JPH04502853A - Primitive cell colony stimulating factor and lymphohematopoietic progenitor cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Primitive cell colony stimulating factor and lymphohematopoietic progenitor cell 1n standing The present invention provides cell populations useful for bone marrow transplantation and cytokines involved in proliferation of bone marrow cells. Regarding.

good love 11 The ability to initiate and regulate hematopoiesis is of great importance in both veterinary and human medicine It is. Various diseases, immune disorders and malignant tumors are associated with destruction within the hematopoietic system. It's good. Many of these disorders result in defects in the individual when triggered to differentiate. Repopulating the hematopoietic system using overcoming progenitor cells can be relieved and/or cured by Currently performed in humans The most common replacement therapy is bone marrow transplantation. However, this form of treatment It is painful for both recipients, and for the recipient, especially if the implant is allogeneic. It is very dangerous if Before performing a bone marrow transplant, the donor and recipient must be HLA-tied. Approximately half of allogeneic bone marrow transplant recipients, even if matched for It causes sexual graft disease (GVHD). Current treatments for GVHD are incomplete. The disease can be disfiguring and/or fatal. Therefore, GVHD The use of bone marrow transplantation is dangerous due to severe immunodeficiency disorders and severe aplastic anemia. and limited to patients with fatal diseases such as malignant tumors.

The potential benefits of bone marrow transplantation have encouraged research into the causes and prevention of GVHD. It is. Animal studies have shown that GVHD is caused by donor 1928 bulbs. I understand. After removing 1928 bulbs from the donor bone marrow inoculum (“transplant”), The incidence of GVHD in mice, dogs and monkeys was reduced.

In human trials, the number of 1928 cells in bone marrow was reduced by various methods, but bone marrow recipients Other possible hematopoietic diseases that cannot prevent or cure GVHD Treatment methods include treating individuals with one or a combination of colony-stimulating factors. It's a method. During the process of blood cell formation, a small number of self-renewing stem cells become lineage-restricted. This lineage-committed progenitor cell then proliferates and differentiates. Circulating mature blood cells are produced, and this blood cell formation process is dependent on a group of glycoprotein hormones. It is maintained throughout life by long factors. These hormones are The aggregate is known as the C3F.

Ward 11” Shimine 1st dose The term colony-stimulating factor refers to the ability of CSF to stimulate progenitor cells of various hematopoietic cell lineages. This led to in vitro observations of the formation of distinct colonies of recognizable mature cells. It originates from The various factors are prefixed to indicate the main type of colony produced. , defined according to its function. 5 types of CSF genes in mice and humans The gene has been cloned and a recombinant form of the protein has been produced and purified. Ru. In addition to these factors, multipotent CSF (multi-CSF), 11 granular macros Phage C3F (GM-CSF), Granulocyte C3F (GCSF), Makrof These include phage C5F (MCSF) and erythropoietin. These factors For a review of the characteristics of offspring, the main target cells are described by 5 Leff, J. Cl1n Invest-, vol. 79, p. 1549, 1987.

15dan 1jo It is an object of the present invention to provide an alternative and/or alternative to bone marrow transplantation as a treatment for hematopoietic disorders. or to provide an auxiliary method.

Non-adherent (NA) cell lines are derived from cultures of bone marrow stroma isolated from various species. It was caused by The first NA cell line was developed from a monolayer culture of bovine stromal cells. I let it happen. Culture supernatant obtained from the parental bovine bone marrow cell line can be used to develop other bovine bone marrow cultures. Morphologically similar NA cell lines were obtained from bone marrow cultures and from bovine bone marrow.

NA cell lines can also be derived from other species, including human, porcine, mouse, and avian bone marrow. It was also induced by cell culture supernatant factors in troma cell cultures. something unexpected However, from the analysis of morphology, function, and phenotype, the obtained NA cells are very have primitive properties, and in fact appear to be multipotent, alternative hematopoietic progenitor cells. . These cells also appear to home in hematopoietic tissues, making them useful for treating hematopoietic disorders. Can be used.

Primitive cell colony stimulating factor, which is a protein factor The offspring (PC-CSF) are derived from NA cell culture and bone marrow-derived stromal cell culture. was identified in and isolated from the supernatant. These PC-CSF , particularly stimulates the proliferation of NA cells. These factors are therefore important for e.g. Relief in cell proliferation, including defects in fusion and double lobes, in individuals undergoing treatment present in an individual or as part of a treatment program for a hematopoietic disorder. to treat hematopoietic disorders by expanding lymphohematopoietic stem cells that are injected into No.-Available.

Accordingly, one embodiment of the present invention provides purified protocell colony stimulating factor (PC -CS F).

Other aspects of the invention provide recombinant vectors that feed PC-C3F and These are cells transformed with -.

Yet another aspect of the invention is purified PC-CSF-α.

Yet another aspect of the invention is purified PC-C3F-β.

Yet another embodiment of the invention comprises substantially mature lymphoid cells and myeloid cells. It is a cell suspension containing multipotent lymphohematopoietic progenitor cells (NA cells) that do not have .

Yet another embodiment of the invention is a suspension of human-derived NA cells, in which The cells are immunologically reactive with a monoclonal antibody directed against the My10 antigen. Rather, this monoclonal antibody has ATCC accession number HB-8483. Produced by hybridoma cell lines.

Another aspect of the invention provides a monoclonal antibody that is immunologically reactive with bone marrow-derived NA cells. Hybridomas that produce antibodies and the substances produced by these hybridomas It is a clonal antibody.

Yet another aspect of the invention provides a step of providing a cell suspension containing NA cells; Cells are grown in a medium containing C-CSF and other components essential for cell growth. growing said cell suspension under conditions that promote proliferation; removing NA from the cell culture; (NA) A step of isolating and recovering a population of cells; and PC-CS F, and conditions that promote cell growth in a medium containing other components essential for the growth of cells expanding said isolated cells; This is a method for producing cell populations.

Still other aspects of the invention include providing a cell suspension from hematopoietic tissue of an individual; contacting the cell suspension with a monoclonal antibody against NA cells; This antibody recognizes antigens on NA cells, but it also recognizes antigens on more mature lymphoid cells and myeloid cells. does not recognize antigens on cells; and from the cell suspension, cells to which the antibody has bound isolating and recovering cells containing multipotent lymphohematopoietic progenitor cells. This is a method for producing cell populations.

Other embodiments of the invention provide a suspension of NA cells in a pharmaceutically acceptable medium. Process: injecting an individual with a dose of NA cells sufficient to overcome the hematopoietic disorder. A method of treating an individual for disorders of the hematopoietic system, including: The individual is a pharmaceutically acceptable excipient at a dosage sufficient to stimulate the proliferation of NA cells in an individual. Treatment may be by injection of a solution of PC-CSF in a formulation.

Yet another aspect of the invention provides for the dissolution of PC-CSF in a pharmaceutically acceptable excipient. supplying fluid; and alleviating the symptoms of the disorder by proliferating the desired cells. administering to the individual a pharmacologically effective dose of pc-csF that , a method of treating an individual for a disorder that is alleviated by cell proliferation. The individual is , the hematopoietic disorder is treated by being given a dose of PC-CSF that alleviates the hematopoietic disorder. Ru. The individual is given a dose of PC-CSF that stimulates wound healing so that the wound heals. be treated. Individuals were administered a dose of pc-csF that stimulated regeneration of the dineuron. Niniron's super thick disorder is treated.

"blood! 4 clothes Figure 1 is a graph showing the cytotoxic effect of bovine NK cells on bovine NA cells. . Figures IA and IB show the absence and presence of IL-2, respectively.

FIG. 2 is a graph showing the cytotoxic effect of human NK cells on human NA cells. . Figures 2A and 2B show the absence and presence of IL-2, respectively.

Figure 3 is a graph showing the cytotoxic effect of mouse NK cells on mouse NA cells. be. Figures 3A and 3B show the absence and presence of IL-2, respectively.

FIG. 4 is a chart showing the induction of NA cell lines.

Figure 5 is a chart showing the effect of PC-CSF-containing supernatant on NA cell proliferation. It is.

FIG. 6 is a chart showing the system of the purification method for PC-C3F.

杢”self 1Δ left J and 1 ta ■, support The term "individual" as used in this application refers to vertebrates, particularly mammalian or avian mammals. includes livestock, sport animals, primates and humans, but does not include It is not limited to these.

The term "primordial cell colony stimulating factor ("PC-CSF")" used in this application term refers to factors derived from each individual, which include adhesive and and/or allow non-adherent cells to grow for long periods of time. Individuals in whom PC-CSF is induced is a vertebrate, preferably a mammal, more preferably a cow, a mouse , pig or human species. The term 'PC-CSF' refers to bone marrow straw stimulate the appearance and proliferation of non-adherent bone marrow cells in bone marrow cultures and/or whole bone marrow cultures. is meant to include analogs and fragments of factors that exhibit biological activity. This biological Activity is detected, for example, by the assays described in Examples 13 and 16 below.

“Purified PC-C3F” specifically coexists with cellular components and naturally with PC-CSF. PC-CSF is meant free of other soluble products. Like PC-C3F Methods for purifying polypeptides are known in the art and described below. .

As used in this application, the term "essentially free" refers to cellular components and soluble means at least about 50% free of product, preferably cellular components and more preferably at least about 70% free of cellular components and soluble products. Meaning at least about 90% free of soluble products.

As used in this application, the term "culture" refers to (unless otherwise specified) refers to a bone marrow cell culture that produces F. and is an immunomodulatory cytokine. may produce a factor of "Cultures" are bone marrow derived adherent cells and/or It may also contain non-adherent cells. The terms cell “culture” and cell “line” are used interchangeably. used in exchange. Cell cultures or cell lines include individual cells, harvested cells, and refers to various embodiments including, but not limited to, cultures containing cells derived from cell lines of Not determined. The term "derived from" means descendant or issue. do. Spontaneous or induced changes in karyotype occur during storage or transfer It is known in the art that this may occur. Therefore, the cell line-derived A cell culture is a cell culture that is not identical in character to the original cell or culture. includes such variations.

A "multipotent lymphohematopoietic progenitor cell" is a cell that exhibits the following characteristics. This cell is a bone Cells derived from medullary stromal culture.

