JP7654259B2 - Method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells from pluripotent stem cells - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) CiRA Retreat 2018, October 29, 2018 (2) Website of the proceedings of the 60th ASH Annual Meeting & Exposition, November 1, 2018 (3) 60th ASH Annual Meeting & Exposition, December 3, 2018 (4) The 81st Annual Meeting of the Japanese Society of Hematology, Abstract Download Site (http://www.jshem.or.jp/81/download.html), September 19, 2019 (5) The 81st Annual Meeting of the Japanese Society of Hematology, October 11, 2019

The present invention relates to a method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells from pluripotent stem cells.

For the treatment of blood-related diseases such as leukemia, it is extremely important to stably expand and supply the necessary amount of blood cells. For this reason, many researchers have attempted to efficiently expand hematopoietic stem cells or hematopoietic progenitor cells. In recent years, methods have been investigated in which hematopoietic stem cells or hematopoietic progenitor cells are induced from pluripotent stem cells (PSCs) such as iPS cells and ES cells, and then used.

The present inventors have developed a method (commonly referred to as the "PSC-Sac method") for obtaining sac-like structures containing a large number of hematopoietic progenitor cells (HPCs) by culturing pluripotent stem cells on feeder cells using a differentiation medium supplemented with vascular endothelial growth factor (VEGF) (Patent Document 1, Non-Patent Document 1). Compared with other hematopoietic progenitor cell differentiation induction methods, this method is superior in that it uses only VEGF as a cytokine and is easy to operate, but there is room for improvement in that the number of hematopoietic progenitor cells obtained is small, and a large number of culture dishes and a corresponding amount of medium are required to ensure the number of cells required for various assays. The present inventors have also found that most of the hematopoietic progenitor cells obtained by the PSC-Sac method have an embryonic phenotype. When considering application to regenerative medicine, hematopoietic progenitor cells with a phenotype closer to that of an adult are desirable.

WO2009/122747

Takayama, et al. Blood. 111(11): 5298-5306, 2008

The present invention aims to provide a method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells (improved PSC-Sac method) in which the number of hemogenic endothelial cells and/or hematopoietic progenitor cells per culture dish is significantly greater than that produced by the conventional PSC-Sac method. The present invention also aims to provide a method for producing hemogenic endothelial cells, hematopoietic progenitor cells, and blood cells that have a phenotype closer to that of an adult.

In order to solve the above problems, the present invention includes the following inventions.
[1] A method for producing hematopoietic progenitor cells by culturing pluripotent stem cells in a hematopoietic progenitor cell differentiation medium containing vascular endothelial growth factor, characterized in that a basic fibroblast growth factor and a transforming growth factor β signaling inhibitor are added to the hematopoietic progenitor cell differentiation medium, or an apelin receptor agonist is added to the hematopoietic progenitor cell differentiation medium.
[2] The method for production described in [1] above, further comprising adding heparin to the hematopoietic progenitor cell differentiation medium to which a basic fibroblast growth factor and a transforming growth factor β signaling inhibitor have been added.
[3] The method for producing the present invention described in [2], further comprising adding an apelin receptor agonist.
[4] The manufacturing method described in [1] above, characterized in that at least one selected from the group consisting of basic fibroblast growth factor, transforming growth factor β signaling inhibitor, and heparin is further added to the hematopoietic progenitor cell differentiation medium containing an apelin receptor agonist.
[5] The manufacturing method described in [4], characterized in that basic fibroblast growth factor, transforming growth factor β signaling inhibitor, heparin and an apelin receptor agonist are added to the hematopoietic progenitor cell differentiation medium.
[6] The method according to any one of [1] to [5] above, wherein the pluripotent stem cells are cultured on feeder cells.
[7] The method for producing a hematopoietic progenitor cell according to any one of [1] to [3] and [5], wherein a hematopoietic progenitor cell differentiation medium containing a basic fibroblast growth factor and a transforming growth factor β signaling inhibitor is used during the middle stage of the culture period.
[8] The method for production according to any one of [2], [3], or [5] above, wherein a hematopoietic progenitor cell differentiation medium containing heparin is used during the middle stage of the culture period, or during the middle stage and thereafter.
[9] The production method according to any one of [1] to [5], wherein a hematopoietic progenitor cell differentiation medium containing an apelin receptor agonist is used during the initial and subsequent stages of the culture period, or during the middle stage, or during the middle and subsequent stages of the culture period.
[10] The method according to any one of [1] to [9] above, wherein the obtained hematopoietic progenitor cells include hematopoietic progenitor cells having a fetal and/or adult phenotype.
[11] A method for producing blood cells, comprising culturing hemopoietic endothelial cells and/or hematopoietic progenitor cells produced by the production method according to any one of [1] to [10] above under conditions suitable for inducing differentiation of blood cells.
[12] The method of producing blood cells described in [11] above, wherein the obtained blood cells include blood cells having a fetal and/or adult phenotype.

The present invention can provide a method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells in which the number of hemogenic endothelial cells and/or hematopoietic progenitor cells per culture dish is significantly larger than that of the conventional PSC-Sac method. Therefore, while the conventional PSC-Sac method requires the use of a large number of culture dishes and the media required therefor to obtain hemogenic endothelial cells and/or hematopoietic progenitor cells, the method of the present invention can obtain them using a small number of culture dishes and the media required therefor, thereby significantly reducing the cost and workload.
Furthermore, the present invention can provide a method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells, including hemogenic endothelial cells and/or hematopoietic progenitor cells having fetal and/or adult phenotypes. Therefore, blood cells obtained by inducing differentiation from the hemogenic endothelial cells and/or hematopoietic progenitor cells obtained by the production method of the present invention also have fetal and/or adult phenotypes, making it possible to provide blood cells that are more suitable for human regenerative medicine.

FIG. 1 shows the yield of hemogenic endothelial cells from cells harvested on day 9 in the conventional PSC-Sac method, improved PSC-Sac method-1, and improved PSC-Sac method-2. FIG. 1 shows the yield of hemogenic endothelial cells from cells harvested on day 9 in the conventional PSC-Sac method, improved PSC-Sac method-1, and improved PSC-Sac method-2. FIG. 1 shows the expression levels of hematopoiesis-related genes in cells recovered on day 9 using the conventional PSC-Sac method, the improved PSC-Sac method-1, and the improved PSC-Sac method-2. (A) is the result for GFI1b, (B) is the result for ERG, (C) is the result for SPI1, (D) is the result for Runx1c, and (E) is the result for TAL1. FIG. 1 shows the expression levels of hematopoiesis-related genes in cells recovered on day 9 using the conventional PSC-Sac method and the improved PSC-Sac method-1. (A) is the result for GFI1b, (B) is the result for ERG, (C) is the result for SPI1, (D) is the result for Runx1c, and (E) is the result for TAL1. FIG. 1 shows the yield of hematopoietic progenitor cells from cells collected on day 14 in the conventional PSC-Sac method, improved PSC-Sac method-1, and improved PSC-Sac method-2. FIG. 1 shows the yield of hematopoietic progenitor cells from cells collected on day 14 in the conventional PSC-Sac method and the improved PSC-Sac method-1. FIG. 1 shows the yield of hematopoietic progenitor cells from cells collected on day 14 in the conventional PSC-Sac method, improved PSC-Sac method-1, and improved PSC-Sac method-2. FIG. 1 shows the results of FACS analysis of CD34+CD38+ cells in hematopoietic progenitor cells from cells collected on day 14 in the conventional PSC-Sac method and the improved PSC-Sac method-1. FIG. 1 shows the results of measuring the yield of hematopoietic progenitor cells when the components added to the medium in the improved PSC-Sac method-1 were added alone or in any combination. FIG. 1 shows the results of measuring the number of CD235-positive cells in hematopoietic progenitor cells obtained by the conventional PSC-Sac method and hematopoietic progenitor cells obtained by the improved PSC-Sac method-1, which were cultured in erythroid differentiation medium, on day 21. FIG. 1 shows the results of comparing the numbers of embryonic red blood cells (ε+γ-β-) in red blood cells obtained by the conventional PSC-Sac method and those obtained by the improved PSC-Sac method-1. This figure compares the number of fetal-type red blood cells in red blood cells obtained by the conventional PSC-Sac method and the improved PSC-Sac method-1. The left figure shows the results for early fetal-type red blood cells (ε+γ+β+), and the right figure shows the results for late fetal-type red blood cells (ε-γ+β+). FIG. 1 shows the results of confirming the presence of adult-type red blood cells (ε-γ-β+) in the red blood cells obtained by the improved PSC-Sac method-1. Hematopoietic progenitor cells obtained by the conventional PSC-Sac method and hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 were each cultured in megakaryocyte/platelet differentiation medium, and the numbers of megakaryocytes and platelets in the cells on day 10 were measured. (A) shows the result for the megakaryocyte count, and (B) shows the result for the platelet count. FIG. 1 shows a comparison of the results of colony assays of hematopoietic progenitor cells obtained by the conventional PSC-Sac method and those obtained by the improved PSC-Sac method-1. FIG. 1 shows the results of inducing differentiation of hematopoietic progenitor cells obtained by the conventional PSC-Sac method and hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 in a lymphocyte differentiation induction system, and measuring the number of CD4+CD8+ T cells. FIG. 1 shows the results of measuring the percentage of CD45-positive cells in hematopoietic progenitor cells obtained by the conventional PSC-Sac method and in hematopoietic progenitor cells obtained by improved PSC-Sac method-1. FIG. 1 shows the results of FACS analysis of CD34+CD43+ cells in hematopoietic progenitor cells collected on day 14 in the conventional PSC-Sac method and the improved PSC-Sac method-1 under feeder-free conditions.

