JP6861405B2 - How to make hematopoietic cells - Google Patents

Description

The present invention relates to techniques for the supply of hematopoietic cells in primates. More specifically, the present invention relates to a method for producing hematopoietic cells from pluripotent stem cells of primate animals, and a chimeric non-human animal for producing the hematopoietic cells and a method for producing the same.

In 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 on patients with diseases such as aplastic anemia, and in the patient's body. , Treatment is performed by producing blood cells from transplanted hematopoietic stem cells. However, it is difficult to provide a stable supply of hematopoietic stem cells suitable for the patient to be transplanted, which is necessary for hematopoietic stem cell transplantation. Therefore, in order to ensure a stable supply of hematopoietic stem cells, attempts were made to transuterinely transplant human hematopoietic stem cells into pig fetuses and amplify human hematopoietic progenitor cells in the pig body. The detection was extremely small (Non-Patent Document 1).

In vitro differentiation of required cells from pluripotent stem cells such as embryonic stem cells (hereinafter, also referred to as ES cells) and induced pluripotent stem cells (hereinafter, iPS cells) for a stable supply of hematopoietic stem cells. Is being researched.

As an example of an attempt to differentiate pluripotent stem cells into hematopoietic stem cells in vitro, phenotype of final hematopoietic stem cells from mouse ES cells introduced with HoxB4, a homeotic selector gene related to self-renewal of hematopoietic stem cells. (Non-Patent Document 2), a technique for differentiating mouse ES cells into hematopoietic precursor cells (Non-Patent Documents 3 to 5), and red-throated monkey ES cells are cultured on mouse S17 stroma cells for hematopoietic stemming. Examples thereof include a technique for differentiating into stem cells (Non-Patent Document 6). However, differentiation of hematopoietic stem cells from human iPS cells is still difficult, and its realization is a matter of hematology.

Therefore, in recent years, embryonic stem cells of primate animals are maintained under conditions suitable for inducing differentiation of hematopoietic cells, and the obtained cells are transplanted into a fetus in the womb of a pregnant sheep to grow the fetus and give birth. A method for producing hematopoietic cells of primate animals, which obtains hematopoietic cells of primate animals from the sheep obtained by growing the pups that have reached the above, has been studied (Patent Documents 1 and 2). Then, there is a demand for a more efficient method for producing hematopoietic cells optimized for human iPS cells.

International Publication No. 2005/019441 Japanese Unexamined Patent Publication No. 2004-350601

Hiromitsu Nakauchi, 1 other person, "3. Establishment of human blood chimeric pig and its application", "117th Japanese Association of Medical Sciences Symposium Record Collection Stem Cell and Cell Therapy- [III] Tissue / Organ Stem Cell: Basic Development for Clinical Application (2) Held from August 4th to 6th, 2000, p99-106 [online], Internet URL: http://www.med.or.jp/jams/symposium/kiroku/117/pdf/1170099.pdf Michael Kyba et al. , Cell, 109, April 5,2002, p. 29-37 Brit M. Johannsson et al. , Molecular and Cellular Biology, 15, No. 1, January 1995, p. 141-11 Toru Nakano, "5. Differentiation of embryonic stem cells into blood cells", edited by Takashi Yokota, special edition of experimental medicine "Forefront of stem cell research and its application to regenerative medicine, elucidation of developmental and differentiation mechanisms, and clinical practice" Published September 25, 2001, Vol. 19, No. 15, p1966-1971 Toru Nakano et al. , Science, 265, August 19, 1994, p. 1098-1101 Fei Li et al. , Blood, 98, July 15, 2001, p. 335-342

An object of the present invention is to efficiently supply hematopoietic cells of humans and other primate animals.

One aspect of the present invention is
The first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells,
The second step of 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, and the second step.
The third step of obtaining the hematopoietic cells of the primate from the body of the animal obtained by raising the pups obtained by the fetal birth, and
It is a method for producing hematopoietic cells of a primate animal having.

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

In the present invention, there may be a step of genetically modifying pluripotent stem cells before the first step.
In the present invention, an animal different from the primate animal may be selected from cows, 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 fetal hematopoietic organ of an animal different from the primatological 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 of an animal different from the chimeric non-human animal may be administered to the pups obtained by the birth of the fetus.

In the present invention, the step of suppressing the proliferation of the endogenous hematopoietic cells of the fetal may be further included before the second step.
In the present invention, in the step of suppressing the proliferation of the endogenous hematopoietic cells of the fetal, a chemotherapeutic agent or an immunosuppressive agent may be administered to the fetal, or radiation may be applied to the fetal.

In the present invention, busulfan may be administered to the fetal in the step of suppressing the proliferation of the endogenous hematopoietic cells of the fetal.
Another aspect of the present invention is
The first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells,
The second step of 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,
The third step of raising the pups obtained by the fetal birth to obtain a chimeric animal that produces the hematopoietic cells of the primatological animal, and the third step.
It is 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 inducing differentiation into hematopoietic cells.
In the present invention, there may be a step of genetically modifying pluripotent stem cells before the first step.

Another aspect of the present invention is
It is a chimeric non-human animal that produces somatic cells of the primatological animal and has somatic cells different from those of the primatological animal.

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

Another aspect of the present invention is
The hematopoietic cells in the body of a chimeric non-human animal having a step of administering lymphocytes of an animal different from the chimeric non-human animal to a chimeric non-human animal having a hematopoietic cell derived from a primate animal. It is a method of increasing the proportion of hematopoietic cells derived from primate animals.

The lymphocytes are preferably MHC non-restrictive lymphocytes.
The lymphocytes are preferably NK cells, γδT cells, or NKT cells.
The chimeric non-human animal may be obtained by transplanting hematopoietic stem cells derived from humans, apes or monkeys into cattle, pigs, horses, sheep or goats.

In the present invention, there may be further a step of obtaining hematopoietic cells from the chimeric animal.
Another aspect of the present invention is
This is a therapeutic method using hematopoietic cells of a primatological animal, which comprises a step of administering the hematopoietic cells obtained by the production method of the present invention to the primatological animal.

Another aspect of the present invention is
The first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells,
The second step of 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, and the second step.
The third step of obtaining the hematopoietic cells of the primate from the body of the animal obtained by raising the pups obtained by the fetal birth, and
The fourth step of administering the hematopoietic cells to the primate animal, and
It is a therapeutic method using hematopoietic cells of a primate animal, which is characterized by having.

