Method for producing hematopoietic cells from pluripotent stem cells

Abstract

A method for producing a hematopoietic cell from a pluripotent stem cell, the method including a first step for culturing naïve pluripotent stem cells and inducing differentiation into yolk sac cells, a second step for culturing primed pluripotent stem cells and inducing differentiation into mesoderm cells, and a third step for co-culturing the mesoderm cells obtained in the second step and the yolk sac cells obtained in the first step and thereby inducing differentiation into hematopoietic cells.

Description

Technical Field

[0001] The present invention relates to a method for producing hematopoietic cells from pluripotent stem cells. The present invention also relates to a method for producing blood cells such as red blood cells and microglia via hematopoietic cells from pluripotent stem cells.

Background Art

[0002] As methods for inducing differentiation of pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into hematopoietic cells, methods using formation of embryoid bodies and addition of cytokines (Non-Patent Documents 1 to 3), co-culture methods with stromal cells derived from different species (Non-Patent Document 4), etc. have been reported so far. However, the former requires many cytokines and thus is costly, and the latter uses heterologous cells, so there are concerns about the safety of the obtained hematopoietic cells. On the other hand, a method is known in which mesoderm cells are induced to differentiate from pluripotent stem cells and then further induced to differentiate into hematopoietic cells (Patent Documents 1 and 2). However, in order to be able to stably supply a large amount of blood cells, further improvement of the method for inducing differentiation into blood cells is required, and the development of new technologies suitable for medical applications has been demanded.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] An object of the present invention is to provide a method for efficiently producing hematopoietic cells using pluripotent stem cells. [Means for Solving the Problems]

[0006] The present inventors conducted intensive studies to solve the above problems. As a result, by using naive pluripotent stem cells and primed pluripotent stem cells, inducing differentiation of naive pluripotent stem cells into yolk sac-like cells via primitive endoderm, inducing differentiation of primed pluripotent stem cells into mesoderm cells, and co-culturing the obtained mesoderm cells with yolk sac-like cells, it was found that mesoderm cells can be efficiently induced to differentiate into hematopoietic cells, and the obtained hematopoietic cells can be induced to differentiate into specific cells such as erythrocytes and microglia, thereby completing the present invention.

[0007] The gist of the present invention is as follows. [1] A method for producing hematopoietic cells from pluripotent stem cells, comprising: a first step of culturing naive pluripotent stem cells to induce differentiation into yolk sac-like cells; a second step of culturing primed pluripotent stem cells to induce differentiation into mesoderm cells; a third step of inducing differentiation of the mesoderm cells obtained in the second step into hematopoietic cells by co-culturing with the yolk sac-like cells obtained in the first step; a method comprising. [2] The first step is A step of inducing differentiation of naive pluripotent stem cells into primitive endoderm cells, and, A step of inducing differentiation of primitive endoderm into yolk sac-like cells, A method for producing hematopoietic cells according to [1], comprising these steps. [3] The step of inducing differentiation of naive pluripotent stem cells into primitive endoderm cells is either overexpressing the GATA gene in naive pluripotent stem cells, or culturing naive pluripotent stem cells in a medium containing one or more selected from BMP (Bone morphogenetic protein), FGF4 (Fibroblast growth factor 4), and PDGF (Platelet-Derived Growth Factor), IL-6 (Interleukine-6), a TGFβ inhibitor, a Wnt signal inhibitor, and retinoic acid. A method for producing hematopoietic cells according to [2]. [4] The step of inducing differentiation of primitive endoderm cells into yolk sac-like cells is performed by adherent culture of primitive endoderm using a serum-free medium. A method for producing hematopoietic cells according to [2] or [3]. [5] The second step is performed by culturing primed pluripotent stem cells in a serum-free medium containing BMP, bFGF (basic fibroblast growth factor), and activin. A method for producing hematopoietic cells according to any one of [1] to [4]. [6] The third step is performed using a serum-free medium. A method for producing hematopoietic cells according to any one of [1] to [5]. [7] A method for producing hematopoietic cells according to any one of [1] to [6], wherein the pluripotent stem cells are induced pluripotent stem cells. [8] A method for producing hematopoietic cells according to any one of [1] to [7], wherein the pluripotent stem cells are human pluripotent stem cells. [9] A step of producing hematopoietic cells by any one of the methods of [1] to [8], and, A step of inducing differentiation of hematopoietic cells into blood cells, A method for producing blood cells, comprising these steps.

[10] A method for producing blood cells according to [9], wherein the blood cells are red blood cells. The step of producing hematopoietic cells by any one of the methods [1] to [8], and The step of inducing differentiation of hematopoietic cells into microglia, A method for producing microglia, comprising the above steps.

[12] Hematopoietic cells produced by any one of the production methods [1] to [8].

[13] Blood cells produced by the production method described in [9] or

[10] .

[14] Microglia produced by the production method described in

[11] .