In particular, from non-adherent cells whose proliferation is induced by PC-CSF in the culture supernatant. cells. Morphologically, these cells are of the monocyte-macrophage lineage. is similar to. Phenotypically, these cells contain T cell-specific antigens and MHC clusters. Lacks antigens, macrophage/monocyte markers, and B cell markers . Even if these cells are treated with IL-2, they become killer cells activated by lymphokines. It's hard to break out. Treatment with IFNγ, INFα, and TNFα does not affect these cells. directing to C5a, a strong chemotactic peptide, without inducing the production of reactive oxygen species. It does not induce sexual migration either. The cells were treated with NFγ, INFα and TNFα. However, it does not induce cell proliferation or change in cell morphology. multiplication Nocturnal lymphoid hematopoietic stem cells are home to hematopoietic tissue. 2, giving rise to undifferentiated colonies according to the CFU-S assay. ′Multipotent Li The terms "neoplastic hematopoietic progenitor cells" and "nonadherent cells" are used interchangeably.

The term "clone" as used herein refers to the progeny of a single cell.

The terms “recombinant host cell” and “host cell” refer to recombinant vectors or other vectors. microbial cells that can be or have been used as receptors for means a cell or eukaryotic cell and includes the progeny of the first cell that has been transferred or transformed It will be done. The progeny of one parent cell may differ in morphology and genome due to accidental or deliberate mutations. Regarding mu DNA Aiura body or total DNA complement, it is not necessary to refer to the original parent cell. It will be understood that they are not exactly the same. Nucleus encoding the desired peptide sufficiently similar to the parental cell, characterized by relevant properties such as the presence of leotide sequences. Progeny of a parent cell that are similar are included in the progeny referred to in this definition and are covered by the term above. Ru.

The term "transformation" as used includes, for example, direct uptake methods, transduction methods and Regardless of the insertion method, such as hybridization or cross-fertilization, means the insertion of

Exogenous polynucleotides may be present, for example, in non-integrating vectors such as plasmids. The polynucleotide encoding PC-C3F in this application is retained in the host genome or integrated into the host genome. The term "recombinant polynucleotide" used to characterize nucleotides is a genome, cDNAs semi-synthetic or polynucleotide of synthetic origin and these polynucleotides, depending on their origin or processing, are (1) All polynucleotides that are joined together naturally or in the form of a library. and/or (2) naturally associated polypeptides. Linked to a polynucleotide other than a nucleotide.

The term "polynucleotide" as used refers to ribonucleotides or dinucleotides. Refers to a polymeric form of oxyribonucleotide nucleotides having a certain length. do. This term refers only to the primary structure of the molecule. Therefore, double-stranded and single-stranded Includes DNA, double-stranded and single-stranded RNA. Also, for example, methylation and/or or cap-modified polynucleotides, and unmodified polynucleotides. Contains punch lines.

A “replicon” is a genetic element such as a plasmid, chromosome, or virus. and these elements behave as autonomous units of polynucleotide replication within cells. i.e. it can reproduce under its own control.

A "vector" is a vector to which other polynucleotide segments are linked and which A replicon that performs segment replication and/or expression.

"Control sequences" are the polynucleotides necessary to effect the expression of the coding sequences to which they are linked. means a nucleotide sequence. The nature of these control sequences varies depending on the host organism. Become. In prokaryotes, such control sequences are generally promoters, ribbon-like It has a joining part and a terminator. In eukaryotes, such regulation is generally Control sequences include promoters, terminators, and in some instances enhancers. I have it. The term ``control sequences'' refers to at least one control sequence that must be present for expression. This often means that it contains all the elements that are needed, such as a leader array. It may also contain additional elements whose presence is advantageous.

"Operably linked" means such a relationship that the components are capable of functioning in their intended manner. This means that they are placed side by side.

A control sequence that is “operably linked” to a coding sequence is a control sequence that is “operably linked” to a coding sequence. are linked in such a way that expression of the sequences is achieved under conditions compatible with the control sequences. .

An “open reading frame” is the region of a polynucleotide sequence that encodes a polypeptide. However, this region may represent part or all of the coding sequence. .

A “coding sequence” refers to the sequence of mRNA that when placed under the control of appropriate regulatory sequences A polynucleotide sequence that is transcribed into a polypeptide and/or translated into a polypeptide. be. The boundaries of the coding sequence are the translation initiation codon at the 5′-end and the 3°- Determined by the translation stop codon at the end. The coding sequence includes mRNA, c. Can include, but are not limited to, DNA and recombinant polynucleotide sequences. Not determined.

The term "immunologically identifiable with/as" means that the antigen is This means that an epitope is present in a cell antigen that is present in a given type of cell.

Immunological identity is measured by antibody binding and/or competition in binding. However, these methods are known to those skilled in the art and are discussed below.

The term "epitope" as used herein refers to an antigenic determinant of a polypeptide. The epitope can be composed of three amino acids in a unique conformation It is. Generally an epitope will have at least 5 such amino acids and more It is generally composed of at least 8 to 10 such amino acids.

Antigens are antigens that antibodies recognize specific epitopes contained within their polypeptides. is "immunologically reactive" with an antibody if it binds to that antibody. immunological reaction Responsiveness refers to binding to an antibody, more specifically the kinetics of binding to an antibody and/or the kinetics of binding to an antibody. Competition using known antigens containing epitopes for which the body has specificity as competitors It can be determined by the conjunction. Determine whether the antigen is immunologically reactive with the antibody Methods for doing so are known in the art.

The term "polypeptide" refers to the amino acid sequence encoded within the genome. product and does not mean a product having a specific length. Therefore, the peptide Polypeptides, oligopeptides, and proteins are included within the definition of polypeptide.

The term also refers to polypeptides such as glycosylation, acetylation, phosphorylation, etc. It does not imply modification after the expression of the code.

The term "therapy" as used herein means prophylaxis and/or treatment.

The term “hematopoietic system disorders” as used refers to disorders resulting from: Ru. That is, for example, pure lineage neoplasms of hematopoietic tissues (myeloid neoplasms and lymphocytic neoplasms) differentiation processes involved in hematopoiesis, such as immunodeficiency diseases and lymphopenia (including Gram changes; essential, such as human alpha-thalassemia and sickle cell disease Genetic defects associated with mutations in structural genes for proteins; including autoimmune diseases Genetic defects related to immune reactivity, especially in humans such as systemic lupus erythematosus, GVHD autoimmune hemolytic anemia, multiple sclerosis, myasthenia gravis, inflammatory bowel disease and collagen diseases, exemplified by atopic eczema, which are associated with abnormal T cell subsets. It is a related autoimmune disease.

Nee J” begging I bile Unless otherwise specified, the present invention relates to cell biology, cell culture, methods of the art, unless otherwise specified. Using conventional methods of nutrition, molecular biology, microbiology, recombinant DNA, and immunology and implement it. Such methods are well explained in the literature. For example, the following There is o　Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloni ng, Volumes I and ■ (edited by D, N, Glover, 1985); Oli gonucleotide 5ynthesis (M, J, Ga1tli collection, 1984); Nuclelc Ac1d Hybridization (B, D. Bames and S. J. Higginsii, 1984) ; Transcription And Translastiom (B , D. Hames and S. J. Higgins (eds., 1984); Cu1 true of Animal Ce1ls (R, 1, Freshney, Alan R, Li5s, Inc., 1987); 1m+aobiliz ed Ce1ls, And Enzya+es (IRL Press.

1986); B. Perbal, A. Practical Guide To Mo1ecular C1゜ning (1984); reatisa, Methods In Enzy+gology (Acad emic Press, Inc., USA, Niyok); Gene Tr ansfer Vectors For Mammalian Ce11s (J , H. Miller and M. P. Calosil collection, Cold Spring Harbor Laboratory, 1987); Methods In Enzymology, volumes 154 and 155 (edited by Wu et al.); I +mmunochemica1Methods In Ce1l And Mo 1ecular Biology (Mayer and Walker collection, A Academic Press, London, UK, 1987) and Handb ook of Experimental l++++*unollogy I -I V@ (D, M.

Weir and C. Blackvel, eds., 1986).

All patents, patent applications and publications mentioned in this application are hereby incorporated by reference. Both of these are incorporated by reference into this application.

n, A11l and Novel cell lines containing lymphohematopoietic progenitors are derived from bone marrow cultures from several species. isolated from These cells, also called “nonadherent” (NA) cells, Show characteristics. These cells are primitive in morphology. These cells have an original morphology. It is original. These cells include lymphoid cells and myeloid cells, as shown in the Examples. cells and lack certain cell surface markers that indicate stem cell characteristics of these cell lines. There is. Under in vitro conditions, the following lymphokines, namely interleukin 2 ( I L-2), γ interferon (IFN-γ), α interferon (IF These cells, regardless of the presence of N-α) and α-cell necrosis factor (TNF-α), The cysts do not differentiate into lymphoid or myeloid cells. However, NA cells When injected into one species, it will home in the hematopoietic tissue (e.g. bone marrow) within the inoculated individual. It seems that Additionally, the cells are known to measure hematopoietic potential. In the CFU-S assay, the hematopoietic activity of multipotent stem cells Show potential. Therefore, these cells appear to be multipotent progenitor cells. be.

Stromal cells and NA cells apparently do not differentiate into NA cells (replicate). secretes factors that cause One of these factors is that mouse NA cells in culture It seems to be secreted, but the apparent molecular weight is about 20-25 kd, and It was named colony stimulating factor α'' (PC-C3F-α).

The other factor appears to be a product of murine bone marrow stromal cultures, but is an apparent molecule. The amount is approximately 60-70kd, and this factor is combined with protocell colony stimulating factor β (PC- C3F-β) (margin below) L　B　PC-C3F The activity of PC-C3F is monitored by its effect on inducing proliferation of NA cells. Can be done. Two suitable assays for PC-C5F activity are described in the Examples. (See Examples 15 and 16. These examples include NA cells and stromal cells. and a clonogenic assay method with (indicated respectively). Interestingly, PC-C3F isolated from one species , which appears to be able to induce proliferation of NA cells from other species.