Prior to the first step, there may be a step of genetically modifying pluripotent stem cells.
In the present invention, the therapeutic diseases of the above-mentioned treatment methods are 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, Buerger's disease, and other hereditary diseases (immunodeficiency, Fanconi anemia, hemophilia, salacemia, sickle redemia, leukodystrophy, etc. It may be any of mucopolysaccharidosis, etc.).

According to the method of the present invention, hematopoietic cells can be more efficiently produced from pluripotent stem cells of humans and other primate animals.

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. It is a figure which shows. FIG. 2 is a diagram illustrating that Brachyury, Etv2, Scl, Runx, PDGFRα and CD34 are expressed, whereas CD43 and CD45 are not expressed in human iPS cells cultured in vitro. FIG. 3 is a diagram showing the presence of activated Notch and CD45 in the liver and bone marrow of sheep embryos 2 months after cell group transplantation. FIG. 4 is a fluorescence microscope image of the liver of a sheep fetus 1 month after transplantation of a human cell group and the bone marrow of a fetus of a sheep 2 months after transplantation of a cell group. FIG. 5 shows the results of FACS analysis of the human cell group after culturing the iPS cells in the differentiation medium (right figure) and the human cell group after culturing the iPS cells in the differentiation medium in Example 1. , The result of analysis by FACS after removal of CD34-expressing cells using a magnetic bead-binding monoclonal antibody against CD34 (left figure) is shown. FIG. 6 is a graph showing changes in the human hematopoiesis ratio in a sheep when human lymphocytes are intravenously administered to a sheep transplanted with a human cell group.

The inventors of the present application cultured pluripotent stem cells for a short period of time under conditions suitable for inducing differentiation of hematopoietic cells, and transplanted the obtained cell group into the fetal body of an animal different from the primate animal. , The present invention has been completed by finding that hematopoietic cells that can be used for transplantation medicine can be efficiently produced from pluripotent stem cells of the primate animals.
I. Method for Producing Hematopoietic Cells of Primatological Animals The present invention, in one embodiment,
A first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells, a first step of obtaining a cell group containing CD34-negative cells, and a first step obtained in the first step. The second step of transplanting at least a part of the cell group into the fetus of an animal different from the primate animal, and the primate animal from the body of the animal obtained by raising the puppies obtained by the fetus at birth. Provided is a method for producing a hematopoietic cell of a primate animal, which comprises a third step of obtaining a hematopoietic cell of the above.

In the present invention, the "hematopoietic cell" is intended to be a hematopoietic stem cell or a group of cells formed by differentiating from the hematopoietic stem cell. The cell group formed by differentiating from hematopoietic stem cells is, but is not limited to, cells having properties such as erythroblasts, myelocytes, megakaryocytes, and lymphocytes.

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

The therapeutic effect can be exerted by transplanting the hematopoietic cells of the present invention into a site of a disease or condition that requires the construction of a hematopoietic system or the action of hematopoietic cells. Therefore, the present invention is characterized in that, in another aspect, a therapeutically effective amount of the hematopoietic system cells of the present invention is supplied to a site of a disease or condition that requires the construction of a hematopoietic system or the action of the hematopoietic cells. It relates to a method for treating a disease that requires the construction of a hematopoietic system or the action of hematopoietic cells. In yet another aspect, the invention relates to the use of hematopoietic cells of the invention for the construction of hematopoietic systems or the treatment of diseases requiring the action of hematopoietic cells.

(1) First step "A step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells"
In this method, first, according to the conventional knowledge in the art, a group of primatological animal cells including CD34-negative cells is formed in vitro from pluripotent stem cells of the primatological animal.

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

In the present invention, the term "pluripotent stem cell" is intended to be a general term for stem cells having the ability to differentiate into cells of any tissue (pluripotency). Although iPS cells are used in the examples described later in the present specification, the pluripotent stem cells that can be used in the method of the present invention are not limited to iPS cells, but include biological organs and tissues of mammals. Includes all pluripotent stem cells with traits similar to embryonic stem cells, derived from cells, bone marrow cells, blood cells, as well as embryonic and fetal cells. In this case, traits similar to embryonic stem cells are specific to embryonic stem cells, such as the expression of embryonic stem cell-specific genes and the ability to differentiate endoderm, mesodermal, and ectoderm into all germ layers. It can be defined with cell biological properties.

Specific examples of cells that can be propagated by the method of the present invention include, but are not limited to, artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and embryonic stem cells (embryonic stem cells). EG cells), germ stem cells (GS cells) and the like. As the pluripotent stem cells in the present invention, ES cells and iPS cells are preferable. iPS cells are particularly preferable because they have no ethical problems. Any known pluripotent stem cell can be used, and for example, pluripotent stem cells proliferated by the method described in International Publication WO2009 / 123349 (PCT / JP2009 / 057441) can be used.

As used herein, "induced pluripotent stem cells" and "iPS cells" are ES cells obtained by introducing transcription factor genes such as Oct3/4, Sox2, Klf4, and c-Myc into somatic cells. It means a cell having pluripotency similar to that of. Therefore, iPS cells, like ES cells, can be expanded indefinitely while maintaining pluripotency (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, RIKEN.

Alternatively, iPS cells may be prepared with reference to the description in the following documents. For example, induced pluripotent stem cells are described in the literature related to the group of Professor Shinya Yamanaka of Kyoto University (Cell, 131, pp. 861-872, 2007; Nat, Biotechnology. 26, 101-106, 2008) and Thomason of Wisconsin University. It can be prepared according to the method described in the literature (Science 318, 1917-1920, 2007) relating to the group of.

Specifically, at least one gene among Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and LIN28 is introduced into any somatic cell, and a gene specific to pluripotent stem cells is introduced. It can be produced by detecting the expression of or protein and selecting these.

Similar to ES cells, iPS cells prepared in this manner can be cultured together with basic fibroblast growth factor in the presence of growth-inactivated mouse fibroblasts or cells that can replace them, and ES It can be used as a pluripotent stem cell as well as a cell.

In the art, conditions suitable for inducing differentiation of pluripotent stem cells into hematopoietic stem cells include, for example, conditions for culturing in the presence of type IV collagen, and the presence of α-minimum essential medium (α-MEM). For hematopoietic stem cells under various conditions such as conditions for culturing on hematopoietic stem cells, conditions for culturing on feeder cells suitable for hematopoietic differentiation, conditions for adhering to dishes after embryonic body preparation, and conditions for any combination of these conditions. Conditions such as the type, concentration, and period of cytokines to be added for inducing differentiation can be mentioned.