[15] A medicament comprising the hematopoietic cells described in [9], the blood cells described in

[13] , or the microglia described in

[14] . [Advantages of the Invention]

[0008] According to the method of the present invention, hematopoietic cells can be induced without using heterogeneous cells, and the use of serum can also be avoided. Therefore, safe hematopoietic cells suitable for medical applications can be provided. Furthermore, by establishing iPS cells from a single donor and producing naive-type iPS cells and primed-type iPS cells therefrom to induce differentiation of yolk sac-like cells and mesoderm cells, it is possible to obtain hematopoietic cells derived from a single individual. Therefore, uniform and high-quality hematopoietic cells can be provided. In addition, by performing a further differentiation induction step, specific cells such as red blood cells and microglia can be efficiently obtained. Since the red blood cells obtained by the method of the present invention include adult-type red blood cells, they are useful for transfusion therapy and the like. [Brief Description of the Drawings]

[0009]

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Embodiments for Carrying Out the Invention

[0010] The method for producing hematopoietic cells of the present invention comprises: a first step of culturing naive pluripotent stem cells to induce differentiation into yolk sac-like cells; a second step of culturing primed pluripotent stem cells to induce differentiation into mesoderm cells; a third step of co-culturing the mesoderm cells obtained in the second step with the yolk sac-like cells obtained in the first step to induce differentiation into hematopoietic cells. It includes these steps.

[0011] <Pluripotent Stem Cells> In the present invention, pluripotent stem cells are stem cells that have pluripotency to differentiate into many cells existing in a living body and also have a proliferative ability, and any cells induced into primitive endoderm are included. Pluripotent stem cells are not particularly limited, and examples include embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), pluripotent cells (Muse cells) derived from cultured fibroblasts and bone marrow stem cells, and the like. Preferred pluripotent stem cells are iPS cells and ES cells. The origin of the pluripotent stem cells is preferably derived from a mammal, more preferably from a primate, and even more preferably from a human.

[0012] Methods for producing iPS cells are known in the art and can be produced, for example, by introducing reprogramming factors into any somatic cells. Here, reprogramming factors include, for example, genes or gene products such as 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, etc. These reprogramming factors can be used alone or in combination. Examples of 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, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 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, R.L. Judson et al., (2009), Nat. Biotechnol., 27: 459 - 461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S 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 combinations described therein are exemplified.

[0013] Somatic cells include, without limitation, somatic cells of a fetus (fetal), somatic cells of a newborn (neonatal), and somatic cells of a mature healthy or diseased individual, and also include any of primary cultured cells, subcultured cells, and established cell lines. Specifically, somatic cells are, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, etc., (2) tissue progenitor cells, (3) blood cells (peripheral blood cells, cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells, and differentiated cells such as fat cells, etc.

[0014] <Naive pluripotent stem cells> Naive pluripotent stem cells are pluripotent stem cells with properties similar to pre-implantation embryos, and specifically have the following characteristics (Cytometry Research 27(1):19 - 24, 2017). They show a dome-shaped colony morphology, and the colony size is smaller than that of the primed type. As markers, they express one or more of CD75, KLF4, and TFCP2L1. The genome is demethylated.

[0015] Naive pluripotent stem cells can be created, for example, by the following methods. Method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158 : 1254-1269, 2014) Method using 5iLFA conditions (Theunissen et al., Cell Stem Cell. 2016 Oct 6; 19(4):502-515.) Method using an HDAC (histone deacetylase) inhibitor (Guo, G. et al. (2017). Development 144(15): 2748-2763.) Also, it can be obtained by culturing primed pluripotent stem cells using a commercially available medium for preparing naive pluripotent stem cells such as t2iLGo (Ndiff227 [Takara Bio, Cat. Y40002]).

[0016] <Primed pluripotent stem cells> Primed pluripotent stem cells are pluripotent stem cells with properties similar to the epiblast of post-implantation embryos. General induced pluripotent stem cells and human ES cells obtained by introducing reprogramming factors into somatic cells fall under this category and have not been subjected to the above-mentioned naive conversion treatment. Primed pluripotent stem cells have the following characteristics. They show a flat colony morphology, and the colony size is larger than that of the naive type. As markers, CD75, KLF4, and TFCP2L1 are negative. The genome is methylated.

[0017] <First Step> In the first step, naive pluripotent stem cells are cultured to induce differentiation into yolk sac-like cells. The first step preferably includes a step of inducing differentiation of naive pluripotent stem cells into primitive endoderm cells and a step of inducing differentiation of the primitive endoderm into yolk sac-like cells.

[0018] <Primitive endoderm cells> The primitive endoderm is classified as an extraembryonic cell, expresses an extracellular matrix, and is a cell that supports epiblast cells. It is characterized by the expression of one or more primitive endoderm markers such as GATA3, GATA4, GATA6, SOX17, FOXA2 (Forkhead Box A2), HNF4A (Hepatocyte Nuclear Factor 4 Alpha), CER1 (Cerberus 1), OTX2 (Orthodenticle Homeobox 2), PDGFRA (Platelet Derived Growth Factor Receptor Alpha), COL4A1 (alpha-1 subunit of collagen type IV), and SPARC (Secreted protein acidic and rich in cysteine).

