WO2005056778A1

WO2005056778A1 - Method of regulating the differentiation of hematopoietic stem cells or proliferating the same

Info

Publication number: WO2005056778A1
Application number: PCT/JP2004/018860
Authority: WO
Inventors: Kazuo Todokoro; Hiroyuki Satofuka; Yoshikazu Kuwahara
Original assignee: Riken
Priority date: 2003-12-11
Filing date: 2004-12-10
Publication date: 2005-06-23
Also published as: JPWO2005056778A1

Abstract

It is intended to provide a method of regulating hematopoietic stem cells in the undifferentiated state or proliferating the same, an agent for regulating the differentiation of hematopoietic stem cells or proliferating the same, and so on. More specifically speaking, it is intended to provide a method of regulating the differentiation of hematopoietic stem cells or proliferating the same characterized by comprising culturing the hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a; a hematopoietic stem cell culture system characterized by containing (a) feeder cells, (b) one or more proteins selected from Wnt2 and Wnt5a and (c) or more hematopoietic factors or cell stimulating factors but being free from serum; an agent for regulating the differentiation of hematopoietic stem cells or proliferating the same characterized by containing one or more proteins selected from Wnt2 and Wnt5a; and so on.

Description

Specification

Method for inhibiting differentiation or expanding hematopoietic stem cells

The present invention relates to a method for inhibiting or proliferating hematopoietic stem cells, a method for producing hematopoietic stem cells, a hematopoietic stem cell obtained by the method, a culture system used in the culture method, and a method for screening for a substance that regulates the growth or differentiation of hematopoietic stem cells. The present invention relates to an agent for suppressing differentiation or proliferating hematopoietic stem cells. Background art

Recently, a protein called Wnt has been actively studied in relation to hematopoietic stem cells. For example, We is sman and colleagues have reported that Wnt signals may be able to amplify stem cells (Nature, VOL. 423, 2003, pp 409-414). In this report, we show that Wnt familly is important for stem cell expansion and maintenance because it can induce the expression of HoxB4 and Notch, which is required for stem cell self-renewal. Role indicated. Matthews et al. Also show that Wnt5a and Wnt10b are expressed in mouse fetal liver, that Wnt10b also expresses hematopoietic stem cells, that Wnt5a or Wnt10a. It has been reported that the culture supernatant of cells expressing b increased stem cells about 5-fold (BLOOD, VOL. 89, 1997, pp. 3624-3635). Hoffman et al. Express Wnt2b (which corresponds to Wnt13), Wnt5a and Wnt10b in human fetal bone marrow, and Wnt5a is expressed in human hematopoietic stem cells. (Non-Patent Document 1; BLOOD, VOL. 92, No. 9, 1998, pp 3189-3202). However, it has not been shown whether these Wnts promote self-renewal of hematopoietic stem cells in an in vitro culture system, and hematopoiesis is not performed in fetal bone marrow in the first place. Bhatia et al. Reported that hematopoietic stem cells cannot be expanded in vitro with Wnt5a, but that stem cells were expanded about three-fold when a culture supernatant containing Wnt5a was administered to mice (Non-Patent Document 2: PNAS, VOL. 100, No. 6, 2003, pp 342 2-3 4 2 7). This document reports that Wnt has almost no effect of expanding stem cells in vitro.

Also, Nusse et al. Reported that Wnt 3a was successfully purified as a soluble protein, and that Wnt 3a alone was able to expand hematopoietic stem cells by about 6-fold (Nature, VOL. 2 0 3, pp 4 4 8 — 4 5 2). However, in this experiment, the stem cells have differentiated, and not only pure stem cells are amplified. The degree of amplification is about 1.7 times, which is not practical. These reports indicate whether Wnt actually functions alone as a self-renewal factor for somatic stem cells, whether Wnt has the ability to self-renew all somatic stem cells, and whether autocrine Questions such as how to explain the contradiction of self-renewal in, and which member of the Wnt family controls which somatic stem cells (specificity) have not yet been elucidated.

Disclosure of the invention

As described above, various studies have been conducted on Wnt proteins in relation to the proliferation of hematopoietic stem cells.However, which Wnt proteins actually have a practical level of inhibiting differentiation or proliferating hematopoietic stem cells is considered. The method of suppressing undifferentiated or proliferating hematopoietic stem cells using such a protein has not been known yet. Therefore, it is possible to specify a protein having the ability to inhibit differentiation or proliferation of hematopoietic stem cells, and to suppress the differentiation or proliferate hematopoietic stem cells using the protein, to treat various blood-related diseases. Or it is effective for research.