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

Hematopoietic stem cell transplantation is divided into bone marrow transplantation, cord blood transplantation, and peripheral blood stem cell transplantation depending on the origin of the hematopoietic stem cell to be transplanted. In each case, a small amount of hematopoietic stem cells in the transplanted cells are engrafted, and it is expected that the effect of continuing to support hematopoiesis for a long period of time is expected. If there is a surface antigen specific to 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 producing hematopoietic stem cells from human iPS cells, the focus was also 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 present in the CD34-negative fraction in humans 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, hematopoietic stem cells were found to be present in both humans and the CD34 negative fraction. Therefore, in the present invention, a group of cells 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. The "CD34-negative cell" included in this cell group is a cell in which CD34 is not present on the cell surface. This cell group is not limited, but for example, CD34-positive cells may be a part of the cell group under conditions suitable for inducing differentiation of pluripotent stem cells of primate animals into hematopoietic cells. It can be obtained by culturing until it appears.

The abundance ratio of CD34-positive cells is not particularly limited, but the cell group of pluripotent stem cells of primate animals is, for example, 50% or less and 30% of the cell group under conditions suitable for inducing differentiation into the hematopoietic system. Hereinafter, it is preferable to culture until 10% or less, 7% or less, or 6% or less of the cells differentiate into CD34-positive cells.

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

The cell group of pluripotent stem cells of primate animals is preferably cultured for a minimum of 2 days, and more preferably for 4 days, under conditions suitable for inducing differentiation into hematopoietic stem cells. It is preferable to culture for 6 days, for the longest, for 12 days, more preferably for 10 days, and even more preferably for 8 days. It is possible to culture within a range in which any number of the shortest culture days described above and any number of days of the longest culture described above are combined. In addition, it is most preferable to incubate for 6 days.

In addition, the cell group of pluripotent stem cells of primate animals should be cultured under conditions suitable for inducing differentiation into hematopoietic stem cells until cells expressing the Brachyury gene or protein of the early mesophyll marker appear. Is preferable, and it is more preferable to culture until cells expressing the PDGFRα gene or protein, which is a mesenchymal marker, appear, and cells expressing the Etv2 gene or protein that induces the expression of the vascular endothelial cell gene appear. It is more preferable to culture until cells expressing the gene or protein of Scl, which is a hematopoietic factor, appear.

In embryonic development, it is known that Brachyury, PDGFRα, Etv2, and Scl are expressed in this order in the mesoderm. 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_0031181.3 or NM_001270484.1.

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 invagination of the archenteron (Takakura Net al., J Histochem Cytochem. 1997 Jun; 45 (6): 883-93.). Human PDGFRα can be searched by accession number NM_006206.4.

Etv2 (ets variant 2) is an essential factor in the development of blood and endothelial cells and is known to be expressed transiently during development (Misato Hayashi et al., Exp Hematol. Volume 40, Issue 9, September 2012, Pages 738-750.e11). Etv2 can be searched by accession numbers NM_014209.3, NM_00130090974.1 or NM_001304549.1.

Scl (Stem cell leukemia) is known to play an essential role in the differentiation of hematopoietic cells in the early mesoderm (Ismailoglu I et al., Exp Hematol. 2008 Dec; 36 (12): 1593- 603. doi: 10.016 / j.expem.2008.07.005.Epub2008Sep21.). Scl can be searched by accession numbers NM_001287347.2, NM_001290403.1, NM_001290404.1, NM_001290405.1, NM_001290406.1, or NM_0031899.5.

Prior to transplantation into the body of an animal, preferably prior to the first step, there may be a step of genetically modifying a cell population of pluripotent stem cells of a primatological animal. The gene modification treatment is not particularly limited, but is, for example, a gene transfer technique using a viral vector such as a lentiviral vector, a gene transfer technique using a plasmid vector derived from adeno-associated virus (AAV) or herpes virus, and ZFN (Zinc Finger). It can be performed by genome editing techniques such as Nuclease), TALEN (Transmission activator-like effector nucleose), and CRISPR / Cas method. By having this step, it is possible to prepare a blood line cell that can be used for treating an individual having a disease caused by a gene mutation. Diseases caused by gene mutations are not particularly limited, but are, for example, X-SCID, ZAP70 deficiency, Jak3 deficiency, Bloom syndrome, Wiscott-Aldrich syndrome, ADA deficiency, chronic granulomatosis, Fanconi anemia, thalassemia, etc. Intended for sickle cell anemia, leukodystrophy, hemophilia, mucopolysaccharidosis, etc.

(2) Second step "A step of 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 primate animals containing CD34-negative cells is used. , Transplant into the fetus of an animal different from the primate.

In the present invention, what kind of animal is transplanted with a cell group containing a large amount of CD34-negative cells derived from the pluripotent stem cell of the primate animal, as long as it is different from the primate animal which is the animal species from which the pluripotent stem cell is derived. It may be an animal species. For example, when the animal species from which pluripotent stem cells are derived is human, industrial animals such as cows, pigs, horses, sheep and goats may be used in addition to primate animals other than humans. it can. Considering the ease of feeding animals, it is preferable to use industrial animals, and in the present invention, it is more preferable to use sheep or pigs.

Transplantation into the fetus of an animal transplanted with a cell group containing CD34-negative cells derived from pluripotent stem cells of the primate animal is performed from outside the body of the pregnant mother of the animal as a transplantation site in the fetal body, a hematopoietic organ, For example, intrahepatic (or more strictly, intrahepatic parenchymal) is desirable, but is not limited to this, and it can be performed by puncturing and injecting transplantation cells into the abdominal cavity, intracardiac, intraumbilical cord, or the like. ..

Transplant surgery, 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 before transplantation.
− Anesthetize the mother and
− Fix the sheep in the proper position for the procedure and
-When transplanting cells into the abdominal cavity or hepatic parenchyma by various operations, for example, after opening the abdomen with a midline incision in the lower abdomen, the uterine mother It can be done by flipping it out of the abdominal cavity.

A method of transplanting cells into an intrauterine fetus is, for example, an incision in the myometrium of an animal, driving the fetus into an exposed peritoneum while preserving it, and transplanting it directly to the fetal transplant site through a transparent peritoneum. The cells for puncture are injected, and the myometrium, peritoneal muscle layer, and skin are sutured and closed. Alternatively, without incising the uterus, the cells for transplantation may be punctured and injected from above the uterine wall under ultrasonic guidance into the transplantation site of the fetus, and the peritoneal muscle layer and the skin may be sutured and closed.

