WO2017038958A1 - Method for preparing hematopoietic cells - Google Patents

Abstract

The invention pertains to a method for preparing hematopoietic cells of primates, characterized by including: a first step for culturing a cell group of pluripotent stem cells of a primate under conditions suited to inducing differentiation into hematopoietic cells, and obtaining a cell group including CD-34 negative cells; a second step for transplanting at least some of the cell group obtained in the first step into the fetus of an animal different from the primate; and a third step for obtaining hematopoietic cells of the primate from the body of the animal obtained by raising the offspring obtained by the birth of the fetus.

Description

Method for producing hematopoietic cells

The present invention relates to a technique for supplying hematopoietic cells of primate animals. More specifically, the present invention relates to a method for producing hematopoietic cells from primate pluripotent stem cells, a chimeric non-human animal that produces the hematopoietic cells, and a method for producing the same.

At the current clinical practice of regenerative medicine, hematopoietic stem cell transplantation represented by bone marrow transplantation, peripheral blood stem cell transplantation, umbilical cord blood stem cell transplantation, etc. is performed for patients with diseases such as aplastic leukemia. Treatment by producing blood cells from transplanted hematopoietic stem cells has been performed. However, it is difficult to stably supply hematopoietic stem cells that are necessary for hematopoietic stem cell transplantation and that are suitable for the patient to be transplanted. Therefore, in order to provide a stable supply of hematopoietic stem cells, human hematopoietic stem cells were transplanted transuterine into pig fetuses to try to amplify human hematopoietic progenitor cells. Detection was extremely small (Non-patent Document 1).

Differentiating necessary cells in vitro from pluripotent stem cells such as embryonic stem cells (hereinafter also referred to as ES cells) and induced pluripotent stem cells (hereinafter referred to as iPS cells) for stable supply of hematopoietic stem cells Research is being conducted on.

As an example of in vitro differentiation from pluripotent stem cells to hematopoietic cells, the phenotype of the final hematopoietic stem cells from mouse ES cells into which HoxB4, a homeotic selector gene related to self-renewal of hematopoietic stem cells, was introduced. (Non-patent Document 2), a technique for differentiating mouse ES cells into hematopoietic progenitor cells (Non-Patent Documents 3 to 5), and rhesus monkey ES cells are cultured on mouse S17 stromal cells, and hematopoietic Examples include a technique for differentiating into stem cells (Non-patent Document 6). However, the differentiation of hematopoietic stem cells from human iPS cells is still difficult, and its realization is a challenge for hematology.

Therefore, in recent years, embryonic stem cells of primates are maintained under conditions suitable for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into a fetus in the uterus of a pregnant sheep, the fetus is grown and born. A method for producing hematopoietic cells of primate animals has been studied, in which hematopoietic cells of primate animals are obtained from sheep obtained by growing lambs that have reached the target (Patent Documents 1 and 2). There is a need for a more efficient method of producing hematopoietic cells optimized for human iPS cells.

International Publication No. 2005/019441 JP 2004-350601 A

An object of the present invention is to efficiently supply hematopoietic cells of primates such as humans.

One embodiment of the present invention provides:
Culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for induction of differentiation into hematopoietic cells, to obtain a cell group containing CD34 negative cells;
A second step of transplanting at least a part of the cell group obtained in the first step into a fetus of an animal different from the primate,
A third step of obtaining hematopoietic cells of the primate animal from the body of the animal obtained by raising a puppy obtained by birth of the fetus;
A method for producing hematopoietic cells of primates having

In the present invention, in the first step, the primate pluripotent stem cell group may be cultured for 2 to 12 days under conditions suitable for induction of differentiation into hematopoietic cells.
In the present invention, in the first step, the primate pluripotent stem cell group is expressed with Brachyury, PDGFRα, Etv2, or Scl gene or protein under conditions suitable for induction of differentiation into hematopoietic cells. May be cultured until cells appear.

In this invention, you may have the process of carrying out a gene modification process with respect to a pluripotent stem cell before said 1st process.
In the present invention, the animal different from the primate animal may be selected from cattle, pigs, horses, sheep or goats.

In the present invention, in the second step, at least a part of the cell group obtained in the first step may be transplanted into a hematopoietic organ of a fetus of an animal different from the primate animal.
In the present invention, the primate animal may be a human.

In the present invention, the pluripotent stem cell may be an iPS cell.
In the present invention, lymphocytes from animals different from the chimeric non-human animal may be administered to pups obtained by birth of the fetus.

In this invention, you may further include the process of suppressing the proliferation of the endogenous hematopoietic system cell of the said fetus before said 2nd process.
In the present invention, in the step of suppressing the growth of endogenous hematopoietic cells in the fetus, a chemotherapeutic agent or an immunosuppressive agent may be administered to the fetus, or radiation may be irradiated to the fetus.

In the present invention, busulfan may be administered to the fetus in the step of suppressing proliferation of endogenous hematopoietic cells of the fetus.
Another aspect of the present invention is:
Culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for induction of differentiation into hematopoietic cells, to obtain a cell group containing CD34 negative cells;
A second step of transplanting at least a part of the cell group obtained in the first step into a fetus of an animal different from the primate,
A third step of obtaining a chimeric animal for producing a hematopoietic cell of the primate by nurturing a pup obtained from the birth of the fetus;
A method for producing a chimeric non-human animal that produces hematopoietic cells of a primate animal.

In the present invention, in the first step, the cells may be cultured for 2 to 12 days under conditions suitable for induction of differentiation into hematopoietic cells.
In this invention, you may have the process of carrying out a gene modification process with respect to a pluripotent stem cell before said 1st process.

Another aspect of the present invention is:
A chimeric non-human animal that produces blood cells of a primate animal and has somatic cells different from the primate animal.

The chimeric non-human animal may be produced by the above-described method for producing a chimeric non-human animal that produces hematopoietic cells of a primate animal.
Another aspect of the present invention is:
It is a hematopoietic cell obtained from the above chimeric non-human animal.

Another aspect of the present invention is:
About the hematopoietic cells in the body of the chimeric non-human animal, comprising the step of administering lymphocytes of an animal different from the chimeric non-human animal to the chimeric non-human animal having hematopoietic cells derived from a primate animal This is a method for increasing the ratio of hematopoietic cells derived from primates.

It is preferable that the lymphocyte is an MHC non-restricted lymphocyte.
The lymphocyte is preferably an NK cell, a γδT cell, or an NKT cell.
The chimeric non-human animal may be obtained by transplanting hematopoietic stem cells derived from humans, apes or monkeys to cows, pigs, horses, sheep or goats.

In the present invention, the method may further comprise a step of obtaining hematopoietic cells from the chimeric animal.
Another aspect of the present invention is:
A therapeutic method using hematopoietic cells of a primate animal, comprising a step of administering hematopoietic cells obtained by the production method of the present invention to the primate animal.

Another aspect of the present invention is:
Culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for induction of differentiation into hematopoietic cells, to obtain a cell group containing CD34 negative cells;
A second step of transplanting at least a part of the cell group obtained in the first step into a fetus of an animal different from the primate,
A third step of obtaining hematopoietic cells of the primate animal from the body of the animal obtained by raising a puppy obtained by birth of the fetus;
A fourth step of administering the hematopoietic cells to the primate animal;
A therapeutic method using hematopoietic cells of a primate animal.

