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WO1995025164A1 - Cellules mesenchymateuses a diffusion hematogene - Google Patents

Cellules mesenchymateuses a diffusion hematogene Download PDF

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Publication number
WO1995025164A1
WO1995025164A1 PCT/US1994/002850 US9402850W WO9525164A1 WO 1995025164 A1 WO1995025164 A1 WO 1995025164A1 US 9402850 W US9402850 W US 9402850W WO 9525164 A1 WO9525164 A1 WO 9525164A1
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Prior art keywords
cells
blood
borne
mammalian blood
fibroblasts
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PCT/US1994/002850
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English (en)
Inventor
Anthony Cerami
Richard J. Bucala
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Picower Institute for Medical Research
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Picower Institute for Medical Research
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Priority to KR1019960705117A priority Critical patent/KR100352057B1/ko
Priority to NZ265435A priority patent/NZ265435A/en
Priority to EP94913910A priority patent/EP0759070A4/fr
Priority to PCT/US1994/002850 priority patent/WO1995025164A1/fr
Priority to JP7523995A priority patent/JPH09510348A/ja
Priority to AU65884/94A priority patent/AU693441B2/en
Publication of WO1995025164A1 publication Critical patent/WO1995025164A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)

Definitions

  • the present invention relates to a population of blood-borne mammalian cells that express a unique profile of surface markers that includes certain markers typical of connective tissue fibroblasts, and are referred to herein as "blood- borne mesenchymal cells.” In particular, it relates to the isolation, characterization and uses of such blood-borne mesenchymal cells.
  • the cells of the present invention can be distinguished from peripheral blood leukocytes by their distinct size, morphology, cell surface phenotype and biologic activities, and are likewise distinguishable from connective tissue fibroblasts by other surface phenotypic markers. These cells proliferate in culture, and in vivo, as demonstrated in animal models, are capable of migrating into wound sites from the blood. Therefore, such blood-borne mesenchymal cells may have a wide range of applications, including, but not limited to, the promotion of wound healing, tissue remodeling, and for gene therapy.
  • WOUND HEALING A wound can be considered a physical interruption in the normal architecture of tissues, which can result from physical or chemical causes, such as burns, abrasions, cuts and surgical procedures.
  • a cutaneous wound may severely compromise an individual's ability to resist infectious agents, rendering the individual susceptible to opportunistic infections, in addition to pain and discomfort. Therefore, it is highly desirable to develop agents and methods for using them to promote a rapid wound healing response.
  • a wound healing is a complex process of events involving both humoral and cellular elements, and which occurs over a time period of days to weeks.
  • wound healing depends on the interactions between specific cell types, cytokines and extracellular matrix (Clark, 1989, Curr. Opinion Cell Biol. 1:1000).
  • a first step in wound healing involves the action of blood-borne cells known as platelets. These cells aggregate at wound sites and form a temporary barrier that prevents blood loss. Platelets achieve this function by secreting thrombin, which catalyzes blood clot formation, and other factors, which serve to attract other cells into the damaged area.
  • Blood-borne neutrophils and monocytes migrate into the wound site. These cells function in part by neutralizing invading microorganisms and secreting enzymes that clear away the initial clot.
  • macrophages play a primary role by secreting a variety of inflammatory cytokines such as tumor necrosis factor (TNF) , the interleukins such as IL-1, IL-6, IL-8, transforming growth factor-j8 (TGF-3) , etc., and growth factors such as epidermal growth factor (EGF) , fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) , that serve to combat infection and recruit additional cell types.
  • TNF tumor necrosis factor
  • TGF tumor necrosis factor
  • cytokines known to be capable of promoting chemotaxis and cellular proliferation.
  • cytokines include PDGF, TGF/3 and FGF (Pierce et al., 1989, J. Cell. Biol. 109:429; Rappolee et al., 1988, Science 241:708).
  • New blood vessels form to support and nourish the newly established tissue.
  • the major cellular mediators of wound repair include blood-borne platelets and leukocytes such as neutrophils and monocytes which become macrophages as they migrate into the wound area. These blood-derived leukocytes combat infection, and secrete cytokines and growth factors.
  • fibroblasts in the surrounding connective tissues also grow into the site of injury to provide additional cytokines and extracellular matrix proteins.
  • a blood-borne population of fibroblast-like cells that possesses the capability of participating in and enhancing wound healing processes had never been described.
  • the present invention relates to mammalian blood-borne mesenchymal cells involved in wound healing, methods of isolating the cells, and methods of using the cells in promoting wound healing processes and tissue remodeling.