The present invention provides a method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells. The method for producing hemogenic endothelial cells and/or hematopoietic progenitor cells of the present invention is a method for producing hematopoietic progenitor cells by culturing pluripotent stem cells in a hematopoietic progenitor cell differentiation medium containing vascular endothelial growth factor (hereinafter referred to as "VEGF"), characterized in that basic fibroblast growth factor (hereinafter referred to as "bFGF") and a transforming growth factor-β (hereinafter referred to as "TGF-β") signal inhibitor are further added to the hematopoietic progenitor cell differentiation medium containing VEGF, or an apelin receptor agonist is further added to the hematopoietic progenitor cell differentiation medium containing VEGF (hereinafter referred to as "the production method of the present invention").

The manufacturing method of the present invention may use a hematopoietic progenitor cell differentiation medium containing VEGF, bFGF and a TGF-β signal inhibitor, or may use a hematopoietic progenitor cell differentiation medium containing VEGF, bFGF, a TGF-β signal inhibitor and heparin, or may use a hematopoietic progenitor cell differentiation medium containing VEGF, bFGF, a TGF-β signal inhibitor, heparin and an apelin receptor agonist.

In addition, the production method of the present invention may use a hematopoietic progenitor cell differentiation medium containing VEGF and an apelin receptor agonist, or may use a hematopoietic progenitor cell differentiation medium further containing one or two selected from the group consisting of bFGF, a TGF-β signal inhibitor, and heparin. In one embodiment, the hematopoietic progenitor cell differentiation medium contains VEGF, bFGF, a TGF-β signal inhibitor, heparin, and an apelin receptor agonist.

Hemogenic endothelium (HE) produced by the production method of the present invention is a vascular endothelial cell that has the ability to differentiate into blood cells, and is defined in the present specification as [CD34+ CD31+ CD73- CD144+ CD117+ KDR+ CD41- CD43- CD45-]. In the present specification, + means positive and - means negative for the markers.

Hematopoietic progenitor cells (HPCs) produced by the manufacturing method of the present invention are cells at a precursor stage to final differentiation into blood cells, and are defined in this specification as [CD34+ CD43+].

Pluripotent stem cells that can be used in the production method of the present invention are stem cells that have pluripotency, that is, the ability to differentiate into all cells present in a living body, and also have the ability to proliferate, and include, but are not limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem (GS) cells, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, pluripotent cells derived from cultured fibroblasts and bone marrow stem cells (Muse cells), etc. Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

(A) Embryonic stem cells
ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (eg, blastocysts) such as humans and mice, and have pluripotency and the ability to proliferate through self-renewal.

ES cells are embryo-derived stem cells that originate from the inner cell mass of a blastocyst, which is an embryo at the eight-cell stage of a fertilized egg, or after the morula stage. They have the ability to differentiate into any cell that makes up an adult, known as pluripotency, and the ability to proliferate by self-renewal. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and subsequently ES cell lines were established in humans, monkeys, and other primates (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 can be established using methods known in the art. For example, ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. Cells can be maintained by subculture using a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. USA 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.

A culture method for producing ES cells may be a method known in the art. For example, DMEM/F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR (KnockOut Serum Replacement, Invitrogen) and 4 ng/ml bFGF may be used as the culture medium, and human ES cells may be maintained at 37°C in a humidified atmosphere of 2% CO2/98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). ES cells may be passaged every 3 to 4 days, and passage may be performed using, for example, 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR.

ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3/4, and Nanog as indicators. In particular, human ES cells can be selected using the expression of gene markers such as OCT-3/4, NANOG, and ECAD as indicators (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452).

As mouse ES cells, various mouse ES cell lines established by inGenious, Inc., RIKEN, etc. are available. As human ES cells, various human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis are available. For example, ES cell lines include NIH's CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc., WisCell Research Institute's WA01(H1) strain, WA09(H9) strain, and RIKEN's KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. In addition, KhES-1 strain, KhES-2 strain, KhES-3 strain, and KthES11 strain are available from the Institute for Virus Research and Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

(B) Spermatogonial stem cells Spermatogonial stem cells are pluripotent stem cells derived from the testis, and are the source of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various lineages of cells, and have the property that, for example, when transplanted into mouse blastocysts, chimeric mice can be produced (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012). They can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and spermatogonial stem cells can be obtained by repeated passage under the same culture conditions as ES cells (Takebayashi, M. et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Extra), pp. 41-46, Yodosha, Tokyo, Japan).

(C) Embryonic germ cells Embryonic germ cells are cells that are established from primordial germ cells during the fetal period and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (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 artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors in the form of DNA or protein into somatic cells and have almost the same properties as ES cells, such as pluripotency and the ability to proliferate through self-renewal (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 composed of a gene specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or non-coding RNA, or a low molecular weight compound. Examples of genes contained in the reprogramming factor include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, and these reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/101084, WO2009/101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, W O2009/157593, WO2010/009015, WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO 2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO 2010/111409, WO 2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

The above-mentioned reprogramming factors include histone deacetylase (HDAC) inhibitors [e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, etc., and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene))], MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., BIX-01294, small molecule inhibitors such as Suv39hl, Suv39h2, SetDBl and nucleic acid expression inhibitors such as siRNA and shRNA for G9a), L-channel calcium agonist (e.g. Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g. LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (e.g. siRNA and shRNA for p53), ARID3A inhibitors (e.g. siRNA and shRNA for ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Signaling (e.g. soluble These factors also include factors used for the purpose of improving establishment efficiency, such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, and DMRTBl, and in this specification, factors used for the purpose of improving these establishment efficiencies will not be distinguished from reprogramming factors.

When the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (e.g., HIV-derived TAT and polyarginine), or microinjection.

On the other hand, in the case of the form of DNA, it can be introduced into somatic cells by, for example, vectors such as viruses, plasmids, and artificial chromosomes, lipofection, liposomes, microinjection, and the like. Examples of viral vectors include retroviral vectors, lentiviral vectors (Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, and Sendai virus vectors (WO 2010/008054). Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As the plasmid, a plasmid for mammalian cells can be used (Science, 322:949-953, 2008). The vector can contain control sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc., so that the nuclear reprogramming substance can be expressed, and further, if necessary, can contain a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, etc., a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG, etc. In addition, the above-mentioned vector may have a LoxP sequence before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor bound thereto, in order to excise both of them after introduction into somatic cells.

In addition, when it is in the form of RNA, it may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

Examples of culture media for inducing iPS cells include DMEM, DMEM/F12, or DME culture media containing 10-15% FBS (these culture media can further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate), or commercially available culture media such as culture media for culturing mouse ES cells (TX-WES culture medium, Thrombo-X), culture media for culturing primate ES cells (culture medium for primate ES/iPS cells, ReproCell), and serum-free pluripotent stem cell maintenance medium (e.g., mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)).

As an example of a culture method, for example, somatic cells are contacted with reprogramming factors in DMEM or DMEM/F12 culture medium containing 10% FBS at 37°C in the presence of 5% CO2 and cultured for about 4 to 7 days, after which the cells are replated onto feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) and cultured in a bFGF-containing culture medium for primate ES cell culture from about 10 days after contact between the somatic cells and the reprogramming factors, and iPS-like colonies can be generated about 30 to 45 days or more after the contact.

Alternatively, ES-like colonies can be generated after about 25 to about 30 days or more by culturing on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM culture medium (which can further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) at 37°C and in the presence of 5% CO2. Desirably, somatic cells to be reprogrammed themselves (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010/137746) or extracellular matrix (e.g., Laminin-5 (WO2009/123349) and Matrigel (BD)) are used instead of feeder cells.