Using conventional methods of protein isolation, PC-C8F is enriched and released into NA cells. Other proteinaceous components of the conditioned medium can be substantially eliminated. of protein Purification methods are well known to those skilled in the art, eg, purification of components by precipitation. Separation methods: e.g. ion exchangers, adsorption methods including adsorption to affinity substances, dye ligands Separation by chromatography and immunosorbents; and in solution by gel filtration separation, such as electrophoresis, isoelectric focusing, isotachophoresis, liquid phase partitioning and ultrafiltration methods. See 5copes (1987). PC-C3F The methods by which it can be purified are described in the Examples.

Although PC-C3F can be isolated from conditioned media and hematopoietic tissues, it is difficult to isolate it using recombinant DNA methods. It can also be synthesized using In these methods, the set encoding PC-C3F is The replacement sequence is inserted into an expression vector so that the control sequences co-codify PC-C3F. is operably linked to a recombinant sequence encoding the recombinant sequence. host cells with this recombinant vector. transformed to express recombinant PC-C3F under appropriate growth conditions.

The recombinant sequence encoding PC-C5F is isolated using 9 molecular biologically known methods. It may also be derived from a sequence. For example, cDNA or genomic library scripts. or when mRNA is present in relatively high abundance. Isolated by reverse transcription of isolated mRNA encoding C-C3F.

Probes prepared as follows for library screening were: Used as a probe to isolate mRNA that is translated into the desired protein can do. NA cells can be lysed and the RNA separated from their DNA. be separated. Both parts where poly(A) is accumulated contain mRNA, but next Separation can be performed using an oligo-dT or oligo-dU column.

Important mRNAs were further purified using electrophoresis and no-in methods, and labeled with 32P. identified by a probe that has been identified. Once the mRNA is identified, from the 9 preparative gel It can be cut out.

The sequence encoding PC-C5F was extracted from mR using a commercially available reverse transcriptase. It can be prepared from rnRNA by reverse transcription of NA. obtained The cDNA is cloned and preferably sequenced. Next, the recombinant cDN A is inserted into an expression vector, and the expression control sequence encodes pc-cSF. operably linked to the array.

In another method, cDNA and/or genomic libraries are can be prepared from pulp cells, preferably from NA cells and/or stromal cells. , more preferably prepared from NA cells. How to prepare cDNA library , known in the art. For example, Maniatis et al., 198 2, and DNA Cloning Volumes I and 2, 1985.

The type of library constructed detects sequences encoding PC-C3F. Live cells that can be screened with oligonucleotide probes designed to For example, a library constructed in λgtlO.

To screen the library, a protein encoding a fragment of PC-C3F was used. probe that is the complement of a sequence encoding a lobe or a fragment of PC-C3F. To construct. This probe detects the sequence of PC-C5F by hybridization. preferably from about 9 to about 120 nucleotides. , more preferably has a length of about 30 to about 60 amino acids. recombinant porine The nucleotide probe is constructed from the amino acid sequence of a polypeptide fragment of PC-C3F. will be built. Using purified PC-C3F to determine the binding of a portion of the molecule can do. The sequenced portion is It is large enough. Methods for determining the amino acid sequence of polypeptide fragments are known to those skilled in the art. Generally the protein in question is.

Specifically cleaved into small peptides for ease of analysis. specific cut Detachment can be accomplished by chemical or enzymatic methods. chemical method Examples of cutting methods include cyanogen bromide, 0-iodosobenzoate (o-i odos.

benzoate), hydroxylamine and 2-nitro-5-thiocyanobe treatment with nizoate. As an example of a method using proteolytic enzymes Includes treatment with trypsin, clostripain and staphylococcal protease. be caught. The amino acid composition of the identified fragment can be determined and the fragment then 1 For example, amino acid analysis can be performed by automated Edman degradation using a sequencer. I can. Sequencing equipment is commercially available, and sequencing should be performed according to the manufacturer's instructions. Therefore, it can be implemented. The peptide fragment of PC-C3F has overlapping Tup fragments are generally prepared in two ways by which they can be analyzed.

Based on the observed sequence of the peptide derived from PC-C5F, the amino acid sequence was A recombinant protein encoding PC-C3F by back-translation into the nucleotide sequence polynucleotide probes can be designed. These probes include Consider using the preferred codons of the species from which PC-C5F was isolated. considered.

Screening of a library of sequences encoding PC-C3F was performed using Mani atis et al. (1982) and DNA Cloning volumes I and II ( This can be done using the method described in (1985). containing that array Once a clone with the Clone and sequence. Only part of the sequence encoding PC-C3F If isolated, the isolated sequence contains overlapping fragments of sequences. can be used to construct probes that detect clones in the original library. I can do it.

These sequences are then cloned and sequenced. This method uses PC-C It can be repeated until all sequences encoding 5F are defined.

Next, a recombinant polynucleotide encoding PC-C3F is inserted into an expression vector. and the expression vector is used to transform host cells. General people listed above The law is set out below.

Recombinant host cells are selected by cloning and the desired protein is expressed. cultured under the following conditions. If the protein is not released, harvest the cells and , lysed according to conventional methods and the protein isolated according to conventional methods. Ta If protein has been released, extract it from the nutrient medium used. can be done.

As discussed earlier, when determining the sequence of a polypeptide, cDNA and/or or prepare and probe genomic libraries and sequence clones. When constructing an expression vector and transforming cells, General methods are known in the art and the practices in which these methods are described. A test guide is available. However, as a general guide, such a method and several sources of information currently available on materials useful in doing this. It is described below.

ff, c, 1. Host and Expression Control Sequences Both eukaryotic and prokaryotic host cells , the desired coding if appropriate control sequences are used that are compatible with the designated host. It can be used to express sequences. Among prokaryotic hosts, E. coli (E, Co 11) is most often used. The expression control sequence for prokaryotes is the promoter and optionally an operator moiety and a liposome binding site. prokaryotic cells Transfer vectors compatible with the host are typically those derived from, for example, pBR322.

This pBR322 confers resistance to ampicillin and tetracycline. This is a plasmid containing an operon. Furthermore, various pUC vectors also It has a sequence that provides a biological resistance marker. These markers select This method is used to obtain satisfactory transformants.

Commonly used prokaryotic control sequences are β-lactamase (penicillinase) and lactose promoter system (Chang et al. 1977), tryptopha (trp) promoter system (G.

Eddell et al. 1980), and the λ-inducible PL promoter and N gene. Gene liposome binding site (ShiIIIatake et al., 19 Chirus sp. (B ac i 11us) or Pseudomus.

Other prokaryotic hosts with corresponding regulatory sequences, such as strains of nas), can also be used. Wear.

Eukaryotic hosts include yeast and mammalian cells in culture media systems. Satucharomyces ・Saccharomyces 5erevisiae and Satsuka Romyces carlsbergensis (Sacchar.

myces carlsbergensis) is the most commonly used yeast. host and a convenient fungal host. Vectors compatible with yeast have nutritional requirements confer prototrophy on sex mutants or resistance to heavy metals on wild-type strains A marker that allows the selection of satisfactory transformants by giving have. Yeast-compatible vectors include a 2 micron origin of replication (Qro ach et al., 1983), a combination of CEN3 and AR3I, or a host Using other methods to ensure replication, such as sequencing that incorporates the appropriate fragment into the cell's genome It is possible. Yeast vector control sequences are known in the art and include glycolytic enzymes. Promoter for the synthesis of ()less et al., 1968; Holland et al., 1978) and the 3-phosphoglycerate kinase promoter ( Hitzeman, 1980). Enolase gene origin Terminator () Rolland, 198 1 year) is also included. A particularly useful control system is glyceraldehyde 3-phosphate Promoter of dehydrogenase (GAPDH) or alcohol dehydrogenase promoter (ADH), terminator derived from GAPD) - and, if secretion is desired, a control consisting of a leader sequence from yeast α-factor. It is a system. Additionally, the transcriptional regulatory region and the transcriptional initiation region may be operably linked; These are not naturally linked in wild type organisms.

Mammalian cell lines available as hosts for expression are known in the art; Many available from the American Type Culture Collection (ATCC). Contains many permanently proliferating cell lines, including Hela cells, Chinese muster ovary (CHD) cells, newborn hamster kidney 11 (BH) cells, etc. There are numerous cell lines. Suitable promoters for mammalian cells are also known in the art. and simian virus 40 (SV40) (Fiers, 1978). , Rous sarcoma virus (RSV), adenovirus (ADV) and bovine papilloma Viral promoters such as the viral promoter from the virus (RPV) can be mentioned. Mammalian cells also require terminator sequences and polyA addition sequences. , also includes enhancer sequences that increase expression, and sequences that cause gene amplification. is also desirable. These sequences are known in the art. Replicates in mammalian cells Suitable vectors to use include viral replicon or NANBV epitope. Sequences that ensure integration of suitable sequences encoding the loop into the host genome are mentioned. Ru.

n, C, 2, transformation Transformation can be any known method of introducing polynucleotides into host cells, e.g. If the polynucleotide is loaded into a virus, host cells are transduced with the virus. methods, and methods by direct incorporation of polynucleotides. used The transformation method used depends on the host being transformed. For example, E. coli host Transformation of cells with λgtll containing the BB-NANBV sequence was performed as described below. This will be discussed in examples. Bacterial enrichment by direct incorporation generally involves calcium chloride. Treatment with rubber or rubidium chloride is used (Cohen, 1977). 2 years; Maniatis, 1982). Yeast traits by direct uptake The conversion was carried out using the method of former nnen et al. (linnen et al., 1978). can be done. Mammalian transformation by direct uptake is described by GrahaIII and Van der Eb's calcium phosphate precipitation method (Graham and Van der Eb (1978), or various known variations of this method. I can do it.

n, C, 3, Vector construction Methods known in the art are used for vector construction. site-specific DNA Cleavage is carried out under conditions generally specified by the manufacturer of commercially available restriction enzymes. This is done by treatment with restriction enzymes. Generally, about 1 μg of plasmid is also Alternatively, the DNA sequence can be prepared in approximately 20 microliters of buffer containing 1 unit of enzyme. Cleavage is performed by incubating at 37° C. for 1 to 2 hours. system After incubation with limiting enzymes, proteins were washed with phenol/chloroform. removed by extraction and recovered by precipitating the DNA with ethanol. do. The cut fragment is εnzymoIogy, Vol. 65, pp. 499-560. , 1980, polyacrylamide was also used. Alternatively, they can be separated using agarose gel electrophoresis.