[0019] <Step of inducing differentiation of naive pluripotent stem cells into primitive endoderm cells> The method for inducing the differentiation of naive pluripotent stem cells into primitive endoderm is not particularly limited, and any method can be used, including known methods. For example, the method of overexpressing the GATA gene (such as GATA6 and GATA4) in naive pluripotent stem cells disclosed in WO 2019 / 093340, or culturing naive pluripotent stem cells in a medium containing one or more (preferably all) selected from BMP (such as BMP4, BMP2, or BMP6), FGF4, PDGF, IL-6, TGFβ inhibitors (such as SB431542, SB202190 (R.K. Lindemann et al., Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009146408), etc.), Wnt signal inhibitors (such as XAV939, IWP-1, IWP-2, IWP-3, IWP-4, IWR-1, 53AH, KY02111 (available from Abcam, Sigma-aldrich, etc.)), and retinoic acid. The types and concentrations of these cytokines and drugs at the time of use are disclosed in WO 2019 / 093340.

[0020] For the medium used to induce the differentiation of primitive endoderm cells, a medium obtained by adding factors necessary for differentiation induction to naive pluripotent stem cell maintenance media such as t2iLGo, 5iL / AF, tt2iLGo, Ndiff227, etc. can be used, and it is preferable to use a serum-free medium, but a serum medium may also be used in the case of induction by overexpression of the GATA6 gene. The culture is preferably adherent culture, and it can be carried out by culturing using a culture vessel coated with an extracellular matrix such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, etc.

[0021] In the process of inducing the differentiation of primitive endoderm cells, the culture temperature conditions for culturing naive pluripotent stem cells are not particularly limited. For example, about 37°C to about 42°C, preferably about 37°C to about 39°C are preferred. Also, the number of culture days is not particularly limited as long as primitive endoderm cells can be obtained. For example, it is at least 1 day or more, preferably 2 to 5 days.

[0022] After performing the primitive endoderm induction step, it is preferable to perform an operation of selecting and concentrating the primitive endoderm. By performing the concentration operation, the next yolk sac-like cell differentiation step can be efficiently performed. The selection can be performed using the expression of one or more of the primitive endoderm-specific markers as described above as an index. As a reagent used for selecting primitive endoderm cells from a cell population containing primitive endoderm cells, any reagent having specific affinity for the above primitive endoderm marker may be used, such as an antibody, an aptamer, a peptide, or a compound that specifically recognizes it. Preferably, it is an antibody or a fragment thereof. Examples of methods for selecting primitive endoderm cells include, for example, a method using a flow cytometer. Also, a method of sedimentation using an antibody bound to a carrier, a method of magnetically selecting cells using magnetic beads (e.g., MACS), a method using a cell sorter with a fluorescent label, or a method using a carrier (e.g., a cell concentration column) to which an antibody or the like is immobilized, etc. are exemplified.

[0023] <Step of inducing the differentiation of primitive endoderm into yolk sac-like cells> The primitive endoderm obtained as described above can be further cultured to differentiate into yolk sac-like cells.

[0024] <Yolk sac-like cells> Yolk sac-like cells are a cell population containing visceral endoderm cells, yolk sac endoderm cells, and extraembryonic mesoderm cells, and are characterized by the expression of 3 or more, preferably 4 or more, particularly preferably all markers selected from AFP, BMP4, FOXA1, COL6A1, SPARC, FOXF1, SNAI2 as shown in Figure 2.

[0025] After sorting (separating) the primitive endoderm as described above, the cells are seeded again in a culture vessel coated with an extracellular matrix such as laminin, and yolk sac-like cells can be obtained by continuing adherent culture. The medium is not particularly limited, but a serum-free medium is preferred. The number of days of culture is not particularly limited as long as yolk sac-like cells can be obtained. For example, it is at least 1 day or more, preferably 3 to 5 days.

[0026] <Second step> In the second step, primed pluripotent stem cells are cultured to induce differentiation into mesodermal cells.

[0027] Mesodermal cells are a cell group that constitutes the mesoderm and, during development, have the ability to form the coelom and the mesothelium lining it, muscle, skeleton, skin dermis, connective tissue, heart and blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels and spleen, kidneys and ureters, and gonads (testes, uterus, gonadal epithelium). Mesodermal cells are indicated by the expression of one or more markers such as, for example, T (synonymous with Brachyury), VEGF receptor-2 (KDR), FOXF1, FLK1, BMP4, MOX1, SDF1, PDGFRA, and CD34. Preferably, they are cells that express KDR and PDGFRA.

[0028] The method for inducing differentiation of primed pluripotent stem cells into mesodermal cells is not particularly limited, and any method can be used, including known methods. For example, the methods disclosed in Patent Documents 1 and 2 can be mentioned. Also, a method of culturing primed pluripotent stem cells in a serum-free medium containing BMP (such as BMP4, BMP2, or BMP6), bFGF, and activin, as described below, can be mentioned. The use concentration of these cytokines is 1 to 100 ng / ml.

[0029] For the medium used for inducing the differentiation of mesodermal cells, a medium obtained by adding factors necessary for inducing differentiation to a normal pluripotent stem cell medium such as StemPro can be used. It is preferable to use a serum-free medium, but in the case of induction by overexpression of the GATA6 gene, a serum medium may also be used. Adherent culture is preferred for the culture, and it can be carried out by culturing using a culture vessel coated with an extracellular matrix such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, etc.

