TW200540270A - Medium for feeder-free differentiation and feeder-free differentiation method from primate embryonic stem cell - Google Patents

Abstract

This invention is related to provide a means whereby primate embryonic stem cells can be differentiated into blood cells or vascular endothelial cells substantially free from contamination with heterogenic animal cells, infection with a virus originating in a heterogenic animal, etc. Namely, a medium for feeder-free differentiation which contains a stroma cell adaptation medium; a culture system for feeder-free differentiation which comprises the above medium for feeder-free differentiation and a culture container coated with an extracellular matrix; a method of feeder-free differentiation from primate embryonic stem cells into blood cells, a method of feeder-free differentiation from primate embryonic stem cells into vascular endothelial cells, a method of producing blood cells, a method of producing vascular endothelial cells and a method of producing finger-like cells each using the above medium for feeder-free differentiation; and blood cells, vascular endothelial cells and finger-like cells obtained by these methods are provided.

Description

200540270 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a medium for differentiation and a culture system for making primate embryonic stem cells into blood cells or vascular endothelial cells to form blood cells or vascular endothelial cells. Differentiation method, blood cell or vascular endothelial cell manufacturing method, and cells obtained therefrom. More specifically, the present invention relates to a culture medium for feeder-free differentiation that makes primate embryonic stem cells into blood cells or vascular endothelial cells, a culture system for feeder-free differentiation, a method for differentiation without feeder cells, blood cells or Method for producing vascular endothelial cells and cells obtained thereby. [Previous technology] Embryonic stem cells were established in mice as early as the 1980s, and are mainly used to make disease model animals. As m is a versatile cell that differentiates into various cells, it has basic materials that can be used for organ production in the fields of regenerative medicine or transplantation medicine. Therefore, the establishment of human embryonic stem cells has been expected. Then in 1998, embryonic stem cells were established in the United States. Research into the differentiation of embryonic stem cells from experimental animal models such as mice is ongoing (see Patent Document 1). '' On the other hand, since blood cells that are the basis of immunity attack foreign bodies that invade the body, NK cells have effects on cancer and the like, and hematopoietic stem cells have effects on leukemia and so on. In addition, hematopoietic stem cells can cope with various diseases based on the plasticity of their tissues. Transdifferentiati on into necessary cells, etc. ', so it is of great importance medically. In addition, for example, if a large number of blood cells can be produced from embryonic stem cells by industrial production, the contribution to medical treatment I00097.doc 200540270 will be immeasurable. ^ ^ The right is red blood cells used for blood transfusion, etc.… hunting for differentiation techniques from embryonic stem cells and deciding AIDS, Hepatitis C, and other problems when donating blood ^ The problem of turbulence in the archeology of the Tujiaren Temple. In addition, the immune system that has been damaged during chemotherapy such as dysfunction can be used to strengthen the immune system with low white blood cells such as θ neutrophils to strengthen the immune system, so it can also solve problems such as internal infections in hospitals. In addition, because blood cells and natural healing powers are also strengthened, they have a great effect on the medical industry and the medical industry.

However, because the technique of differentiating blood from embryonic stem cells (especially from primate embryonic stem cells) is extremely difficult, such research is currently rare in the world. Previously, with regard to the differentiation of embryonic stem cells into blood cells, for example, there have been attempts to use stromal cell lines (that is, O1 > 9 cells) as feeder cells to grow and culture with embryonic stem cells (for example, refer to non-patent literature 丨). . Related differentiation methods are to differentiate embryonic stem cells into mesoderm cells by seeding them on 0p9 cells, or to differentiate embryonic stem cells into embryoid bodies (EBs) and differentiate into After the mesoderm cells are treated with trypsin or the like, the mesoderm cells are detached, seeded again on OP9 cells, and cultured to differentiate them into blood cells. [Patent Document 1] International Publication No. 99/20741 [Non-Patent Document 1] Nakano T. et al., "In vitro development of early red blood cells and completely red blood cells derived from dissimilar precursor cells (Irl in vitro development of primitive and definitive erythrocytes from different precursors) ", Science, Vol. 272, p. 722 ~ p. 724 100097.doc 200540270 (1996) [Problems to be Solved by the Invention] The first-oriented system of the present invention provides _ species can Carry out at least one kind of culture medium for differentiation without cultivating cells 'which allows primate embryonic stem cells to differentiate into blood cells or vascular endothelial cells' without being accompanied by the incorporation of heterogeneous animal cells and derived from heterogeneous animals Viral infection, etc., to differentiate with good regenerability, and to obtain high purity blood cells or vascular endothelial cells with high efficiency. The present invention provides a kind of feeder-free differentiation culture system, which enables primate embryonic stem cells to differentiate into blood cells without substantially being accompanied by a mixture of heterogeneous cells and viruses derived from heterogeneous animals. The primate embryonic stem cells are differentiated into gold cells through high stringency control. In addition, the third aspect of the present invention provides a method for differentiation of primate embryonic stem cells into blood cells without feeding cells, which can at least differentiate primate embryonic stem cells into blood cells without realizing them. Sexually accompanied by the incorporation of heterogeneous animal cells and diseases derived from heterogeneous animals: infections, etc., or the primate embryonic stem cells differentiate into blood cells, etc. through high stringency control. Furthermore, the fourth aspect of the present invention provides a feeder-free cell differentiation method for making primate embryonic stem cells into vascular endothelial cells, which allows primate embryonic stem cells to differentiate into blood ... with a heterogeneous animal The incorporation of cells and the origin of == toxic infections, etc., allow primate embryonic stem cells to differentiate into vascular endothelial cells and the like through high-rigidity control. A fifth aspect of the present invention is to provide a method for producing blood cells, which can obtain blood cells of high purity, and continuously obtain blood cells and the like for a long time with excellent reproducibility. Furthermore, 100097.doc 200540270 of the present invention provides a method for producing vascular endothelial cells which can obtain high-purity vascular endothelial cells and the like. A seventh aspect of the present invention is to provide a blood cell or a vascular endothelial cell which exhibits uniform properties. The eighth aspect of the present invention provides a method for manufacturing finger cells, which can more effectively obtain a large number of fingers that are differentiated into blood cells, vascular endothelial cells, and the like. A finger cell that can supply blood cells, vascular endothelial cells, etc. suitable for clinical purposes and the like. Other subjects of the present invention can be understood from the disclosure of this specification and the like. [Summary of the invention] That is, the main points of the present invention are: (1) a medium for differentiation without cultured cells, which is characterized by containing a stromal cell acclimation medium; (2) a medium for differentiation without feed cells as described in (1) above, It further contains a medium component for maintaining primate embryonic stem cells, serum, and cytokines; (3) The medium for feeding cell differentiation without feeding as described in (2) above, wherein the medium is used for maintaining the primate embryo The components of the culture medium of sexual stem cells are those containing Iskov's modified Dubeco medium; (4) The medium for feeding cell differentiation without feeding cells as described in (2) or (3) above, wherein the cytokine is selected from the group consisting of blood vessels At least one of the group consisting of endothelial growth factor, bone morphogenetic protein, stem cell factor, Flt3-ligand, interleukin-6, interleukin-3, and granule colony-stimulating factor community; (5) as described above (1 ) To (4) of the culture medium for no-feeder cell differentiation, wherein the stromal cell line is irradiated stromal cells, 100097.doc 200540270 (6) as in any one of (1) to (4) above no The medium for feeding cell differentiation is "the stromal cell domestication medium" obtained by performing a process containing the following steps: A) a step of culturing in a medium suitable for maintaining stromal cells without forming a fusion, B) a step of culturing the cells obtained in the above step in a medium suitable for maintaining stromal cells and embryonic stem cells, and C) a step of removing stromal cells from the culture obtained in step (B) above (7) The medium for differentiation without feeder cells according to any one of (1) to (5) above, wherein the stromal cell domestication medium is obtained by performing a process comprising the following steps: A ') suitable for The step of culturing stromal cells in a medium that does not form a fusion, B ') irradiating the cells obtained in step (a) with radiation, and culturing the cells obtained in a medium suitable for maintaining stromal cells and embryonic stem cells Steps of cells' and C,) Step (8) of removing stromal cells from the culture obtained in step (B,) above-a kind of pseudotrophic cells A culture system, characterized in that it contains the medium for feeding-free cell differentiation according to any one of the above (1) to (7) and a culture container covered with extracellular matrix; (9) the feeding-free cell of (8) above Culture system for differentiation, wherein the extracellular matrix is collagen; 100097.doc -10- 200540270 (10) The culture system for differentiation of non-feeding cells according to (9) above, wherein the collagen is type IV collagen; (11) a kind of Feedless cell differentiation method for primate embryonic stem cells to become blood cells, characterized in that the primate embryogenicity is cultured in a feedless cell differentiation medium according to any one of the above to the above Stem cells; (12) A method for differentiation of primate embryonic stem cells into blood cells as described in (11) above, wherein the primate embryonic stem cells are placed in a cell covered with extracellular matrix In a culture container, in a state of no feeder cells, the cells are cultured in the medium for feeder-free cell differentiation as described in any one of (1) to (7) above to form finger_nke cells, and then continue. (13) A method for differentiation of non-feeding cells that allows primate embryonic stem cells to become vascular endothelial cells, which is characterized in that: primate embryonic stem cells are placed between extracellular spaces In the culture container covered by the substance, in a state of no nutrition and cells, culture in the medium for feeding cell differentiation as described in any one of (1) to (7) above to produce finger cells Then, the finger cells are moved to a culture medium covered with extracellular matrix with a new medium, and further cultured, so that the finger cells generate vascular endothelial cells; (14) A method for producing blood cells, which It is characterized in that: primate embryonic stem cells are placed in a culture container covered with extracellular interstitial material, and in the state of no feeder cells, they are differentiated from the feeder-free cells of any one of (1) to (7) above Cultured in a culture medium to differentiate the embryonic stem cells into blood cells, and then the blood cells are separated and separated; 100097.doc 200540270 (15) The method for producing blood cells as described in (14) above Wherein, the culture medium for culturing cell differentiation according to any one of the above items ⑴ to ⑺ is added to the culture container after the blood cells are stripped, and then cultured in the state without culturing cells to obtain blood cells; (16 ) — A type of blood cell, which is characterized in that it can be obtained by the manufacturing method of the above (ι sentence or 0 fantasy); (17) — a method of making vascular endothelial cells, which is characterized in that ... The stem cells are placed in a culture container covered with extracellular interstitial substance, and cultured in the state without feeder cells in the medium for feeder-free differentiation of any one of (1) to (7) above to generate Finger cells, the finger cells are transferred to a new culture medium-covered extracellular mesenchymal culture container, and further cultured to differentiate the embryonic stem cells into vascular endothelial cells, and isolate the vascular endothelial cells; (18) A vascular endothelial cell, characterized in that it can be obtained by the manufacturing method of the above (17); (19) A method of manufacturing finger cells, characterized in that the primates The embryonic stem cells are cultured in a culture vessel covered with extracellular mesenchyme in a state of no silver-cultured cells, and cultured in the culture medium for feeder-free cell differentiation according to any one of (1) to (7) above; and (20) A finger cell characterized in that it can be obtained by the manufacturing method of (9) above. [Effects of the Invention] The medium for feeder-free cell differentiation according to the present invention will produce the following excellent effects: the primate 100097.doc -12- 200540270 can be differentiated in the absence of heterogeneous animal cells It is a blood cell, but it is not substantially accompanied by the incorporation of heterogeneous animal cells and infections derived from animal viruses. Furthermore, according to the medium for non-feeder cell differentiation of the present invention, the following excellent effects can be achieved: differentiation with excellent regenerative properties can be performed, and blood cells or vascular endothelial cells can be obtained with high efficiency and high purity. According to the culture system for non-junker cell differentiation of the present invention, the following excellent effects will be produced: the primate embryonic stem cells are differentiated into blood cells through the control of high stringency in the absence of heterogeneous animal cells, Without substantial concomitant incorporation of heterologous animal cells and virus infection from heterologous animals, and the method of non-trophoblastic differentiation of primate embryonic stem cells into blood cells according to the present invention, the following will be produced: Excellent effect: Differentiation can be implemented without substantial concomitant incorporation of heterogeneous animal cells and infections derived from heterogeneous animal viruses', so that primate embryonic stem cells can be differentiated into blood cells through highly stringent control. Furthermore, the method for differentiation of primate embryonic stem cells into vascular endothelial cells according to the present invention without feeder cell differentiation will have the following excellent effects: · Differentiate primate embryonic stem cells through highly stringent control Become vascular endothelial cells without substantial concomitant incorporation of xenogeneic animal cells and infection from xenobiotic animal viruses. In addition, according to the method for producing blood cells of the present invention, excellent effects can be obtained in that high-purity blood cells can be obtained with excellent reproducibility for a long period of time. Furthermore, the method for producing vascular endothelial cells according to the present invention will produce an excellent effect of obtaining high-purity vascular endothelial cells. The blood cells or vascular endothelial cells according to the present invention show high purity and homogeneity, and are shown to be suitable for use as blood transfusion blood applications and 100097.doc 200540270 materials for hemorrhage treatment or local blood flow improvement, and transplantation. Materials, materials and other properties. According to the method for manufacturing finger cells of the present invention, the following excellent effects can be produced: • The effect is greater and the finger cells can be effectively differentiated into blood cells, vascular endothelial cells and the like. According to the finger cells of the present invention, the following excellent effects can be produced: Blood cells, vascular endothelial cells, and the like suitable for clinical purposes can be supplied. [Embodiment] Φ The present invention is based on the following insights of the present inventors: the primate embryonic stem cells are placed in a state of stromal cell acclimation medium, and the culture container is covered with extracellular matrix By culturing the cells, they can be differentiated into blood cells even in the absence of feeder cells. Therefore, according to the present invention, it is possible to provide a method and method for making primate embryonic stem cells into blood cells or vascular endothelial cell differentiation without feeding cells, which is a method that is substantially accompanied by Incorporation of heterologous animal cells and differentiation of infections originating from heterologous animal viruses, differentiation through highly stringent control, highly reproducible production of blood cells or vascular endothelial cells, high efficiency of blood cells or vascular endothelial cells Manufacturing, manufacturing of pure blood cells or vascular endothelial cells, etc. Examples of the primate embryonic stem cells include, for example, cynomolgus monkey embryonic stem cells [Suemori (Η ·), etc., "" Establishment of Stem Cells (Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Rhesus monkey embryonic stem cells [Thomson, J. A. et al., "Isolation of a primate embryonic stem cell line ·" ", Proc. Natl. Acad. Sci, USA, Vol. 92 , P. 7844 to p. 7848 (1995)], velvet monkey embryonic stem cells [Thomson, J_A, etc. ', plurip〇tent cell lines derived from common marmoset blastocysts ·) ", Biolol · Repr〇d ·, p. 55, p. 254 ~ p. 259 (1996)], human embryonic stem cells [Thomson, JA ·, etc.," embryonic stem cell lines derived from human embryo cells ( Embryonic stem cell lines derived from blastocysts ·) ", Science, 282, pp. 1145 ~ 1147 (1998); Reubinoff, Β · Ε ·, etc., derived from human embryonic embryonic stem cells; somatic cell differentiation in vitro ( Embryonic stem cell lines from human blastosysts: somatic differentiation in vitro.), Nat. Biotech., Pp. 399 to 404 (2000)] and so on. One aspect of the present invention relates to a feeder-free medium for differentiation (i.e., a 'blood-differentiation medium for feeder-free embryonic stem cells') characterized in that it contains a stromal cell acclimation medium. The non-feeder cell differentiation culture gene of the present invention contains a stromal cell domestication medium, so it can exhibit the following excellent effects, allowing primate embryonic stem cells to differentiate into blood cells without using heterogeneous animal cells as feeder cells. As a result, blood cells can be obtained without substantial concomitant incorporation of heterogeneous animal cells, infection by a virus derived from a heterogeneous animal, and the like. Blood cells obtained by culturing using the medium for non-fertility differentiation of the present invention are separated from the above-mentioned culture container, and then cultured by adding a new 100097.doc -15- 200540270 silver culture cell differentiation medium to generate blood cells Excellent effect. Therefore, the root differentiation medium has excellent effects. The blood cells that can be regenerated and can exhibit amazing performances according to the present invention are excellent in non-feeding cell-free blood cells and that the "feed-free differentiation medium according to the present invention contains the above-mentioned remuneration medium" and thus will be exhibited. It can obtain excellent effects of blood cells or vascular endothelial cells with high efficiency and high purity.

