for example, Glassgow'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
TesR1 medium (Life Technologies), Essential 8 (Life Technologies), Essential 6 (Life Technologies), Stepro34SFM medium (Life Technologies) and a mixed medium thereof are included. The basal medium may include, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, as needed. , Collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. may contain one or more serum substitutes, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, It may also contain one or more substances such as vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts. A preferred basal medium is a basal medium that does not contain TGFβ, and examples of such a medium include Essential 6.

Regarding the culture conditions in the process of producing blood vessel precursor cells, the culture temperature is not particularly limited, but is about 30 to 40 ° _C , preferably about 37 ° _C. The concentration is preferably about 2-5%. The culture period is 2 days or more, preferably 2 to 3 days.

<Kit>
In another embodiment of the invention, a kit for producing mesoderm progenitor cells from pluripotent stem cells and / or a kit for producing blood vascular progenitor cells from mesoderm progenitor cells is included. The kit contains a culture medium, an additive, a culture vessel, or the like used in each step of producing the above-mentioned mesoderm progenitor cells. The kit may further include a document or instruction manual describing the manufacturing process procedure.

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited thereto.

Cells and Cultured Human ES cells (KhES1) were received from the Institute for Frontier Medical Sciences, 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 four factors into cord blood mononuclear cells by an episomal vector (Okita K, et al, Nat Methods. 8, 409-412, 2011) (referred to as CB-iPSC).

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 subculture is a CTK solution (
The cells were dissociated by treatment with 0.25% trypsin (Life technologies), 0.1% collagenase IV (Life technologies), 20% KSR, and 1 mM CaCl ₂ ) at room temperature for about 30 seconds, and the existing method (Suemori, Suemori, SNL cells were removed by H. et al. Biochemical and Biophysical Research Communications 345, 926932 (2006)).

Induction of differentiation of pluripotent stem cells into mesoderm progenitor cells Human ES cells were seeded on a plate coated with LM511 (Biolamina) at a density of 5 colonies / cm ² and cultured with 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 growth to colony diameter of 750 μm. And cultured for 2 days to obtain KDR-positive mesoderm progenitor cells.

Induction of differentiation of mesodermal progenitor cells into blood vascular progenitor cells The medium of mesodermal progenitor cells obtained by the above method was aspirated and removed, and 2 μM SB431542 (STEMGENT), 80 ng / mL VEGF (RSD), 50 ng / The cells were replaced with 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 vascular progenitor cells.

Evaluation of blood vascular progenitor cells The medium of blood vascular progenitor cells obtained by the above method was 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 cells were replaced with a medium and cultured for 2 days.

Further, the medium was removed by suction, 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. The same medium was replaced on the second day of culture.

As a result, it was confirmed that the adhered cells differentiated like endothelial cells (Fig. 1). Furthermore, when the obtained cells were collected and FACS analysis was performed, 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 showing the above (Fig. 2B) was obtained.

From this result, it was confirmed that the cells obtained from the pluripotent stem cells by the above-mentioned method are mesodermal progenitor cells having an ability to induce mesenchymal cells such as endothelial cells and hematopoietic cells, and further, among them. It was confirmed that the cells obtained from the embryonic progenitor cells were blood vascular progenitor cells having the ability to induce endothelial cells and hematopoietic cells.

Similarly, using CB-iPSC, a blood vascular progenitor cell population was prepared via mesoderm progenitor cells by the above method. CD34-positive cells were isolated from this blood cell progenitor cell population using MACS (Magnetic Cell Separator) (Milteny), seeded on Matrigel (BD Biosciences), and differentiated by the same method as above. It was confirmed that the adhered cells were differentiated like endothelial cells (Fig. 3A), and when the floating cells were stained with Meigimza, it was confirmed that blood cell lineage cells were present (Fig. 3B).

From this result, it was shown that the CD34-positive fraction in the day 4 cells induced to differentiate by the above method was blood vessel progenitor cells.

Similar results were obtained when LM511 was replaced with LM511E8 (iMatrix-511, Nippi) in the induction of differentiation into mesoderm progenitor cells.

INDUSTRIAL APPLICABILITY According to the present invention, blood vessel progenitor cells can be produced from pluripotent stem cells such as ES cells and iPS cells. Blood vascular precursor cells can induce blood line cells or vascular endothelial cells, which can be used in the production of blood preparations or in the field of revascularization medicine.

Claims (4)

A method for producing blood vessel progenitor cells from mesoderm progenitor cells.
Mesoderm progenitor cells on culture equipment coated with laminin 511 or laminin 511 E8
A method comprising a step of adhesive culture in a culture medium containing VEGF, SCF, bFGF and TGFβ inhibitor. The method according to claim 1, wherein the culture solution is a culture solution containing no TGFβ. The method of claim 1 or 2, wherein the TGFβ inhibitor is SB431542. The method according to any one of claims 1 to 3, wherein the step is carried out for 2 days or more.

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