JP2008050355A - Pharmaceutical compositions and methods for mobilizing hematopoietic stem cells into proliferation, differentiation and / or circulatory system - Google Patents

Abstract

The present invention provides a pharmaceutical composition for mobilizing hematopoietic stem cells, CFU-S or CFU-C into the proliferation, differentiation and / or circulatory system. The present invention also provides a method for proliferating and / or differentiating hematopoietic stem cells in vitro. Furthermore, the present invention provides a pharmaceutical composition for treating and / or preventing a cytopenic condition.
The pharmaceutical composition of the present invention comprises a plasminogen activator and / or a nucleic acid encoding it. The present invention also includes a step of culturing in a culture medium containing a plasminogen activator.
[Selection figure] None

Description

  The present invention relates to a pharmaceutical composition for mobilizing hematopoietic stem cells, CFU-S or CFU-C into the proliferation, differentiation and / or circulatory system. The present invention also relates to a method for proliferating and / or differentiating hematopoietic stem cells, CFU-S or CFU-C in vitro. Furthermore, the present invention relates to a pharmaceutical composition for treating and / or preventing a cytopenic condition.

  Stem cells reside in specialized niches where they are maintained through self-renewal and can be mobilized into the circulation and produce offspring, which progeny undergo terminal differentiation (non-patented) Literature 1-3). Therefore, hematopoietic stem cells are cells having the ability to differentiate into all blood cells such as red blood cells, white blood cells, and megakaryocytes (platelets). Since each cell of the blood cell line has a unique life span, hematopoietic stem cells continuously divide and proliferate in order to continue supplying these blood cells. As described above, hematopoietic stem cells can be differentiated into cells of all blood cell lineages, and thus are expected to be used for prevention and treatment of cytopenia or regenerative medicine.

  We have previously demonstrated that release of KitL mediated by matrix metalloproteinase-9 (MMP-9) regulates stem cell recruitment from the bone marrow (BM) niche (non-patented). Reference 1). Therefore, it is known that hematopoietic stem cell recruitment is caused from MMP-9 through KitL. Here, recruiting is used in the sense of regenerative proliferation of hematopoietic stem cells and is a concept different from mobilization into the circulatory system.

  So far, G-CSF or the like is known as a substance that exhibits advantageous effects on stem cells such as mobilization of stem cells. However, their long-term safety is unknown. Therefore, other factors that are highly safe and cause stem cell mobilization have been sought.

  Plasmin is one of endoproteases and is known to act physiologically on fibrin formed in blood vessels and decompose it. Plasmin is also produced from plasminogen (Plg), an inactive proenzyme, by tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and other serine proteases. (Non-Patent Document 4).

tPA is synthesized by endothelial cells (Non-patent document 5), monocytes (Non-patent document 6), megakaryocytes (Non-patent document 7) and mast cells (Non-patent document 8). tPA is a classic fibrinolytic factor that converts Plg into active proteolytic plasmin, and is clinically used for the treatment of myocardial infarction without showing significant side effects (Non-Patent Document 9- 10).
B. Heissig et al. , Cell 109, 625 (May 31, 2002) F. Arai et al. , Cell 118, 149 (Jul 23, 2004) L. M.M. Calvi et al. , Nature 425, 841 (Oct 23, 2003) K. Dano et al. , Adv Cancer Res 44, 139 (1985). E. G. Levin, D.D. J. et al. Losutoff, J Cell Biol 94, 631 (Sep, 1982) P. H. Hart, G. F. Vitti, D.C. R. Burgess, D.C. K. Singleton, J.A. A. Hamilton, J Exp Med 169, 1509 (Apr 1, 1989) C. Brisson-Jeanneau, L.M. Nelles, E.M. Rouer, Y. et al. Sultan, R.A. Benaroous, Histochemistry 95, 23 (1990) C. Silber et al. , J Immunol 162, 1032 (Jan 15, 1999) R. G. Wilcox, Am J Cardiol 78, 20 (Dec 19, 1996) H. R. Andersen et al. , N Engl J Med 349, 73 3 (Aug 21, 2003) T.A. H. Bugge, M.M. J. et al. Flick, C.I. C. Daugherty, J .; L. Degen, Genes Dev 9, 794 (Apr 1, 1995) K. Hattori et al. , J Exp Med 193, 1005 (May 7, 2001) K. Hattori et al. , Nat Med 8, 841 (Aug, 2002) K. Hattori et al. , Blood 97, 3354 (Jun 1, 2001) T.A. M.M. Dexter, T.W. D. Allen, L.M. G. Lajtha, J Cell Physiol 91, 335 (Jun, 1977) Yepes, et al (2002). J Clin Invest 109, 1571-1578 Hu, K.K. , Et al (2006) J Biol Chem 281, 2120-2127. S. M.M. Camiolo, S.M. Thorsen, T .; Astrup, Proc. Soc. Exp. Biol. Med. 138, 277 (Oct, 1971) M.M. Hoylerts, D.H. C. Rijken, H.M. R. Lijnen, D.C. Collen, JBiol Chem 257, 2912 (Mar 25, 1982) B. Heissig et al. , Hematology 10, 247 (Jun, 2005) J. et al. Zhang et at (2003). Nature 425, 836-841. S. Thorsen, P.M. Glas-Greenwalt, T.W. Astrop, Thromb Diae Haemorr 28, 65 (Aug 31, 1972) A. L. Copley, Biorheology 21, 135 (1984)

  The present invention provides pharmaceutical compositions and methods for mobilizing hematopoietic stem cells, CFU-S or CFU-C into the proliferation, differentiation and / or circulatory system. Specifically, the pharmaceutical composition comprises a plasminogen activator and / or a nucleic acid encoding it. Furthermore, the present invention provides a pharmaceutical composition for treating and / or preventing a cytopenia comprising plasminogen activator and / or a nucleic acid encoding the same.

  As a result of diligent research to solve the above problems, the present inventors discovered that tissue-type plasminogen activator (tPA) mobilizes hematopoietic stem cells into proliferation, differentiation and / or circulation. The present invention has been conceived.

  Specifically, the present invention is based on the finding that by administering tPA to mice, hematopoietic stem cells, etc. were proliferated, differentiated and mobilized into the circulatory system via plasmin, MMP-9, KitL.

It has been unexpected for a person skilled in the art that the present invention has been conceived in vivo with many obstructive factors.
(1) A pharmaceutical composition for mobilizing hematopoietic stem cells into proliferation, differentiation and / or circulation
(I) Pharmaceutical composition using plasminogen activator The present invention provides a plasminogen activator for proliferating and / or differentiating and / or mobilizing the plasminogen activator into the circulatory system.

The present invention also provides a method of administering a plasminogen activator to proliferate and / or differentiate and / or mobilize hematopoietic stem cells into the circulatory system.
The present invention provides a pharmaceutical composition for proliferating and / or differentiating and / or mobilizing hematopoietic stem cells into the circulatory system. The pharmaceutical composition of the present invention comprises a plasminogen activator.

  The present invention also provides a method of administering a pharmaceutical composition for proliferating and / or differentiating and / or mobilizing hematopoietic stem cells into the circulatory system. The pharmaceutical composition used for the administration method of the present invention comprises a plasminogen activator.

  The present invention also provides for the manufacture of a pharmaceutical composition for treating and / or preventing a plasminogen activator, a reduced state of blood cells, or for mobilizing hematopoietic stem cells or hematopoietic progenitors into the circulatory system. Provide the use of.

The present invention is supported by, for example, Examples 5, 12 and 15 described later.
The “hematopoietic stem cell” refers to a cell having the ability to reconstruct hematopoiesis over a long period of several months or more, having self-renewal ability and multipotency to differentiate into all blood cells. Previous studies have revealed that hematopoietic stem cells are contained in very small amounts in bone marrow, peripheral blood, and umbilical cord blood, and can be collected from these. To date, methods for sorting hematopoietic stem cells are known. For example, Lin-, c-kit +, sca-1 + cells can be sorted from bone marrow, peripheral blood, and umbilical cord blood based on cell surface antigens expressed by hematopoietic stem cells. It is certain that hematopoietic stem cells are present in the Lin-, c-kit +, and sca-1 + fractions. The “hematopoietic precursor” refers to a cell that has been differentiated from a hematopoietic stem cell and has not yet undergone terminal differentiation into erythrocytes and leukocytes. This includes CFU-S and CFU-C.

  “Proliferate” means that cells increase in cell number by cell division without losing their original properties. The number of cells can be counted visually by using, for example, a blood cell counter on the cell suspension. Various cell counting devices and hemocytometers are commercially available, and the cells may be counted by using them.

  “Differentiate” means that one cell type changes to another cell type. Therefore, not only terminal differentiation such as erythrocytes, leukocytes and lymphocytes, but also changes to lineage limited stem cells (such as CFU-S and CFU-C) that are not fully differentiated are within the category of differentiation. In general, the differentiation state of blood cells can be identified by cell morphology and cell surface expressed antigen.

  “Mobilize” means that hematopoietic stem cells and hematopoietic progenitor cells move into the circulatory system. Currently used hematopoietic stem cells for hematopoietic stem cell transplantation and in vitro proliferation of hematopoietic stem cells are obtained by collection from bone marrow, cord blood, and collection from peripheral blood. Among these, collection from peripheral blood has attracted attention because it has a small burden on patients and a sufficient amount of hematopoietic stem cells is available. However, since hematopoietic stem cells are usually present in the stem cell niche in the bone marrow and not in the peripheral blood, it is necessary to mobilize hematopoietic stem cells into the circulatory system. According to the pharmaceutical composition of the present invention, hematopoietic stem cells can be mobilized in the circulatory system.

  As used herein, the “plasminogen activator” can include any protein as long as it has a function of converting plasminogen to plasmin. For example, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are known as plasminogen activators. It is also known that plasminogen is converted to plasmin by other serine proteases (eg, streptokinase).

  Human tissue-type plasminogen activator (tPA) is a single-chain glycoprotein consisting of 562 amino acids (including signal peptide), which is limited to plasmin etc. and is bound by a disulfide bond. It is known to be activated). Both single-stranded and double-stranded forms can degrade plasminogen to plasmin. In the present invention, it is important that tPA has a function of degrading plasminogen into plasmin. Therefore, in the present invention, tPA includes both single-stranded and double-stranded types. Further, tPA may be a recombinant modified so as to be easily produced as long as it has a function of degrading plasminogen into plasmin. As tPA recombinant proteins, Monteplase (Product name: Clearacter (Eisai, Tokyo, Japan)), Alteplase (Product name: Activacin (Kyowa Hakko), Product name: Glutopa (Mitsubishi Pharma)), Pamiteplase (Product name: Solinase) (Astellas)), nateplase (product name: Millizer (Nippon Schering), etc., and as tPA cell culture protein, nasalplase (product name: thrombolinase (Benesis, Mitsubishi Pharma)) is the other tPA. Tisokinase (product name: plus beta (Asahi Kasei), product name: Happase Kowa (Kowa)), batroxobin (product name: defibrase (Tohyo Pharmaceutical Co., Ltd.)) and the like are known.

  Human urokinase-type plasminogen activator (uPA) is produced as a precursor consisting of 411 amino acid residues. Thereafter, the precursor is cleaved at the 156th site by plasmin or the like, and the C-terminal chain of the cleaved site becomes active uPA. In the present invention, it is important that uPA has a function of degrading plasminogen into plasmin. Therefore, in the present invention, the term uPA includes both the precursor type and the active type after cleavage. The uPA may be a recombinant modified so that it can be easily produced as long as it has a function of degrading plasminogen into plasmin.