[0030] In the step of inducing the differentiation of mesodermal cells, the culture temperature conditions for culturing the naive pluripotent stem cells are not particularly limited. For example, about 37°C to about 42°C, preferably about 37°C to about 39°C are preferred. Also, the number of culture days is not particularly limited as long as mesodermal cells can be obtained. For example, it is at least 1 day or more, preferably 2 to 5 days.

[0031] After performing the mesodermal cell induction step, it is preferable to perform an operation of sorting and concentrating the mesodermal cells. By performing the concentration operation, the next co-culture step (step 3) can be efficiently performed. The sorting can be performed using the expression of one or more of the mesodermal cell-specific markers as described above as an index.

[0032] <Step 3> In step 3, the mesodermal cells obtained in step 2 are co-cultured with the yolk sac-like cells obtained in step 1 to induce differentiation into hematopoietic cells.

[0033] After sorting (separating) the mesodermal cells as described above, they are seeded on the yolk sac-like cells obtained above and cultured adherently to obtain hematopoietic cells. The medium is not particularly limited, but a serum-free medium is preferred. The number of culture days is not particularly limited as long as hematopoietic cells can be obtained. For example, it is at least 1 day or more, preferably 5 to 25 days, more preferably 8 to 14 days.

[0034] <Hematopoietic cells> In the present invention, hematopoietic cells refer to cells that have the ability to produce mature blood cells such as T cells, B cells, red blood cells, platelets, eosinophils, monocytes, neutrophils, basophils, macrophages, etc., and have the ability of self-renewal. Note that hematopoietic cells also include hematopoietic progenitor cells and hematopoietic stem cells. These cells are detected by the expression of one or more markers such as KDR, CD34, CD43, CD90, and CD117, and preferably CD34 and CD43 are positive. Further, CD235a may be positive. In particular, the yolk sac is the site of primary hematopoiesis, and embryonic erythrocytes can be produced by using this culture system, and furthermore, fetal erythrocytes or adult erythrocytes can also be produced.

[0035] That is, in the method of the present invention, a differentiation induction step for maturing the hematopoietic cells obtained in the third step into blood cells such as T cells, B cells, red blood cells, platelets, eosinophils, monocytes, neutrophils, basophils, macrophages, etc. may be performed to produce blood cells. For example, in order to induce differentiation of hematopoietic cells into red blood cells, an erythrocyte differentiation induction step may be performed. The erythrocyte differentiation induction step can employ known methods. For example, a step of culturing in a medium containing erythropoietin (EPO) is exemplified. The concentration of EPO is, for example, 1 to 100 U / mL. The culture period may be a period sufficient for the induction of red blood cells, for example, 1 day or more, preferably 5 days or more.

[0036] Moreover, fetal macrophages can be produced, and these constitute the microglia of the brain. Here, the central nervous system is composed of neurons and three types of glial cells. In particular, microglia are known as immune cells in the central nervous system, and it is known that macrophages produced in the yolk sac migrate into the bloodstream and become central microglia. In the central nervous system, microglia with immune functions are said to gather around degenerated nerves or show abnormal activity. Therefore, it is also involved in chronic inflammation in the central nervous system, and is also speculated to be one of the causes of Parkinson's disease and Alzheimer's disease. Currently, drug discovery is actively carried out as a treatment target. The genetic disease, Nasu-Hakola disease, is also known to be an abnormality of microglia. Therefore, by the method of the present invention, it is considered effective for the treatment of central nervous system diseases and central inflammatory diseases by producing macrophages.

[0037] In addition, in the method of the present invention, a differentiation induction step for maturing the hematopoietic cells obtained in the third step into microglia may be performed to produce microglia. Microglia also differentiate by continuing co-culture, but a known method may be adopted for the microglia differentiation induction step. For example, a step of culturing in a medium containing MCSF (macrophage colony-stimulating factor) or GCSF (granulocyte colony-stimulating factor) is exemplified. The concentration of MCSF or GCSF is, for example, 1 ng / ml to 1 mg / ml. Also, IL-34 may be added as necessary. The culture period may be a period sufficient for the induction of microglia, but for example, it is 1 day or more, preferably 5 days or more.

[0038] <Pharmaceutical containing hematopoietic cells, blood cells or microglia> The present invention provides a pharmaceutical containing hematopoietic cells or blood cells produced by the method of the present invention, for example, a therapeutic agent for blood diseases. The present invention also provides a pharmaceutical containing microglia produced by the method of the present invention, for example, a therapeutic agent for central nervous system diseases. The hematopoietic cells, blood cells or microglia obtained by the method of the present invention may be derived from the patient himself / herself who is the subject of the treatment, or may be derived from other individuals. Preferably, they are derived from the patient himself / herself who is the subject of the treatment. When the hematopoietic cells, blood cells or microglia are derived from other individuals, from the viewpoint of avoiding rejection reactions, it is preferable to collect somatic cells from another person with the same HLA type.