In the present invention, the fetus of an animal transplanted with the cell group containing the CD34-negative cells derived from the pluripotent stem cells of the primate animal in this way is born, and the pups are grown in the body of the animal. In, the cell group derived from the primate animal is differentiated and proliferated, and as a result, hematopoietic cells of the primate animal are obtained. By transplanting a cell group containing the CD34-negative cells derived from the primate animal into the body of the animal, the cell group containing the CD34-negative cells derived from the pluripotent stem cells of the primate animal can be obtained in the body of the animal. On the other hand, further differentiation / induction into hematopoietic cells is induced, and as a result, the latter half of the process of producing hematopoietic cells from pluripotent stem cells of primate animals is realized.

The hematopoietic stem cells derived from the primate animal thus induced and proliferated in the body of the animal are more suitable tissues than the pups after the fetus of the animal transplanted with the cell group is born, for example, liver, bone marrow, etc. Tissues were collected to obtain cells for primary culture, and the obtained cells were subjected to (1) hematopoietic colony assay, and (2) the hematopoietic colonies formed in (1) above were specific to primate animals. It can be evaluated by examining the expression of a typical marker gene.

The hematopoietic colony assay of (1) is described in, for example,
i) Obtain liver tissue biopsy, bone marrow and other tissues from pups.
ii) The obtained tissue was subjected to appropriate treatment according to the tissue such as trypsin treatment, DNaseI treatment, lysis treatment, etc. to obtain cells.
iii) The obtained cells were 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, stirred, and stirred.
v) Then, the medium containing the obtained cells is injected into a dish and cultured under the conditions of _{37 ° C. and 5% by volume CO 2 for 14 days.}

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

In the method of the present invention, a step of suppressing endogenous hematopoiesis of an animal fetal is performed before transplanting a cell group containing CD34-negative cells derived from the pluripotent stem cells of the primate animal into the fetal animal. Can be further included. To suppress the endogenous hematopoiesis that occurs in the body of an animal and promote the production of hematopoietic cells from a group of cells containing CD34-negative cells derived from the pluripotent stem cells of the exotic primate that are subsequently transplanted. Can be done. Intrinsic hematopoiesis in the body of a pup can be suppressed, for example, by administering a chemotherapeutic agent or an immunosuppressant to the fetal, or by irradiating the fetal with radiation, eg, to the fetal. On the other hand, it can be carried out by administering an antitumor agent such as busulfan, fludarabine, melphalan, or cyclophosphamide to cause cell death in hematopoietic stem cells. The chemotherapeutic agent or immunosuppressant is preferably administered to the fetus before transplantation via the maternal vein. The dose of the chemotherapeutic agent or the immunosuppressant and the irradiation amount of the radiation may be determined by 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 example, in the case of sheep generally bred in Japan.

In the step of transplanting a cell group containing CD34-negative cells derived from the pluripotent stem cells of the primate animal into the embryo of the animal, the lymphocytes of the primate animal may be transplanted to the pups after birth. By transplanting the lymphocytes of the primate animal, it is possible to enhance the induction and proliferation of the hematopoietic stem cells of the primate animal transplanted into the body of the pup.

The lymphocyte may be an MHC (major histocompatibility complex) non-binding lymphocyte, but NK cells, γδT cells, or NKT cells are preferably used.

Lymphocytes are not limited to those collected from individuals from which the cells to be transplanted are derived. For example, any lymphocyte collected from a species other than the animal species to which the cell group is transplanted can be used.

The administration conditions of lymphocytes are not particularly limited. For example, lymphocytes may be administered via the maternal vein before the pups are born, but it is preferably administered intravenously immediately after the pups are born, and is optional after birth. It may be infused repeatedly at the time of.

(3) Third step "Step of obtaining hematopoietic cells of a primate animal from the body of an animal obtained by raising a pup that was obtained by fetal birth"
Separation of hematopoietic cells in primates, for example,
1) Liver, bone marrow, blood (peripheral blood, umbilical cord blood), thymus, spleen, etc. are collected from the animals obtained as described above.
2) Obtain a cell group from the obtained organ by an appropriate method.
3) It can be obtained by separating hematopoietic cells derived from primate animals 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 immune beads, or the like is performed.

Examples of the "marker specific to a primate animal" may be a marker that crosses a primate and does not cross a recipient animal, and examples thereof include HLA-ABC, β2-microglobulin, and CD45.

Evaluation of hematopoietic cells of primates obtained by the production method of the present invention includes, for example, CD45, TER119, α4-integrin, VE-cadherin, c-kit, Sca-1, CD34, CD13, CD14, GPIIb / IIIa. , CD3, CD4, CD8, sIgM, CD19, etc. 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 primatological animal and a method for producing the same.

The chimeric non-human animal that produces the hematopoietic cells of the primatological animal of the present invention is
The first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells,
The second step of 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, and the second step.
The third step of raising the pups obtained by the fetal birth to obtain a chimeric animal that produces the hematopoietic cells of the primatological animal, and the third step.
It can be produced by a method for producing a chimeric non-human animal that produces hematopoietic cells of a primate animal, which is characterized by having.

In another embodiment, the chimeric non-human animal of the present invention is an animal having somatic cells different from those of the primatological animal, which can produce blood cells of the primatological animal.
Prior to the first step, pluripotent stem cells may be genetically modified by the method described above. Chimeric non-human animals can be produced that produce bloodline cells that can be used to treat diseases caused by genetic mutations.

Since the chimeric non-human animal of the present invention has cells derived from primate animals 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 lineage cells can be transplanted into the donor animal 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. Moreover, since the blood line cells can be cultured in the chimeric non-human animal until the transplantation date, the blood line 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 chimeric non-human animals of the present invention.

The blood lineage cells obtained from the chimeric non-human animal of the present invention are primate animal cells derived from pluripotent stem cells transplanted into the chimeric non-human animal, and if the MHC matches, the cells were transplanted into the primate animal. If there is no rejection. Therefore, it can be used for the treatment of primate animals, such as transplantation into primate animals from which pluripotent stem cells transplanted into chimeric non-human animals are derived.
IV. Therapeutic method Another aspect of the present invention is a therapeutic method using hematopoietic cells of a primatological animal, which comprises a step of administering the hematopoietic cells obtained by the production method of the present invention to the primatological animal.

