Culture medium and blood cell clone, and method for producing same

Abstract

This culture medium is for multiplying blood cells capable of dividing, and contains a compound (I) represented by a formula or a physiologically acceptable salt thereof. Formula (I). In the formula, R1, R2 and R3 are each H, a linear or branched C1-C4alkyl, bromine, or iodine; R4 is a linear or branched C1-C4 alkyl, bromine or iodine; R5 is oxygen, methylene or a direct bond; R6 is hydrogen or-NH2; and R7 is hydrogen or -COOH. Note that R6 and R7 are not simultaneously hydrogen.

Description

Culture medium, clone of blood cells, and method for producing the same

[0001] The present invention relates to a culture medium and a method for cloning hematopoietic stem cells (HSCs) and other blood cells capable of cell division, and a method for producing the same, particularly using the thyroid hormone thyroxine and its metabolites.

[0002] Patent Document 1 discloses a cell culture kit. In this kit, thyroid hormone or its metabolite, triiodo-L-thyronine, is selected as the unstable factor (claim 9). The unstable factor does not induce differentiation of cells during culture. Hematopoietic progenitor cells are selected as the cells (claim 23).

[0003] Patent Document 2 discloses a method for preparing a T cell composition from stem cells, in which cell aggregates containing stromal cells and stem cells are cultured in a medium (claim 1), and the medium contains triiodo-L-thyronine (claim 12).

[0004] According to Non-Patent Document 1, the biological activity of thyroid hormones is mainly due to triiodo-L-thyronine (T ３ ) binds to its nuclear receptor, which then acts as a ligand-inducible transcription factor to regulate target gene expression in a hormone-dependent manner. Kruppel-like factor 9 (KLF9) is primarily downregulated in hypothyroid mutants. KLF9 is also directly regulated by thyroid hormone. Furthermore, knockdown of klf9 in zebrafish embryos inhibits hematopoietic development, including erythrocyte maturation and T lymphopoiesis.

[0005] As described above, Patent Document 1 is a technology for culturing hematopoietic progenitor cells using triiodo-L-thyronine. Patent Document 2 is a technology for differentiating into T lymphocytes using triiodo-L-thyronine. Non-Patent Document 1 is a technology for inhibiting the formation of T lymphocytes by knocking down the triiodo-L-thyronine receptor. Neither technology expands hematopoietic stem cells while suppressing their differentiation.

[0006] Turning our attention to compounds other than triiodo-L-thyronine, it is known that mouse hematopoietic stem cells are expanded by polyvinyl alcohol (PVA) (Non-Patent Document 2), and that human hematopoietic stem cells are expanded by compounds such as UM171, Soluplus (trademark, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, PCL-PVAc-PEG), cell-permeable phosphopeptide 740Y-P, and butyzamide (Non-Patent Documents 3 and 4).

[0007] Furthermore, technologies for inhibiting the differentiation of hematopoietic stem cells include Patent Document 3 and Non-Patent Document 5. Conventionally, hematopoietic stem cells rapidly proliferate and differentiate in vitro. In contrast, when the conditions of extremely low cytokine concentration, low oxygen, and extremely high fatty acid concentration are met, both cell proliferation and differentiation are inhibited. This inhibits the proliferation and differentiation of hematopoietic stem cells for one month, i.e., hematopoietic stem cells can be maintained in a quiescent state suitable for transplantation.

[0008] JP 2014-533113 A JP 2018-533364 A JP 2019-201599 A

[0009] Ying Zhang, Yuanyuan Xue, Chunwei Cao, Jiaojiao Huang, Qianlong Hong, Tang Hai, Qitao Jia, Xianlong Wang, Guosong Qin, Jing Yao, Xiao Wang, Qiantao Zheng, Rui Zhang, Yongshun Li, Ailing Luo, Nan Zhang, Guizhi Shi, Yanfang Wang, Hao Ying, Zhonghua Liu, Hongmei Wang, Anming Meng, Qi Zhou, Hong Wei, Feng Liu, Jianguo Zhao, “Thyroid hormone regulates hematopoiesis via the TR-KLF9 axis”, Blood, 2017, 130 (20), pages 2161-2170, [online], [retrieved on 2023-03-13] Retrieved from<URL: https: / / doi.org / 10.1182 / blood-2017-05-783043>

[0010] Wilkinson, AC, Ishida, R., Kikuchi, M. et al. "Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.", Nature, 2019, 571, pages 117-121, [online], [retrieved on 2023-03-24] Retrieved from<URL: https: / / doi.org / 10.1038 / s41586-019-1244-x>

[0011] Fares, Iman, Chagraoui Jalila, Gareau Yves, Gingras Stephane, Ruel Rejean, Mayotte Nadine, Csaszar Elizabeth, Knapp David J. H. F., Miller Paul, Ngom Mor, Imren Suzan, Roy Denis-Claude, Watts Kori L., Kiem Hans-Peter, Herrington Robert, Iscove Norman N., Humphries R. Keith, Eaves Connie J., Cohen Sandra, Marinier Anne, Zandstra Peter W., Sauvageau Guy, “Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal”, Science, 2014-09-19, 345(6203), pages 1509-1512, [online], [retrieved on 2023-03-20] Retrieved from <URL: https: / / doi.org / 10.1126 / science.<1256337>Sakurai, M., Ishitsuka, K., Ito, R. et al. Chemically defined cytokine-free expansion of human haematopoietic stem cells. Nature, 2023, 615, pages 127-133, [online], [retrieved on 2023-04-19] Retrieved from <URL:https: / / doi.org / 10.1038 / s41586-023-05739-9>

[0012] Kobayashi Hiroshi, Morikawa Takayuki, Okinaga Ayumi, Hamano Fumie, Hashidate-Yoshida Tomomi, Watanuki Shintaro, Hishikawa Daisuke, Shindou Hideo, Arai Fumio, Kabe Yasuaki, Suematsu Makoto, Shimizu Takao; Takubo, Keiyo, “Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo”, Cell Reports, 2019-07-02, 28 (1), pages 145-158, [online], [retrieved on 2023-03-13] Retrieved from <URL: https: / / doi.org / 10.1016 / j.celrep.2019.06.008>

[0013] Bolger Michael and Jorgensen Eugene, “Molecular interactions between thyroid hormone analogs and the rat liver nuclear receptor, Partitioning of equilibrium binding free energy changes into substituent group interactions” The Journal of biological chemistry, 255, 21, 10271-8, [online], [retrieved on 2023-03-27] Retrieved from <URL: https: / / doi.org / 10.1016 / S0021-9258(19)70460-8>

[0014] Grazia Chiellini, James W. Apriletti, Hikari Al Yoshihara, John D. Baxter, Ralff CJ Ribeiro, Thomas S. Scanlan, “A high-affinity subtype-selective agonist ligand for the thyroid hormone receptor, Chemistry & Biology”, Volume 5, Issue 6, 1998, Pages 299-306, [online], [retrieved on] 2023-03-27] Retrieved from<URL: https: / / doi.org / 10.1016 / S1074-5521(98)90168-5> Lorenzini L, Nguyen NM, Sacripanti G, Serni E, Borso M, Saponaro F, Cecchi E, Simoncini T, Ghelardoni S, Zucchi R and Saba A (2019) Assay of Endogenous 3,5-diiodo-L-thyronine (3,5-T2) and 3,3′-diiodo-L-thyronine (3,3′-T2) in Human Serum: A Feasibility Study. Front. Endocrinol. 10:88. [online], [retrieved on 2023-06-08] Retrieved from<URL: https: / / doi.org / 10.3389 / fendo.2019.00088>

[0015] An objective of the present invention is to provide a means suitable for expanding hematopoietic stem cells and other blood cells capable of cell division while suppressing their differentiation.

[0016] <1> A medium for expanding blood cells capable of cell division, comprising compound (I) of the following formula or a physiologically acceptable salt thereof: ...(I) In the formula, R １ , R ２ and R３ are each hydrogen, straight-chain or branched C １ -C ４ alkyl, bromine, and iodine; in one embodiment, R １ , R ２ and R ３ None of R is hydrogen; ４ is a linear or branched C １ -C ４ alkyl, bromine, or iodine; R ５ is oxygen, methylene, or a direct bond; R ６ is hydrogen or -NH ２ and R ７ is hydrogen or —COOH, provided that R ６ and R ７ None of the medium is hydrogen.