In a preferred embodiment of the invention, the plasminogen activator is tPA.
In a further preferred embodiment of the invention, tPA is selected from the group consisting of the following proteins:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating effect; and
(C) a protein comprising an amino acid sequence having a deletion, substitution, and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating action.

  Here, the plasminogen activation action refers to an action of converting plasminogen to plasmin. SEQ ID NO: 1 is the amino acid sequence of wild-type human tPA. The amino acid sequence of SEQ ID NO: 1 represents the full-length sequence, and includes a signal peptide that is unnecessary for the physiological activity of tPA (considered as 1-23 amino acid residues). Therefore, a sequence obtained by removing the signal peptide from the sequence of SEQ ID NO: 1 is also included in tPA.

  In the present invention, as described above, tPA refers to a protein having a function of converting plasminogen to plasmin. Accordingly, a protein comprising an amino acid sequence having 95% or more identity to SEQ ID NO: 1 and having a plasminogen activating action, and deletion or substitution of one or several amino acid residues, and / or Proteins that contain amino acid sequences with additions and have a plasminogen activating action are also within the category of tPA.

  In the present invention, substitution of one or several amino acids of tPA is preferably performed by conservative substitution. As used herein, conservative substitution refers to substitution of an amino acid residue with an amino acid residue having biologically similar characteristics. Examples of conservative substitutions are replacing one aliphatic residue (Ile, Val, Leu, or Ala) with another aliphatic residue, and replacing one polar residue with another polar residue. (Eg, substitution between Lys and Arg; Glu and Asp; or Gln and Asn), or substitution of one aromatic residue (Phe, Trp, or Tyr) with another aromatic residue . Further examples of conservative substitutions include substitutions of amino acids outside the active domain, and substitutions of amino acids that do not change the secondary and / or tertiary structure of tPA. Those skilled in the art use, for example, site-directed mutagenesis methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, (2001), etc. by well-known genetic engineering techniques. Thus, it is possible to make a desired deletion, insertion or substitution.

  In the present invention, “several” of “amino acid sequence having deletion, substitution and / or addition of several amino acid residues” means 2 to 9, preferably 2 to 7, more preferably Means 2 to 5, particularly preferably 2 or 3.

  In a preferred embodiment of the present invention, tPA has a plasminogen activating action and is 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 98% or more, most preferably SEQ ID NO: 1. Has an identity of 99% or more.

  The percent amino acid identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by Needleman, S .; B. And Wunsch, C.I. D. (J. Mol. Biol., 48: 443-453, 1970) and determined by comparing sequence information using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). May be. Preferred default parameters for the GAP program include: (1) Henikoff, S and Henikoff, J. et al. G. (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992), scoring matrix, blossum 62; (2) 12 gap weights; (3) 4 gap length weights; And (4) no penalty for end gaps.

  Other programs for sequence comparison used by those skilled in the art may also be used. The percent identity can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997). The program can be used on the Internet from the website of National Center for Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ). Various conditions (parameters) for identity search by the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values.

  It is a well-known fact to those skilled in the art that even proteins having the same function may have different amino acid sequences depending on the varieties from which they are derived. As long as it has a plasminogen activation effect, tPA may also include such homologues and variants of the amino acid sequence of SEQ ID NO: 1. In addition to the human tPA of SEQ ID NO: 1, for example, the presence of a gene encoding a protein exhibiting the same activity in mice (M. musculus), chickens (Gallus gallus), medaka (Oryzia latipes) and the like is known.

  The pharmaceutical composition of the present invention is preferably administrable with one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are formulated using pharmaceutically acceptable diluents, fillers, adjuvants, excipients, and vehicles for the route of administration, and suitable dispersing, wetting, and suspending agents. It may be an aqueous or oily suspension.

  Pharmaceutically acceptable carriers are generally sterile and free of pyrogens and contain water, oils, solvents, salts, sugars, and other carbohydrates, emulsifiers, buffers, antimicrobials, and chelating agents. May include. The particular pharmaceutically acceptable carrier and ratio of active compound to carrier will be determined by the solubility and chemical properties of the composition, the mode of administration, and standard pharmaceutical practice.

  The compositions described herein can be contained in vials, bottles, tubes, syringe inhalers or other containers for single or multiple administration. Such containers can be made from glass or polymeric materials such as polypropylene, polyethylene, or polyvinyl chloride. Preferred containers may include a sealing system, such as a rubber stopper that can be penetrated by a needle to withdraw a single dose and then resealed when the needle is withdrawn.

  The plasminogen activator or nucleic acid encoding it is administered to the patient in an appropriate manner. For example, a plasminogen activator or a nucleic acid encoding the same or a pharmaceutical composition comprising the same is intravenous, transdermal, intradermal, intraperitoneal, intramuscular, intranasal, epidural, oral, topical, subcutaneous Or any other suitable technique. Preferably, it is administered intravenously or intraperitoneally. The optimal pharmaceutical formulation can be readily determined by one skilled in the art depending on the intended route of administration, delivery format and desired dosage.

The amount of tPA in the pharmaceutical composition of the present invention depends on age, the condition to be treated, the body weight, the desired duration of treatment, the method of administration, and other parameters. Effective doses are routinely determined by a physician or other qualified medical professional. While not limited, a typical effective dose is approximately 1 × 10 ³ IU / kg to approximately 5 × 10 ⁶ IU / kg body weight. In a preferred embodiment, the dose for intraperitoneal injection is approximately 1 × 10 ⁵ -5 × 10 ⁶ IU / kg. Also in a preferred embodiment, the dose for intravenous injection is 5 × 10 ³ -5 × 10 ⁵ IU / kg.

“IU” indicates an international unit.
(Ii) Pharmaceutical composition using nucleic acid encoding plasminogen activator The present invention relates to a nucleic acid encoding a plasminogen activator for mobilizing hematopoietic stem cells into proliferation, differentiation and / or circulatory system provide.

The invention also provides a method of administering a nucleic acid encoding a plasminogen activator for mobilizing hematopoietic stem cells into the proliferation, differentiation and / or circulatory system.
The present invention provides a pharmaceutical composition for mobilizing hematopoietic stem cells into the proliferation, differentiation and / or circulatory system. The pharmaceutical composition of the present invention comprises a nucleic acid encoding a plasminogen activator.

  The present invention also provides a method of administering a pharmaceutical composition for proliferating, differentiating and / or mobilizing hematopoietic stem cells into the circulatory system. The pharmaceutical composition used for the administration method of the present invention comprises a nucleic acid encoding a plasminogen activator.

  The present invention also provides a pharmaceutical composition for treating and / or preventing nucleic acid encoding a plasminogen activator, or for declining blood cells, or for mobilizing hematopoietic stem cells or hematopoietic progenitors into the circulatory system. Provide use for the manufacture of.

In a preferred embodiment of the invention, the nucleic acid encoding the plasminogen activator is a nucleic acid encoding tPA.
In a further preferred embodiment of the invention, the nucleic acid encoding tPA is selected from the group consisting of the following nucleic acids:
(A) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(B) a nucleic acid comprising a base sequence having at least 95% identity with the base sequence of SEQ ID NO: 2 and encoding a protein having a plasminogen activation effect;
(C) a nucleic acid encoding a protein comprising a base sequence having a deletion, substitution and / or addition of one or several nucleic acids from the base sequence of SEQ ID NO: 2 and having a plasminogen activating effect; and ,
(D) a nucleic acid that contains a base sequence that can hybridize with the base sequence of SEQ ID NO: 2 under highly stringent conditions and encodes a protein having a plasminogen activating action.

  Here, SEQ ID NO: 2 is a nucleic acid sequence encoding wild type human tPA. SEQ ID NO: 2 represents the nucleotide sequence of wild-type human tPA cDNA, which is a sequence encoding a signal peptide unrelated to the function of converting plasminogen of tPA to plasmin (considered as the nucleotide sequence of 1-69). Included). Therefore, a sequence obtained by removing the sequence encoding the signal peptide from the sequence of SEQ ID NO: 2 is also included in the nucleic acid encoding tPA.

  In the present invention, as described above, tPA refers to a protein having a function of converting plasminogen to plasmin. Therefore, a nucleic acid encoding a protein containing a base sequence having 95% or more identity to SEQ ID NO: 2 and having a plasminogen activating action, deletion or substitution of one or several amino acid residues, And / or a nucleic acid encoding a protein having an amino acid sequence having an addition and having a plasminogen activating action, and a base sequence capable of hybridizing with the base sequence of SEQ ID NO: 2 under highly stringent conditions, and Nucleic acids that encode proteins with plasminogen activating activity are also within the category of nucleic acids that encode tPA.

  “Stringent conditions” means moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Chapter 6-7, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5 Pre-wash solution of% SDS, 1.0 mM EDTA, pH 8.0, about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C. , Other similar hybridization solutions such as Stark's solution) and, for example, about 40 ° C.-60 ° C., 0.5-6 × SSC, 0.1% SDS wash conditions Use of. Preferably, moderately stringent conditions include hybridization conditions (and wash conditions) at about 50 ° C. and 6 × SSC. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA.

In general, these conditions include hybridization at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, about 65 ° C., 6 × SSC to 0.2 × SSC, preferably 6 × SSC, More preferably 2 × SSC, most preferably 0.2 × SSC hybridization) and / or washing, eg hybridization conditions as described above, and approximately 65 ° C.-68 ° C., 0.2 × SSC, 0 Defined with 1% SDS wash. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes after hybridization is complete.

In order to achieve the desired degree of stringency by applying the basic principles known to those skilled in the art and governing the hybridization reaction and duplex stability, as further described below, the wash temperature It should be understood that the wash salt concentration can be adjusted as needed (see, eg, Sambrook et al., 2001). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the length of the hybrid can be determined by juxtaposing the nucleic acid sequences and identifying the region or regions with optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be the length of less than 50 base pairs, not should be 5-25 less ℃ than hybrid melting temperature (T _m), T m is determined by the following equation Is done. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (A + T base number) +4 (G + C base number). For hybrids longer than 18 base pairs, T _m = 81.5 ° C. + 16.6 (log ₁₀ [Na ⁺ ]) + 41 (molar fraction [G + C]) − 0.63 (% formamide) −500 / n, where N is the number of bases in the hybrid, and [Na ⁺ ] is the sodium ion concentration in the hybridization buffer (1 × SSC [Na ⁺ ] = 0.165 M). Preferably, each such hybridizing nucleic acid is at least 8 nucleotides (or more preferably at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 1% of the length of the nucleic acid to which it hybridizes (more preferably at least 25%, or at least 50%, or at least 70%, and most preferably at least 80) At least 50% (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least with the nucleic acid to which it hybridizes 95%, at least 97.5%, or at least 99%, and sequence identity and most preferably at least 99.5%). Here, sequence identity, as described in more detail above, by comparing the sequences of hybridizing nucleic acids aligned in parallel to maximize identity with overlapping portions while minimizing sequence gaps. It is determined.