The present inventors have conducted extensive studies and found that adult mouse bone marrow stromal cells express Wnt2 and Wnt5a, and adult human bone marrow stromal cells express Wnt2, Wnt5a and Wnt5a. b was expressed, and Wnt 2 was found to be most frequently expressed. The present inventors have continued their research based on this new finding and found that the use of Wnt2 or Wnt5a can suppress or proliferate the differentiation of hematopoietic stem cells. The present invention has been completed. That is, the present invention provides the following methods for inhibiting and differentiating or proliferating hematopoietic stem cells, a method for producing hematopoietic stem cells, a method for producing hematopoietic stem cells, a culture system used in the culture method, and a method for expanding hematopoietic stem cells. Also, the present invention provides a method for screening a substance that regulates differentiation, an agent for suppressing differentiation or proliferating hematopoietic stem cells, and the like.

(1) A method for inhibiting differentiation or expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.

(2) The method according to (1) above, wherein the culture is performed in the presence of one or more hematopoietic factors or cell stimulating factors, and in the absence of serum.

(3) The method according to (2), wherein the culture is performed in the presence of one or more hematopoietic factors selected from SCF and TPO.

(4) adding one or more proteins selected from Wnt2 and Wnt5a to a culture system, or culturing on a feeder cell expressing the protein;

The method according to (3).

(5) A hematopoietic stem cell obtained by the method according to any one of (1) to (4).

(6) A method for producing hematopoietic stem cells, comprising a step of culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.

(7) (a) a feeder cell, (b) one or more proteins selected from Wnt2 and Wnt5a, and (c) one or more hematopoietic or cell stimulating factors and no serum. A hematopoietic stem cell culture system, characterized in that:

(8) The culture system according to (7), wherein the feeder cells include C 127 cells that can survive without serum.

(9) Use of feeder cells that can survive in the absence of serum; (a) Add a test substance to a culture system that contains feeder cells and hematopoietic stem cells and is free of serum. (B) culturing hematopoietic stem cells in a culture system in which the test substance is present; and (c) hematopoietic stem cells in the presence of the test substance as compared to the absence of the test substance. A method for screening a substance that regulates the proliferation or differentiation of hematopoietic stem cells, comprising a step of measuring a change in the ratio and / or number of cells.

(10) An agent for suppressing or proliferating hematopoietic stem cell differentiation, which comprises one or more proteins selected from Wnt2 and Wnt5a. (11) The agent for suppressing or proliferating hematopoietic stem cell differentiation according to (10), further comprising a hematopoietic factor or a cell stimulating factor.

(12) The agent for suppressing or proliferating hematopoietic stem cell differentiation according to (11), wherein the agent includes one or more hematopoietic factors selected from SCF and TP0 as the hematopoietic factors.

By using the hematopoietic stem cell differentiation suppressing or proliferating method of the present invention, it is possible to culture and proliferate hematopoietic stem cells in vitro and ex vivo. In addition, the use of the hematopoietic stem cell differentiation-inhibiting or proliferating agent of the present invention allows hematopoietic stem cells to be expanded in vivo or outside of the body while remaining undifferentiated. Improvement of illness, prevention of infectious diseases caused by lymphopenia, treatment of bone marrow diseases such as bone marrow dysplasia and myelosuppression, treatment of bone marrow diseases such as leukemia, severe renal impairment, and bone marrow suppression, derived from genetic diseases It can be used for in vitro culture of recombinant stem cells at the time of treatment of hypocytosis and gene therapy.

Further, according to the screening method of the present invention, it is possible to screen for a substance that regulates the proliferation or differentiation of hematopoietic stem cells, so that diseases at the level of hematopoietic stem cells can be elucidated and new therapeutic methods and therapeutic agents developed. Useful for BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail. 1. A method for inhibiting or expanding hematopoietic stem cells, and a method for producing hematopoietic stem cells

According to a first aspect of the present invention, there is provided a method for inhibiting differentiation or expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a. (Hereinafter, also referred to as “culture method”). Here, “hematopoietic stem cells” include all cells including granulocyte cells (mye 1 oid), lymphocyte cells (1 ymph oid), erythroid cells (erythroid), megakaryocyte cells (me gakaryocyte), etc. Refers to cells that have the ability to differentiate into blood cells and have the ability to self-renew. Cultivation of hematopoietic stem cells in vitro requires the co-presence of some hematopoietic factors or cell stimulating factors, and in the absence of such factors hematopoietic stem cells Cells die quickly. On the other hand, in the presence of such factors, hematopoietic stem cells easily differentiate. Hematopoietic stem cells lose their ability to self-renew when differentiated. To suppress the differentiation of hematopoietic stem cells means to increase the abundance ratio and / or the number of hematopoietic stem cells having self-renewal ability under conditions that normally allow differentiation of hematopoietic stem cells to proceed. Proliferation (or expansion) of hematopoietic stem cells means that hematopoietic stem cells that retain self-renewal ability increase, that is, self-renewal. An increase in cells differentiated from hematopoietic stem cells corresponds to proliferation of hematopoietic stem cells. do not do.