[0017] <2> (1) R ５ is oxygen or methylene, R ６ is hydrogen or -NH ２ and R ７ is hydrogen or —COOH, provided that R ６ and R ７ or (2) R ５ is a direct bond, and R ６ is hydrogen, and R ７ is -COOH, the above medium.

[0018] <3> Compound (I) is Thyroxine (T ４ ), 3,3',5-Triiodo-L-thyronine (T ３ ), 3,3',5'-triiodothyronine (rT ３ ), 3,3'-Diiodo-L-thyronine (3,3'-T ２ ), 3,5-Diiodo-L-thyronine (3,5-T ２ ), 3-Iodo-L-thyronine (3-T １ ), 3-Iodo-L-thyroacetic acid (3-T １ A.A.), 3-Iodo-L-thyronamine (3-T １ A.M.), Sobechirom, 3,3',5-Triiodo-L-thyroacetic acid (3,3',5-T ３ AA) and 3,5-Diiodo-L-thyroacetic acid (3,5-T ２ AA), and preferably thyroxine (T ４ ), 3,3',5-Triiodo-L-thyronine (T ３ ), 3,3',5'-triiodothyronine (rT ３ ), 3,3'-diiodo-L-thyronine (3,3'-T ２ ), 3,5-diiodo-L-thyronine (3,5-T ２ ) and sobetirom, 3,3',5-triiodo-L-thyroacetic acid (3,3',5-T ３ AA) and 3,5-diiodo-L-thyroacetic acid (3,5-T ２ AA).

[0019] <4> R １ is hydrogen, and R ２ and R ３ One of the groups is hydrogen, straight or branched C １ -C ４ The medium as described above, wherein the alkyl group is either bromine or iodine, and the other is hydrogen.

[0020] <5> Compound (I) is 3,5-diiodo-L-thyronine (3,5-T ２ ), and 3,3'-diiodo-L-thyronine (3,3'-T ２ ) The above medium.

[0021] <6> The medium as described above, wherein the blood cells are collected from an aged donor, or the blood cells are collected from umbilical cord blood.

[0022] <7> The above medium for expanding the blood cells without mixing them with blood cells at other differentiation stages.

[0023] <8> The above-mentioned medium for expanding the blood cells and further expanding differentiated blood cells generated in the process of expanding the blood cells.

[0024] <9> The above medium, wherein the blood cell is any one of: hematopoietic stem cells that have not yet differentiated into pluripotent progenitor cells; pluripotent progenitor cells that have already completed differentiation from hematopoietic stem cells but have not yet differentiated into progenitor cells of differentiated blood cells; and differentiated blood cells that have already completed differentiation from the progenitor cells.

[0025] <10> The medium described above, wherein the medium is a serum-free medium and contains compound (I), and the compound (I) is, for example, 1 fM to 1 mM, preferably 1 pM to 1 mM, preferably 1 nM to 1 mM, preferably 0.1 μM to 1 mM, or the medium is a serum-free medium and contains compound (I) in an amount of 0.1 μM to 1 mM.

[0026] <11> Compound (I) of the following formula or a physiologically acceptable salt thereof is applied to dividing blood cells in vitro, while the blood cells are expanded: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, straight-chain or branched C １ -C ４ alkyl, bromine, and iodine; in one embodiment, R １ , R ２ and R ３ None of R is hydrogen; ４ is a linear or branched C １ -C ４ alkyl, bromine, or iodine; R ５ is oxygen, methylene, or a direct bond; R ６ is hydrogen or -NH ２ and R ７ is hydrogen or —COOH, provided that R ６ and R ７ A method for producing clones of blood cells, none of which is hydrogen.

[0027] <12> A clone of a blood cell capable of cell division obtained by applying compound (I) of the following formula or a physiologically acceptable salt thereof to a blood cell capable of cell division in vitro: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, straight-chain or branched C １ -C ４ alkyl, bromine, and iodine; in one embodiment, R １ , R ２ and R ３ None of R is hydrogen; ４ is a linear or branched C １ -C ４ alkyl, bromine, or iodine; R ５ is oxygen, methylene, or a direct bond; R ６ is hydrogen or -NH ２ and R ７ is hydrogen or —COOH, provided that R ６ and R ７ None of the seeded blood cells are hydrogen clones, wherein the seeded blood cells are taken from an aged donor.

[0028] <13> A drug to be added to a serum medium or a serum-free medium for expanding blood cells capable of cell division in vitro, comprising compound (I) of the following formula or a physiologically acceptable salt thereof as an active ingredient: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, straight-chain or branched C １ -C ４ alkyl, bromine, or iodine; R ４ is a linear or branched C １ -C ４ alkyl, bromine, or iodine; R ５ is oxygen, methylene, or a direct bond; R ６ is hydrogen or -NH２ and R ７ is hydrogen or —COOH, provided that R ６ and R ７ None of the drugs are hydrogen.

[0029] The present invention provides a means suitable for expanding hematopoietic stem cells and other blood cells capable of cell division while suppressing their differentiation.

[0030] The graph shows the total cell number in each colony. The graph shows the mean fluorescence intensity (MFI) of CD48 staining in each colony. The graph shows the change in cell number during culture. The figure shows an overview of the chimerism experiment. The graph shows the results of chimerism analysis. The graph shows the number of hematopoietic stem cells in each colony. The graph shows the results of chimerism analysis. The graph shows the proportion of each type of blood cell in the mouse. The graph shows the proportion of EPCR-positive cells (EPCR+HSC) after culturing HSCs in the presence of a carboxylic acid derivative of thyronine. The graph shows the number of cells when various blood cell lineage cells were cultured for 4 weeks in the presence of T2.

[0031] <Culture medium containing compounds>

[0032] One aspect of this embodiment is a culture medium for expanding hematopoietic stem cells and other blood cells capable of cell division. The culture medium is, for example, a liquid culture medium. The culture medium contains compound (I) of the following formula or a physiologically acceptable salt thereof:

[0033] ...(I)

[0034] R １ , R ２ and R ３ are each hydrogen, straight-chain or branched C １ -C ４ In one embodiment, R is any of alkyl, bromine, and iodine, such as any of methyl, ethyl, isopropyl, and iodine. １ , R ２ and R ３ None of R can be hydrogen. ４ is a linear or branched C １ -C ４R is any of alkyl, bromine, and iodine. For example, R is any of methyl, ethyl, isopropyl, and iodine. ５ is oxygen, methylene, or a direct bond; R ６ is hydrogen or -NH ２ and R ７ is hydrogen or —COOH, where R ６ and R ７ None of R can be hydrogen. ５ is oxygen, and R ６ is hydrogen, and R ７ is -COOH. For example, R ５ is methylene, and R ６ Ga-NH ２ and R ７ is -COOH.

[0035] In one embodiment, R ５ is oxygen or methylene, R ６ is hydrogen or -NH ２ and R ７ is hydrogen or —COOH. ６ and R ７ In another embodiment, none of R ５ is a direct bond, and R ６ is hydrogen, and R ７ is —COOH.

[0036] Preferably, R １ is hydrogen, and R ２ and R ３ One of the groups is hydrogen, straight or branched C １ -C ４ R is any one of alkyl, bromine and iodine, for example, methyl, ethyl, isopropyl and iodine, and the other is hydrogen; ４ is a linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine, for example, any one of methyl, ethyl, isopropyl and iodine; ５ is oxygen, methylene, or a direct bond; R ６ is hydrogen or -NH ２ and R７ is hydrogen or —COOH, where R ６ and R ７ None of R can be hydrogen. ５ is oxygen, and R ６ is hydrogen, and R ７ is -COOH. For example, R ５ is methylene, and R ６ Ga-NH ２ and R ７ is -COOH.