Other examples include a method of culturing using a serum-free medium (Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106:15720-15725). Furthermore, to increase the efficiency of establishment, iPS cells may be established under low oxygen 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 culture medium is replaced with fresh medium once a day from the second day of culture onward. The number of somatic cells used for nuclear reprogramming is not limited, but is in the range of about 5 x 10 ³ to about 5 x 10 ⁶ cells per 100 cm ² culture dish.

iPS cells can be selected based on the shape of the colonies they form. On the other hand, if a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are reprogrammed (e.g. Oct3/4, Nanog) is introduced as a marker gene, the established iPS cells can be selected by culturing them in a culture medium containing the corresponding drug (selection culture medium). In addition, if the marker gene is a fluorescent protein gene, iPS cells can be selected by observing them under a fluorescent microscope, if the marker gene is a luminescent enzyme gene, by adding a luminescent substrate, and if the marker gene is a chromogenic enzyme gene, by adding a chromogenic substrate.

The term "somatic cells" as used herein refers to any animal cell (e.g., mammalian cell, including human cell) except germline cells such as eggs, oocytes, and ES cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (baby) somatic cells, neonatal (baby) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

Furthermore, when iPS cells and/or cells differentiated therefrom are used as a source of transplant cells, it is desirable to use somatic cells with the same or substantially the same HLA genotype as the recipient individual, from the viewpoint of preventing rejection. Here, "substantially the same" HLA type means that the HLA genotype matches to an extent that the transplanted cells can survive when transplanted, for example, somatic cells with an HLA type that matches the main HLA (three loci: HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C).

As the artificial pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. may be used. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, Nips-B2 line, etc., and Kyoto University's AK5 line, TkDN-Sev2 line, 692D2 line, 253G1 line, 201B7 line, 409B2 line, 454E2 line, 606A1 line, 610B1 line, 648A1 line, 1231A3 line, 1390D4 line, and 1390C1 line. Alternatively, clinical grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines prepared using these cell lines, etc. may be used.

(E) ES cells derived from cloned embryos by nuclear transfer (ntES cells)
The ntES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have almost the same properties 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). In other words, ntES (nuclear transfer ES) cells are ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate ntES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and ES cell generation technology (mentioned above) is used (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (special issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal, and the egg can be initialized by culturing for several hours.

(F) Multilineage-differentiating Stress Enduring cells (Muse cells)
Muse cells are pluripotent stem cells produced by the method described in WO2011/007900. More specifically, Muse cells are pluripotent cells obtained by trypsinizing fibroblasts or bone marrow stromal cells for a long period of time, preferably 8 or 16 hours, followed by culture in suspension; these cells are positive for SSEA-3 and CD105.

The medium used in the production method of the present invention is a medium prepared by adding VEGF to a medium capable of inducing differentiation into hematopoietic progenitor cells. Such a medium can be prepared by adding factors necessary for differentiation induction to a basal medium used for culturing animal cells, and further adding VEGF. Examples of basal media include IMDM medium, Medium 199 medium, EMEM medium, αMEM medium, DMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures of these.

The basal medium may contain serum or may not contain serum. The basal medium may contain, as necessary, substances such as albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture, Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, growth factors, small molecule compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts.

The VEGF added to the medium may be human VEGF, VEGF of a non-human animal, or a functional variant thereof. In one embodiment, the VEGF added to the medium is human VEGF. VEGF may be natural or recombinant. For example, commercially available VEGF may be used. The concentration of VEGF may be, for example, 0.1 ng/mL to 1000 ng/mL, 1 ng/mL to 100 ng/mL, 5 ng/mL to 50 ng/mL, or 10 ng/mL to 30 ng/mL. In one embodiment, the concentration of VEGF is 20 ng/mL.

The bFGF added to the medium may be human bFGF, bFGF of a non-human animal, or a functional variant thereof. In one embodiment, the bFGF added to the medium is human bFGF. The bFGF may be natural or recombinant. For example, commercially available bFGF may be used. The concentration of bFGF may be, for example, 1 ng/mL to 1000 ng/mL, 5 ng/mL to 500 ng/mL, 10 ng/mL to 100 ng/mL, or 40 ng/mL to 60 ng/mL. In one embodiment, the concentration of bFGF is 50 ng/mL.

A TGF-β signal inhibitor is a substance that inhibits TGF-β signal transduction mediated by the binding of TGF-β to a TGF-β receptor. Examples of TGF-β signal inhibitors added to the medium include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, and LY580276. In one embodiment, the TGF-β signal inhibitor added to the medium is SB431542. When SB431542 is used as a TGF-β signal inhibitor, the concentration may be, for example, 0.1 μM to 100 μM, 1 μM to 50 μM, or 5 μM to 20 μM. In one embodiment, the concentration of SB431542 is 10 μM.

Commercially available heparin sodium can be used as the heparin added to the medium. The concentration of heparin may be, for example, 0.1 U/mL to 100 U/mL, 1 U/mL to 50 U/mL, or 5 U/mL to 20 U/mL. In one embodiment, the concentration of heparin is 10 U/mL.

Apelin receptor agonists are also called APJ receptor agonists. The apelin receptor agonist may be any substance that has agonist activity against the APJ receptor, and examples of such substances include peptides, polypeptides, apelin derivatives, and low molecular weight compounds that have agonist activity against the APJ receptor. In one embodiment, the apelin receptor agonist used is apelin. The apelin added to the medium may be human apelin, apelin of an animal other than human, or a functional variant thereof. In one embodiment, the apelin added to the medium is human apelin. Apelin may be natural or recombinant. For example, commercially available apelin 36 and apelin 13 may be used. When apelin 13 is used as the apelin receptor agonist, the concentration may be, for example, 1 ng/mL to 1000 ng/mL, 5 ng/mL to 500 ng/mL, 10 ng/mL to 100 ng/mL, or 40 ng/mL to 60 ng/mL. In one embodiment, the concentration of apelin 13 is 50 ng/mL.

In the PSC-Sac method previously developed by the present inventors (hereinafter referred to as the "conventional PSC-Sac method"), it is believed that when pluripotent stem cells are cultured in a hematopoietic progenitor cell differentiation medium containing VEGF, they differentiate into mesodermal cells within a few days, after which hemogenic endothelial cells appear and differentiate into hematopoietic progenitor cells.

In the production method of the present invention, bFGF and a TGF-β signal inhibitor may be used in the middle of the culture period. The middle of the culture period is preferably a period when mesodermal cells are present. Specifically, assuming that the day when the culture of pluripotent stem cells is started in a hematopoietic progenitor cell differentiation medium containing VEGF (hereinafter simply referred to as "hematopoietic progenitor cell differentiation medium") is set as day 0 (the same applies hereinafter), the hematopoietic progenitor cell differentiation medium containing bFGF and a TGF-β signal inhibitor may be used in any period within the range of days 3 to 8. The start of use of the hematopoietic progenitor cell differentiation medium containing bFGF and a TGF-β signal inhibitor may be the second, third, fourth, or fifth day. The start of use is, for example, the third or fourth day. In one embodiment, it is the fourth day. The end of use of the hematopoietic progenitor cell differentiation medium containing bFGF and a TGF-β signal inhibitor may be the eighth, seventh, sixth, or fifth day. The end of use is, for example, the seventh or sixth day. In one embodiment, it is the seventh day.

In the production method of the present invention, heparin may be used in the middle of the culture period, or in the middle or later period. The middle of the culture period is preferably a period when mesodermal cells are present. Specifically, the start of use of the heparin-containing hematopoietic progenitor cell differentiation medium may be on the second, third, fourth, or fifth day. The start of use is, for example, on the third or fourth day. In one embodiment, it is on the fourth day. When used in combination with bFGF and a TGF-β signal inhibitor, it may be started to be used simultaneously with the bFGF and the TGF-β signal inhibitor. The end of use of the hematopoietic progenitor cell differentiation medium containing heparin is not particularly limited, and it may be used until the end of the culture period, or it may be ended at any time. The end of use may be on the 11th, 10th, 9th, 8th, 7th, 6th, or 5th day. The end of use is, for example, on the 10th or 9th day. In one embodiment, it is on the 10th day.

In the production method of the present invention, the apelin receptor agonist may be used at the beginning or later of the culture period, in the middle period, or in the middle period or later. The middle period of the culture period is preferably a period when mesodermal cells are present. Specifically, the start of use of the hematopoietic progenitor cell differentiation medium containing the apelin receptor agonist may be day 0, day 1, day 2, day 3, day 4, or day 5. The start of use is, for example, day 3, day 4, or day 5. In one embodiment, it is day 4. The end of use of the hematopoietic progenitor cell differentiation medium containing the apelin receptor agonist is not particularly limited, and it may be used until the end of the culture period, or it may be ended at any time. The end of use may be day 11, day 10, day 9, day 8, day 7, day 6, or day 5. The end of use is, for example, day 7 or day 6. In one embodiment, it is day 7.

