WO2005019441A1 - 霊長類動物の胚性幹細胞から造血系細胞への分化方法 - Google Patents
霊長類動物の胚性幹細胞から造血系細胞への分化方法 Download PDFInfo
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- WO2005019441A1 WO2005019441A1 PCT/JP2004/012456 JP2004012456W WO2005019441A1 WO 2005019441 A1 WO2005019441 A1 WO 2005019441A1 JP 2004012456 W JP2004012456 W JP 2004012456W WO 2005019441 A1 WO2005019441 A1 WO 2005019441A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/103—Ovine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/106—Primate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
Definitions
- the present invention relates to a technique for supplying hematopoietic cells, differentiated cells and the like of primates. More specifically, the present invention relates to a method for differentiating primate embryonic stem cells into hematopoietic cells, a hematopoietic cell obtained by the differentiation method, and a method for producing a chimeric hedge that produces hematopoietic cells of a primate animal. . Background art
- hematopoietic stem cell transplantation represented by bone marrow transplantation, peripheral blood stem cell transplantation, umbilical cord blood stem cell transplantation, etc. is performed, and blood cells are produced in the patient's body It is.
- transplantation to human hematopoietic stem cells transuterine in the fetus is an attempt to amplify hematopoietic progenitor cells in the body [Nakauchi Keiko, et al. 1 Name, "3. Establishment of human blood chimera and its application", “Records of the 1st 17th Annual Meeting of the Japan Medical Society, Stem Cell and Cell Therapy 1 [III] Tissue, Organ Stem Cell: Basic Development for Clinical Application (2) 2000 August 4-6, 1999, p 99—106 [online], Internet
- the differentiation method described in the document of Michael Caiba et al. Has a drawback in that the application range of the obtained hematopoietic stem cells may be limited because ES cells into which Hox B4 gene has been introduced are used. .
- the present invention provides a stable supply of primate hematopoietic cells, and the introduction of a foreign gene into an embryonic stem cell of a primate animal without performing a genetic engineering operation.
- the present invention relates to a method for differentiation of embryonic stem cells of primates into hematopoietic cells, which enables at least one of differentiation of embryonic stem cells of animals into hematopoietic cells.
- the present invention provides a stable supply of primate hematopoietic cells, a large amount of primate hematopoietic cells, and a stable supply of platelets for blood transfusion of primates.
- the present invention relates to a method for producing hematopoietic cells of primate animals, which enables at least one of stably supplying CD 3 4 + cells for transplantation of primate animals. Furthermore, in another aspect, the present invention relates to hematopoietic cells obtained by the production method. In still another aspect, the present invention relates to a method for producing a chimeric hedge that can stably supply primate hematopoietic cells. That is, the gist of the present invention is
- Embryonic stem cells of primates are maintained under conditions suitable for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into the fetus in the womb of a pregnancy hedge, and the fetus is grown and born.
- a primate characterized in that hematopoietic cells of a primate animal are obtained from a hydge obtained by administering a site force-in specific to a primate animal to a puppy that has reached the A method for differentiating embryonic stem cells from hematopoietic cells into hematopoietic cells,
- step (I) the differentiation method according to [2] above, wherein the embryonic stem cells of a primate animal are maintained on a feeder cell in the presence of bone morphogenetic protein_4.
- the present invention relates to a method for producing a chimeric hedge that produces hematopoietic cells of a primate animal, characterized by administering a specific cytokine to a primate animal and further allowing the pup to grow.
- Fig. 1 shows cells obtained by maintaining primate embryonic stem cells under conditions suitable for inducing differentiation of hematopoietic cells.
- Panel (A) shows undifferentiated embryonic stem cells and the scale bar shows lOOm.
- Panel (B) shows the cell schema during culture in the presence of BMP-4 on P9 cells.
- Panel (C) shows cells obtained by maintaining embryonic stem cells in hematopoietic differentiation-inducing conditions, and scale bar is 100 m.
- Fig. 2 is a diagram showing the results of detection of monkey / 32-microglobulin in Hedge fetus obtained after transplantation.
- panel (A) shows the results for fetal liver and panel (B) shows the results for bone marrow.
- FIG. 3 shows the results of detection of monkey] S 2 -microglobulin in peripheral blood or bone marrow before and after human SCF was administered to Hedge.
- the upper panel shows the results of PCR, and the lower panel shows the results of Southern plot hybridization.
- M is a molecular weight marker
- lanes 1 to 3 are negative control (water)
- lane 4 is Hedge DNA alone
- lane 5 to: L 0 is 0.0001% (cell count), 0.
- lane 1 1 is monkey DNA alone
- Lane 12 is no sample
- Lane 13 to Lane 17 are peripheral blood derived samples before administration of human SCF, 6 days after administration, 1 day after administration, 3 days after administration, and 40 days after administration
- Lane 18 -22 show the bone marrow-derived samples before human SCF administration, 6 days after administration, 1 day after administration, 3 days after administration, and 40 days after administration, respectively. Acts on monkeys Shows that monkey cells appeared in the peripheral blood and bone marrow blood of the Hedge by administration of human SCF, which does not act on Hedge.
- embryonic stem cells are treated under the conditions for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into a fetal fetus in the uterus of a pregnant hidge, so that the post-natal hidge is essentially a monkey. Based on the inventors' surprising finding that hematopoietic cells of the origin are produced.
- the present invention relates to a method for differentiating primate embryonic stem cells into hematopoietic cells.
- non-human mammals for example, pigs, horses, monkeys, etc.
- chimeric non-human mammals can be produced with a chimera rate of at least 1%, and hematopoietic cells can be produced.
- the method for differentiating primate embryonic stem cells from hematopoietic cells of the present invention comprises maintaining primate embryonic stem cells under conditions suitable for inducing differentiation of hematopoietic cells, and then obtaining the resulting cells as pregnancy hedges. Transplanted into the fetus in the uterus of the uterus, and then obtaining hematopoietic cells of a primate animal from the born Hedge.
- the method for differentiating primate embryonic stem cells from hematopoietic stem cells of the present invention comprises maintaining primate embryonic stem cells under conditions suitable for inducing differentiation of hematopoietic cells.
- the obtained cells are transplanted into a fetus in the womb of a pregnancy hedge, the fetus is grown, and a citrus force specific to a primate is administered to the pup pup that has reached birth, and the pup
- It is a method characterized in that hematopoietic cells of a primate animal are obtained from a hidge obtained by growth.
- the differentiation method of the present invention has one major feature in maintaining embryonic stem cells of primate animals under conditions suitable for induction of hematopoietic cell differentiation (hereinafter also referred to as hematopoietic differentiation induction conditions).