Specifically, 'the primate embryonic stem cells are cultured by using the feeder-free cell differentiation medium of the present invention, for example, as shown in FIG. 7E, all cells are differentiated substantially in the same morphology. Cells obtained by differentiation showed substantially ⑽% positive for cd45 cells' specifically expressed in blood cells. Therefore, the non-feeder cell differentiation medium according to the present invention can differentiate embryonic stem cells into blood cells with extremely high efficiency. Among them, although it is difficult to produce cd34 + CD45 + hematopoietic cells from embryonic stem cells, it is only about 5% [for example, in Chadwick et al. 'Bl00d' Vol. 102, p. 906 ~ In the method, the production rate of CD34 + CD45 + m cells from human embryonic stem cells is 7.5 ± G.5%] 'However, according to the medium for culturing cell differentiation according to the present invention, it can exert a specific expression on hematopoietic stem cells. Cd34, can obtain the excellent effect of cells showing approximately 1GG% positive. Furthermore, 'Although the differentiation of embryonic stem cells into vascular endothelial cells was previously performed in a system different from blood cells, according to the culture medium for feeder-free differentiation of the present invention, surprisingly, the following excellent effects can be obtained. !! The finger cells generated when the stem cells differentiate into blood cells are placed in 100097.doc -16-200540270, and are further cultured with the new feeder-free cell differentiation medium described above, thereby obtaining vascular endothelial cells.

In the present invention, the above-mentioned so-called Γ-cylinder ru ^ ^ ^ L n month without meals "means that no feeding cells are used, or the condition that no feeding cells exist. The so-called blood cells may be all blood cells. Specifically, as the blood cells, for example, hematopoietic stem cells 0102, lymphoblastic stem cells, "Tianyu 0133, and lymphoblastic dendritic cells" are shown in the differentiation system diagram of autologous stem cells as blood cells represented by I. Precursor cells, lymphoblastic dendritic cells 03, T-lymphocyte precursor cells 0H) 4, T cells-5, B-lymphocyte precursor cells B, B-cells 107, Leukocytes 0, Nk precursors "Tian I 0109 NK cells 〇11 〇, bone marrow stem cells 〇i34, bone marrow dendritic cell precursor cells 0111, bone marrow dendritic cells 0112, mast cell precursor cells, mast cells 0114, basophilic spheroids Precursor cells 0115, Basophils 0116, Eosinophils 0117, Eosinophils 08, Granular spheres sf cell line precursors 0119 m Precursor cells mononuclear spheres 0121, Giant cells 〇22, Osteoclast precursor cell 0123 Moonbreaker cell 0124, neutrophil precursor cell 〇25, neutrophil 0126, megakaryocyte precursor cell 〇127, megakaryocyte 〇128, platelet MM, pre-small red blood cell precursor cell. 13〇 Late small erythroid lineage progenitor cells 〇131, 0132 and other red blood cells. In addition, the above-mentioned "blood cells" include precursor cells of hematopoietic stem cells; all forms of blood cells existing in all differentiation processes from the final differentiation of hematopoietic stem cells to peripheral blood. The above-mentioned acclimation medium (ie, the culture supernatant) refers to a nutrient supernatant obtained by culturing cells. In particular, the chemical culture used in the present invention 100097.doc • 17-200540270 may contain factors produced by stromal cell lines during culture.

The above stromal cell line supporting blood cell line is preferable, and examples thereof include OP9 cell line, S17 cell line (mice stromal cells in mice), and MS-5 cell line (mice stromal cells in mice). In the present invention, if the cells have the ability to support blood cells equivalent to the stromal cells described above, they can also be used from mouse embryo yolk sac, mouse embryo mouth mouth liver, mouse Primary cultured cells isolated from the hematopoietic tissue of the embryonic aorta reproductive middle kidney region (a〇rta-g0nad-mesonephros; AGM), para-aortic-splanchnopleura (PsP) along the embryonic aorta To replace the stromal cell lines described above. When using these cell lines and initial culture, the following types of serum may be replaced with bovine serum, or other animal serum such as horse serum, etc. The concentration of the serum may be changed. Moreover, although the differentiation from embryonic stem cells to finger cells requires the above-mentioned culture medium supernatant, the differentiation from embryonic stem cells to CD45-positive hematopoietic stem cells is not necessarily the entire process, and may also depend on the condition of the finger cells. For the differentiation of positive hematopoietic stem cells, the above culture supernatant is temporarily not used. In the present invention, the stromal cells can be either cells that have been irradiated with radiation or radiation, or can be untreated cells. Knife adjustment can be performed by irradiating radiation onto the above-mentioned substrate strain. For example, by radiating radiation to the above-mentioned Kibei, Takashi, and Tho strains, the proliferation rate of the above-mentioned F-cell line can be slowed, and the nutrient content contained in the culture fluid towel used for the culture of the stromal cell line can be reduced. Consumption rate. It also has the following advantages: · Reduces the generation of δ-radiation decomposition products excreted from the stromal strain I, and can extract embryonic stem fines at a higher rate. 100097.doc -18 · 200540270 cells differentiate into blood cells or vascular endothelium The nutrients and the above-mentioned nutritional components produced by the above-mentioned beauty strains necessary for cells are necessary for cells. Furthermore, in the present invention, other methods that can control the proliferation rate can be implemented instead of irradiation with radiation. Furthermore, when the culture is performed in a manner that does not control the proliferation rate, the culture medium supernatant is recovered after the fusion is formed, and the factors required for differentiation into blood cells or vascular endothelial cells are not damaged (for example, by dialysis) , Supplement the nutritional components consumed by the stromal cell line, and remove the surplus medium obtained by the nutrients and metabolic decomposition products produced by the stromal cell line. Furthermore, in order to stably inhibit the proliferation rate of the stromal cells, the radiation 1 of the radiation is preferably 40 Gy or more, and more preferably 50 Gy or more. On the other hand, in the above-mentioned stromal cell acclimation medium, the above-mentioned stromal cells necessary for fully exerting the effects of differentiation of embryonic stem cells into blood cells and vascular endothelial cells and sufficient payment for differentiation of embryonic stem cells into blood cells or vascular endothelial cells In terms of the generated factors, the radiation dose is 90 Gy or less, preferably 80 Gy or less, more preferably 70 Gy or less, and particularly preferably 60 Gy or less. For example, an X-ray irradiation device (trade name: MBR-1520R-3, manufacturer: Yuet Li Medical Co., Ltd. (English name: mtacM Medical)