[0037] In another aspect of this embodiment, compound (I) is a thyroid hormone or a mimetic compound thereof. Compound (I) is, for example, any of the following:

[0038] Thyroxine (T ４ ),

[0039] 3,3',5-Triiodo-L-thyronine (T ３ ),

[0040] 3,3',5'-triiodothyronine (rT ３ ),

[0041] 3,3'-Diiodo-L-thyronine (3,3'-T ２ ),

[0042] 3,5-Diiodo-L-thyronine (3,5-T ２ ) 3-Iodo-L-thyronine (3-T １ ), 3-Iodo-L-thyroacetic acid (3-T １ A.A.), 3-Iodo-L-thyronamine (3-T １ A.M.),

[0043] Sobetirome,

[0044] 3,3',5-Triiodo-L-thyroacetic acid (3,3',5-T ３ AA) and

[0045] 3,5-Diiodo-L-thyroacetic acid (3,5-T ２ AA).

[0046] Compound (I) is preferably thyroxine (T ４ ), 3,3',5-Triiodo-L-thyronine (T ３ ), 3,3',5'-triiodothyronine (rT ３ ), 3,3'-diiodo-L-thyronine (3,3'-T ２ ), 3,5-diiodo-L-thyronine (3,5-T ２ ) and sobetirom,

[0047] By culturing hematopoietic stem cells in a medium containing compound (I), the cells can be expanded while suppressing their differentiation, particularly differentiation into pluripotent progenitor cells (hematopoietic progenitor cells) and subsequent differentiation along cell lineages. Expansion results in the generation of a large number of clones of the seeded hematopoietic stem cells. In this respect, a preferred compound (I) is 3,5-diiodo-L-thyronine (3,5-T ２ ), and 3,3'-diiodo-L-thyronine (3,3'-T ２ ) are either of these diiodo-L-thyronine (T ２ ) is particularly effective in inhibiting the differentiation of hematopoietic stem cells, while promoting the proliferation of hematopoietic stem cells like other compounds. Similar effects are also observed on other blood cell types capable of cell division.

[0048] In producing compound (I), free thyroid hormone may be extracted from blood. Compound (I) can be chemically synthesized according to known methods, for example, the method described in Non-Patent Document 6. For example, sobetirom may be synthesized as described in Non-Patent Document 7.

[0049] In one embodiment, the concentration of compound (I) in the medium is 1 fM to 1 mM, preferably 1 pM to 1 mM, preferably 1 nM to 1 mM, preferably 0.1 μM to 1 mM. The concentration of compound (I) in the medium may be any of 1, 2, 5, 10, 20, 50, and 100 μM.

[0050] <Drugs applied to blood cells>

[0051] Another aspect of this embodiment is a drug applied to hematopoietic cells capable of cell division in order to expand the cells in vitro. The drug contains compound (I) of the above formula or a physiologically acceptable salt thereof as an active ingredient. The drug may be a medium additive. The drug may be added to serum medium or serum-free medium. The drug may be applied to the cells before expanding the cells in the medium.

[0052] <Basal medium>

[0053] The composition of the basal medium to which compound (I) is to be added is not particularly limited, as long as it is capable of easily suppressing the differentiation of hematopoietic stem cells and other blood cells capable of cell division, in other words, it is capable of easily maintaining the undifferentiated state. In one embodiment, the basal medium is a serum-free medium. The medium to which compound (I) is added is also a serum-free medium. In another embodiment, the basal medium is a serum-free medium.

[0054] The basal medium supplemented with Compound (I), particularly the serum medium, contains Compound (I) in an amount of 0.1 μM to 1 mM. This range of concentration is, for example, the same as that of human free thyroxine (T ４ ), for example, 0.8 to 1.8 (ng / dl). ２ The blood concentration of 3,3'-diiodo-L-thyronine (3,3'-T) has been shown to be 78±5 pM. ２ The blood concentration of Compound (I) added as an active ingredient to serum culture media is shown to be 253±29 pM. The range of concentrations of Compound (I) added as an active ingredient to serum culture media is much higher than this.

[0055] The basal medium may be any medium used for ordinary cell culture, a medium prepared for the culture of hematopoietic stem cells, etc. Examples of media used for ordinary cell culture include αMEM, DMEM, Ham's F12 medium, DMEM / F12 medium, RPMI1640 medium, IMEM, McCoy's 5A medium, and EMEM. Examples of media used for culturing hematopoietic stem cells include S-Clone medium (e.g., SF-O3 medium, manufactured by EIDIA), StemPro34 (manufactured by Invitrogen), Stemline (manufactured by Sigma-Aldrich), Stemline II (manufactured by Sigma-Aldrich), X-VIVO 10 (manufactured by Lonza), X-VIVO 15 (manufactured by Lonza), X-VIVO 20 (manufactured by Lonza), HPGM (manufactured by Lonza), StemSpan H3000 (manufactured by Stem Cell Technology), StemSpan SFEM (Serum-Free Expansion Medium, manufactured by Stem Cell Technology), and QBSF-60 (manufactured by Quality Biologicals). In this embodiment, these media may be mixed appropriately and used. These basal media can also be suitably used for other blood cells capable of cell division.

[0056] The basal medium according to this embodiment is not limited to the above examples, but is preferably SF-O3 medium, αMEM, or DMEM / F12 medium, more preferably SF-O3 medium, because they have a proven track record of use in culturing hematopoietic stem cells. Furthermore, as shown in the Examples below, StemSpan Serum-Free Expansion Medium can also be suitably used for human hematopoietic stem cells. These basal media can also be suitably used for other blood cells capable of cell division.

[0057] <Other medium additives>

[0058] In one embodiment, the basal medium is supplemented with "stem cell factor (SCF)." SCF is a type of hematopoietic factor (cytokine). Specifically, it is a ligand for the tyrosine kinase receptor encoded by the KIT gene locus. SCF is also referred to as KIT ligand or KITLG. Human-derived SCF is a protein consisting of the amino acid sequence set forth in NCBI Reference Sequence: NP_000890 or NP_003985. Mouse-derived SCF is a protein consisting of the amino acid sequence set forth in NCBI Reference Sequence: NP_001334085 or NP_038626.

[0059] In one embodiment, the concentration of SCF is between 10 and 1,000 ng / mL. The concentration may be any of 20, 50, 100, 200, and 500 ng / mL.

[0060] In one embodiment, thrombopoietin (TPO) is added to the basal medium. TPO is a type of hematopoietic factor (cytokine). Specifically, it is a glycoprotein involved in the proliferation and differentiation of platelet precursor cells (megakaryocytes). TPO is also called THPO, thrombopoiesis-stimulating factor (TSF), megakaryocyte colony-stimulating factor (MK-CSF), megakaryocyte-stimulating factor, or megakaryocyte-potentiating factor. Human-derived TPO is a protein consisting of the amino acid sequence set forth in the NCBI reference sequences: NP_000451, NP_001171068, NP_001171069, NP_001276926, or NP_001276927. Mouse-derived TPO is a protein consisting of the amino acid sequence set forth in the NCBI reference sequences: NP_001166976, NP_001276823, NP_001276825, or NP_033405.

[0061] In one embodiment, the concentration of TPO is 10 to 1,000 ng / mL. The concentration may be any of 20, 50, 100, 200, and 500 ng / mL.

[0062] In one embodiment, the basal medium is supplemented with "low density lipoprotein (LDL)." In one embodiment, the concentration of LDL is 10 to 1,000 ng / mL. The concentration may be any of 20, 50, 100, 200, and 500 ng / mL.

[0063] In one embodiment, "Flt3-ligand (Fms-like tyrosine kinase 3 ligand)" is added to the basal medium. In one embodiment, the concentration of Flt3-ligand is 10 to 1,000 ng / mL. The concentration may be any of 20, 50, 100, 200, and 500 ng / mL.

[0064] In one embodiment, the basal medium is supplemented with "low density lipoprotein (LDL)." In one embodiment, the concentration of LDL is 10 to 1,000 ng / mL. The concentration may be any of 20, 50, 100, 200, and 500 ng / mL.