Another aspect of the present invention is a first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells. The second step of 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, and the raising of the pups obtained by the fetus at birth. A primate animal hematopoietic system, which comprises a third step of obtaining the primate hematopoietic cells from the body of the animal and a fourth step of administering the hematopoietic cells to the primate. It 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 the treatment of diseases requiring transplantation of hematopoietic cells, and the diseases include, for example, aplastic anemia, leukemia, immunodeficiency (X-SCID, ZAP70 deficiency, Jak3 deficiency). Diseases, ADA deficiency, chronic granulomatosis, Bloom syndrome, Wiscott-Aldrich syndrome, etc.), ischemic heart disease such as myocardial infarction, obstructive arteriosclerosis, Buerger's disease, and other hereditary diseases (Fanconi anemia, blood friend) Diseases, salacemia, sickle redemia, leukodystrophy, mucopolysaccharidosis, etc.).

Non-limitingly, the present invention includes the following aspects.
[Aspect 1] A first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells.
The second step of 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, and the second step.
The third step of obtaining the hematopoietic cells of the primate from the body of the animal obtained by raising the pups obtained by the fetal birth, and
A method for producing hematopoietic cells of a primate animal having.
[Aspect 2] The primate animal according to aspect 1, wherein in the first step, a cell group of pluripotent stem cells of a primate animal is cultured for 2 to 12 days under conditions suitable for inducing differentiation into hematopoietic cells. How to make hematopoietic cells.
[Aspect 3] In the first step, a gene or protein of Brachyury, PDGFRα, Etv2, or Scl is expressed in a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells. The method for producing hematopoietic cells of a primate animal according to aspect 1 or 2, wherein the cells are cultured until they appear.
[Aspect 4] The method for producing a hematopoietic cell of a primatological animal according to any one of aspects 1 to 3, which comprises a step of genetically modifying pluripotent stem cells before the first step.
[Aspect 5] Preparation of hematopoietic cells of the primatological animal according to any one of aspects 1 to 4, wherein an animal different from the primatological animal is selected from a cow, a pig, a horse, a sheep, or a goat. Method.
[Aspect 6] In any one of aspects 1 to 5, 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 animal different from the primatological animal. The method for producing hematopoietic cells of a primatological animal according to the section.
[Aspect 7] The method for producing a hematopoietic cell of a primate animal according to any one of aspects 1 to 6, wherein the primate animal is a human.
[Aspect 8] The method for producing a hematopoietic cell of a primate animal according to any one of aspects 1 to 7, wherein the pluripotent stem cell is an iPS cell.
[Aspect 9] The primate according to any one of aspects 1 to 8, wherein lymphocytes of an animal different from the chimeric non-human animal are administered to the pups obtained by the fetal birth. How to make animal hematopoietic cells.
[Aspect 10] The primate animal hematopoietic cell according to any one of aspects 1 to 9, further comprising a step of suppressing the proliferation of the fetal endogenous hematopoietic cells prior to the second step. How to make.
[Aspect 11] The primate according to aspect 10, wherein in the step of suppressing the proliferation of endogenous hematopoietic cells of the fetal, a chemotherapeutic agent or an immunosuppressant is administered to the fetal or radiation is applied to the fetal. Method for producing hematopoietic cells of similar animals.
[Aspect 12] The method for producing a primate hematopoietic cell according to aspect 11, wherein busulfan is administered to the fetal in the step of suppressing the proliferation of the endogenous hematopoietic cells of the fetal.
[Aspect 13] A first step of culturing a cell group of pluripotent stem cells of a primate animal under conditions suitable for inducing differentiation into hematopoietic cells to obtain a cell group containing CD34-negative cells.
The second step of 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, and the second step.
The third step of raising the pups obtained by the fetal birth to obtain a chimeric animal that produces the hematopoietic cells of the primatological animal, and the third step.
A method for producing a chimeric non-human animal that produces hematopoietic cells of a primate animal.
[Aspect 14] The method for producing a chimeric non-human animal according to Aspect 13, wherein in the first step, the chimeric non-human animal is cultured under conditions suitable for inducing differentiation into hematopoietic cells for 2 to 12 days.
[Aspect 15] The method for producing a chimeric non-human animal according to aspect 13 or 14, further comprising a step of genetically modifying pluripotent stem cells before the first step.
[Aspect 16] A chimeric non-human animal having somatic cells different from that of the primatological animal, which produces blood cells of the primatological animal.
[Aspect 17] The chimeric non-human animal according to Aspect 16 produced by the method according to any one of Aspects 13 to 15.
[Aspect 18] Hematopoietic cells obtained from the chimeric non-human animal according to aspect 16 or 17.
[Aspect 19] Hematopoiesis in the body of a chimeric non-human animal, which comprises a step of administering lymphocytes of an animal different from the chimeric non-human animal to a chimeric non-human animal having a hematopoietic cell derived from a primate animal. A method for increasing the proportion of hematopoietic cells derived from the primate animal.
20. The method of aspect 19, wherein the lymphocyte is an MHC non-restrictive lymphocyte.
[Aspect 21] The method according to aspect 20, wherein the lymphocyte is an NK cell, a γδ T cell, or an NKT cell.
[Aspect 22] The chimeric non-human animal is obtained by transplanting human, apes or monkey-derived hematopoietic stem cells into a cow, pig, horse, sheep or goat, any one of aspects 19 to 21. The method described in the section.
[Aspect 23] A method for producing a hematopoietic cell, further comprising a step of obtaining a hematopoietic cell from the chimeric animal, as opposed to the method according to any one of aspects 19 to 22. 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 although step 1 (in vitro culture) can differentiate human iPS cells toward hematopoietic stem cells to a certain stage, sufficient differentiation cannot be obtained by step 1 alone.
(1) Preparation of feeder cells A stroma cell line prepared from a neonatal calvaria of a (C57BL / 6 × C3H) F2-op / op mouse, which is a dysfunctional mouse of a macrophage colony stimulating factor, and is a preadipocyte line. OP9 cells in medium for OP9 cells {composition per 1250 ml: α-MEM [Gibco, Catalog No. 12000-022] 980 ml, fetal bovine serum 250 ml (final concentration 20%), penicillin (100 U / ml) ) -Streptomycin (100 μg / ml) 10 ml, 200 mM L-glutamine solution 10 ml}, cultured at _{37 ° C. under 5% CO 2 conditions.}

Then, the cells adhered in the culture medium and grown to the confluent or immediately before the confluent were washed twice with phosphate buffered saline. Then, 0.1% trypsin-EDTA (2 ml / 10 cm dish) was added to the cells on the dish after washing, and the cells were kept warm in an incubator for 5 minutes.