Corporation), with a tube voltage of 150 kV and a tube current of 20 mA, the filter was set to 0.5 AL + 0.1 Cu, a culture plate of matrix cells was set in the test material room, and then irradiation was performed to perform radiation irradiation. In addition, in the non-feeding cell differentiation medium of the present invention, other methods such as irradiation with radiation and controlling the proliferation rate can be used to slow down the proliferation rate of the substrate 100097.doc • 19 · 200540270 cells to produce an acclimatization medium. . The r: the secret medium used in the present invention can also be an artificial medium with the same composition as that of the culture medium obtained by cultivating the above-mentioned base 1; The so-called "cultivated culture medium obtained from cultured stromal cells: with the same component J" refers to the components or derivatives contained in the cultured culturing substrate obtained by culturing the stromal cells described above, and the relevant components can be appropriately determined by: (Such as mass analysis, various chromatography, NMR, etc.) for analysis and determination. The above-mentioned acclimatization medium of stromal cells can be obtained by containing, for example, a process: w, ⑷ in a medium suitable for maintaining stromal cells, so as not to form a fusion (B) the step of culturing the cells obtained in the above step 置于 in a medium suitable for maintaining stromal cells and embryonic stem cells, and (C) from The step of removing stromal cells from the culture obtained in the above step (B) and 'when the base f cells are irradiated with radiation', the above-mentioned stromal cell acclimation medium can be obtained by a process containing, for example, the following steps: The step of maintaining the culture of stromal cells in a culture-free manner (ie, before fusion), (B ') irradiation and radiation Steps of culturing the cells obtained in (a) above, placing the cells obtained in a medium suitable for maintaining stromal cells and embryonic stem cells, and (C,) the cells obtained from step (B,) above Steps to remove stromal cells in culture 100097.doc -20- 200540270

Specific examples of the "medium medium for maintaining stromal cells" are, for example, OP9 cells. For example, OP9 cell culture medium composition may include: α-MEM, 20 vol% heat-deactivated bovine embryo serum, cellophyl alcohol, 1.6 mM L-amidamine, final concentration 100u / ml penicillin, final concentration 100μδ / ΐη1 streptomycin}, OP9 cell culture medium 2-1 {composition: heart] \ ^] \ 4 , 20 vol% heat-deactivated bovine fetal serum, 0.1 mg mercaptoethanol, 1 mM L-amidamine, final concentration 10 u / ml penicillin, final concentration w pg / ml streptomycin}, and the like. Examples of the above-mentioned culture medium suitable for maintaining stromal cells and embryonic stem cells include the following: P9 cell culture medium 2 (composition: Iskov-modified Dubco medium, 5 vol% heat-deactivated cattle Embryo serum, β-fluorenyl alcohol, 2 mM L-amidamine, opium cell culture medium 2-2 {Composition: Iskov's modified Dubeco medium, 15% volume heat to deactivate bovine embryos Serum, 0 mM mercaptoethanol, 3 mM L-glutamine, 5 μM hydrocortisone, final concentration 10 u / mi penicillin, final concentration 10 Pg / ml streptomycin} and the like. When producing the above-mentioned acclimatization medium, the culture conditions of the stromal cells can be appropriately set depending on the type of the cells to be used, and for example, conditions of 37 t and 5 vol% co2 can be mentioned. More specifically, when OP9 cells are used, the above-mentioned stromal cell acclimation condition is to use the above-mentioned OP9 cell culture medium ^ or ^ on a 100111 culture plate to 37. (:, Cultured in 5 vol% Co2, subcultured every i ~ 2 days, in a P9 cell plate that has formed 60 ~ 70% confluence, with 100097.doc -21 · 200540270 of 46 Gy 7 rays After irradiating for about 10 minutes, incubate at 37 ° C, 5% by volume for 4 hours, then use phosphate buffered saline or isotonic solution without endotoxin or "suitable for maintaining stromal cells and embryonic stem cells. "P9 cell culture medium", "P9 cell culture medium 1-1 or 2-1", and P9 cells were washed. To this dish, 10 ml of the above OP cell culture medium 1-2 or 2-2 was added, and at 37 ° C, After culturing in 5 vol% co2 for 12 hours, the supernatant was obtained from the culture by centrifugation and filtration. One embodiment of the culture medium for feeder-free differentiation of the present invention contains the above-mentioned leftover culture medium. 2. Culture medium components (eg, culture fluid) for maintaining primate embryonic stem cells, serum, and cytokine-free medium for differentiation of feeder cells. In addition, the domestication medium in related embodiments may also contain an oxidation inhibitor. As the "to maintain the primate embryonicity The "cell culture component" may be a specific medium commonly used to maintain primate embryonic stem cells, and specific examples include, for example, Iskov's modified Dubeck medium (IMDM / F12), etc. Those who maintain embryonic stem cells include, for example, human serum, bovine serum, bovine embryo composition, horse serum, etc. In addition, in terms of not causing a difference in the success or failure of serum batch culture, the culture of embryonic stem cells It is better to perform the serum batch inspection. The so-called batch inspection refers to determining the suitability with the above-mentioned embryonic stem cells by performing a culture experiment. Furthermore, in the present invention, the batch size of serum suitable for each embryonic stem cell line is also It can be different. In addition, the present invention can also use the same ingredients as the serum. The cytokine used in the present invention can be used to differentiate embryonic stem cells. 100097.doc -22- 200540270 is a blood cell and / or vascular endothelial cell. Factors are not particularly limited, and examples include: stem cell factor (SCF), granulocyte community stimulating factor (G-CSF), granulocyte macrophage community Stimulating factors (gm_csf, B. macrophage cell community stimulating factor (M-CSF), erythropoietin (EpO), platelet growth factor (TPO), Flt3 ligand (FL), interleukin (IL) (for example ...) Interleukin-3, interleukin-6, interleukin-15, interleukin, etc.), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP; for example, BMP-4, etc.), tumor suppressor M, Acidic and basic fibroblast growth factors (acidic FGF, basic FGF), angiopoietin family (such as angiopoietin and angiopoietin-2), etc. The above-mentioned G-CSF has the function of enhancing the production of neutrophils. In addition, EPO (erythropoietin) induces the production of red blood cells that have the function of transporting oxygen. Also, TPO (platelet growth factor, platelet growth factor) induces the production of megakaryocytes and platelets that have the effect of proliferating and hemostasis of hematopoietic stem cells (coagulating blood and preventing bleeding). In addition, melanin-15 induces natural killer cells (NK cells) that attack cancer cells and the like. Another aspect of the present invention relates to a culture system for non-atrophoblast differentiation, comprising a culture medium for non-feeder cell differentiation and a culture container covered with extracellular matrix. A major feature of the feeder-free cell differentiation culture system of the present invention is that it contains the feeder-free cell differentiation medium of the present invention and a culture container covered with extracellular matrix. Therefore, according to the culture system for non-feeding differentiation according to the present invention, excellent effects can be exhibited: in the absence of xenobiotic cells, primate embryonic stem cells can be differentiated into blood cells under the control of a high degree of tightness without Will be substantially accompanied by the incorporation of heterogeneous animal cells, 100097.doc -23- 200540270, and virus infections originating from heterogeneous animals. The above-mentioned extracellular matrix can include: collagen, laminin, fibronectin, and urinary acid secreted from other cells to promote the adhesion of cells. ^ Inducing high-efficiency differentiation into blood cells and vascular endothelial cells Lu, on the other hand, is better. Examples of the knee source include ιν-type collagen. … Furthermore, as described above, the coating of the extracellular interstitial culture container can be performed by the conventional φφ method. X. In the present invention, it can be considered that the above-mentioned extracellular interstitial coating (such as collagen coating) is a three-dimensional mesh structure. In this way, the embryonic stem cells contact the collagen even on the side or the top surface. Thereby increasing the interaction and information exchange with collagen, the survival rate and differentiation rate can be improved. The culture container may be a container generally used for cell culture. The culture system for non-feeder cell differentiation of the present invention is suitable for the differentiation of primate embryonic stem cells into gold fluid cells. Another aspect of the present invention relates to a method for the differentiation of feeder cells from primate embryonic stem cells into blood cells (hereinafter referred to as the method for feeder cell-free blood cell differentiation), which is characterized in that: The primate embryonic stem cells are cultured in a culture medium in a state of non-regulatory cells to differentiate the embryonic stem cells into blood cells. One of the major features of the feeder-free blood cell differentiation method of the present invention is that the feeder-free cell differentiation medium of the present invention is used. Therefore, according to the method for differentiation of blood cells without feeder cells of the present invention, the following excellent effects can be exhibited: Since the feeder-free cell differentiation medium of the present invention is used, primate embryonic stem cells can be implemented as blood cells. Differentiation and 100097.doc • 24-200540270 will not be substantially accompanied by the incorporation of xenobiotic cells and virus infection from xenobiotics. In addition, the method for differentiation of arsenic-free blood cells of the present invention has the following characteristics, which uses the culture medium for feeder-free differentiation of the present invention and a culture container covered with extracellular matrix. Therefore, according to the method for differentiation of non-feeding blood cells of the present invention, it is possible to exert an excellent effect of controlling primate embryonic stem cells to differentiate into blood cells through a higher degree of control. In the feed cell-free blood cell differentiation method of the present invention, specifically, 'the primate embryonic stem cells are placed in a culture container covered with extracellular matrix, and in the state of no feed cells, the The cells are cultured in a feeder cell differentiation medium to generate finger cells (fin # r_iike cell), and further culture is performed to generate blood cells. The above-mentioned "fingerMike cell" refers to a cell that has a finger or joint-like structure as shown in Fig. 7A. In addition, the related "finger cells" are cells that appear after culturing undifferentiated primate embryonic stem cells in the medium for feeding-free cell differentiation of the present invention. When cynomolgus embryonic stem cells are used, It is a cell that appears from day 1 to day 7 (average 3 days). It has the ability to differentiate into either a blood cell line or a vascular endothelial cell line, and it has the activity as a so-called "angioblast" Cell group. In addition, it is positive for CD 1 51, which is well-known as a marker of vascular endothelial cells, but it is a marker for hematopoietic vascular endothelial (hematopoietic endothelium) that exhibits "angioblast" activity in petit mongolia. Ve-cadherin is negative and can locate a new type of 100097.doc -25- 200540270 cell group that shows angioblast activity in primates. It can also be considered that the finger cells may be immature cells in various tissues due to the grain-i which is an undifferentiated mesoderm marker. · ⑽ is positive, so it has the characteristics of tissue flying, flying, and field cells. In addition, the above-mentioned "vasoblast" activity refers to the ability of one or more cells to differentiate into blood cell lines or vascular endothelial cells from culture conditions. The present invention also includes a method for producing finger cells, which is characterized in that: primate embryonic stem cells are placed in a culture container covered with extracellular interstitial matter in the state of culturing cells as described above The culture medium for culturing cells is differentiated. Furthermore, this month / month also contains the related finger-shaped fine monthly bag. The finger cell line of the present invention is obtained by placing primate embryonic stem cells in a K-culture container covered with extracellular mussels, in a state without feeder cells, and in a feeder-free cell differentiation matrix of the present invention. Cells obtained by culturing. The expression of the above-mentioned finger cells (for example, the form of a finger or a joint-like structure) 5 1 Flk 1 c-kit, VE-cadherin, and the like can be classified as "and separated. Specifically, for example, Cell classification using flow cytometry using antibodies with the above-mentioned markers (such as CD15 m, c, t, VE_ about adhesion, etc.), cell classification using magnetic beads that hold the antibody, etc. Isolate target finger cells. The above finger cell lines provide, for clinical and basic research purposes, for example, blood cells and vascular endothelial cells that differentiate cells from the pedigree to the downstream. That is, δ can be provided as a matter of course. As a cell therapy material for disease groups with blood diseases and vascular lesions, and in a variety of tissues with increased stem cells or bone marrow precursor cells, by cell fusion or in the group 100097.doc • 26- 200540270 = Transdifferentiati () n), and a broader organization can provide effective materials for general treatment of regenerative medicine. In the latter, of course, it can also be used for related primates Effective materials for basic research on the differentiation and development of blood cells and vascular endothelial cells, especially in the analysis of the early hematopoietic institutions (which cannot be used to analyze the use of T organisms in mice) after the research of Jiu Luoluo so far, finger cells can provide Makes molecular-level analysis easy, correct, and general.