In one embodiment, in the manufacturing method of the present invention, cells are cultured in a hematopoietic progenitor cell differentiation medium supplemented with bFGF, a TGF-β signal inhibitor, and heparin when mesodermal cells are present. In another embodiment, cells are cultured in a hematopoietic progenitor cell differentiation medium supplemented with bFGF, a TGF-β signal inhibitor, heparin, and an apelin receptor agonist when mesodermal cells are present. The period when mesodermal cells are present is from the 3rd to the 8th day, and the start of use of the above medium may be the 2nd, 3rd, 4th, or 5th day. The start of use is, for example, the 3rd or 4th day. In one embodiment, it is the 4th day. The end of use may be the 8th, 7th, 6th, or 5th day. The end of use is, for example, the 7th or 6th day. In one embodiment, it is the 7th day. Note that heparin and an apelin receptor agonist may be contained in the medium thereafter.

The production method of the present invention may use a culture system that uses feeder cells, or may use a culture system that does not use feeder cells (feeder-free). In one embodiment, a culture system that uses feeder cells is used. There is no particular limitation on the feeder cells, as long as they are cells that can be used in a feeder cell culture system for pluripotent stem cells. In one embodiment, interstitial cells are used as the feeder cells. For example, mouse fetal fibroblasts can be used. Specific examples include C3H10T1/2 and OP-9. Cells whose proliferation has been inhibited by radiation treatment, mitomycin C treatment, etc. may be used as the feeder cells.

In the production method of the present invention, when starting the culture of pluripotent stem cells in a hematopoietic progenitor cell differentiation medium, the culture may be started in a hypoxic environment. The hypoxic environment may have an oxygen partial pressure lower than the atmospheric oxygen partial pressure (21%), and the oxygen partial pressure may be 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, or 10% or less. The lower limit of the oxygen partial pressure may be 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more. It is preferable that the culture in the hypoxic environment is ended at the same time as the end of the use of the hematopoietic progenitor cell differentiation medium containing bFGF and a TGF-β signal inhibitor. Specifically, it may be the 8th day, the 7th day, the 6th day, or the 5th day. In one embodiment, it is the 7th day or the 6th day. In another embodiment, it is the 7th day. After the end of the culture in the hypoxic environment, it is preferable to perform the culture at a higher oxygen partial pressure. For example, it may be 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more. In one embodiment, the culture is carried out at atmospheric oxygen partial pressure of 21%.

In the production method of the present invention, the end time of the culture period is not particularly limited as long as it is after the 8th day. The end time of the culture period may be before the 18th day, before the 17th day, before the 16th day, or before the 15th day. If the culture is terminated and the cells are collected on the 8th to 10th day, for example, on the 9th day, cells containing a large number of hemogenic endothelial cells can be collected. If the culture is terminated and the cells are collected on the 11th to 18th day, for example, on the 13th, 14th, 15th, or 16th day, cells containing a large number of hematopoietic progenitor cells can be collected.

The method for recovering the cells at the end of the culture is not particularly limited, and any method that can recover hemogenic endothelial cells and/or hematopoietic progenitor cells induced to differentiate from pluripotent stem cells may be used. For example, a method may be used in which the cells on the bottom of the dish, including the feeder cells, are peeled off using a cell scraper, the sac-like structures that are subsequently formed are collapsed, the cells within the sacs are dispersed, and the resulting cell suspension is passed through a cell strainer and recovered in an appropriate tube (see Examples).

Most of the red blood cells differentiated from hematopoietic progenitor cells obtained by the conventional PSC-Sac method had an embryonic phenotype, but it has been confirmed that the red blood cells differentiated from the hemogenic endothelial cells and hematopoietic progenitor cells obtained by the production method of the present invention have a fetal and/or adult phenotype. Blood cells induced to differentiate from hemogenic endothelial cells and/or hematopoietic progenitor cells having a fetal and/or adult phenotype have a phenotype closer to that of an adult, and are therefore considered to be suitable for application in regenerative medicine. The fact that the hemogenic endothelial cells and hematopoietic progenitor cells have a phenotype closer to that of an adult can be confirmed based on an analysis of the hemoglobin expression pattern after erythrocyte differentiation. It may also be determined by the presence of CD34+CD38+ cells in the obtained hematopoietic progenitor cells.

The present invention provides a method for producing blood cells, which comprises culturing hemogenic endothelial cells and/or hematopoietic progenitor cells produced by the above-mentioned production method of the present invention under conditions suitable for inducing differentiation of blood cells. Blood cells include red blood cells, granulocytes, monocytes/macrophages, lymphocytes, megakaryocytes/platelets.

"Conditions suitable for inducing differentiation of blood cells" may be, for example, conditions in which a combination of appropriate differentiation-inducing factors is added to the medium depending on the type of blood cells of interest. Examples of differentiation-inducing factors include TPO, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, EPO, GM-CSF, Flt3 ligand, and heparin. Conditions for inducing differentiation into red blood cells and into megakaryocytes and platelets include, for example, the conditions described in the Examples. In addition, appropriate conditions for inducing differentiation into blood cells may be selected and used from among known conditions for inducing differentiation into blood cells.

The obtained blood cells include blood cells having fetal and/or adult phenotypes. For example, it can be confirmed that red blood cells obtained from hemogenic endothelial cells and hematopoietic progenitor cells obtained by the production method of the present invention have fetal and/or adult phenotypes by expression analysis of ε-globin/γ-globin/β-globin (see Examples).

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

Example 1: Production of hematopoietic progenitor cells by the improved PSC-Sac method
1. Materials and Methods 1-1 Pluripotent stem cells (hereinafter referred to as "PSCs")
(1) Cell Lines Human iPS cell lines 692D2, AK5, and TkDN-Sev2, and human ES cell lines KhES-3 and H1 were used. 692D2 is a human iPS cell line established by the method described in Okita K, et al, Stem Cells 31(3): 458-466 (2013) and received from the Center for iPS Cell Research and Application, Kyoto University. AK5 is a human iPS cell line established by the method described in Nakagawa et al., Scientific Reports, 4: 3954, 2014 and received from the Center for iPS Cell Research and Application, Kyoto University. TkDN-Sev2 is a human iPS cell (DN-SeV2) described in Nakamura et al., Cell Stem Cell. 2014 Apr 3;14(4):535-48. KhES-3 was obtained from the Institute for Virus Research and Frontier Medical Sciences, Kyoto University. H1 was obtained from the WiCell Research Institute.

(2) Maintenance culture of PSCs
PSCs were maintained and cultured using mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in Dulbecco Modified Eagle's Medium/Nutrient Mixture F-12 Ham (Sigma-Aldrich) containing 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 20% KnockOut ^™ Serum Replacement (Gibco/Thermo Fisher), and 5 ng/mL bFGF (Wako).

1-2 PSC-Sac method (1) Feeder cells Mouse stromal cell line C3H10T1/2 was purchased from the Riken BioResource Research Center and used as feeder cells. After mitomycin C treatment, the cells were seeded on a 0.1% gelatin-coated dish (Φ10 cm) and cultured overnight at 37°C under 5% _O2 conditions.

(2) Hematopoietic progenitor cell differentiation medium
Iscove's modified Dulbecco's medium (IMDM, Sigma) containing 15% FBS (Sigma or Gibco), Pen Strep Glutamine (100X) (Gibco), ITS supplement (10 μg/mL insulin, 5.5 μg/mL transferrin, 6.7 ng/mL sodium selenite) (Thermo Fisher), 50 μg/mL ascorbic acid (Sigma), 0.45 mM monothioglycerol (Sigma), and 20 ng/mL recombinant human VEGF (Wako) was used as the hematopoietic progenitor cell differentiation medium. In the improved PSC-Sac method-1 and improved PSC-Sac method-2, bFGF (Wako), SB431542 (Wako), heparin (AY Pharmaceuticals) or apelin 13 (Cayman) were added to the hematopoietic progenitor cell differentiation medium, as shown in the table below.

