JP2001275679A - Hematopoietic stem cell-growing agent containing macrophage migration-inhibitory factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hematopoietic stem cell-growing agent capable of efficiently growing a human hematopoietic stem cell and a hematopoietic precursor cell in vitro, more than in the past, and is obtain the hematopoietic stem cell- growing agent capable of being utilized as a therapeutic agent for dyshemopoiesis in the radiotherapy and chemotherapy for various hematopoietic organ diseases or cancer, capable of being utilized in the mass production of hemocyte, in the improvement in the transduction efficiency of a gene into the hematopoietic cell on a gene therapy and further being effectively utilized as a diagnostic and an examination drug. SOLUTION: This hematopoietic stem cell-growing agent is obtained by including a macrophage migration-inhibitory factor. The macrophage migration- inhibitory factor may comprise a polypeptide containing either of the amino acid sequence represented by the sequence number 1 to 2 (see specification), a peptide which contains an amino acid sequence obtained by substituting, deleting or adding one or several numbers of amino acids to either of the amino acid sequence represented by the sequence number 1 or 2 and exhibits hematopoietic stem cell growth activity of their modified substances.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a hematopoietic stem cell growth factor having a macrophage migration inhibitory factor as a hematopoietic stem cell growth factor having hematopoietic stem cell survival activity, proliferation activity, and differentiation activity. And a hematopoietic stem cell proliferating agent that can be used as a therapeutic agent for hematopoietic failure during chemotherapy. The hematopoietic stem cell proliferating agent of the present invention can also be used for mass production of blood cells and for improving the efficiency of introducing genes into hematopoietic stem cells during gene therapy, and can also be used as diagnostics and test agents.

[0002]

2. Description of the Related Art Hematopoietic stem cells have the ability to differentiate into all mature blood cells (pluripotency) and the ability to replicate cells having the same ability as self (self-renewal ability), and support hematopoiesis for a long time. Continue to do. Hematopoiesis in human organisms is generally considered to be regulated by direct interactions between cells and various substances present in body fluids (ie, humoral hematopoietic regulators). Direct interactions between cells include interactions between hematopoietic stem cells, hematopoietic progenitor cells committed to differentiate into specific blood cells, and stromal cells, the hematopoietic microenvironment surrounding them.

[0003] A variety of humoral hematopoietic regulators have been found in the last decade or so due to advances in biotechnology. For example, erythropoietin (Lin et al., Proc. N
atl. Acad. Sci. U. S. A. , 82:75
80 (1985)), granulocyte colony stimulating factor (Nagata et al., EMBO J, 5: 575 (1986)), granulocyte macrophage colony stimulating factor (Miyatake et al., EMB)
OJ, 4: 2561 (1985)), macrophage colony stimulating factor (Wong et al., Science, 23
5: 1504 (1987)), thrombopoietin (Sauvage et al., Nature, 369: 533 (199).
4)), stem cell growth factor (Anderson et al., Cell,
63: 235 (1990)), Fms-like tyrosine kinase 3 ligand (Limanet et al., Cell, 75: 115).
7 (1993)), a leukemia inhibitory factor (Stal et al., J. Am.
Biol. Chem. , 265, (15): 8833
(1990)), interleukin 1 (Clark et al., N
ucleic Acids Res. , 14,: 789
7 (1986)), interleukin 3 (Dolthers et al., Gene, 55: 115 (1987)), interleukin 6 (Yaskawa et al., EMBO J, 6: 2939).
(1987)), Interleukin 11 (Paul et al., P.
rc. Natl. Acad. Sci. U. S. A. ,
87: 7512 (1990)), interleukin 12
(Wolf et al., J. Immunol. 146: 3074.)
(1991)). None of these cytokines can proliferate hematopoietic stem cells alone by themselves. To proliferate to some extent, it is necessary to use two or more, preferably three or more in combination. Therefore, it is desired to provide a hematopoietic growth factor capable of proliferating hematopoietic stem cells more efficiently.

The macrophage migration inhibitory factor is a lymphokine first discovered as a protein that inhibits the migration of guinea pig macrophages (Bloom et al., Science, 153: 80 (1966); David et al., Proc. Natl. Acad.Sci.
U. S. A. 56:72 (1966)). Expression of macrophage migration inhibitory factor activity is associated with delayed-type hypersensitivity and cell immunity in animal models and humans (Bloom et al., Science, 153: 80 (196)
6); David et al., Proc. Natl. Acad.
Sci. U. S. A. 56:72 (1966); David et al., Prog. Allergy, 16: 300
(1972); Rocklin et al. Engl. J. Me
d. 282: 1340 (1970)). In addition, macrophage migration inhibitory factor activity is detected in leukocyte culture supernatants upon rejection of murine allografts (Aluascari et al., Nature, 205: 916 (1965);
Harrington et al., Cell. Immunol. , 30: 2
61 (1977)) and in the synovial fluid of patients with rheumatoid polyarthritis (Odink et al., Nature, 330: 80 (1).
987)) It has also been found in various sites of chronic inflammation (Blumeister et al., Lymphokine Re).
s. 3: 236 (1984)). The expression of the macrophage migration inhibitory factor at the site of inflammation suggests that the macrophage migration inhibitory factor contributes as a mediator that regulates the macrophage function in host defense.

[0005] Human macrophage migration inhibitory factor is 19
Cloned in 1989 (Weiser et al., Proc.
Natl. Acad. Sci. U. S. A. 86: 7
522 (1989)). The mouse macrophage migration inhibitory factor was also cloned (Bernhagen et al., Nature, 365: 756 (1993)).