You may have the process of carrying out a gene modification process with respect to a pluripotent stem cell before said 1st process.
In the present invention, the therapeutic diseases of the above treatment methods are aplastic anemia, leukemia, immunodeficiency (X-SCID, ZAP70 deficiency, Jak3 deficiency, ADA deficiency, chronic granulomatosis, Bloom syndrome, Wiscott-Aldrich). Syndrome), ischemic heart disease such as myocardial infarction, obstructive arteriosclerosis, Buerger's disease, and other inherited diseases (immunodeficiency, Fanconi anemia, hemophilia, thalassemia, sickle cell anemia, leukodystrophy, Any of mucopolysaccharidosis etc.).

By the method of the present invention, hematopoietic cells can be more efficiently produced from pluripotent stem cells of primates including humans.

FIG. 1 shows the results of FACS analysis of PDGFRα (mesoderm marker), CD34 (hematopoietic stem cell marker), CD43 (pan blood cell marker) and CD45 (pan blood cell marker) in human iPS cells when cultured in vitro. FIG. FIG. 2 is a diagram for explaining that Brachyury, Etv2, Scl, Runx, PDGFRα and CD34 are expressed in human iPS cells cultured in vitro, whereas CD43 and CD45 are not expressed. FIG. 3 shows the presence of activated Notch and CD45 in the liver and bone marrow of sheep fetuses two months after cell group transplantation. FIG. 4 is a fluorescence microscopic image of the liver of an ovine fetus 1 month after transplantation of human cell groups and the bone marrow of an ovine fetus 2 months after transplantation of cell groups. FIG. 5 shows the result of FACS analysis of a group of human cells after iPS cells were cultured in a differentiation medium in Example 1 (right diagram), and the group of human cells after iPS cells were cultured in a differentiation medium. FIG. 4 is a view showing the results (left figure) of FACS analysis after removal of CD34-expressing cells using a magnetic bead-binding monoclonal antibody against CD34. FIG. 6 is a graph showing changes in the ratio of human hematopoiesis in the sheep when human lymphocytes are intravenously administered to sheep transplanted with human cell groups.

The inventors of the present invention cultured a pluripotent stem cell for a short period of time under conditions suitable for induction of hematopoietic cell differentiation, and transplanted the obtained cell group into the fetus of an animal different from the primate animal. The inventors have found that hematopoietic cells that can be used for transplantation medicine can be efficiently produced from the pluripotent stem cells of the primate animals, and have completed the present invention.
I. In one aspect, the present invention relates to a method for producing a hematopoietic cell of a primate animal.
A first group of primate pluripotent stem cells cultured under conditions suitable for induction of differentiation into hematopoietic cells, a cell group containing CD34 negative cells, and the first process obtained A second step of transplanting at least a part of a cell group into a fetus of an animal different from the primate, and the primate from the body of the animal obtained by growing a pup obtained by birth of the fetus A method for producing hematopoietic cells of primates, comprising a third step of obtaining the hematopoietic cells.

In the present invention, the term “hematopoietic cell” means a hematopoietic stem cell or a cell group obtained by differentiation from the hematopoietic stem cell. The cell group that can be differentiated from hematopoietic stem cells is not limited, and examples thereof include cells having properties such as erythroblasts, myeloid cells, megakaryocytes, and lymphocytes.

The hematopoietic cells obtained in the present invention are essentially cells derived from primates and are suitable for transplantation to primates. In the present invention, if human cells are used for transplantation into animals, blood cells that can be transplanted into humans can be produced. iPS cells can be used to create animals with “own” blood cells.

The 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, the present invention, in another aspect, is characterized by supplying a therapeutically effective amount of the hematopoietic cells of the present invention to a site of a disease or condition that requires the construction of the hematopoietic system or the action of hematopoietic cells. The present invention relates to a method for treating a disease requiring construction of a hematopoietic system or an action of hematopoietic cells. In yet another aspect, the present invention relates to the use of hematopoietic cells of the present invention for the treatment of diseases requiring the construction of hematopoietic systems or the action of hematopoietic cells.

(1) 1st process "The process of obtaining the cell group containing the CD34 negative cell which culture | cultivates the cell group of the pluripotent stem cell of a primate animal on the conditions suitable for the differentiation induction to a hematopoietic system cell."
In this method, first, in accordance with conventional knowledge in the art, primate cell groups including CD34-negative cells are formed in vitro from primate pluripotent stem cells.

The method for producing hematopoietic cells of the present invention is a method for the purpose of efficiently producing human hematopoietic cells used mainly for treating blood-related diseases in humans.
The source cell for producing hematopoietic cells is not particularly limited as long as it is a pluripotent stem cell of a primate animal. Preferably, they are derived from humans, apes (chimpanzees, gorillas or orangutans), monkeys (baboons, macaques, marmosets, etc.) and the like. More preferably, it is derived from a human.

In the present invention, the term “pluripotent stem cell” is intended to be a generic term for stem cells having the ability to differentiate into cells of any tissue (pluripotency). In the examples described later in this specification, examination is carried out using iPS cells. However, pluripotent stem cells that can be used in the method of the present invention are not limited to iPS cells, but can also be used in mammalian organs and tissues. Examples include all pluripotent stem cells derived from cells, bone marrow cells, blood cells, and embryonic and fetal cells and the like and having traits similar to embryonic stem cells. In this case, traits similar to embryonic stem cells are specific to embryonic stem cells, such as the expression of genes specific to embryonic stem cells and the ability to differentiate into all germ layers of endoderm, mesoderm, and ectoderm. It can be defined with cell biological properties.

Specific examples of cells that can be grown by the method of the present invention include, but are not limited to, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ stem cells ( EG cells), germ stem cells (GS cells) and the like. Note that ES cells and iPS cells are preferred as the pluripotent stem cells in the present invention. iPS cells are particularly preferred for reasons such as no ethical problems. Any known pluripotent stem cell can be used. For example, pluripotent stem cells grown by the method described in International Publication WO2009 / 123349 (PCT / JP2009 / 057041) can be used.

“Artificial pluripotent stem cells” and “iPS cells” in this specification are ES cells obtained by introducing genes for transcription factors such as Oct3 / 4, Sox2, Klf4, and c-Myc into somatic cells. It means a cell having differentiation pluripotency similar to. Therefore, iPS cells can be increased without limitation while retaining differentiation pluripotency, as in ES cells (Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007). The established human iPS cell line is available from, for example, Kyoto University and RIKEN.

Alternatively, iPS cells may be prepared with reference to the following literature. For example, induced pluripotent stem cells can be obtained from literature related to the group of Professor Shinya Nakayama (Cell, 131, pp. 861-872, 2007; Nat, Biotechnol. 26, 101-106, 2008) and Thomson at the University of Wisconsin. It can be produced according to the method described in the literature (Science 318, 1917-1920, 2007) relating to this group.

Specifically, at least one gene of Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and LIN28 is introduced into any somatic cell, and the gene is specific for pluripotent stem cells. Or by detecting the expression of proteins and selecting them.

The iPS cells produced in this manner can be cultured with basic fibroblast growth factor in the presence of proliferation-inactivated mouse fibroblasts or cells that can replace them, in the same manner as ES cells. It can be used as a pluripotent stem cell as well as a cell.

In this technical field, conditions suitable for inducing differentiation from pluripotent stem cells to hematopoietic stem cells include, for example, conditions for culturing in the presence of type IV collagen, presence of α-minimum essential medium (α-MEM) and the like. For hematopoietic stem cells for various conditions, such as conditions for culturing in vitro, conditions for culturing on feeder cells suitable for hematopoietic differentiation, conditions for adhering to a dish after embryoid body preparation, and conditions for any combination thereof. Conditions such as the type, concentration, period, etc. of cytokine to be added for inducing differentiation are mentioned.