[0065] In one embodiment, the basal medium may or may not contain a compound selected from the group consisting of UM171, Soluplus, 740Y-P, and butizamide. When UM171 is added, the concentration is, for example, 3.5 to 350 nM. The concentration may be any of 7, 17.5, 35, 70, or 175 nM.

[0066] Cited from Non-Patent Document 3

[0067] In one embodiment, the basal medium is supplemented with or without polyvinyl alcohol (PVA). In one embodiment, the basal medium is free of PVA, UM171, Soluplus, 740Y-P, butizamide, and other non-biologically derived components.

[0068] The medium of this embodiment may further contain well-known and commonly used medium additives in addition to those described above. Examples of such medium additives include functional proteins (insulin, transferrin, lactoferrin, etc.), lipids other than fatty acids (cholesterol, etc.), reducing agents (2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides (nucleosides, cytidine, adenosine 5'-monophosphate, hypoxanthine, thymidine, etc.), metal salts (iron (III) nitrate, iron (II) sulfate, copper sulfate, sulfite, etc.), and the like. lead, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenious acid), organic compounds (para-aminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putrescine, pyruvic acid, lactic acid, triiodothyronine, etc.), antibiotics (penicillin, streptomycin, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), pH indicators (phenol red, etc.), but are not limited to these.

[0069] One aspect of this embodiment is a culture medium kit comprising two or more of the above-mentioned compound (I), a basal medium, and a medium additive, separated from one another. By using the culture medium kit, fresh medium can be prepared in a timely manner.

[0070] <Culture environment>

[0071] It is preferable to culture hematopoietic stem cells under an oxygen concentration lower than the oxygen concentration in normal air, for example, 20% by volume. Under such hypoxic conditions, the oxygen concentration in the gas in contact with the culture medium during culture is preferably 1 to 15% by volume, more preferably 1 to 10% by volume, and even more preferably 1 to 5% by volume. The carbon dioxide concentration in the gas is typically 1 to 10% by volume, preferably 2 to 5% by volume. Culture under such a low oxygen concentration can be carried out, for example, by culturing in a commercially available hypoxic incubator. The culture temperature is not particularly limited, but is typically 30 to 40°C, preferably approximately 37°C. These culture conditions can also be used suitably for other hematopoietic cells capable of cell division.

[0072] Known culture vessels can be used appropriately for culturing hematopoietic stem cells. The culture vessel may be non-adhesive. The culture vessel may be coated with a cell-adhesive substance, for example, a cell-supporting substrate such as ECM, Matrigel, gelatin, or collagen. Hematopoietic stem cells may be cultured in either non-adhesive or adherent culture. Examples of non-adhesive culture include dispersed culture, aggregated suspension culture, and suspension culture on a carrier. These culture vessels and culture formats can also be suitably used for other blood cells capable of cell division.

[0073] It is preferable to select a value suitable for cell expansion as the number of cells to be seeded in a unit amount of medium. For example, 3 × 10 cells per mL of medium. ０ From 3 x 10 ６ The number of cells was 3 x 10 per mL of medium. １ , 1×10 ２ , 3 x 10 ２ , 1×10 ３ , 3 x 10 ３ , 1×10 ４ , 3 x 10 ４ , 1×10 ５ , 3 x 10 ５ , 1×10 ６ One piece is fine.

[0074] When changing the medium, 1 / 2 to 1 / 4 of the cells were left, and the number of cells in the medium was 3 x 10 per mL of medium. ０ From 3 x 10 ６ The differentiated hematopoietic stem cells may be removed from the medium at a frequency of once every 1 to 10 medium changes, preferably once every 3 to 4 medium changes. At this time, the hematopoietic stem cells may be purified by processing the cells with a cell sorter. These culture formats can also be suitably used for other blood cells capable of cell division.

[0075] In one embodiment, hematopoietic cells are expanded without being mixed with hematopoietic cells at other stages of differentiation. In one embodiment, hematopoietic cells are expanded, and differentiated hematopoietic cells resulting from the expansion of hematopoietic cells are also expanded. In one embodiment, the hematopoietic cells are hematopoietic stem cells that have not yet differentiated into multipotent progenitor cells. In another embodiment, the hematopoietic cells are multipotent progenitor cells that have already completed differentiation from hematopoietic stem cells and have not yet differentiated into progenitor cells of differentiated hematopoietic cells. In one embodiment, the hematopoietic cells are differentiated hematopoietic cells that have already completed differentiation from their progenitor cells. In one embodiment, differentiated hematopoietic cells are, for example, monocytes, macrophages, lymphocytes, neutrophils, basophils, and eosinophils. Lymphocytes are, for example, T lymphocytes and B lymphocytes.

[0076] In one embodiment, hematopoietic stem cells are cultured without being mixed with other cells. Hematopoietic stem cells are expanded without being mixed with other cells, such as bone marrow stromal cells. In another embodiment, hematopoietic stem cells are expanded while being mixed with other cells, such as bone marrow stromal cells. These culture formats can also be suitably used for other blood cells capable of cell division.

[0077] In one embodiment, the length of the culture period is from 1 day to 6 months, for example, 1, 2, 4, 6, or 13 weeks. In one embodiment, the passage interval is from 5 to 30 days, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. Passaging may be unnecessary. In one embodiment, the interval between medium changes is from 2 to 10 days, for example, 4, 5, 6, or 7 days. Medium changes may be unnecessary.

[0078] In one aspect, a culture kit is provided that includes a medium supplemented with compound (I) or a medium kit containing compound (I). In addition to the medium kit, the culture kit includes an incubator, a hypoxic incubator, and other cells. In one aspect, the culture kit further includes a reagent for confirming that the hematopoietic stem cells maintain their undifferentiated state. Such reagents include antibodies that recognize the markers described below, labeled secondary antibodies that recognize the antibodies, and magnetic beads bound to any of these antibodies. Furthermore, the kit can include instructions for use that describe the conditions for expanding hematopoietic stem cells. These kit formats can also be suitably used for other blood cells capable of cell division.

[0079] <Preparation of hematopoietic stem cells and other blood cells capable of cell division to be expanded>

[0080] From one perspective, "hematopoietic stem cells" are cells that have the pluripotency to differentiate into all blood cell lineages of blood cells and are capable of self-replication while maintaining this pluripotency. From one perspective, "blood cell" is a cell of a cell lineage that can differentiate from hematopoietic stem cells. From one perspective, blood cell cells capable of cell division are blood cells excluding red blood cells and platelets. Multipotent progenitor cells differentiate from hematopoietic stem cells. Progenitor cells of each differentiated blood cell, for example, granulocyte progenitor cells, differentiate from these progenitor cells. Each differentiated blood cell, for example, white blood cells, differentiates from these progenitor cells.

[0081] The animal species from which hematopoietic stem cells are derived, i.e., the donor, is not particularly limited, but is preferably a vertebrate, and more preferably a mammal. Examples of mammals include, but are not limited to, primates such as humans, monkeys, and marmosets; rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, sheep, cattle, goats, and horses; and carnivores such as dogs and cats. The hematopoietic stem cells according to this embodiment are preferably hematopoietic stem cells derived from rodents such as mice or primates such as humans, and more preferably hematopoietic stem cells derived from humans. The same applies to other blood cells capable of cell division.

[0082] The age of the donor is not particularly limited. Applying the method of this embodiment to hematopoietic stem cells collected from an aged donor, particularly primary cultured hematopoietic stem cells, is expected to functionally rejuvenate these hematopoietic stem cells. Here, the "aged donor" may be an adult. For example, in the case of humans, an adult may be 20 to 100 years old, and may be further narrowed to 40 to 100 years old. However, if the donor is of childbearing age or age, the phrase "hematopoietic stem cells collected from a donor" excludes hematopoietic stem cells collected from the side of the donor's placenta closer to the fetus or newborn, such as hematopoietic stem cells derived from umbilical cord blood. Because hematopoietic stem cells derived from umbilical cord blood are considered to be derived from fetal or newborn cells rather than maternal cells, they may not apply to hematopoietic stem cells expected to be rejuvenated by the treatment of this embodiment. Furthermore, the scope of "aged donor" may be narrowed to older individuals. When the donor is a mouse, the aged donor may be, for example, an individual between 80 weeks of age and the age of death due to lifespan. When the donor is a human, the age of the aged donor may be, for example, 56 to 100 years old. In one aspect, hematopoietic stem cells derived from an aged donor have a reduced ability to differentiate into cells of the T lymphocyte lineage compared to hematopoietic stem cells derived from a younger donor. In one aspect, rejuvenating hematopoietic stem cells means increasing their ability to differentiate into cells of the T lymphocyte lineage. Thus, no method has been known to restore the function of hematopoietic stem cells that declines with age. Hematopoietic stem cells collected from an aged donor may be rejuvenated and then autologously transplanted. The same applies to other blood cells capable of cell division.