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 recovered 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) under 37 ° C. and 5% CO ₂ conditions. The cells were cultured until they became confluent.

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

The resulting cells, gelatin-coated 6cm culture dish [Falcon (FALCON) Co., Ltd., trade name: Tissue Culture Dish] on, the on OP9 cell medium, 1: 2 (about 1 × 10 ⁵ cells / ml per culture ) Was sown. The OP9 cells prepared as described above were used as feeder cells in the hematopoietic differentiation-inducing culture.
(2) Hematopoietic differentiation culture of iPS cells Human Oct3 / 4, human Sox2, human Klf4 and human c-Myc were added to mononuclear cells collected from adult peripheral blood using a Ficoll-Hypaque (manufactured by GE Healthcare) solution. A human iPS cell was prepared by gene transfer using a Sendai virus vector (manufactured by ID Pharma) equipped with. Human iPS cells are prepared by adding 2 bFGF (manufactured by Wako Pure Chemical Industries, Ltd.) to 500 ml of a medium for primate ES cells [Primate ES Cell Medium (manufactured by Reprocell)) on mouse embryonic fibroblasts treated with mitomycin C. It was maintained in 5.5 μg (final concentration 5 ng / ml) added]. Human iPS cells collected in 1 ml of stripping solution were subjected to a medium for differentiation [composition per 100 ml: IMDM (Iscover's Modified Dulvecco's Medium) 84 ml, 8% horse serum 8 ml, 8% bovine fetal 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)] Suspended in 5 ml. The obtained cell suspension was seeded on the feeder cells in the hematopoietic differentiation-inducing culture. The culture was carried out in an incubator under 37 ° C. and 5% CO _{2 conditions.} The differentiation medium was changed once every two days.

After culturing for 6 days, the old medium was aspirated and removed and the cells were washed with phosphate buffered saline. Then, 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. Then, the bottom of the dish was tapped at room temperature to exfoliate the cell colonies. The obtained cell group was used for transplantation into a fetal sheep.
<Relative expression level analysis of genes>
Using the RNA of hematopoietic differentiated cells prepared as described above as a sample, we attempted to detect the brachyury, Etv2, Gata2, Scl, Runx1A, and Runx1B genes. First, cDNA was synthesized from total RNA using an RNA PCR kit (manufactured by TakaRa) according to the protocol. The sequences of the primers for each gene detection are as follows: Brachyury primer set is 5'-ACAAAGAGAGAGAGGAACCCG-3'and 5'-AGGATAGAGATTTGCAGGTGGACA-3', Etv2 primer set is 5'-CAGAAGGCTTCATCATCGCATCGCATCGCATCGCATCGCATCGCATCG , GATA2 primer set is 5'-AAGGACTTGGAGACTTGGTGGTC-3'and 5'-GGCTATTTCAGAGAGGAAGCCA-3', Scl primer set is 5'-ATGCCTTCCATGTTCACCACCA-3'and 5'-TGGAGATACCGCCGCCACT -CCGAGAACCTCGAAGACATC-3'and 5'-GCTGTGTCTTCTCTCTGCAT-3', the primer set of Runx1b was 5'-CGACTCTCAACGGCACCCGA-3'and 5'-ATGGCCGACATGCCGATAGCC-3'. The PCR amplification conditions after the RT reaction were a combination of 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, human iPS cells first express brachyury required for mesoderm lineage differentiation, then Etv2 required for vascular / blood lineage differentiation, and then a group of genes required for hematopoietic system, that is, 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), and CD45 (pan-blood cell marker) antibody. After adding the antibody solution to the cell suspension and reacting at 4 ° C. for 20-60 minutes, the fluorescently labeled cells were analyzed using a FACS Accuri flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in FIG.

The observed surface markers are PDGFRα (mesoderm marker), CD34 (hematopoietic stem cell marker), CD43 (pancytopenia marker), and CD45 (pancytopenia marker). It is said that PDGFRα, CD34, CD43, and CD45 are expressed in this order in 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 observed. That is, it can be seen that the iPS cells do not differentiate into hematopoietic stem cells in the in vitro culture because some elements necessary for hematopoietic differentiation are lacking in the in vitro culture (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 sequence. So far, the process of development into hematopoietic cells has been faithfully reproduced. However, the pancytopenia markers CD43 and CD45 have not been expressed since then. It is considered that multiple factors are deficient in vitro. Therefore, in Example 2, the cells of Day 6 were transplanted into the liver of an animal fetal. The fetal liver is the original hematopoietic tissue, and it is thought that all the factors necessary for differentiating into hematopoietic cells are present here.

Example 2: By adding step 2 (transplantation into animal fetal liver) in addition to step 1 (in vitro culture), differentiation of human iPS cells into hematopoietic stem cells is induced, and further migration from liver to bone marrow. Indicates that a "movement" will occur.

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

<Analysis of liver and bone marrow of fetal sheep after transplantation of cell group>
The liver of the sheep embryo 1 month after the cell group transplantation and the bone marrow of the sheep embryo 2 months after the cell group transplantation were collected and stained with anti-activated Notch antibody, human CD34 antibody and anti-human CD45 antibody for observation. Fluorescence microscope images are shown in FIGS. 3 and 4.

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

Activation of Notch signaling is considered essential for the maintenance of hematopoietic stem cells, but activation of human Notch was confirmed in the fetal liver of sheep 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 in the fetal liver of sheep 2 months after transplantation, the cells were stained with this antibody. It can be seen that there are cells. That is, it shows that the human Notch signal was activated for the first time in the fetal liver of the sheep.

Engraftment of human CD34-positive cells, engraftment of human CD45-positive cells, and activated human Notch signal were also observed in the fetal bone marrow of sheep 2 months after transplantation. That is, human hematopoietic stem cells migrated from the fetal sheep liver to the bone marrow. It is shown that the migration of hematopoietic tissue from the liver to the bone marrow during this period is a hematopoietic development process characteristic of mammals, and this process faithfully traces it (Figs. 3 and 4).

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

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

Maternal sheep were anesthetized with intravenous xylazine injection (0.03 mg / kg body weight). Sheep, lay on his back on the operating table, after securing the limb was performed surgery under general anesthesia under O _{2 /} air / sevoflurane of spontaneous breathing. The surgery was performed with a clean operation. After laparotomy with a midline incision in the lower abdomen, the uterus was inverted into the abdominal cavity. The injection site of the cells for transplantation was within the hepatic parenchyma of the fetus.