[0006] Thereafter, various properties of macrophage migration inhibitory factor have been elucidated. For example, macrophage migration inhibitory factor is a major component of secretory granules in adrenocorticotropic hormone-producing cells of the anterior pituitary gland. Also,
Macrophage migration inhibitory factors are thought to play a central role in the host response to endotoxin shock (Bernhagen et al., Natur.
e, 365: 756 (1993)). In addition, the macrophage migration inhibitory factor has a post-translational glycosylation inhibitory activity of an immunoglobulin E binding factor, and is referred to as a "glycosylation inhibitory factor" (Mikayama et al., Proc. N).
atl. Acad. Sci. U. S. A. , 90:10
056 (1993)). Furthermore, the expression of macrophage migration inhibitory factor mRNA has been observed in fetal eye lenses and fibroblasts activated by growth factors (Wiistow et al., Proc. Natl. Acad. S.).
ci. U. S. A. 90: 1272 (1993); Lanahan et al., Mol. Cell. Biol. , 12:39
19 (1992)). Macrophage migration inhibitory factor is also expressed in human melanoma cells and is involved in its tumor cell growth and angiogenesis (Shimizu et al., Bioc).
hem. Biophys. Res. Com. , 264:
751 (1999)). However, there has been no report on the physiological effect of macrophage migration inhibitory factor on stem cells.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a hematopoietic stem cell proliferating agent capable of proliferating human hematopoietic stem cells and hematopoietic progenitor cells outside the body more efficiently than before. A further object of the present invention is that it can be used as a therapeutic agent for various hematopoietic organ diseases, hematopoietic failure during radiotherapy and chemotherapy for cancer, and can be used for mass production of blood cells,
An object of the present invention is to provide a hematopoietic stem cell proliferating agent that can be used for improving the efficiency of introducing genes into hematopoietic stem cells during gene therapy and can be further used as a diagnostic agent and a test agent.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, activated the proliferation of hematopoietic stem cells in the culture supernatant of mouse myeloma cells (NS-1 cells). It was found that the protein was present. We have:
By separating, purifying, and identifying this protein,
The present inventors have clarified that this protein is a macrophage migration inhibitory factor, which is a novel hematopoietic stem cell growth factor, and completed the present invention by utilizing their hematopoietic stem cell growth activity.

[0009] The hematopoietic stem cell proliferating agent of the present invention contains a macrophage migration inhibitory factor. In one embodiment, the macrophage migration inhibitory factor is a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, wherein one or several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 1. It may be a polypeptide having an amino acid sequence and having hematopoietic stem cell proliferation activity, or a modified form thereof.

[0010] In one embodiment, the macrophage migration inhibitory factor is a polypeptide having the amino acid sequence represented by SEQ ID NO: 2, wherein one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 are substituted, deleted or deleted. It may be a polypeptide having an added amino acid sequence and having hematopoietic stem cell proliferation activity, or a modified form thereof.

[0011] The method for expanding hematopoietic stem cells or hematopoietic progenitor cells of the present invention includes a step of expanding hematopoietic stem cells or hematopoietic progenitor cells in a non-human animal or in vitro using any of the above-mentioned agents for expanding hematopoietic stem cells.

In one embodiment, a foreign gene may be introduced into hematopoietic stem cells or hematopoietic progenitor cells in the above-mentioned expansion step.

The method for expanding human tissue stem cells or tissue progenitor cells of the present invention comprises the step of expanding human tissue stem cells or tissue progenitor cells in a non-human animal or in vitro using any one of the above-mentioned agents for expanding hematopoietic stem cells. .

The diagnostic or test agent for a blood system disease of the present invention includes any of the above-mentioned hematopoietic stem cell proliferating agents.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the practice of the present invention, unless otherwise indicated, protein separation and analysis methods and assay methods known in the art are employed. I. Definitions Terms used for describing the present invention will be described below. The macrophage migration inhibitory factor, as described above, is a lymphokine first discovered as a protein that inhibits the migration of guinea pig macrophages.
In the present invention, the term "macrophage migration inhibitory factor"
Is not limited to polypeptides of the so-called macrophage migration inhibitory factor, but also includes polypeptides having substantial homology in amino acid sequence to this polypeptide, and modified versions of any of these polypeptides . Examples of homologous polypeptides include species variants and allelic variants.

The human macrophage migration inhibitory factor contains the amino acid sequence of SEQ ID NO: 1 in the sequence listing. The mouse macrophage migration inhibitory factor includes the amino acid sequence of SEQ ID NO: 2 in the sequence listing.

Obviously, human-derived polypeptides are preferred for the purpose of human disease or treatment and in the expansion of human hematopoietic stem cells. However, homologous polypeptides from other mammals can also be used depending on the purpose. Furthermore, comparison with a polypeptide derived from another mammal is important in obtaining a variant in which the desired activity of a polypeptide derived from human is retained.

The macrophage migration inhibitory factor used in the present invention is not necessarily limited by the above-mentioned specific sequences, and one or several amino acids are deleted, substituted or added to these sequences. A homologous polypeptide having an amino acid sequence and retaining a desired activity (ie, a homolog of a macrophage migration inhibitory factor) is also included as a subject. Here, the mutation of up to "several" amino acids is not necessarily intended to be limited to a specific upper limit as long as a desired activity is obtained, but typically is not more than about 60 amino acids, preferably about It may range up to 40, more preferably up to about 20. Examples of amino acid additions include the addition of one amino-terminal Met residue.

Conservative substitution of amino acids is one of the preferred means for obtaining homologous polypeptides. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,
Glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Sequence identity (homology) between two amino acid sequences is determined by optimizing residue matches, introducing gaps where necessary. Polypeptides having substantial amino acid sequence homology to human macrophage migration inhibitory factor typically have at least about 60%, preferably at least about 70%, as compared to the amino acid sequence of human macrophage migration inhibitory factor. More preferably, it can be expressed as a polypeptide having at least about 80%, even more preferably at least about 90% or more homology. Software for homology determination is readily available and can be found, for example, in Gene Works (Inte
lligenetics Inc. ) Etc. are available.

The modified form of the macrophage migration inhibitory factor used in the present invention is a polypeptide having a sequence identical or homologous to the above-mentioned sequence, wherein the amino acid side chain, amino terminal or carboxyl terminal is modified, and Polypeptides that retain the desired activity are included. The homolog and the modified form of the macrophage migration inhibitory factor are collectively referred to as a functional equivalent. Examples of the above modifications include polypeptides in which the amino terminus is acylated (eg, acetylated), and polypeptides in which the carboxy terminus is amidated or esterified.