When the primate pluripotent stem cell group is a human iPS cell line or ES cell, for example, BMP-4, SCF, IL-3 can be used as cytokines used for conditions suitable for inducing differentiation into blood cells. IL-6, VEGF, G-CSF, Flt-3 ligand, EPO, and TPO can be used.

Hematopoietic stem cell transplantation is classified into bone marrow transplantation, umbilical cord blood transplantation, and peripheral blood stem cell transplantation depending on the origin of the hematopoietic stem cells to be transplanted. In all cases, a small number of hematopoietic stem cells in the transplanted cells are engrafted, and an effect of continuing to support hematopoiesis over a long period of time is expected. If there is a surface antigen specific for hematopoietic stem cells, hematopoietic stem cells can be concentrated using this as an index. CD34 is well known as this surface antigen. That is, human hematopoietic stem cells are CD34 positive. When creating hematopoietic stem cells from human iPS cells, the focus has been on how to efficiently induce differentiation of CD34 positive cells. On the other hand, mouse hematopoietic stem cells are said to be CD34 negative. If hematopoietic stem cells are also present in the CD34 negative fraction as in the case of mice, it is not always necessary to focus only on CD34 positive cells when producing hematopoietic stem cells from human iPS cells. As demonstrated in the Examples, it was found that hematopoietic stem cells exist in both human and CD34 negative fractions. Therefore, in the present invention, a cell group containing human CD34-negative cells is transplanted into an animal body. That is, in the present invention, the “cell group containing CD34 negative cells” is transplanted into the fetus of an animal different from the primate animal in the second step described later. “CD34 negative cells” included in this cell group are cells in which CD34 does not exist on the cell surface. Although this cell group is not limited, for example, a CD34 positive cell is a part of the cell group in a condition suitable for inducing differentiation into hematopoietic cells. It can be obtained by culturing until it appears.

The abundance ratio of CD34 positive cells is not particularly limited. For example, the pluripotent stem cell group of a primate animal is subjected to conditions suitable for inducing differentiation into the hematopoietic system, for example, 50% or less, 30% of the cell group. In the following, it is preferable to culture until 10% or less, 7% or less, or 6% or less of cells differentiate into CD34 positive cells.

In the present invention, the method for determining the proportion of CD34 positive cells is not particularly limited. For example, the cell group is stained using an anti-CD34 antibody, and the number of CD34 positive cells in the cell group and the CD34 negative are determined using flow cytometry. The number of cells can be measured and determined based on the number of measurements.

The pluripotent stem cell group of primate animals is preferably, but not limited to, cultivated for a minimum of 2 days, more preferably 4 days under conditions suitable for inducing differentiation into hematopoietic stem cells. Preferably, the culture is more preferably 6 days, the longest is preferably 12 days, more preferably 10 days, and even more preferably 8 days. It can culture | cultivate by the number of days within the range which combined arbitrary days among said shortest culture days, and arbitrary culture days among said longest culture days. Moreover, it is most preferable to culture for 6 days.

The primate pluripotent stem cell population should be cultured under conditions suitable for induction of differentiation into hematopoietic stem cells until cells expressing the Brachyury gene or protein, an early mesoderm marker, appear. It is more preferable to culture until cells expressing PDGFRα gene or protein, which is a mesoderm marker, appear, and cells expressing Etv2 gene or protein that induces expression of vascular endothelial cell gene appear It is more preferable to culture until a cell expressing the Scl gene or protein, which is a hematopoietic factor, appears.

In embryo development, it is known that mesoderm is expressed in the order of Brachyury, PDGFRα, Etv2, and Scl. Brachyury is a transcription factor belonging to the T box family and is a protein essential for mesoderm formation and cell differentiation into the mesoderm system (YH Edwards et al, Genome Res. 1996. 6: 226). 233). Human Brachyury can be searched by accession number NM_003181.3 or NM_0012704484.1.

PDGFR (Platelet-Derived Growth Factor receptor) is a protein expressed on the cell surface of various mesenchymal cells and has tyrosine kinase activity. There are α and β receptors with similar amino acid sequences. It is known that it is expressed in the paraxial mesoderm at the time of gastrulation (Takakura N et al., J Histochem Cytochem. 1997 Jun; 45 (6): 883-93.). Human PDGFRα can be searched by the accession number NM_006206.4.

Etv2 (ets variant 2) is an essential factor for the development of blood and endothelial cells and is known to be temporarily expressed during development (Misato Hayashi et al., Exp Hematol. Volume 40, Issue 9, September 2012, Pages 738-750.e11). Etv2 can be searched by the accession number NM_014209.3, NM_001300974.1, or NM_0013044549.1.

Scl (Stem cell leukemia) is known to play an essential role in the differentiation of hematopoietic cells in the early mesoderm (Ismaloglu I et al., Exp Hematol. 2008 Dec; 36 (12): 1593-). 603. doi: 10.016 / j. Ephem. 2008.7.0.005. Epub 2008 Sep 21.). The Scl can be searched by the accession number NM_001287347.2, NM_001290403.1, NM_001290404.1, NM_001290405.1, NM_0012904406.1, or NM_003189.5.

Before transplanting into the body of an animal, preferably, prior to the first step, there may be a step of genetically modifying a cell group of pluripotent stem cells of a primate animal. The gene modification treatment is not particularly limited. For example, gene transfer technology using a viral vector such as a lentivirus vector, gene transfer technology using a plasmid vector derived from an adeno-associated virus (AAV) or herpes virus, ZFN (Zinc Finger) Nuclease), TALEN (Transscript activator-like effector nuclease), and genome editing techniques such as CRISPR / Cas method. By having this step, blood cells that can be used to treat an individual having a disease caused by a gene mutation can be produced. The disease caused by the gene mutation is not particularly limited, for example, X-SCID, ZAP70 deficiency, Jak3 deficiency, Bloom syndrome, Wiscott-Aldrich syndrome, ADA deficiency, chronic granulomatosis, Fanconi anemia, thalassemia, Intended for sickle cell anemia, leukodystrophy, hemophilia, mucopolysaccharidosis, etc.

(2) Second step “Transplanting at least a part of the cell group obtained in the first step into the fetus of an animal different from the primate animal”
In the present invention, for the purpose of further differentiating and inducing a cell group containing CD34 negative cells derived from pluripotent stem cells of the primate animal into a hematopoietic system, a cell group of a primate animal containing CD34 negative cells is selected. And transplanted to a fetus of an animal different from the primate animal.

In the present invention, any animal that transplants a cell group containing a large amount of CD34-negative cells derived from pluripotent stem cells of the primate animal as long as it is different from a primate animal that is a species derived from pluripotent stem cells. For example, when the animal species derived from pluripotent stem cells is human, it is possible to use industrial animals such as cattle, pigs, horses, sheep and goats in addition to non-human primates. it can. Considering the ease of supply of animals, it is preferable to use industrial animals, and in the present invention, it is more preferable to use sheep and pigs.

Transplantation into the fetus of an animal transplanted with a group of cells containing CD34 negative cells derived from pluripotent stem cells of the primate animal, from the outside of the pregnant mother of the animal, as a transplantation site in the fetus, a hematopoietic organ, For example, in the liver (or, more strictly, in the liver parenchyma) is desirable, but is not limited thereto, and may be performed by puncture injection of transplanted cells into the abdominal cavity, heart, umbilical cord, or the like. .