There were two cell transplantation methods: I) uterine incision method and II) ultrasonic guide method. In the case of the uterine incision method, the myometrium of the sheep is incised, the sheep fetus is driven into the exposed egg membrane while being preserved, and the cells for transplantation are used with a 23 G needle in the fetal abdominal cavity under direct vision through the transparent egg membrane. 1 × 10 ⁶ to 1 × 10 ⁸ cells) were punctured and injected, and the myometrium, peritoneal muscle layer, and skin were sutured and closed. ^{In the case of the ultrasonic-guided method, cells (1 × 10 6} to 1 × 10 ⁸ cells) are punctured and injected into the parenchyma of the fetal sheep from above the uterine wall under ultrasonic guidance without incising the uterus. Then, the peritoneal muscle layer and the skin were sutured and closed. After the surgery, antibiotics (mycillin sol) were injected intramuscularly. Then, the extubation was performed, and the awakening of anesthesia of the sheep was confirmed.
<Obtaining a sample>
Peripheral blood and bone marrow were collected from sheep transplanted with human iPS cell-derived cell groups at a pace of about once every two weeks after birth. As a control, peripheral blood and bone marrow were collected from sheep transplanted with a group of cells in the umbilical cord at a pace of about once every two weeks after birth. The collected samples were placed in sterile conical tubes, shaken and stored at room temperature, and cryopreserved within one day.
<Calculation method of human hematopoiesis ratio>
For the colony culture of hematopoietic progenitor cells from the specimen, the above-mentioned methylcellulose medium for human cells, MethoCult ^TM GF ^+, was used. Harvested and isolated bone marrow cells 35mm culture dish (Cat No.1008, FALCON) and cultured at a concentration of 5-10X10 ⁴ in 1ml methylcellulose medium in _{(37 ℃, 5% CO 2} in air). After 10-14 days, the formation of myelocyte / macrophage colonies (CFU-GM), erythroblast colonies (CFU-E) and mixed colonies (CFU-Mix) on the dish was observed. Individual colonies were transferred into 50 μl distilled water (manufactured by Nippon Gene) in a 96-well plate, treated at 99.0 ° C. for 5 minutes and then at 55.0 ° C. for 1 hour at 20 μg / ml Proteinase K (manufactured by Takara). The protein was removed and the DNA was eluted. 5 μl of each sample was used for PCR analysis. For each well of the 96-well plate, nested PCR was performed targeting a human-specific ND5 gene sequence (sheep ND5 cannot be detected) using 250 ng of DNA as a template. The outer primer sets for the ND5 sequence are 5'-ACTGAGCCACAACCCAAACA-3'and 5'-CTGCTCGGGCGTACCATCAA-3', and the inner primer sets are 5'-TTCATCCCTGTTAGCATTGTTCG-3'and 5'-GTTGGAATAGGTGTG. For the PCR reaction, Master Cycler (Eppendorf) was used, and each PCR cycle consisted of three steps of denaturation, annealing, and elongation. 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 was used as a negative control, methyl cellulose and normal sheep blood sample DNA used in colony analysis were used, and human DNA was used as a positive control. The amplified product (135 bp) was mixed with Loading Buffer (manufactured by Wako) at a ratio of 10: 1 and electrophoresed on a 2% agarose gel containing ethidium bromide (manufactured by GIBCO). A 100b Plus DNA Ladder (manufactured by Invtrogen) was used as a molecular weight marker. The gel after electrophoresis was visualized under ultraviolet irradiation. The chimera rate of an individual was calculated by the ratio of the number of detected wells of human ND5 to the number of detected wells of β-actin.
<Result>
Total number of transplanted cells under each condition, number of CD34 positive cells, number of CD34 negative cells, ratio of CD34 negative cells, human hematopoiesis ratio, and number of CD34 positive cells required to obtain 1% human hematopoiesis ratio (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 fetal sheep. As a result, four animals were born, and the human hematopoietic chimera rate was 2.3 to 6.3%. On the other hand, as a positive control, human cord blood CD34 cells were transplanted into 6 sheep fetuses, and the postnatal human hematopoietic chimera rate was 1.1 to 2.3%. Human iPS cells had a higher chimera rate than the original hematopoietic stem cells present in cord blood. However, when the number of transplanted cells is viewed, for example, in terms of the number of CD34-positive cells, the chimera rates cannot be simply compared because they differ 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 have almost the same hematopoietic remodeling ability as cord blood. It was.

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

From this result, the cell group obtained by culturing pluripotent stem cells under conditions suitable for inducing differentiation into hematopoietic stem cells includes hematopoietic stem cells, which are more immature in differentiation stage than hematopoietic stem cells. It is thought that cells that induce differentiation into hematopoietic stem cells are included.

Human hematopoietic cells were detected even 16 months after transplantation in the sheep transplanted with the cell group obtained by culturing pluripotent stem cells under conditions suitable for inducing differentiation into blood lineage cells.
From the following points, it is considered that human hematopoiesis in the sheep body can be reproduced with extremely high efficiency, which is comparable to the original hematopoietic stem cells called human umbilical cord blood stem cells.
(1) Culture method We devised a culture method in which 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 pancytopenia markers CD43 and CD45 should appear. The cells were transplanted on the 6th day of culture. If the pancytopenia markers CD43 and CD45 are delayed from the point where they should appear, such as after the 12th day of culture, the timing of hematopoietic differentiation is considered to be missed. 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), but the timing of transplantation is 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 the CD34-positive fraction, and in fact, CD34-positive cell transplantation is also performed clinically. However, similarly in mice, hematopoietic stem cells are present in the CD34 negative fraction. This will be demonstrated in the next example.
(3) Pretreatment of fetuses One of the reasons is that human iPS-derived cells were transplanted after administration of busulfan as a pretreatment.
(4) Transplantation site The cells were transplanted into the fetal liver, that is, into the hematopoietic tissue.

Example 4: Transplantation of only human iPS cell-derived CD34-negative cells Conventionally, mouse hematopoietic stem cells were said to be CD34-negative, while human hematopoietic stem cells were said to be CD34-positive. In this example, we show that hematopoietic stem cells are present in both humans and the CD34 negative fraction.
<Transplantation of cells into fetal sheep and sampling from sheep>
<Removal of CD34-positive cells in the cell group obtained in <iPS cell hematopoietic differentiation culture> of Example 1>
Using a magnetic bead reagent for cell separation and a purified column (magnetic bead-binding monoclonal antibody against CD34 (manufactured by Milteny Biotech) and a purified column (manufactured by Milteny Biotech), <iPS cell hematopoietic differentiation culture of Example 3 >, The cell surface CD34-expressing cells were removed from the cell group obtained in.