  The percent nucleic acid identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, this comparison is made by comparing sequence information using a computer program. A typical preferred computer program is the Wisconsin package, version 10.0 program “GAP” from the Genetics Computer Group (GCG; Madison, Wis.) (Devereux et al., 1984, Nucl. Acids Res. 12: 387). By using this “GAP” program, in addition to comparing two nucleic acid sequences, two amino acid sequences can be compared, and a nucleic acid sequence and an amino acid sequence can be compared. Here, the preferred default parameters of the “GAP” program are: (1) GCG implementation of a unitary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), and Schwartz and Dayhoff supervised “Atlas of Polypeptide Sequence and Structure” National Biomedical Research Foundation, pages 353-358, 1979, Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986 weighted amino acid comparison matrix; or other comparable comparison matrix; (2) 30 penalties for each gap of amino acids and one additional penalty for each symbol in each gap; or each of the nucleotide sequences Includes 50 penalties for gaps and an additional 3 penalties for each symbol in each gap; (3) no penalty to end gaps; and (4) no maximum penalty for long gaps. Other sequence comparison programs used by those skilled in the art include, for example, the National Medical Library website: http: // www. ncbi. nlm. nih. gov / blast / bl2seq / bls. The BLASTN program available for use by html, version 2.2.7, or the UW-BLAST 2.0 algorithm can be used. Standard default parameter settings for UW-BLAST 2.0 can be found at the following Internet site: http: // blast. Wustl. It is described in edu. In addition, the BLAST algorithm uses a BLOSUM62 amino acid scoring matrix and the selection parameters that can be used are: (A) Segments of query sequences with low compositional complexity (Woughton and Federhen SEG program (Computers and Chemistry, 1993); Woton and Federhen, 1996, “Analysis of compositionally biased regions in sequence data bases” or 71: 66. , A segment consisting of short-periodic internal repeats (Cl including a filter for masking verie and States (determined by the XNU program of Computers and Chemistry, 1993), and (B) a threshold of statistical significance for reporting a fit to the database sequence, or According to the statistical model of E-score (Karlin and Altschul, 1990), the expected probability of a match that is simply found by chance; if the statistical significance due to a match is greater than the E-score threshold, this fit is Not reported); the preferred E-score threshold value is 0.5 or, in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e 30,1e-40,1e-50,1e-75, or a 1e-100.

  In a preferred embodiment, the nucleic acid encoding tPA includes a protein encoded by a nucleic acid sequence having one or several deletions, substitutions, and / or additions from the base sequence of SEQ ID NO: 2. “Several” is preferably 2 to 20, more preferably 2 to 10, more preferably 2 to 5, and most preferably 2 or 3. In a preferred embodiment, one or several deletions and / or additions do not alter the conformation of the protein secondary structure or higher by shifting the frame of codon reading and frameshifting. However, deletions and / or additions that cause a frameshift can be tolerated if the site where the frameshift occurs is close to the 3 'end of the tPA coding sequence and has a small effect on the overall protein structure and / or function. A person skilled in the art can make a desired deletion, insertion or substitution by a well-known genetic engineering technique using, for example, site-directed mutagenesis described in Sambrook et al. (2001) (described above) and the like. Is possible.

  The pharmaceutical composition of the present invention can be administered by inserting a nucleic acid encoding a plasminogen activator into a vector. The “vector” in the present invention is any one as long as it introduces a nucleic acid encoding a plasminogen activator into hematopoietic stem cells and the like and expresses a desired gene product in the cells. Also good.

  In a preferred embodiment, the vector is a plasmid, virus, phage or the like, and in a more preferred embodiment is a plasmid or virus. The plasmid may be any plasmid that can express the plasminogen activator encoded by the nucleic acid. In a preferred embodiment, the plasmid has a constitutively expressed promoter or a promoter that is expressed only in hematopoietic stem cells or the like. It is also possible to introduce such a plasmid into a hematopoietic stem cell or the like in vitro and deliver it into a living body by intravenous injection or the like. Methods for introducing a plasmid into a hematopoietic stem cell or the like in vitro are widely known, and examples thereof include a calcium phosphate method, a lipofection method, and an electroporation method. Reagents used for the lipofection method are commercially available, for example, Effectene (Qiagen), LipofectAMINE (Invitrogen) and the like.

  Suitable plasmids for the pharmaceutical composition of the present invention include, but are not limited to, pcDNA3, pMXs-IRES-GFP, pMYs-IRES-GFP, and suitable promoters include, for example, CMV and LTR. is there. Moreover, the virus which can be used is not specifically limited. However, in a preferred embodiment, a known virus such as adenovirus, retrovirus or lentivirus is used as the virus. Such virus production methods are known. The virus may be delivered directly into the living body, or may be introduced into hematopoietic stem cells or the like in vitro, and the hematopoietic stem cells delivered to the living body.

(2) A pharmaceutical composition for proliferating, differentiating and / or mobilizing blood cells that can differentiate from CFU-S, CFU-C and / or these cells into the circulatory system The present invention relates to CFU differentiated from hematopoietic stem cells. -Cells that can differentiate into hematopoietic cells such as S, CFU-C, CFU-GEMM, CFU-E, CFU-GM, preferably CFU-S, CFU-C and / or blood cells that can differentiate from these cells Provides a pharmaceutical composition for growth, differentiation and / or mobilization into the circulatory system . The pharmaceutical composition comprises a plasminogen activator and / or a nucleic acid encoding it. This is derived from the finding of the present invention that the number of CFU-S and CFU-C in peripheral blood was increased by administering tPA to mice. The present invention is also supported by Examples 5, 12, 15 and the like.

  In the pharmaceutical composition of the present invention, “plasminogen activator” and “nucleic acid encoding plasminogen activator” are used to “(1) mobilize hematopoietic stem cells into proliferation, differentiation and / or circulation. The same as the definition of “pharmaceutical composition”.

  “CFU-S (colony forming unit-splene)” is an extremely early cell differentiated from a hematopoietic stem cell, and is a lineage-limited stem cell that differentiates into a specific blood cell. CFU-S can also proliferate by self-replication. Since CFU-S progeny can differentiate into many cell types, CFU-S is believed to have pluripotency. Specific examples of blood cells that can be differentiated from CFU-S include erythrocytes, platelets, granulocytes (neutrophils, eosinophils and basophils), macrophages and lymphocytes.

  “CFU-C (colony forming unit-culture)” is a cell further differentiated from CFU-S, and is a lineage-restricted stem cell that can differentiate into a plurality of types of blood cells. Similar to CFU-S, CFU-C can also grow by self-replication. Specific examples of blood cells that can differentiate from CFU-C include granulocytes (neutrophils, eosinophils, and basophils) and macrophages.

  According to the pharmaceutical composition of the present invention, the number of CFU-S and CFU-C cells can be proliferated, differentiated and / or mobilized into the circulatory system, and the number of differentiable blood cells can be increased from these cells. One skilled in the art will recognize that this can be done.

The pharmaceutical composition of the present invention preferably increases CFU-S and CFU-C cell numbers.
(3) Method for proliferating and / or differentiating hematopoietic stem cells, CFU-S or CFU-C in vitro The present invention relates to a method for proliferating and / or differentiating hematopoietic stem cells, CFU-S or CFU-C in vitro. provide. The method includes culturing the cells in a culture medium containing a plasminogen activator. This method is based on the findings of the present invention that the addition of tPA to Lin-cells sorted from mice dramatically increased the number of CFU-C cells. This method is also supported by Examples 9, 12, 14 and the like.

  In the method of the present invention, “plasminogen activator” and “nucleic acid encoding plasminogen activator” are “(1) pharmaceuticals for mobilizing hematopoietic stem cells into proliferation, differentiation and / or circulation” This is the same as the definition of “composition”.

  “Culture medium” refers to a culture solution for culturing cells. A culture medium that can be used for hematopoietic stem cells is required to maintain the undifferentiated nature of hematopoietic stem cells. Such culture media are known in the art. For example, any known medium such as StemPro-34 Serum Free Medium (Invitrogen) or X-VIVO-15 can be used.

“Culturing” refers to maintaining and growing cells in a culture medium. Media and additives for culturing cells such as hematopoietic stem cells and undifferentiated hematopoietic progenitor cells, and culturing methods are known in the art. However, when culturing hematopoietic stem cells and undifferentiated hematopoietic progenitor cells, the cells are preferably cultured while maintaining the undifferentiated state of these cells. Additives used for such purposes include cytokines such as SCF (stem cell factor) and TPO (thrombopoietin). If desired, a cytokine such as Flt3 ligand and / or IL-6 may be added. These cytokines may be recombinants modified so that they can be easily produced if they have their physiological functions. The culture is desirably performed in a CO ₂ incubator at 5% CO ₂ and 37 ° C.

  In one embodiment of the present invention, hematopoietic stem cells, CFU-S or CFU-C can be cultured with feeder cells. Such feeder cells include stromal cells, preferably MS-5 cells.

  According to the method of the present invention, as shown in FIG. 4D, the number of CFU-C cells increased. Therefore, in a preferred embodiment, the pharmaceutical composition of the present invention increases the number of CFU-C cells by 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, more preferably 2.4 or more. It is possible.

(4) Pharmaceutical composition for treating and / or preventing a cytopenia The present invention provides a pharmaceutical composition for treating and / or preventing a cytopenia. The pharmaceutical composition comprises a plasminogen activator and / or a nucleic acid encoding it.

  In the method of the present invention, “plasminogen activator” and “nucleic acid encoding plasminogen activator” are “(1) pharmaceuticals for mobilizing hematopoietic stem cells into proliferation, differentiation and / or circulation” This is the same as the definition of “composition”.

  A pharmaceutical composition comprising a plasminogen activator or a nucleic acid encoding it is administered to a patient in an appropriate manner. For example, a plasminogen activator or a nucleic acid encoding the same or a pharmaceutical composition comprising the same is intravenous, transdermal, intradermal, intraperitoneal, intramuscular, intranasal, epidural, oral, topical, subcutaneous Or any other suitable technique. Preferably, it is administered intravenously or intraperitoneally. The optimal pharmaceutical formulation can be readily determined by one skilled in the art depending on the intended route of administration, delivery format and desired dosage.

  With the pharmaceutical composition of the present invention, the decreased state of blood cells is treated and / or prevented. The decreased state of blood cells consists of, for example, the following groups: decreased state of blood cells (white blood cells, red blood cells and platelets) after chemotherapy / radiotherapy for hematopoietic tumors, etc. Diseases such as hematopoietic failure. However, the present invention is not limited to these, and if the number of blood cells is smaller than usual, it becomes a subject to be treated or prevented by the pharmaceutical composition of the present invention.

Here, “treatment and / or prevention” includes treatment and / or prevention of the above-mentioned diseases (decreased blood cells) of mammals, particularly humans, and includes, for example, the following (a) to (c):
(A) preventing the occurrence of a disease or condition in a patient who may have a predisposition to the disease or condition but has not yet been diagnosed;
(B) inhibit disease symptoms, ie prevent or delay its progression;
(C) To alleviate disease symptoms, i.e., cause a regression of the disease or symptoms, or reverse the progression of symptoms.

  In addition, the aspect and dosage described in “(1) Pharmaceutical composition for mobilizing hematopoietic stem cells into proliferation, differentiation and / or circulation” are the pharmaceutical composition for treatment and / or prevention of the present invention. It is also applicable to.

  The pharmaceutical composition of the present invention makes it possible to treat a patient whose blood cells are in a decreased state and to prevent a decreased blood cell state. Plasminogen activator is a classic fibrinolytic factor that has been used clinically without any significant side effects. Therefore, the above effect can be achieved without causing side effects.