- the differentiation method of the present invention in order to maintain embryonic stem cells of primate animals under the conditions for inducing hematopoietic differentiation, when undifferentiated embryonic stem cells are transplanted as they are, from the undifferentiated embryonic stem cells, Despite the fact that differentiation into hematopoietic cells does not substantially occur, according to the differentiation method of the present invention, it is surprising that the embryonic stem cells of the primate can be differentiated into hematopoietic cells. Show the effect.
- the primate embryonic stem cell since the primate embryonic stem cell is maintained in the hematopoietic differentiation induction condition, when the primate embryonic stem cell is transplanted into a Hedge fetus, Fetal fetus [strictly speaking, after birth,
- the differentiation method of the present invention comprises a primate embryonic stem cell to differentiate into a hematopoietic cell, and the primate embryonic stem cell induces differentiation into a foreign gene (eg, a homeotic selector).
- a foreign gene eg, a homeotic selector
- the differentiation method of the present invention has one major feature in that a fetus fetus in the womb of a pregnant fetus is used.
- the above-mentioned Hedge fetus is used. It exhibits an excellent effect that it can be differentiated into hematopoietic cells derived from.
- the differentiation method of the present invention since the above-mentioned Hedge fetus is used, it is possible to obtain an excellent effect of increasing the chimera ratio with a primate animal. Therefore, according to the differentiation method of the present invention, it is possible to obtain an excellent effect that hematopoietic cells essentially derived from primate animals can be obtained more efficiently in the body of the hidge.
- the differentiation method of the present invention since the fetal fetus in the uterus of the pregnant hige is used, immune tolerance to the transplanted cells is obtained, and after birth, the embryonic stem cells of the primate animal are further obtained. It has an excellent feature that it can be additionally transplanted. Therefore, according to the differentiation method of the present invention, hematopoietic cells derived essentially from primates can be supplied more efficiently and stably.
- the differentiation method of the present invention also includes transplanting cells obtained by maintaining embryonic stem cells of primate animals under the above-mentioned conditions for inducing hematopoietic differentiation into a fetus fetus in the uterus of a pregnancy hedge.
- the differentiation method of the present invention cells obtained by maintaining embryonic stem cells of primate animals under the conditions for inducing differentiation of hematopoietic system are transplanted into primate animals in the womb fetus in the womb of pregnancy hedge.
- the derived hematopoietic cells can be supplied efficiently and stably.
- the differentiation method of the present invention is a method for transplanting cells obtained by maintaining embryonic stem cells of primate animals under the conditions for inducing differentiation of the hematopoietic system to a fetus in a womb of a pregnant ledge.
- hematopoietic cells derived from primates can be supplied more efficiently and stably.
- a chimeric animal can be obtained with surprisingly high efficiency by using cells maintained for 6 to 8 days, preferably 6 days, under conditions for inducing hematopoietic differentiation. It has an excellent effect of being able to achieve a surprisingly high chimera rate.
- “primate animals” refers to humans, monkeys, and the like. Examples of the monkeys include power cynomolgus monkeys, lion monkeys, dihon monkeys, marmoset, and the like.
- embryonic stem cells (hereinafter also referred to as ES cells) refers to undifferentiated cells having multipotency and self-renewal ability.
- primate embryonic stem cells used in the differentiation method of the present invention include CMK 6 cells.
- Such embryonic stem cells can be maintained and supplied by using cells such as mouse embryo fibroblasts that have stopped dividing and growing in a medium for ES cells as feeder cells.
- the medium for ES cells may be any medium suitable for embryonic stem cells depending on the type of embryonic stem cells.
- the composition per 20 Om 1 is DMEM / F 12 163 ml, guinea pig fetal serum 30 ml (final concentration 15%), L 2 ml monoglutamine (final concentration 2 mM), penicillin (100 U / ml) —streptomycin (100 g / m 1) 2 ml 1, 2 ml non-essential amino acid solution, 2-mercaptoethanol 1 ml (final concentration 0 ImM)
- the maintenance and proliferation of the undifferentiated state of the embryonic stem cell varies depending on the type of embryonic stem cell, and can be performed, for example, by culturing under conditions such as the presence of leukemia inhibitory factor (LIF).
- LIF leukemia inhibitory factor
- the culture conditions for the maintenance and proliferation of undifferentiated state of embryonic stem cells varies depending on the type of embryonic stem cells, for example, a gas phase of 5% by volume C_ ⁇ 2,. 36 to 38 ° C, Preferably, the temperature is maintained at 37 ° C.
- the feeder cells used for maintaining and proliferating the undifferentiated state of the embryonic stem cells vary depending on the cell type, for example, D 10 medium (DMEM medium containing 10% by volume of fetus serum) Maintenance, proliferation, etc. can be performed.
- the culture conditions of the feeder single cell varies depending on the type of cell, for example, a gas phase of 5-body volume% C_ ⁇ 2, 36 to 38 ° C, preferably, and the like to be maintained at 37 ° C for .
- examples of hematopoietic cells include, for example, hematopoietic stem cells and cell groups obtained by differentiation from the hematopoietic stem cells, that is, cells having properties such as erythroblasts, myelocytes, megakaryocytes, and lymphocytes. Specific examples include erythroblasts and myelocytes.
- conditions suitable for inducing differentiation of hematopoietic cells include, for example, conditions for culturing in the presence of type IV collagen, one minimal essential medium (one MEM), etc.
- conditions for culturing in the presence feeder suitable for hematopoietic differentiation, conditions for culturing on a single cell, conditions for adhering to a dish after creation of embryoid bodies, and conditions for any combination thereof .
- the cells obtained by maintaining embryonic stem cells of primate animals under conditions suitable for inducing differentiation of hematopoietic cells are evaluated for hematopoietic colony forming ability and expression of hematopoietic surface markers.
- the cells are differentiated cells that are negative or weakly positive.
- Such cells may be so-called mesoderm cells or cells close to the state, properties, etc. of the mesoderm cells.
- cells obtained by maintaining the embryonic stem cell line CMK6 derived from the power cynomolgus monkey for about 6 days under conditions suitable for induction of differentiation of hematopoietic cells can be mentioned.
- the hematopoietic colony forming ability is evaluated by, for example, colony assembly described below.