In the present invention, "having hematopoietic stem cell proliferating activity" means, by definition, substantially the same conditions as in Example 16 below (the concentration of macrophage migration inhibitory factor is 20%).
ng / ml), the CD34 antigen is positive and the CD38 antigen is negative (hereinafter CD34 + CD3
This means that the cell number is larger than the cell number shown in the control of Example 16, typically about 110% or more, preferably about 200% or more.

"Hematopoietic stem cells" are pluripotent cells capable of differentiating not only into bone marrow cells such as erythrocytes, leukocytes and megakaryocytes, but also into all blood systems including lymphoid systems such as T cells and B cells. A cell that is capable of self-proliferation. Hematopoietic stem cells are characterized by being positive for the CD34 antigen and negative for the CD38 antigen (CD34 + CD38-). Even more immature hematopoietic stem cells are CD34 antigen positive and CD38, CD33, CD19,
And CD2 antigen are negative (CD34 + CD38−
33-19-2-). "Hematopoietic progenitor cells" refer to cells that have been determined to differentiate into a particular cell lineage of the blood system but are capable of self-proliferation by division. Hematopoietic progenitor cells are characterized by both CD34 and CD38 antigens being positive (CD34 + CD38 +).

"Tissue stem cells" refer to cells that have the ability to self-renew with division and have the ability to generate cells having different properties at the same time, and include hematopoietic stem cells, stem cells of the epidermal basement membrane, and stem cells of the small intestinal mucosal epithelium. And all types of stem cells, including nervous system stem cells. “Tissue progenitor cells” refer to cells that have not yet differentiated and matured into a particular tissue. II. Macrophage migration inhibitory factor having hematopoietic stem cell proliferation activity In the present invention, macrophage migration inhibitory factor is used as an active ingredient of a hematopoietic stem cell proliferating agent. Macrophage migration inhibitory factors can be isolated from natural sources, produced using recombinant DNA technology, or chemically synthesized.

When the macrophage migration inhibitory factor is isolated from a natural source, it can be purified, for example, as follows. First, myeloma cells (for example, P3-NSI / 1-
Ag4-1 (abbreviation: NS-1) cell) is cultured, and a hematopoietic stem cell proliferating active protein is separated and purified from the obtained culture supernatant. "Myeloma cells" are cancerous cell lines that have the ability to grow permanently. Myeloma cells are known for a variety of uses, for example, Koehler and Milstein describe the myeloma cell P3-X63-
Ag8 (abbreviated X63) cells were fused with lymphocytes from mouse spleen sensitized with sheep red blood cells to produce B cell hybridomas (Kohler et al., Nature,
256: 495 (1975)). X63 cells are BAL
A cell obtained by selecting a cell population that is 8-azaguanine resistant and cannot grow in a medium containing hypoxanthine, aminopterin and thymidine by cloning from immunoglobulin G1 kappa chain-producing myeloma cells derived from B / c mice. Is a stock. NS-1 cells are myeloma cells derived from X63 cells and are known to synthesize only immunoglobulin G1 kappa chains but not to secrete them (Kohler et al., Eur. J. Immunology, 6: 511).
(1976)).

The present inventors have surprisingly found that the NS-1 cell culture supernatant has a hematopoietic stem cell proliferating activity. Therefore, the hematopoietic stem cell growth-activating factor in the present invention can be isolated from the culture supernatant of NS-1 cells or the culture supernatant of other myeloma cells showing similar activity. The separation and purification of the hematopoietic stem cell proliferating activity protein found in the myeloma cell culture supernatant is started by preparing a large amount of a myeloma cell culture supernatant exhibiting hematopoietic stem cell proliferating activity. The culture of the cells may be performed according to the usual cell culture method. For example, in an animal cell culture medium containing an appropriate ratio of fetal calf serum (FCS), C
Incubate in O ₂ incubator.

In order to separate and purify proteins from the culture supernatant, it is necessary to reduce contaminating proteins as much as possible. For this purpose, it is preferable to replace the medium with a serum-free medium immediately before the content of the hematopoietic stem cell growth-active protein increases most, continue the culture, and then collect the culture supernatant. The collected culture supernatant can be passed through a filter to remove cell debris.

In order to separate and purify the hematopoietic stem cell proliferating protein from the obtained culture supernatant, an ordinary protein separation / purification method can be used. An ultrafiltration membrane may be used to concentrate the culture supernatant. The concentrated culture supernatant is applied to a gel filtration column using HPLC, and a fraction having hematopoietic stem cell proliferation activity is collected.
The obtained active fraction is applied to a reverse phase HPLC column. From the eluted fraction, a fraction having hematopoietic stem cell proliferation activity is collected. The fraction thus obtained has sufficient purity and can be subjected to amino acid sequence analysis using a protein sequencer without further purification. Thus, hematopoietic stem cell proliferating protein found in the myeloma cell culture supernatant can be identified. III. Confirmation of Hematopoietic Stem Cell Proliferation Activity Next, an example of a method for confirming the hematopoietic stem cell proliferation activity of the macrophage migration inhibitory factor will be described. Hematopoietic stem cells prepared from cord blood (CD34 + CD38-33-19-2-cells)
Are plated on microtiter plates.
As a medium used for the proliferation of hematopoietic stem cells, in the test group, a medium (stem cell growth factor / interleukin 6 / An interleukin 3 medium) is prepared, and a medium containing a sample containing a polypeptide of interest (eg, human macrophage migration inhibitory factor) to be assayed for hematopoietic stem cell proliferation activity is used. In the control group, a medium obtained by adding 2% FCS-RPMI1640 to a stem cell growth factor / interleukin 3 medium is used instead of the sample containing the target polypeptide.
After culturing them at about 37 ° C. for about 10 to 30 days, the cells are treated with a CD34-FITC antibody (a fluorescein isothiocyanate dye is bound to the CD34 antibody), and the cells are taken up by FACScan for a certain period of time. The ratio of CD34 + cells is determined, the viable cells are measured by the trypan blue method, and the number of viable cells is multiplied by the ratio of CD34 + cells to calculate the number of CD34 + cells. In this way, hematopoietic stem cell proliferation activity of the target polypeptide can be confirmed. IV. Hematopoietic stem cell proliferating agent and use thereof The hematopoietic stem cell proliferating agent of the present invention is provided as a composition containing a macrophage migration inhibitory factor having hematopoietic stem cell proliferating activity and any medium that does not substantially inhibit the activity. Here, “macrophage migration inhibitory factor” is used in a broad sense, and in addition to normal macrophage migration inhibitory factor,
It is meant to include homologs and modifications of the macrophage migration inhibitory factors described above. Hematopoietic stem cell proliferating agents for administration to humans will typically contain, in addition to an effective amount of a macrophage migration inhibitory factor, any pharmaceutically acceptable excipient known to those of skill in the art. Examples of excipients include lactose,
Corn starch, magnesium stearate, alum and the like.