  Hereinafter, the present invention will be described by way of examples. However, the examples are for illustrative purposes and do not limit the present invention. The scope of the present invention is determined based on the description of the scope of claims. Furthermore, those skilled in the art can easily make corrections and changes based on the description of the present specification.

Examples were carried out by the following methods unless otherwise specified.
Statistical analysis results are expressed as mean ± SEM (standard error mean). Data were analyzed using an unpaired two tail student t-test. A P value of <0.05 was considered significant.

Materials and methods
Animals . Mice that were backcrossed for 21 generations to C57BL / 6J background were heterozygously mated to obtain age-matched (6-8 weeks old) and gender-matched Plg + / + and Plg − / − mice. (Non-Patent Document 11). MMP-9 + / + mice and MMP-9 − / − mice after backcrossing to CD1 mice> 10 times were used (Non-patent Document 1). Mice were maintained in a filtered sterile air Thorensten apparatus. Animal treatment was approved by the in-house animal experiment committee at Juntendo University.

Bone marrow suppression model . Plg + / + and Plg − / − mice or MMP-9 + / + and MMP-9 − / − mice were injected with myelosuppressant 5-FU (250 mg / kg body weight, Kyowa Hakko Kogyo, Tokyo, Japan). . Mice were injected intraperitoneally with human recombinant tPA (Eisai, courtesy of Tokyo, Japan) or PBS at a concentration of 1.25 × 10 ⁶ IU / kg body weight daily. Survival was monitored daily. Blood was drawn at the indicated times.

BM transplantation research . BM cells obtained from either Plg + / + (Ly5.2) or C57BL / 6 (Ly5.1) mice were lethally irradiated (9.5 Gy) C57BL / 6 (Ly5.1) mice. Intravenous injection. Peripheral blood was drawn at different time points and peripheral blood mononuclear cells (PBMC) were isolated. PBMC were stained for Ly5.1-PE and Ly5.2-FITC (BD Biosciences Pharmingen) and analyzed by fluorescence activated cell sorting (FACS).

Administration of growth factors in vivo . SDF-1 (400 ng / mouse, Pepro Tech NJ, twice daily injection) or VEGF (300 ng / mouse, Pepro Tech NJ, once daily injection) resuspended in 100 μl 0.5% BSA was added to Plg + / + Mice and Plg − / − mice were administered intraperitoneally for 5 days. The control group was similarly injected with 100 μl of 0.5% BSA. Blood and plasma samples were collected at different time points.

Peripheral blood analysis . At indicated time points, blood was collected from mice by retroorbital bleeding using heparinized capillaries. White blood cell (WBC) counts were measured. PBMCs were isolated from heparinized blood after centrifugation on a discontinuous gradient using Lympholyte-M (Cedarlane, Ontario, Canada). Plasma samples were stored at −80 ° C. until further analysis.

CFU-S assay . After starting treatment with recombinant tPA, mobilized PBMCs were obtained on day 1, day 2, day 3, day 5 and day 10. For each time point, three recipient syngeneic mice were irradiated with 9 Gy from a ¹³⁷ Cs gamma source at a dose rate of ˜0.90 Gy / min to prevent endogenous spleen colony production. Within ⁵ hours after completion of irradiation, 10 ⁵ PBMCs were administered i.v. to the tail vein of irradiated mice. v. Injected. Mice were sacrificed after 12 days and the spleen was removed and fixed in Buan fixative. Next, the number of spleen colonies visible with the naked eye was scored (Non-patent Document 12).

Histological evaluation . Deparaffinized BM sections were stained with hematoxylin and eosin (H & E) or MSB trichrome staining (collagen stains blue, while cytoplasm and erythrocytes stain red / orange and nuclei stain black). Furthermore, as described (Non-Patent Document 1), BM sections were also stained for MMP-9 (7-11C). As for fibrin (fibrinogen), deparaffinized BM sections were stained (Non-patent Document 11), and then stained with Alexa 488-labeled secondary antibody (Molecular Probes, Inc).

  BM sections were also pretreated with hyaluronidase, blocked with skim milk, and incubated for 1 hour with rabbit anti-human Plg polyclonal antibody (H-90, Santa Cruz, 1/100 dilution). After catalyzed signal amplification II system (Dako), slides were expressed in DAB and counterstained with H & E.

1 × 10 ⁷ BMMC from competitive transplantation experiments Plg + / + or Plg − / − mice were mixed with 2 × 10 ⁷ CD45.1 cells and comparative contributions to hematopoiesis using antibodies to CD45.2, GR-1, B220 or CD3 It was then evaluated by analyzing the% contribution of the three donor-derived lines in PBMC.

In other experiments, Ly-5.1 mice irradiated with lethal doses of radiation were treated with 10, 50 or 100 KSL (c-Kit + / Sca-1 + / lin-) from Plg + / + and Plg − / − mice. Cells (10 recipient mice per cell enrichment) were administered with 5 × 10 ⁴ CD45.1 normal BM competitor cells. The ratio of CD45.2 in PB was then assayed 7 months after transplantation.

Identical genetic background of 1x10 ⁵ , 4x10 ⁴ or 2.5x10 ⁴ BM cells from long-term transplanted Plg + / + or Plg-/-mice (CD45.2 +) irradiated with a lethal dose of radiation (900cGy) CD45.1 mice were transplanted by tail vein administration. In another series of experiments, 1 × 10 ⁵ , ⁴ × 10 ⁴ or 2.5 × 10 ⁴ BM cells from CD45.1 mice were treated with Plg + / + or Plg − / − with the same genetic background irradiated with a lethal dose of radiation. (CD45.2 +) transplanted into recipient mice. The percent donor contribution was calculated by counting CD45.1 and CD45.2 on peripheral blood leukocytes in recipient mice 90 days after transplantation. Six mice from each recipient group were analyzed. Comparison between treatment groups was performed using a two-tailed Student's t-test and the data plotted as mean (M) ± standard error (SEM).

In vivo tPA-treated mice received human recombinant tPA or PBS intraperitoneally at a concentration of 1.25 × 10 ⁶ IU = 1 mg / kg (body weight) daily for a total of 3 days. (The recombinant tPA dose is 10 mg / kg in human clinical practice). Blood and plasma samples were collected at each time point.

In vitro assay
Lin-cell-separated femurs and tibias were flushed to obtain BM cells from mice. For Lin - cell separation, cells were stained with a commercially available antibody cocktail (Stem Cell Technologies, Vancouver). After passing through a 2MACS column (Miltenyi Biotec), Lin-cells were stained with antibodies against c-Kit and Sca-1. FACS analysis revealed that more than 90% of the separated Lin-cells were c-Kit / Sca-1 double positive cells.

Murine BM cells derived from Plg + / + and Plg − / − BM cells (1 × 10 ⁶ ) isolated after treatment with / without murine BM culture 5-FU were added to serum-free medium (X-VIVO-15). I left it overnight. The culture supernatant was analyzed by enzyme electrophoresis.

Suspension culture (without stromal cell support)
In the serum-free medium, with or without recombinant KitL (2 μg / ml, PeproTech), in the presence / absence of recombinant tPA (5 μg / ml, Cleartor®, Eisai, Japan) BM-derived Lin − cells (1.0 × 10 ⁵ ) from Plg + / + and Plg − / − mice were cultured overnight. Cells were then harvested, labeled with c-Kit-FITC antibody (Becton Dickinson) and analyzed for c-Kit expression by FACS.

1 × 10 ⁵ Lin − cells from the BM of stromal cell line cultured Plg + / + mice were placed in culture together with a confluent layer of stromal cells (MS-5). Recombinant tPA (5 μg / ml) was added to the culture daily. KitL (2 μg / ml) was added to the culture every other day. After 7 days, adherent cells were harvested using trypsin (Sigma) and pooled with non-adherent cells. The total number of hematopoietic cells was counted. The cells were then subjected to a hematopoietic precursor assay.

In another set of experiments, 1.2 × 10 ⁵ Lin − cells were isolated from Plg + / + mouse BM and in the presence of recombinant tPA (5 μg / ml, Cleartor®, Eisai, Japan). With / without blocking antibody to c-Kit (clone ASK, final concentration 0.5 μg / ml), blocking antibody to SCF (KitL) (final concentration 0.5 μg / ml, R & D Systems) in the absence of Co-cultured with MS-5 stromal cells with / without PAI-1 (2 μg / ml, Calbiochem) or daily with or without a final concentration of 1 nM MMP inhibitor (CGS27023A, Novartis, Basel, Switzerland). After 5 days, the culture was terminated and adherent and non-adherent cells were collected. After counting hematopoietic cells, the cells were subjected to hematopoietic precursor assay.

  In another set of experiments, MS-5 stromal cells were cultured in serum-free medium in the presence / absence of tPA (5 μg / ml, Cleartor®, Eisai, Japan). Cell supernatants were stored at −30 ° C. and then analyzed for KitL release by ELISA.

Long-term culture progenitor cell assay 20% horse serum (Gibco) and stromal layer (MS-5) cultures in long-term culture medium containing hydrocortisone (10 ⁻⁶ mol, Sigma) were added to Plg + / + mice and Plg − / -Mouse-derived BM cells were added. Every 5 days, half of the medium was supplemented with fresh long-term culture medium. After 3 weeks in culture, adherent and non-adherent cells were collected, the number of hematopoietic cells was measured, and the cells were subjected to a hematopoietic progenitor cell assay. After measuring the number of CFU-C, the absolute number of long-term cultured progenitor cells was calculated by dividing the total number of CFU-C by 4.

Commercially available containing hematopoietic precursor assay mIL-3 (10 ng / ml), murine KitL (50 ng / ml), human IL-6 (10 ng / ml) and human erythropoietin (3 units / ml) PBMC (10 ⁵ cells / plate) or Lin-cells (5 × 10 ³ cells / plate) were seeded in triplicate in 1 ml of a methylcellulose-based assay (Methocult, Stem Cell Technologies, Vancouver). On day 7, scoring was performed with an inverted microscope (40 × magnification).

Side population cell analysis The following filter sets: 405/30 bandpass (BP) to detect Hoechst blue, 585 / 42BP to detect Hoechst red, and 440-longpass dichroic filter to separate Hoechst red from Hoechst blue Total BM cells were subjected to fluorescence activated cell sorting (FACS; BD Biosciences) using MoFlo (DakoCytomation). A subset of cells treated with verapamil confirmed the identity of the SP gate.

Substrate enzyme electrophoresis (substrate zymography)
After a single injection of 5-FU, BM cells (1 × 10 ⁶ ) were collected from Plg + / + and Plg − / − mice and cultured overnight in serum-free medium. The supernatant was treated with gelatin-agarose beads to concentrate the gelatinase and processed through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) containing 0.1% gelatin. After incubation in 2.5% Triton X-100 for 1 hour at room temperature and rinsing in cold water, low salt collagenase buffer (50 mmol / l Tris, pH 7.6, 0.2 mol / l NaCl, 5 mmol / l CaCl ₂ and 0.2% v / v Brij-35) at 37 ° C. for 14-18 hours. After staining the gel for 30 minutes with a solution containing 10 ml of 0.2% Coomassie blue stock and 190 ml of a destaining agent (DW, methanol and glacial acetic acid, 6: 3: 1) and left at room temperature for 1 hour, A band of gelatin dissolving activity was visualized. Pro MMP-2 standard (Oncogene Research Products, Cambridge) was used as a positive control and to determine the molecular weight of the gelatin lysis band.