For example, if the animal is a sheep,
-Pregnant sheep on the 40th to 85th day of pregnancy (maturity 147 days) should be accustomed to the breeding environment at the time of transplantation 1 week to 10 days in advance.
− Anesthetize the mother,
-Fix the proper position for the treatment of sheep,
-To perform transplantation of cells for transplantation at the transplantation site with a clean operation, for example, when transplanting cells into the abdominal cavity or liver parenchyma, the uterine maternal body is opened after abdominal midline incision. It can be performed by turning outside the abdominal cavity.

The cells are transplanted into the intrauterine fetus by, for example, dissecting the myometrium of the animal, driving the fetus into the exposed egg membrane, and transplanting it directly into the fetal transplant site through the transparent egg membrane. The cells for use are punctured and injected, and the myometrium, peritoneal muscle layer, and skin are sutured and closed in layers. Alternatively, without incising the uterus, transplantation cells may be punctured and injected into the transplantation site of the fetus under the ultrasound guide from the uterine wall, and the peritoneal muscle layer and skin may be closed and closed.

In the present invention, the fetus of the animal transplanted with the cell group containing the CD34 negative cells derived from the pluripotent stem cell of the primate animal is born, the pup is grown, and the body of the animal is The primate animal-derived cell group is differentiated and proliferated to obtain hematopoietic cells of the primate animal. By transplanting a cell group containing CD34-negative cells derived from the primate animal into an individual body of the animal, a cell group containing CD34-negative cells derived from the pluripotent stem cells of the primate animal in the body of the animal. On the other hand, further differentiation / induction into blood cells is caused, and as a result, the latter half of the process of producing hematopoietic cells from primate pluripotent stem cells is realized.

The hematopoietic stem cells derived from the primate thus induced and propagated in the body of the animal are born from the fetus of the animal transplanted with the cell group, and then the appropriate tissue such as the liver, bone marrow, etc. (1) Hematopoietic colony assay is performed on the obtained cells. (2) Hematopoietic colonies formed in (1) above are specific to primates. Can be assessed by examining the expression of specific marker genes.

The (1) hematopoietic colony assay is, for example,
i) From a pup, obtain a tissue biopsy of the liver, tissue such as bone marrow,
ii) The obtained tissue is subjected to appropriate treatment according to the tissue such as trypsin treatment, DNase I treatment, lysis treatment, etc. to obtain cells,
iii) The obtained cells are suspended in 10% FBS (fetal bovine serum) -IMDM to obtain a cell suspension.
iv) The obtained cell suspension and a methylcellulose medium {for example, trade name: MethoCult GF + [manufactured by StemCell Technologies, catalog number: ST-04435] etc.} are mixed and stirred.
v) Subsequently, the medium containing the obtained cells is poured into a dish and cultured under conditions of 37 ° C. and 5% by volume CO ₂ for 14 days.

In addition, the expression of the marker gene specific to the primate animal of the above (2) is performed by extracting DNA from the colony formed by the hematopoietic colony assay by a conventional method, and for the obtained DNA, for example, primate animal It can be evaluated by detecting the gene by hybridization using a probe specific to a specific marker gene, PCR using a specific primer pair, or the like. Alternatively, hematopoietic colonies may be assessed by immunostaining with antibodies against antigens specific for primates.

In the method of the present invention, the step of suppressing endogenous hematopoiesis in the fetus of the animal before performing the step of transplanting the cell group containing CD34 negative cells derived from the pluripotent stem cells of the primate animal into the fetus of the animal. Further can be included. Promoting the generation of hematopoietic cells from a cell group containing CD34-negative cells derived from exogenous primate pluripotent stem cells to be transplanted thereafter while suppressing endogenous hematopoiesis occurring in the animal body Can do. Endogenous hematopoiesis in the pup's body can be suppressed, for example, by administering a chemotherapeutic or immunosuppressive agent to the fetus or by irradiating the fetus with radiation, for example, in the fetus. On the other hand, an antitumor agent such as busulfan, fludarabine, melphalan, or cyclophosphamide can be administered to cause hematopoietic stem cells to undergo cell death. The chemotherapeutic agent or immunosuppressive agent is preferably administered via the maternal vein to the fetus before transplantation. The dose of the chemotherapeutic agent or immunosuppressive agent and the dose of radiation may be determined depending on the sensitivity of the fetus and are not particularly limited. The dose of busulfan is not particularly limited, but may be 3 to 5 mg / kg for sheep generally bred in Japan, for example.

In the step of transplanting a cell group containing CD34-negative cells derived from the pluripotent stem cells of the primate animal into the fetus of the animal, primate lymphocytes may be transplanted into the pup after birth. When the lymphocytes of the primate animal are transplanted, the induction and proliferation of the hematopoietic stem cells of the primate animal transplanted into the puppy body can be enhanced.

The lymphocytes may be MHC (major histocompatibility complex) non-restricted lymphocytes, but NK cells, γδ T cells, or NKT cells are preferably used.

Lymphocytes are not limited to those collected from the individual from which the transplanted cell population is derived. For example, lymphocytes collected from species other than animal species that receive transplantation of cell groups can be used.

Lymphocyte administration conditions are not particularly limited, but may be administered, for example, via the maternal vein before the pups are born, but are preferably administered intravenously immediately after the pups are born. It may be infused repeatedly at the same time.

(3) Third step “a step of obtaining hematopoietic cells of a primate animal from the body of an animal obtained by breeding a pup obtained from the birth of a fetus”
Separation of primate hematopoietic cells, for example,
1) From the animal obtained as described above, liver, bone marrow, blood (peripheral blood, umbilical cord blood), thymus, spleen, etc. are collected,
2) Obtain a cell group from the obtained organ by an appropriate method,
3) It can be obtained by separating hematopoietic cells derived from primates from the obtained cell group.

In 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 primates” may be any marker that crosses primates and does not cross recipient animals, and includes HLA-ABC, β2-microglobulin, CD45, and the like.

Evaluation of hematopoietic cells of primate animals obtained by the production method of the present invention is, for example, CD45, TER119, α4-integrin, VE-cadherin, c-kit, Sca-1, CD34, CD13, CD14, GPIIb / IIIa , CD3, CD4, CD8, sIgM, CD19 and the like can be used as an index.
II. Chimeric animals By the method of the present invention, chimeric non-human animals that produce hematopoietic cells of primate animals can be produced. Therefore, in one aspect, the present invention relates to a chimeric non-human animal that produces hematopoietic cells of a primate animal and a method for producing the same.

A chimeric non-human animal that produces hematopoietic cells of the primate animal of the present invention,
Culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for induction of differentiation into hematopoietic cells, to obtain a cell group containing CD34 negative cells;
A second step of transplanting at least a part of the cell group obtained in the first step into a fetus of an animal different from the primate,
A third step of obtaining a chimeric animal for producing a hematopoietic cell of the primate by nurturing a pup obtained from the birth of the fetus;
It can be produced by a method for producing a chimeric non-human animal that produces hematopoietic cells of primates.

In another aspect, the chimeric non-human animal of the present invention is an animal having a somatic cell different from that of the primate animal capable of producing primate blood cells.
Before the first step, a pluripotent stem cell may be subjected to a genetic modification treatment by the method described above. Chimeric non-human animals that produce blood cells that can be used to treat diseases caused by genetic mutations can be created.

Since the chimeric non-human animal of the present invention has cells derived from a primate animal in an immunologically tolerant state, the chimeric non-human animal has conditions suitable for inducing differentiation of hematopoietic cells. Can be maintained in the body for any period of time. Therefore, the obtained blood system cells can be transplanted to donor animals at any time.