Wnt2 and Wnt5a used in the present invention are known proteins, and their amino acid sequences and the nucleotide sequences of genes encoding the proteins can be obtained from the following sequence databases. The human Wnt2 gene and the human Wnt5a gene are registered in GenBank as Accession No. NM-003391 and Accession No. NM-003392, respectively. Human Wnt2 protein and human Wnt5a protein are registered as NC cession No. NP- 0 03382 and Ac cession No. NP- 003383 with NCB I Entrez Protein DataBase, respectively. It has been. The mouse Wnt2 gene and mouse Wnt5a gene are registered in GenBank as Accession No. BC026373 and Accession No. BC018425, respectively. Mouse Wnt 2 protein and mouse Wnt 5a protein were registered with NCB I Entrez Protein DataBase as Ac cession on No. P 21 552 and Ac cession on No. A AH 18425, respectively. ing. Wnt2 and Wnt5a used in the present invention may be derived from any species, but are preferably derived from humans. In addition, a mutant protein comprising an amino acid sequence in which a deletion, substitution, insertion, and / or addition has occurred to the amino acid sequence of a human protein, as long as the protein has the same activity as human Wnt 2 and Wnt 5a. Can be used in the same way as Wnt 2 and Wnt 5 a. The mutation site and number of amino acids are not particularly limited as long as the mutant protein retains the same activity as human Wnt2 and Wnt5a, but the number of mutations is usually within several tens of amino acids (for example, within 60 amino acids). ), Preferably within 10 amino acids, more preferably 1 to several (eg 1 to 6), even more preferably It is 1-3, more preferably 1-2. Wnt2 and Wnt5a used in the present invention can be prepared from cells or tissues expressing the protein, and can be prepared using a peptide synthesizer (for example, peptide synthesizer-1433A, Applied Biosystems Japan). (Produced by K.K.) or a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes. However, from the viewpoint of its purity, production by genetic engineering techniques and recombinant proteins are preferred. The Wnt2 and Wnt5a used in the present invention can be used in their own form or in the form of a fusion protein with another kind of protein, and can further convert the protein into various forms. It is possible. For example, processing by various methods known to those skilled in the art, such as various chemical modifications to proteins, binding to polymers such as polyethylene glycol, binding to insoluble carriers, sugar chain modification, lipid modification, and the like can be considered.

The hematopoietic stem cells used in the present invention can be collected from, for example, fetal liver, bone marrow, peripheral blood, umbilical cord blood, and the like of mammals such as humans and mice, but the source of collection is not limited as long as hematopoietic stem cells are included. The preparation and isolation can be carried out by a usual method using a marker of a target hematopoietic stem cell as an index. For example, in mice, of the cells that are negative for cell differentiation antigen (Linege) and positive for c-kit and Sea-1 and that show CD34 antigen negative to weakly positive, the characteristics of hematopoietic stem cells Have been found (Osawa, M., Science, 27-3: 242, 1996). The CD34 antigen is known as a marker for human hematopoietic stem cells. In particular, the more undifferentiated markers are known to be CD34 antigen positive, CD38 antigen negative, and cell differentiation antigen negative (Bhatiaeta 1. Sci. USA 94, 5320-5325, 1997). In some cases, a nucleated cell derived from human or mouse bone marrow or a stem cell fraction can be directly used for culture without isolating hematopoietic stem cells. For example, as a stem cell fraction derived from mouse bone marrow or the like, SP (si depopulation) cell fraction (which contains about half of hematopoietic stem cells and is considered to contain other tissue stem cells) can also be used. . According to the present invention, the hematopoietic stem cells or hematopoietic stem cell fraction collected as described above are cultured in the presence of one or more proteins selected from Wnt2 and Wnt5a. The cells can be cultured in vitro using known cell culture techniques (for example, see Shinsei Kagaku Kenkyusho 18 Cell Culture Techniques (edited by The Japanese Biochemical Society, published by Tokyo Chemical Dojin, 1989)). Hematopoietic stem cells can be cultured using an appropriate medium in a culture dish, flask, or bioreactor capable of mechanically controlling the composition and pH of the medium. The appropriate medium used for the culture is not particularly limited as long as the survival and proliferation of hematopoietic stem cells are not inhibited. For example, SF-02 medium (Sanko Junyaku), 〇pti-MEM medium (GI BCO BRL), MEM medium ( GIB CO BRL), DMEM medium (GI BCO BRL), I MDM medium (GIBC

0 BRL), PRM I 1640 medium (GI BCO BRL), RD medium (RPM

1 1640: DMEM = 1: 1 [V / V mixed medium]. Culture systems usually contain additional components such as insulin, transferrin, lactoferrin, 2-mercaptoethanol, ethanolamine, sodium selenite, and HE PES, serum (eg, fetal calf serum, human serum). , Poma serum), monothioglycerol, sodium pyruvate, polyethylene glycol, various vinamines, various amino acids, and various growth factors are added as needed.