The sticky-end cleavage fragment is made by adding the appropriate deoxynucleotide triphosphate present in the mixture. In the presence of acid (aNTP), E. coli DNA polymerase I (Flenow) This can be used to create a smooth residual limb. Treatment with S1 nuclease can also be used, Any single DNAfi portion is hydrolyzed.

The ligation reaction was performed using standard buffer and temperature conditions using T4 DNA IJ gauze and AT Execute using P. The ligation reaction of sticky ends requires ATP and gauze. less than for blunt-end ligation reactions. Add the vector fragment to the ligation mixture. When used as a bacterial alkaline phosphatase (BAP) or bacterial alkaline phosphatase (BAP), The vector is religated by treatment with intestinal alkaline phosphatase to remove the 5'-phosphate. It is often prevented from occurring.

Alternatively, restriction enzyme digestion of undesired fragments may be used to prevent ligation.

Use the ligation mixture to transform a suitable cloned host such as E. coli. and then selecting satisfactorily transformed cells, e.g. by antibiotic resistance, Then screen for the correct structure.

Prepared using a reported automated oligonucleotide synthesizer. As desired, The synthesized strands were synthesized using standard conditions for the reaction, ``polynucleotide in the presence of P-ATP''. Labeling with 32P may be achieved by treatment with tidokinase.

DNA sequences include DNA sequences isolated from cDNA libraries, but , for example in Zoller (1982). Site-directed mutagenesis Modifications can be made by known methods, including methods. Simply put, the DN to be modified A is - packaged into the phage as a heavyweight sequence and modified as a primer. It is complementary to a portion of the desired DNA and contains the desired modification part in its own sequence. Double-stranded DNA is synthesized by DNA polymerase using synthetic oligonucleotides. Converted to A. Using the obtained double-stranded DNA, a host bacterium that retains the phage Transform. The transformed bacterial culture contains copies of each phage. Obtain plaques by plating on agar. Theoretically a new plaque 50% contain phages with mutated sequences, and the remaining 50% contain phage with the original sequence. Has a column. Plaque replicas are able to hybridize with the correct strand. However, unmodified sequences cannot be labeled at temperatures and conditions that do not allow hybridization. form a hybrid with the attached synthetic probe. Identified by hybridization The resulting sequence is recovered and cloned.

The hybridization NA library with the n,C,5,probe was probed using the method of Unstein and Hogness, 1975. obtain. Briefly, in this method, the DNA to be probed is immobilized on a loin filter, denatured, then 0-50% formamide; 0.75M NaCl, 75mM sodium citrate, bovine serum albumin and potassium Rivinylpyrrolidone and Ficoll each at 0.02% (wt/v), 50mM Sodium phosphate (p) 16.5>, 0.1% SOS, and 100 μg/ml Prehybridization is performed with a buffer containing DNA denatured with a carrier of cormorant. The percentage of formamide in the above buffer and the prehybridization step. The time and temperature conditions for the subsequent hybridization step are determined by the required stringency: 1. I came over Ru. Oligomeric probes that require low stringency conditions generally have low percent medium formamide, used at low temperatures and long hybridization times. c　 As many as 30 nucleotides, such as those derived from DNA or genomic sequences. Probes containing more than 40% are generally heated at higher temperatures, e.g. 2° C. and a high percentage of formamide, for example 50%. P Following rehybridization, 5°-322 labeled oligonucleotide probes The lobes are added to the buffer and the filter is then hybridized in this mixture. Incubate under pad formation conditions. After cleaning, the treated filter is automatically The positions of probes that were subjected to radiography to form hybrids are shown. original Use the DNA at the corresponding position on the agar plate as the super-thickness of the desired DNA. Ru.

The usual vector construction method is to use a ligation mixture to OI or other suitable host is transformed, and satisfactorily transformed cells are treated with antibiotics. Selected by resistance or other markers. Then the plus from the transformed cells The medium was prepared according to the method of Clewell et al., 1969, and then Loramphenicol amplification (Clewell, 1972) is performed. That D The NA is isolated and analyzed, usually by restriction enzyme analysis and/or sequencing. Ru. Sequencing was performed using the dideoxy method of Sanger et al. (Sanger et al., 1977). 7), as well as the method described in Messing et al., 1981, or Ma Xam et al., 1980. Sometimes I look at areas with a lot of GC. The problem of crowded bands is discussed in Barr et al., 1986. is overcome using T-deaziguanosine according to the method described in .

Preparation of antibodies against Il, D, NA cells using NA cells obtained from different species. Both polyclonal and monoclonal antibodies can be produced. polyclonal When you want antibodies, use a selected mammal (e.g. mouse) that is xenogeneic to the NA cells of interest. , rabbit, goat, horse, etc.) with NA cells or lysed NA cell preparations. , preferably in the presence of an adjuvant. Serum from immunized animals are collected and processed according to known methods. Polyclonal antibody against NA cells If the serum it contains contains antibodies against other cell types, polyclonal Antibodies can be purified by immunoaffinity chromatography. Polik Methods for producing and processing local antisera are known in the art. For example l See 1layer and Walker, 1987.

Monoclonal antibodies directed against specific epitopes on NA cells can also be used in this technique. It can be easily produced by one skilled in the art. by hybridoma General methods for making monoclonal antibodies are well known. Immortal antigen-antibody Cell lines can be obtained by cell fusion method or by direct transformation of B'J lymphocytes with cancer DNA. , or prepared by other methods such as those introduced by the Epstein-Parr virus. can be done. For example, M. 5chreier et al., 1980; Ha Mmmerling et al., 1981 HKennett et al., 1980 , U.S. Patent No. 4.341.761: U.S. Patent No. 4.399.121: U.S. Patent No. 4.427.7 No. 83; No. 4.444.877: No. 4,452, 570: No. 4.466, 9 No. 17: No. 4.472.500; No. 4.491.632; and No. 4.49 See No. 3.890. Panel of monoclonal antibodies produced against NA cells using known methods including, for example, flow cytometry. can be screened for specificity for cells. Especially that scree Numerous methods include those listed in Tables 1 and 2 below. Examples include testing antibodies against mature cell types. More mature cell types An antibody specific for NA cells is selected in that it does not bind to. On top of that, antibodies Also screened for various characteristics, i.e. isotype, epitope affinity, etc. can be done.

Both monoclonal and polyclonal antibodies induced against NA cells , this method is useful for identifying and isolating these cells from bone marrow or other lymphohematopoietic cells. To separate NA cells from more mature cells in the organ, antibodies, preferably A method using monoclonal antibodies is being considered. Generally contains hematopoietic cells. A cell suspension prepared from a tissue (e.g. bone marrow, pancreas, bursa) , with a preparation of anti-NA cell antibodies. Cells to which the antibody has been bound are transferred to cells in the art. The cells are separated from unbound cells by means known to those skilled in the art.

Various methods are known for separating antibody-bound cells from unbound cells. example For example, the antibody-cell complex can be labeled, so that the cell can detect the presence of the label. The cells are separated using a mechanical cell sorter that detects the presence of cells. Fluorescence-labeled cell sorter is based on this technology. Well known in the field. In a preferred embodiment, the anti-stem cell antibody is attached to a solid support. connected to the body. A variety of solid supports are known to those skilled in the art, and In addition to galose beads, polystyrene beads, hollow fiber membranes, and plastic pellets are also available. An example is a bird dish. Antibody-bound cells are removed from the cell suspension onto a solid support. They can be removed from the cell suspension simply by physical separation.

Select cytophoresis is used to produce cell suspensions from hematopoietic organs. ive cytophoresjs) can be used.

For example, bone marrow can be obtained from a donor by any suitable means.

The obtained bone marrow can be used as desired by primarily using the recovered cells. can be processed separately. Suspension of bone marrow cells recognizes antigen on NA cells can be brought into physical contact with a monoclonal antibody, which is bound to the solid phase be done. Incubation of antibodies bound to solid phase with bone marrow cell suspension The exact conditions and duration will depend on a number of factors specific to the system used. deer The selection of appropriate conditions is within the skill of the art.

After binding for an appropriate time, unbound cells are eluted with physiological buffer. Or wash it off. The bound cells are then separated from the solid phase by a suitable method. Ru. For example, bound cells can be eluted by vigorous agitation. a Alternatively, the “spacer” sequence that binds the solid phase and antibody can be cleaved or deleted with an enzyme. It can be eluted by

Both eluted and concentrated portions of cells are then washed with buffer, followed by regular cryopreservation. method to cryopreserve the implant in a viable state for later use or Immediately inject into recipient intravenously. Instead, cells are placed in cell culture medium for later use. May be maintained with nutrients. Appropriate conditions for culturing cells are within the skill of the art. The conditions under which NA cells are maintained in culture are described in the Examples.

The above cell suspension of NA cells is suitable for use in progenitor cell transplantation methods and for those skilled in the art. It can be used in such therapeutic methods as well as in obvious methods. For example, pharmaceutically acceptable Cell populations suspended in excipients can be added to individuals in need of cell replacement therapy. Directly administered intravenously in an amount sufficient to reconstitute the hematopoietic and/or immune systems of can do. Pharmaceutically acceptable excipients include 9 e.g. saline. a physiologically buffered isotonic salt solution, and glucose or dextrose. contains an isotonic solution of water. Other components of the cell suspension are as follows for PC-C3F: It may be one listed in . The precise effective amount can be readily determined by one of ordinary skill in the art. This will, of course, depend on the exact symptoms being treated with the therapy.

However, in many applications, the 172 to 11 Amounts containing approximately the same number of NA cells should be suitable.

A method of treating a hematopoietic disorder in an individual includes treating the individual with PC-C5F. Ru. Treatment with PC-C3F may be alone or with e.g. Other treatments such as cell replacement or cell expansion may also be combined.

PC-C3F may be prepared as an injectable solution, such as a liquid solution or suspension. : manufactured in solid form suitable for liquid solution or suspension in preparation prior to injection. It's okay. The formulation may be emulsified or the protein may be encapsulated in liposomes. You can also set it to . Active PC-C5F contains pharmaceutically acceptable and active ingredients. Often mixed with compatible excipients.