[0029] The hematopoietic stem cell proliferating agent of the present invention can be prepared according to a method known in the art. The hematopoietic stem cell proliferating agent of the present invention may be in any form. The hematopoietic stem cell proliferating agent of the present invention can be a solid, such as a tablet, pill, capsule, granule; or a liquid, such as an aqueous solution and suspension. When the hematopoietic stem cell proliferating agent of the present invention is orally administered as a tablet, usually, excipients such as lactose, corn starch, and magnesium stearate can be used. When the hematopoietic stem cell proliferating agent of the present invention is orally administered as a capsule, usually, excipients such as lactose and dried corn starch can be used. For oral administration in an aqueous suspension, the macrophage migration inhibitory factor may be used in combination with an emulsion or suspension. Aqueous suspensions may optionally contain sweetening and flavoring agents. When the hematopoietic stem cell proliferating agent of the present invention is injected intramuscularly, intraperitoneally, subcutaneously, and intravenously, a macrophage migration inhibitory factor is dissolved in a sterilized solution to prepare a buffer, and the pH is adjusted to an appropriate value. . When the hematopoietic stem cell proliferating agent of the present invention is administered intravenously, the proliferating agent is preferably isotonic.

By using the hematopoietic stem cell proliferating agent of the present invention, a large amount of tissue stem cells or tissue progenitor cells including hematopoietic stem cells or hematopoietic progenitor cells can be proliferated. For example, a hematopoietic stem cell or hematopoietic progenitor cell can be proliferated in vitro using the hematopoietic stem cell proliferating agent of the present invention to produce large quantities of blood cells. The effective amount of the hematopoietic stem cell proliferating agent and the conditions for producing and collecting blood cells can be appropriately selected by those skilled in the art.

Furthermore, the hematopoietic stem cell proliferating agent of the present invention can be used for treating various hematopoietic organ diseases such as immunosuppressive disorders, and patients in whom an increase in blood cells is desired, such as hematopoietic failure due to cancer radiotherapy and chemotherapy. Can be used.
In therapeutic applications, the hematopoietic stem cell proliferating agent of the present invention comprises Re
minton's PharmaceuticalS
citenses, Mack Publishing (Easton, Pa.) in the form of conventional polypeptide formulations. For example, the hematopoietic stem cell proliferating agent of the present invention can be administered by parenteral administration such as oral administration, intravenous administration, intramuscular injection, intraperitoneal injection, and subcutaneous injection. It is also possible to replenish these polypeptides into amniotic fluid. Preferably, these polypeptides can be administered by injection.

When the hematopoietic stem cell proliferating agent of the present invention is administered to humans, the daily dose is usually determined in consideration of the patient's symptoms, severity, individual differences in sensitivity, body weight, age and the like. It can be determined appropriately by the trader. The hematopoietic stem cell proliferating agent of the present invention may be administered once a day, or may be administered several times a day.

In gene therapy, it is known that the efficiency of introducing a target gene depends on how many cells are dividing in the process of introducing the gene into cells. Therefore, a very high gene transfer efficiency can be obtained by introducing a gene while proliferating hematopoietic stem cells or hematopoietic progenitor cells using the hematopoietic stem cell proliferating agent of the present invention.

Further, the hematopoietic stem cell proliferating agent of the present invention can be used as it is as a diagnostic or test agent for blood system diseases (eg, leukemia, myeloma, anemia, etc.).

[0035]

EXAMPLES Hereinafter, the macrophage migration inhibitory factor as a hematopoietic stem cell proliferating agent of the present invention will be described more specifically.

Example 1 Preparation of Hematopoietic Stem Cells from Human Umbilical Cord Blood Ficoll from human umbilical cord blood (approximately 50 ml using heparin as an anticoagulant) provided by gynecology and informed consent A mononuclear cell fraction was prepared by a method (using Amersham Pharmacia Biotech Co., Ltd., Ficoll Pack) and stored frozen. This was freeze-thawed at the start of the assay and then 15% FCS-RPMI
The cells were washed with 1640 (manufactured by Niken Biomedical Research Institute Co., Ltd.), and the cells were treated with a CD34-FITC antibody (a fluorescin isothiacinate dye bound to the CD34 antibody),
CD38-PE antibody (CD38 antibody with phycoerythrin dye attached), CD33-PE antibody (CD33
Phycoerythrin dye bound to antibody), CD1
Each of the 9-PE antibody (CD19 antibody with phycoerythrin dye bound thereto) and CD2-PE antibody (CD2 antibody with phycoerythrin dye bound) was treated with each antibody (manufactured by Immunotech Co.), and FACStar was performed. (Manufactured by Becton Dickinson) by using hematopoietic stem cells CD34 + C
D38-33-19-2-cells were sorted.