[0039] The medicament of the present invention may contain hematopoietic cells, blood cells or microglia alone, or may contain a buffer solution, an antibiotic, other pharmaceutical additives, etc., and any cells (multiple are also possible) other than hematopoietic cells together with the hematopoietic cells, blood cells or microglia.

[0040] The medicament containing the hematopoietic cells or blood cells of the present invention is widely effective as a therapeutic agent for blood diseases. Specifically, the target diseases include, but are not limited to, congenital anemia, aplastic anemia, autoimmune anemia, myelodysplastic syndrome (MDS), granulocytopenia, lymphocytopenia, thrombocytopenia, hematopoietic cell reduction associated with various cancers or tumors, hematopoietic cell reduction associated with cancer chemotherapy or radiotherapy, acute radiation syndrome, delayed recovery of hematopoietic cells after bone marrow / umbilical cord blood / peripheral blood transplantation, hematopoietic cell reduction associated with blood transfusion, leukemia (including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)), malignant lymphoma, multiple myeloma, myeloproliferative diseases, hereditary blood diseases, etc.

[0041] The medicament containing the microglia of the present invention is widely effective as a therapeutic agent for central nervous system diseases. Specifically, the target diseases include, but are not limited to, Parkinson's disease, Alzheimer's disease, Nasu-Hakola disease, etc.

[0042] The route of administration of the medicament of the present invention to a patient is not particularly limited. For example, it is administered to a patient by transplantation. In that case, it can be carried out in the same manner as the conventionally performed bone marrow transplantation or umbilical cord blood transplantation. Further, for example, administration forms such as intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal, intramedullary, and intracerebral can also be exemplified. In the case of a medicament containing hematopoietic cells or blood cells, a preferred route of administration can be intravenous administration or intramedullary administration. In the case of a medicament containing microglia, a preferred route of administration can be intracerebral administration.

[0043] The dosage / transplantation amount of the medicament of the present invention to a patient cannot be uniquely determined because it varies depending on the type of condition to be treated, symptoms and severity of the disease, patient age, sex or weight, administration method / transplantation method, etc. However, a physician can appropriately determine an appropriate dosage / transplantation amount by considering the above situations.

Examples

[0044] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following aspects.

[0045] Material and method Cell culture Human prime-type pluripotent stem cell (PSC) lines (H9ES (embryonic stem) cells, H1ES cells, AdiPS cells) were maintained on γ-irradiated MEFs using the Conventional condition (referred to as F12 / KSR) (Dulbecco’s modified Eagle medium [DMEM / F12; Nacalai Tesque, Cat.08460-95], 20% [v / v] KSR [Thermo Fisher Scientific, Cat. 10828028], nonessential amino acids [NEAA; Thermo Fisher Scientific, Cat. 11140-050], 4 ng / ml recombinant human bFGF [bFGF; Oriental Yeast, Cat. NIB 47079000], 0.1 mM 2-mercaptoethanol [Sigma-Aldrich, Cat.M3148]). Cells were dissociated and passaged every 5 - 7 days using Dissociation Buffer (DB; 0.025% Trypsin [Thermo Fisher Scientific, Cat. 15090-046], 1mg / ml Collagenase IV [Thermo Fisher Scientific, Cat. 17104-019], 20% KSR, 1mM CaCl2) by peeling them off into small clamp-like pieces. Alternatively, they may be maintained in feeder-free media (such as Stemfit, mTeSR, E8, DEF-CS, etc.) containing FGF, TGFB, Activin, etc.

[0046] Human naive pluripotent stem cell lines (H9ES cells, H1ES cells, derived from AdiPS cells) were maintained on MEF using t2iLGo (Ndiff227 [Takara Bio, Cat. Y40002], 1 μM PD0325901 [PD03; Tocris, Cat.4192], 1 μM CHIR99021 [CH; Sigma-Aldrich, Cat.SML1046], 10 ng / ml Recombinant human LIF [hLIF; Peprotech, Cat.300-05], 3 μM Go6983 [Go; Tocris, Cat.2285]). Cells were detached and passaged every 3 - 5 days using Accutase (Sigma-Aldrich, Cat.A6964). The following, any induction method, establishment method, and maintenance method can induce primitive endoderm and hematopoiesis.