- the surface markers include CD45, TER 1 19, ⁇ 4 —integrin, VE—forced doherin, S ca—1, CD 34, CD 13, CD 14, GPIIb Ilia, CD 3, CD4, CD 8, s I gM CD 19, c-Kit (CD 1 17), Thy-1 (CD 90), CD 31, HLA-ABC,
- feeder cells used for maintaining hematopoietic differentiation-inducing conditions include stromal cell lines deficient in Mcphage phage stimulation factors, such as mouse bone marrow cell lines deficient in macrophage stimulation factors, Examples thereof include cells obtained by mitomycin C treatment or X-ray treatment of mouse yolk sac cell line lacking macrophage stimulation factor. As a stromal cell line deficient in the macrophage stimulation factor,
- the mouse bone marrow cell line includes an S 17 cell line (Colins (Co 1 ins) et al., J. Immuno 1., 1987, Vol. 138, ⁇ 82-1087),
- the mouse yolk sac cell line is C 166 cell line [One
- the feeder cell used for maintaining the hematopoietic differentiation induction condition is a stromal cell line deficient in macrophage stimulation factor, such as ⁇ -MEM medium (Minimum Essential Medium Dial). It can be prepared by seeding in a gelatin-coated culture vessel containing. One feeder cell should be seeded to the extent that it covers the culture vessel without any gaps.
- the culture conditions of the feeder single cell varies depending on the type of cells used, when the scan Toroma cell lines ⁇ _P 9 cells, for example, in 5% C0 2 in the gas phase, preferably, 36 to 38 ° C, in particular Preferably, the temperature is maintained at 37 ° C.
- the feeder cells used when maintaining the hematopoietic differentiation induction conditions are specifically: When using P9 cells as feeder cells,
- ⁇ P 9 cell culture medium for example, composition per 1250 ml: ⁇ -MEM (Gibco), catalog number: 12000—022) 980 ml, fetal bovine serum 250 ml (final concentration 20%) in Benishirin (100 UZm 1) one streptomycin (100 gZm 1) 10m l, 200 mM L- dull evening Min solution 10 ml ⁇ , and cultured at 37 ° C, 5% C0 2 under, (1) Wash the cells that have adhered to the culture medium and have grown to confluence or just before, twice with phosphate buffered saline,
- the pregnancy hedge used in the present invention is 40 days or more after pregnancy, preferably 50 days or more, more preferably 60 days or more from the viewpoint of safety, for example, prevention of miscarriage. From the viewpoint of obtaining a sufficient engraftment rate of transplanted cells, it is desirable that it is 85 days or less after pregnancy, preferably 75 days or less, and more preferably 60 days or less. Therefore, it is desirable that the fetus in the uterus is 50 to 70 days of gestation.
- a differentiation method of the present invention more specifically,
- (Ii) A method comprising the step of transplanting the cells derived from the primate animal obtained in the step (I) to a fetus in the uterus of a pregnancy hedge and growing the fetus until birth.
- step (I) it is desirable to seed the primate embryonic stem cells so as to increase the density from the viewpoint of further differentiation.
- the cells are cultured on a feeder cell in a medium corresponding to embryonic stem cells of a primate animal.
- the medium may be changed as appropriate.
- the feeder one on cells, embryonic stem cells of a primate is a 5% C0 2 in the gas phase, preferably, 36 to 38 ° C, particularly preferably at 37 ° C for It is desirable to maintain.
- CMK 6 force cynomolgus embryonic stem cell line
- differentiation medium Composition per 100 ml: I MDM (I scove's Modulated Dulbecco's Medium) 84 ml, 8% ⁇ Ma serum 8m 1, 8% ⁇ shea calf serum 8m 1, 5X 10- 6 M hydrocortisone 0. 18 / ig, BMP-4 2 g ( final concentration 2 O ngZml), SCF (final concentration 2
- IL 1 3 2 g final concentration 20 ng Zm 1), IL—61 PL g (final concentration 1 O ngZml), VEGF 2 g (final concentration 20 ngZm 1), G—CSF 2 g (final concentration 20 ng / m 1), F 1 t-3 ligand 2 g (final concentration 10 ng / m 1), EPO 200U (2U / m 1)) liquid and against the 5 X 10 5 cells in feeder one on the cells, appropriate amount of to cell seeding, then include 37 ° C, 5% C_ ⁇ you cultured for 6 days at 2 under conditions.
- the cells obtained by performing step (I) correspond to the “cells obtained by maintaining primate embryonic stem cells under conditions suitable for induction of hematopoietic cell differentiation”. To do.
- embryonic stem cells for example, the power bicyno embryonic stem cell line CMK6, It is desirable to maintain the conditions suitable for inducing differentiation of hematopoietic cells on a day, preferably 6 days. This also enables surprisingly high efficiency and rapid differentiation of embryonic stem cells into hematopoietic cells.
- step (I) from the viewpoint of obtaining hematopoietic cells more efficiently, preferably, in addition to the hematopoietic differentiation induction conditions, —It is desirable to maintain primate embryonic stem cells in the absence of leukemia inhibitory factors on the cells.
- the cells obtained by carrying out the step (I) are then transplanted into a fetus in the uterus of a pregnancy hedge, and the fetus is grown until birth [step.
- Cells used for transplantation are treated with trypsin.
- 0.1% BSA (Ushi Serum Albumin) ZHBS S (Hank's Balanced Salt Solution) is used to improve the engraftment rate, the rate of chimera, etc.
- 1 ⁇ 10 6 cells or more preferably IX 10 6 cells to 1 ⁇ 10 9 cells, preferably 1 ⁇ 10 7 cells to 1 ⁇ 10 9 cells.
- step (II) cells are transplanted into the fetus by, for example, puncturing and injecting cells for transplantation into the liver (or more strictly, within the liver parenchyma), the abdominal cavity, the heart, the umbilical cord, etc. It can be done by.
- Transplant surgery is, for example,
- the cells can be transplanted into the intrauterine fetus by, for example, incising the myometrium of the hidge, driving the fetal fetus into the exposed egg membrane, and then directing the fetal transplant site through the transparent egg membrane. Then, the cells for transplantation are injected into the puncture, and the myometrium, peritoneal muscle layer, and skin are combined and closed in layers. Or, without incising the uterus, ultrasonic gazing from the uterine wall Under the id, the transplantation cells are punctured and injected into the transplantation site of the fetus, and the peritoneal muscle layer and skin are closed and closed.
- step (II) after the transplant operation, the pregnancy hedge is raised until birth, for example, under the same conditions as normal breeding.
- step ( ⁇ ) namely, the birth Hidge
- hematopoietic cells that are essentially derived from primates
- a suitable tissue such as liver, bone marrow, etc.
- hidge a birth puppy
- the hematopoietic colony formed in (1) can be evaluated by examining the expression of a marker gene specific for a primate animal.
- the hematopoietic colony assembly of (1) is, for example,
- tissue is treated appropriately according to the tissue such as trypsin treatment, DNase treatment, lysis treatment, etc. to obtain cells,
- the obtained cells are suspended in 2% by weight FBS (Huy Fetal Serum) and IMDM to obtain a cell suspension.