[0083] The tissue from which hematopoietic stem cells are derived is not particularly limited as long as the cells are present, but is preferably a hematopoietic tissue, such as bone marrow, umbilical cord blood, peripheral blood, or liver. The same applies to other blood cells capable of cell division.

[0084] Hematopoietic stem cells may also be induced from pluripotent stem cells such as iPS cells or ES cells. Such pluripotent stem cells may be so-called universal cells, cells that have been made defective in antigen presentation by HLA class 1 molecules. Hematopoietic stem cells induced from such universal pluripotent stem cells are transplanted into a recipient. Hematopoietic stem cells that are universal cells are less susceptible to immune rejection after transplantation. The same applies to other blood cells capable of cell division.

[0085] Hematopoietic stem cells are usually isolated from the above tissues using as an indicator surface markers that are specifically expressed in the cells (hematopoietic stem cell-specific markers) or surface markers that are not expressed. Regarding the presence or absence of expression of such markers, human-derived hematopoietic stem cells are usually CD34-positive (+) cells or CD34-positive (+) CD38-negative (-) CD90-positive (+) CD45RA-negative (-) cells, and preferably CD34 ＋ CD38 － CD90 ＋ CD45RA － CD49f ＋ In another embodiment, the hematopoietic stem cells derived from a human are CD34 ＋ CD38 － CD90 ＋ CD45RA － EPCR ＋ Furthermore, mouse-derived hematopoietic stem cells are usually CD150 ＋ CD48 － Flt3 － LSK, preferably CD150 ＋ CD48 － CD41 ― CD34 － Flt3 － In another embodiment, the hematopoietic stem cells derived from a human are CD150 ＋ CD48 － EPCR ＋ LSK is a cell that is a Lineage. － Sca-1 ＋ c-Kit ＋ In one embodiment, the hematopoietic stem cells to be expanded have not yet differentiated into hematopoietic progenitor cells.

[0086] When cells in the hematopoietic tissue are bound together by stroma, they are first dissociated into individual cells by treatment with protease, collagenase, etc. On the other hand, when individual cells are separated, as in blood tissue, such as peripheral blood, they are subjected to the isolation of hematopoietic stem cells described below without any cell separation treatment. The same applies to other blood cells capable of cell division.

[0087] The isolation method for hematopoietic stem cells is not particularly limited, and can be carried out by appropriately using known methods. For example, as shown in the Examples below, cells having the characteristics of the hematopoietic stem cell-specific markers can be isolated by using antibodies against the above-mentioned hematopoietic stem cell-specific markers and devices such as a cell sorter or magnetic beads. This isolation process purifies the hematopoietic stem cells. Such purification may also be carried out during the subculture of the hematopoietic stem cells. The same applies to other blood cells capable of cell division.

[0088] <Cloning of hematopoietic stem cells and other blood cells capable of cell division>

[0089] One aspect of this embodiment is the in vitro production of clones of hematopoietic stem cells and other blood cells capable of cell division. In this method, compound (I) or a physiologically acceptable salt thereof is applied to the hematopoietic stem cells, causing the hematopoietic stem cells to expand. One aspect of the application of compound (I) or a salt thereof is culturing the hematopoietic stem cells in a medium containing compound (I) or a salt thereof. This production method can provide a large amount of clones that maintain undifferentiated state. The same applies to other blood cells capable of cell division.

[0090] Another aspect of this embodiment is a clone of a hematopoietic stem cell obtained by expanding the hematopoietic stem cell as described above. In this embodiment, a clone refers to a group of identical cells that share a common ancestor. A clone may be derived from a single ancestor, i.e., monoclonal. A clone may be derived from a small number of ancestors, i.e., oligoclonal. A clone may be derived from a large number of ancestors, i.e., polyclonal. These clones maintain undifferentiated status in a state similar to or younger than their ancestors. The same applies to other blood cell lines capable of cell division.

[0091] In one embodiment, the progenitor hematopoietic stem cells, i.e., the hematopoietic stem cells before administration of compound (I) or a physiologically acceptable salt thereof, are from an aged donor. Such clones have a higher ability to differentiate into the T lymphocyte lineage than their progenitors. The same is true for other blood cells capable of cell division.

[0092] In one embodiment, the clones include cells that have differentiated into hematopoietic progenitor cells or cells of a subsequent cell lineage. In one embodiment, the proportion of clones that maintain an undifferentiated state is 30% to 100%, for example, 50% or more or 70% or more. The proportion of clones that maintain an undifferentiated state can be identified using a cell sorter with an antibody against the marker. In one embodiment, the marker is expressed specifically in hematopoietic stem cells and not in differentiated cells. In another embodiment, the marker is not expressed specifically in hematopoietic stem cells and is expressed in differentiated cells. The same applies to other blood cells capable of cell division.

[0093] Whether a clone functions as a hematopoietic stem cell can be examined by transplanting the clone into an immunodeficient mouse, such as a NOG mouse or a NOD / SCID mouse. The cloned cells to be examined are transplanted into the mouse, and the migration or engraftment, pluripotency, and bone marrow reconstitution ability of the transplanted cells are detected. Based on these detection results, it can be confirmed that the clone maintains its function as a hematopoietic stem cell. Furthermore, the bone marrow of an immunodeficient mouse after successful transplantation can be collected and transplanted again into another immunodeficient mouse, i.e., a secondary transplant, to measure the self-renewal ability and long-term bone marrow reconstitution ability of the transplanted cells. The same applies to other blood cell lines capable of cell division.

[0094] Implants and Pharmaceutical Compositions

[0095] The clones are useful in cell therapy and regenerative medicine. In one embodiment, the clones are transplanted into a recipient whose bone marrow hematopoietic capacity is impaired. In one embodiment, the clones can engraft in the bone marrow, restoring the bone marrow's hematopoietic capacity. The clones can be used as grafts for hematopoietic stem cell therapy, replacing grafts used in bone marrow transplantation and umbilical cord blood transplantation. In one embodiment, the clones are used in pharmaceutical compositions. The same applies to other blood cells capable of cell division.

[0096] The pharmaceutical composition of this embodiment can be prepared as a parenteral preparation such as an injection, suspension, or infusion by mixing with a pharmaceutically acceptable carrier according to conventional means. Examples of pharmaceutically acceptable carriers that can be included in such parenteral preparations include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride), etc. The preparations may also be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride), stabilizers (e.g., serum albumin, polyethylene glycol), preservatives, antioxidants, etc.

[0097] Furthermore, clones are effective in treating various diseases, including leukemia, which requires bone marrow transplantation, etc. Therefore, hematopoietic stem cells maintained by the method of this embodiment are effective for diseases accompanied by a decrease in hematopoietic stem cells and / or a decrease in hematopoietic function, diseases accompanied by hematopoietic dysfunction, a decrease in immune cells, an increase in immune cells, diseases accompanied by autoimmunity, immune dysfunction, and ischemic diseases. The same applies to other blood cells capable of cell division.