Western Blot Analysis Western blot using plasminogen antibody (clone E-14, Santa Cruz) and visualized using ECL chemiluminescence (Amersham Pharmacia-Biotech) before and after 5-FU treatment. The collected plasma samples from Plg + / + and Plg − / − mice were analyzed.

FACS analysis Plg + / + and Plg − / − mouse BM cells after 5-FU treatment were incubated with CD11b-FITC and GR1-PE antibodies and analyzed by FACS. After fixing the cells with ethanol and labeling with propidium iodide, FACS was used to analyze BM cell ploidy.

  All BM cells were labeled with Sca-1-FITC for cell cycle analysis. Labeled BM cells were fixed in ice-cold ethanol for 30 minutes. After treatment with RNase (Sigma, MO), cells were stained with propidium iodide (Molecular Probes, Oregon). The DNA content of SCA-1 + cells was determined by flow cytometry (Non-patent Document 1).

  In another series of experiments, Lin-cells were isolated from BM, Sca-1-FITC stained, and propidium iodide was performed prior to cell cycle analysis. For side population cell analysis, all BM cells were subjected to cell activated cell sorting (FACS; BD Bioscience) using MoFlo with the following filter set: 405/30 for detecting Hoechst blue Bandpass (BP), 585 / 42BP for detecting Hoechst red, and 440-long pass dichroic filter for separating Hoechst red from Hoechst blue. A subset of cells treated with verapamil confirmed the identity of the SP gate.

KitL and immunoassays for pro-MMP-9 and active MMP-9 KitL (SCF) and pro-MMP-9 were measured using commercially available ELISA (R & D Systems) in plasma or culture supernatant. MMP-9 activity assay Active MMP-9 was analyzed using a Biotrak ^TN- based ELISA (Amersham Biosciences, UK).

Culture supernatant (a) BM cells (1 × 10 ⁶ ) derived from 5-FU-treated Plg + / + and Plg − / − mice were collected and cultured overnight in serum-free medium. (B) MS-5 cells were grown to confluence in 6-well plates (Falcon) and then exposed to tPA (5 μg / ml, Cleartor®) in serum-free medium overnight. (C) BM-derived lin + cells (1 × 10 ⁵ ) from Plg + / + mice in serum-free medium overnight in the presence / absence of recombinant tPA (5 μg / ml, Cleartor®) Cultured. Culture supernatants of these cultures were collected. Supernatants (a)-(c) were analyzed by substrate zymography or ELISA.

  Alternatively, confluent MS-5 cells or primary confluent stromal cells can be obtained at a final concentration of PAI-1 (2 μg / ml, calbiochem) or 1 nM in the presence of tPA (5 μg / ml, Cleartor®). Cultured daily with / without MMP inhibitor (CGS27023A, Novartis, Basel, Switzerland). Cell supernatants were stored at −30 ° C. and then analyzed for KitL release by ELISA.

RNA extraction Total RNA was extracted from 4-week-old LTC stroma and MS-5 cells by acid guanidine thiocyanate-phenol-chloroform extraction using Trizol reagent.

RT-PCT analysis RT-PCT was performed using One Step RNA PCR kit (AMC) (Takara Shuzo, Japan) as recommended by the manufacturer. All probes were tested using the β-actin primers 5′-TGGAATCCTGTGGGCATCCATGAAAC-3 ′ (sense strand) and 5′-TAAAACGCAGCTCAGATAACAGTCCG-3 ′ (antisense strand) to test the integrity of the total RNA extract, Sorted in advance. These primers yielded a 349 bp product. The primer sequences for tPA are 5'-CTGAGGTCACAGTCCAAGCAATGT-3 '(sense primer) and 5'-GCTCACGAAGATGATGGTGTAAAG-3' (antisense primer), resulting in a 564 bp product. The primer sequences for plasminogen are 5'-TGTGGGCCTCTAAAGATGGAACTCC-3 '(sense primer) and 5'-GACAAGGGGAACTCGCTGGATGGCTA-3' (antisense primer), resulting in a 268 bp product.

Statistical analysis results were expressed as mean ± SEM (standard error). Data was analyzed using an unpaired two-sided Student's t-test. A P value of <0.05 was considered significant.

Example 1
In this example, Plg + / + and Plg − / − mice were used to confirm that Plg is a very important component of the stem cell niche and a potential upstream target for MMP activation.

The present inventors utilized a mouse in which the Plg gene was homozygously disrupted (Non-patent Document 11) and a model of stress hematopoiesis. Is a cell cycle cytotoxic agent 5-fluorouracil (5-FU) bone marrow suppression induced is actively but cell cycle resulted in apoptosis of HSC and progenitor cells circulating in the HSC of G ₀ phase There was no effect (Non-Patent Document 13). All Plg − / − mice that received 5-FU died by 10 days after 5-FU injection, while Plg + / + mice survived 5-FU treatment (FIG. 1A). Circulating white blood cells (WBC) and platelets showed recovery (FIGS. 1B, C). Regeneration of total BM cells CD11b + / GR-1 + bone marrow cells and immature megakaryocyte progenitors in 2N and 4N phases occurred in Plg + / + mice but not in Plg − / − mice (FIG. 1D, E, F, G). However, high differentiation (> N16) more differentiated megakaryocyte cells accumulated in Plg − / − mice (FIG. 1G). These data indicate that stress-induced megakaryocyte and bone marrow cell regeneration / maturation was blocked in the absence of Plg.

During hematopoietic regeneration, quiescent stem cells (G ₀ / G ₁ ) enter the cell cycle. Under steady state conditions, Plg − / − mice had more BM-derived Sca-1 + cells. This is a rough estimate of the number of stem cells in the G ₀ / G ₁ phase of the cell cycle when compared to the wild type counterpart (FIG. 1H). Myelosuppression shifted the wild type stem cell fraction to the S phase of the cell cycle, thereby promoting hematopoietic regeneration. However, this cell cycle transition did not occur in Plg-deficient cells (FIG. 1H), suggesting that BM regeneration was impaired in Plg − / − mice because cell cycle progression was blocked. .

Example 2
In this example, whether or not Plg is important for stem cell proliferation or the like was examined using a gene-deficient mouse other than Plg.

  When uPA, tPA (n = 5, data not shown) or urokinase plasminogen activator receptor (uPAR) (n = 6, data not shown) single deficient mice were treated with 5-FU, Plg − In contrast to the mice, the mice did not die. However, one third of u-PA / tPA double-deficient mice died within a few days after 5-FU injection (data not shown). BM recovery promotes the shift of quiescent stem cells to the cell cycle through activation by tPA and / or u-PA and assists in differentiation into a cell-specific lineage, during which Plg is required This is suggested by these data.

Example 3
In this example, changes in the expression levels of MMP-9, KitL, and the like during the BM regeneration period after administration of 5-FU were examined.

  Western blotting of plasma of 5-FU treated wild type animals showed that Plg was converted to plasmin during the BM regeneration phase (FIG. 2A). Plg activation can also lead to MMP activation. We observed an increase in pro-MMP-2 and pro-MMP-9, particularly active MMP, in the supernatant of cultured BM cells obtained from Plg + / + animals 5 and 10 days after 5-FU treatment. An increase of −9 was found (FIG. 2B). On the other hand, these were not as high in Plg − / − mice. However, immunoreactive MMP-9 and MMP-9 activation was attenuated in the BM of Plg − / − mice treated with 5-FU (FIGS. 2B, C). Similarly, high levels of active MMP-9 and pro-MMP-9 were detectable in the plasma of Plg + / + mice treated with 5-FU compared to Plg − / − mice (FIG. 2D, FIG. 5A). These data are associated with MMP activation in BM cells during the regeneration phase after bone marrow suppression.

  If plasmin activates MMPs, the plasma level of KitL (Non-patent Document 1), a stem cell active cytokine controlled by MMP-9, should be low in Plg − / − mice after 5-FU treatment It is. This was indeed the case (FIG. 2E). Daily injection of recombinant KitL partially restored the survival of Plg − / − mice after bone marrow suppression (FIG. 2F) and promoted BM cell recovery (FIGS. 2G, H). This allowed us to relate the lack of hematopoietic recovery in Plg − / − mice to impaired KitL release. Thus, the hematopoietic recovery deficit in Plg − / − mice can be explained in part by the inability to produce sufficient amounts of soluble KitL necessary to replenish the hematopoietic niche.

Example 4
In this example, using factors that increase active MMP-9, it was examined whether Plg caused stem cell mobilization via MMP.

  If plasmin activates MMPs, Plg − / − mice should have impaired stem cell recruitment by these factors. Indeed, in Plg − / − mice, recombinant SDF-1 induces (non-patent document 14), active MMP-9 (FIG. 3A), pro-MMP-9 (FIG. 5B), WBC (FIG. 3B) and hematopoietic progenitors. The increase in body (FIG. 3C) was missing. Similarly, when angiogenic factor VEGF was administered, as previously reported (12), in Plg + / + mice, plasma pro-MMP-9 (Fig. 5C) and active MMP-9 (Fig. 5D) and WBC count (FIG. 3D), hematopoietic progenitors (FIG. 3E) increased, but not so much in Plg − / − mice. Incubation of MS-5 stromal cells with VEGF overnight upregulated tPA gene expression by RT-PCR (data not shown). These data suggest that chemokines and angiogenic factors promote stem cell mobilization by activating both Plg (possibly via tPA) and MMPs.

Example 5
In this example, it was confirmed whether or not hematopoietic stem cells or precursors can be mobilized into the circulation or hematopoietic stem cells can be expanded by administering tPA to mice.

When wild type mice are injected daily with recombinant tPA, hematopoietic progenitors (colony forming unit cells, CFU-C) (FIG. 3F) and more immature hematopoietic stem cells (CFU-spleen, CFU-S) are recruited into the circulation. (FIG. 3G) and BM cell cellularity was increased (FIG. 3H). These data suggest that tPA treatment increases BM cell proliferation. Indeed, more BM Sca-1 + cells entered the S phase of the cell cycle in the BM of mice treated with tPA compared to animals treated with carrier (FIG. 3I). In the absence of Plg, cell cycle progression from G ₀ / G ₁ phase to S phase was prevented in vivo (FIG. 1H). These data suggest that the tPA-Plg axis regulates hematopoietic cell cycle progression.

Example 6
In this example, it was confirmed that tPA-mediated cell mobilization is dependent on MMP-9 activation.

  Specifically, when recombinant tPA was administered, MMP-9 in BM was up-regulated (FIG. 3J). However, when 5-FU was administered to MMP-9-deficient mice simultaneously with recombinant tPA, BM regeneration was reduced (FIG. 3K). A synthetic broad spectrum MMP inhibitor (MPI) also blocked tPA-mediated precursor and stem cell mobilization (data not shown). This indicates that the effect of tPA is mediated through plasmin-mediated MMP-9 activation.