According to the chimeric non-human animal of the present invention, blood cells derived from a specific individual can be easily amplified in the animal body. In addition, since blood cells can be cultured in chimeric non-human animals until the day of transplantation, blood cells can be easily maintained.
III. Hematopoietic cells obtained from chimeric non-human animals Another aspect of the present invention is hematopoietic cells obtained from the chimeric non-human animals of the present invention.

The blood cell obtained from the chimeric non-human animal of the present invention is a primate cell derived from a pluripotent stem cell transplanted into the chimeric non-human animal. If the MHC matches, it is transplanted into the primate animal. In case there is no rejection. Therefore, it can be used for the treatment of primates such as transplantation to primates derived from pluripotent stem cells transplanted to chimeric non-human animals.
IV. Treatment Method Another aspect of the present invention is a treatment method using hematopoietic cells of a primate animal, comprising a step of administering hematopoietic cells obtained by the production method of the present invention to the primate animal.

Another aspect of the present invention is a first step of obtaining a cell group containing CD34-negative cells, wherein a cell group of primate pluripotent stem cells is cultured under conditions suitable for induction of differentiation into hematopoietic cells. A second step in which at least a part of the cell group obtained in the first step is transplanted into a fetus of an animal different from the primate, and a pup obtained from the birth of the fetus A primate hematopoietic system comprising a third step of obtaining hematopoietic cells of the primate animal from the body of the animal, and a fourth step of administering the hematopoietic cells to the primate animal. This is a treatment method using cells.

The administration method in the administration step in the present invention is not particularly limited, and a known method can be used.
The treatment method can be used for treatment of a disease requiring transplantation of hematopoietic cells, and examples of the disease include aplastic anemia, leukemia, immunodeficiency (X-SCID, ZAP70 deficiency, Jak3 deficiency). , ADA deficiency, chronic granulomatosis, Bloom syndrome, Wiscott-Aldrich syndrome, etc., ischemic heart disease such as myocardial infarction, obstructive arteriosclerosis, Burger disease, and other genetic diseases (Fanconi anemia, hemophilia) Disease, thalassemia, sickle cell anemia, leukodystrophies, mucopolysaccharidosis, etc.).

In the present specification, more specific aspects of the invention described above will be described in more detail below in the form of examples.

Example 1: It is shown that human iPS cells can be differentiated to a certain stage toward hematopoietic stem cells by step 1 (in vitro culture), but step 1 alone does not provide sufficient differentiation.
(1) Preparation of feeder cells A stromal cell line prepared from a neonatal calvaria of a (C57BL / 6 × C3H) F2-op / op mouse, which is a dysfunctional mouse of macrophage colony-stimulating factor, and a preadipocyte cell line OP9 cells were prepared in a medium for OP9 cells {Composition per 1250 ml: α-MEM (Gibco, catalog number: 12000-022) 980 ml, fetal calf serum 250 ml (final concentration 20%), penicillin (100 U / ml ) -Streptomycin (100 μg / ml) 10 ml, 200 mM L-glutamine solution 10 ml}, and cultured under conditions of 37 ° C. and 5% CO ₂ .

Thereafter, the cells that had adhered in the culture and grown to confluence or immediately before that were washed twice with phosphate buffered saline. Next, 0.1% trypsin-EDTA (2 ml / 10 cm dish) was added to the cells on the dish after washing, and the mixture was incubated for 5 minutes in an incubator.

The medium for OP 9 cells was added to the dish, and the medium containing the cells was collected in a 50 ml conical tube. The medium containing the collected cells was centrifuged at 1500 rpm for 5 minutes. The obtained cells were seeded in the OP9 cell medium at 1: 4 to 1: 5 (about 2.5 × 10 ⁵ cells to 4 × 10 ⁵ cells, and at 37 ° C. under 5% CO ₂ condition. Cultured until confluent.

The obtained cells were cultured on a 10 cm dish (FALCON) for 2 to 4 hours in 10 ml of the OP9 cell medium containing mitomycin C at a final concentration of 10 μg / ml to stop cell division and proliferation. 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, 1 mM EDTA) to obtain a cell suspension, and the cell suspension was centrifuged to obtain cells.

The obtained cells were placed in the above-mentioned culture medium for OP9 cells on a gelatin-coated 6 cm culture dish (trade name: Tissue Culture Dish, manufactured by FALCON) 1: 2 (about 1 × 10 ⁵ cells / ml per medium). ) Sowing. The OP9 cells prepared as described above were used as feeder cells in the hematopoietic differentiation induction culture.
(2) Hematopoietic differentiation culture of iPS cells Human Oct3 / 4, human Sox2, human Klf4 and human c-Myc were added to mononuclear cells recovered from adult peripheral blood using Ficoll-Hypaque (manufactured by GE Healthcare). The gene was introduced using a Sendai virus vector (manufactured by ID Pharma Co., Ltd.) loaded with human iPS cells. Human iPS cells were obtained by injecting 2 FGF (manufactured by Wako Pure Chemical Industries) into 500 ml of primate ES cell medium (Prime ES Cell Medium (manufactured by Reprocell)) on mouse embryo fibroblasts inactivated by mitomycin C. 0.5 μg (final concentration 5 ng / ml) added]. Human iPS cells collected in 1 ml of the stripping solution were differentiated into differentiation medium (100 ml composition: IMDM (Iscove's Modified Dulbecco's Medium) 84 ml, 8% horse serum 8 ml, 8% fetal bovine serum 8 ml, 5 × 10 -6M hydrocortisone 0.18 μg, BMP-4 2 μg (final concentration 20 ng / ml), SCF 2 μg (final concentration 20 ng / ml), IL-3 2 μg (final concentration 20 ng / ml), IL-6 1 μg (final concentration 10 ng / ml) ml), VEGF 2 μg (final concentration 20 ng / ml), G-CSF 2 μg (final concentration 20 ng / ml), Flt-3 ligand 2 μg (final concentration 10 ng / ml), EPO 200 U (2 U / ml), TPO 4 μg (final concentration) 40 ng / ml)] was suspended in 5 ml. The obtained cell suspension was seeded on feeder cells in the hematopoietic differentiation induction culture. The culture was performed in an incubator under conditions of 37 ° C. and 5% CO ₂ . The differentiation medium was changed once every two days.

After 6 days of culture, the old medium was aspirated and removed, and the cells were washed with phosphate buffered saline. Subsequently, 1 ml of 0.1% trypsin EDTA / PBS (−) was added to the cells on the dish, and the cells were cultured at 37 ° C. for 3 to 4 minutes. Thereafter, the bottom of the dish was tapped at room temperature to detach the cell colonies. The obtained cell group was used for transplantation into sheep fetuses.
<Relative expression analysis of genes>
Detection of brachyury, Etv2, Gata2, Scl, Runx1A, and Runx1B genes was attempted using the RNA of hematopoietic differentiated cells prepared as described above as a sample. First, cDNA was synthesized from total RNA using RNA PCR kit (TaKaRa) according to the protocol. The sequence of each gene detection primer is as follows. , GATA2 primer set is 5'-AAGGACTTGGAGAACTTGGGTGTC-3 'and 5'-GGCTATTTCAGAGAGGAGACCCA-3', Scl primer set is 5'-ATGCCTTCCTCATGTTCACCACCA-3 ' -CCGAG ACCTCGAAGACATC-3 'and 5'-GCTGTGTCTTCCTCCTGCAT-3', the primer set of Runx1b was '5'-ATGGCCGACATGCCGATGCC-3 with the'5'-CGACTCTCAACGGCACCCGA-3. The PCR amplification conditions after the RT reaction were 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds for 45 cycles. The results are shown in the table below.