In the culture method of the present invention, hematopoiesis can be more effectively achieved by adding a hematopoietic factor or a cell stimulating factor, or both to a culture system, in addition to one or more proteins selected from Wnt2 and Wnt5a. It can suppress stem cell differentiation and Z or self-renewal. Here, the term “hematopoietic factor or cell stimulating factor” broadly means a factor that stimulates hematopoietic cells to self-renew, proliferate, differentiate, survive, migrate, and the like. The hematopoietic factor or cell stimulating factor used here is not particularly limited as long as it does not inhibit the survival and proliferation of hematopoietic stem cells. Examples of preferably used hematopoietic factors or cell stimulating factors include, for example, SCF (stem cell growth factor), ΤΡ〇 (thrompopoetin), IL-3 (interleukin-13), and IL-11 (interleukin). 1 1), GM—CSF (granulocyte / macrophage colony stimulating factor; granulocyte / macrophage colony—st imu latin ng factor), G—CSF (granulocyte colony stimulating factor) Offspring; granulocyte colony—st imu lati ng f actor), TGF- / 3 (transforming growth factor 1/3), MIP-1α, F 1 t 3 / F 1 k 2—1 ig and, EPO (erythropoietin), Notch ligand (Jagge d family, Delta family), Tie2 ligand (angiobotin), BMP4, F1k2 / F1k3 ligand, FL, bFGF, Oncos Evening M, IL 6 / s IL 6R, EGF and LIF. The hematopoietic factor or cell stimulating factor used in the present invention includes proteins derived from mammals such as humans and mice produced by genetic recombination techniques, peptide synthesis methods, cell culture methods, and the like. Furthermore, the hematopoietic factor or the cell stimulating factor used herein is such that a part (for example, 1 to several (for example, 1 to 6), preferably 1 to 3) of the amino acid sequence is substituted, inserted, added and substituted. Even a mutant protein which has been deleted can be used as long as it retains its activity. Alternatively, a feeder cell expressing each factor may be used.

The amount of hematopoietic or cell stimulating factor added to the culture system is generally factor-specific, but the factor is usually in the hematopoietic stem cell culture medium at about 1 ng Zm1 to about 100 ng / m1. , Preferably at a concentration of about 5 ng / ml to about 50 ng / ml, more preferably at a concentration of about 5 ng / ml to about 30 ng / ml. In the culture method of the present invention, one or more proteins selected from Wnt2 and Wnt5a and one or more proteins selected from various hematopoietic factors or cell stimulating factors can be added to the culture system. Hematopoietic stem cells may be co-cultured on a single feeder expressing proteins and factors. The feeder cells preferably used here include bone marrow cells or bone marrow stromal cells whose growth ability has been lost by X-ray treatment or the like, cells derived from AGM regions, fibroblasts, cells derived from fetal liver, mesenchymal stem cells, Vascular endothelial cells, preadipocytes and the like can be used. According to a particularly preferred embodiment of the present invention, the culture is performed in a serum-free medium without adding serum to the culture system. A feeder cell that is particularly preferably used in a serum-free medium includes C127 cells. In addition, the C127 cells which can be used in the present invention and can survive, long-term culture, and / or proliferate in a serum-free medium can be obtained, for example, as follows. First, C127 cells were treated with DMEM containing 1% FCS + 5% KSR for 1 month. Select the cells that will survive, and then expand the cells that survive in DMEM containing 0.1% FCS + 5% KSR. C127 cells capable of surviving, long-term culture, and Z or proliferating in serum-free medium are then cloned by limiting dilution of cells that have been amplified with complete serum-free DMEM + 5% KSR.

Expression of one or more proteins selected from Wnt2 and Wnt5a in a feeder cell and various hematopoietic factors or cell stimulating factors include DNA encoding the amino acid sequence of those proteins or factors. A recombinant vector may be prepared and the recombinant vector may be introduced into a feeder cell.