[0047] Naive H9 was established by a method using an HDAC inhibitor (Guo, G. et al. (2017). Development 144(15): 2748 - 2763.). Prime H9 was detached into single cells using trypsin / EDTA (Nacalai Tesque, Cat. 32777 - 15), and 1×10 5 cells / cm 2 were seeded onto MEF in F12 / KSR medium supplemented with 10 μM Y-27632 (Wako, Cat.034-24024). From the next day, cells were cultured with cRM1 (Ndiff, 1 μM PD03, 10 ng / ml hLIF, 1 mM Valproic acid sodium salt [VPA; Sigma-Aldrich, Cat.P4543]) for 48 hours, and then maintained with cRM2 (Ndiff, 1 μM PD03, 10 ng / ml hLIF, 2 μM Go, 2 μM XAV939 [Sigma-Aldrich, Cat.X3004]). Around passage 3 - 5, most cells form colonies showing a dome-shaped morphology. In some experiments, naive H9 was established using 5iLFA conditions (Theunissen et al., Cell Stem Cell. 2016 Oct 6;19(4):502-515.). Prime H9 was detached into single cells with trypsin / EDTA, and 5 cells / cm 2 were seeded onto MEF in F12 / KSR medium supplemented with 10 μM Y-27632. From the next day, the medium was changed to 5iLFA medium (Ndiff, 1 μM PD03, 1 μM CH, 1 μM WH-4-023 [A Chemtek H620061], 0.5 μM SB590885 [R and D 2650], 10 μM Y-27632, 10 ng / ml hLIF, 20 ng / ml Activin A [R&D, Cat.388-AC], 8 ng / ml bFGF) and the culture was continued. Most cells formed colonies showing a dome-shaped morphology. Naive H1 PSC and AdiPS were established by a method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158 : 1254-1269, 2014). Plasmids capable of inducing overexpression by DOX were electroporated and introduced into H1 and AdiPS (H1 NK2, AdiPS NK2). The plasmid was incorporated with a gene (neomycin resistance) serving as a drug resistance marker, and after introduction into cells, drug selection was performed using Geneticin (Thermo Fisher Scientific, Cat.10131035) to select cells into which the plasmid was introduced. Prime H1 NK2 and AdiPS NK2 were detached into single cells with trypsin / EDTA, and 1×10 5 cells / cm 2The cells were seeded onto MEF in F12 / KSR medium supplemented with 10 μM Y-27632 (Wako, Cat.034-24024). The next day (day1), 1 μg / ml doxycycline hyclate (Dox; Sigma-Aldrich, Cat.D9891) was added. From Day2, it was switched to 2iL (1 μM PD03, 1 μM CH, LIF) + Dox medium and cultured for about one week. Thereafter, it was established by switching to and maintaining in t2iLGo.

[0048] Primitive endoderm induction After dissociating and collecting naive PSCs cultured on MEF with Accutase, they were seeded onto gelatin-coated dishes and cultured at 37 °C for 1 - 2 hours in t2iLGo supplemented with 10 μM Y-27632 (ROCK inhibitor) to remove MEF. Thereafter, they were resuspended in each induction medium and induction was started simultaneously with seeding. When inducing by overexpression of the GATA6 gene, 1×10 5 cells / cm 2Cells seeded with an inducible GATA6 expression plasmid were used. As the induction medium, either serum medium (Glasgow Minimum Essential Medium [GMEM; Sigma-Aldrich, Cat.G5154], 15% FBS [Thermo Fisher Scientific, Cat. 10437028], 2 mM L-Glutamine [Thermo Fisher Scientific, Cat. 25030081], 1 mM Sodium Pyruvate [Thermo Fisher Scientific, Cat.11360-070], NEAA, 0.1 mM 2-mercaptoethanol) or a medium prepared by adding 0.1% bovine serum albumin (BSA; Wako, Cat.012-23881), 50 μM 2-mercaptoethanol, 25 ng / ml recombinant human FGF4 (FGF4; Peprotech, Cat.100-31), and 1 μg / ml heparin sodium (Wako, Cat. 081-00131) to StemClone SFO-3 (Aidenia, Cat.SS1303) was used. 0.1 μg / ml Dox was added from 48 hours after the start of induction until the end. When inducing with a compound, 5×10 4 cells / cm 2Cells were seeded. As the induction medium, Ndiff227 medium supplemented with 25 ng / ml FGF4, 1 μg / ml heparin sodium, 10 - 200 ng recombinant human BMP-4 (BMP-4; R&D, Cat.314-BP), 10 ng / ml recombinant human PDGF-AA (PDGF-AA, Peprotech, Cat.100-13A), 10 ng / ml recombinant human IL-6 (IL-6; Oriental Yeast, Cat.47066000), 1 μM XAV939, 3 μM A83-01 (Tocris, Cat. 2939), and 0.1 μM retinoic acid (RA; Sigma-Aldrich, Cat.R2625) was used. IL-6 was added 48 hours after the start of induction. In some experiments, instead of BMP-4, 10 - 500 ng / ml recombinant human BMP-2 (BMP-2; Oriental Yeast, Cat.47304000) or 50 ng / ml recombinant human BMP-6 (BMP-6; Peprotech, Cat.120-06) was used.

[0049] Yolk sac-like cell induction from primitive endoderm On the third day after induction to primitive endoderm, positive cells were purified by flow cytometry using a PDGFRA antibody. Then, 2×10 5 cells / cm 2 were seeded on iMatrix-511 silk (MAX, Cat.892021). Culturing was continued using Ndiff227 or Essential6 (E6: Gibco, Cat.A1516401) medium.