Example 7
In this example, it was confirmed that Plg is important for the release of KitL.
If Plg is important for KitL release and tPA converts Plg to plasmin, an increase in KitL plasma levels should be found in animals treated with tPA. This is actually seen in Plg + / + mice after tPA treatment (FIG. 3L), whereas in Plg − / − mice treated with tPA, KitL plasma levels were only slightly elevated (FIG. 5E) (FIG. 5E). FIG. 4N). These data suggest that tPA improves KitL release in a plasminogen-dependent manner. If the Plg pathway is important for BM stem cell proliferation and differentiation, Plg − / − mice may also be defective. Plg − / − mice have a reduced number of c-Kit ^highly expressed Lin− cells, which reflects a higher number of immature cells compared to wild type controls (FIG. 4A). The c-Kit intensity of Lin- (mostly c-Kit ^high ) expressing cells was significantly reduced after KitL treatment, resulting in the accumulation of c-Kit ^low expressing cells. Treatment with tPA slightly reduced c-Kit expression on lin-Plg + / + cells, but had no effect on lin-Plg − / − cells. On the other hand, when lin- (mostly c-Kit ^high ) expressing cells were incubated with tPA and KitL, the c-Kit intensity decreased dramatically in Plg + / + cells, resulting in accumulation of c-Kit ^low expressing cells. This phenomenon was not observed in Plg − / − cells. These data show for the first time that plasminogen activation alters c-Kit / KitL expression and signaling, possibly thereby altering the fate of stem cells.

Example 8
In this example, the influence of Plg on the number of stem cells was confirmed by examining side population cells of Plg + / + and Plg − / − mice.

  The inventors evaluated murine side population cells (SP cells) as a measurement of the number of murine HSCs. Indeed, the number of SP cells in Plg − / − mice was lower than in Plg + / + mice (FIG. 5F). Stem cell deficiency in Plg − / − mice is confirmed in vitro by showing that Plg − / − BM cells produce fewer initiating cells (LTC-IC) than their wild-type counterparts. (FIG. 5G). Furthermore, in the bone marrow transplantation assay, the leukocyte antigen Ly5.2 or Plg − / − BM cells compete with a transplant containing BM cells expressing Ly5.1. Ly5.1 + / Ly5.2 + cell ratios analyzed after weeks and 17 weeks were higher in Plg − / − mice compared to wild type controls (1.0, 1.0 vs. 0.1, respectively). 4 and 0.4). Taken together, these data indicate that the number of HSCs in Plg − / − mice is less than in Plg + / + controls.

Example 9
In this example, it was examined in vitro whether tPA modulates stem cell microenvironment / stem cell niche in addition to affecting stem cell number.

  The long-term culture system (15) developed by Dexter for murine BM is a classic assay that determines the components of the microenvironment. In long-term culture, primitive hematopoietic cells associate with the stromal layer and are typically capable of producing bone marrow progenitors and mature granulocytes for many weeks using appropriate culture conditions.

  tPA improved Lin-cell survival and proliferation in culture in the presence of the MS-5 stromal cell line (FIG. 4B). tPA and KitL synergized in these stromal cell-based cultures to increase precursor number (cell proliferation). Taken together, these data imply that tPA can modulate progenitor cell growth by promoting the release of stromal-derived factor (s) in a Plg-dependent manner.

Example 10
In this example, it was investigated whether BM cells can produce Plg or Plg activator. As a result, it was confirmed that immunoreactive Plg and immunoreactive MMP-9 were expressed by administration of tPA.

  Plg mRNA was detectable only in liver cells and not in freshly isolated BM Lin− or lin + cells or cultured BM stroma (FIG. 4H). On the other hand, tPA is strongly expressed in BM stromal cells and cultured MS-5 stromal cells of both Plg + / + and Plg − / − mice, and isolated fresh Plg + / + and Plg − / -Weakly expressed in both mouse lin + or Lin- cells.

  Since tPA can activate Plg, we concluded that tPA can activate MMP in BM stromal cells as well. Activated MMP-9 and MMP-2 were also zygotes in freshly isolated lin + cells (FIG. 4J) from MS-5 (FIG. 4I) and Plg + / + animals incubated with recombinant tPA. Detected by graphy. Immunoreactive Plg can be detected 1 hour after injection of recombinant tPA very close to the sinusoidal BM vessels (FIG. 4K), while hematopoietic and stromal cells in BM of animals treated with tPA are Immunoreactive MMP-9 was expressed (FIG. 4L).

Example 11
In this example, it was confirmed that the release of KitL was increased by the tPA treatment.
One potential stroma-derived factor is KitL. Therefore, the present inventors treated adhesive stromal cells (MS-5) and the like (Non-patent Document 1) expressing KitL with tPA during the serum-free culture. tPA treatment significantly increased KitL release from MS-5 stromal cells (FIG. 4C) and primary BM stromal cells (FIG. 4M). When MS-5 stromal cells were treated with PAI-1, a natural inhibitor of tPA, KitL release was lower than in the control supernatant. These data suggested that MS-5 cells also expressed tPA and maintained a steady state release of KitL. In fact, RT-PCR found tPA mRNA in MS-5 cells (data not shown). Importantly, tPA-mediated release of KitL from stromal cells occurred via MMP activation: Addition of an MMP inhibitor completely inhibited KitL release from stromal cells. These data supported our initial data (FIG. 5E), which showed that tPA promotes the release of KitL in vivo by analyzing the plasma of animals treated with tPA for KitL.

Example 12
In this example, in order to evaluate whether tPA can act as a hematopoietic growth factor, HSPC (hematopoietic stem / progenitor cell) increase was evaluated both in vitro and in vivo. It was confirmed that the cells grew.

  In vivo tPA administration significantly increased the number of BM cells in wild type controls, but this was attenuated in both Plg-deficient and MMP-9-deficient mice (FIG. 6A). tPA is the number of immature spleen colony forming units (CFU-S) in treated animals (FIG. 6B), long-term culture-initiating cells (LTC-IC, FIG. 6C), and BM cells C-Kit + / Sca-1 + / lin- (KSL) cell ratio (FIG. 6D) was increased. Long-term administration of tPA continuously maintained the same number of BM cells as the above-mentioned steady value (FIG. 6E). These data indicate that tPA can act as a hematopoietic growth factor that affects precursor growth and cell differentiation.

To test whether tPA-mediated hematopoietic cell proliferation and differentiation is dependent on KitL in vivo, we treated Kit-L deficient S1 / S1 ^d mice and WBB6F1 + / + control mice with tPA. BM cell viability (FIG. 6F) and number of immature CFU-S progenitor cells (FIG. 6G) increased by 2 days after tPA administration in WBB6F1 + / + animals, but not in S1 / S1 ^d mice. Taken together, these experiments showed that tPA promotes hematopoietic cell differentiation and proliferation by releasing KitL provided by BM stromal cells / BM niche cells.

Example 13
In this example, whether Plg and MMP-9 are necessary for tPA-mediated BM reconstitution was examined, and it was confirmed that Plg and MMP-9 were necessary for the effect of tPA hematopoietic reconstitution.

  To solve the question of whether tPA-mediated hematopoiesis requires Plg and / or MMP-9, we treated Plg and MMP deficient mice with 5-FU and daily with recombinant tPA. Processed with or without. However, tPA did not improve survival in Plg-deficient mice (FIG. 7A), or recovery of WBC (FIG. 7B), and number of BM cells (FIG. 7C). Interestingly, strong Plg / plasmin staining was detected by immunohistochemistry after administration of recombinant tPA to the osteoblast niche, a region close to the bones of Plg wild type mice (FIG. 7D).

  Similarly, recovery of BM cells (FIG. 7E) or WBC (FIG. 3K) was delayed in 5-FU treated MMP-9 deficient mice and could not be rescued by daily administration of recombinant tPA. Hematopoietic recovery was delayed and resulted in approximately 70% death of MMP-9 − / − mice, which could not be prevented by tPA administration (FIG. 7F). Thus, the effect of tPA on hematopoietic reconstitution after myelosuppression requires the presence of both Plg and MMP-9.

Example 14
In this example, to elucidate whether the observed effect of tPA on hematopoietic cells is mediated directly or indirectly (eg, via stromal cells in the BM niche) by tPA. In addition, we examined the effect of tPA on hematopoietic cell expansion in vitro. As a result, tPA was confirmed to cause proliferation of hematopoietic stem cells in vitro by using stromal cells as feeder cells.

  tPA improved Lin-cell proliferation only in stromal cell-based cultures (MS-5 feeder layer) (FIG. 8A), but not without the feeder layer (FIG. 8B). Synergistically, tPA and KitL increase the number of precursors in cultures based on these stromal cells. A comparable number of precursor colonies appeared in culture with Plg + / + or Plg − / − lin cells (FIGS. 8A, 3J and K). These data indicate that tPA can regulate the growth of precursor cells by promoting the release of stromal-derived factors such as KitL.

Example 15
In this example, it was confirmed whether c-Kit / KitL signaling after tPA treatment promotes cell proliferation.

  If c-Kit / KitL signaling after tPA treatment promotes cell proliferation, blocking signaling should prevent tPA-induced precursor growth / proliferation. Addition of neutralizing antibodies to c-Kit and / or KitL prevented tPA-mediated CFU-C production from Lin-cells in stromal cell-based cultures (FIG. 4D). Similarly, adding PAI-1 to these cultures abolished tPA-mediated CFU-C production. Since tPA also activates MMPs through plasmin production in BM stromal cells, the question arises whether tPA-mediated stem cell proliferation is dependent on MMP activation. In support of our in vivo data, when synthetic MPI was added, precursor production was completely blocked and tPA-mediated precursor growth was prevented. These data suggest that tPA can regulate stem cell fate via plasmin-mediated activation of MMPs and KitL release.

Example 16
In this example, since tPA had an effect in the presence of stromal cells, it was examined whether the hematopoietic defect observed in Plg-deficient mice was due to stromal / niche defects. The results showed that the presence of Plg in the BM niche is important for reducing hematopoietic cell differentiation and proliferation in Plg − / − mice.

  When CD45.1 donor cells were transplanted into Plg + / + or Plg − / − recipients, donor cell contribution to PB leukocytes at day 80 post transplant was lower in Plg − / − mice (FIG. 8C). This was particularly evident with limiting cell dilution and was associated with decreased mouse survival 80 days after transplantation of donor cells in Plg − / − recipient mice (FIGS. 8D and 8E). . These data suggest that Plg deletion in BM niche deficiency is important for reduced hematopoietic cell differentiation and proliferation in Plg − / − mice.

Example 17
In this example, it was shown that the presence of Plg in the BM niche is important for BM reconstruction.

In order to overcome the problem of reduced cell proliferation of stem cells in a Plg-deficient microenvironment, we have generated a large number of BM cells (2 × 10 ⁷ cells / mouse) from Plg − / − or Plg + / + mice, A lethal dose of radiation was transplanted into irradiated Plg − / − or Plg + / + mice. Under these conditions, all mice survived and showed a transplant after 4 weeks as determined by white blood cell count (WT cells transplanted into WT mice were 7933 ± 945 / μl (blood) , 7133 ± 1815 for KO cells transplanted into WT mice, 6400 ± 1697 for WT cells transplanted into KO mice, and 7700 ± 1556 / μl (blood) for KO cells transplanted into KO mice. The chimeric mice were then treated with myelosuppressant 5-FU. Plg-deficient mice transplanted with either Plg + / + or Plg − / − BM donor cells died in the first 2 weeks after induction of myelosuppression (FIG. 8F; n = 9 per group). In contrast, all Plg wild type mice transplanted with Plg + / + or Plg − / − BM donor cells survived (n = 9 per group), which is important for Plg in the non-hematopoietic compartment BM niche. Emphasize the role. The inventors then examined whether the reduced viability and differentiation of stem cells within the Plg-deficient BM niche was due to reduced production of KitL, which resulted in decreased cell proliferation and differentiation. Interestingly, lower KitL levels were detected in Plg − / − recipient chimeric mice compared to Plg + / + recipient controls (FIG. 8G). These data highlight the importance of KitL release from the Plg component of the BM niche for BM reconstitution after myelosuppression with 5-FU.