[0098] Specific examples of diseases related to hematopoietic function include blood cancers such as acute or chronic leukemia, chronic granulomatous disease, severe combined immunodeficiency syndrome, adenosine deaminase (ADA) deficiency, agammaglobulinemia, immunodeficiency syndromes such as Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, and acquired immunodeficiency syndrome (AIDS), C3 deficiency, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, Gaucher's disease, and Mueller's disease. Examples of such conditions include lysosomal storage diseases such as copolysaccharidosis, adrenoleukodystrophy, Fanconi syndrome, aplastic anemia, granulocytopenia, lymphopenia, thrombocytopenia, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, Kasabach-Merritt syndrome, malignant lymphoma, Hodgkin's disease, multiple myeloma, chronic liver damage, autoimmune hemolytic anemia, myelodysplastic syndrome, polycythemia vera, polycythemia erythrocytosis, essential thrombocythemia, myeloproliferative disorders, and spinal cord injury due to trauma or the like.

[0099] The transplant may be either allogeneic or autologous. For example, in cases where bone marrow suppression occurs as a side effect of chemotherapy, radiation therapy, or the like in a patient with solid cancer, bone marrow can be collected before the treatment, and clones produced by the method of this embodiment can be returned to the patient after the treatment, allowing for early recovery from damage to the hematopoietic system. This method allows for more powerful chemotherapy, etc., and improves the therapeutic effect.

[0100] Furthermore, the clone contained in the pharmaceutical composition of this embodiment may be a clone of a cell that has been genetically modified for gene therapy. Genetic modification may also be performed on the clone. The gene to be modified may be selected depending on the target disease and is not particularly limited, but examples include genes for hormones, cytokines, receptors, enzymes, polypeptides, etc. Furthermore, genetic modification can be performed by those skilled in the art using known techniques, such as gene transfer using viral vectors, genome editing, knockout methods, siRNA methods, shRNA methods, ribozyme methods, and antisense methods. Furthermore, such treatments using genetic modification of hematopoietic stem cells can be applied to the treatment of congenital genetic diseases. Examples of such treatments include the introduction of an ADA gene for adenosine deaminase (ADA) deficiency, the introduction of a γc chain gene for X-linked severe combined immunodeficiency (X-SCID), the introduction of a gp91-phox gene for chronic granulomatous disease, and the introduction of a glucocerebrosidase gene for Gaucher disease. The same applies to other hematopoietic cells capable of cell division.

[0101] Furthermore, this embodiment provides a method for treating a disease related to hematopoietic function, which comprises administering to a patient a clone obtained in this embodiment or a pharmaceutical composition containing the clone as an active ingredient.

[0102] The clones, pharmaceutical compositions, and diseases related to hematopoietic function are as described above. The method of administration of the cells and other substances varies depending on the age, weight, sex, condition, etc. of the recipient, but can be administered via any of the following routes: oral administration or parenteral administration (e.g., intravenous administration, arterial administration, or topical administration). The dose of the clone also varies depending on the age, weight, sex, condition, etc. of the recipient, but can be adjusted appropriately by one skilled in the art.

[0103] <1. Contents of the Example>

[0104] Hereafter, hematopoietic stem cells (HSCs) are referred to as HSCs. Mouse and human hematopoietic stem cells are expanded using thyroid hormones (T4 and T3) and their metabolites, rT3 and T2 (3,3'-Diiodo-L-thyronine and 3,5-Diiodo-L-thyronine). HSCs can be expanded simply by adding thyroid hormones or their metabolites to conventional HSC culture media. Furthermore, aging HSCs can be functionally rejuvenated.

[0105] <2. Comparison with conventional technology>

[0106] (1) Regarding conventional technology: Although HSC expansion technology is thought to be useful in gene and cell therapy and regenerative medicine, it has not been established until very recently. Molecules such as PVA and UM171, as mentioned above, can expand either mouse or human HSCs. These molecules do not naturally exist in vivo. In other words, physiological HSC expansion factors have not been identified. Furthermore, HSC function declines with age, but there is no method for restoring this function using external factors.

[0107] (2) Differences from conventional techniques: This example can be applied to both mouse and human HSCs. The proportion of HSCs in all expanded cells is maintained at a higher level than with conventional expansion techniques. Furthermore, this example is not limited to simple expansion techniques. This example also restores the function of aging HSCs. That is, in this example, (i) the engraftment rate of HSCs transplanted into the living body is improved, and (ii) a significant improvement in lymphocyte production capacity is observed. In other words, functional rejuvenation of HSCs is observed.

[0108] Furthermore, unlike PVA and UM171, thyroid hormones or their metabolites are physiological factors. Therefore, the biological availability of these compounds and mimic compounds is high. Furthermore, cell expansion is simple, requiring only the addition of these compounds to the culture medium, and the cost of expansion is low. However, the combined use of PVA or UM171 with thyroid hormones or their metabolites is not excluded from one aspect of the present invention.

[0109] (3) Applications of the Examples: A physiological expansion factor for HSCs has been identified. This will lead to improvements in transplantation techniques for hematopoietic stem cells obtained from umbilical cord blood. Furthermore, the methods of the Examples are expected to lead to improvements and the development of new gene and cell-based therapeutic techniques. Furthermore, a technique for functionally rejuvenating aging HSCs using external factors is provided.

[0110] <3-1. Short-term culture of HSCs>

[0111] Methods: By processing a population containing mouse hematopoietic stem cells using a cell sorter, we identified cells with the surface phenotype CD150. + CD48 - EPCR + Lineage - Sca-1 + c-Kit + These purified cells were then enriched for mouse HSCs. These purified cells are hereafter referred to as mouse HSCs. Three hundred mouse HSCs were cultured for one week at 37°C under 5% CO2 in a medium containing SF-O3 (Sanko Junyaku), 100 ng / mL SCF, and 100 ng / mL TPO. The number of cells in each colony that emerged after culture was counted. All subsequent culture experiments were performed at 37°C under 5% CO2. The concentrations of various thyroid hormones and their metabolites were all 10 μM. The control group (Ctl) did not contain any thyroid hormones or their metabolites.

[0112] Results: The thyroid hormone and its metabolite groups were compared with the control group (Ctl). The graph in Figure 1 shows the average total cell number in each colony. The circles indicate the counts in each colony. The error bars indicate the standard deviation. Ctl on the horizontal axis represents the absence of thyroid hormone and its metabolites. The graph in Figure 2 shows the mean fluorescence intensity (MFI) of CD48 staining in each colony. The circles indicate the measurements in each colony. The error bars indicate the standard deviation.

[0113] In Figures 1 and 2, T4 is thyroxine (T ４ ) T3 represents 3,3',5-triiodo-L-thyronine (T ３ ) rT3 represents 3,3',5'-triiodothyronine (rT ３) 3,3'T2 represents 3,3'-diiodo-L-thyronine (3,3'-T ２ ) 3,5T2 represents 3,5-diiodo-L-thyronine (3,5-T ２ ) 3',5'T2 represents 3',5'-diiodo-L-thyronine (3',5'-T ２ ) T1AA stands for 3-iodothyroacetic acid (3-T １ T1AM stands for 3-iodothyronamine (3-T １ AM). L-T0 represents L-thyronine (T0). DL-T0 represents the racemic form of thyronine.

[0114] As shown in Figure 1, the use of thyroid hormones and their metabolites, T4, T3, rT3, 3,3'-Diiodo-L-thyronine (3,3'-T2), and 3,5-Diiodo-L-thyronine (3,5-T2), resulted in a high number of HSCs after culture. Furthermore, as shown in Figure 2, the use of these compounds maintained low levels of CD48 expression. CD48 is known as a marker for cells differentiated from HSCs. Of the compounds, 3,3'-T2 had the greatest suppression of CD48 expression.

[0115] <3-2. Long-term culture of HSCs>

[0116] Methods: Thirty mouse HSCs were cultured long-term in a medium containing SF-O3, SCF 100ng / mL, TPO 100ng / mL, and 3,3'-T2 10μM (hereafter referred to as mouse T2 medium). The number of HSCs was recorded at each time point. Mouse HSCs in the control group were cultured in mouse T2 medium, but without 3,3'-T2. The first medium change was performed one week after the start of culture, and thereafter, the medium was changed every 4-5 days. By leaving 1 / 2 to 1 / 4 of the total cells during the medium change, the total number of cells in the medium was kept at 3 x 10 6 The concentration was kept below 100 cells / mL. Differentiated blood cells were removed from the medium every 3 to 4 medium changes. The cells were purified using a cell sorter to obtain a mouse HSC fraction. The culture was continued for a long period of time.