[0050] Mesoderm cell induction The induction from primed PSCs to mesoderm cells was performed by slightly modifying a previously reported method (Sturgeon. et al. (2014). Nat Biotechnol 32(6): 554-561.). Primed H9 cells were detached into single cells with trypsin / EDTA (Nacalai Tesque, Cat.32777-15), seeded onto gelatin-coated dishes, and cultured at 37 °C for 1-2 hours in F12 / KSR supplemented with 10 μM Y-27632 (ROCK inhibitor) to remove MEFs. Then, the cells were resuspended in differentiation induction medium (StemPro34 [Invitrogen, Cat. 10639011], 2 mM L-glutamine [Thermo Fisher Scientific, Cat. 25030081], 4×10 4 M monothioglycerol [Sigma-Aldrich, Cat.M6145], 150 μg / ml transferrin [Roche, Cat. 10652202001], 50 μg / mL ascorbic acid), and differentiation induction using suspension culture was started simultaneously with seeding. Day0-1: 10 ng / ml BMP4 and 10 μM Y27632 were added to the differentiation induction medium. Day1-3: 10 ng / ml BMP4, 8 ng / ml ActivinA, and 5 ng / ml bFGF were added to the differentiation medium. The suspended cells were seeded at a cell concentration of 2.4×10 5 cells / well in an Elplasia plate (Kurare, Cat.RB 500 400 NA). On the 3rd day of induction, mesoderm cells were purified using PDGFRA and KDR antibodies.

[0051] Method for inducing hematopoietic cells using primitive endoderm The purified mesoderm cells were seeded at 7.5×10 3 cells / cm 2 onto yolk sac-like cells induced from primitive endoderm and cultured in differentiation induction medium. Five days after the start of co-culture, the cells were collected and analyzed by flow cytometry. Also, hematopoietic cells were purified using the hematopoietic cell markers CD34 and CD43, and RNA was collected.

[0052] FACS analysis / sorting Primitive endoderm-like cells and mesoderm cells were detached and collected into single cells using Accutase. Subsequently, blocking was performed for 30 minutes on ice using HBSS (Thermo Fisher Scientific, Cat.14185052) supplemented with 1% BSA (Sigma-Aldrich, Cat.A2153). Then, antibodies were added in each combination and incubated on ice for 30 minutes. When using biotinylated antibodies, after washing, Streptavidin-APC (Biolegend, Cat.405207) was added and incubated on ice for 20 minutes. BD LSR Fortessa (BD) was used for FACS analysis and FACS AriaII (BD) was used for sorting. Also, Flow Jo V10.2 software was used for data analysis.

[0053] [Table 1]

[0054] Reverse Transcription Quantitive Real-time PCR Total RNA was extracted using the RNeasy kit (Qiagen, Cat.74106), and cDNA was synthesized from 1000 ng of RNA using SuperScriptIV (Thermo Fisher Scientific, Cat.18090050) and oligo-dT primers. PowerUP Sybr Green Master Mix (Thermo Fisher Scientific, Cat.A25743) was used for real-time PCR, and QuantStudio3 (Thermo Fisher Scientific) was used for PCR amplification. Analysis after real-time RT-PCR reaction was performed using QuantStudio Design&Analysis Software v1.4.1.

[0055] Erythroid induction After purifying hematopoietic cells induced by co-culture with yolk sac-like cells or OP9 by FACS sorting, approximately 2.5×10^4 cells were seeded into a 96-well plate in erythroid induction medium (differentiation induction medium + 4 U / ml recombinant human erythropoietin [hEPO; Calbiochem, Cat.329871] + 100 ng / ml recombinant human SCF [R&D, Cat.252-SC] + 5 ng / ml recombinant human IL-3 [R&D, Cat. 203-IL]), and differentiation induction was initiated. On the 14th day of erythroid differentiation induction, the cells were collected, and the expression of hemoglobin was confirmed by qPCR.

[0056] Microglia induction Yolk sac-like cells and mesodermal cells induced from primitive endoderm were co-cultured in differentiation induction medium for 12 days, and then replaced with N2B27 medium containing 10 ng / mL of M-CSF or G-CSF and cultured for an additional 21 days. In addition, after microglia induction, IL-34 (10 ng / mL) was added to the medium from the 10th day onwards.

[0057] Results <Analysis results of yolk sac-like cells> The cells induced to differentiate into yolk sac (Figure 1) were collected, and gene expression was confirmed by qPCR. The results are shown in Figure 2. It can be seen that the primitive endoderm cells before induction hardly express yolk sac marker genes, while the cells after induction strongly express yolk sac marker genes.

[0058] <Analysis results of hematopoietic cells> Figure 3 shows the results of analyzing cells derived from mesodermal cells induced by co-culturing mesodermal cells with yolk sac-like cells by flow cytometry. CD34 and CD43 were used as hematopoietic cell markers, and CD235a was used as a hematopoietic cell marker in the early embryo. In addition, as a control, the results of samples seeded with mesodermal cells on laminin-coated plates were shown. Co-culture with yolk sac-like cells enabled the induction of hematopoietic cells co-expressing CD34 and CD43. Moreover, since the induced hematopoietic cells were CD235a-positive, they were considered to be hematopoietic cells found in the early embryo. When endodermal cells were cultured in laminin-coated dishes, no hematopoietic cells were induced, indicating that co-culture with yolk sac-like cells is important.