Example 2 Construction of Hematopoietic Stem Cell Growth Factor Detection System Using Human Cord Blood Hematopoietic Stem Cells CD34 + CD38-33-19-2 of human cord blood collected in Example 1
-Cells were plated on 96-well U-type plates (Coaster) (50 cells / 100 [mu] l / well). The medium used was 15% FCS-RPMI1640.
In addition, stem cell growth factor (25 ng / m
l), interleukin 6 (20 ng / ml), and interleukin 3 (20 ng / ml), and 20 μl (/ well) of a sample to be measured for hematopoietic stem cell proliferation activity was added. 37
After liquid culture at 14 ° C for 14 to 27 days, the number of viable cells was measured by the trypan blue method (trypan blue was manufactured by Dainippon Pharmaceutical Co., Ltd.). Cell counting was performed using a hemocytometer. CD34 +
The number of cells and the number of CD34 + CD38− cells were calculated as follows. First, cells after liquid culture were transferred to CD34-F
ITC antibody (manufactured by Immunotec) and CD38-PE
Staining was performed with an antibody (manufactured by Immunotech) (50 ng of antibody to 10 ⁵ cells). Stained cells were washed with PBS
(-) (Phosphate-buffered sa
line, pH 7.2; washed twice with Niken Biomedical Research Institute, Inc. (containing 1% FCS and 0.01% sodium azide), and then propidium iodide (Sigma-Aldrich Japan Co., Ltd.) Concentration 10 μg /
The cells were stained by adding to a volume of ml. The stained cells are analyzed (flow cytometry) with a flow cytometer FACScan (manufactured by Becton Dickinson) to determine the CD34 + cell ratio and the CD34 + CD38− cell ratio, and the CD34 + cell count is calculated by multiplying the viable cell count by the ratio. And the number of CD34 + CD38- cells was calculated.

Example 3 Mouse Myeloma Cell Culture and Preparation of Culture Supernatant Mouse Myeloma Cell (NS
-1) Culture was performed using 15 ml of 15% FCS-RPMI1640 medium so that the concentration of cells was 1.0 × 10 ⁵ / ml.
Suspended in a culture dish (Greiner)
(Diameter 10 cm), and culture was started at 37 ° C. in a 5% CO ₂ incubator. Culture 0, 1, 2, 3,
On days 4, 5, 6, 7 and 8, culture supernatants were collected and
The supernatant passed through a 22 μm filter was collected.

Example 4 Preparation of NS-1 Cell Proliferation Curve NS-1 Cell Culture 0, 1, 2, 3, 4, 5, 6, 7
On the 8th and 8th days, the number of living cells and the number of dead cells were measured by the trypan blue method. As a result of the measurement, the live cell concentrations were 1 × 10 ⁵ cells / ml, 4 × 10 ⁵ cells / ml, and 10 × 1
0 ⁵ pieces / ml, 17 × 10 ⁵ pieces / ml, 18 × 10 ⁵ pieces / ml
ml, 14 × 10 ⁵ / ml, 10 × 10 ⁵ / ml, 5
× 10 ⁵ / ml, 2 × 10 ⁵ / ml. Dead cell concentration, 0.05 × 10 ⁵ cells /Ml,0.1×10 ⁵ pieces /
ml, 0.5 × 10 ⁵ / ml, 1 × 10 ⁵ / ml, 4
× 10 ⁵ / ml, 10 × 10 ⁵ / ml, 13 × 10 ⁵
Pieces / ml, 17 × 10 ⁵ pieces / ml, 20 × 10 ⁵ pieces / ml
Met. As a result, it was found that the number of viable cells was maximized on the fourth day of culture.

Example 5 Determination of NS-1 Cell Culture Supernatant Recovery Time NS-1 cells were cultured and cultured at 0, 1, 2, and 3.
The culture supernatant collected on days 3, 4, 5, 6, 7, and 8 was evaluated using the hematopoietic stem cell growth factor detection system using the human cord blood hematopoietic stem cells constructed in Example 2. 20 μl of each culture supernatant was added to a 96-well U-type well. C
The number of D34 + cells was 362, 550, 153, 115 and 93 on days 4, 5, 6, 7 and 8 of culture, respectively. As a result, it was found that the supernatant on the fifth day of culture had the most hematopoietic stem cell proliferation activity.

(Example 6: Examination of concentration dependency of hematopoietic stem cell proliferation activity of NS-1 cell culture supernatant) Example 3
The concentration dependence of the hematopoietic stem cell proliferation activity of the NS-1 cell culture supernatant collected in the above was examined. Culture was performed using a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells.
The NS-1 cell culture supernatant on the day was diluted 2 times, 4 times, 8 times, 16 times, 32 times, 6 times
Hematopoietic stem cell proliferation activity was evaluated by adding 4-fold dilution, 128-fold dilution and 256-fold dilution. The number of CD34 + cells was 238, 144, 74, 104, 4
5, 1, 0, 11, and 10. CD
The number of 34 + CD38− cells was 107, 36, respectively.
, 44, 37, 17, 1, 1, 0, 2, and 1. Thus, the proliferation of hematopoietic stem cells is NS-1
It was almost dependent on the concentration of the cell culture supernatant.

(Example 7: Large-scale preparation of NS-1 cell culture supernatant) NS-1 cells were cultured in a 50% 15% FCS-RPMI1640 medium at a concentration of 1.0 × 10 ⁵ / ml.
l and suspended in a culture flask (Greiner, 1
50 cm ² ). In a 5% CO ₂ incubator,
The culture was started at 37 ° C., and the culture supernatant 5 days after the culture was collected, and the supernatant passed through a 0.22 μm filter (manufactured by Nippon Millipore) was collected. A total of 3,620 ml of the culture supernatant was collected.

Example 8 Purification of NS-1 Cell Culture Supernatant by Gel Filtration 15 ml of the NS-1 cell culture supernatant collected in Example 7 was subjected to an ultrafiltration filter having a molecular weight cut off of 10,000 (German filter). After concentrating the solution to 1 ml with a Sephacryl S-200 column (manufactured by Amersham Pharmacia Biotech) (diameter 10 m).
m, height 19.1 cm). For the equilibration buffer, RPMI1640 medium (manufactured by Nissui Pharmaceutical) was used.
The eluted fraction was collected. The eluted fraction was evaluated for hematopoietic stem cell proliferation activity using a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells constructed in Example 2, and an activity was observed in a fraction having a molecular weight of around 20,000. A 15% FCS-RPMI1640 medium stored at 37 ° C. for 5 days was similarly gel-filtered with a Sephacryl S-200 column, and evaluated using the same hematopoietic stem cell growth factor detection system as a control. Was rarely observed. From the above, it was found that hematopoietic stem cell growth activating factor was present in the culture supernatant fraction having a molecular weight of about 10,000 to 20,000.