[0117] When calculating the number of HSCs, we took into account the dilution of HSCs during medium changes. Specifically, we first determined the number of HSC clones generated from the initial HSCs in the medium at the time of counting, and then calculated the number of expanded HSCs by converting the dilution factor. This takes into account the fact that HSC culture involves cell expansion, and therefore cell numbers tend to diverge over time. For example, continuing to culture all expanded cells may require 1 L, 2 L, or even more of medium. However, depending on the intended use of the cultured cells, the number of cells obtained with tens to hundreds of mL of medium may be sufficient. Therefore, even if we initially started with 30 HSCs, after expansion, we discarded a portion of the cells to retain only the required number of cells. For example, at the time of medium change, half of the cells could be discarded and only the remaining half could be continued. This allows us to limit the amount of medium used for cell culture to the required number of cells. For example, at a certain point in culture, we may be culturing "15 initial HSCs," i.e., a dilution factor of 2. Alternatively, this could be "a situation where an initial HSC equivalent to one cell is being cultured," i.e., a dilution factor of 30. If the culture further progresses and half of the cells are discarded, this would be "a situation where an initial HSC equivalent to 0.5 cells is being cultured," i.e., a dilution factor of 60. By repeatedly leaving only a portion of the cells, the number of HSCs in the initial culture is diluted to a decimal point. In this example, the number of HSCs that could have been expanded if the culture had originally started with 30 HSCs is calculated. For example, suppose 10 HSCs thought to be derived from one HSC are detected in the medium after replacement. In this case, the number of HSCs in the initial culture was 30, so the dilution factor is 30. Therefore, it can be calculated that 30 HSCs have expanded to 10 x 30 = 300 HSCs.

[0118] Results: The graph in Figure 3 shows the change in cell number during culture. Each point represents the average result of independent trial experiments. Error bars represent standard deviation. In the mouse T2 medium group, the number of HSCs rapidly increased compared to the control group, and this trend continued. In other words, the HSC pool expanded, and this trend continued. In the control group, 99.7% of all cells had died about 6 weeks into culture. At this point, culture of the control group cells was discontinued. From this point on, culture of only the mouse T2 medium group cells continued. About 3 months after the start of culture, the number of cells was about 5 x 10 of the number of cells at the start of culture. 7 increased to twice as much.

[0119] <3-3. Functional evaluation of cultured HSCs>

[0120] Methods: Figure 4 shows an outline of the chimerism experiment. Donor mice (Dnr, Ubc-GFP mice) expressing GFP under the Ubc (Ubiquitin C) promoter were bred. In the donor mice, blood cells other than red blood cells and platelets are Ly5.2 positive. HSCs collected from the donor mice were purified. 100 of these HSCs (GFP + HSCs were cultured in mouse T2 medium for two weeks. Separately, recipient mice (Ly5.1 mice) were prepared after irradiating with 8.5 Gy of radiation (rd). In the recipient mice, all blood cells except red blood cells and platelets are Ly5.1 positive.

[0121] Whole cells after culture i.e. GFP + Total cells derived from 100 HSCs were cultured in a 2000-well plate, and 4 × 10 bone marrow mononuclear cells (MNCs) derived from Ly5.1 mice were cultured in a 2000-well plate. 5 The total number of cells after culture was more than 100, and these cells were expanded by culture in mouse T2 medium. The control group (Ctl) Ly5.1 mice were given 100 fresh GFP cells purified by cell sorter. + HSCs were transplanted together with MNCs. + The HSCs were not cultured in mouse T2 medium. After one month of growth, peripheral blood was collected from each recipient mouse and further fractionated.

[0122] Donor-derived chimerism was calculated from the frequency of Ly5.2-positive cells and Ly5.1-positive cells in the peripheral blood. This calculated chimerism indicated the granulocyte (Gr-1) abundance in each recipient mouse. + or Mac-1 + ), B lymphocytes (B220 + ), T lymphocytes (CD4 + or CD8 + ), red blood cells (Ter119 + ), platelets (CD41 + The efficiency of generating donor cells was verified for each fraction of peripheral blood, with respect to blood cells other than red blood cells and platelets (Ly5.2). + cell count) / {(Ly5.2 + Number of cells) + (Ly5.1 + On the other hand, the chimerism of donor-derived cells for red blood cells and platelets was calculated using the formula (GFP + number of cells) / {(GFP + number of cells) + (GFP - The calculation was carried out using the formula:

[0123] Results: The graph in Figure 5 shows the results of chimerism analysis. Circles indicate the actual chimerism (%) in each recipient. Error bars indicate standard deviation. *** indicates a P value below the significance level of α = 0.001. In the group of cells cultured in mouse T2 medium (T2 group on the horizontal axis of the graph), the progeny of donor mouse HSCs were superior to the progeny of MNCs in all blood cell types compared to the control group (Ctl on the horizontal axis of the graph).

[0124] <3-4. Expansion of human umbilical cord blood HSCs>

[0125] Methods: Human umbilical cord cells provided by a cord blood bank under ethical approval were purified using a cell sorter to isolate HSCs (CD34 + CD38 - CD90 + CD45RA - EPCR +) were obtained. Hereafter, cells with similar surface phenotypes are referred to as human HSCs. Medium containing StemSpan™ Serum-Free Expansion Medium (STEMCELL Technologies), 100 ng / mL human SCF, 100 ng / mL human Flt3-ligand, 100 ng / mL human TPO, 10 μg / mL human LDL, and 35 nM UM171 was dispensed into well plates. 100 human HSCs were seeded into each well. 10 μM 3,3'-T2 was added to the medium (T2+ group). Hereafter, this medium containing 3,3'-T2 is referred to as human T2 medium. The control group (T2-) medium contained DMSO instead of 3,3'-T2. The control group (T2-) medium contained the above ingredients, including 35 nM UM171. The first medium change was performed one week after the start of culture. Thereafter, 3 / 4 of the medium was changed every three days. Two weeks after the start of culture, the number of human HSCs was counted using a flow cytometer.

[0126] Results: The graph in Figure 6 shows the number of hematopoietic stem cells in each colony. Circles indicate the counts in each colony. Error bars indicate standard deviation. * indicates a P value below the significance level of α=0.05. We found that human HSCs can be expanded more efficiently by culturing in human T2 medium. We also found that thyroid hormone metabolites can promote expansion in addition to the promotion of expansion by UM171.

[0127] 3-5. Expansion and rejuvenation of HSCs derived from aged mice

[0128] Method: A chimerism experiment was performed following the procedure shown in Experimental Example 3 and Figure 4. An 80-week-old mouse was selected as the donor mouse Dnr. 300 HSCs derived from the aged mouse were used per recipient mouse. HSCs were cultured in mouse T2 medium for 4 weeks. One month after HSC transplantation, peripheral blood was collected from the recipient mouse. Granulocytes (Gr-1 + or Mac-1 + ), B lymphocytes (B220 + ), T lymphocytes (CD4 + or CD8 +Chimerism derived from the donor mice was verified in the same manner as in Experiment 3.

[0129] Results: The graph in Figure 7 shows the results of chimerism analysis. Circles indicate the actual chimerism (%) for each recipient. Error bars indicate standard deviation. *** indicates a P value below the significance level of α = 0.001. One month after transplantation, the group transplanted with cells cultured in mouse T2 medium (T2 on the horizontal axis of the graph) showed that the progeny of donor mouse HSCs were superior to the progeny of MNCs in all blood types compared to the control group (Ctl on the horizontal axis of the graph).

[0130] The graph in Figure 8 shows the percentage of each blood cell type in mouse blood. Error bars indicate standard deviation. Aged HSCs are characterized by a granulocyte-predominant hematopoiesis (myeloid bias). Therefore, in the control (Ctl) group, granulocytes competed with lymphocytes, i.e., the sum of T and B lymphocytes. In contrast, in the group transplanted with aged HSCs cultured in mouse T2 medium, lymphocytes predominated over granulocytes. T2-treated aged HSCs were shown to undergo lymphocyte-predominant hematopoiesis similar to that of young HSCs. The effect of T2 treatment was not simply an improvement in HSC expansion; rather, T2 treatment functionally rejuvenated the properties of HSCs.