* \ For example, by using material-specific antibodies, ultra-pure probes, and primer pairs Λ #, the performance of the above-mentioned morphogenetic cells on VE, vascular endothelial cell-specific markers, adhesion markers, CD151 and other markers was performed. Analysis can be determined by showing VE-cadherin-negative CD151 positive. For example, by using the above-mentioned "medium-suitable medium for maintaining stromal cells and embryonic stem cells", or by cultivating the above-mentioned finger cells according to different circumstances, a culture medium to which various cytokines are added to obtain active proliferation. In addition, after the finger cells are detached from the culture container using membrane protease and the like, and the above-mentioned medium is supplemented in the same culture container to continue culturing, it is easy to proliferate the finger cells in a reproducible manner, so it can be used in the same culture container. Culture and maintain for at least 2 months. Hematopoietic stem cells or hematopoietic precursor cells can be obtained by the method for differentiation of blood cells without feeder cells according to the present invention, which are cells that are hematopoietic line specific markers cd34 and CD45 (CD34 positive CD45 positive cells). The CD34-positive and CD45-positive cells obtained by the method for differentiation of blood cells without feeder cells of the present invention exhibit a relatively high nucleus / cytoplasm and have a prominent nucleoli nature. In addition, in a community analysis using a semi-solid 100097.doc -27- 200540270 medium containing methyl cellulose and the like, a mother cell colony was formed efficiently (plating efficiency> 80%) (see FIG. 17) ), Showing properties as undifferentiated hematopoietic cells with very high hematopoietic capacity. Therefore, it is possible to differentiate from the above-mentioned CD34-positive and CD45-positive cells into hematopoietic stem cells, lymphoblastic stem cells, lymphocytic dendritic cell precursor cells, lymphoblastic dendritic cells, T-lymphocyte precursor cells, τ cells, and B-lymphocyte precursor cells. , B cells, cytoblasts, NK precursor cells, NK cells, bone marrow stem cells, bone marrow dendritic cell precursors, bone marrow dendritic cells, mast cell precursor cells, mast cells, basophilic precursor cells, basophils Spheroids, eosinophil precursors, eosinophils, granulosa macrophage precursors, macrophage precursors, monocytes, macrophages, osteoclast precursors, osteoclasts, neutrophils Precursor cells, neutrophils, megakaryocyte precursor cells, megakaryocytes, platelets, precursor cells of early small red blood cell lines, precursor cells of late small red blood cell lines, red blood cells and other cells. When primate embryonic stem cells are seeded in the above-mentioned feeder-free cell differentiation medium, the undifferentiated embryonic stem cell group maintained at the center of the undifferentiated embryonic stem cell community is removed from the culture plate, and type IV collagen is used. When the covered 6-well plate is used as a culture container covered with extracellular interstitial material, it is better to seed in each well so as to become lxlO1 to lxl03 cells, and it is better to sow in the form of 102 cells. x Nutrient conditions for primate embryonic stem cells in the medium for non-feeder cell differentiation can be appropriately set according to the type of primate embryonic stem cells used. For example, 37 ° C, 5 vol% C02 Conditions, etc. The & culture medium 'can be derived from the primate embryonic stem cells 100097.doc -28-200540270, and is suitable to be exchanged for a new medium without feeder cell differentiation. The above-mentioned finger cells are generated after the culture of primate embryonic stem cells in a culture medium without feeder cells has begun for several days (for example, in the case of cynomolgus monkey embryonic stem cells, about the third day), After the fusion is formed, round cells will be generated from the above finger cells. From the viewpoint of preventing the depletion of nutrients after the appearance of the above-mentioned round cells, it is preferable to change the culture medium after 2 days. The above-mentioned round cells obtained by the method for differentiation of feeder cell-free blood cells of the present invention can be used to analyze stem cell markers CD34, hematopoietic lines, for example, by using conventional methods such as specific antibodies, specific probes, and primer pairs. The expression of the specific marker CD45 and other markers can be confirmed by showing CD34 positive CD45 positive. Another aspect of the present invention is a feeder-free cell differentiation method (hereinafter referred to as a no-feeding and cell vascular endothelial cell differentiation method) for making primate embryonic stem cells into hemorrhagic endothelial cells, which is characterized by: Animal embryonic stem cells are placed in a culture container covered with extracellular mesenchyme, cultured in a feeder-free state in the feeder-free cell differentiation culture medium of the present invention to make finger cells, and the finger-like cells are formed. The cells are transferred to a new culture medium, and further cultured in a culture container covered with extracellular interstitial material, so as to generate vascular endothelial cells. One of the major features of the method for differentiation of vascular endothelial cells without feeder cells of the present invention is that the culture medium for feeder-free cell differentiation according to the present invention is used. Therefore, according to the method for differentiating vascular endothelial cells of the present invention, 100097.doc -29- 200540270 can exert the following excellent effects, which can allow primate embryonic stem cells to differentiate into vascular endothelial cells without substantial Accompanied by the incorporation of heterologous animal cells, and the infection of viruses derived from heterologous animals. In the method for differentiation of vascular endothelial cells without the proud cells of the present invention, the steps for generating finger cells are the same as the above-mentioned method for differentiation of blood cells without feeder cells. Finger cells can be separated from a culture container covered with extracellular matrix by using a conventional method (such as trypsin treatment) for separating cells from a culture container. The isolated finger cells are transferred to a culture container which is placed in a new non-feeder cell differentiation medium and covered with an extracellular matrix, and further cultured. The culture conditions of the finger cells can be the same as those of the embryonic stem cells in the above-mentioned method for differentiation of feeder-free blood cells, and examples thereof include conditions of 37 ° C and 5% by volume of CO2. According to the non-feeding vascular endothelial cell differentiation method of the present invention, blood and endothelial cells can be obtained as negative for hematopoietic line specific markers 匸 34 and 匸 45, and for endothelial cell specific markers VE -Cadherin-positive cells (CD34-negative CD45-negative VE-cadherin-positive cells). The above-mentioned CD34-negative, CD45-negative VE-cadherin-positive cell line is in the shape of a crane stone. In addition, the above-mentioned CD34-negative CD45-negative VE-cadherin-positive cells can be subcultured in a new plate for several months, and thus exhibit excellent properties of stable proliferation. Specifically, the above-mentioned cobblestone-like CD34-negative CD45-negative VE_cadherin-positive cells are, for example, cells that can be subcultured for several months in an OP9 domesticated medium containing cytokines, and can be used in vitro 100097.doc -30- 200540270 Proliferation since. Therefore, with the related cells, the excellent effect of preparing a large number of grafts can be exerted. In addition, by culturing the cobblestone-like CD34-negative CD45-negative VE-cadherin-positive cells in a culture medium suitable for vascular endothelial culture, it is possible to obtain cord-like cells that form a lumen of a blood vessel. Related rope-shaped cells with vascular lumen can be used, for example, to modulate functional cells before transplantation. Hematogenous endothelial cells obtained by the method for differentiation of vascular endothelial cells without feeder cells of the present invention, for example, by conventional methods using specific antibodies, specific probe primer pairs, and the like, analyze, for example, hematopoietic line specific markers CD34 and CD45, endothelial cell-specific markers VE_cadherin and other markers can be confirmed by showing CD34 negative CD45 negative VE-cadherin positive. Another aspect of the present invention relates to a method for manufacturing a blood cell, which is characterized in that primate embryonic stem cells are placed in a culture container covered with extracellular interstitial substance in a state of no feeding cells. The culture medium for feeder cell differentiation without φ was cultured to differentiate the embryonic stem cells into 'blood cells', and finally the blood cells were separated and separated. One of the major characteristics of the method for producing a blood cell of the present invention is to use the culture medium for feeding cell-free differentiation of the present invention. Therefore, according to the method for producing a blood cell of the present invention, it is possible to exert excellent effects of obtaining high-purity blood cells without substantially accompanying the incorporation of heterogeneous animal cells and diseases derived from heterogeneous animals. One of the major characteristics of the method for producing a blood cell of the present invention is that primate embryonic stem cells are placed in a culture container covered with extracellular matrix. 100097.doc 31 200540270 The culture medium for feeder-free cell differentiation according to the present invention Cultivate. Therefore, according to the method for producing a blood cell of the present invention, it is possible to exert an excellent effect of allowing primate embryonic stem cells to differentiate into vascular endothelial cells under highly tight control. In addition, another embodiment of the method for producing a blood cell of the present invention is obtained by adding the culture medium for feeder-free cell differentiation of the present invention to a culture container after blood cell separation, and further culturing in a state without feeder cells. Method of blood cells. According to the method for producing blood cells according to the related embodiment, the blood cells can be obtained by adding the culture medium for feeding-free cell differentiation of the present invention to the culture container after the blood cells are separated, and further culturing without feeding. Therefore, according to the method for producing blood cells of the present invention, the excellent effect of obtaining blood cells with long-term sustainability and excellent reproducibility will be exhibited. The method for producing blood cells of the present invention can be carried out in the same order as the method for differentiation of acellular cells of the present invention. Furthermore, in the method for producing a blood cell of the present invention, the CD34-positive and CD45-positive cells can be further differentiated under appropriate conditions according to the type of target blood cells, and the number of cells in the medium for feeder-free differentiation can be appropriately changed. Cytokines. In the method for producing blood cells of the present invention, examples of producing blood cells with various cytokines include the differentiation of τ into g_csf and GM-CSF granule cells, and mononuclear spheres / giants of GM-CSF and M-CSF. Phage differentiation, NK cells becoming IL-15, erythrocyte differentiation, TPO-derived megakaryocyte / platelet differentiation, 100097.doc -32- 200540270 IL-4 and GM-CSF dendritic cells Differentiation. The target blood cells in the method for producing a blood cell of the present invention can be classified and separated, for example, by the expression of specific markers such as CD34 and CD45 in the blood cells as indicators. Specifically, for example, by using an antibody having the above-mentioned label (for example, CD34, CD45, etc.), cell classification by flow cytometry, and cell classification using magnetic beads holding the antibody, etc., the target can be separated. Blood cells. Furthermore, in the method for producing a blood cell of the present invention, the cells are cultured in a feeder-cell-free differentiation medium obtained from fibroblast cell-line stromal cells such as OP9 cells, and then in a bone marrow cell line from MS-5 cells and the like. By culturing the feeder-free cell differentiation medium obtained from stromal cells, giant pupae cells can be produced. Blood cells obtained by related manufacturing methods are also included in the scope of the present invention. The blood cell line of the present invention is obtained by the manufacturing method of the present invention, and therefore has excellent properties such as incoherent mixed human cells with heterologous animal cells, and infections derived from heterologous animal viruses. Furthermore, since the gold liquid cell line of the present invention is obtained by the production method of the present invention, it exhibits high purity and uniformity. θ Here, the blood cells of the present invention can be used as materials for manufacturing blood for transfusion, blood for blood transfusion, and materials for basic research on hematopoietic mechanisms. From the beginning of 4 days in the middle of the Ming Dynasty, 1, ~~, 'can also be maintained in a medium such as a cell storage tank (manufactured by Shici Science Co., Ltd.) and other special solutions for cold storage under nitrogen cold conditions. . 100097.doc -33-200540270 Another aspect of the present invention relates to a method for manufacturing vascular endothelial cells, which is characterized in that primate embryonic stem cells are placed in a culture container covered with extracellular mesenchyme and fed without culture. In a state, the medium is cultured in the feeder-free medium for differentiation of the present invention to generate finger cells, and the finger cells are moved to a culture container covered with extracellular matrix placed in a new medium for further development. The embryonic stem cells are cultured to differentiate into vascular endothelial cells, and the vascular endothelial cells are isolated. The method for manufacturing vascular endothelial cells of the present invention is characterized in that: primate embryonic stem cells are placed in a culture container covered with extracellular matrix, and in the state of no feeding cells, The medium for feeding cell differentiation is cultured so as to generate finger cells. Therefore, according to the method for producing a vascular endothelium according to the present invention, vascular endothelial cells can be obtained without substantial concomitant incorporation of heterogeneous animal cells and infection by a virus derived from a heterogeneous animal. The method for producing a vascular endothelial cell according to the present invention is characterized in that: the finger cells are transferred to a culture container covered with extracellular substance and further cultured in a new medium. Therefore, according to the method for producing vascular endothelial cells according to the present invention, the following excellent effects can be exhibited: Surprisingly, vascular endothelial cells can be obtained due to a high degree of strict control, thereby obtaining vascular endothelial cells of pure purity. ° In the method for producing vascular endothelial cells of the present invention, for example, the expression of markers such as cadherin and PECAM (CD31) can be used as indicators to classify and isolate vascular endothelial cells. Specifically, for example, the cell classification of 100097.doc -34- 200540270 of a flow cytometer can be performed by using ^ for the above-mentioned antibodies of VE-cadherin, PECAM (CD31) and other markers, and by using the antibody Isolation of vascular endothelial cells by cell classification of magnetic beads. Vascular endothelial cells obtained by a related manufacturing method are also included in the scope of the present invention. Since the vascular endothelial cell according to the present invention is obtained by the production method of the present invention, it exhibits excellent properties such as the absence of substantial incorporation of heterogeneous animal cells, infection by a heterogeneous animal virus, and the like. Furthermore, since the vascular endothelial cells of the present invention are obtained by the production method of the present invention ', they exhibit a purity and uniformity. Therefore, according to the vascular endothelial cells of the present invention, they can be used as materials in related basic research such as vascular injury treatment or materials for improving local blood flow, transplant materials and the manufacture of these materials, as well as management of the development and differentiation mechanism of endothelium. . According to the vascular endothelial cell of the present invention, for example, three-dimensional vascular structure can be obtained by culturing in collagen or the like. The vascular endothelial cells of the present invention can be maintained in a culture medium such as a cell cold storage solution such as a cell storage tank (manufactured by Shici Science Co., Ltd.) and under nitrogen cold conditions. Furthermore, this #Mingzhi Wuyi culture cell differentiation medium and the method of using 苴 are not limited to the differentiation technology of embryonic stem cells into blood, and can be used as an extension to various cell differentiation technologies according to the type of cytokines added. application. The following describes the present invention in detail based on examples, but the present invention is not limited to those examples. 100097.doc -35- 200540270 [Example 1] Preparation of stromal cell domestication medium