(Example 9: Purification of gel filtration fraction of NS-1 cell culture supernatant by reverse phase HPLC) The active fraction obtained by gel filtration obtained in Example 8 was subjected to reverse phase HPLC. YMC-Pack PROTE as a reversed-phase HPLC column
IN-RP (manufactured by YMC Corporation, length 250m
m, inner diameter 4.6 mm), and LC-10A manufactured by Shimadzu Corporation was used as the HPLC apparatus. Elution trifluoroacetic acid (TFA) - was carried out in a system of acetonitrile (CH ₃ CN). That is, solution A contains 0.1% TFA (pH 2.0),
Solution B contains 80% CH ₃ CN-0.1% TFA (pH 2.
0) using a linear gradient from 0% concentration of solution B for the first 5 minutes, from 0% to 70% for the next 70 minutes, and from 70% to 100% for the next 10 minutes Eluted with a linear gradient in the same manner. The eluted fraction was evaluated by a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells. As a result, an activity was observed in a fraction at a retention time of about 60 minutes and a fraction at a retention time of about 63 minutes.

(Example 10: NS-1 by serum-free culture)
Cell Culture and Preparation of Culture Supernatant) In order to prepare a culture supernatant that is easier to purify than a medium containing 15% FCS, an NS-1 cell culture method by serum-free culture was established. NS-1 cells at a concentration of 1 × 10 ⁵ / ml in 15% F
14. 40 ml of CS-RPMI 1640 medium, diameter 14.
The culture was started in a 5 cm dish (manufactured by Greiner). On day 4 of culture, a new 15% FCS-RPMI164
The medium was replaced with 40 ml of medium 0, and the cells were further cultured for one day. Then, the plate was washed twice with PBS (-), and the RPM without serum was used.
The cells were transferred to 40 ml of I1640 medium and cultured in a fresh dish for one day. Next, the culture was passed through a 0.22 μm filter to remove cell debris, and the culture supernatant was recovered. The collected culture supernatant was subjected to evaluation in a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells to confirm that the activity was present.

Example 11 Purification of NS-1 Cell Serum-Free Culture Supernatant by Gel Filtration
One cell serum-free culture supernatant (20 ml) was subjected to fractionation molecular weight of 10,000
Zero ultrafiltration filter (German Sciences)
2 ml of the concentrated culture supernatant obtained by concentration in
It was applied to a Kgel G3000SW _XL column. As the equilibration buffer, a 50 mM phosphate buffer containing 0.3 M salt was used, and the eluted fractions were collected. The eluted fraction was evaluated by a hematopoietic stem cell growth factor detection system using human umbilical cord blood hematopoietic stem cells. As a result, the fraction having a molecular weight of about 10,000 to 20,000 showed activity.

(Example 12: Purification of gel filtration fraction of NS-1 cell serum-free culture supernatant by reverse phase HPLC) Example 1
14 ml of the active fraction obtained by gel filtration obtained in step 1 was subjected to reverse phase HP
Subjected to LC. As a reversed phase HPLC column, YMC-P
HPL using ACK PROTEIN-RP (manufactured by YMC Corporation, length 250 mm, inner diameter 4.6 mm)
As the C apparatus, LC-10A manufactured by Shimadzu Corporation was used. Elution is T
It was carried out in a system of FA-CH ₃ CN. That is, 0.
1% TFA (pH 2.0), 80% CH ₃ CN in solution B
Using 0.1% TFA (pH 2.0), the first 15
The eluate was eluted with a linear gradient from 0% concentration of solution B to 0% to 70% in the next 70 minutes, and similarly from 70% to 100% in the next 10 minutes. CH ₃ CN concentration around 47% (retention time 71 minutes, 72 minutes, 74 minutes total 3)
Was recovered and re-chromatographed on the same column again to purify the same.

(Example 13: Confirmation of amino terminal amino acid sequence of purified sample)
The three samples obtained by purification by re-chromatography of Pack PROTEIN-RP were subjected to a protein sequencer (Applied Biosystems).
Procise Sequencer: Model
492) and analyzed. For 71 minutes,
Amino-terminal analysis of 25 amino acid residues was consistent with mouse cyclophilin B. Those at 72 minutes corresponded to mouse cyclophilin A. At 74 minutes, it was thought that its main constituent protein could not be degraded by Edman. However, as a minor constituent protein, it corresponded to mouse macrophage migration inhibitory factor. For a sample with a retention time of 74 minutes, a peptide map using tosylphenylalanyl chloromethyl ketone (TPCK) -trypsin was prepared and analyzed. The peptide at the amino terminus could not be Edman-degraded. Corresponded to mouse cyclophilin A. This protein is mouse cyclophilin A
-Like protein was considered.

(Example 14: Separation of mixture in a sample with a retention time of 74 minutes) In Example 13, a mouse macrophage migration inhibitory factor, which is a minor constituent protein, and a main constituent were prepared in a sample with a retention time of 74 minutes. Since it was found that mouse cyclophilin A-like protein, which is a protein, was mixed, separation of both was attempted. Purification of several lots of NS-1 cell culture supernatant was performed by the methods of Examples 10, 11 and 12, and reversed phase HPL
Examination of the chromatographic pattern of C revealed that in some lots, a peak corresponding to a peak at a retention time of 74 minutes was split into two peaks. When a peak having a slow retention time was analyzed with a protein sequencer, this peak was found to be a mouse macrophage migration inhibitory factor (FIG. 1). Similarly, peptide mapping using TPCK-trypsin and analysis with a protein sequencer also revealed that the peak with the earlier retention time was the mouse cyclophilin A-like protein.