- FBS Human Fetal Serum
- the expression of the marker 1 gene specific to the primate animal of (2) above is carried out by extracting DNA from colonies formed by hematopoietic colony assembly by a conventional method. It can be evaluated by detecting the gene by hybridization using a probe specific to a marker gene specific to a mammal, PCR using a specific primer pair, or the like. Alternatively, hematopoietic colonies may be assessed by immunostaining with antibodies against antigens specific to primates.
- the cells obtained in step (I) may be further transplanted into the fetus fetus obtained in the above step (II), that is, more strictly, to the birth pups. Good.
- the primate hematopoietic cells can be supplied efficiently and stably.
- a cyto force-in specific to a primate animal or the like may be administered to the birth pup Hedge obtained in the step (ii).
- a cyto force-in specific to a primate animal or the like may be administered to the birth pup Hedge obtained in the step (ii).
- the site force-in includes stem cell factor (SCF), basic fibroblast growth factor (bFGF), oncostin M (OSM), f1k2Zf1k3 ligand, interleukin-1 and interleukin-1.
- SCF stem cell factor
- bFGF basic fibroblast growth factor
- OSM oncostin M
- f1k2Zf1k3 ligand interleukin-1 and interleukin-1.
- Leukin-3 granulocyte colony stimulating factor (G-CSF)
- chemokine erythropoietin, thrombopoietin, etc., and any combination thereof.
- the cells are maintained for 6 to 8 days, preferably 6 days under conditions suitable for induction of differentiation of hematopoietic cells, and the obtained cells are transplanted into a fetus in the uterus of a pregnancy hedge. Pups that have been born, grown, and born, Surprisingly, the administration of tocaine has an excellent effect of achieving a high chimera rate.
- Evaluation of hematopoietic cells of primates obtained by the differentiation method of the present invention is, for example,
- CD45 TER 1 19, 0! 4 _integrin, VE—force doherin, c-Kit, Sca_l, CD 34 (stem cell marker), CD 13 (myelocyte marker one), CD 14 (myelocyte marker one) ), GpIIb / IIIa (megakaryocyte marker), CD3 (T cell marker), CD 4 (T cell marker), CD8 (T cell marker), s I gM (B cell marker), CD 19 (B cell) It can be performed by using the presence or absence of expression of a marker as an index.
- the differentiation method of the present invention can be used for the production of hematopoietic cells of primates.
- the present invention relates to a method for producing hematopoietic cells of a primate animal.
- the method for producing hematopoietic cells of the primate animal of the present invention comprises:
- step (III) The fetus born in the above step (II), that is, more precisely, a puppy sheep is administered with a site force-in specific to a primate animal, and is further obtained by growing the pup ledge. Separating the differentiated primate hematopoietic cells from the primate embryonic stem cells
- the production method of the present invention has one major feature in maintaining primate embryonic stem cells under conditions for inducing hematopoietic differentiation. Therefore, according to the production method of the present invention, in order to maintain embryonic stem cells of primate animals under the hematopoietic differentiation induction conditions, when the undifferentiated embryonic stem cells are transplanted as they are, the undifferentiated embryonic stem cells From hematopoietic cells In spite of the fact that differentiation does not occur substantially, surprisingly, it exhibits an excellent effect that hematopoietic cells can be produced from embryonic stem cells of the primate.
- the differentiation method of the present invention when embryonic stem cells of a primate animal are transplanted into a Hedge fetus, the embryonic stem cell is transferred to the Hedge fetus (ie, strictly, after birth, As a result, it is possible to more effectively use the internal environment of Hidge), and thus, it exhibits an excellent effect of stably supplying hematopoietic cells of primates.
- the production method of the present invention has one major feature in that the fetus fetus in the uterus of the pregnant hige is used.
- the rate of the chimera with the primate is increased, whereby hematopoietic cells of the primate are derived from the embryonic stem cells of the primate. It exhibits an excellent effect of being able to supply a large amount of hematopoietic cells.
- an embryonic stem cell for example, a cynomolgus monkey embryonic stem cell line CMK6 etc.
- CMK6 cynomolgus monkey embryonic stem cell line
- hematopoietic for 6 to 8 days, preferably 6 days.
- primate embryonic stem cells can be additionally transplanted into a birth pup Hedge, and transplanted with a primate cytokine that does not act on the Hedge.
- Primate embryonic stem cells or hematopoietic cells that are essentially derived from primates can be selectively stimulated, so that hematopoietic cells that are essentially derived from primates are more efficient and stable. Can be supplied.
- blood transfusion platelets and transplanted CD 3 4 + cells can be stably supplied.
- step (III) the hematopoietic cells of the primate animal differentiated from the embryonic stem cells of the primate animal are separated from the fetus born in the step (II), that is, the pup ledge.
- step (III) it is desirable that the fetus hedge fetus, that is, the fetus hige, is an individual within one year after birth from the viewpoint of chimera rate.
- step (III) it is preferable to administer the cytokine or the like specific to a primate to the born puppy and further grow the puppy.
- the step (( The cells obtained in I) may be further transplanted.
- step (III) the separation of primate hematopoietic cells is, for example,
- liver (peripheral blood, umbilical cord blood), thymus, spleen, etc.
- step 3 for example, flow cytometry using a marker specific to a primate animal or an antibody against the marker, a method using immunobeads, and the like are performed.
- the “marker specific to a primate animal” may be any marker that crosses a primate and does not cross a hedge, such as HLA-ABC, jS 2 -microglobulin, and CD45.
- Evaluation of primate hematopoietic cells obtained by the production method of the present invention can be performed by, for example, CD45, TER 1 19, 4 integrin, VE-cadherin, c_kit :, Sca_ l, CD34, CD 13, CD 14, GP II b / IIIa, CD 3, CD 4, CD 8, s Ig M, CD 19 and the like can be used as indicators.
- the present invention relates to a primate hematopoietic cell obtained by the production method of the present invention.
- Such hematopoietic cells are not particularly limited, but are expected to be used as, for example, transplant materials for angiogenesis therapy, and can be applied to ischemic diseases such as myocardial infarction.
- the hematopoietic cell of the present invention is, as described above, a hematopoietic stem cell and a cell group obtained by differentiation from the hematopoietic stem cell, that is, an erythroblast, a myeloid cell, a megakaryocyte system, a lymphocyte, etc. Examples of such cells include erythroblasts and myelocytes.
- the hematopoietic cell of the present invention is essentially a cell derived from a primate animal, and is a cell into which a foreign gene has not been introduced at the time of production, and is therefore suitable for transplantation to a primate animal.
- a therapeutic effect can be exerted by transplanting the hematopoietic cell of the present invention to a site of a disease or condition that requires the construction of the hematopoietic system or the action of the hematopoietic cell. Therefore, in another aspect, the present invention is characterized by supplying a therapeutically effective amount of the hematopoietic transcellular of the present invention to a site of a disease or condition that requires the construction of a hematopoietic system or the action of hematopoietic cells.