  • the invention is based, in part, on the
  • a distinct population of relatively large, spindle-shaped, fibroblast-like cells can be isolated and cultured from the human and murine peripheral blood. Phenotypic analysis of these cells with antibodies specific for various known cell- markers reveals that they are of mesenchymal origin, as they express typical fibroblast markers such as collagen, vimentin and fibronectin. In cell culture, the large spindle-shaped cells co-exist with small round cells that also display a fibroblast-like phenotype. Thus, these mesenchymal cells are distinguishable from peripheral blood leukocytes by their cell size, morphology and unique phenotype.
  • blood-borne mesenchymal cells Because of the correspondence of this profile of surface markers to fibroblasts rather than known blood cell types, these cells are referred to herein as "blood-borne mesenchymal cells.”
  • the invention is described by way of examples in which human blood-borne mesenchymal cells are isolated, cultured and their cell surface phenotype characterized. In vitro, the cultured mesenchymal cells expand in numbers in response- to granulocyte-macrophage colony stimulating factor (GM-CSF) in a dose-dependent manner. In vivo, a corresponding murine cell population is observed to migrate into wound chambers that have been experimentally-implanted into animals.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • a wide variety of uses of the blood-borne mesenchymal cells, and factors produced by these cells, are encompassed by the invention described herein, particularly to improve wound healing, including, but not limited to, cutaneous wounds, corneal wounds, wounds of epithelial-lined organs, resulting from physical abrasions, cuts, burns, chronic ulcers, inflammatory conditions and the like, as well as from any surgical procedure.
  • the mesenchymal cells may be genetically engineered to express one or more desired gene products.
  • the engineered cells may then be administered in vivo (e.g.. either returned to the autologous host or administered to an appropriate recipient) to deliver their gene products locally or systemically.
  • FIG. 1 Forward and side scattering of incident light during cytofluorography demonstrating the increase in size and granularity of cell populations after time in culture.
  • FIG. 1A Freshly isolated peripheral blood lymphocytes before culture.
  • FIG. IB Cells recovered after 4 weeks in culture.
  • FIG. 2 Proliferation of blood-borne mesenchymal cells in response to granulocyte macrophage-colony stimulating factor.
  • the present invention relates to mammalian blood-borne mesenchymal cells, to methods of isolating and characterizing the cells, and to methods of using the same for a variety of applications including but not limited to wound healing and gene therapy.
  • the present invention provides for methods of enriching and/or purifying mesenchymal cells from peripheral blood or other physiological sources of these cells.
  • the biologic activity of these cells may allow for their uses in settings where absolute purity is not achieved.
  • the mesenchymal cells of the invention may be isolated from any tissue where they reside or from which they may mature, including but not limited to the bone marrow, fetal liver, or embryonic yolk sac.
  • blood-borne mesenchymal cells may be isolated by separation based on the presence or absence of specific cell surface markers.
  • These techniques may include flow cytometry using a fluorescence activated cell sorter or biotin-avidin or biotin-streptavidin separations using biotin-conjugated to marker-specific polyclonal or monoclonal antibodies and avidin or streptavidin bound to a solid support such as affinity column matrix or plastic surfaces, magnetic separations using antibody-coated magnetic beads, destructive separations such as antibody plus complement or antibody coupled to cytotoxins or radioactive isotopes for the removal of undesirable cell populations.
  • mesenchymal cells may be isolated by procedures involving repetitive density gradient centrifugation, lectin chromatography, affinity chromatography involving positive selection and negative selection, or a combination thereof.
  • Positive selection methods may utilize affinity chromatography with antibodies directed to mesenchymal cell-specific surface markers. For example, most mononuclear cells may be depleted first from the blood after density gradient centrifugation and plastic adhesion, then an antibody to vimentin antigen can be used to positively select for mesenchymal cells.
  • Negative selection includes modifications of the protocol disclosed herein, infra. In essence, a mesenchymal cell preparation may be reacted with one or more antibodies directed at cell surface antigens not expressed by mesenchymal cells for their removal.
  • Antibodies to any T cell, B cell, monocyte, natural killer (NK) cell, dendritic cell and granulocyte markers may be used.
  • examples of such antibodies include anti-CD3, anti-CD4, anti-CD5, anti-CD8, anti- ⁇ /S and anti-75 T cell receptor specific for T cells; anti-CD12, anti-CD19 and anti-CD20 specific for B cells; anti-CD14 specific for monocytes; and anti-CD16, and anti-CD56 specific for NK cells. These antibodies may be applied in any combination repeatedly or in a sequential manner for the enrichment of mesenchymal cells.