Example 15: Confirmation of Hematopoietic Stem Cell Proliferation Activity of Purified Sample CD34 + CD, immature hematopoietic stem cells
Using the 38-cell amplification as an index, the activity of the mouse macrophage migration inhibitory factor fraction obtained in Example 14 was examined using the hematopoietic stem cell growth factor detection system constructed in Example 2. As a control, 2% FCS-RPMI1640 was used.
Cells cultured in 5 wells of 96 wells were mixed, and the number of viable cells contained therein was measured by the trypan blue method described in Example 2. The number of CD34 + cells and the number of CD34 + CD38- cells were also calculated by the method described in Example 2. The concentration of the mouse macrophage migration inhibitory factor fraction was not accurately determined, but was about 2 to 20 ng / ml. Result is,
Total viable cell count, CD34 + cell count, and CD34 +
Both CD38-cell numbers were amplified as compared to the control group (representative results are shown in FIGS. 2 and 3). The total viable cell count was 18.8-fold compared to the control group, the CD34 + cell count was 3.8-fold compared to the control group, and CD34 + CD38.
-The number of cells was amplified 3.4 times compared with the control. This indicates that the mouse macrophage migration inhibitory factor has human cord blood hematopoietic stem cell proliferation activity.

Example 16 Confirmation of Hematopoietic Stem Cell Proliferation Activity of Escherichia coli Type Human Macrophage Migration Inhibitory Factor Hematopoietic stem cells (CD34 + CD38− cells) prepared from human umbilical cord blood were plated on a 96-well U-type plate in 5 wells each ( 200 cells / 100 μl / well). The medium used for the growth of hematopoietic stem cells was 15% F in the test plot.
CS-RPMI1640 contains stem cell growth factor (25 ng / ml) as a cytokine, interleukin 6 (2
0 ng / ml), and interleukin 3 (20 ng
/ Ml) was added to the culture medium (stem cell growth factor / interleukin 6 / interleukin 3 medium), and Escherichia coli human macrophage migration inhibitory factor (R &
D Systems Inc. (human macrophage migration inhibitory factor, 2% FCS-RPMI1640)
(Final concentration of human macrophage migration inhibitory factor is 20 ng / well).
ml). In the control group, instead of the above sample containing human macrophage migration inhibitory factor,
20% of 2% FCS-RPMI1640 in stem cell growth factor / interleukin 6 / interleukin 3 medium
(/ Well) The added medium was used. This experiment was performed with triplicate. After culturing at 37 ° C. for 14 days and 27 days, 5 wells were combined into one, and the number of viable cells contained therein was measured by the trypan blue method described in Example 2. CD34 + cell count and CD34 + CD38
-The number of cells was also calculated by the method described in Example 2. As a result, the viable cell count, CD34 + cell count, and CD3
In each of the 4 + CD38-cell numbers, the number of cells was increased in the test group (human macrophage migration inhibitory factor-added group) as compared with the control group (FIGS. 4 and 5). In the case of FIG.
The total viable cell count was 25.3 times that of the control group,
4+ cell count 13.4 fold compared to control and CD3
4 + CD38− cells were amplified 10.5-fold compared to the control. In the case of FIG. 5, the total number of living cells is 21.3 times as compared with the control group, the number of CD34 + cells is 18.0 times as compared with the control group, and the number of CD34 + CD38− cells is 15.3 times as compared with the control group. Amplification was 2-fold. This means
This shows that E. coli human macrophage migration inhibitory factor has human cord blood hematopoietic stem cell proliferation activity.

[0052]

According to the present invention, a hematopoietic stem cell proliferating agent containing a macrophage migration inhibitory factor is provided. This hematopoietic stem cell proliferating agent can be used as a therapeutic agent for hematopoietic failure during various hematopoietic organ diseases, cancer radiotherapy and chemotherapy, can be used for mass production of blood cells, Can be used to improve the efficiency of introduction of the drug, and can be further used as a diagnostic agent and a test agent.

[0053]

[Sequence List] SEQUENCE LISTING <110> Kaneka Corporation <120> Promotion Agents for Hematopoietic Stem Cells Containing Macrophage 211 <1> T2 <1> T1 <4> T1 <1> T2 <1> T2 <1> T1 <1> T2 <1> T1 <1> T2 <1> T1 <1> T2 213> Homo sapiens <400> 1 Met Pro Met Phe Ile Val Asn Thr Asn Val Pro Arg Ala Ser Val Pro 1 5 10 15 Asp Gly Phe Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly 20 25 30 Lys Pro Pro Gln Tyr Ile Ala Val His Val Val Pro Asp Gln Leu Met 35 40 45 Ala Phe Gly Gly Ser Ser Glu Pro Cys Ala Leu Cys Ser Leu His Ser 50 55 60 Ile Gly Lys Ile Gly Gly Ala Gln Asn Arg Ser Tyr Ser Lys Leu Leu 65 70 75 80 Cys Gly Leu Leu Ala Glu Arg Leu Arg Ile Ser Pro Asp Arg Val Tyr 85 90 95 Ile Asn Tyr Tyr Asp Met Asn Ala Ala Ser Val Gly Trp Asn Asn Ser 100 105 110 Thr Phe Ala 115 <210> 2 <211> 114 <212> PRT <213> Mus sp . <400> 2 Pro Met Phe Ile Val Asn Thr Asn Val Pro Arg Ala Ser Val Pro Glu 1 5 10 15 Gly Phe Leu Ser Glu Leu Thr Gln Gln Leu Ala Gln Ala Thr Gly Lys 20 25 30 Pro Ala Gln Tyr Ile Ala Val His Val Val Pro Asp Gln Leu Met Thr 35 40 45 Phe Ser Gly Thr Asn Asp Pro Cys Ala Leu Cys Ser Leu His Ser Ile 50 55 60 Gly Lys Ile Gly Gly Ala Gln Asn Arg Asn Tyr Ser Lys Leu Leu Cys 65 70 75 80 Gly Leu Leu Ser Asp Arg Leu His Ile Ser Pro Asp Arg Val Tyr Ile 85 90 95 Asn Tyr Tyr Asp Met Asn Ala Ala Asn Val Gly Trp Asn Gly Ser Thr 100 105 110 Phe Ala

[Brief description of the drawings]

FIG. 1 shows a purified chromatographic pattern of a gel-filtered fraction of a serum-free culture supernatant of NS-1 cells by reversed-phase HPLC (PROTEIN-RP), in which a fraction of a mouse macrophage migration inhibitory factor is clearly shown. FIG. FIG. 1 shows the absorption at 214 nm on the vertical axis and reverse-phase HPLC on the horizontal axis.
(PROTEIN-RP) Indicates retention time (minutes). The absorbance at 214 nm reflects the amount of protein contained in each fraction.