- the present invention relates to a method for treating a disease requiring the construction of a hematopoietic system or the action of hematopoietic cells.
- the present invention relates to the use of the hematopoietic cells of the present invention for the treatment of diseases requiring the construction of hematopoietic systems or the action of hematopoietic cells.
- Diseases that require the construction of the hematopoietic system or the action of hematopoietic cells are not particularly limited.
- ischemic heart diseases such as aplastic anemia, leukemia, immunodeficiency, and myocardial infarction, obstructive arteriosclerosis Symptom, Birja's disease, etc.
- the “therapeutically effective amount” may be an amount sufficient to construct the hematopoietic system or to recognize the effect of the hematopoietic cell, and refers to a disease or condition that requires the construction of the hematopoietic system or the action of the hematopoietic cell.
- the degree can be set as appropriate according to the body weight, age, physical strength, progress of construction of the hematopoietic system, and the like.
- the hematopoietic cell of the present invention may be supplied to a site of a disease or condition that requires the construction of the hematopoietic system or the action of the hematopoietic cell, for example, by puncture injection or the like. You may give it.
- the therapeutic effect can be improved by appropriate means (eg, fiberscope, ultrasound, CT, MRI observation, angiography, measurement of disease-specific marker in serum, etc.) It can be evaluated as an indicator that improvement is expressed.
- appropriate means eg, fiberscope, ultrasound, CT, MRI observation, angiography, measurement of disease-specific marker in serum, etc.
- the present invention relates to the use of the hematopoietic cell of the present invention for the manufacture of a medicament for the treatment of a disease requiring the construction of a hematopoietic system or the action of a hematopoietic cell.
- a chimeric wedge that produces hematopoietic cells of primates can be produced. Therefore, in another aspect, the present invention relates to a method for producing a chimeric hedge that produces hematopoietic cells of a primate animal.
- embryonic stem cells of a primate animal are maintained under conditions suitable for inducing differentiation of hematopoietic cells, and the obtained cells are used in a pregnant hidge.
- One major characteristic is that a specific cytokine is administered to a primate animal, which is transplanted into a fetus in the uterus and born, and then the pupa is grown.
- a chimeric hedge capable of supplying primate hematopoietic cells more stably can be obtained.
- the chimeric hedge obtained by the method for producing a chimeric hedge of the present invention has cells derived from primates in an immunologically tolerant state
- the chimeric hedge contains hematopoietic cells. Maintain the cells in conditions suitable for differentiation induction, and Can be transplanted to.
- the chimeric hedge obtained by the method for producing a chimeric hedge of the present invention can selectively differentiate into hematopoietic cells derived from primates in the body by administering a specific cytokine to a primate animal. Can irritate.
- mice embryo fibroblasts BALB / c AJ c 1 (supplied by CLEA Japan, Inc.) up to 5 passages in D 10 medium (composition: 10% FCS, DMEM) containing mitomycin C at a final concentration of 10 g / ml
- D 10 medium composition: 10% FCS, DMEM
- mitomycin C concentration of 10 g / ml
- the treated cells were washed with phosphate buffered saline.
- the washed cells were treated with trypsin ⁇ EDTA (0.05% trypsin, ImM EDTA) to obtain a cell suspension, and the cell suspension was centrifuged to obtain cells.
- the obtained cells were suspended in D10 medium and seeded in a gelatin-coated 6 cm culture dish [FALCON, trade name: T issuecu 1 turedish] so as to be 1 ⁇ 10 6 cells. Cells obtained by culturing until they adhere to the dish were used as feeder cells.
- ES cell CMK 6 derived from force cynomolgus monkeys that had been cryopreserved was added to ES cell medium [composition per 200 ml: DMEM—F 12 (GI BCO) 163 ml, ushi fetal serum 30 ml ( Final concentration 15%), L-Daltamin 2ml (final concentration 2mM), Penicillin (100 U / m 1) — Sulevutmycin (10 O ⁇ gZml) 2ml, Non-essential amino acid solution [GIBCO (GI BC Ii) manufactured by the company] 2 ml, 2-mercaptoethanol 1 ml (final concentration 0 lm M)] 5 ml.
- the obtained cell suspension was seeded on the feeder cell. Incidentally, the culture became rows 37 ° C, 5% C_ ⁇ incubator two conditions. The ES cell medium was changed once every two days.
- the old medium was aspirated and removed, and the cells were washed with phosphate buffered saline. Subsequently, 1 ml of 0.25% trypsin / HBSS (Hanks balanced salt solution) was added to the cells on the dish, and the cells were incubated at 37 ° C. for 3 to 4 minutes. Then, the bottom of the dish was tapped at room temperature to detach the cell colonies.
- trypsin / HBSS Hops balanced salt solution
- undifferentiated cell colonies were recovered from the cell colonies by pipetting with a 5 ml pipet using the ES cell medium.
- the collected cells were suspended in the ES cell medium, and 5 ml of the obtained cell suspension was seeded on a feeder cell on a 6 cm dish. Thereafter, the cells were passaged every 2-4 days depending on the size of the colony.
- the obtained undifferentiated ES cells were used for transplantation into Hedge fetuses. Such undifferentiated ES cells were used for the following induction of hematopoietic differentiation.
- Example 2 Induction of hematopoietic differentiation of monkey embryonic stem cells
- the OP 9 cell culture medium was added to the dish, and the medium containing the cells was collected in a 50 ml 1 conical tube. The medium containing the collected cells was centrifuged at 1500 rpm for 5 minutes. The obtained cells were seeded at 1: 4 to 1: 5 (approximately 2.5 ⁇ 10 5 cells to 4 ⁇ 10 5 cells) in the above OP 9 cell culture medium at 37 ° C. and 5% CO 2 conditions. Incubated until confluent.
- the obtained cells were cultured on a 10 cm dish (manufactured by FALCON) on the OP 9 cell medium 10 ml 1 containing mitomycin C at a final concentration of 10 gZm 1 for 2 to 4 hours. Mitotic growth was stopped and inactivated. Subsequently, the medium containing mitomycin C was removed. The treated cells were washed with phosphate buffered saline. The washed cells were treated with trypsin ⁇ EDTA (0.05% trypsin, ImM EDTA) to obtain a cell suspension, and the cell suspension was centrifuged to obtain cells.
- trypsin ⁇ EDTA 0.05% trypsin, ImM EDTA
- the obtained cells were gelatin-coated 6 cm culture dish (FALCON, trade name: T issue Culture Dish). On the above OP 9 cell culture medium, 1: 2 (about per medium) 1 x 10 5 cells / ml) did.