OP9 cells as stromal cells were placed in OP9 cell culture medium 1-1 {composition: α-ΜΕΜ [manufactured by Invitrogen], 20 vol% heat-deactivated bovine fetal serum [PAA Laboratories GmbH], 1 mM β-pore Ethyl alcohol [manufactured by Sigma Chemical], 1.6 mM L-amidamine [manufactured by Invitrogen], final concentration 100 U / ml penicillin [manufactured by Invitrogen], final concentration 100 pg / ml streptomycin [Invitrogen] Manufacturing;)}, and cultured on a 10 cm culture plate in a CO 2 incubator at 37 ° C. and 5% by volume of CO 2. Op9 cells were maintained in a manner that did not form a confluence through subcultures once every about 3 days. Specifically, the culture fluid was removed from the OP9 cells to be fused at a weight of 0.25. / 〇 的 Trypsin-HBSS (Hanks balanced salt solution,

Hanks balanced salt solution) and react for about 2 minutes to remove the cells. After washing the cells in the OP9 cell culture medium described above, the cell density is reduced to about a fraction, and the cells are suspended again. In the above medium. The cells were seeded in a 100-diameter melon culture dish, and cultured in a CO2 incubator at 37 ° C and 5% by volume of CO2. In the cultivation of the above-mentioned medium, the culture plate was irradiated with radiation (15 kV '2G mA, 60 Gy, 1 G minutes) before the fusion was formed. Thereafter, a 0.25% by weight trypsin-depleted solution (soh-ion) was added to the culture plate, a pipetting operation was performed, and the cells were detached. Trypanocysts were removed from the solution containing the obtained cells, and cultured in qP9 cell culture ^ 2 {Composition: Iskov's Modified Dubco Medium (Iskov, s 100097.doc • 36- 200540270 modified Dulbecco's medium; IMDM) [manufactured by Sigma Chemical Co., Ltd.], 15% by weight of heat-deactivated bovine fetal serum [PAA Laboratories GmbH], 1 mM β-benzyl ethanol (manufactured by Sigma Chemical Co., Ltd.), 2 mM L-amidamine Cells were washed once with amine [manufactured by Invitrogen]}. Thereafter, the obtained cells were suspended in the above-mentioned OP9 cell culture medium 1-2. The obtained cell suspension was placed on a 6-well flat-bottomed microtiter plate in a manner of 3 × 104 cells per well, and placed in a C02 incubator at 37 ° C, 5% by volume of 0: 0. 2 Cultivate. The culture supernatant was collected 1 day, 2 days, and 3 days later, and a new OP9 cell culture medium 1-2 was added. The above-mentioned culture supernatant was used as an OP9 acclimatizing medium for non-feeder cell differentiation. [Example 2] Differentiation of undifferentiated primate embryonic stem cells into blood cells 1 Subsequent undifferentiated monkey embryonic stem cells were used as primate embryonic stem cells, and were derived from mice by modulation Embryonic fibroblasts are used as feeder cells. In the above-mentioned undifferentiated monkey embryonic stem cell culture plate, a 0.25% by weight trypsin-HBSS solution was added, and then the cells were detached and suspended in a differentiation induction medium 丨 -y Composition: The above non-feeder cell differentiation was domesticated with OP9 Medium i, final concentration of 20 ng / mli vascular endothelial growth factor (VEGF), final concentration of 20 ng / mi2 bone morphogenetic protein-4 (BMP-4), 20 ng of stem cell factor (SCF), final concentration of iOOn # FU3-ligand}. The obtained cell suspension was seeded in a collagen-coated plate (a 6-well microtiter plate covered with mouse ιν-type collagen manufactured by Becton Dickinson, Inc., and then the above-mentioned embryonic stem cells were placed in 100097.doc -37 · 200540270 Culture in an incubator at 37 ° C, 5 vol./0 〇2. Within 4 to 5 days after the start of the culture, it can be observed that slightly elongated cells are arranged in the longitudinal direction and have a structure like the "chuan" In addition, FIG. 2 shows the colony derived from embryonic stem cells on the 7th day after the start of the culture. On the 7th day after the start of the culture, the medium was changed to a differentiation medium 1-2 {Composition: The above-mentioned P9 acclimatization medium without pseudoculture cells differentiation 1. VEGF with a final concentration of 20 ng / ml, and a final concentration of 20 ng / m] ^ BMP-4, SCF with a final concentration of 20 ng / ml, and a final concentration of 20 ng / ml? 10 ligand, final concentration between 10 ng / ml interleukin-6 (IL_6), the final concentration is called / such as interleukin-3 (IL-3), final concentration 1 ng / ml of granulocyte macrophage colony stimulating factor (GM-CSF)} and continue the culture. 10th day after the start of the culture On the 14th day, cell proliferation masses appeared around the "Chuan" -shaped structure, showing differentiation to the blood line. 14 days after the start of the culture, the medium 1-2 was changed to a medium for inducing differentiation 1 -3 0.99 acclimation medium for cultured cell differentiation 1, SCF at a final concentration of 20 ng / ml, Flt3-ligand at a final concentration of 20 ng / ml, IL-6 at a final concentration of 10 ng / ml, and final concentration IL-3 at 1 ng / ml and GM-CSF at a final concentration of 1 ng / ml} and continue the culture. As shown in Figure 3, complete differentiation into blood cells was observed on the 18th day after the start of the culture Then, 1 ml trypsin solution was added to the culture plate in an amount of 0.25% by weight, followed by vigorous pipetting operation, and cultured at 37t to recover the cells. The recovered cells were phosphate-buffered saline (pBS) After washing, 1x10 cells were combined with primary antibodies such as CD34, CD45 and other markers on ice, ie 100097.doc -38- 200540270 combined with anti-CD34 antibody [manufactured by BD Biosciences] or FITC-conjugated anti-CD45 antibody [ BD Biosciences company] reaction for 30 minutes. Thereafter, the amount was used by The product name is FACSCalibin [manufactured by BD Biosciences]. The expression of each marker was analyzed. The results are shown in Figs. 4 and 5. Fig. 4 shows the ratio of CD34-positive cells. Fig. 5 shows the ratio of CD45-positive cells. As shown in Figures 4 and 5, CD45-positive cells were 70.60% and CD34-positive cells were 92.60%. On average, CD45-positive cells are 30-50%, and CD34-positive cells are 70-90%. [Example 3] Preparation of OP9 acclimatization medium for non-feeder cell differentiation 2 OP9 cells were placed in OP9 cell culture medium 2-1 {Composition: α-ΜΕΜ [manufactured by Invitrogen], 20 vol% heat of selected batches Deactivated bovine fetal serum [PAA Laboratories GmbH], 0.1 mM β-Weiyl ethanol [manufactured by Simga Chemical], 1 mM L-glutamine (manufactured by Invitrogen), final concentration of 10 U / ml Cyanogenin [manufactured by Invitrogen], streptomycin [manufactured by Invitrogen]] with a final degree of 10 pg / ml, and placed on a 10 cm incubator in a CO2 incubator at 37t, 5 Cultivation is performed at (02% by volume). Subculture is performed every 1 to 2 days before fusion is formed. At the time of subculture, the culture supernatant is first drawn from the culture on the culture plate, and the cells on the culture plate are absorbed. 0.2 ml of trypsin / 0.02 wt% of a £ 0 buta solution [manufactured by GIBC0, catalog number: 23200-072] was added, and 2 ml was cultured at 37 ° C for 2 to 3 minutes. Thereafter, new OP9 was added. Cell culture 100097.doc • 39- 200540270 Culture medium 2-18 ml, sufficient The suspension was centrifuged and the supernatant was removed. Thereafter, the OP9 cells were suspended at 1 to adjust the cell concentration to about 1/3 / 3-1 / 6 of the above culture. 〇ml of new OP9 cell culture medium 2-1. The obtained cell suspension was transferred to a 10 cm culture plate, and cultured in a CO2 incubator at 37 ° C and 5% by volume of CO2. Then, a disc of 60-70% confluent OP9 cells formed in a 10 cm culture dish was placed on a focal table 290 mm under the trade name: MBR-1520R-3 (manufactured by Hitachi Medical Co., Ltd.) at 46 Gyi 7 The radiation was irradiated for 4 hours. After that, OP9 cells were washed with phosphate-buffered physiological saline (PBS), and 10 ml of OP cell culture medium 2-2 was added to the dish. {Composition: Iskov modified Du 15% by weight of heat deactivated bovine embryo serum [paa] manufactured by Beco Medium (IMDM) [Invitrogen]