(3) Conventional PSC-Sac method After removing the medium from PSCs during maintenance culture and washing with PBS, cell detachment solution [2.5% Trypsin (Gibco), 100 mM CaCl ₂ , KnockOut ^™ Serum Replacement (Gibco) prepared in PBS] was added and incubated at 37°C. The cell detachment solution was aspirated and hematopoietic progenitor cell differentiation medium was added, and PSC colonies were detached and crushed into clumps using a Pipetman P-1000 (Gilson). A certain amount of the resulting PSC clump suspension was evenly seeded on C3H10T1/2 feeder cells and culture was started under 37°C and 5% O ₂ conditions (Day 0). On Day 4 (Day 4), the medium was completely replaced and culture was continued under 37°C and 21% O 2 conditions. On Day 7 (Day 7), the medium was completely replaced and culture was continued under 37°C and 21% O ₂ conditions. On day 10 (Day 10), the medium was not removed, but fresh medium (approximately 70% of the volume) was added, and the culture was continued under conditions of 37°C and 21% _O2 .

On days 9 and 14, cells were harvested as follows: First, all cells on the bottom of the dish, including feeder cells, were detached using a cell scraper. Next, the medium was aspirated with a pipette, and the formed sac-like structure (Sac) was collapsed by pressing it against the bottom of the dish, dispersing the cells inside the Sac. The cells and medium were harvested, and the dish was washed twice with PBS to harvest the remaining cells. The cell suspension was passed through a 40 μm cell strainer, and the cells were collected in a 50 mL tube and centrifuged (400 G, 5 min, room temperature). The supernatant was removed, and the cells were suspended in PBS containing 3% FBS. A portion was taken for cell counting, and the remaining cell suspension was used for subsequent analysis. Cell counting was performed using a hemocytometer after trypan blue staining.

(4) Improved PSC-Sac method-1
A PSC clamp suspension was prepared in the same manner as in the conventional PSC-Sac method, seeded on C3H10T1/2 feeder cells, and culture was started under 37°C and 5% _O2 conditions (Day 0). On Day 4 (Day 4), the medium was replaced in its entirety, and VEGF, bFGF, SB431542, and heparin were added to the medium, and culture was continued under 37°C and 5% _O2 conditions. On Day 7 (Day 7), the medium was removed except for a portion, and hematopoietic progenitor cell differentiation medium containing only VEGF was added, and culture was continued under 37°C and 21% _O2 conditions. Heparin was added at this time. On Day 10 (Day 10), the medium was not removed, and hematopoietic progenitor cell differentiation medium containing only VEGF (approximately 50% volume) was added, and culture was continued under 37°C and 21% _O2 conditions. On days 9 (Day 9) and 14 (Day 14), cells were harvested using the same procedure as for the conventional PSC-Sac method, a portion was taken for cell counting, and the remaining cell suspension was used for subsequent analysis.

(5) Improved PSC-Sac method-2
A PSC clump suspension was prepared in the same manner as in the conventional PSC-Sac method, seeded on C3H10T1/2 feeder cells, and culture was started under 37°C and 5% _O2 conditions (Day 0). On Day 4 (Day 4), the entire medium was replaced, and VEGF, bFGF, SB431542, heparin, and apelin were added to the medium, and culture was continued under 37°C and 5% _O2 conditions. On Day 7 (Day 7), the medium was removed except for a portion, and hematopoietic progenitor cell differentiation medium containing only VEGF was added, and culture was continued under 37°C and 21% _O2 conditions. At this time, heparin was added in the amount of the medium added. On Day 10 (Day 10), the medium was not removed, and hematopoietic progenitor cell differentiation medium containing only VEGF (approximately 50% volume) was added, and culture was continued under 37°C and 21% _O2 conditions. On days 9 (Day 9) and 14 (Day 14), cells were harvested using the same procedure as for the conventional PSC-Sac method, a portion was taken for cell counting, and the remaining cell suspension was used for subsequent analysis.

1-3 Analysis of Hemogenic Endothelium (HE) (1) Measurement of Hemogenic Endothelium (HE) Yield in Cells Collected on Day 9 from the Start of the PSC-Sac Method Hemogenic endothelial cells were defined as [CD34+ CD31+ CD73- CD144+ CD117+ KDR+ CD41- CD43- CD45-]. Cells collected on day 9 were stained with anti-CD34, anti-CD31, anti-CD73, anti-CD144, anti-CD117, anti-KDR, anti-CD41, anti-CD43, and anti-CD45 antibodies, and subjected to FACS Verse ^TM (BD bioscience) to determine the percentage of hemogenic endothelial cells as defined above. The yield of hemogenic endothelial cells in each PSC-Sac method was calculated from the number of live cells measured separately and the percentage of HE obtained by FACS analysis.

(2) Expression analysis of hematopoietic-related genes in cells harvested on day 9
From the cells harvested on day 9, mRNA was extracted using miRNeasy Mini Kit (QIAGEN) according to the attached protocol. Then, cDNA was synthesized using ReverTra Ace ^R qPCR RT Master Mix (TOYOBO) and gDNARemover (TOYOBO). Real-time PCR was performed using StepOnePlus ^TM (Thermo Fisher Scientific) and SYBR ^R Premix Ex Taq ^TM II (Takara). Primer sets for GFI1b, SPI1, and TAL1 were determined using Universal ProbeLibrary for Human (https://qpcr.probefinder.com/input.jsp?organism=h_sap). Primer set for ERG was referred to "Tomlins et al, Science, 310(5748):644-648, 2005". The primer set for Runx1c was selected from the literature (Petter, et al., Blood, 111(1): 122-131, 2008).

1-4 Analysis of hematopoietic progenitor cells (HPCs) (1) Measurement of hematopoietic progenitor cell (HPC) yield in cells collected on day 14 after the start of the PSC-Sac method Hematopoietic progenitor cells were defined as [CD34+ CD43+]. Cells collected on day 9 were stained with APC conjugated CD43 monoclonal antibody (eBio84-3C1) (eBioscience/Thermo Fisher) and Brilliant Violet 421 anti-human CD34 antibody (BioLegend) and subjected to FACS Verse ^TM (BD bioscience) to determine the percentage of hemogenic endothelial cells as defined above. The yield of hematopoietic progenitor cells in each PSC-Sac method was calculated from the number of live cells measured separately and the percentage of HPCs obtained by FACS analysis.

(2) Analysis of CD34+CD43+ cells in hematopoietic progenitor cells on day 14 (2-1) Isolation and recovery of hematopoietic progenitor cells
CD34+ cells were isolated from the cells collected on day 14 by magnetic-activated cell sorting (MACS) using PE/Cy7-labeled anti-CD34 antibody (BioLegend) and anti-PE microbeads (Miltenyi Biotec). The collected cells were then stained with APC-labeled CD43 monoclonal antibody (eBio84-3C1) (eBioscience/Thermo Fisher) and subjected to FACS AriaII ^TM (BD Biosciences) to collect CD34+CD43+ cells. The collected hematopoietic progenitor cells were more than 90% pure.

(2-2) FACS analysis In addition to the antibody staining described above in (2-1), CD34+CD43+ cells were stained with PE/Cy7-labeled anti-CD38 antibody (BioLegend) and subjected to FACS Verse ^™ (BD Biosciences) to analyze CD34+CD38+ cells.

2. Results 2-1 Analysis of hemogenic endothelial cells (HE) (1) Comparison of hemogenic endothelial cell yields Figure 1 shows the results of FACS analysis of the hemogenic endothelial cell yields of cells collected on day 9 from the conventional PSC-Sac method, the improved PSC-Sac method-1, and the improved PSC-Sac method-2. Figure 1 shows the results when KhES-3, 692D2, AK5, and H1 were used as PSCs. If the yield per dish of the conventional PSC-Sac method (Conv. in the figure) is taken as 1, the yield of the improved PSC-Sac method-1 (Revised in the figure) was 28.7 times that of the conventional PSC-Sac method, and the yield of the improved PSC-Sac method-2 (R+Apln in the figure) was 38.4 times that of the improved PSC-Sac method, showing a significant increase in the yield of hemogenic endothelial cells. The results when AK5 was used as PSCs are shown in Figure 2. Similarly, when AK5 was used, the yield of hemogenic endothelial cells was significantly increased by the improved PSC-Sac method-1 and the improved PSC-Sac method-2. Furthermore, when H1 was used as PSC, the yield of hemogenic endothelial cells was increased by about two-fold by the improved PSC-Sac method-2 compared to the improved PSC-Sac method-1.