Suitable excipients include water, saline, dextrose, glycerin, Ethanol, etc. and combinations thereof.

Additionally, if desired, the PC-C3F formulation may contain wetting agents or emulsifying agents, and p It may also contain small amounts of auxiliary substances such as H buffers. Typically, this preparation includes e.g. It is administered parenterally, for example by intravenous, subcutaneous or intramuscular injection. other Other suitable formulations for administration include suppositories and, in some cases, oral formulations. used. Suitable materials for these formulations are known to those skilled in the art. . PC-C3,F is administered in a manner compatible with the dosage form and in a therapeutically effective amount.

The dosage will depend on the hematopoietic disorder and the recipient being treated.

Although PC-C3F may be administered in a single dose regimen, it is better to administer it in a multiple dose regimen. preferable. Multiple-dose therapy refers to initial treatment in 1 to 10 separate doses, followed by at intervals of time necessary to maintain, improve and/or intensify the therapeutic response. Another method of administration. The dosing regimen may also be tailored, at least in part, to the needs of one individual. and the judgment of the doctor.

The following examples are provided to demonstrate certain embodiments of the invention. child These examples are for illustrative purposes only and are not intended to limit the scope of the claimed invention. It doesn't come out.

Bone marrow cultures were derived from bone marrow aspirated from two Holstein calves. . Bone marrow cells were collected from the iliac crest of two anesthetized calves (both younger than 1 year of age). and collected in 2x citric acid solution. The obtained bone marrow sample was cultured in minimal essential medium (MEM). ) for 15 minutes at 2.000 rpI11. Centrifuged. The resulting cell pellet was resuspended in MEM and the cell suspension The solution was layered onto Ficoll Hyback density gradient solution for 30 min at room temperature (rt). The mixture was centrifuged at 2.00 rpm. Following centrifugation, at the gradient interface Collect the cells, dilute with 111EM, and centrifuge again for 10 minutes at 1.000 rpm. I let go. The resulting cells were further washed twice. After the final wash, the cells were Resuspended in RPMI-1640 supplemented with % fetal bovine serum (PBS). child The cell suspension was adjusted to a final cell concentration of 2×10′ cells/d. Then this The bone marrow cell suspension was placed in a 15 m In 1 volume of growth medium (RPMI + 10% FBS), add 1X10' cells/plate. It was inoculated at a concentration of Next, this culture plate was placed in an air atmosphere containing 5% CO□. The cells were incubated at 37° C. in a humidified warmer below. The two cultures obtained They were named B, BM, 1 and B, 8M, 2.

During the first 2 to 3 weeks of culture, a population of adherent cells that have grown on the surface of the vetri dish will appear. It was. When these cells are in a fused state, the T75cof file is injected as follows. The cells were transferred to a Lasco and subcultured. Place the vetri dish containing the confluent monolayer of adherent cells in the bag. Versene (registered trademark of EDTA) trypsin solution (5% ) until excess growth medium was removed. After the second wash, 2.0 Add the above Versen-trypsin solution of r111 and incubate the plate at 37°C. Incubate for 5 minutes and inspect the plate to see if cells have separated from the plate surface. 1. Add 10 ml of RPM1 containing 10% FBS to the plate. , and then stirring to obtain a single cell suspension. The obtained cell suspension The suspension was centrifuged at 1.00 rpm for 8 minutes, and the supernatant was discarded. The obtained fine Resuspend the cell pellet in fresh growth medium and adjust the cell number to 2 x 10” cells/ml. It was knotted. This cell suspension was then mixed into 40 cells of culture medium at 4X10' cells/flask. It was placed in a sterile T 75CJl flask to achieve the desired concentration. Next, this cell is 1 as an adherent population using Dulbecco's minimum essential medium supplemented with 0% fetal bovine serum. Successive subcultures were performed.

B, BM, 1 and B, BM, 2 adherent cell populations were not passaged As a result of further proliferation for 2 months, a duplicate population of NA cells was formed on the surface of the adherent cells. I was able to propagate it. Cells in both bottles were subcultured and stored8. A portion of 8M, 2 was stored in liquid nitrogen. B, 8M, 2 are adhesive (strobe) (Ma) cells and NA cells were maintained in culture as a mixed population.

During initial culture and maintenance of NA cells, NA cells are physically attached to the stromal monolayer. I found out that it was glued. Allows NA cells to separate without physical adhesion placed in a chamber that can only “communicate” through soluble factors. 1. However, the NA cells died. But there is a stroma below the chamber. Small colonies of NA cells developed in the area.

Initial attempts to grow NA cell populations without stroma were unsuccessful. But the most By utilizing the conditioned medium from the first B, BM, 1 culture, the straw A NA cell line containing no cells was isolated and maintained in culture. These cell lines are It comes from several species. In some cases, i.e. human and porcine bone marrow cultures. For culture, to obtain NA cell cultures, bone marrow cultures are grown in culture from bovine bone marrow cultures. It was necessary to treat the cells twice with the culture medium. In these cases, treatment with conditioned medium lasts for 4 days. Structural analysis of intercellular cell lines is performed using thin-section electron microscopy. I used it. Stromal cells and NA cells were prepared separately for testing.

NA cells were washed thoroughly, removed from the culture by pipetting, and strained. Rho-7 cells were removed by trypsinization. Cell pellets with 2,5% O containing gel taraldehyde, 1M phosphate buffer (PBS) pH 7.1 The cells were pre-fixed in 1% 0 SOJ PBS and post-fixed in 1% 0 SOJ PBS. Ultra-thin sections with uranyl acetate It was stained with lead citrate and examined in a Philips electron microscope at 60 kV.

Stromal cells removed by trypsinization contain numerous cells of varying length and thickness. It seems to have a rippled membrane due to filipodia. be. The cell body was 20-25 millimicrons in diameter. The nucleus is the nuclear layer It is highly pleomorphic and euchromatic, with narrow bands of heterochromatin along the It was. A large jin was usually present. The cytoplasm is electron-dense and contains many intracellular organelles. It looked ugly. The cellular organelles have a well-developed Golgi region, a very dense matrix. Elongated mitochondria with trix, polymorphic with granular and filamentous contents It contains several vesicles and numerous rough endoplasmic reticulum (RER). RER expands Contains dense proteinaceous material. fibers in many cells Patches of fibrous material occupied the peripheral area of the cytoplasm. This is the “adhesive device” for cells. It constitutes the government.

NA cells in their initial culture form have progranulocytic characteristics with a small number of mitotic cells. The cells were of uniform size and morphology, with the exception of rare cells. NA cell size The sections were approximately 12-16 millimicrons in diameter. This cell is round or bilobed It appears to have a shaped nucleus, which is usually euchromatic and has a large membrane. It has connected ren. Its cytoplasm contained relatively few organelles; The intracellular organ consists of a small number of chains of RER, and the number fluctuates and expands greatly. Composed of many mitochondria and a few small electron-dense granules and multivesicular bodies It had been. However, there are many free liposomes and polyliposomes in the cytoplasm. Since it exists, the electron density seems to be high.

NA cells in the second induced culture were grown as described in Example 2. These are all monocytes, although they appear to be more heterogeneous in size and morphology. They resembled cells of the macrophage lineage. The shape of the nucleus is similar to that of the primary culture. However, the nucleus was often rich in heterochromatin. The cytoplasm is It is characterized by an abundance of isolated liposomes and polyliposomes, and even more The vesicles were fully equipped with intracellular organelles. A prominent Golgi region is observed in many cells. , this region was composed of large stacks of flattened vesicles and continuous vesicles. R E! R is also more abundant in these cells than in the cells of the initial culture system, and is Signs of activation (exo/endo, auto) are evident within some cells. Ta. Cellular heterogeneity somewhat reflects differences in cell age more than differences in differentiation pathways. It's showing. The reason is that signs of aging also sometimes appear in cells, and these This is because the phenotypic morphology of the cells appears to be homologous (see Example 4 and Table 1).

(Margin below) Example 4 were studied using semi-quantitative membrane fluorescence and flow cytometry (IJ-analysis).

These cells are NA cell line.

The phenotypes of two bovine cell lines B, BM, 1 and B, BM, 3 were compared to the T cell sub- set, MHC class I and class II antigens, macrophages/monocytes, null cells ( Panel of monoclonal antibodies specific for non-T/non-B), # and B lymphocytes It was analyzed using Panel of monoclonal antibodies, Pull, Washington, USA Mann, Washington State University, W. C. l1avis. anti-corrosion used The bodies and their specificities are shown in Table 1.

Flow cytometry analysis was performed as follows. Pellet bone marrow cells 1.00 Or pm for 8 minutes), 0.2% gelatin (Sigma Chem , Co, St. Louis, Missouri, USA), 1mM sodium azide, and and 2.0% normal rabbit serum (Cooper biomedical, Pediatrics, USA). Phosphate buffered saline (PBS G) containing (Malvern, NC) resuspended at a concentration of 2XIQ' in To study the surface phenotype, 50 μl The cell suspension of 0 μl and incubated for 30 minutes at 4°C. Cells in ice-cold PBS G FITC-labeled F (a b’) 2 goat anti-mouse immunoglobulin (heavy and light chain specific, Cooper Biomedical, Malvern, PA, USA) and 100 μl of Both were incubated at 4°C for 30 minutes. After incubation, cells were transferred to PB Washed three times with SG and fixed in a 2% solution of formaldehyde in PBS G.

The resulting cell preparations were kept in the dark at 4°C until analyzed. Cell aggregates are Fc Immediately before performing the analysis, a 62-millimeter nylon monofilament mesh <Small Parts Inc., Miami, Florida, USA) and removed from all samples. Non-specific labeling due to Fc receptor (PcR) binding Appropriate controls were added for detection. In these controls, bone Myelin cells respond to FITC-labeled F(ab')z alone or to an unrelated antigen. specific, isotype-matched monochromes Reacted with local antibodies.

The FITC-labeled bone marrow cells were transferred to Courter Electronics. Ltd. EPIC3V flow cytometer. 20,000 thin Data were collected from the cells and analyzed for patterns of monoclonal antibody reactivity. Before Two-parameter analysis of azimuth angle light scattering (FALS) versus 90° light scattering (Li2O) was used to remove dead cells and debris from the population under analysis. Fluorescence data, fluorescence Both scatter plots of FALS and histograms of fluorescence versus cell number and and collected. The percentage of positive cells was calculated using the immune program (CoulterIl!Ie ctronics Ltd, M-mouth ADS System, Version Determined using 3.1).