Laboratories GmbH], 0.1 mM β-mercaptoacetate [manufactured by Sigma Chemical Co., Ltd.], 3 mM L-glutamine (manufactured by Invitrogen Corporation), 5 // Μ hydrocortisone, final concentration is 丨〇u / ml of penicillin [manufactured by Invitrogen], streptomycin [manufactured by Invitrogen] at a final concentration of 10 Å / ...}. Thereafter, the oop9 cells were cultured in a CO2 incubator at 37 ° C, 5% by volume of CO2 for 12 hours, and the culture supernatant of the first round was recovered. Thereafter, a new OP cell culture medium 2-2 was added to the remaining OP9 cells and cultured in the same manner as described above. After 2 hours, the second culture supernatant was recovered. The obtained supernatant was recovered and immediately filtered through a filter with a pore size of 22.22 μm to obtain 100097.doc -40-200540270 OP exposed medium 2 for feeder-free cell differentiation. Furthermore, the culture medium 2 used for the differentiation of OP9 was stored under passage. [Example 4] Differentiation of primate embryonic stem cells into blood cells 2 (1) Maintenance of primate embryonic stem cells undifferentiated Maintenance of the MEF disc was used to remove embryos and fetuses with a gestational age of 12.5 to 13.5 days In mice, the brain, limbs, internal organs (digestive tract, liver, kidney, lung), and tail were removed, and only the body tissue was recovered. Thereafter, the obtained body and stem tissues were cut finely with dissecting scissors, and then repeated several times up and down in the inside of the 18G needle for further fine cutting. The obtained cut tissue was placed in a 50 ml volume tube containing 5 m] [capacity glass beads, and then 0.25 wt% trypsin solution was added for 10 m. Then, the cut tissue was placed under 3rc. Stirring was performed every 10 minutes and culture was performed. After 30 minutes, 40 ml of DMEM containing 10% by volume of bovine embryo fetal serum was added to the above tube, and the cell suspension was recovered. The cell suspension was removed from the cell suspension through a sterilization screen (40 μm holes, manufactured by BD Falcon, trade name: Cell Strainer) to obtain a cell stock solution. The obtained cells were frozen and stored as a frozen cell stock solution. The frozen cell stock solution was thawed and cultured in a DMEM medium containing 10% by volume of bovine embryo fetal serum at 37 ° C and 5% by volume of CO2. After one pass, at the time of becoming confluent, mitomycin c [manufactured by Sigma] was added at a final concentration of 10 mg / 1, and cultured at 37 ° C for 3 hours. Thereafter, the cells were washed three times with pBs and recovered 'to obtain mitomycin C-treated embryonic fibroblasts. 100097.doc • 41-200540270 Then, the mitomycin C was used to treat embryonic fibroblasts in a manner of 1 x 106 cells per plate, and seeded in 6 cm plates at 37 ° C, 5 ° C. CO2 was cultured in volume% to obtain MEF discs for maintaining embryonic stem cell undifferentiated cells. Furthermore, the cultivation of related MEFs should be started more than 8 hours before seeding embryonic stem cells. (2) Differentiation and culture of primate embryonic stem cells The cynomolgus monkey embryonic stem cell CMK-6 strain was used as a primate cell. The supernatant was removed from the culture plate of the cynomolgus monkey embryonic stem cells, and the embryonic stem cells were washed once with PBS. To the washed embryonic stem cells on the plate, 0.25 wt ° / 0 trypsin solution was added at 1 m and the embryonic stem cells were cultured at 37 ° C for 1 minute. Immediately thereafter, the side of the disc was tapped to suspend the embryonic stem cells. Add 1 ml of embryonic stem cell undifferentiated maintenance medium to the suspension of embryonic stem cells {Composition: MEM / F12 medium [Invitrogen Corporation], selected volume of 20% by volume heat deactivated bovine embryo fetal serum (FBS) 〔 PAA Laboratories GmbH], a test fibroblast growth factor (bFGF) manufactured by Invitrigen [8], and a final concentration of 10 ng / ml bone formation protein (BMP-4) [R & D System Co., Ltd.], manufactured by Leukemia Inhibitory Factor (LIF) (Chemicon International Co., Ltd.) at a final concentration of 1000 U / ml, 0.1 mM β-Mercaptoethanol [Sigma Chemical Co., Ltd.], and 1 mM L-amidamine Amines [Invitrogen Corporation], final concentration of 10 U / ml penicillin [Invitrogen Corporation], final concentration 10 pg / ml Streptomycin [Invitrogen]] to inactivate trypsin. 100097. doc -42- 200540270 Thereafter, the embryonic stem cells were recovered in another step; ^ ^ ^ ... 3. The cells were sufficiently suspended in the above-mentioned medium for maintaining the differentiation of embryonic stem cells and applied. The supernatant was removed by centrifugation. The obtained cells were suspended in a new 2Gmj [] of the above-mentioned embryonic stem cell undifferentiated feedstock; MEF for embryonic stem cell undifferentiated maintenance obtained in (1) Panzhi: On the basis of the white fibroblast embryonic fibroblasts, each of the seven-seeded embryos and the stem cell suspensions were sown in 4 pans, and placed in [ο] incubator, The culture was performed at 37 t, 5% by volume of co2. Thereafter, embryonic stem cells were subcultured every 2 to 3 days, and the maintenance culture was terminated after about 2 weeks. Using a phase-contrast optical microscope [manufactured by Olympus Co., Ltd.] (Trade name: 1X70) Observe the cells directly. Regarding the above cells, analyze the performance of markers such as SSEAd and SSEA_4 located on the cell surface in the following manner. Add 0.25 wt% trypsin 1 to the dish after the culture. After incubating at 37 ° C for 1 minute, only the embryonic stem cells were detached by quickly tapping the side of the disc. The recovered cells were washed with an isotonic solution containing a divalent ion chelating agent such as EDTA. Detach cells simultaneously by pipetting After that, 1 × i 06 cells were reacted on ice with primary antibodies for markers such as SSEA-1, SSEA-4, etc. The primary antibodies were anti-SSEA-1 antibodies [manufactured by Chemicon International Co., Ltd.] or anti- SSEA-4 antibody [manufactured by Chemicon International Co., Ltd.]. Then, when analyzing the performance of SSEA-1, an anti-mouse IgM antibody [manufactured by ICN Biomedicals Co., Ltd.] was used as a secondary antibody, and when analyzing the performance of SSEA-4, an anti-mouse mouse igG antibody [icN Biomedicals Co., Ltd. was used. [Production] As a secondary antibody, a secondary 100097.doc -43- 200540270 antibody reaction was performed. Thereafter, the expression of each marker was analyzed using a trade name: FACSCalibiir [manufactured by BD Biosciences]. As a result, as shown in FIG. 6A, the cultured cells formed a flat circular colony comprising a single type of cell group having a polygonal cytoplasm and a large nucleus. Also, as shown in panel B of FIG. 6, the above-mentioned cells showed a higher SSEA-4 expression level and a lower SSEA] expression level. Therefore, it is considered that most of the cells maintained in an undifferentiated state after culture. (3) The differentiation-inducing community from primate embryonic stem cells to blood cells (hematopoietic cells). A micro-processed Pasteur pipette (microcapillary) at the front end will succeed the embryonic stem cell community on the second day. Roughly placed in the crack. Then, using a microcapillary, a group of undifferentiated embryonic stem cells located in the center of the colony was attracted and removed under a light microscope. This can substantially eliminate the incorporation of mouse embryonic fibroblasts. On the other hand, 5 ml of differentiation induction medium 2 {composition: the above-mentioned OP9 acclimatization medium without feeder cells for differentiation, 2 blood vascular endothelial growth factor (VEGF) at a final concentration of 20 ng / mi, and a final concentration of 20 ng / mi of BMP-4, 20 ng of stem cell factor (SCF), final ligand of i_ng / mi 3_ligand, final concentration of 10 ng / ml2IL_6, final concentration of 20 ng / ml2iL_3) added In a type IV collagen-coated -6 well plate [BD Biosciences], a culture plate for inducing embryonic stem cell differentiation is prepared in advance. The group of the above-mentioned undifferentiated embryonic stem cells was partially separated by smooth pipetting operation, and the colonies were divided into groups! Wells are 1x102 cells,