  Binding of Plg to fibrin enhances Plg activity (Non-patent Documents 18 and 19). Immunohistochemical staining of BM sections from wild-type animals after bone marrow suppression showed that fibrin, as well as Plg, was detected very close to the osteoblasts inside the bone, a region recently called the osteoblast niche. (Non-patent Documents 20 and 21) (FIG. 8H). Fibrin deposition was observed in Plg − / − BM after myelosuppressive stress and to a lesser extent in Plg + / + mice. These data suggest that tPA, Plg and fibrin associate together within the osteoblast niche, resulting in ordered local activity of the fibrinolytic complex after, for example, bone marrow suppression (FIG. 8I). This fibrinolytic complex, in turn, can further activate cascades of other proteases such as MMPs and make the growth factor bioavailable and help promote cell recruitment and differentiation.

Example 18
In this example, in order to understand which cell components are dependent on Plg to maintain in vivo long-term hematopoiesis, we have derived from Plg − / − mice or Plg + / + mice. Chimeras were developed by transplanting BM of BM into lethal irradiated Plg − / − mice or Plg + / + mice.

  All mice showed transplantation after 5 weeks, as determined by blood count. Two-thirds of Plg-deficient mice (n = 3 per group) transplanted with either Plg + / + or Plg − / − BM donor cells died within 2 months. In contrast, all Plg wild type mice transplanted with either Plg + / + or Plg − / − BM donor cells survived (n = 3 per group), and host Plg in the stromal compartment plays a very important role It was stressed to have. Five weeks after BM transplantation, plasma KitL levels were similar to those in Plg + / + recipient mice, independent of transplanted cells (Plg + / + donor cells 220 ng / ml vs. Plg − / − donor cells 231 ng / ml). Met. On the other hand, plasma KitL levels were lower after transplantation of Plg + / + donor cells (136 ng / ml) or Plg − / − donor cells (156 ng / ml) into Plg − / − recipient mice. These data emphasize that Plg has a very important role in the control of hematopoiesis through regulation of the BM stromal niche, and tPA releases growth factors such as KitL, and tPA and KitL Consistent with our current model that both can regulate the stem cell niche / microenvironment by regulating the stem cell pool.

Example 19
In this example, the effects of two systems, fibrinogen / fibrin and Plg / plasmin, on the stem cell niche were examined.

  tPA is an inefficient activator of Plg in the absence of fibrin, but in the presence of fibrin, the activation of Plg is greatly enhanced (Non-Patent Documents 18, 19 and 22). Fibrin is deposited along the BM surface (Non-patent Document 22). We have shown that fibrin is extensively deposited in Plg − / − BM after myelosuppressive stress, as determined by MSB trichrome staining, but to a lesser extent in Plg + / + mice. Found (FIG. 4E). After bone marrow suppression, fibrin could be detected by immunohistochemical staining in the BM region where stem cells were resident (FIG. 4F): During the initial recovery period (3 days after bone marrow suppression), the stem cells were in close proximity to osteoblasts. Thus (osteoblast niche) is resident and at later time points (> 6 days after myelosuppression), more commonly seen in the central region of the bone marrow cavity, referred to as the vascular niche (20). Our data suggest that the balance between the two opposing systems of fibrinogen / fibrin versus Plg / plasmin in BM can control the stem cell pool.

  Cell migration within the hematopoietic microenvironment is very important for blood formation and blood cell function. Our results show that plasminogen activation by tPA is an important regulator of this function in hematopoietic cells (FIG. 4G). Plasminogen activation by tPA, accompanied by continuous activation of MMPs, plays a unique physiological role, particularly with respect to stem cell migration, proliferation and differentiation. We conclude that a cascade with activation of two distinct protease systems plays a very important role in regulating its escape from the stem cell pool and stem cell niche.

  TPA is also expressed by BM cells, and Plg is found in a niche similar to fibrin (FIG. 8I). Fibrin localizes fibrinolytic complexes containing Plg and tPA in regions of the BM niche where HSPC is thought to be present. Activation of the Plg / plasmin system can activate another important protease cascade, MMP. Activation of MMP-9 releases growth factors such as KitL, which in turn promote cell growth and differentiation within the stem cell niche. This process serves to replenish hematopoietic cells and peripheral blood within the BM. The involvement of the fibrinolytic system in the control of adult hematopoiesis represents a new paradigm with important implications for pathophysiology and cancer treatment and regenerative medicine.