(2) Expression analysis of hematopoietic-related genes in hemogenic endothelial cells The expression levels of hematopoietic-related genes in cells collected on day 9 from the conventional PSC-Sac method, improved PSC-Sac method-1, and improved PSC-Sac method-2 were analyzed by quantitative PCR, and the results are shown in Figure 3. Figure 3 shows the results when KhES-3, 692D2, and AK5 were used as PSCs. (A) shows the results for GFI1b, (B) for ERG, (C) for SPI1, (D) for Runx1c, and (E) for TAL1, and the relative expression levels are shown when the expression level from the conventional PSC-Sac method (C in the figure) is set to 1. The expression levels of all hematopoietic-related genes were significantly increased in the improved PSC-Sac method-1 (R in the figure) and improved PSC-Sac method-2 (R+A in the figure) compared to the conventional PSC-Sac method. In (D) and (E), expression was higher in the improved PSC-Sac method-2 than in the improved PSC-Sac method-1. Figure 4 shows the results of comparing the conventional PSC-Sac method (Conv. in the figure) and the improved PSC-Sac method-1 (Revised in the figure) using KhES-3 as PSCs. (A) shows the results for GFI1b, (B) for ERG, (C) for SPI1, (D) for Runx1c, and (E) for TAL1. Expression of all hematopoietic-related genes was significantly higher in the improved PSC-Sac method-1 than in the conventional PSC-Sac method. Furthermore, the improved PSC-Sac method-1 and the improved PSC-Sac method-2 were performed using 692D2 as PSCs. In the improved PSC-Sac method-2, expression of SPI1 was similar to that in the improved PSC-Sac method-1, and expression of the four genes other than SPI1 was increased.

2-2 Analysis of hematopoietic progenitor cells (1) Comparison of hematopoietic progenitor cell yields Figure 5 shows the results of FACS analysis of the hematopoietic progenitor cell yields of cells collected on day 14 using the conventional PSC-Sac method, improved PSC-Sac method-1, and improved PSC-Sac method-2. Figure 5 shows the results when KhES-3 and 692D2 were used as PSCs. If the yield per dish of the conventional PSC-Sac method (Conv. in the figure) is taken as 1, the yield of the improved PSC-Sac method-1 (Revised in the figure) was 65.3 times that of the conventional PSC-Sac method, and the yield of the improved PSC-Sac method-2 (R+Apln in the figure) was 101.9 times that of the conventional PSC-Sac method, indicating a significant increase in the yield of hematopoietic progenitor cells. This result means that the conventional PSC-Sac method required 100 dishes and the necessary medium to obtain hematopoietic progenitor cells, whereas the improved PSC-Sac method-2 required only one dish and the necessary medium to obtain hematopoietic progenitor cells, and it was revealed that the cost and the amount of work can be significantly reduced. Furthermore, the results of comparing the conventional PSC-Sac method (Conventional in the figure) with the improved PSC-Sac method-1 (Revised in the figure) using TkDN-Sev2 and AK5 as PSCs are shown in Figure 6. Similarly, when these cells were used, the improved PSC-Sac method-1 significantly increased the yield of hematopoietic progenitor cells. Figure 7 shows the results when 692D2 and KhES-3 were used as PSCs, respectively.

(2) Comparison of CD34+CD38+ hematopoietic progenitor cells Figure 8 shows the results of FACS analysis of CD34+CD38+ cells in hematopoietic progenitor cells from cells collected on day 14 by the conventional PSC-Sac method and the improved PSC-Sac method-1. Figure 8 shows the results when KhES-3 was used as PSC. There were almost no CD34+CD38+ cells (0.41%) in hematopoietic progenitor cells obtained by the conventional PSC-Sac method (Conventional in the figure), but the presence of CD34+CD38+ cells (9.70%) was confirmed in hematopoietic progenitor cells obtained by the improved PSC-Sac method (Revised in the figure). CD34+CD38+ hematopoietic progenitor cells are cells found in human umbilical cord blood and human adult bone marrow, and the hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 were considered to be closer to fetal and adult types than the hematopoietic progenitor cells obtained by the conventional PSC-Sac method. A similar tendency was observed when AK5 was used as PSC.

Example 2: Production of hematopoietic progenitor cells by adding various components
The yield of hematopoietic progenitor cells was measured when the components added to the medium in the improved PSC-Sac method-1 were added alone or in any combination.

2-1 Materials and Methods The hematopoietic progenitor cell differentiation medium and differentiation induction procedures used in this example are described below.
● Addition of bFGF In the improved PSC-Sac method-1 of Example 1, the medium from day 4 (Day 4) to day 7 (Day 7) was the hematopoietic progenitor cell differentiation medium of Example 1 supplemented with 50 ng/mL of bFGF, and differentiation was induced in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no heparin was added to the amount added in the medium change on day 7 (Day 7), and the cells were collected on day 14 (Day 14).

●Addition of SB431542 In the improved PSC-Sac method-1 of Example 1, the medium used from day 4 (Day 4) to day 7 (Day 7) was the hematopoietic progenitor cell differentiation medium of Example 1 supplemented with 10 μM SB431542, and differentiation was induced in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no heparin was added to the amount added in the medium change on day 7 (Day 7), and the cells were harvested on day 14 (Day 14).

● Addition of heparin In the improved PSC-Sac method-1 of Example 1, a hematopoietic progenitor cell differentiation medium of Example 1 supplemented with 10 U/mL heparin was used as the medium from day 4 (Day 4) to day 7 (Day 7), and differentiation was induced in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no additional heparin was added to the amount added in the medium change on day 7 (Day 7), and the cells were harvested on day 14 (Day 14).

Addition of bFGF and SB431542 In the improved PSC-Sac method-1 of Example 1, a hematopoietic progenitor cell differentiation medium of Example 1 supplemented with 50 ng/mL bFGF and 10 μM SB431542 was used as the medium from day 4 (Day 4) to day 7 (Day 7), and differentiation was induced in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no heparin was added to the amount added in the medium change on day 7 (Day 7), and the cells were collected on day 14 (Day 14).

Addition of bFGF and heparin In the improved PSC-Sac method-1 of Example 1, a hematopoietic progenitor cell differentiation medium of Example 1 supplemented with 50 ng/mL bFGF and 10 U/mL heparin was used as the medium from day 4 (Day 4) to day 7 (Day 7), and differentiation was induced in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no additional heparin was added to the amount added in the medium change on day 7 (Day 7), and the cells were collected on day 14 (Day 14).

● Addition of SB431542 and heparin In the improved PSC-Sac method-1 of Example 1, the medium used from day 4 (Day 4) to day 7 (Day 7) was the hematopoietic progenitor cell differentiation medium of Example 1 supplemented with 10 μM SB431542 and 10 U/mL heparin, and differentiation was induced in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no additional heparin was added to the amount added in the medium change on day 7 (Day 7), and the cells were harvested on day 14 (Day 14).

Addition of bFGF, SB431542 and heparin Differentiation induction was carried out in the same manner as in the improved PSC-Sac method-1 of Example 1, except that no heparin was added to the amount of medium added during the medium change on Day 7 (Day 7), and the cells were harvested on Day 14 (Day 14).

Hematopoietic progenitor cell yield was measured using the same method as described in Examples 1, 1-4(1).

2-2 Results The results are shown in Figure 9. Figure 9 summarizes the results when KhES-3 and 692D2 were used as PSCs. Figure 9 shows the ratio of the yield of hematopoietic progenitor cells under each condition, assuming that the yield of hematopoietic progenitor cells per dish in the conventional PSC-Sac method (C in the figure) is 1. Note that the data for the conventional PSC-Sac method (C in the figure), improved PSC-Sac method-1 (FSH+H in the figure), and improved PSC-Sac method-2 (FSH+H+Apln in the figure) are the same as those in Figure 5. As shown in Figure 9, when bFGF and SB431542 were added from days 4 to 7 (FS in the figure), and when bFGF, SB431542, and heparin were added from days 4 to 7 (FSH in the figure), a significant increase in the yield of hematopoietic progenitor cells was observed, although this was not as high as that of the improved PSC-Sac method-1 and the improved PSC-Sac method-2.

Example 3: Analysis of erythrocytes differentiated from hematopoietic progenitor cells produced by the PSC-Sac method
1. Materials and Methods 1-1 Separation and Recovery of Hematopoietic Progenitor Cells CD34+CD43+ cells were recovered as hematopoietic progenitor cells from the cells recovered on day 14 by the conventional PSC-Sac method and the improved PSC-Sac method-1. The method for recovering CD34+CD43+ cells was the same as that described in Example 1, 1-4(2)(2-1).

1-2 Induction of Differentiation from Hematopoietic Progenitor Cells to Erythrocytes (1) Erythroid Differentiation Medium The hematopoietic progenitor cell differentiation medium used in Example 1 was prepared by removing VEGF from the medium and adding 5 IU/mL erythropoietin (Kyowa-Kirin), 50 ng/mL human stem cell factor (R&D systems), 10 ng/mL thrombopoietin (R&D systems), 50 μM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich), and 50 μM Trolox (Cayman) to the medium, which was then used as the erythroid differentiation medium.