- the cells prepared as described above were used as feeder cells in the following hematopoietic differentiation induction culture.
- Example 1 the force recovered in 1 ml of 0.25% trypsin ZHBS S was obtained from the medium for differentiation ES cell line CMK6 [differentiation medium [composition per 10 ml: IMDM 84 ml, 8% horse serum 8 ml] 8% urine fetal serum 8m 1, 5 X 10 " 6 M hydrocortisone 0.18 g, BMP—42 g (final concentration 2031871111), SCF 2 jug (final concentration 20 ng / m 1), IL 3 2 g (final concentration 20 ng / m 1), IL— 6 lg (final concentration 10 ng Zml), VEGF 2 g (final concentration 20 ng Zml), G—CSF 2 g (final concentration 20 ng / m 1), F 1 t.3 ligand 2 g (final concentration 10 ng Zml), EPO 200U (final concentration 2 UZml)] and suspended in 5 ml, and the resulting cell suspension was obtained in Example 2
- Fig. 1 The obtained cells (Fig. 1) were used for the following transplantation as hematopoietic differentiation-inducing cells.
- the cells on the feeder cell are fed together with the feeder cell.
- the cells were collected in a 50 ml 1-volume conical tube containing 20 ml of the differentiation medium and centrifuged at 800 rpm for 4 minutes.
- the supernatant was removed by suction, and the cells were suspended in a 5 Oml conical tube containing 2 Oml of the differentiation medium to obtain a cell suspension.
- Pregnancy hedges purchased from Japan Lamb, a handling company for experimental hedges, were used. Transplantation was scheduled on the 60th day of pregnancy, and we were accustomed to the breeding environment at the time of transplantation, 1 week to 10 days before transplantation. The fetus was confirmed by abdominal ultrasonography.
- Hedge Maternal was anesthetized with Keyumin intramuscular injection (15 mg / kg body weight). Hidge was laid on his back on the operating table, the limbs were fixed, and then endotracheal intubation was performed. Under spontaneous anesthesia, the patient underwent general anesthesia with 0 2 / air Z-halothane.
- the operation was performed with clean operation. After laparotomy with a midline incision in the lower abdomen, the uterus was turned into the abdominal cavity.
- the transplanted cells were injected in two locations: I) in the fetus and II) in the liver parenchyma.
- Cell transplantation was performed in two ways: I) hysterectomy and i) ultrasound guided method.
- I) hysterectomy incision is made in the myometrium of the hidge, the sheep fetus is driven into the exposed egg membrane while being preserved, and transplanted with a 23 G needle into the abdominal cavity of the fetus directly through the transparent egg membrane
- the cells for use (1 ⁇ 10 6 to 1 ⁇ 10 8 cells) were punctured and injected, and the myometrium, peritoneal muscle layer, and skin were closed in layers.
- the uterus wall is Under ultrasound guidance from above, the transplanted cells (1 X 10 7 to 1 X 10 8 cells) are punctured and injected into the fetal parenchyma of the Hedge fetal liver, and the peritoneal muscle layer and the skin are closed and closed in layers. did.
- antibiotics mycillin sol
- Hedgee's anesthesia was confirmed.
- Induction of anesthesia with 0 2 Z-air halothane was started in the mother of Hedge by spontaneous respiration by oral fistula. After 15 minutes, the cesarean section was performed on the maternal body, two fetuses from one uterus were removed, and the tissue was collected and processed.
- the body weight of the fetus transplanted with cells was 950 g, and the weight of the control fetus was 1040 g.
- umbilical vein blood (umbilical cord blood sample) and umbilical vein blood (peripheral blood sample) were collected with the umbilical cord connected to the mother.
- the amount of blood collected is 10 ml for the cord blood sample of the transplanted fetus, 2 Om1 for the peripheral blood sample of the transplanted fetus, 2 Oml for the cord blood sample of the control fetus, and 2 Oml for the peripheral blood sample of the control fetus. did.
- the umbilical cord was dissected, and then a force neuron was inserted into the umbilical vein with a 6 G atom tube. As the umbilical artery could not be inserted with force neuret, the cut end was opened. A reflux solution (Cold Lactec 500 ml) was connected to the force neulet on the umbilical vein side, and reflux was started by infusion. The umbilical artery side was used as a blood removal route, and the blood flow was continued until blood flowed out (approximately 1000 ml in total).
- the abdomen was opened, and the partial liver and femur were collected as a primary culture sample by a clean operation.
- the organ was removed.
- the removed organs were placed in petri dishes on ice.
- the collected tissues are shown below.
- Liver including bile duct, liver parenchyma, hepatic artery, portal vein region
- Cartilage epiphyseal cartilage
- Peripheral blood (umbilical artery blood)
- Umbilical cord blood (umbilical vein venous)
- Bone marrow samples were decalcified.
- one specimen of the 4% PFA-fixed sample was sequentially maintained in 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol for 1-2 hours, and 100% ethanol. Maintain for 1 to 2 hours, then maintain with 100% ethanol for 1 to 2 hours, and further maintain with 1% 100% ethanol for dehydration, soak in benzene (twice for 30 minutes), then soak in paraffin (45 minutes x 2 times) to obtain paraffin-embedded sections after fixation with 4% PFA.
- the other 4% PFA-fixed sample was placed in Cliomold 1 (Tissuetek, Miles, Elkhart, IN, USA) and embedded and frozen. The embedding and freezing were carried out by embedding with OCT compound (Tissuetek) and freezing with liquid nitrogen. This sample was used as a specimen for frozen sections after fixation.
- the remaining one specimen was also placed in Cryomold No. 1 and embedded and frozen.
- the embedding / freezing was carried out by embedding with OCT compound and freezing with liquid nitrogen. This sample was used as a specimen for a fresh frozen section.
- peripheral blood heparin blood collected
- bone marrow collected in heparin raw diet (10 units Zm 1)
- umbilical cord blood heparin blood collected
- peripheral blood and bone marrow were collected approximately once a month after birth. Specifically, after birth, about 20 ml of peripheral blood (heparin blood collection) is collected every 2 weeks during the first few months, and bone marrow (collected during heparin diet) is collected every month. An appropriate amount was collected. The collected sample was placed in a sterile conical tube and stored at room temperature with shaking, and nucleated cells were separated and stored frozen within one day. For hematopoietic tissue and cells such as liver, umbilical cord blood, bone marrow, and peripheral blood, leukocyte fractions are separated by hemolysis, and are used as DNA extraction samples, RNA extraction samples, primary culture samples, and FACS analysis samples. Alternatively, it was stored frozen in liquid nitrogen.