According to Step 1 of the present invention, brachyury necessary for mesodermal differentiation is first expressed from human iPS cells, then Etv2 required for vascular / blood differentiation is expressed, and then a gene group necessary for hematopoietic system, namely Gata2 , Scl, Runx1A, Runx1B were expressed in a wavy manner.
<FACS analysis>
FACS analysis was performed on the differentiated cells as described above using anti-human PDGFRα (mesoderm marker), CD34 (hematopoietic stem cell marker), CD43 (pan blood cell marker), CD45 (pan blood cell marker) antibody. The antibody solution was added to the cell suspension and reacted at 4 ° C. for 20-60 minutes, and then the fluorescence-labeled cells were analyzed using a FACS Accuflow cytometer (Becton Dickinson, San Jose, Calif.). The results are shown in FIG.

The observed surface markers are PDGFRα (mesoderm marker), CD34 (hematopoietic stem cell marker), CD43 (pan blood cell marker), and CD45 (pan blood cell marker). It is said that PDGFRα, CD34, CD43, and CD45 are expressed in the order of development. As is clear from FIG. 1, the expression of CD34 was certainly seen from day 4. However, almost no expression of CD43 and CD45 was seen. That is, it can be seen that iPS cells do not differentiate into hematopoietic stem cells in culture in vitro because some elements necessary for hematopoietic differentiation are lacking in culture in vitro (FIG. 1).

Fig. 2 illustrates the above data. Brachyury, Etv2, Scl, and Runx are expressed in a wavy manner, and PDGFRα and CD34 are also expressed in order. So far, the process of development into hematopoietic cells has been faithfully reproduced. However, CD43 and CD45 which are pan blood cell markers are not expressed thereafter. Multiple factors are considered to be lacking in the test tube. Therefore, in Example 2, Day 6 cells were transplanted into the liver of an animal fetus. The fetal liver is an original hematopoietic tissue, and it is thought that all the factors necessary for differentiation into hematopoietic cells are provided here.

Example 2: In addition to step 1 (in vitro culture), addition of step 2 (transplantation into animal fetal liver) induces differentiation of human iPS cells into hematopoietic stem cells, and further transition from liver to bone marrow Indicates that a “moving” will occur.

<Hematopoietic differentiation culture of iPS cells and preparation of transplanted cells>
Hematopoietic human iPS cells obtained in Example 1 (2) on the 6th day of culture were used as transplanted cells.

<Analysis of liver and bone marrow of sheep fetus after transplantation of cells>
Liver of sheep fetus 1 month after cell group transplantation and bone marrow of sheep fetus 2 months after cell group transplantation were collected and stained with anti-activated Notch antibody, human CD34 antibody and anti-human CD45 antibody and observed. The fluorescence microscope images are shown in FIGS.

FIG. 4 shows that CD45 positive cells were observed one month after transplantation of human iPS-derived cells into sheep fetal liver. The cell group was CD45-negative before transplantation (left figure in FIG. 4), indicating that it became CD45-positive in the sheep fetal liver.

It is thought that activation of Notch signal is essential for maintenance of hematopoietic stem cells, but activation of human Notch was confirmed in sheep fetal liver 2 months after transplantation. When stained with an antibody that recognizes only human activated Notch, as shown in FIG. 3, stained cells were not detected before transplantation, but were stained with this antibody in the sheep fetal liver two months after transplantation. You can see that there are cells. That is, it shows that the human Notch signal was activated for the first time in the sheep fetal liver.

Also in sheep fetal bone marrow 2 months after transplantation, engraftment of human CD34-positive cells, engraftment of human CD45-positive cells and activated human Notch signal were observed. That is, human hematopoietic stem cells migrated from sheep fetal liver to bone marrow. During this period, the hematopoietic tissue moves from the liver to the bone marrow is a hematopoietic development process characteristic of mammals, indicating that this process faithfully traces this (FIGS. 3 and 4).

In summary, it was found that CD45 of human cells was positively converted in the sheep fetal liver, the Notch signal was activated, and further it moved to the bone marrow. It can be said that human hematopoietic stem cells grow in sheep.

Example 3: It shows that human hematopoietic stem cells can be efficiently produced in the postnatal sheep when a cell group containing CD34 negative cells is transplanted.
<Hematopoietic differentiation culture of iPS cells and preparation of transplanted cells>
The hematopoietic human iPS cells obtained in Example 1 (2) on the 6th day of culture were used as cells for transplantation.
<Preparation of CD34 positive cell group in umbilical cord>
As frozen human umbilical cord blood cells, those purchased from Riken Cell Bank were used. Mononuclear cell fractions were obtained by overlaying on Ficoll-Hypaque (GE Healthcare) solution, and magnetically labeled anti-human CD34 antibody (Miltenyi Biotech) was reacted according to the protocol to separate and collect CD34 positive cells. The obtained cell group was used for transplantation into a sheep fetus and used as a control.
<Transplantation of cells into fetal sheep>
Pregnant sheep purchased from Japan Lamb, an experimental sheep handling company, was used. Transplantation was scheduled on the 60th day of pregnancy and 1 week to 10 days before transplantation, the animals were accustomed to the rearing environment at the time of transplantation. In addition, fetuses were confirmed by abdominal ultrasonography. Six days before the transplantation, busulfan was administered at 3 mg / kg in maternal weight in maternal vein.

Sheep mothers were anesthetized by intravenous xylazine injection (0.03 mg / kg body weight). The sheep was laid on its back on the operating table, the limbs were fixed, and the operation was performed under general anesthesia with O ₂ / air / sevoflurane under spontaneous breathing. The operation was performed with a clean operation. After laparotomy with the lower abdominal midline incision, the uterus was turned into the abdominal cavity. The transplanted cells were injected into the fetal liver parenchyma.