FIG. 1A shows that a single dose of myelosuppressant 5-FU was administered i.p. to plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9). v. The evaluation of the survival of mice after administration is shown. FIG. 1B shows that a single dose of myelosuppressant 5-FU was administered i.p. to plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9). v. The number of WBC after administration is shown. FIG. 1C shows that plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9) received a single dose of myelosuppressant 5-FU i. v. The platelet count after administration is shown. FIG. 1D shows that a single dose of myelosuppressant 5-FU was administered i.p. to plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9). v. The total number of BM mononuclear cells (BMMC) per thigh after administration is shown. FIG. 1E shows that plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9) received a single dose of myelosuppressant 5-FU i. v. Shown is H & E staining of BM sections on the indicated days after dosing. Magnification x200. FIG. 1F shows that a single dose of myelosuppressant 5-FU was administered i.p. to plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9). v. Following administration, BM cells were stained for bone marrow markers CD11b-FITC and Gr-1-PE and analyzed by FACS (upper panel). The absolute number of CD11b + / GR-1 + BM cells per thigh is shown (lower panel; p <0.05). FIG. 1G shows that plasminogen (Plg) + / + mice (n = 9) and Plg − / − mice (n = 9) received a single dose of myelosuppressant 5-FU i. v. Cell ploidy after administration is shown by FACS. FIG. 1H shows the DNA content of BM cells at the indicated time points after 5-FU treatment. (N = 3 / group and time point). ^* P <0.05. FIG. 2A shows that a single dose of 5-FU was administered i.v. to Plg + / + and Plg − / − mice. v. The results of detecting Plg and plasmin by Western blot analysis after injection (each group, n = 12) are shown (pooled plasma from 3 animals per time point). FIG. 2B shows BM cells (from 3 mice per time point) obtained at different time points after 5-FU injection and after gelatin culture in serum-free medium overnight, by gelatin enzyme electrophoresis. The expression levels of pro-MMP-9 (103 kDa), active MMP-9 (86 kDa), pro-MMP-2 (72 kDa) and active MMP-2 (62 kDa) are shown. FIG. 2C shows immunohistochemistry of BM sections for pro-MMP-9 9 days after 5-FU injection. Magnification x200. There is positive staining in the BM compartment of Plg + / + mice, but no positive staining in Plg − / − mice. FIG. 2D shows that a single dose of 5-FU was administered i.p. to Plg + / + and Plg − / − mice. v. The amount of active MMP-9 in plasma obtained from peripheral blood (PB) after injection is shown. FIG. 2E shows that a single dose of 5-FU was administered i.d. to Plg + / + and Plg − / − mice. v. The amount of KitL in plasma obtained from peripheral blood (PB) after injection is shown. FIG. 2F shows that Plg + / + and Plg − / − mice are left untreated (n = 8), or recombinant KitL daily i.d. v. In combination with injection, these mice received a single 5-FU. v. Injection (n = 6). The survival of the treated animals was determined daily (n = 6, p <0.05). FIG. 2G shows that Plg + / + and Plg − / − mice are left untreated (n = 8), or recombinant KitL daily i.d. v. In combination with injection, these mice received a single 5-FU. v. Injection (n = 6). The total number of BM cells per thigh is shown. FIG. 2H shows that Plg + / + and Plg − / − mice are left untreated (n = 8), or recombinant KitL daily i.d. v. In combination with injection, these mice received a single 5-FU. v. Injection (n = 6). H & E staining of BM sections 10 days after 5-FU treatment is shown. Magnification x200. ^* P <0.05. FIG. 3A shows that Plg + / + mice and Plg − / − mice received recombinant SDF-1 i.d. p. The active MMP-9 in plasma is shown after injection (each group, n = 6 mice). FIG. 3B shows that Plg + / + and Plg − / − mice were treated with recombinant SDF-1 i. p. The number of WBC after injection (each group, n = 6 mice) is shown. FIG. 3C shows that Plg + / + and Plg − / − mice were injected with recombinant SDF-1 i.d. p. The number of CFU-C mobilized after injection (each group, n = 6 mice) is shown. (N = 3 per group). Mobilized PBMC were evaluated in a colony assay. FIG. 3D shows that Plg + / + and Plg − / − mice were treated with recombinant VEGF daily i.d. p. Number of WBC after injection (n = 6 mice per group) is shown. FIG. 3E shows that Plg + / + and Plg − / − mice were treated with recombinant VEGF daily i.d. p. The number of CFU-C in PBMC after injection (n = 6 mice per group) is shown. FIG. 3F shows that C57BL / 6 mice received recombinant tPA or carrier control daily i.p. p. The number of circulating CFU-C after injection (n = 9 per group) is shown. FIG. 3G shows that C57BL / 6 mice received recombinant tPA or carrier control daily i.p. p. The pluripotency of mobilized cells was assessed by CFU-S after injection (n = 9 per group). FIG. 3H shows that C57BL / 6 mice receive recombinant tPA or carrier control i. p. The number of BMMC per thigh after injection (n = 9 per group) is shown. FIG. 3I shows that C57BL / 6 mice received recombinant tPA or carrier control i. p. After injection (n = 9 per group), BM Lin − cells are labeled with propidium iodide and the cell cycle is analyzed (2 independent experiments, 5 mice / time point). FIG. 3J shows immunohistochemistry for MMP-9 in BM sections 2 days after the start of tPA treatment (magnification x200). FIG. 3K shows that MMP-9 + / + and MMP-9 − / − mice were injected with a single dose of 5-FU followed by daily injections of recombinant tPA or carrier (n = 6 per group). , Indicates the number of WBC. FIG. 3L shows that C57B1 / 6 mice received recombinant tPA or carrier daily i.p. p. Plasma KitL levels after injection are shown (n = 9). ^* P <0.05. FIG. 4A shows that Lin − cells from Plg + / + and Plg − / − mice were cultured overnight in the presence / absence of tPA with / without KitL (n = 3) after c− Shown are the results of staining cells with Kit antibody and analyzing by FACS. FIG. 4B shows that cells were harvested after culturing Lin-cells from Plg + / + mice on MS-5 stromal cells with / without KitL and in the presence / absence of tPA for 7 days, and The results of the precursor assay are shown (n = 6). FIG. 4C: Confluent MS-5 stromal cells were treated with recombinant tPA, PAI-1 or CGS27023A in serum-free medium for 24 hours. 24 hours later, the results of analyzing the supernatant for released KitL by ELISA are shown (n = 2). FIG. 4D: Lin − cells from Plg + / + mice were cultured on confluent MS-5 stromal cells in serum-free medium (n = 3). Antibodies against tPA, PAI-1, c-Kit or KitL, or MPI (CGS27023A) were added to the culture for 5 days. The cells are then collected and the results subjected to a precursor assay are shown. FIG. 4E shows MSB three-color staining (arrow: blue fibrin deposition) of BM sections 10 days after 5-FU treatment of Plg + / + and Plg − / − mice. Magnification x200. FIG. 4F shows that Plg + / + and Plg − / − mice were treated i. v. Shown is immunohistochemical staining for fibrin (green fluorescence) in BM sections from each mouse after treatment. FIG. 4G: FIG. 4G shows an overview of the signal cascade in the present invention. tPA converts plasminogen to plasmin and promotes the production of MMP-2 and MMP-9, thereby releasing KitL. Activation of the tPA / plasminogen pathway increased KitL production. FIG. 4H shows BM stroma, MS-5 cells (lane 4) in liver (lane 1; positive control for tPA), 4 week old Plg + / + mice (lane 2) and Plg − / − mice (lane 3). ), Freshly isolated BM-derived Lin − cells from Plg + / + mice (lane 5) or Plg − / − mice (lane 6), and Plg + / + mice (lane 7) and Plg − / −. Shown are RT-PCR for tPA and Plg in freshly isolated BM-derived lin + cells from mice (lane 8). MS-5 cells were cultured overnight with or without serum-free tPA. FIG. 4I is a diagram obtained by analyzing zymography for MMP-2 and MMP-9. Lin + BMMC from Plg + / + mice were cultured overnight with and without serum-free tPA. FIG. 4J is a diagram obtained by analyzing zymography for MMP-2 and MMP-9. FIG. 4K shows immunohistochemistry for Plg in BM sections of Plg + / + mice 3 days after the start of tPA treatment (magnification x200). FIG. 4L shows immunohistochemistry for MMP-9 in BM sections of Plg + / + mice 3 days after the start of tPA treatment (magnification x200). Plg + / + primary BM stromal cells were cultured in serum-free medium in the presence of recombinant tPA, recombinant PAI-1 or MPI (CGS27023A) with or without tPA. FIG. 4M is a diagram in which this culture supernatant was collected and analyzed for KitL by ELISA. Plg + / + and Plg − / − mice received a single tPA or PBS intraperitoneally on days 0-2. Blood was collected at each time point after tPA administration. Plasma samples were analyzed for KitL by ELISA (n = 6; ELISA was performed twice). FIG. 5A shows pro-MMP-9 after treatment of Plg + / + and Plg − / − mice with 5-FU by a single injection on day 0. FIG. 5B shows pro-MMP-9 after treatment of Plg + / + and Plg − / − mice with recombinant SDF-1 twice daily for 5 days. FIG. 5C shows pro-MMP-9 after treatment of Plg + / + and Plg − / − mice with recombinant VEGF daily for 5 days. FIG. 5D shows the results of analyzing plasma samples for active MMP-9 after VEGF injection at the indicated time points (n = 3 per group and time point). FIG. 5E shows i. p. Injections treated Plg + / + and Plg − / − mice, and 3 days later, blood was drawn. The results of analyzing plasma samples for KitL by ELISA are shown (n = 2). FIG. 5F shows the results of analysis of total BM cells from Plg + / + and Plg − / − mice after verapamil treatment using MoFlo (n = 3, typical experiment). One of these). FIG. 5G shows the results of subjecting BM cells derived from Plg + / + mice and Plg − / − mice to long-term culture (LTC) and measuring the number of LTC progenitor cells (LTC-IC) per thigh. ^* P <0.05. FIG. 6A shows the results of counting BM cells by administering recombinant tPA intraperitoneally on day 0-2 (short term effect) to Plg-deficient mice, MMP-9-deficient mice and littermate control mice. FIG. 6B shows the results of analysis of Plg + / + and Plg − / − BM cells by CFU-S assay (n = 9). FIG. 6C shows the results of analyzing BM cells of Plg + / + and Plg − / − cells by LTC-IC assay (n = 9). FIG. 6D shows the result of analyzing BM cells of Plg + / + and Plg − / − cells by FACS to confirm the presence of KSL cells (n = 9). FIG. 6E shows the results of administering recombinant tPA to Plg + / + and Plg − / − mice on day 0-5 (long-term effect) and counting the number of BM cells (n = 9). FIG. 6F shows the number of BM cells per thigh when S1 / S1 ^d mice and WBB6F1 mice were treated with / without tPA (n = 5). FIG. 6G shows the number of CFU-S per 10 ⁵ cells when S1 / S1 ^d mice and WBB6F1 mice were treated with / without tPA. FIG. 7A shows Plg + / + and Plg − / − mice with a single 5-FU administration followed by daily administration of recombinant tPA or vehicle, with Plg + / + (n = 9 per group) and Plg − / -Shows the survival rate of mice (n = 9 per group). FIG. 7B shows the number of WBCs when Plg + / + and Plg − / − mice were administered daily with recombinant tPA or carrier following a single 5-FU administration. FIG. 7C shows the number of BM cells per thigh when Plg + / + and Plg − / − mice were administered a single 5-FU followed by daily recombinant tPA or carrier. FIG. 7D shows a staining diagram in which BM sections of Plg + / + mice co-administered with or without tPA were stained for Plg 2 days after 5-FU treatment (brown staining). Plg staining in the BM region is also referred to as an osteoblast niche. FIG. 7E shows the number of BM cells per thigh when MMP-9 + / + and MMP-9 − / − mice were administered a single 5-FU followed by daily recombinant tPA or carrier. FIG. 7F shows the survival rate when MMP-9 + / + and MMP-9 − / − mice were administered daily with recombinant tPA or carrier following a single 5-FU administration. n = 12. ^* P <0.05. Lin − cells from Plg + / + mice were cultured on MS-5 stromal cells with / without KitL in the presence / absence of tPA. FIG. 8A shows the results of the precursor assay after 7 days (n = 6). tPA-mediated cell and precursor growth did not occur without the presence of a stromal feeder layer. Lin − cells from Plg + / + mice were cultured in suspension culture in the presence / absence of tPA with / without KitL. FIG. 8B shows the number of cells (left) and the results of the precursor assay when harvested after 4 days (n = 3). BM cells from donor mice with the same genetic background as CD45.1 were transplanted into Plg + / + or Plg − / − CD45.2 + recipient mice irradiated with a lethal dose of limited cell dilution (n = 6). ). FIG. 8C shows the evaluation of the contribution of mouse PB cell donors (CD45.1 + cells) 80 days after transplantation. BM cells from donor mice with the same genetic background as CD45.1 were transplanted into Plg + / + or Plg − / − CD45.2 + recipient mice irradiated with a lethal dose of limited cell dilution (n = 6). ). FIG. 8D shows the survival rate of CD45.1 recipient mice that received Plg + / + BM cells (n = 9). BM cells from donor mice with the same genetic background as CD45.1 were transplanted into Plg + / + or Plg − / − CD45.2 + recipient mice irradiated with a lethal dose of limited cell dilution (n = 6). ). FIG. 8E shows the survival rate of CD45.1 recipient mice that received Plg − / − BM cells (n = 8). Plg + / + or Plg − / − mice irradiated with a lethal dose of radiation were transplanted with BM cells from Plg + / + or Plg − / − donor mice on day 16 prior to 5-FU administration (in each group, n = 9). A single 5-FU was administered on day 0 after BM cell transplantation. FIG. 8F shows the survival rate of transplanted mice ( ^* p <0.05 compared to the survival rate of Plg + / + recipient mice with Plg − / −). Plg + / + or Plg − / − mice irradiated with a lethal dose of radiation were transplanted with BM cells from Plg + / + or Plg − / − donor mice on day 16 prior to 5-FU administration (in each group, n = 9). A single 5-FU was administered on day 0 after BM cell transplantation. And blood was collect | recovered at each time after 5-FU administration. FIG. 8G shows the results of analyzing KitL by ELISA of plasma samples. Plg + / + or Plg − / − mice irradiated with a lethal dose of radiation were transplanted with BM cells from Plg + / + or Plg − / − donor mice on day 16 prior to 5-FU administration (in each group, n = 9). A single 5-FU was administered on day 0 after BM cell transplantation. Then, Plg + / + and Plg − / − mice were administered 5-FU intravenously on day 0. FIG. 8H is the result of immunohistochemistry of fibrin (green fluorescence) and Plg (brown staining) in BM sections from Plg + / + and Plg − / − mice treated with 5-FU. TPA-mediated activation of Plg results in plasmin production after induction of hematopoietic stress in the BM niche. This results in activation of MMPs, particularly MMP-9, thereby releasing / sharing chemokines / cytokines such as KitL. These factors can not only promote stem cell recruitment, proliferation, improve BM reconstitution, but also control cell recruitment. ^* P <0.05.

Claims (11)

  A pharmaceutical composition for proliferating, differentiating and / or mobilizing hematopoietic stem cells, CFU-S or CFU-C into the circulatory system, comprising a plasminogen activator and / or a nucleic acid encoding it Pharmaceutical composition.   The pharmaceutical composition of claim 1, wherein the plasminogen activator is a tissue-type plasminogen activator (tPA). 2. The pharmaceutical composition of claim 1, wherein the plasminogen activator is selected from the following group:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating effect; and
(C) a protein comprising an amino acid sequence having a deletion, substitution, and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating action. 2. The pharmaceutical composition of claim 1, wherein the nucleic acid encoding the plasminogen activator is selected from the following group:
(A) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(B) a nucleic acid comprising a base sequence having at least 95% identity with the base sequence of SEQ ID NO: 2 and encoding a protein having a plasminogen activation effect;
(C) a nucleic acid encoding a protein comprising a base sequence having a deletion, substitution and / or addition of one or several nucleic acids from the base sequence of SEQ ID NO: 2 and having a plasminogen activating effect; and ,
(D) a nucleic acid that contains a base sequence that can hybridize with the base sequence of SEQ ID NO: 2 under highly stringent conditions and encodes a protein having a plasminogen activating action. A method of proliferating and / or differentiating hematopoietic stem cells, CFU-S or CFU-C in vitro, comprising culturing the cells in a culture medium containing a plasminogen activator.   6. The method of claim 5, wherein the plasminogen activator is tissue type plasminogen activator (tPA). 6. The method of claim 5, wherein the plasminogen activator is selected from proteins consisting of the following sequences:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating effect; and
(C) a protein comprising an amino acid sequence having a deletion, substitution, and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating action. A pharmaceutical composition for treating and / or preventing a cytopenia comprising the plasminogen activator and / or a nucleic acid encoding the same.   9. The pharmaceutical composition of claim 8, wherein the plasminogen activator is tissue type plasminogen activator (tPA). The pharmaceutical composition of claim 8, wherein the plasminogen activator is selected from proteins consisting of the following sequences:
(A) a protein comprising the amino acid sequence of SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating effect; and
(C) a protein comprising an amino acid sequence having a deletion, substitution, and / or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 and having a plasminogen activating action. 9. The pharmaceutical composition of claim 8, wherein the nucleic acid encoding the plasminogen activator is selected from nucleic acids consisting of the following sequences:
(A) a nucleic acid comprising the base sequence of SEQ ID NO: 2;
(B) a nucleic acid comprising a base sequence having at least 95% identity with the base sequence of SEQ ID NO: 2 and encoding a protein having a plasminogen activation effect;
(C) a nucleic acid encoding a protein comprising a base sequence having a deletion, substitution and / or addition of one or several nucleic acids from the base sequence of SEQ ID NO: 2 and having a plasminogen activating effect; and ,
(D) a nucleic acid that contains a base sequence that can hybridize with the base sequence of SEQ ID NO: 2 under highly stringent conditions and encodes a protein having a plasminogen activating action.

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