  • the cells may be removed by adsorption to a solid surface coated with an anti-mouse antibody column, as the majority of monoclonal antibodies directed at cell surface markers are of mouse origin, or if the antibodies are conjugated with biotin, the antibody-bound cells can be removed by an avidin-coated surface; or if the antibodies are conjugated to magnetic beads, the cells expressing antigens recognized by the antibodies can be removed in a magnetic field.
  • adsorption to a solid surface coated with an anti-mouse antibody column as the majority of monoclonal antibodies directed at cell surface markers are of mouse origin, or if the antibodies are conjugated with biotin, the antibody-bound cells can be removed by an avidin-coated surface; or if the antibodies are conjugated to magnetic beads, the cells expressing antigens recognized by the antibodies can be removed in a magnetic field.
  • blood-borne cells are detectable immunochemically in the peripheral blood, and may be purified to homogeneity by various procedures.
  • the cells in short-term cultures fall into two distinct morphological profiles, a "round" cell type and
  • spindle-shaped fibroblast-like cell type In these short term cultures, the round cells appear to be a mixture of lymphocytes and a small round cell type which, like the spindle-shaped cells, demonstrate a fibroblast-like phenotype. Long-term culture appears to enhance the growth of the mesenchymal cells, i.e. , both the round and spindle-shaped cells which display the fibroblast-like phenotype, until they become the dominant cell type jLn vitro.
  • the small round mesenchymal cells may represent the mitotically active stage of the spindle-shaped mesenchymal cells. Thus, it appears that the initial population of lymphocytes present in the short-term cultures, i.e..
  • the cells of the present invention are characterized to be of mesenchymal origin primarily because of their unusual cell surface phenotype for blood-derived cells. In particular, these cells express vimentin, fibronectin, collagen I and III, which are typical markers for fibroblasts. Conversely, these cells do not express cytokeratin, von Willebrand's factor, desmin, laminin and smooth muscle cell ⁇ -actin, all of which are commonly used markers for epithelial, endothelial or smooth muscle cells.
  • peripheral blood leukocytes such as CD3, CD4, CD8, and CD56 are also not present on the blood-borne mesenchymal cells. Interestingly, these cells are positive for CD34, which is a marker on hematopoietic stem cells, suggesting that the mesenchymal cells described herein may be bone marrow-derived.
  • the mesenchymal cells of the present invention are larger and more granular than peripheral blood leukocytes when assessed by forward and side scattering of incident light during cytofluorography. They exhibit a unique spindle-shaped morphology which is typical for fibroblasts, but atypical for other blood-derived cells. Hence, taken collectively, the blood-borne mesenchymal cells appear to be a distinct cell type which is different from all previously described cell populations from the blood, based on their cell size, cell surface phenotype, and morphological properties.
  • Isolated blood-borne mesenchymal cells proliferate in vitro in culture media for extended periods of time using standard culture techniques that are well known to those skilled in the art.
  • serum-enriched medium should be used, and more preferably medium containing 20% fetal bovine serum should be used, e.g. see Section 6.1.1, infra. It has been shown that their growth may be further enhanced by the addition of GM-CSF.
  • short term cultures derived from peripheral blood lymphocytes contain a contaminating population of lymphocytes, whereas cells positive for fibroblast markers predominate in the long term cultures.
  • GM-CSF accelerates the time course over which the fibroblast- like cells dominate the culture.
  • isolated cells may be engineered to express endogenous GM-CSF to sustain their long-term growth in an autocrine fashion (See Section 5.4, infra) .
  • Continuous cell lines or clones generated in this manner may facilitate further isolation of cell surface markers and cytokines and the genes encoding therefor.
  • Long-term culture of blood-borne mesenchymal cells may be performed in tissue culture flasks, roller bottles, bioreactor systems and any culture methods known in the art. In fact, these mesenchymal cells may respond to a number of other conventional cytokines and growth factors.
  • a major impediment in the current attempts to achieve stable integration of foreign genes in eukaryotic host cells of different organs is the inability of most of these cells to proliferate in vitro. Since the mesenchymal cells proliferate in vitro. especially in response to GM-CSF, these cells may be ideal candidates as recipients for the introduction of exogenous genes in culture.
  • cytokines normally synthesized by the blood-borne mesenchymal cells a number of other cytokine or adhesion molecule genes may be engineered into these cells to further augment their ability to promote and accelerate wound healing and tissue remodeling, or to deliver products of any gene introduced into the mesenchymal cell for therapeutic purposes.