The cell group thus obtained was analyzed by FACS. The results are shown in the left figure of FIG. On FACS, 0.0% of CD34-positive cells in the cell group. From this, it can be seen that the obtained cell group contained only CD34-negative cells.

The cell group containing only the CD34-negative cells was transplanted into the fetal sheep in the same manner as in Example 3, and the human hematopoietic ratio was analyzed in the same manner as in Example 3. The table below 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 required 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 hematopoietic cells includes cells whose differentiation stage is more immature than hematopoietic cells. It is thought that it is.

Among the cells in the cell group obtained by culturing pluripotent stem cells under conditions suitable for inducing differentiation into hematopoietic cells, sheep transplanted with a cell group containing only a 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 a sheep transplanted with a group of cord blood-derived cells In this example, it is shown that human hematopoiesis in a sheep is enhanced when lymphocytes are transplanted into a sheep.
<Preparation of lymphocytes in the umbilical cord>
When transplanting umbilical cord blood stem cells, lymphocytes are usually removed as unnecessary components, but in this 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 a lymphocyte culture medium supplemented with CD3 / 28 magnetic beads (Dinal) (RPMI-added with 30 U / ml of human recombinant IL-2). After culturing in 1640 (manufactured by Invitrogen) for 14 days, the obtained cell group was used for injection into postnatal sheep. The lymphocytes used for injection include NK cells, γδ T cells and NKT cells.
<Effect of infusion of human lymphocytes into sheep transplanted with cord blood-derived cells>
After birth, sheep transplanted with umbilical cord-derived cells ^{were intravenously administered with 4.8 × 10 7} / kg or 10.2 × 10 ⁷ / kg of human lymphocytes. Then, peripheral blood and bone marrow were collected from this sheep at a pace of once every two weeks. The human hematopoiesis ratio (human CFU rate (%)) was examined for the collected samples. The results are shown in FIG. 6 and Table 5.

Eight weeks after administration, the human hematopoietic ratio increased. Since it took as long as 8 weeks from the administration of human lymphocytes to increase the human hematopoietic ratio, it is considered that human hematopoietic stem cells increased as a result of the influence of lymphocytes on the immunological mechanism. It was.

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

［配列表］

[Sequence list]

Claims (19)

A cell group of pluripotent stem cells of a primate animal is cultured under conditions suitable for inducing differentiation into hematopoietic cells and expressing CD34, and CD34-positive cells are removed to obtain a cell group containing only CD34-negative cells. First step and
The second step of 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, and the second step.
The third step of obtaining the hematopoietic cells of the primate from the body of the animal obtained by raising the pups obtained by the fetal birth, and
Have,
Here, the primate animal is a human.
How to make hematopoietic cells in primates. The hematopoietic system of a primate animal according to claim 1, wherein in the first step, the cell group of pluripotent stem cells of the primate animal is cultured for 2 to 12 days under conditions suitable for inducing differentiation into hematopoietic cells. How to make cells. In the first step, cells expressing the Brachyury, PDGFRα, Etv2, or Scl gene or protein appear under conditions suitable for inducing differentiation of pluripotent stem cells of primate animals into hematopoietic cells. The method for producing hematopoietic cells of a primate animal according to claim 1 or 2, which is cultured until the cells are grown. The method for producing a hematopoietic cell of a primatological animal according to any one of claims 1 to 3, which comprises a step of genetically modifying pluripotent stem cells before the first step. The method for producing hematopoietic cells of a primate animal according to any one of claims 1 to 4, wherein an animal different from the primate animal is selected from a cow, a pig, a horse, a sheep, or a goat. The invention according to any one of claims 1 to 5, 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 animal different from the primatological animal. How to make hematopoietic cells in primatological animals. The method for producing a hematopoietic cell of a primate animal according to any one of claims 1 to 6, wherein the pluripotent stem cell is an iPS cell. The hematopoiesis of a primate animal according to any one of claims 1 to 7, wherein lymphocytes of an animal different from the chimeric non-human animal are administered to the pups obtained by the fetal birth. Method for producing lineage cells. The method for producing a hematopoietic cell of a primate animal according to any one of claims 1 to 8, further comprising a step of suppressing the proliferation of the endogenous hematopoietic cell of the fetus before the second step. .. The primate animal according to claim 9, wherein in the step of suppressing the proliferation of endogenous hematopoietic cells of the fetus, a chemotherapeutic agent or an immunosuppressive agent is administered to the fetus, or radiation is applied to the fetus. Method for producing hematopoietic cells. The method for producing hematopoietic cells of a primate animal according to claim 10, wherein busulfan is administered to the fetus in the step of suppressing the proliferation of the endogenous hematopoietic cells of the fetus. A cell group of pluripotent stem cells of a primate animal is cultured under conditions suitable for inducing differentiation into hematopoietic cells and expressing CD34, and CD34-positive cells are removed to obtain a cell group containing only CD34-negative cells. First step and
The second step of 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, and the second step.
The third step of raising the pups obtained by the fetal birth to obtain a chimeric animal that produces the hematopoietic cells of the primatological animal, and the third step.
Have,
Here, the primate animal is a human.
A method for producing a chimeric non-human animal that produces hematopoietic cells of a primate animal. The method for producing a chimeric non-human animal according to claim 12, wherein in the first step, the chimeric non-human animal is cultured under conditions suitable for inducing differentiation into hematopoietic cells for 2 to 12 days. The method for producing a chimeric non-human animal according to claim 12 or 13, which comprises a step of genetically modifying pluripotent stem cells before the first step. Hematopoietic cells in the body of a chimeric non-human animal, which comprises a step of administering lymphocytes of a primate animal different from the chimeric non-human animal to a chimeric non-human animal having a hematopoietic cell derived from a primate animal. A method for increasing the proportion of hematopoietic cells derived from the primate animal.
15. The method of claim 15, wherein the lymphocyte is an MHC non-restrictive lymphocyte. 16. The method of claim 16, wherein the lymphocytes are NK cells, γδT cells, or NKT cells. The method according to any one of claims 15 to 17, wherein the chimeric non-human animal is obtained by transplanting human-derived hematopoietic stem cells into a cow, pig, horse, sheep or goat. A method for producing hematopoietic cells, further comprising a step of obtaining hematopoietic cells from the chimeric animal, as opposed to the method according to any one of claims 15 to 18.

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