Cell transplantation was performed in two ways: I) hysterectomy and II) ultrasonic guide. In the case of hysterotomy, the myometrium of the sheep is incised, the sheep fetus is driven into the egg membrane exposed while being preserved, and the cells for transplantation (with a 23G needle in the abdominal cavity of the fetus under direct vision through the transparent egg membrane) 1 × 10 ⁶ to 1 × 10 ⁸ cells) were injected by puncture, and the myometrium, peritoneal muscle layer and skin were sutured and closed in layers. In the case of the ultrasonic guide method, cells (1 × 10 ⁶ to 1 × 10 ⁸ cells) are punctured and injected with a 23 GPTC needle into the fetal liver parenchyma under ultrasound guidance from the uterine wall without incising the uterus. Then, the peritoneal muscle layer and the skin were closed in layers. After the operation, antibiotics (mycillin sol) were injected intramuscularly. Subsequently, the tube was extubated, and anesthesia awakening of the sheep was confirmed.
<Acquisition of sample>
Peripheral blood and bone marrow were collected from a sheep transplanted with human iPS cell-derived cell groups at a rate of about once every two weeks after birth. As a control, peripheral blood and bone marrow were collected from a sheep transplanted with cells in the umbilical cord at a rate of about once every two weeks after birth. The collected sample was put in a sterile conical tube, stored at room temperature with shaking, and stored frozen within one day.
<Calculation method of human hematopoietic ratio>
For colony culture of hematopoietic progenitor cells from the specimen, the above-described methylcellulose medium for human cells, MethoCult ^™ GF ^+, was used. The harvested and separated bone marrow cells were cultured at a concentration of 5-10 × 10 ⁴ in 1 ml methylcellulose medium (37 ° C., 5% CO ₂ in air) in a 35 mm culture dish (Cat No. 1008, FALCON). After 10-14 days, formation of myeloid / macrophage colonies (CFU-GM), erythroid colonies (CFU-E) and mixed colonies (CFU-Mix) on the dish was observed. Individual colonies were transferred into 50 μl of distilled water (manufactured by Nippon Gene) in a 96-well plate, treated at 99.0 ° C. for 5 minutes, and then 20 μg / ml Proteinase K (manufactured by Takara) at 55.0 ° C. for 1 hour. Protein was deproteinized and DNA was eluted. 5 μl of each sample was used for PCR analysis. For each well of a 96-well plate, nested PCR was performed using 250 ng of DNA as a template and targeting a human-specific ND5 gene sequence (sheep ND5 cannot be detected). The outer primer set for the ND5 sequence is 5'-ACTGAGCCCCAACCCAAACA-3 'and 5'-CTGCTCGGGCGTATCATCAA-3', and the inner primer set is 5'-TTCATCCCTGTAGCATTTGTCG-3 'and 5'-GTTGGAATAGGTTGTTAGTAG. For the PCR reaction, MasterCycler (Eppendorf) was used, and each PCR cycle was composed of three steps of denaturation, annealing and extension, and the outer PCR amplification conditions were 95 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 30 seconds. The combination of seconds was 25 cycles. The inner PCR amplification conditions were 95 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 30 cycles. The reaction solution was prepared by mixing DNA and primers in a buffer containing Taq of Takara Taq DNA Polymerase (RR001B, Takara) according to the protocol. Distilled water (manufactured by Nippon Gene Co., Ltd.) used for preparing the reaction solution, methylcellulose and normal sheep blood sample DNA used in the colony analysis were used for the negative control, and human DNA was used for the positive control. The amplified product (135 bp) was mixed with Loading Buffer (manufactured by Wako) at 10: 1 and electrophoresed on a 2% agarose gel to which ethidium bromide (15585-011, manufactured by GIBCO) was added. As the molecular weight marker, 100b Plus DNA Ladder (manufactured by Invitrogen) was used. The gel after electrophoresis was visualized under ultraviolet irradiation. The chimera rate of the individual was calculated by the ratio of the number of wells in which human ND5 was detected to the number of wells in which β-actin was detected.
<Result>
The total number of transplanted cells under each condition, the number of CD34 positive cells, the number of CD34 negative cells, the ratio of CD34 negative cells, the human hematopoietic ratio, and the number of CD34 positive cells necessary to obtain a human hematopoietic ratio of 1% (number of CD34 positive cells) ÷ Human hematopoiesis ratio) was examined. The results are shown in the table below.

Human iPS-derived cells were transplanted into 7 sheep fetuses. As a result, 4 were born, and the human hematopoietic chimera rate was 2.3 to 6.3%. On the other hand, human umbilical cord blood CD34 cells were transplanted into 6 sheep fetuses as a positive control, and the human hematopoietic chimera rate after birth was 1.1 to 2.3%. A higher chimera rate was obtained with human iPS cells than with the original hematopoietic stem cells present in cord blood. However, when the number of transplanted cells is viewed as, for example, the number of CD34 positive cells, this chimera rate cannot be simply compared because it differs between the two groups. In order to quantitatively compare the two, the number of CD34 cells required to achieve a human hematopoietic ratio of 1% was calculated, and it was found that human iPS cells had almost the same hematopoietic remodeling ability as compared with cord blood. It was.

As described above, by transplanting a group of cells containing CD34-negative cells obtained by culturing pluripotent stem cells under conditions suitable for induction of differentiation into blood cells, a smaller amount can be obtained. It was found that more human hematopoietic cells can be obtained using the number of CD34 positive cells.

From this result, the cell group obtained by culturing pluripotent stem cells under conditions suitable for induction of differentiation into blood cells, blood cells that have an immature differentiation stage than hematopoietic stem cells, It is considered that cells that induce differentiation into hematopoietic stem cells are included.

Human hematopoietic cells were detected 16 months after transplantation in sheep transplanted with a group of cells obtained by culturing pluripotent stem cells under conditions suitable for induction of differentiation into blood cells.
From the following points, it is considered that human hematopoiesis in sheep can be reproduced with extremely high efficiency comparable to the original hematopoietic stem cells called human umbilical cord blood stem cells.
(1) Cultivation method Brachyury, Etv2, Scl, and Runx are expressed in a wavy manner, while PDGFRα and CD34 are also expressed in order, and before the point where the panblood cell markers CD43 and CD45 should appear. It was transplanted on the 6th day of culture. It seems that the timing of hematopoietic differentiation is missed if it lags behind the point at which the panblood cell markers CD43 and CD45 should appear, such as after 12 days of culture. Although there was an example of transplantation on the 17th day of culture (Blood.2006 Mar 1; 107 (5): 2180-2183.doi: 10.1182 / blood-2005-05-1922), the timing of transplantation was late. it is conceivable that.
(2) Transplanted cells Cells containing a CD34 negative fraction were transplanted. Conventionally, human hematopoietic stem cells are considered to be in a CD34-positive fraction, and in fact, CD34-positive cell transplantation is also performed clinically. However, in the mouse as well, hematopoietic stem cells are present in the CD34 negative fraction. This is demonstrated in the following example.
(3) Pretreatment of fetus One of the reasons is that human iPS-derived cells were transplanted after busulfan was administered as a pretreatment.
(4) Transplantation site Transplantation of cells into the fetal liver, that is, into the hematopoietic tissue.

Example 4: Example of transplantation of only human iPS cell-derived CD34 negative cells Conventionally, mouse hematopoietic stem cells were said to be CD34 negative, whereas human hematopoietic stem cells were said to be CD34 positive. This example shows that hematopoietic stem cells are present in both human and CD34 negative fractions.
<Transplantation of cells into the fetus of a sheep and sampling from the sheep>
<Removal of CD34-positive cells in the cell group obtained in <Hematopoietic differentiation culture of iPS cells> in Example 1>
Example 3 <Hematopoietic differentiation culture of iPS cells in Example 3 using a magnetic bead reagent for cell separation and a purification column (a magnetic bead-binding monoclonal antibody against CD34 (Miltenyi Biotech) and a purification column (Miltenyi Biotech)) > The cell surface CD34-expressing cells were removed from the cell group obtained in>.

The cell group obtained by this was analyzed by FACS. The results are shown in the left figure of FIG. On FACS, CD34 positive cells in the cell group was 0.0%. This shows that the obtained cell group contained only CD34 negative cells.

The cell group containing only CD34-negative cells was transplanted into sheep fetuses in the same manner as in Example 3, and the human hematopoietic ratio was analyzed in the same manner as in Example 3. The following table shows the total number of transplanted cells, the number of CD34 positive cells, the number of CD34 negative cells, the human hematopoietic ratio, and the number of CD34 positive cells necessary to obtain a human hematopoietic ratio of 1%.

From this result, the cell group obtained by culturing pluripotent stem cells under conditions suitable for inducing differentiation into blood cells includes cells that are immaturely differentiated more than hematopoietic cells. It is thought that.

Among the cells in the cell group obtained by culturing pluripotent stem cells under conditions suitable for inducing differentiation into blood cells, a sheep transplanted with a cell group containing only the CD34 negative cell group, Human hematopoietic cells were detected 3 months after transplantation. This means that hematopoietic stem cells are present in the CD34 negative fraction.