[0131] <3-6. Replacement with thyroid hormone derivatives>

[0132] Culturing mouse-derived HSCs in medium containing the thyroid hormone derivative sobetirom (manufacturer: MedChem Express, product code: HY-14823) promoted HSC expansion and inhibited HSC differentiation.

[0133] 3-7. Expansion of mouse-derived HSCs in serum-free medium

[0134] Instead of the serum-containing mouse T2 medium, mouse-derived cells were cultured in serum-free medium containing PVA and T2. This serum-free medium also promoted HSC expansion, while suppressed HSC differentiation. The optimal concentration of T2 was 0.1 μM.

[0135] <3-8. Carboxylic acid derivatives of thyronine>

[0136] As shown in Figure 1, T1AA, or 3-iodothyroacetic acid (3-T １ T1AA tended to increase cell proliferation, and T1AA tended to decrease CD48 expression, as shown in Figure 2. Therefore, we investigated the effects of carboxylic acid derivatives of other compounds.

[0137] Figure 9 is a graph showing the percentage of EPCR-positive HSCs (EPCR+HSCs) after culturing HSCs in the presence of a carboxylic acid derivative of thyronine. T3 is 3,3',5-triiodo-L-thyronine (T ３ ) T3 acetic acid represents 3,3',5-triiodo-thyroacetic acid (T ３ 35T2 represents 3,5-diiodo-L-thyronine (3,5-T ２ ) 35T2 acetic acid stands for 3,5-diiodo-thyroacetic acid (3,5-T ２ 33T2 represents 3,3'-diiodo-L-thyronine (3,3'-T ２ In all groups, the proportion of EPCR+ HSCs was increased compared to the control group that received only DMSO.

[0138] <3-9. Amplification of other blood cells>

[0139] The effects of the compounds on the expansion of blood cells other than hematopoietic stem cells were examined.

[0140] Figure 10 shows the cell counts after 4 weeks of culture of blood cells in the presence of T2. DMSO alone was added to the control group. Differentiated blood cells are c-Kit-negative cells. MPP3, MPP2, and MPP1 are multipotent progenitor cells known as hematopoietic progenitor cells. EPCR-HSCs are EPCR-negative HSCs. EPCR-HSCs are slightly less functional than EPCR-positive HSCs. EPCR+HSCs are EPCR-positive HSCs. EPCR+HSCs are the most undifferentiated group of HSCs.

[0141] As shown in Figure 10, in the group where EPCR HSCs were cultured in the presence of T2, EPCR HSCs expanded more than in the control group (DMSO). The expansion rate was 204-fold. In addition to EPCR HSCs, other cells derived from EPCR HSCs, such as EPCR-HSCs, MPP3, MPP2, and MPP1, and differentiated blood cells, also expanded. The compound of this embodiment, which is effective in expanding hematopoietic stem cells, also contributes to the expansion of blood cells other than HSCs, such as multipotent progenitor cells and differentiated blood cells. The compound of this embodiment is effective in expanding EPCR HSCs, and increases in EPCR-HSCs, multipotent progenitor cells (e.g., MPP3, MPP2, and MPP1), and differentiated blood cells originating from EPCR HSCs were observed, similar to normal hematopoiesis. This demonstrates the establishment of a method for promoting hematopoietic stem cell expansion.

[0142] The present invention is not limited to the above and can be modified as appropriate within the scope of the spirit of the present invention. This application claims priority based on Japanese Patent Application No. 2023-102048, filed on June 21, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0143] "Ctl": control group, "Dnr": donor, "HSC": hematopoietic stem cells, "MNC": (bone marrow) mononuclear cells, "T2": group of cells cultured in mouse T2 medium, "rd": radiation

Claims

1. A medium for expanding blood cells capable of cell division, comprising a compound (I) of the following formula or a physiologically acceptable salt thereof: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ４ is a linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ５ is any one of oxygen, methylene and a direct bond; R ６ is hydrogen or -NH ２ And R ７ is hydrogen or -COOH, where R ６ and R ７ None of the medium is hydrogen.

2. (1) R ５ is oxygen or methylene; R ６ is hydrogen or -NH ２ And R ７ is hydrogen or -COOH, where R ６ and R ７ or (2) none of R ５ is a direct bond, R ６ is hydrogen; R ７ The medium according to claim 1 , wherein is —COOH.

3. Compound (I) is Thyroxine (T ４ ), 3,3',5-Triiodo-L-thyronine (T ３ ), 3,3',5'-Triiodothyronine (rT ３ ), 3,3'-Diiodo-L-thyronine (3,3'-T ２ ), 3,5-Diiodo-L-thyronine (3,5-T ２ ), 3-Iodo-L-thyronine (3-T １ ), 3-Iodo-L-thyroacetic acid (3-T １ A.A.), 3-Iodo-L-thyronamine (3-T １ A.M.), Sobechirom, 3,3',5-Triiodo-L-thyroacetic acid (3,3',5-T ３ AA) and 3,5-Diiodo-L-thyroacetic acid (3,5-T ２ The medium according to claim 1 , wherein the medium is selected from the group consisting of α- and β-amino acids, ... and β-amino acids.

4. R １ is hydrogen; R ２ and R ３ One of the groups is hydrogen, linear or branched C １ -C ４ 2. The medium according to claim 1, wherein the aryl group is any one of alkyl, bromine and iodine, and the others are hydrogen.

5. Compound (I) is 3,5-diiodo-L-thyronine (3,5-T ２ ), and 3,3'-diiodo-L-thyronine (3,3'-T ２ The medium according to claim 4, wherein the medium is any one of the above.

6. The medium of claim 1, wherein the blood cells are harvested from an aged donor, or the blood cells are harvested from umbilical cord blood.

7. The medium according to claim 1, wherein the blood lineage cells are expanded without being mixed with blood lineage cells at other stages of differentiation.

8. The medium according to claim 1, for expanding the blood cell lineage cells and further expanding differentiated blood cell lineage cells generated during the expansion of the blood cell lineage cells.

9. The medium described in claim 1, wherein the blood cell is any one of: hematopoietic stem cells that have not yet differentiated into pluripotent progenitor cells; pluripotent progenitor cells that have already completed differentiation from hematopoietic stem cells and have not yet differentiated into precursor cells of differentiated blood cells; and differentiated blood cells that have already completed differentiation from their precursor cells.

10. The medium according to claim 1, wherein the medium is a serum-free medium and contains compound (I), or the medium is a serum medium and contains 0.1 μM to 1 mM of compound (I).

11. Compound (I) of the following formula or a physiologically acceptable salt thereof is applied in vitro to a blood cell capable of cell division, thereby amplifying the blood cell: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ４ is a linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ５ is any one of oxygen, methylene and a direct bond; R ６ is hydrogen or -NH ２ And R ７ is hydrogen or -COOH, where R ６ and R ７ A method for producing clones of blood cell lines, none of which are hydrogen.

12. A clone of a blood cell capable of cell division obtained by applying a compound (I) of the following formula or a physiologically acceptable salt thereof to the in vitro amplification of the blood cell capable of cell division: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ４ is a linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ５ is any one of oxygen, methylene and a direct bond; R ６ is hydrogen or -NH ２ And R ７ is hydrogen or -COOH, where R ６ and R ７ none of the seeded blood cells are hydrogen; the seeded blood cells are taken from an aged donor; a clone.

13. A drug to be added to a serum medium or a serum-free medium for expanding blood cells capable of cell division in vitro, comprising the compound (I) of the following formula or a physiologically acceptable salt thereof as an active ingredient: ...(I) In the formula, R １ , R ２ and R ３ are each hydrogen, linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ４ is a linear or branched C １ -C ４ R is any one of alkyl, bromine and iodine; ５ is any one of oxygen, methylene and a direct bond; R ６ is hydrogen or -NH ２ And R ７ is hydrogen or -COOH, where R ６ and R ７ None of the drugs are hydrogen.

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