(2) Culture C3H10T1/2 feeder cells were prepared as described in 1-2(1) of Example 1. The collected CD34+CD43+ cells were suspended in erythroid differentiation medium, and 0.7× ¹⁰⁵ cells were seeded on the C3H10T1/2 feeder cells and cultured for 21 days.

1-3 Measurement of CD235-positive cell count Cells were collected on the 21st day after the start of culture in erythroid differentiation medium. The cells in the culture dish were transferred to a 15 mL tube using a Pipetman P-1000 (Gilson), and the culture dish was washed with PBS and the remaining cells were transferred to the tube. The cells were centrifuged (600G, 8 minutes, room temperature), the supernatant was removed, and the cells were suspended in PBS containing 3% FBS. A portion was taken for cell counting, stained with trypan blue, and the number of live cells was measured using a hemocytometer. The remaining cells were stained with BV510 Mouse Anti-human CD235a (BD Biosciences) and subjected to FACS Verse ^TM (BD bioscience) to determine the percentage of CD235-positive cells. The number of CD235-positive cells in each PSC-Sac method was calculated from the number of live cells measured previously and the percentage of cells measured by FACS analysis.

1-4 Analysis of hemoglobin expression pattern in CD235 positive cells (erythrocytes) Cells were harvested 21 days after the start of culturing in erythrocyte differentiation medium in the same manner as in 1-3 above, and a portion was stained with trypan blue to measure the number of viable cells. The remaining cells were fixed in 4% PFA (Wako) for 15-60 minutes, then replaced with chilled 90% methanol (Nacalai Tesque) and fixed for 10-30 minutes. The medium was then replaced with 0.5% Saponin/PBS (MP biomedicals) and incubated for 10 minutes to perform cell membrane permeabilization. The medium was replaced with PBS containing 3% FBS and the cells were suspended, then stained with FITC-labeled hemoglobin ε antibody (Fitzgerald), PE-labeled hemoglobin γ antibody (Santa Cruz), Alexa Fluor 647-labeled hemoglobin β antibody (Santa Cruz), and Pacific Blue anti-human CD235ab Antibody (BioLegend). The cells were then subjected to FACS Verse ^TM (BD bioscience) to determine the percentage of CD235-positive cells (red blood cells) with ε-positive, γ-negative, β-negative (hereinafter also referred to as "ε+γ-β-") hemoglobin, ε-positive, γ-positive, β-positive (hereinafter also referred to as "ε+γ+β+") hemoglobin, ε-negative, γ-positive, β-positive hemoglobin (hereinafter also referred to as "ε-γ+β+"), or ε-negative, γ-negative, β-positive (hereinafter also referred to as "ε-γ-β+") hemoglobin.

2. Results 2-1 Number of CD235-positive cells Hematopoietic progenitor cells obtained by the conventional PSC-Sac method and the improved PSC-Sac method-1 were cultured in erythroid differentiation medium, and the number of CD235-positive cells in the cells on day 21 was measured. The results are shown in Figure 10. Figure 10 shows the results when AK5 was used as PSC. If the number of CD235-positive cells per dish in cells induced to differentiate from hematopoietic progenitor cells using the conventional PSC-Sac method (C in the figure) was set to 1, the number of CD235-positive cells in cells induced to differentiate from hematopoietic progenitor cells using the improved PSC-Sac method-1 (R in the figure) was about 7 times higher.

2-2 Analysis of hemoglobin expression patterns It is known that the amount of globin expressed in human red blood cells changes according to the developmental stage (Wood WG, Br. Med. Bull. 32, 282. 1976). Embryonic red blood cells express ε globin and show the ε+γ-β- phenotype. Fetal red blood cells express γ globin and β globin simultaneously, with early fetal red blood cells showing the ε+γ+β+ phenotype and late fetal red blood cells showing the ε-γ+β+ phenotype. With birth, the amount of γ globin expression decreases, and adult red blood cells show the ε-γ-β+ phenotype.

Figure 11 shows the results of comparing the number of embryonic red blood cells (ε+γ-β-) in red blood cells obtained using the conventional PSC-Sac method and the improved PSC-Sac method-1. Figure 11 shows the results when AK5 was used as the PSC. If the number of embryonic red blood cells obtained using the conventional PSC-Sac method (C in the figure) is set to 1, the number of embryonic red blood cells obtained using the improved PSC-Sac method-1 (R in the figure) was approximately 0.04, making it clear that there were almost no embryonic red blood cells.

Figure 12 shows the results of comparing the number of fetal-type red blood cells in red blood cells obtained by the conventional PSC-Sac method and the improved PSC-Sac method-1. Figure 12 shows the results when AK5 was used as the PSC. The left side shows the results for early fetal-type red blood cells (ε+γ+β+), and the right side shows the results for late fetal-type red blood cells (ε-γ+β+). For early fetal-type red blood cells (ε+γ+β+), if the number of embryonic red blood cells obtained by the conventional PSC-Sac method (C in the figure) is set to 1, the number of embryonic red blood cells obtained by the improved PSC-Sac method-1 (R in the figure) was approximately 2.7. For late fetal-type red blood cells (ε-γ+β+), if the number of embryonic red blood cells obtained by the conventional PSC-Sac method (C in the figure) is set to 1, the number of embryonic red blood cells obtained by the improved PSC-Sac method-1 (R in the figure) was approximately 4.7. These results demonstrated that red blood cells obtained by the conventional PSC-Sac method contained more embryonic-type red blood cells, while red blood cells obtained by the improved PSC-Sac method-1 contained more fetal-type red blood cells.

The presence of adult-type red blood cells (ε-γ-β+) in the red blood cells obtained by the improved PSC-Sac method-1 was confirmed and is shown in Figure 13. Figure 13 shows the results when 692D2 was used as the PSC.

Example 4: Analysis of megakaryocytes and platelets differentiated from hematopoietic progenitor cells produced by the improved PSC-Sac method
1. Materials and Methods 1-1 Separation and Recovery of Hematopoietic Progenitor Cells Using the same method as in Example 3, CD34+CD43+ cells were recovered as hematopoietic progenitor cells from the cells recovered on day 14 by the conventional PSC-Sac method and the improved PSC-Sac method-1.

1-2 Induction of Differentiation from Hematopoietic Progenitor Cells to Megakaryocytes and Platelets (1) Megakaryocyte/Platelet Differentiation Medium The hematopoietic progenitor cell differentiation medium used in Example 1 was prepared by removing VEGF from the medium, and adding 50 ng/mL thrombopoietin (R&D systems), 50 ng/mL human stem cell factor (R&D systems), 25 U/mL heparin (AY Pharma), 15 μM KP457 (Kaken Pharmaceutical), 0.75 μM SR1 (Calbiochem), and 10 μM Y-27632 (Wako), and the resulting medium was used as the megakaryocyte/platelet differentiation medium.

(2) Culture C3H10T1/2 feeder cells were prepared as described in 1-2(1) of Example 1. The collected CD34+CD43+ cells were suspended in megakaryocyte/platelet differentiation medium, and 0.5× ¹⁰⁵ cells were seeded on the C3H10T1/2 feeder cells and cultured for 10 days.

1-3 Measurement of megakaryocyte count Cells were collected on the 10th day after the start of culture in megakaryocyte/platelet differentiation medium. The cells in the culture dish were transferred to a 15 mL tube using a Pipetman P-1000 (GILSON), and the culture dish was washed with PBS and the remaining cells were transferred to the tube. The cells were centrifuged (400 G, 8 min, room temperature), the supernatant was removed, and the cells were suspended in PBS containing 3% FBS. A portion was taken for cell counting, and after trypan blue staining, the number of live cells was measured using a hemocytometer. The remaining cells were stained with APC/Cy7-labeled anti-human CD41 antibody (Biolegend) and PE-labeled anti-human CD42b antibody (Biolegend), and subjected to FACS Verse ^TM (BD bioscience), and the content ratio of megakaryocytes as CD41-positive CD42b-positive cells was calculated. The number of megakaryocytes in each PSC-Sac method was calculated from the number of live cells measured previously and the content ratio by FACS analysis.

1-4 Measurement of platelet count Platelets were collected on the 10th day after the start of culture in megakaryocyte/platelet differentiation medium. The inside of the culture dish was stirred using a Pipetman P-1000 (GILSON) to uniformly disperse the platelet precipitate in the medium, and 100 μL was then taken. APC-labeled anti-human CD41 antibody (Biolegend) and PE-labeled anti-human CD42b antibody (Biolegend) were added and stained at room temperature, and 400 μL of 3% FBS-containing PBS was added to dilute the mixture, and 450 μL of the diluted mixture was transferred to BD Trucount ^TM Tubes (BD Biosciences). The number of beads measured by FCM based on the unique number of beads in the BD Trucount ^TM Tubes and the number of CD41-positive and CD42b-positive cells were used to calculate the number of platelets in the original culture dish.