Example 5: Infusion of lymphocytes into sheep transplanted with cord blood-derived cells This example demonstrates that human hematopoiesis in sheep is enhanced when lymphocytes are transplanted into sheep.
<Preparation of lymphocytes in umbilical cord>
When transplanting umbilical cord blood stem cells, lymphocytes are usually removed as unnecessary components, but in the present example, the lymphocytes were used without being discarded. Specifically, after separating CD34 positive cells from human umbilical cord blood, the remaining cells were cryopreserved. After the birth of a sheep transplanted with CD34 positive cells, the cryopreserved cells were thawed and lymphocyte culture medium supplemented with CD3 / 28 magnetic beads (Dynal) (RPMI-supplemented with 30 U / ml human recombinant IL-2) was added. 1640 (manufactured by Invitrogen)) for 14 days, the obtained cell group was used for injection into a sheep after birth. The lymphocytes used for injection include NK cells, γδT cells, and NKT cells.
<Infusion effect of human lymphocytes on sheep transplanted with cord blood-derived cells>
After the sheep transplanted with umbilical cord-derived cells were born, 4.8 × 10 ⁷ / kg or 10.2 × 10 ⁷ / kg of human lymphocytes were intravenously administered. Then, peripheral blood and bone marrow were collected from this sheep at a rate of once every two weeks. The collected samples were examined for human hematopoiesis ratio (human CFU rate (%)). The results are shown in FIG.

The human hematopoietic ratio increased 8 weeks after administration. It is considered that human hematopoietic stem cells increased as a result of lymphocytes affecting the immunological mechanism, etc., because it took 8 weeks from the administration of human lymphocytes to increase the human hematopoietic ratio. It was.

Example 6: Infusion of lymphocytes into sheep transplanted with human iPS cell-derived cell groups In this example, lymphocytes were infused into sheep transplanted with human iPS cell-derived cell groups obtained in Example 3. , Indicating that human hematopoiesis in sheep is enhanced.
<Preparation of human lymphocytes>
After the sheep transplanted with human iPS cell-derived cells were born, peripheral blood mononuclear cells from the same person from which the iPS cells were derived were added to lymphocyte culture medium (human recombinant IL) supplemented with CD3 / 28 magnetic beads (Dynal). -MI was added with RPMI-1640 (manufactured by Invitrogen) to which 30 U / ml-2 was added for 14 days, and the obtained cell group was used for injection into sheep after birth. The lymphocytes used for the injection include NK cells, γδT cells and NKT cells.
<Infusion effect of human lymphocytes into sheep transplanted with human iPS cell-derived cells>
After the sheep transplanted with human iPS cell-derived cells were born, 9.3 × 10 ⁷ / kg human lymphocytes were intravenously administered. Bone marrow was collected from this sheep 8 weeks after human lymphocyte injection. The collected samples were examined for human hematopoiesis ratio (human CFU rate [%]). The results are shown in Table 6. Similar to Example 5, the ratio of human hematopoiesis increased.

Claims (23)

1. Culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for induction of differentiation into hematopoietic cells, to obtain a cell group containing CD34 negative cells;
   A second step of transplanting at least a part of the cell group obtained in the first step into a fetus of an animal different from the primate,
   A third step of obtaining hematopoietic cells of the primate animal from the body of the animal obtained by raising a puppy obtained by birth of the fetus;
   A method for producing a hematopoietic cell of a primate animal, comprising:
2. The primate hematopoietic system according to claim 1, wherein in the first step, the primate pluripotent stem cell group is cultured for 2 to 12 days under conditions suitable for induction of differentiation into hematopoietic cells. Cell production method.
3. In the first step, a cell that expresses Brachyury, PDGFRα, Etv2, or Scl gene or protein appears under conditions suitable for inducing differentiation into hematopoietic cells from a group of primate pluripotent stem cells. The method for producing hematopoietic cells of primate animals according to claim 1 or 2, wherein the cells are cultured until they are cultured.
4. The method for producing hematopoietic cells of a primate animal according to any one of claims 1 to 3, further comprising a step of genetically modifying pluripotent stem cells before the first step.
5. The method for producing hematopoietic cells of a primate animal according to any one of claims 1 to 4, wherein the animal different from the primate animal is selected from cattle, pigs, horses, sheep or goats.
6. 6. The method according to claim 1, wherein in the second step, at least a part of the cell group obtained in the first step is transplanted into a hematopoietic organ of an embryo different from the primate animal. Of producing hematopoietic cells of primate animals.
7. The method for producing hematopoietic cells of a primate animal according to any one of claims 1 to 6, wherein the primate animal is a human.
8. The method for producing hematopoietic cells of primate animals according to any one of claims 1 to 7, wherein the pluripotent stem cells are iPS cells.
9. The hematopoiesis of a primate animal according to any one of claims 1 to 8, wherein lymphocytes of an animal different from the chimeric non-human animal are administered to a puppy obtained by birth of the fetus. A method for producing a cell line.
10. The method for producing hematopoietic cells of a primate animal according to any one of claims 1 to 9, further comprising a step of suppressing the growth of endogenous hematopoietic cells of the fetus before the second step. .
11. The primate animal according to claim 10, wherein in the step of suppressing the growth of endogenous hematopoietic cells in the fetus, a chemotherapeutic agent or an immunosuppressive agent is administered to the fetus, or radiation is irradiated to the fetus. A method for producing hematopoietic cells.
12. The method for producing hematopoietic cells of a primate animal according to claim 11, wherein busulfan is administered to the fetus in the step of suppressing the growth of endogenous hematopoietic cells in the fetus.
13. Culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for induction of differentiation into hematopoietic cells, to obtain a cell group containing CD34 negative cells;
    A second step of transplanting at least a part of the cell group obtained in the first step into a fetus of an animal different from the primate,
    A third step of obtaining a chimeric animal for producing a hematopoietic cell of the primate by nurturing a pup obtained from the birth of the fetus;
    A method for producing a chimeric non-human animal that produces hematopoietic cells of a primate animal.
14. The method for producing a chimeric non-human animal according to claim 13, wherein in the first step, the cells are cultured for 2 to 12 days under conditions suitable for induction of differentiation into hematopoietic cells.
15. The method for producing a chimeric non-human animal according to claim 13 or 14, further comprising a step of genetically modifying pluripotent stem cells before the first step.
16. A chimeric non-human animal that produces blood cells of a primate animal and has somatic cells different from the primate animal.
17. The chimeric non-human animal according to claim 16, produced by the method according to any one of claims 13 to 15.
18. A hematopoietic cell obtained from the chimeric non-human animal according to claim 16 or 17.
19. About the hematopoietic cells in the body of the chimeric non-human animal, comprising the step of administering lymphocytes of an animal different from the chimeric non-human animal to the chimeric non-human animal having hematopoietic cells derived from a primate animal A method for increasing the ratio of hematopoietic cells derived from primates.
20. The method according to claim 19, wherein the lymphocytes are MHC non-restricted lymphocytes.
21. The method according to claim 20, wherein the lymphocyte is an NK cell, a γδT cell, or an NKT cell.
22. The chimeric non-human animal according to any one of claims 19 to 21, wherein the chimeric non-human animal is obtained by transplanting a hematopoietic stem cell derived from a human, an ape or a monkey to a cow, pig, horse, sheep or goat. the method of.
23. 23. A method of producing hematopoietic cells, further comprising a step of obtaining hematopoietic cells from the chimeric animal, compared to the method according to any one of claims 19 to 22.

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