This is the result of flow cytometry analysis of B, BM, 1 and B, BM, 3. These cell lines do not express T- or B-cell markers and are monocyte/macrophocytes. It was also found that no phage markers were expressed. These cell lines also have MHC It also lacks expression of class I (IA) antigen and T4 helper cell antigen. But All cells express MHC class I antigens.

Immunocytochemical studies of these cells were performed to further characterize these cells. went. Immunocytochemical demonstration of surface membrane and cytoplasmic phenotypic markers: 1. Bi Ohmann, J., Histochea+, Cytoch Em, vol. 35, pp. 627-633, 1987. I put it away and went. Simply put, white blood cell antigens are immunized with monoclonal antibodies. panel (Table 1), appropriately dilute the antibody in question, and dilute it with poly-Li-lysine. Cytospin preparations were incubated on coated slides for 1 hour. Detected by incubation. After washing three times, rabbit anti-mouse I g and incubation was repeated for 30 minutes, followed by washing. Arca The lyphosphatase anti-alkaline phosphatase complex is then added. final cleaning Afterwards, slides were washed with substrate (0°2M) Lys buffer containing phosphate (2MgAs-M). solution pH 8, 2, 1mM lidocaine, and 1mg fast TR salt) was incubated. After counterstaining with methyl green, cell preparations were stained with aqueous gelatin. It was placed in latin and examined under an optical microscope.

The results of the phenotypic analysis using immunocytochemical methods are shown in Table 1. These results are , Consistent with the flow cytometry analysis results, B, BM, 1 and B, BM , 3 cells contain T cell markers, M) Ic class ■ antigens, B cell markers, and and were shown to lack some macrophage markers. However, In contrast to raw cytometric analysis, immunocytochemical methods were used for 18A, CA 137A and Cl116A were also present. Immunocytochemical methods are used to detect intracellular antibodies. Flow cytometry detects surface-bound antigens, whereas flow cytometry detects surface-bound antigens. monitor only those antigens that have been detected. The detected difference is due to this in the specificity of the two methods. may be related to differences in

Table 1 874 null cell phenotype of cultured bovine bone marrow cells determined by immunocytochemical method 99. ONT BL8A Granulocytes, monocytes 92.7' NT subset T-lymphocytes Subset (?) DH59B Blood monocyte 97.9 N7M718 Mature macrophage 0.0 NTT) 114B MHC class n O, 00H42A MHC class n o ,o.

CH128A T-lymphocyte (E-roset) 0.0 5.5'R1 (1 2A MHC class 1 5.2' 100' BIg73 B-lymphocyte (Ig) I) <20GS5A B+T lymphocytes? 0? subset BAQ44 B-Lymphocyte? ? H18A Monocyte-subset (?) 100 NTR1 (16A MHC class I-/? 100bB29A Pan T-lymphocyte ONT1134A Single Globule-Subset 95.7 NTC) 116A Monocyte-Subset 94. ON TJL-227riv7y-di<1' NT subpopulation (mature cells) E4CT-lymphocyte subset 0,0 111TE40CO,ONT )1tlH27 granulocytes (neutrophils) 0. ONTB4Q4A non-T non-B cells <1 'NTBAS9A B-lymphocyte? NT 1134A M) Ic class n o, o.

(1987) of cytospin on poly-lysine-coated slides. stained using a mouse monoclonal antibody conjugate (the latter two reagents were AKOPATTS a/w, Denmark, Grosdrop). Cromo The compound was naphthol-XM-phosphate. At least 100 cells are scanned. Each ride was counted.

Only 4 antigens are expressed intracellularly. Only giant cells were positive.

もWeak antigen reaction (low staining intensity).

6 Weak suspicious reactions in/on some cells.

6 Positive cells present only in giant cells.

car, B cell markers and several tumor cell lines.

The function that identifies a cell as a member of a lineage is first incubated. Ability to migrate to activated bovine serum, a tumor cell marker, even in non-baked cells (chemotaxis).

It was. That is, IFN-7, IFN-α, and TNF-α affect the number of colonies. No changes in cell morphology or colony type were observed.

One of the main roles of natural killer effector (NK) cells is to is to regulate hematopoiesis by eliminating or modulating the competent stem cell population. <<Barlozzaci et al., 1987). NK cells to bone marrow cells The cell-lethal effect on the I watched it. This method uses NA bone marrow cells (B, BM, 3.) IU, BM, 2 and MU, BM, and 2) were labeled with Cr and cultured in human, bovine, and mouse lungs. This method is used as a target for NK bound to peripheral blood lymphocytes derived from . This effector cell (NK) in the presence and absence of recombinant IL-2 The cells were assayed for cytolytic activity.

As can be seen from the results shown in Figures 1.2 and 3, cows, leeches, and to a lesser extent The respective bone marrow cell lines of the burr mouse are derived from putative bovine, human and murine NK. Serves as a target for non-MllC-restricted cytolytic reactions by cells. This thin Cytolytic reactions induce NK cells to be immunized with IL-2 for 48 hours before interacting with adherent bone marrow cells. Time processing significantly accelerates this.

Pretreatment of bone marrow cells with recombinant IL-2 also allows these cells to have their own or Notably, it does not induce effector functions against tumor targets like 562. Worth it.

Mouse NA bone marrow cells, Mll, BM, 2 were labeled with 32p and intravenously injected into mice. inoculated through. After an appropriate amount of time has passed after inoculation, the mice are killed and exposed to placed on the rum. Substances labeled with 32p by developing exposed X-ray film was examined to determine whether it was present in the hematopoietic tissues of inoculated mice.

As a result, we found the following. In other words, the amount of 32p in mouse bone marrow can be detected within 2 hours. It became possible to detect most of the labeled cells within 30 hours, and the labeled cells were found in mouse hematopoietic tissues. It was shown that he had returned to the nest.

Measure the hematopoietic potential of multipotent stem cells by CF[I-S assay.

CPU-5 assays were performed on lethally irradiated mice. B.A. A dose of V irradiation of 1200 rad/whole body (300 rad/min) to LB/C mice. was irradiated with radiation. This dose has been determined in advance to be the LD5° dose. Ta. A small amount of about 1000 MU, BM, and 1 piece was applied to the irradiated mouse. When the cells were inoculated, a large number of them occurred in the lungs. 103 or Mice injected with 108 cells each received 10 cells per mouse after 13 days. Formed more than 30 colonies.

General histomorphological appearance of lungs of inoculated mice suggests immune activity Ta. This classification has well-developed secondary follicles and thick, cellular periarteriolar lymph nodes. Based on PALS (PALS). When this expansion of the ``white pancreas'' occurs, Concomitant reduction of the red pancreas occurred.

The small group of blastoid cells is 2 to 4 times larger than the follicular IJ lymphocytes, but is the main component of the white pancreas. During the pancreatic phase, it extends deep into the pancreatic parenchyma or directly beneath its capsule. So small cell colonies are separated by non-lymphoid structures (connective tissue or endothelium) It was not clearly separated from normal lung parenchyma. these small colonies The cells inside appear homologous and contain abundant protoplasm and large orthochromatic nuclei. Ta. Mitosis varied in frequency.

Large subcapsular colonies were also present within the am of the inoculated mice. Blast-like cells Unlike the small colonies of vesicles, this large subcapsular colony is more heterogeneous; It had mitotic cells and pyknotic cells. Nuclear pyknosis is generally an indicator of cell degeneration. Ru. This colony is partially demarcated by endothelium/connective tissue, and is divided into small arteries or were well vascularized with small veins. Sometimes coagulative necrosis is present in the center of the colony. there was. Large colonies have some resemblance to lymph node parenchyma (paracortical area) However, the most distinctive feature of this colony is that it is undifferentiated.

The lungs of some inoculated animals contain groups of two or more megakaryocytes. there was. The locations of these colonies are clearly different from the locations of the “undifferentiated” colonies described above. It was unrelated.

NA bone marrow cells promote autocrine self-augmentation by producing soluble factors. It appears to regulate its own replication by reproduction. This method of duplication is and is involved as the first step in tumorigenesis and metastasis (Lang et al. , 1985). To determine whether NA bone marrow cells are tumorigenic, B, BM, 3 or Htl, BM, 2 or M for thymic nude mice [I, BM, 2-derived cells were inoculated into the intestinal cavity or subcutaneously.

All three cell lines generated tumors, but tumor generation was independent of the route of inoculation. Was. Tumor cells were collected from both solid tumors and the peritoneum and recultured in vitro. I succeeded in that.

As controls, cell lines B, BM, 3 and Hll, BM, 2 were Scan for retroviruses using both microscopy and assays for reverse transcriptase. Cleaned.

The method for measuring reverse transcriptase activity is as follows. Supernatant obtained from bone marrow cell line The solution is subjected to ultracentrifugation to capture potential retroviruses in the resulting pellet. did. After reconstituting the pellet in a small volume of buffer (RT buffer), Wu et al. roc, Nati, Acad, Sci, USA, Volume 70, 1289-1302 The analysis was carried out according to the method described in J. P., 1973.

The results of both analyzes by electron microscopy and assay for reverse transcriptase He was negative about viral infection.

(Margin below) Large m Kashio’s on the ground: NA's and's 1 Geki B, BM, 1 After the culture reaches a confluent state, remove the culture by 0.22 microns. Condition by passing the supernatant of the culture through a standard filter. A medium was obtained. This conditioned medium was added to bone marrow stromal cultures obtained from other calves. Ta. This bone marrow stromal culture was prepared as described in Example 1. Ta. After treatment, NA cells were observed in these cultures 4-6 days later. conditioned medium Without it, it usually takes about two months for NA cells to develop. treated with conditioned medium The NA cell lines derived from bovine bone marrow stromal cultures were named B, BM, and 3.

Shigosho U Factors in the conditioned medium from cultures of bovine stromal cells may be present in NA bone marrow from other species. to determine whether it has activity in stimulating cell emergence and proliferation. Bone marrow stromal cultures from pigs and turkeys were treated with conditioned media. all In some cases, conditioned media reduced the time required for NA cells to appear.