  • genetic engineering of the cells involves isolating blood-borne mesenchymal cells from an individual, transferring a gene of interest into these cells, confirming stable integration and expression of the desired gene products.
  • Such genetically engineered cells may be transplanted into the same, or an HLA-matched, or otherwise suitable patient and/or used-as a source of factors and/or genes encoding factors made by the cells.
  • mesenchymal cells isolated by the procedures described in Section 6, infra. may be used as recipients in gene transfer experiments.
  • the cells may be grown in culture prior to, during, and after introduction of an exogenous gene. The proliferative activity of these cells may be enhanced by GM-CSF.
  • any cloned gene may be transferred using conventional techniques, including, but not limited to, microinjection, transfection and transduction.
  • One method of gene transfer utilizes recombinant viruses, such as retroviruses or adenoviruses.
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g.. the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome (e.g...
  • region El or E3 will result in a recombinant virus that is viable and capable of expressing the gene product in infected mesenchymal cells (e.g. , see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 3655-3659) .
  • the vaccinia virus 7.5K promoter may be used. (e.g. , see, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et al. , 1982, Proc. Natl. Acad. Sci.
  • Vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements are also candidates (Sarver, et al., 1981, Mol. Cell. Biol. 1: 486). Shortly after entry of this DNA into cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene.
  • a retroviral genome can be modified for use as a vector capable of introducing and directing the expression of any gene of interest in the blood-borne mesenchymal cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349-6353).
  • High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • the mesenchymal cells can be transformed with a cDNA controlled by appropriate expression control elements (e.g.. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker confers resistance to the selection and allows cells to stably integrate the recombinant DNA into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered mesenchymal cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817) genes.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare, et al. , 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg,
  • neo which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150: 1)
  • hygro which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147) genes.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • Blood-borne mesenchymal cells may be isolated from the peripheral blood and expanded in culture for a variety of therapeutic purposes, including but not limited to the enhancement of wound healing.
  • Isolated blood-borne mesenchymal cells which are purified or partially enriched with or without exogenous genes may be directly applied to external wound sites including, but not limited to, severe wounds, burns, cuts, abrasions, chronic ulcers and inflammatory diseases of skin. Cosmetic applications of these cells are also within the scope of this invention.
  • the cells may be directly applied to damaged tissues or organs such as those resulting from trauma or in the course of surgery, for the repair of internal organs, e.g.. gastric mucosa, cardiac tissue, bone, and vascular tissue, as well as tissues that are difficult to heal by traditional methods, e.g.. joint cartilage, ligaments, tendons, and neural tissue.
  • damaged tissues or organs such as those resulting from trauma or in the course of surgery, for the repair of internal organs, e.g.. gastric mucosa, cardiac tissue, bone, and vascular tissue, as well as tissues that are difficult to heal by traditional methods, e.g.. joint cartilage, ligaments, tendons, and neural tissue.
  • the cells may be administered to patients via any of a number of routes, including but not limited to intravenous, intramuscular, subcutaneous, intradermal, etc., for the treatment of external or internal visceral injuries.
  • routes including but not limited to intravenous, intramuscular, subcutaneous, intradermal, etc.
  • the mesenchymal cells will migrate in vivo to the site of the wound where they can enhance wound healing.
  • Genetically engineered mesenchymal cells may be used in this fashion to deliver gene products to the site of the wound; e.g.. genes for Factor VIII, growth factors, etc. may be useful in this regard.
  • methods of administration which do not allow for migration may allow the mesenchymal cells, genetically engineered or otherwise, to take up residence at the site of administration where they can deliver gene products to the local environment, and/or systemically.
  • protocols may be designed to inhibit or remove these cells jLn vivo such as by the administration of monoclonal antibodies to specific surface markers, in the treatment of chronic diseases with noted fibrosis, particularly conditions of excessive fibroses such as myelofibroses, histiocytoses, hepatic cirrhosis, keloid formation, scleroderma, etc.
  • the inhibition of migration of these cells into wound sites may prevent excessive scar formation.
  • the blood-borne mesenchymal cells may be quantified in peripheral blood samples obtained from individuals. Abnormally low or high concentrations of such cells (as compared to values determined for healthy individuals) may be correlated with diseases or disorders.
  • the quantification of the blood-borne mesenchymal cells may be accomplished by morphological analysis, biological activities, or preferably by immunochemical means. For example, antibodies specific for vimentin, fibronectin, collagen I, or collagen III may be used individually or in combination for the detection and quantification of these cells.
  • polyclonal and monoclonal antibodies which recognize novel antigenic markers expressed by the blood-borne mesenchymal cells.