The cultivation is performed, for example, at a temperature of 33 to 39 (preferably 37 ° C.) under 3 to 6% CO 2 (preferably 5%) for 5 to 50 days. The results of the differentiation inhibition and / or self-renewal of hematopoietic stem cells are described in Herzenberg, LA rweir's Handbook of Experimental Immunology, 5th edition, Blackwell Science Inc. 1997, Spangrude, GJ, Proc. Natl. Acad. Sci. USA, 87 : 7433-7437, 1990; Visser, JMW, Flow cytometry in hematology], Academic Press, p9-29, 1992, etc., and can be confirmed using cell surface antigens as an index. For example, as a marker for human hematopoietic stem cells, at least CD34 antigen positive, preferably CD34 antigen positive, CD38 antigen negative, cell differentiation antigen negative can be used as an indicator of human hematopoietic stem cells. As a means to confirm the presence of hematopoietic stem cells, a transplantation experiment using an irradiated mouse or an in vitro (in vitro) colony formation method (Bradley, TR, J. Exp. Med., 44: 287-299, 1966) can also be used. In the transplantation experiment system using irradiated mice, a bone marrow cell or hematopoietic stem cell-containing fraction isolated from another mouse (Donna 1) is transplanted into a mouse (recipient) irradiated with radiation and damaging the hematopoietic system. After transplantation, the proportion of hematopoietic cells derived from recipients and donors (chimerism) is used as an indicator to confirm the presence of hematopoietic stem cells with long-term bone marrow remodeling ability (Osawa, M., Science, 273: 242-245). Natl. Ac ad. Sci. USA 94, 5320-5325, 1997). In the colony forming method, when hematopoietic stem cells are cultured in a medium supplemented with various cytokins so that various blood cells can appear, hematopoietic progenitor cells in which the differentiation direction has been determined can be a small number or a single cell. Contains only differentiated cells Hematopoietic stem cells that form no colonies but have pluripotency are able to form colonies that include blood cells of multiple differentiation lineages. In particular, the formation of a mixed colony containing red blood cells (CFU-Emix) is regarded as an indicator of hematopoietic stem cells in humans.

The present invention provides a method for producing hematopoietic stem cells, characterized by using the above-described culture method of the present invention. The production method of the present invention is characterized by comprising the step of (a) culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a. The production method of the present invention may further include the following steps, but is not limited thereto, and may appropriately include the steps according to the culture method and culture system of the present invention; (b) -A step of culturing in the presence of at least one or more hematopoietic factors or cell stimulating factors, in the absence of serum, (c) a step of co-culturing hematopoietic stem cells with one feeder cell, and (d) a Wnt t A step of expressing one or more proteins selected from 2 and Wnt5a. 2. Hematopoietic stem cells obtained by the above culture method

The hematopoietic stem cells obtained by the above method of the present invention can be used for hematopoietic stem cell transplantation instead of conventional bone marrow transplantation or cord blood transplantation. For example, for a disease such as an autoimmune disease whose improvement is seen by bone marrow transplantation, the stem cell expansion technology according to the present invention can be used to expand autologous or non-autologous stem cells. Specifically, the hematopoietic stem cells obtained by the method of the present invention can be used in combination with systemic X-ray therapy or advanced chemotherapy for various leukemias. In addition, hematopoietic stem cells are obtained by collecting bone marrow prior to surgery and performing hematopoietic stem cell expansion in vitro, for example, when performing chemotherapy, radiation therapy, or other treatments that cause bone marrow suppression as a side effect in patients with solid cancer. By returning to the patient after the procedure, hematopoietic damage due to side effects can be recovered early, and stronger chemotherapy can be performed.

In addition, the present invention is used to treat a patient with insufficient formation of various blood cells by dividing a patient or another person's hematopoietic stem cells into various blood cells and transferring them into the patient's body. Can be. In addition, the culture obtained by the culture method of the present invention. Blood stem cells can ameliorate hematopoietic failure caused by bone marrow hypoplasia presenting anemia such as aplastic anemia. Other diseases for which hematopoietic stem cell transplantation obtained by the culture method of the present invention is effective include chronic granulomatous disease, double immunodeficiency syndrome, cancer-free magropurinemia, Wisko tt—Aldrich syndrome, acquired Immunodeficiency syndrome (AIDS) and other immune deficiency syndromes, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, Gaucher disease, lysosomal storage disease such as mucopolysaccharidosis, adrenal white matter Degeneration, various cancers or tumors, and the like.

The transplantation of hematopoietic stem cells can be performed in the same manner as conventional bone marrow transplantation or cord blood transplantation, except for the cells used. The graft of the present invention can be used as a composition containing a buffer solution and the like in addition to the cell components containing hematopoietic stem cells grown by the method of the present invention.