- cells were isolated from the liver as follows. Take a sample under aseptic procedure, chop the sample appropriately, and add 50 ml Konica. The sample was collected in a tube, added with 2 Oml of 1% trypsin ZED TA / phosphate buffered saline, suspended well, and cultured at 37 ° C for 10 minutes with shaking. DNase I solution [10 mg DNase I dissolved in 1 ml 0.15M NaC1] 200 1 was added to the resulting product, and 10 1 of 1M MgCl 2 was further added. Incubated at ° C for 10 minutes.
- the product was filtered through a 7 Omm diameter nylon mesh (Falcon, trade name: ce 1 1 strainer), and the same amount of D10 medium as the product was added. Thereafter, the mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed by aspiration. If the pellet is red due to red blood cell contamination, add approximately 10 times the amount of ACK hemolysis medium to the pellet and maintain on ice for 15 to 0 minutes, then at 1500 rpm for 5 minutes at 4 ° Centrifuged at C. The pellet was washed twice with about 40 ml of phosphate buffered saline. The supernatant was removed by aspiration, and the resulting pellet was suspended in a cell panker with 10 6 to 10 7 cell pairs and stored frozen at 80 ° C. or in liquid nitrogen.
- umbilical cord blood, peripheral blood and bone marrow were isolated as follows.
- a sample was collected by aseptic technique in the same manner as heparin blood collection, 35 ml of blood was placed in a 50 ml conical tube, and centrifuged at 1500 rpm for 10 minutes. The plasma was aspirated off and the pellet was dispensed into five 50 ml conical tubes.
- 40 ml (about 10 times the amount of the pellet) of ACK hemolysis medium was added to each conical tube, mixed, and incubated on ice for 15 minutes. After centrifugation at 1500 rpm for 5 minutes at 4 ° C, the supernatant was aspirated off.
- PCR a two-step PCR method in which PCR using an external primer pair followed by PCR using an internal primer pair
- PCR conditions were as follows: reaction solution [composition: H 2 0 30. 7 5 1 1 0 XPCR buffer 5 1, dNTP (2.5 mM each) 4 I, Ex Taq (5 / ⁇ 1) 0.
- C] 3 1 5 '-CATTGTCATGGACTCTGGCGACGG-3' (SEQ ID NO: 4) and C / 32: 5 '-CATCTCCTGCTCGAAGTCTAGGGC-3' (SEQ ID NO: 5) and
- PCR was carried out with the same reaction mixture (30 cycles, 1 minute at 95 ° C, 1 minute at 54 ° C and 1 minute at 72 ° C for 2 minutes).
- the product generated by PCR was subjected to electrophoresis on a 2% agarose gel (containing 0.01% ethidium bromide).
- monkey-derived cells could not be detected in each tissue of Hedge fetuses 1 month after transplantation, and as described in Example 6 below, cells in which hematopoietic differentiation was induced on monkey embryonic stem cells were transplanted Monkey-derived cells were detected only in Hedge's hematopoietic progenitor cells. Therefore, according to this method, it is particularly suitable for the differentiation of primate embryonic stem cells into hematopoietic cells, and it is considered that cell differentiation of other lineages does not occur, and this method has excellent specificity for hematopoietic differentiation. It is suggested.
- Example 6 Analysis of Monkey / Hedge Hematopoietic Chimera
- Hematopoietic colony assembly was performed on cells isolated for primary culture.
- the cryo-tube of the cryopreserved cells was lysed in a 37 ° C thermostat and suspended in a 15m 1 conical tube containing 9m 1 of 2% FB S—I MDM. A portion was used to count the number of cells. Subsequently, the suspension was centrifuged at 1300 rpm for 4 minutes, and the resulting pellet was suspended with 2% FBS-IMDM to 1 ⁇ 10 6 cells Zm 1 to obtain a cell suspension. 2% F Cell suspension of 1 x 10 5 cells Zm 1 was prepared by diluting 10 times with BS—IMDM.
- Methylcellulose medium (trade name: Methocult GF + (manufactured by StemCe 1 1 Tec hn ologies, Kataguchi No .: ST-0443 5)) 4 ml in a 14m 1 polystyrene round bottom tube Then, 400 1 of cell suspension was added, and the cap was closed and shaken well. Then, it was allowed to stand for about 5 minutes until the bubbles disappeared.
- Hedge No: 141 the subject of administration, was 81 days after birth (156 days after transplantation) and weighed about 14 kg at the start of administration.
- Hedge No. 182 to be administered was 12 days after birth (94 days after transplantation), and weighed about 10 kg at the start of administration.
- Peripheral blood and bone marrow were collected on the 6th day after administration, 1 day after the last administration and 3 days after the last administration.
- FACS buffer composition: phosphate buffered saline containing 5% urine fetal serum and 0.05% Na N 3
- the suspension was passed through a 70 m mesh.
- the obtained product was centrifuged at 1500 rpm (490 X g) for 4 minutes, and the supernatant was removed by aspiration. Thereafter, the FACS buffer 450 1 was added to the obtained pellet.
- the obtained solution 150 1 was dispensed into 96 well plates. Note that a sample obtained by adding the FACS buffer 3501 to 1501 of the above solution was used as an antibody negative control.
- the solution in the 96 well plate well was then centrifuged at 1800 rpm (710 X g) for 2 minutes to remove the supernatant.
- PE—Anti—human CD 45 Becton Dates Kinson, BD catalog number: 557059
- F ACS buffer solution 80 1 the product name: PE—Anti—human CD 45 (Becton Dates Kinson, BD catalog number: 557059) 20 1 and the F ACS buffer solution 80 1 were added to the well.
- PE-Anti-uman CD45 the trade name PE-Anti-mouse IgGlK (manufactured by Becton Dickinson, catalog number: 338 15X) was used as an isotype control.
- the resulting mixture was incubated on ice for 30 minutes and then centrifuged at 1800 rpm (710 X g) for 2 minutes to remove the supernatant. Thereafter, the obtained pellet was washed twice with the FACS buffer 1501. The obtained pellet was suspended in the FACS buffer 3501, and the obtained sample was subjected to flow cytometry.
- the leukocyte fraction was separated from the peripheral blood and bone marrow by the hemolysis method as described in Example 4. From the obtained sample, DNA was extracted according to the manufacturer's protocol attached to the kit using the product name: QI Aamp DNA Mini Kit and the product name: QI Aamp DNA Block Mini Kit. Next, the DNA is of a saddle type, and as a primer pair for monkey 32-microglopurin, the CB 2MG-2 F and hB 2MG-5 R are used as an external primer pair, and the CB 2MG-2 F and hB 2MG-3. Nested PCR was performed using R as an internal primer pair. PCR conditions are as follows: reaction solution [composition: H 2 0 3
- a monkey prepared by PCR using a primer having the base sequence shown in SEQ ID NO: 3 or the base sequence shown in SEQ ID NO: 4, and a probe of 32 microglobulin A Southern plot hybridization was performed. The results are shown in the lower panel of Fig. 3.