The NA cell line derived from this treatment is shown in Figure 4.

Colony withdrawal PC-CSF life Conditioned medium derived from stromal cells is a primitive NA bone marrow with potential multipotent functions. Contains PC-CSF that induces normal bone marrow stromal cultures to produce cells. It is prepared as follows.

B, BM, 3ffl cells [figurine (in 75 cm2 flask), stromal cells ( A high temperature chamber filled with gas (air containing 5% CO2) Culture was carried out at 37°C in a humidity incubator.

During this culture period, excess NA cells were removed daily. Stromal cells are in a fused state Once reached, the medium was removed and fresh medium was added. Then remove the medium The cells were incubated again for 24-30 hours. After incubating, remove the supernatant liquid. Collect and centrifuge at 2000 rpm for 10 minutes), then 0.22 μm Filtered using a standard Millipore filter. Store the filtrate at -70°C.

K standing five Assay of PC-CSF 100+ am diameter Petri blood cells grown to a confluent state prepared as follows: of bovine stromal cultures were washed until free of medium and treated as in Example 14. Crude PC-CSF prepared as above was added. Added PC-CSF is not diluted Alternatively, various dilutions ranging from 2 to 256 times were prepared using the 2-fold dilution method. That program The ink was incubated for 4 hours at 37°C in a high humidity incubator filled with gas. It was incubated. After the first incubation, 10ae of growth medium (antibiotics RPMI-1640 supplemented with 10% fetal bovine serum) was added and then The plates were further incubated for at least 14 days. In PC-CSF The treated medium was examined daily for the appearance of colonies of NA cells and the results were recorded. Ta.

To prepare bovine bone marrow stromal cell cultures, animals younger than 1 year of age were anesthetized. Citrated bone marrow samples were collected from the iliac crest or sternum of calves. Bone The spinal cord aspirate was centrifuged at 200 rpm for 15 minutes at room temperature. Remove supernatant liquid , the resulting cell pellet was placed in minimal essential medium (MEM) supplemented with antibiotics. Resuspend. Layer the resulting cell suspension onto a Ficoll Hyback density gradient solution. and centrifuged at 200 ORPM for 30 minutes at room temperature. After centrifugation, Cells at the dispensing interface were collected, washed with a diluted solution of MEM, and then incubated at 11,000 rpm for 8 Centrifuged for minutes. This washing was repeated four times. After the last wash, 10% Resuspend in RPMI-1640 without antibiotics containing fetal bovine serum and count. and diluted to a final concentration of 5 x 1 G' cells/kick.

The resulting cell suspension was transferred to a sterilized 100 wl 11 diameter veterinary tube for tissue culture. , plate at a cell concentration of 5X10' cells/plate in growth medium for 15 minutes and gas The ink was incubated at 37°C in a high-humidity incubator filled with air containing 5% CO2. I bet. Examine the cultures daily for the appearance of colonies of adherent stromal cells. However, colonies appeared every day 7 to 12 days after starting the culture. straw makoro When needles appear, wash the culture until free of NA cells and supplement with growth medium. It was filled. When stromal cell cultures reach a confluent state, trypsin-ED The cell suspension obtained by treatment with TA and dispersion was further transferred to a new 100 mm straight tube. The cells were transferred to a large-sized vetri dish and subcultured. Stromal cultures are usually passaged once a week Inoculated. A portion of the passaged cells was added with 10% fetal bovine serum and 10% DMSO. Stored in liquid nitrogen in storage medium consisting of supplemented RPMI-1640.

The results of the crude PC-CSF assay are shown in Figure 5. Colonies of NA cells are grown in conditioned medium. appeared 4-7 days after treatment with undiluted PC-CSF containing . Figure 5 In , the cross sign indicates the number of colonies detected during the 14th day. Contains conditioned medium In the absence of PC-CSF, no NA colonies were detected at this time. Sara PC-CSF activity was detectable when the conditioned medium was diluted 16 times.

inkstone PC-CSF clonal assay The activity of PC-CSF was measured using a clonogenic assay according to the following method. . This method utilizes sterile techniques, glassware, and culture media. PC-CSF The conditioned medium estimated to contain the Diluted. O, Swt containing 1x106 bone marrow cells was diluted appropriately. Conditioned medium was mixed in a 0.5-liter and 5-liter vial. Bone marrow cells are described below. It was prepared as follows. Each vial above contains 1.6% methylcellulose. RPMI-16401, Oma was added. Mix the contents of the vial and mix 1.0 μm of the sample was added to a Co5tar 35X 10 mm Petri dish. Each app Sei went twice. Tilt the plate gently from front to back and from side to side until the entire surface of the plate is covered. The medium was distributed evenly over the body. Next, place those beet dishes into a 150 mm diameter beet dish. Each large petri dish contains 35 x 10 m of water without a lid. One m Petri dish was placed to maintain humidity. Next, take this large vetri dish. Plates kept at 37°C in an atmosphere containing 5% CO2 were inspected daily for 10 days. and colonies were counted using an inverted microscope.

The bone marrow cell suspension used for the clonogenic assay was prepared from anesthetized calves (1 year old). It was obtained from bone marrow fluid collected by aspirating into 2× citrate solution from the iliac crest of a young (younger) iliac crest.

The bone marrow sample was washed free of citrate with MEM and kept at room temperature. Centrifuged at 200 ORPM for 15 minutes. Re-incubate the generated cell pellet into MEM. and place the cell suspension in a layer on a Ficoll-Hypaque density gradient. and centrifuged at 2QOORPM for 30 minutes at room temperature. Following centrifugation, gradient solution Collect the cells located at the interface of the centrifuged.

Washing was repeated twice and cells were transferred to RPM1-1640 supplemented with 10% fetal bovine serum. resuspended in Adjust this cell suspension to a final concentration of 2XIO' cells/cell. I made it.

The results obtained in the clonogenic assay are as follows. In daily inspection of the plate Colonies with fewer than 10 cells were observed on the third day of culture, and colonies with fewer than 10 cells were observed on the first 3 days of culture. Colonies larger than 15 cells per knee appeared after 4 days of culture. Titrate to P To obtain the final concentration of C-CSF activity, incubate the culture for 10 days. is preferable.

Morphologically, the cells in the resulting colonies were NA cells fed with conditioned medium. -They seem similar. No other types of cell colonies were observed . Furthermore, the following was observed. That is, the supernatant fluid derived from NAA vacuoles is PC-C showed SF activity, but this activity was demonstrated when NA cells were combined with bovine bone marrow stromal cultures. It was observed that this was significantly promoted by incubation. Furthermore, this The promotion of the activity of NA cells is due to the fact that NA cells are isolated from the bone marrow stroma, that is, BO, BM, and 001. BO, BM, 3 (NA cells) incubated with separated stroma ) appears to have occurred even in different species.

Clonogenic assays are significantly more sensitive than methods that measure stromal cell induction. It seems very expensive. Maximal PC-CSF activity utilizes stromal cell induction in the clonogenic assay, the supernatant was obtained at a dilution of l/16. In the case of A, the peak activity for the same supernatant solution is 1/loo to 1/1000 dilution. Obtained at the rate of conversion.

Also, at concentrations that give peak activity of PC-CSF in clonogenic assays, It was found that stromal cell clones were seen to proliferate. Usually, Troma cell clones do not appear until 10 to 14 days after starting the culture. that Therefore, the supernatant of the culture stimulates the proliferation of stromal cells present in the bone marrow culture. It seems to be progressing.

Shigogomame PC-C5F-a to PC-C5F-noF'' PC-CSF-α and PC-CSF -β in mouse stromal cell cultures and mice according to plan A shown in FIG. Purified from conditioned media from SNA cell cultures.

According to this plan, the medium is suitable for stromal cell culture and/or NA. As described in Example 12 above, except that it was derived from A-cell culture. A conditioned medium is obtained. Concentrate approximately 20 μl of conditioned medium to approximately 1,800 μl. Ru. Alternatively, to concentrate the sample, lyophilize the medium and reduce the dry matter. By redissolving the bacteria in double distilled water, a sample concentrated approximately 15 times is obtained. concentrated Samples were filtered through o, 4s micron Zydex filters and 5yn Cropak GPC column (Syr+chro, La Eye, Indiana, USA) HPLC gel filtration on II+Inc, ). The dimensions of this column are 500mm loim (inner diameter), 0.15M NaCl and 0.05M phosphate) Perform isocratic elution using a buffer containing (pH 7,0) to remove the tank from the column. Elute the pack. Determine which of the eluted fractions contain PC-C3F For this purpose, the biological activity of the fractions is determined using the phosphorogenicity assay described above. . )’ The desired fractions with C-C5F activity were collected, lyophilized, and washed with sterile double-distilled water. dissolve in This solution containing PC-CFS was then transferred to a Varian LC5 500 for preparative reverse phase high performance liquid chromatography. sample, Vy dac Protectn C4 reverse phase column (Vyda, California, USA) c 5eparatfon Group) and elute PC-CSF. melt Separation was performed using solution A (0.1% trifluoroacetic acid aqueous solution) for 10 minutes, and then from solution A to B. Change to liquid [water containing 0.1% trifluoroacetic acid and acetonitrile (90:IO)]. Do this for 50 minutes. It has PC-CSF activity by this apheresis. Two main components are obtained. That is, PC- with an apparent molecular weight of about 20 Kd C3F-α and PC-CSF-β having an apparent molecular weight of about 60 to 701:d. be.

The isolated fractions were loaded onto a Vydac Protein C4 reverse phase analytical column (150 HPLC and high performance protein liquid chromatography using (, FPLC).

Based on the chromatographic pattern of the fraction containing PC-CSP activity, PC-C3 F-α was clearly homogeneously purified, and PC-CSF-β was approximately 70~ Estimated to be 80% pure.

The HPLC reverse phase analysis profile of the PC-CSF-β fraction shows that low concentrations of PC-es This suggests that it is contaminated with F-α.

Both PC-CSF-α and PC-CSF-β were tested in the clonogenic assay described above. , was tested for its ability to induce primitive cell colony forming units (PC-CFU). . PC-CFU can be induced along with both PC-CSF. Also PC - The activity of CSF-β, when combined with PC-CSF-α, increases with at least the adduct and can be synergistic.