3. Cell culture system and screening method using the same

According to another embodiment of the present invention, (a) a feeder cell, (1 :) one or more proteins selected from 11 cells and 11c5a, and (c) one or more hematopoietic factors or cells. Provided is a hematopoietic stem cell culture system containing a stimulating factor and containing no serum. In such a serum-free culture system, it is necessary to use a feeder cell that is at least viable in serum-free, preferably long-term culture, and / or is capable of growing. A preferred feeder cell is a C 127 cell that has been prepared to survive in serum free. Such a serum-free culture system can be used for a method of screening for a substance that regulates differentiation or proliferation of hematopoietic stem cells. That is, the method of screening for a substance that regulates the proliferation or differentiation of hematopoietic stem cells of the present invention is characterized by using a feeder cell that can survive in serum-free, and (a) comprises a feeder cell and a hematopoietic stem cell. A step of coexisting (adding or expressing in a feeder cell) a test substance in a culture system characterized by the absence of virulent serum; (b) coexisting a test substance (expressing in an added or feeder cell) Culturing hematopoietic stem cells in a culture system, and (c) hematopoietic stem cells in the presence (addition or expression) of the test substance compared to the absence (non-addition or non-expression) of the test substance Measuring the change in the percentage and / or number of Screening of the present invention In the method, for example, when testing a substance having a hematopoietic stem cell proliferation promoting activity, one or more proteins selected from Wnt2 and Wnt5a are included in the culture system (addition or feeder). Thus, the proliferation promoting activity of the test substance can be more effectively tested in a state where the differentiation of the stem cells is suppressed. If necessary, one or more hematopoietic factors or cell stimulating factors may be included in the culture system. The regulation of proliferation or differentiation means any of growth suppression, growth promotion, differentiation suppression and differentiation promotion, and growth promotion is particularly preferred.

Test substances used in this screening include cell culture supernatants, purified proteins or peptides, synthetic compounds, natural products derived from microorganisms and plants, and factors that promote the growth or differentiation of hematopoietic stem cells. Examples include proteins and biological materials that are expected to be produced by supporting cells. The activity of the test substance can be measured by calculating the ratio and Z or number of hematopoietic stem cells contained in the cells before and after the treatment in the presence and absence of the test substance in the culture system. The proportion and / or number of hematopoietic stem cells contained in the cells can be determined by FACS. The differentiation of hematopoietic stem cells can be measured by FACS or a fluorescent antibody method using an antibody against an antigen specific to the differentiated cells. For example, B220 can be used as a specific antigen for B cells, Gr-1 as a specific antigen for granulocytes, and TER119 as a specific antigen for erythroid cells. In addition, it can be measured by colony assay such as CFU-GM, CFU-Mix, and CFU-S. As a result of such screening, if significant self-renewal / proliferation, proliferation suppression, or differentiation of hematopoietic stem cells is detected, the test substance used for screening is a factor that regulates the growth or differentiation of hematopoietic stem cells. It is determined that there is. Such factors are particularly useful in developing therapeutics for hematological disorders. 4. Hematopoietic stem cell differentiation inhibitor or proliferation agent

According to another aspect of the present invention, a hematopoietic stem cell differentiation inhibitory or proliferating agent containing one or more proteins selected from Wnt2 and Wnt5a (hereinafter, also simply referred to as "hematopoietic stem cell proliferating agent") ) Is provided. The growth agent of the present invention preferably comprises, in addition to one or more proteins selected from Wnt2 and Wnt5a, Alternatively, it contains one or more cell stimulating factors, for example, SCF, TPO or both.

Since the hematopoietic stem cell proliferating agent of the present invention can proliferate hematopoietic stem cells in vivo or outside of the body while undifferentiated, it can be used in the above-mentioned method for culturing and expanding hematopoietic stem cells in vitro, and in addition to radiation therapy. Chemotherapeutic drugs such as anticancer drugs and anticancer drugs to prevent cytopenia caused by lymphopenia, treatment of bone marrow disorders such as bone marrow aplasia and bone marrow suppression, leukemia, severe renal disorders, bone marrow suppression, etc. It can be used for the treatment of bone marrow diseases, the treatment of hypocytosis caused by genetic diseases, the in vitro culture of recombinant stem cells during gene therapy, and the like.

The hematopoietic stem cell proliferating agent of the present invention can be administered orally, parenterally, locally, such as bone marrow, or any other suitable route. The desired dose of the hematopoietic stem cell proliferating agent is 1 to 10 per day as its active ingredient (the Wnt protein and, if necessary, each hematopoietic factor or cell stimulating factor is referred to as “active ingredient”). It is about 0 i GZ kg body weight, preferably about 5 to 500 ^ 71 ^ 8 body weight, but naturally varies depending on the weight and symptoms of the patient and individual administration route. In some cases, a dose lower than the lower limit of the above range may be appropriate, and even if the dose is higher than the above range, if it is divided into several times a day and administered in small amounts, adverse side effects may occur. Sometimes it does not happen.