[0059] Next, hematopoietic cells (CD34+ / CD43+) and vascular endothelial cells (CD34+ / CD43+) were separated from samples obtained after co-culturing mesodermal cells with yolk sac-like cells or OP9 cells using FACS, and gene expression was compared by qPCR. Human ES cells (Day0) and induced mesodermal cells (Day3) were used as comparison targets. As shown in Figure 4, the hematopoietic cells induced by the present invention showed equivalent or higher expression of all hematopoietic cell genes compared to hematopoietic cells induced by OP9.

[0060] Hematopoietic cells (CD34+ / CD43+ / CD235a+) obtained after co-culturing mesodermal cells with yolk sac-like cells or OP9 cells were cultured under conditions for inducing erythroid differentiation, and the type of hemoglobin of the resulting cells was examined. As a result, as shown in Figure 5, it was found that hematopoietic cells induced by co-culture with yolk sac-like cells differentiated into erythrocytes expressing embryonic hemoglobin.

[0061] Mesodermal cells were co-cultured with yolk sac-like cells or OP9 cells for 8 days or 14 days. The resulting hematopoietic cells were cultured for 14 days under conditions for inducing erythroid differentiation, and the resulting cells were analyzed by flow cytometry. As a result, as shown in Figure 6, CD71-positive and CD235-positive erythrocytes could be induced from both hematopoietic cells co-cultured for 8 days and hematopoietic cells co-cultured for 14 days.

[0062] After purifying the CD71-positive, CD235-positive cells obtained above by flow cytometry, the expression of hemoglobin was confirmed. The results are shown in Fig. 7. The CD71-positive, CD235-positive cells obtained by inducing erythroid differentiation after 8 days of co-culture were HBE-predominant yolk sac type (embryonic type) hemoglobin. On the other hand, in the CD71-positive, CD235-positive cells obtained by inducing erythroid differentiation after 14 days of co-culture, the expression of HBE decreased, and fetal-type hemoglobin produced in the fetal liver, spleen, and bone marrow with HBG predominance was expressed. Furthermore, it was found that adult-type hemoglobin expressing HBB also began to be expressed. From the above, it was found that the hematopoietic cells induced to differentiate by the method of the present invention have the ability to induce erythrocytes expressing adult-type hemoglobin.

[0063] In addition, when mesoderm cells were cultured for 3 weeks on yolk sac-like cells using a serum-free medium (differentiation induction medium), as shown in Fig. 8, it was found that CD14+ / CD11b+ macrophages were obtained.

[0064] After culturing mesoderm cells on yolk sac-like cells for 14 days using a serum-free medium (differentiation induction medium), they were further cultured for 25 days in a medium supplemented with IL-34 (10 ng / ml) to G-CSF or M-CSF, and gene expression was confirmed. As a result, as shown in Fig. 9, the expression of genes expressed in microglia increased, and differentiation into microglia was confirmed.

Claims

1. A method for producing hematopoietic cells from pluripotent stem cells, comprising: a first step of culturing naive pluripotent stem cells to induce differentiation into yolk sac-like cells; a second step of culturing primed pluripotent stem cells to induce differentiation into mesodermal cells; a third step of co-culturing the mesodermal cells obtained in the second step with the yolk sac-like cells obtained in the first step to induce differentiation into hematopoietic cells, wherein the yolk sac-like cells are cells characterized by the expression of three or more selected from AFP, BMP4, FOXA1, COL6A1, SPARC, FOXF1, and SNAI2, and the mesodermal cells are cells expressing KDR and PDGFRA; The first step includes a step of inducing differentiation of naive pluripotent stem cells into primitive endoderm cells, and a step of inducing differentiation of primitive endoderm into yolk sac-like cells, wherein the step of inducing differentiation of naive pluripotent stem cells into primitive endoderm cells is performed by overexpressing the GATA gene in naive pluripotent stem cells, or culturing naive pluripotent stem cells in a medium containing one or more selected from BMP (Bone morphogenetic protein), FGF4 (Fibroblast growth factor 4), PDGF (Platelet-Derived Growth Factor), IL-6 (Interleukine-6), TGFβ inhibitor, Wnt signal inhibitor, and retinoic acid; the step of inducing differentiation of primitive endoderm cells into yolk sac-like cells is performed by adherent culture of primitive endoderm using a serum-free medium; the third step is performed by adherent culture using a serum-free medium; The method.

2. The second step is performed by culturing primed pluripotent stem cells in a serum-free medium containing BMP, bFGF (basic fibroblast growth factor), and activin. The method for producing hematopoietic cells according to claim 1.

3. The method for producing hematopoietic cells according to claim 1 or 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

4. The method for producing hematopoietic cells according to any one of claims 1 to 3, wherein the pluripotent stem cells are human pluripotent stem cells.

5. A method for producing blood cells, comprising: a step of producing hematopoietic cells by the method according to any one of claims 1 to 4, and a step of inducing differentiation of hematopoietic cells into blood cells.

6.

7.

8.

9.

10. The method for producing blood cells according to claim 5, wherein the blood cells are red blood cells.

7. A step of producing hematopoietic cells by the method according to any one of claims 1 to 4, and a step of inducing differentiation of the hematopoietic cells into microglia, A method for producing microglia, comprising the steps.

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