1■11 PC-CSF-α and PC-C3F-6 stromal cells only, NA cells only , and conditioned media from cultures of combined stromal and NA cells. were analyzed by vertical polyacrylamide gel electrophoresis in the presence of SDS. the The gel pattern shows a molecular weight comparable to that of PC-CSF-α (i.e. 20-25 Kd ) is derived from NA cells and stromal cells plus NA cells. were shown to be present in the conditioned medium. Molecular weight comparable to PC-CSF-β In particular, proteins with 60 to 70 Kd) are found in stromal cells and stromal cells. present in conditioned medium from cells plus NA cells. This data is 20-25 The Kd protein is a product of NA cells, while the 60-70Kd protein is a product of NA cells. This suggests that it is a product of troma cells.

Weiyu Shi IjEffi Shihi 1 Rebellion The cell lines listed below are licensed under the terms of the Budapest Treaty, United States, Maryland, United States. Parklawn Drive, American Type Culture Collection (ATCC) and has been given the following accession number:

(Margin below) Cell line ATCC number Deposit date BoBm3+80 CRL-9825 September 14, 1988 MoBm2+44 CRL-9826 September 14, 1988 BoBm Stro-v+ CRL-982 7 September 14, 1988 PoBm 2÷12 CRL-9828 September 1988 14th HuBttr2+40 CRL-9829 September 14th, 1988 Japanese application filed in US Once granted and issued as a national patent, all of these restrictions on access to deposits are ultimately removed. It will be canceled. Additionally, while the application is pending, the Commissioner of the Patent Office must 1.14 and U.S. Patent Law (350SC) 1.22. Those who decide to do so will have the right to obtain the deposited materials specified above. A final request five years from the date of request or the period during which the U.S. patent is enforceable, whichever is longer. maintained. These deposits are made for convenience and are Nothing in the description may be necessary to practice the invention.

Indigo The following cited documents were cited when discussing this invention.

Barr et al. (1986) Biotechniques soil: 428°Bar lozzaci et al. (19B?) Proc, Nat'l Acad, Set , USA Dania 691゜ Biofeldt Ohmann (19B?) J, Histochem, Cytoche+++, 35:627゜ Botstein (1979) Gene 8: 17°Broach ( 1981) Molecular Biology of the East On Saccharomyces: 445 (Cold Spring Har bor　Press).

Broach et al. (1983) Meth, Enz, 10Uni 307°Chan g et al. (1977) Nature 198: 1056゜Clewel et al. 1969) Proc, Nat'l Acad, Sci, USA 62: 1 159° Clewell (1972) J, Bacteriol, 110: 667゜Cohen (1972) Proc, Nat'l Acad, Sci , USA 69: 2110° De Boer et al. (1983) Proe, Na tol Acad, Set, USA 292: 128° Fiers et al. (197 8) Nature 273: 113° Goeddel et al. (1980) Nu cleic Ac1ds Res, 8: 4057゜Graha+a and V an der Eb (19711) Vfology 52: 546°G runstein and Hogness (1975) Proc, Nat’ l　Acad,　Set.

USA 7: 3961° Ha++aerling et al. (1981) Monoclonal Antib odis and T-CellHybrfdotas.

He5s et al. (19613) J, Adv, Enzyme Reg, 7: 1 49゜Hinnen et al. (1978) Proc, Nat'l Acad, Se t, 75: 1929° Hitzeman et al. (1980) J, BioJ, Ch ew, 255: 2073゜Holand et al. (197B) Biochemi stry 17: 4900゜Ho1land (1981) J, Biol , Chet 256 2 1385゜Kennett et al. (1980) Mon oclonal Antibodies.

Lae+ea+1i (1970) Nature 227: 680°Lan g et al. (1985) Ce1l 43: 531° Maniatis et al. (198 2) Mo1ecular Cloning: A LaboratoryMa nual (Cold Spring Harbor Press, Cold Spring Harbor.

N, Y, ).

Mayer and Walker eds, (1987) I+wmunoch ei+1cal Methods Ce1l and Mokecular B iology (Acade+gic Press, London).

Maxam et al. (1980) Methods in Enzymology 6 5: 499° Sanger et al. (197?) Proc, Nat'l Acad , Set, USA 74: 5463°5chreier et al. (1980) Hy bridema Techniques.

5copes (1987) Protein Purification, 2 nd ed, (SpringerVerlag).

Shimatake et al. (1981) Nature 292: 128°Wa rner (19g4) DNA 3: 401°Nu and Grossma n (19g?) Methods in Enzya+ology Vol.

154, Recombinant DNA, Part E.

Wu (198?) Methods in Enzy+oology Vol. 155°Reco+*binant DNA, Part F.

Zoller (1982) Nucleic Ac1ds Res, 10: 6487° mouth The NA cells of this invention include PC-CSF production and cell replacement for hematopoietic disorders. There is commercial utility in providing treatment to individuals in need thereof. against NA cells Monoclonal antibodies are useful for the isolation and identification of these NA cells.

PC-CSF may be useful in treating hematopoietic disorders. In particular, PC-CSF is a hematopoietic If the rate is significantly altered, it may have the potential to modulate early immunity and is particularly may have the potential to modulate the immune response to infectious diseases in neonates.

In the latter case, it is important not only for humans but also for the treatment of livestock, including agricultural animals. Ru. In addition, PC-CSF is a stimulant for NA cells, so it can be used in bone marrow transplantation therapy. It can be used to increase therapy.

In addition to the above, PC-CSF can be used in other cases where cell proliferation is desired, such as wounds. It can be used to heal wounds, stimulate the proliferation of nerve cells, especially aged nerve cells.

FIG, IA 5 25 50 to.

E2: T k+:, (%n FIG, IB FIG, 2A Special Table Sen4-502853 (19) + 25 50 to.

E:1 ratio r’t) FIG, 2B 5 25 50 to.

1 25 50 to.

Mitsuomi, X BO, B, M, -1 4) -; + to BO, B, M, -3HLJ, 8. M, -I MLl, B, M, -I PO, B, M, -$BO, B, M, -4HLl, B, M, -2MLl, B, M, -2BO, B, M, -3 (foam $AN゛ lever (1 and 700)) HU, B, M, -2 (緬4'L ”　b〆 + 6°) MLl, B, M, -2 (hair L ゛ level C tθ　 ) po, s, M, -i (SL4'L' and S'+t, -J-r) pC-CS F top charge +, 5 +4 +++4 - + + +1, RPM! -1640+I O%FBS2, U3- 蓼4X*bk* *24QMd’kfRr5 7- (tr=dzt (o, n, s, yc+t-1-, v: Hatu=Motsuj 3. Same as 6 o'clock/Industrial service ＋し 7- 57 crowns 1) 5, Colony 17 Nitrogen embryo J1... 10 Same as A1.

FIG. 5 Upper two i! Heka FIG. 6 April 8, 1991

Claims (20)

[Claims] 1. Purified primitive cell colony stimulating factor (PC-CSF). 2. The purified PC-CSF according to claim 1, which is derived from mouse bone marrow cells. 3. The purified PC-CSF according to claim 1, which is derived from bovine bone marrow cells. 4. Purified PC-CSF according to claim 1, derived from human bone marrow cells. 5. Crude vector encoding PC-CSF. 6. A cell transformed with the recombinant vector according to claim 5. 7. Purified PC-CSF-α. 8. Purified PC-CSF-β. 9. Multipotent lymphoid cells that are substantially free of mature lymphocytes and myeloid cells Cell suspension containing blood progenitor cells (NA cells). 10. A suspension of human-derived NA cells, wherein the NA cells respond to My10 antigen. The monoclonal antibody is not immunologically reactive with the monoclonal antibody. is a hybridoma cell line, ATCC No. produced by HB-8483, Human-derived NA cell suspension. 11. Produces monoclonal antibodies that are immunologically reactive with bone marrow-derived NA cells Hypuridoma. 12. A monoclonal antibody produced by the hybridoma of claim 11. 13. A method for producing a cell population containing NA cells, the method comprising: (a) containing NA cells; (b) supplying a cell suspension containing PC-CSF and other cells essential for cell proliferation; the cell suspension under conditions that promote cell proliferation in a medium containing the following components: Propagation step; (c) isolating a population of NA (NA) cells from the cell culture obtained in step (b); a step of recovering; and (d) in a medium containing PC-CSF and other components essential for cell growth. , a step of growing the cells isolated in step (c) under conditions that promote cell proliferation. ; How to include. 14. A method for producing a cell population containing multipotent lymphohematopoietic progenitor cells, the method comprising: (a) supplying a cell suspension from hematopoietic tissue of an individual; (b) supplying the cell suspension; , contacting with a monoclonal antibody against NA cells, wherein the antibody is directed against the NA cells. Recognizes antigens on A cells, but more mature lymphocytes and myeloid cells does not recognize; and (c) separating and collecting cells to which nuclear antibodies have been bound from the cell suspension; How to include. 15. 1. A method of treating an individual for a disorder of the hematopoietic system, the method comprising: providing a suspension of NA cells in a medium that can be (b) injecting the individual with a dose of NA cells sufficient to overcome the hematopoietic disorder; Process; How to include. 16. further administering to said individual sufficient to stimulate proliferation of NA cells within said individual. by injecting a solution of PC-CSF in a pharmaceutically acceptable excipient at 16. The method of claim 15, wherein the method is treated with: 17. A method of treating an individual for a disorder alleviated by cell proliferation, the method comprising: (a) providing a solution of PC-CSF in a pharmaceutically acceptable excipient; (b) administering to the individual a pharmacologically effective dose of PC-CSF that alleviates the symptoms of the disorder; administering; How to include. 18. the individual is treated for a hematopoietic disorder, and a PC that alleviates the hematopoietic disorder 18. The method of claim 17, wherein a dose of CSF is administered. 19. the individual is being treated for a wound, and the PC stimulates healing of the wound. 18. The method of claim 17, wherein a dose of CSF is administered. 20. the individual is treated for a disorder of neuronal origin and 18. The method of claim 17, wherein a dose of PC-CSF is administered that stimulates the regeneration of .