The active ingredient used in the present invention can be administered alone or together with a pharmaceutically or pharmaceutically acceptable carrier or diluent by any of the above three administration routes. It can be performed once or in several times. More specifically, the proliferating agent of the present invention can be administered in various types of administration forms, for example, tablets, capsules, medicinal drops in combination with various pharmaceutically acceptable inert carriers. Troches, hard candy, powders, sprays, creams, salves, suppositories, jellies, jewels, pastes, lotions, ointments, aqueous suspensions, injections, elixirs, syrups and the like. These carriers include solid diluents or excipients, sterile aqueous media, various non-toxic organic solvents, and the like. Example

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto. (Example 1)

1. Identification of Wnt gene expressed in mouse and human adult bone marrow stromal cells 1.1 Preparation of bone marrow stem cells

Mouse bone marrow cells were injected into C57BL / 6 mice at 8 weeks of age by rapidly pouring phosphate-buffered saline (PBS) containing 2% inactivated fetal calf serum (FCS) into the marrow cavity with a 26-gauge needle. Prepared from mice (Charles River). ¹ Adult human bone marrow cells were obtained from the posterior iliac crest (pos terior il iac crest) of normal donors based on informed consent and institutional ethics committee approval. The cell suspension was placed on Ly hop rep (Nycomed) and centrifuged at 800 g for 20 minutes. Mononuclear cells were collected and washed with PBS containing 2% FCS. Then, the adherent cells were cultured for 10 days in an IMD culture solution (Gibco-BRL) containing 10% FCS. After isolating total RNA by the AGPC method, the type of Wnt expressed was identified by degenerative RT-PCR.

1.2 Degenerative RT-PCR analysis

Reverse transcription polymerase chain reaction (RT-PCR) was performed as follows. First strand (firststrand) cDNA was synthesized using Superscript II (Gibco-BRL). PCR amplification is performed with LA Tag polymerase (Takara) and

5 '-TG (C / T) AA (G / A) TGCCA (C / T) GG (G / C) -3' (SEQ ID NO: 1)

5 '-(G / A) CAGCACCAGTGGAA-3' (SEQ ID NO: 2)

1 cycle at 94 ° C for 1 minute, 1 minute at 94 ° C, 30 seconds at 54 ° C; 60 cycles of 45 seconds at 72 ° C and 1 cycle of 5 minutes at 72 ° C . The purified PCR product was subcloned into pBluescript and the nucleic acid sequence was determined using an ABI 3700 Genetic Analyzer (Applied Biosystems). By this method, Wnt2, Wnt15a in mice and Wnt2, Wnt5a, W in humans It was found that n15b was expressed. In both cases, Wnt 2 was most frequently expressed.

2. Proliferation of stem cell fraction using Wnt

2.1 Preparation of hematopoietic stem cells

Nucleated cells were prepared from bone marrow cells of 7-8 week old lacZ transgenic mice (B6; S129-Gt (R0SA26) Sor) by centrifugation using Lymphoprep and suspended in PBS containing 2% FCS. Cells (1 × 10 ⁶ cells / ml) were transferred to Hoechs t33342 (l.

And Rhodamme l23 (0.1 g / ml). Side population (SP) cells, a fraction rich in hematopoietic stem cells, were isolated on a FACS Vantage SE cell sorter (trademark: Beckton Dickinson Bioscience). Approximately 4000 SP cells were isolated from one mouse. From lacZ transgenic mice, Lin-negative c-Kit-positive Seal-positive CD34-negative or low cell fractions, mouse bone marrow hematopoietic stem cells, were isolated using FACS. Approximately 10,000 cells were isolated from one mouse.

2.2 Self-renewal of stem cells in vitro

The full-length mouse or human Wnt2 and Wnt5a cDNAs were isolated by RT-PCR using K0D + DNA polymerase, and the complete nucleic acid sequence of the Wnt cDNA was identical to the sequence registered in the database overnight. confirmed. Recombinant mouse or human Wnt cDNAs in PCDNA3.1 expression vector, transfect C127 cells using Lipofect Amine 2000 (invitrogen), resistant to G418 (l mg / ml) The stably expressing cells were maintained in a serum-free medium (DMEM + 5% Knockout serum replacement (KSR) (Gibco)). The expression of Wnt has been confirmed by RT-PCR, and expression of Wnt5a, for which some antibodies can be used, was confirmed by Western blot analysis.

After seeding C127 transfectants expressing recombinant Wnt or C127 cells not expressing recombinant Wnt in a 24-well plate, the isolated lacZ transgenic mouse-derived bone marrow hematopoietic stem cells were implanted at 500 / well, and Co-cultured. The medium used was a serum-free medium or a serum-free medium supplemented with TP0 and SCF. The medium was changed every two days, and the cells were collected by trypsin digestion on the sixth day. Cells derived from lacZ transgenic mice are fractionated by fluorescence using fluoroscein di-jQ-galactopyranoside as a substrate, and the percentage of Lin-negative c-Kit-positive Seal-positive cells (hematopoietic stem cells) is calculated. did.