The seeds were selected and planted in the above-mentioned culture plate. Thereafter, the above-mentioned embryonic stem cells were cultured in a constant temperature phase of CO 100097.doc • 44 · 200540270 ′ at 37 ° C. and 5% by volume of CO 2 to induce differentiation. Furthermore, the culture was continued by changing the culture medium every 3 to 4 days. About 3 days after the induction of differentiation, as shown by the arrow in Fig. 7A, these have a relatively fixed width from one end to the other. As shown by the arrow, cells with unique morphology (finger-shaped) appear everywhere. Cell), which shows a joint-like structure that can be thought of as a result of fusion between adjacent cells. The fusion was formed on the 7th to 10th days after the induction of differentiation. Therefore, the medium was changed and the culture was continued. As a result, "round cells" appeared in the form of sprouts from fingers. Starting from the appearance of the above-mentioned round cells, the medium was changed every 2 days and the culture was continued. As a result, the round cells increased slowly, as shown in FIG. 7C. From 1 and a half to 2 weeks later, the slowly attached clusters or mostly suspended round cells originating from the budding mother cells were almost filled. All holes. Add 0.25% by weight trypsin solution to the dish! The cells were pipetted at room temperature, and round cells were recovered. Regarding the relevant cells, Table 5 in Table 5 was analyzed for markers such as CD34 and CD45 on the cell surface in the same manner as in Example 2 above, and the differentiation stage was analyzed. As a result, as shown in FIG. 7 and FIG. D, almost all round cells were positive for stem cell markers (ie, CD34) of various tissues such as hematopoietic cells, neuromeniscus, and endothelial cells, and were specific for being hematopoietic. The marker cD45 was positive. In addition, the morphology after Wright-Giemsa staining was analyzed. As a result, as shown in Fig. 7, the above cells showed a high nuclear / cell f ratio with significant nucleoli, which was consistent with the characteristics of immature hematopoietic cells. . Therefore, 100097.doc -45- 200540270 shows that almost all round cells are CD34 positive and CD45 positive, and almost all of them are hematopoietic stem cells or hematopoietic precursor cells. Furthermore, the above-mentioned "finger cells" and immature mesoderm markers Flk-1, CD34 and C-kit, immature cell markers mainly composed of hematopoietic stem cells, specific markers CD45 for all blood cells, and blood vessels Endothelial cell-specific markers VE-Cadherin and primary antibodies respond to markers such as CD151, which is a vascular endothelial marker, and secondary antibody responses can be continued as appropriate. Next, the expression amount of each marker was analyzed using a brand name: FACSCalibur [manufactured by BD Biosciences]. Furthermore, PE-conjugated anti-CD34 antibody [manufactured by BD Biosciences], FITC-conjugated anti-CD45 antibody [manufactured by BD Biosciences], PE-conjugated anti-VE-cadherin antibody (manufactured by Chemicon International Co., Ltd.), and PE-conjugated anti-CD 133 antibody were used. [Miltenyi Biotech GmbH], PE-conjugated anti-Flk-1 antibody [manufactured by BD Biosciences], PE-conjugated anti-C-kit (CD117) antibody [manufactured by BD Biosciences], and anti-CD151 antibody [manufactured by BD Biosciences] as primary antibodies. As a result, as shown in FIG. 8, the above-mentioned "finger cells" were ve-cadherin-negative Flk-1 positive c-kit-positive CD151 positive. [Example 5] The regeneration of blood cells with residual finger cells was performed at the stage of 7 to 10 days after the induction of differentiation in the above (4) of Example 4 '. In a culture plate containing finger cells in a fusion state, added 0.25 wt% of trypsin solution was pipetted at room temperature, and the finger cells were detached. 100097.doc • 46- 200540270 The results are shown in Figure A of Figure 9. Despite the intense pipetting and trypsin treatment, only a small amount of finger cells remained. 2.5 ml of the above-mentioned differentiation induction culture medium was re-added to the remaining small number of finger cells, and the medium was replaced every 3 to 4 days, and cultured at 37 ° C and 5 vol% C02 in a c02 strange material. As a result, the finger cells proliferated vigorously again. Furthermore, a fusion can be formed in about one week, and round cells appear. Thereafter, the culture was continued by changing the culture medium every 2 days. As a result, as shown in Fig. 9B & c, round cells formed a state that covered the entire well of the culture plate after one and a half weeks to two weeks. Further, by analyzing the surface markers in the same manner as in (3) of Example 4 above, it can be known from the results that these cells are CD34 + CD45 + hematopoietic stem cells / precursor cells. Then, 0.25% by weight of trypsin solution was added to the wells of the above-mentioned culture plate, and a pipetting operation was performed at room temperature, and the finger cells were detached. In the same manner as above, a small amount of the remaining finger cells were cultured again in a new differentiation-inducing medium. As a result, like the above, the finger cells proliferated vigorously. In addition, fusion was formed in about one week, and blood cells were generated again after about one and a half weeks to two weeks. Furthermore, the differentiation of such residual finger cells into blood cells was repeated at least three times and observed. Hematopoietic stem cells / precursor cells could stably grow from the same disk for more than 2 months. [Example 6] In the above Example 3, at the stage of 7 to 10 days after the induction of differentiation, 0.25% by weight of trypsin solution was added to the culture plate, a pipetting operation was performed at room temperature, and the separation was performed. Finger cells. 100097.doc -47- 200540270 was added to the obtained finger cells, and the feedless cell differentiation medium described in Example 3 described above was used to completely resuspend the medium. Thereafter, the supernatant was removed by centrifugation, and the above-mentioned "suitable culture beauty of cultured stromal cells and embryonic stem cells" was used, and dilution was performed so as to be about 1/3 to 1/6 of the original cell density. The obtained cell dilution was placed on a new 6-well plate or a 6-well plate covered with collagen, and cultured at 37 ° C, 5 vol% CO2. As a result, the cells proliferated vigorously, and then the morphology changed to a "pebble-like shape", as shown in Figure A of Figure 10. Within a few days, the cobble-like cells will cover the entire pore. The cobblestone-like cells were treated with 0.02% by weight iEDTA solution and recovered, and the recovered cells were washed with PBS. Thereafter, the primary antibodies of 1χ 抗体 〇ό cells to markers such as hematopoietic-specific markers CD34, CD45, and endothelial cell-specific markers VE · cadherin were reacted on ice for 30 minutes. The situation can continue with a secondary antibody reaction. Next, the performance of each bidder was analyzed using the trade name FACSCalibur [manufactured by BD Biosciences]. Furthermore, PE-conjugated anti-Cd34 antibody [manufactured by Bd Biosciences], FITC-conjugated anti-CD45 antibody [manufactured by bd Biosciences], and anti-VE_cadherin antibody [Chemicon]丨 Co., Ltd.] as the primary antibody. The results are shown in Figure B of Figure 10. The cobblestone-like cells described above were € 1334 negative CD45-negative VE-cadherin. Therefore, it was shown that the finger cells had been differentiated into blood f endothelial cells. In addition, the cobblestone-like cells actively proliferate in vitro and can be subcultured for several months in new discs, so that they can proliferate with stable reproducibility. 100097.doc -48- 200540270 [Example 7] Using trypsin / EDTA [manufactured by GIBCO-BRL Co., Ltd.], the finger cells obtained in the above Example 4 were detached from the disc. Thereafter, the obtained cells (0.5 × 106 to 1 × 106 cells per well) were placed on a 6-well flat-bottomed microtiter plate, and 2.5 ml of an accumulating medium for feeding-free cell differentiation as described in Example 3 above was used. 2 or 2.5 ml of a medium suitable for vascular endothelial culture [trade name: EGM-2 BulletKit (Code B3162), manufactured by Takara Bio] was cultured at 37 C, 5% by volume of CO 2 for 3 days. Obtained cells were observed with a retardation optical microscope (manufactured by Olympus, trade name: IX70). The results of observing the cell morphology obtained after culturing in the above-mentioned O9 acclimation medium 2 for cultureless cell differentiation are shown in FIG. 11, and the cells obtained after culturing in the medium using the above-mentioned brand name: EGM-2 BulletKit were observed. The results of the morphology are shown in FIG. 12. The obtained cells were analyzed for the presence or absence of VE-cadherin using pE-binding anti-VE_cadherin antibody, which is an antibody specific to the vascular endothelial cell marker VE-cadherin. As a result, VE_calcium was obtained from any of the cells obtained after culturing in the above-mentioned culture medium 2 for non-feeder cell differentiation and 9 domestication medium 2 and the cells obtained after culturing using the above-mentioned brand name · EGM_2 BuUetKit. Since the adhesion is positive, we know that it has differentiated into vascular endothelial cells. Further, as shown in FIG. 11, when cultured through the above-mentioned acclimation medium 2 for feeding cell-free differentiation, cobblestone cells were generated from the upper finger cells. As shown in FIG. 12, the above-mentioned product name is used for permeation. · When BuuetKit's medium is cultured, the cells generated from the above-mentioned finger cells are shown in Table I00097.doc -49- 200540270. The rope-like structure that forms the inner cavity of the blood vessel appears. Also, trypsin / edta [manufactured by GIBCO-BRL] was used to detach the obtained cells from the plate, and the same amount or more of OP9 acclimatization medium was added to inactivate trypsin, and then the supernatant was removed by centrifugation, Then, the product name: EGM-2 BulletKit was appropriately diluted and cultured in the product name: EGM-2 BulletKit, and it was confirmed that it was sufficient to carry out the subculture until the second round to self-proliferate, and 3 The successors of the above were not confirmed. [Example 8] Since it was confirmed that the cells obtained in Example 7 were endothelial cells, vascular structure formation analysis was performed as described below. The product name: Put a basal layer [manufactured by bd BioSciences] into a 24-well plate so that 95 μ 成 per well was formed, and incubate at 37 t:% minutes to gelate. In Example 7, the vascular endothelial cells 1 × 104 cells obtained by culturing under the trade name: EGM-2 BulletKit [manufactured by Takara Bio Co., Ltd .: B3162] were suspended in the trade name: Egm-2 Bullet Kit. The medium was 0.5 mi and placed in each well. The cells were cultured in a carbon dioxide culture apparatus at 371, 5 vol% co2 for 2 days and nights, and the cell morphology was often observed with a phase contrast microscope. In addition, as a reference, the same operations were performed using the finger cells and undifferentiated embryonic stem cells' obtained in Example 4 above. Shown in Figure 13 under the trade name: EGM-2

The results of vascular endothelial cells obtained by culturing in BulletKit [manufactured by Takara Bio Co., code: B3 162] are shown in Fig. 14 as results of finger cells' and Fig. 15 are results of undifferentiated embryonic stem cells. 100097.doc -50- 200540270 As a result, as shown in FIG. 13, it can be known that the vascular endothelial cells obtained by culturing in a trade name: EGM-2 BulletKit [manufactured by Xi Liyu Bio Co., Ltd. · B3162] were used for When the formation of vascular structures is analyzed, vascular structures are formed. On the other hand, as shown in FIG. 14, when finger cells are used to analyze the formation of vasculature, the formation of vasculature can be observed in some of them, but there are also clusters that form clusters. . Therefore, it appears that the obtained cells are in an undifferentiated stage compared to mature vascular endothelial cells. As shown in FIG. 15, when undifferentiated embryonic stem cells were used for the analysis of vascular structure formation, almost no vascular structure formation was found. Therefore, from the above results, it is shown that finger cells obtained from embryonic stem cells can be differentiated into vascular endothelial cells by maintaining conditions suitable for endothelial cell culture, thereby forming a vascular structure. [Example 9] MS-5 cells [purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)] as mouse bone marrow stromal cell lines were maintained in [Invitrogen], in which α-ΜΜ contained selected batches. 10% thermal deactivated FBS [manufactured by PAA Laboratories GmbH], 2 mM L-glutamine (manufactured by Invitrogen), 2 mM sodium pyruvate (manufactured by Invitrogen), 10 U / ml penicillin (manufactured by Invitrogen), And 10 pg / ml streptomycin [manufactured by Invitrogen]. These cells were treated with γ-irradiation at 46 Gy in the immediate fusion state. After washing with PBS, the cells were cultured in Iskov's modified Dubco medium (IMDM) [manufactured by Invitrogen Corporation 100097.doc -51- 200540270], and the medium contained 15% of Activating FBS (manufactured by PA A Laboratories GmbH), 0.1 mM β-myelinated ethanol (manufactured by Sigma Chemical), 3 mM L-glutamine (manufactured by Invitrogen), 5 μM hydrocortisone, 10 U / ml cyanogenin [manufactured by Invitrogen] and 10 pg / ml streptomycin [manufactured by Invitrogen]. After further culturing at 37 ° C for 20 hours, the supernatant was recovered and passed through a 0.22 μm disc filter (manufactured by Corning) to obtain a domesticated culture medium. The finger cells obtained in the above Example 4 were further cultured in the above-mentioned MS-5 cell domestication medium containing rhG-CSF for 2 to 3 weeks. By culturing, the round cells became a sheet-like shape and began to detach from the plate as a whole. These free cells were recovered and subjected to Wright-Giemsa staining. The results are shown in FIG. 16A. As a result, as shown in FIG. A, the above-mentioned cells exhibited morphological characteristics of nuclear mature macrophages containing large and irregular cytoplasms and altered configurations. Finally, the above-mentioned cells were fixed on a glass slide using a trade name: Cytospin2 [manufactured by Shandon Co., Ltd.] with an acetone / methanol (1: 3) solution. Thereafter, immunostaining was performed using an anti-CD14 antibody [manufactured by BD Biosciences]. In addition, an isotype control antibody was used as a control. The results are shown in panel B of FIG. As a result, as shown in FIG. B, the above-mentioned cells were positive for the well-known LPS receptor complex component (i.e., CD 14) of monocyte / macrophage-specific markers, and had differentiated into macrophages. Furthermore, the above cells were subjected to reduction analysis of tetrazolium blue 100097.doc -52- 200540270 salt (NBT) exhibiting active oxygen-generating activity to analyze the function of macrophages. 5 × 105 cells were recovered, washed once with PBS, and suspended in 1 ml fractions.