- PCR was performed at the same time under the same conditions as the sample and used for electrophoresis of the product.
- FIG. 3 showed that monkey cells appeared at a rate of about 0.01% in the peripheral blood on the 6th day after SCF administration and in the bone marrow on the 1st, 3rd, and 40th days after the administration.
- a sample for primary culture was prepared from peripheral blood and bone marrow by the same method as in Example 4. The obtained sample for primary culture was cultured for 14 days in a methyl cellulose medium CMe tho Cu 1 t GF + (manufactured by Stem Cell Tecnoh no gies)].
- SEQ ID NO: 1 is the sequence of primer-CB2MG-2F.
- SEQ ID NO: 2 is the sequence of primer-hB2MG-5R.
- SEQ ID NO: 3 is the sequence of primer-hB G-3R.
- SEQ ID NO: 4 is the sequence of primer--C] S1.
- SEQ ID NO: 5 is the sequence of Primer-C jS 2. Industrial applicability
- the present invention it becomes possible to supply a large amount of cells that can be used for the construction of a hematopoietic system or the treatment of a disease or condition that requires the action of a hematopoietic cell.
- the development of means for the treatment of diseases or conditions that require action is expected.
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Abstract
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US10/568,502 US20070124826A1 (en) | 2003-08-25 | 2004-08-24 | Method of differentiation from embryo-stem cell of primate to hematogenous cell |
EP04772412A EP1661984A4 (en) | 2003-08-25 | 2004-08-24 | METHOD FOR DIFFERENTIATING FROM THE EMBRYONAL STEM CELL OF A PRIMATE TO A HEMATOGENIC CELL |
JP2005513388A JP4332527B2 (ja) | 2003-08-25 | 2004-08-24 | 霊長類動物の胚性幹細胞から造血系細胞への分化方法 |
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PCT/JP2004/012456 WO2005019441A1 (ja) | 2003-08-25 | 2004-08-24 | 霊長類動物の胚性幹細胞から造血系細胞への分化方法 |
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US (1) | US20070124826A1 (ja) |
EP (1) | EP1661984A4 (ja) |
JP (1) | JP4332527B2 (ja) |
KR (1) | KR20060087509A (ja) |
CN (1) | CN1842590A (ja) |
TW (1) | TW200519207A (ja) |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2008041370A1 (fr) * | 2006-10-04 | 2008-04-10 | The University Of Tokyo | Structure renfermant des cellules progénitrices hématopoïétiques issues de cellules es et procédé de préparation de cellules sanguines faisant appel à ladite structure |
WO2009122747A1 (ja) * | 2008-04-01 | 2009-10-08 | 国立大学法人東京大学 | iPS細胞からの血小板の調製方法 |
US8787089B2 (en) | 2005-12-15 | 2014-07-22 | Spansion Llc | Semiconductor device and method of controlling the same |
WO2016208532A1 (ja) * | 2015-06-22 | 2016-12-29 | 全国農業協同組合連合会 | 血液キメラ動物の作出法 |
JPWO2017038958A1 (ja) * | 2015-08-28 | 2018-06-14 | 学校法人自治医科大学 | 造血系細胞の作製方法 |
-
2004
- 2004-08-24 US US10/568,502 patent/US20070124826A1/en not_active Abandoned
- 2004-08-24 JP JP2005513388A patent/JP4332527B2/ja active Active
- 2004-08-24 WO PCT/JP2004/012456 patent/WO2005019441A1/ja active Application Filing
- 2004-08-24 CN CNA2004800247009A patent/CN1842590A/zh active Pending
- 2004-08-24 EP EP04772412A patent/EP1661984A4/en not_active Withdrawn
- 2004-08-24 KR KR1020067003500A patent/KR20060087509A/ko not_active Application Discontinuation
- 2004-08-26 TW TW093125624A patent/TW200519207A/zh unknown
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JP5283120B2 (ja) * | 2006-10-04 | 2013-09-04 | 国立大学法人 東京大学 | Es細胞からの造血前駆細胞を内包する構造物、及び該構造物を用いた血球細胞の調製方法。 |
JPWO2008041370A1 (ja) * | 2006-10-04 | 2010-02-04 | 国立大学法人 東京大学 | Es細胞からの造血前駆細胞を内包する構造物、及び該構造物を用いた血球細胞の調製方法。 |
GB2455472B (en) * | 2006-10-04 | 2011-07-20 | Univ Tokyo | Structure enclosing hematopoietic progenitor cells from ES cells and method for preparing blood cells using the same |
US8058064B2 (en) | 2006-10-04 | 2011-11-15 | The University Of Tokyo | Sac-like structure enclosing hematopoietic progenitor cells produced from ES cells and method for preparing blood cells |
WO2008041370A1 (fr) * | 2006-10-04 | 2008-04-10 | The University Of Tokyo | Structure renfermant des cellules progénitrices hématopoïétiques issues de cellules es et procédé de préparation de cellules sanguines faisant appel à ladite structure |
GB2455472A (en) * | 2006-10-04 | 2009-06-17 | Univ Tokyo | Structure enclosing hematopoietic progenitor cells from ES cells and method for preparing blood cells using the same |
WO2009122747A1 (ja) * | 2008-04-01 | 2009-10-08 | 国立大学法人東京大学 | iPS細胞からの血小板の調製方法 |
US8546141B2 (en) | 2008-04-01 | 2013-10-01 | The University Of Tokyo | Method for preparation of platelet from iPS cell |
WO2016208532A1 (ja) * | 2015-06-22 | 2016-12-29 | 全国農業協同組合連合会 | 血液キメラ動物の作出法 |
JPWO2016208532A1 (ja) * | 2015-06-22 | 2018-06-07 | 全国農業協同組合連合会 | 血液キメラ動物の作出法 |
US11432537B2 (en) | 2015-06-22 | 2022-09-06 | The University Of Tokyo | Method for producing blood chimeric animal |
JPWO2017038958A1 (ja) * | 2015-08-28 | 2018-06-14 | 学校法人自治医科大学 | 造血系細胞の作製方法 |
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TW200519207A (en) | 2005-06-16 |
JPWO2005019441A1 (ja) | 2006-10-19 |
EP1661984A4 (en) | 2007-03-21 |
CN1842590A (zh) | 2006-10-04 |
JP4332527B2 (ja) | 2009-09-16 |
KR20060087509A (ko) | 2006-08-02 |
US20070124826A1 (en) | 2007-05-31 |
EP1661984A1 (en) | 2006-05-31 |
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