FIG. 2: NS-1 cells purified from serum-free culture supernatant,
CD34 antigen and CD38 of cells that were cultured and expanded using mouse macrophage migration inhibitory factor fraction for 15 days
The expression status of the antigen is shown by flow cytometry. The vertical axis represents the fluorescence intensity of the CD38-PE antibody, and the horizontal axis represents the fluorescence intensity of the CD34-FITC antibody. The left part of the figure is a control group (2% FCS-RPMI1640), and the right part is a flow cytometry diagram of cells cultured and amplified using a macrophage migration inhibitory factor. Dots indicate cells. The crossed line indicates the vertical and horizontal axes drawn on the flow cytometry diagram obtained by treating the cells cultured and expanded as described above with the mouse immunoglobulin G1-FITC antibody and the mouse immunoglobulin G1-PE antibody. Shown on the axis is a line that separates each positive and negative. That is, the lower right quadrant is C
D34 + CD38-, the upper right quadrant shows CD34 +
CD38 +, upper left quadrant: CD34-CD38
+ And the lower left quadrant is CD34-CD38-
Is shown. Total viable cell count, CD34 + cell count, and C
It can be seen that both D34 + CD38− cell numbers were clearly amplified as compared with the control group.

FIG. 3 is a diagram summarizing human cord blood hematopoietic stem cell proliferation activity of a mouse macrophage migration inhibitory factor fraction purified from a serum-free culture supernatant of NS-1 cells. The vertical axis is cell number
On the abscissa, in order from the left, live cells on day 15, CD34 + cells on day 15, and CD34 + CD on day 15
Shows 38-cells. The black column represents the control, and the white column represents the number of cells cultured using the macrophage migration inhibitory factor. Total viable cell count, 1 compared to control
8.8 times, the number of CD34 + cells is 3.8 compared to the control group.
The number of CD34 + CD38- cells was increased 3.4-fold compared to the control group, indicating that the mouse macrophage migration inhibitory factor has human cord blood hematopoietic stem cell proliferation activity.

FIG. 4. Human cord blood hematopoietic stem cell proliferation activity of E. coli human macrophage migration inhibitory factor (day 14 of culture)
FIG. The vertical axis indicates the number of cells (cells), and the horizontal axis indicates, from the left, live cells on day 14, CD34 + cells on day 14, and CD34 + CD38- cells on day 14. Vertical bars indicate the respective standard deviations. The black column represents the control, and the white column represents the number of cells cultured using the macrophage migration inhibitory factor. The total number of living cells was 25.3 times compared to the control group, the number of CD34 + cells was 13.4 times compared to the control group, and CD34 + CD38.
-Amplified by 10.5 times compared to the control group in cell number,
This shows that E. coli human macrophage migration inhibitory factor has human cord blood hematopoietic stem cell proliferation activity.

FIG. 5: Human cord blood hematopoietic stem cell proliferation activity of E. coli human macrophage migration inhibitory factor (day 27 of culture)
FIG. The vertical axis indicates the number of cells (cells), and the horizontal axis indicates, in order from the left, live cells on day 27, CD34 + cells on day 27, and CD34 + CD38- cells on day 27. Vertical bars indicate the respective standard deviations. The black column represents the control, and the white column represents the number of cells cultured using the macrophage migration inhibitory factor. The total number of living cells was 21.3 times compared to the control group, the number of CD34 + cells was 18.0 times compared to the control group, and CD34 + CD38.
-Amplified 15.2 times in number of cells compared to the control,
This shows that E. coli human macrophage migration inhibitory factor has human cord blood hematopoietic stem cell proliferation activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/52 C12R 1:91) C12N 5/10 C12N 15/00 ZNAA 5/06 A61K 37/02 // (C12N 15/09 ZNA C12N 5/00 B C12R 1:91) E (C12N 5/10 C12R 1:91) C12R 1:91) F term (reference) 4B024 AA01 AA11 BA21 DA02 DA03 GA11 HA11 HA17 4B065 AA91Y AA93X AB01 BA01 BA25 BD14 CA24 CA44 CA46 4C084 AA02 AA06 BA44 CA17 CA18 CA56 DA05 NA14 ZA512 ZB082 ZB222 4H045 AA10 BA10 CA41 DA01 EA24 EA50 FA72 FA74

Claims (7)

[Claims] 1. A hematopoietic stem cell proliferating agent comprising a macrophage migration inhibitory factor. 2. The macrophage migration inhibitory factor is a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, wherein one or several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 1. The hematopoietic stem cell proliferating agent according to claim 1, which is a polypeptide having an amino acid sequence and having hematopoietic stem cell proliferating activity, or a modified form thereof. 3. A polypeptide having an amino acid sequence represented by SEQ ID NO: 2, wherein one or several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 2. The hematopoietic stem cell proliferating agent according to claim 1, which is a polypeptide having an amino acid sequence and having hematopoietic stem cell proliferating activity, or a modified form thereof. 4. A method for proliferating hematopoietic stem cells or hematopoietic progenitor cells in a non-human animal or in vitro using the hematopoietic stem cell proliferating agent according to any one of claims 1, 2 and 3, A method for expanding hematopoietic progenitor cells. 5. The method according to claim 4, wherein a foreign gene is introduced into hematopoietic stem cells or hematopoietic progenitor cells in the expanding step. 6. A human tissue comprising a step of growing a human tissue stem cell or a tissue precursor cell in a non-human animal or in vitro using the hematopoietic stem cell proliferating agent according to any one of claims 1, 2 and 3. A method for expanding stem cells or tissue progenitor cells. 7. A diagnostic or test agent for diseases of the blood system, comprising the hematopoietic stem cell proliferating agent according to any one of claims 1, 2 and 3.