Induction medium. Finally, 1 ml of NBT solution [1 mg NBT (manufactured by Nacalai Tesque) / ml differentiation induction medium] was added, and the cells were cultured in the presence of 100 ng of TPA for 25 minutes. Thereafter, the cells were washed with PBS, and resuspended in 10 μΐ of PBS. The obtained cell suspension was dropped into a slide glass' and covered with a cover glass. Using an optical microscope [manufactured by Olympus, trade name: BX51], the number of νβτ positive cells was δ. Furthermore, 'hl-60 cells of human leukemia cells were used as a negative control, and NB reduction analysis was also performed. The results are shown in FIG. 16C. As a result, as shown in FIG. 16C, the macrophages derived from embryonic stem cells were actually positive for NBT reducing activity. Therefore, it can be seen that self-finger cells have been effective in producing hematopoietic cells that can at least differentiate into functional mature macrophages. [Industrial Applicability] According to the present invention, it is possible to stably provide blood cells and vascular endothelial cells suitable for use in blood for blood transfusion, materials for transplantation, and the like on an industrial scale. Furthermore, since the blood cells and the like of the present invention are also related to the enhancement of natural healing power, it can be expected that the shadows m generated by these medical and medical industries will be reassuring and safe The wealth of the blood used in the round of blood; the creation of the property has the possibility of developing into a huge industry. [Brief description of the figure] FIG. 1 is a system diagram showing the differentiation of blood cells from hematopoietic stem cells. Figure 2 shows the embryonic stem cell community on the 7th day after the start of culture. Scale bar 100097.doc • 53- 200540270 represents 10 μηι 〇 Figure 3 is an enlarged view of the part of the embryonic stem cell community on the 18th day after the start of culture. The scale bar indicates 10 μηι. Figure 4 shows the ratio of CD34-positive cells. Figure 5 shows the ratio of CD45 positive cells. Fig. 6 shows the morphology and marker expression of undifferentiated cynomolgus embryonic stem cells. FIG. A shows the results of observation with a phase difference microscope. In FIG. AA, the scale bar indicates 40 μm, and in FIG.AB, the scale bar indicates 40 μm. Figure B shows the results of cell surface analysis of FACS. Figure B shows the analysis of the performance of "SSEA4" in SSEA-4 as a marker of undifferentiated pluripotency stage (a in Figure B) and "SSEA1" in SSEA-1 as a marker of differentiation stage. The results after the performance (b in Figure B). The black line indicates the staining result of the control IgM or IgG3 of the same type, and the green line indicates the staining of the anti-SSEA-1 antibody or the anti-SSEA-4 antibody. Fig. 7 is a graph showing the results of analyzing differentiation from finger cells to blood cells. Figure A shows the results of observation of finger cells with a phase contrast microscope. The scale bar indicates 40 μηι. Figure B shows the observation results of small finger-shaped cells (indicated by arrows) in the phase contrast microscope. The scale bar indicates 40 μηι. Figure C shows the observation results of a large number of hematopoietic precursor cells generated by densely cultured finger cells through a phase contrast microscope. The scale bar indicates 40 μηι. Figure D shows the results of the cell surface analysis of FACS. Figure a shows the performance of CD34 as a stem cell marker, and b shows the performance of CD45 as a hematopoietic marker. The black line indicates the staining result of the control IgG as the isotype, and the green line indicates the staining result of the anti-CD34 or anti-CD45 antibody. Figure E shows the results of Wright-Giemsa staining of hematopoietic 100097.doc • 54- 200540270 precursor cells. The scale bar indicates 40. FIG. 8 is a graph showing the results of cell surface analysis of finger cells of FACS. The black line indicates the staining result of the isotype control IgG, the green line indicates the anti-CD34 antibody, anti-CD45 antibody, anti-VE-Cadherin ("VE-Cadherin") antibody, and the anti-Flk-1 image is " FLK-1 ") antibody, anti-c-kit antibody, anti-CD133 antibody or anti-CD151 antibody. Fig. 9 is a graph showing the results of observation of the repetitive generation of hematopoietic precursor cells through a phase difference microscope. Panel A shows the residual finger cells after the protease treatment. The scale bar indicates 40 μηι. Figure B is a graph showing the results of observation of cells by a phase contrast microscope after one week of culture, and Figure C is a graph showing the results of observation of cells by a phase contrast microscope after two weeks of culture. Scale bar shows 40 μηη 〇 Figure 10 shows the results of analyzing the differentiation from finger cells to vascular endothelial cells. Figure A shows the results of observing vascular endothelial cells with a phase contrast microscope. The scale bar indicates 40 μm. Figure B shows the results of cell surface analysis of FACS. In Figure B, a indicates CD34, b indicates CD45, and c indicates the expression of VE-cadherin, which is a specific marker for endothelial cells. The black line indicates the staining result of isotype control IgG, and the green line indicates the staining of anti-CD34 antibody, anti-CD45 antibody or anti-VE-4 toxonectin antibody. Fig. 11 is a graph showing the results of observing the morphology of cells obtained after cultured in an OP9 acclimatizing medium for non-chain trophoblast differentiation after the cells were detached by trypsin treatment and the finger cells were transferred to a new culture medium. FIG. 12 is a graph showing the results of observation of 100097.doc -55- 200540270 on cells obtained by culturing finger cells under conditions suitable for the cells observed in Fig. U and further suitable for vascular endothelial cell culture. Figure 13 is a graph showing the results of vascular structure formation analysis using cells obtained by selecting cells under culture conditions suitable for blood f endothelial cells. θ Fig. 14 is a diagram showing the results of using finger cells for vascular structure formation analysis Fig. 15 is a diagram showing the results of using undifferentiated embryonic stem cells for vascular structure formation analysis.

Fig. 16 is a graph showing the results of analysis of differentiation of finger cells into monocytes / macrophages. Panel A shows the results of Wright-Giemsa staining. Figure β shows the results of immunostaining using an anti-CD 14 antibody. Figure (b) shows the results of anti-cd 14 antibodies, and (b) shows the use of isotype control antibodies as controls. Figure c shows the results of the analysis of tetrazine blue reduction activity. (A) of Fig. C is a result of cells derived from finger cells' (b) is a result of using HL60 cells as a control. Fig. 17 is a diagram showing a mother community. [Description of main component symbols]

0101 0102 0103 Embryonic stem cells Hematopoietic stem cells Dendritic cells 0133 0134 Lymphocytic stem cells Bone marrow stem cells 100097.doc -56-

Claims (1)

200540270 10. Scope of patent application: 1. A culture medium for feeder-free cell differentiation, which is characterized by containing a matrix cell culture medium. 2. The medium for feeder-free cell differentiation according to claim 1, further comprising a medium component for maintaining primate embryonic stem cells, serum, and cytokines. 3. The medium for feeder-free cell differentiation according to claim 2, wherein the component of the medium for maintaining the primate embryonic stem cells is the one containing Isco's modified Dubco medium. 4. The medium for feeder-free cell differentiation according to claim 2 or 3, wherein the cytokine is selected from the group consisting of vascular endothelial growth factor, bone morphogenetic protein-4, stem cell factor, FH3-ligand, melanin-6, At least one of the group consisting of interleukin-3 and granulocyte colony-stimulating factor. 5. The feeder-free medium for culture according to any one of claims 1 to 4, wherein the stromal cell is a stromal cell irradiated with radiation. 6. The medium for feeding-free cell differentiation according to any one of the claims 1 to 4, wherein β-β-based cell leftover medium is obtained by performing a process comprising the following steps: Α) suitable for maintenance A step of culturing the stromal cell medium without forming a fusion, B) a step of culturing the cells obtained in the above step (A) in a medium suitable for maintaining stromal cells and embryonic stem cells, and C) A step of removing stromal cells from the culture obtained in the above step (B). 100097.doc 200540270 7. The medium for feeding-free cell differentiation according to any one of claims 1 to 5, wherein the stromal cell brushing medium is obtained by performing a process comprising the following steps: Α) suitable for maintenance The step of culturing the stromal cells in a medium without forming a fusion. B ′) The cells obtained in the above step (a) are irradiated with radiation, and the cells are obtained by culturing in a medium suitable for maintaining stromal cells and embryonic stem cells. A step of cells, and C ') a step of removing stromal cells from the culture obtained in the above step (B'). 8. A culture system for feeder-free cell differentiation, comprising: a culture medium for feeder-free cell differentiation according to any one of claims 1 to 7; and a culture container covered with extracellular matrix. 9-The culture system for feeder-free cell differentiation according to claim 8, wherein the extracellular matrix is collagen. 10. The culture system for feeder-free cell differentiation according to claim 9, wherein the collagen is type IV collagen. Π · A method for the differentiation of non-feeding cells from primate embryonic stem cells to blood cells', characterized in that the method is characterized in that it is cultured in a non-feeding cell differentiation medium according to any one of claims 1 to 7, Cell-like, isolated itchy primate embryonic stem cells. 12. A method for differentiation of primate embryonic stem cells into blood cells as described in claim 11 in which the primate embryonic stem cells are placed in a culture container covered with extracellular matrix, and in Wurao State of culturing cells 100097.doc 200540270 In a culture medium without feeder cell differentiation as in any one of the claims 1 to 7, ^ σ is cultured to form finger-like cells, and then continue to culture To make blood cells. 13 ·-A kind of non-feeding method that makes primate embryonic stem cells become vascular endothelial cells, which is characterized by placing primate embryonic stem cells in a culture container covered with extracellular space f In the state of agar-free cells, and culture in the culture medium for culturing cell differentiation according to any one of item 7, to make finger cells, and then the finger cells The culture was moved to a culture container covered with extracellular matrix with a new medium, and the finger cells were made into vascular endothelial cells. 14. A method for producing blood cells, characterized in that: primate embryonic stem cells are placed in a culture container covered with extracellular space f, in a state of non-regulated cells, as described in item 1 to 7 The non-healthy cells of 7-knife were cultured in a medium for 7-knife to differentiate the embryonic stem cells into blood cells, and then the blood cells were separated and separated. 15. The method for producing a blood cell according to claim 14, wherein the culture vessel in which the blood cells have been peeled off is added with a culture medium for culturing without cells as described in any one of the above claims, and then to silver-free In the state of culturing cells, blood cells are obtained by culturing. 16-A blood cell, characterized in that Iksan was tested and obtained by a manufacturing method such as item 14 or 15. I7. —Production of vascular endothelial cellsAn animal embryonic stem cell is placed in a state covered with no feeding cells. The method is characterized in that the primate is cultured in an extracellular matrix, as in item 1 ~ No. 100097.doc 200540270 of any of 7 is cultured in a culture medium for forging cells to produce finger cells, and the finger cells are moved to a new medium-covered extracellular matrix In a culture container, the embryonic stem cells are cultured to differentiate into vascular endothelial cells, and the vascular endothelial cells are isolated. 18. 19. 20. A vascular endothelial cell ', characterized by being obtained by the production method according to claim 17. A method for manufacturing finger cells, 1. Specially, the primate embryonic stem cells are placed in a culture container covered with extracellular space f, in the state of no feeding cells, as requested ~ 7 The culture medium of any one of the above is cultured. A finger cell characterized by being difficult to obtain. The wrong reason is as requested! 9 The manufacturer 100097.doc