2. Results Hematopoietic progenitor cells obtained by the conventional PSC-Sac method and the improved PSC-Sac method-1 were cultured in megakaryocyte/platelet differentiation medium, and the number of megakaryocytes and platelets in the cells on day 14 were measured. The results are shown in Figure 14. Figure 14 shows the results when AK5 was used as PSC. (A) shows the result of megakaryocyte count, and (B) shows the result of platelet count, which are relative amounts when the number of cells in the conventional PSC-Sac method (C in the figure) is set to 1. The number of megakaryocytes and the number of platelets were significantly higher when differentiation was induced from hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 (R in the figure) than when differentiation was induced from hematopoietic progenitor cells obtained by the conventional PSC-Sac method (C in the figure). These results demonstrated that the hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 had significantly higher differentiation efficiency into megakaryocytes and platelets than the hematopoietic progenitor cells obtained by the conventional PSC-Sac method.

Example 5: Analysis of differentiation of hematopoietic progenitor cells produced by the PSC-Sac method into leukocytes
1. Materials and Methods 1-1 Separation and Recovery of Hematopoietic Progenitor Cells Using the same method as in Example 3, CD34+CD43+ cells were recovered as hematopoietic progenitor cells from the cells recovered on day 14 by the conventional PSC-Sac method and the improved PSC-Sac method-1.

1-2 Colony assay of hematopoietic progenitor cells A colony assay was performed by seeding 1,000 hematopoietic progenitor cells using MethoCult H4434 (Stem Cell Technologies), a medium for hematopoietic colony assay of hematopoietic progenitor cells, according to the attached protocol. The number of granulocyte colonies and monocyte colonies formed was evaluated as the differentiation efficiency of hematopoietic progenitor cells to leukocytes.

1-3 Measurement of the number of CD4+CD8+ T cells
24-well plates coated with StemSpan ^™ Lymphoid Differentiation Coating Material (Stem Cell Technologies) were used and incubated at room temperature according to the attached protocol. 0.2 × ¹⁰⁵ hematopoietic progenitor cells were seeded and cultured for 10 days in StemSpan™ SFEM II medium supplemented with 10 ng/mL TPO, 10 ng/mL SCF, and 5 ng/mL Flt3L (Peprotech), and 20 ng/mL IL-7 (Miltenyi Biotec). Then, the cells were cultured for 11 days in StemSpan ^™ SFEM II medium supplemented with 100 ng/mL TPO, 50 ng/ ^mL SCF, 50 ng/mL Flt3L, 50 ng/mL IL-7, 15 μM SB203580 (Calbiochem), and 30 ng/mL SDF1α (R&D Systems). After trypan blue staining, the number of viable cells was measured using a hemocytometer. The remaining cells were stained with anti-human CD3 antibody (anti-CD3-Alexa Fluor 488 (BioLegend Cat. No. 300415)), anti-human CD5 antibody (anti-CD5-APC (BioLegend Cat. No. 300612)), anti-human CD4 antibody (anti-CD4-PE/Cy7 (BioLegend Cat. No. 357410)), and anti-human CD8 antibody (anti-CD8-PE (BioLegend Cat. No. 344705)), and subjected to FACS analysis to determine the percentage of CD4+CD8+ T cells. The number of CD4+CD8+ T cells in each PSC-Sac method was calculated from the number of viable cells measured previously and the percentage of CD4+CD8+ T cells obtained by FACS analysis.

2. Results (1) Colony assay of hematopoietic progenitor cells The results of the colony assay of hematopoietic progenitor cells are shown in Figure 15. Figure 15 shows the results when AK5 was used as PSC. Comparing the results of the colony assay of hematopoietic progenitor cells obtained by the conventional PSC-Sac method (Conventional in the figure) and the improved PSC-Sac method-1 (Revised in the figure), it was shown that the differentiation efficiency into granulocytes and monocytes was almost the same.

(2) Number of CD4+CD8+ T cells Hematopoietic progenitor cells obtained by the conventional PSC-Sac method and those obtained by the improved PSC-Sac method-1 were induced to differentiate using the lymphocyte differentiation induction system described above, and the number of CD4+CD8+ T cells was measured. The results are shown in Figure 16. Figure 16 shows the results when AK5 was used as PSC. The number of CD4+CD8+ T cells was significantly higher when hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 (R in the figure) were induced to differentiate than when hematopoietic progenitor cells obtained by the conventional PSC-Sac method (C in the figure).

Example 6: Analysis of CD45-positive cells in hematopoietic progenitor cells produced by the improved PSC-Sac method
In the same manner as in Example 3, cells collected on day 14 by the conventional PSC-Sac method and the improved PSC-Sac method-1 were stained with a CD43 monoclonal antibody (eBio84-3C1) (eBioscience/Thermo Fisher), Brilliant Violet 421 anti-human CD34 antibody (BioLegend), and Alexa Fluor 488 anti-human CD45 antibody (Biolegend) and then subjected to FACS analysis to determine the percentage of CD45-positive cells present among CD34-positive CD43-positive hematopoietic progenitor cells.

The results are shown in Figure 17. Figure 17 shows the results when AK5 was used as the PSC. There was a tendency for the percentage of CD45 positive cells among hematopoietic progenitor cells obtained by the improved PSC-Sac method-1 (R in the figure) to be higher than the percentage of CD45 positive cells among hematopoietic progenitor cells obtained by the conventional PSC-Sac method (C in the figure).

Example 7: Comparison of hematopoietic progenitor cells under feeder-free conditions
The conventional PSC-Sac method and improved PSC-Sac method-1 were performed without using feeder cells, which are usually used in the PSC-sac method, and the effects of bFGF, SB431542, and heparin were confirmed. KhES-3 was used as PSC and seeded on a 0.1% gelatin-coated dish (Φ10 cm). The results of FACS analysis of CD34+CD43+ cells in hematopoietic progenitor cells from cells collected on day 14 are shown in Figure 18. Under feeder-free conditions, the conventional PSC-Sac method (Gel-Conv in the figure) produced an extremely low yield of CD34+CD43+ hematopoietic progenitor cells. On the other hand, the improved PSC-Sac method-1 (Gel-Rev in the figure) produced approximately 20 times more CD34+CD43+ hematopoietic progenitor cells than the conventional PSC-Sac method. That is, by using a combination of bFGF, SB431542, and heparin, CD34+CD43+ hematopoietic progenitor cells could be increased in the PSC-sac method, regardless of the presence or absence of feeder cells.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. In addition, all academic literature and patent documents described in this specification are incorporated herein by reference.

This patent application claims priority to Japanese Patent Application No. 2019-196750, the entire contents of which are hereby incorporated by reference into this specification.

Claims (10)

A method for producing hematopoietic progenitor cells by culturing pluripotent stem cells in a hematopoietic progenitor cell differentiation medium containing vascular endothelial growth factor, characterized in that a basic fibroblast growth factor , a transforming growth factor β signaling inhibitor and heparin are added to the hematopoietic progenitor cell differentiation medium. The method according to claim 1 , further comprising adding an apelin receptor agonist. The method according to claim 1 or 2 , wherein the pluripotent stem cells are cultured on feeder cells. The method according to claim 1 or 2 , wherein a hematopoietic progenitor cell differentiation medium containing a basic fibroblast growth factor and a transforming growth factor β signaling inhibitor is used during the middle stage of the culture period. The method according to claim 1 or 2 , wherein a hematopoietic progenitor cell differentiation medium containing heparin is used during the middle stage of the culture period, or during the middle stage and thereafter. The method for production according to claim 1 or 2 , wherein a hematopoietic progenitor cell differentiation medium containing an apelin receptor agonist is used during the initial and subsequent stages of the culture period, or during the intermediate stage, or during the intermediate and subsequent stages of the culture period. The method according to any one of claims 1 to 6 , wherein the obtained hematopoietic progenitor cells include hematopoietic progenitor cells having a fetal and/or adult phenotype. A method for producing blood cells, comprising producing hemopoietic endothelial cells and/or hematopoietic progenitor cells by the production method according to any one of claims 1 to 7 , and culturing the obtained hemopoietic endothelial cells and/or hematopoietic progenitor cells in a medium containing a differentiation-inducing factor . The method of claim 8, wherein the differentiation-inducing factor is at least one selected from the group consisting of TPO, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, EPO, GM-CSF, Flt3 ligand, and heparin. The method of claim 8 or 9 , wherein the obtained blood cells include blood cells having a fetal and/or adult phenotype.