When co-cultured with recombinant Wnt-expressing C127 transfectants or non-recombinant Wnt-expressing C127 cells and mouse bone marrow hematopoietic stem cells, lacZ transgenic after 3 days when cultured in serum-free medium without TPO and SCF Mouse-derived cells were not viable, and hematopoietic stem cells could not be maintained or expanded. When co-cultured with C127 cells that did not express recombinant Wnt, when cultured in serum-free medium supplemented with TPII and SCF, 88% of the recovered jS_Gal-positive cells were in-positive and differentiated cells. Was. 12% were Lin-negative c-Kit-positive Seal-positive cells and were considered to be stem cells. This is almost consistent with the results of conventional stem cell amplification using cytokines. On the other hand, when co-cultured with recombinant Wnt-expressing C127 transfectant and cultured in a serum-free medium supplemented with TPO and SCF, 23% to 27% of the recovered 3-gal-positive cells (mouse Wnt 2 (27%, 25% in mouse Wnt5a, and 23% in human Wnt5a) were hematopoietic stem cells. In the case of co-culture with recombinant Wnt-expressing C127 transfectants, the percentage of hematopoietic stem cells in lacZ transgenic mouse-derived cells surviving the co-culture treatment was higher than that of co-culture with recombinant Wnt-non-expressing C127 cells. As compared to the case, mouse Wnt2 showed 2.3 times, mouse Wnt5a showed 2.1 times, and human Wnt5a showed 1.9 times. In other words, Wnt 2 and Wnt 5a are effective in suppressing the differentiation of hematopoietic stem cells and maintaining their undifferentiated state in the presence of factors that promote hematopoietic stem cell differentiation, such as TPO and SCF. It turned out that. Industrial applicability

Hematopoietic stem cells are differentiated or proliferated in vivo or in vitro without undifferentiation by using the method for suppressing or proliferating hematopoietic stem cells of the present invention, the method for producing hematopoietic stem cells, and the agent for suppressing or proliferating hematopoietic stem cells of the present invention Can be because of It can be used to improve cytopenia, prevent infectious diseases caused by lymphopenia, treat bone marrow disease, and treat bone marrow diseases such as leukemia, severe renal impairment, and bone marrow suppression. In addition, according to the screening method of the present invention, a substance that regulates the proliferation or differentiation of hematopoietic stem cells can be screened, which is useful for elucidating diseases at the level of hematopoietic stem cells and for developing new therapeutic methods and therapeutic agents. is there.

Claims

The scope of the claims

I. A method for inhibiting differentiation or expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.

2. The method according to claim 1, wherein the culturing is performed in the presence of one or more hematopoietic factors or cell stimulating factors and in the absence of serum.

3. The method according to claim 2, wherein the culture is performed in the presence of one or more hematopoietic factors selected from SCF and TP0.

4. The method according to claim 3, wherein one or more proteins selected from Wnt2 and Wnt5a are added to a culture system, or cultured on a feeder cell expressing the protein.

5. A hematopoietic stem cell obtained by the method according to any one of claims 1 to 4.

6. A method for producing hematopoietic stem cells, comprising a step of culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.

7. (a) Feeder cells, (b) one or more proteins selected from Wnt2 and Wnt5a, and (c) one or more hematopoietic or cell stimulating factors and no serum. Characteristic hematopoietic stem cell culture system. ·

8. The culture system according to claim 7, wherein C127 cells that can survive without serum are used as one feeder cell.

9. Use of feeder cells that can survive in serum-free condition. (A) Coexist with test substance in culture system containing feeder cells and hematopoietic stem cells and free of serum. (B) culturing the hematopoietic stem cells in a culture system in which the test substance is present; and (c) comparing the hematopoietic stem cells in the presence of the test substance in comparison with the absence of the test substance. A method for screening a substance that regulates proliferation or differentiation of hematopoietic stem cells, comprising a step of measuring a change in the ratio and / or the number.

10. An agent for inhibiting or proliferating hematopoietic stem cell differentiation, comprising one or more proteins selected from Wnt2 and Wnt5a.

I I. The agent for suppressing or proliferating hematopoietic stem cell differentiation according to claim 10, further comprising a hematopoietic factor or a cell stimulating factor. O 2005/056778

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12. The agent for suppressing or proliferating hematopoietic stem cell differentiation according to claim 11, wherein the agent includes one or more hematopoietic factors selected from SCF and TPO as the hematopoietic factor.