  • Such antibodies may have a variety of uses such as the isolation and characterization of blood-borne mesenchymal cells by affinity chromatography.
  • Various procedures known in the art may be used for the production of antibodies to these mesenchymal cells.
  • Various host animals can be immunized by injection with viable isolated mesenchymal cells, fixed cells or membrane preparations, including but not limited to rabbits, hamsters, mice, rats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to
  • Monoclonal antibodies to novel antigens on these mesenchymal cells may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256. 495-497), and the more recent human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. 80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .
  • Syngeneic, allogeneic, and xenogeneic hosts may be used for injection of blood-borne mesenchymal cells prepared in viable form, or in fixed form, or as extracted membrane preparations thereof. Monoclonal antibodies can be screened differentially by selective binding to mesenchymal cells, but not to other blood cells.
  • Antibody fragments which contain the binding site of the molecule may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • mesenchymal cells may be used as a source for identifying novel cytokines and cell surface accessory molecules and the genes encoding therefor.
  • long-term mesenchymal cell cultures may be established or continuous cell lines may be generated by transforming the cells to tumor cells using a virus or a chemical. Culture supernatants may be directly analyzed by applying them to various cell types or in various animal models, which can then be assayed for the appropriate desired biological response.
  • the cells may be metabolically labelled and their supernatants subjected to biochemical analysis to identify candidate proteins responsible for the observed bioactivity. Additionally, cytokines may be identified by inducing cytokine production in the cells. To this end, the cells may be exposed or contacted with an agent that induces the expression and production of a cytokine. A number of agents known to induce cytokine production in other cells may be useful in this approach. Such agents may include but are not limited to calcium ionophores, endotoxins, phorbol esters, known cytokines, chemokines, growth factors, hormones and/or other mediators.
  • the protein may be purified by a variety of techniques known in the art including but not limited to SDS-preparative gels, ion exchange chromatography, isoelectric focusing gels and other types of chromatography. Purity of the proteins can be verified by SDS-PAGE, quantified by protein assays, their activities confirmed in bioassays, and used as immunogens for the production of polyclonal and monoclonal antibodies.
  • the purified proteins can be further tested in bioassays to stimulate and/or inhibit proliferation and/or differentiation of a variety of indicator cell lines of diverse tissue types. Radiolabelled proteins may also be used to identify their cell surface receptors by methods such as affinity labelling. Specific antibodies to the cytokines may be used to identify and quantify membrane forms and secreted forms of the cytokines, to study their biosynthetic pathways, to affinity purify the proteins and to immunoscreen expression libraries for the molecular cloning of the coding sequences.
  • Pelleted cells were resuspended in 25 ml Dulbecco's Modified Eagle's Medium/20% fetal bovine serum (FBS)/ and 0.1% gentamicin. The cells then were plated onto a 150 mm tissue culture plate. After 24 hours, medium together with non-adherent cells was aspirated and replaced with fresh medium. Medium was replaced with fresh medium weekly and adherent cells enumerated at intervals.
  • FBS fetal bovine serum
  • IMMUNOFLUORESCENCE AND CYTOFLUOROGRAPHY in some cases, cells were seeded into wells that had microscope slide coverslips resting on the bottom of the wells. Spot immunofluorescence was then performed on cells cultured for 4 weeks on 13 mm glass coverslips. For analysis, the slips were removed from the plates, washed twice with PBS, and fixed by immersion in 3.5% formaldehyde for 20 minutes. The cells were washed once with PBS, then immersed for 7 minutes at -20°C in 70% ethanol. The 70% ethanol was replaced with 100% ethanol in which the cells were immersed for an additional 7 minutes at -20°C. The cells then were immersed in 70% ethanol for 5 minutes at -20°C and washed 3x with PBS.
  • Cytofluorography was performed on 4 week cultured cells. Adherent cells were removed by gentle scraping and elutriation. After washing 3 times in PBS and enumeration, the cells were resuspended in 1% BSA in PBS at a concentration of 5X10 6 cells/ml. 3X10 5 cells were aliquoted into polystyrene tubes (10 x 75 mm) and 10 ⁇ l of undiluted primary antibody added for 45 minutes on ice in the dark, then washed 3x in 1% BSA/PBS. Ten ⁇ l of a second antibody-fluorescent dye conjugate was added (if the primary antibody was not directly conjugated to a fluorescent dye) and the cells were incubated for 40 minutes on ice in the dark.
  • cells were incubated as above with two directly conjugated antibodies of different fluorescence properties (i.e., FITC and rhodamine or Texas Red) .
  • the cells were washed 3x in 1% BSA/PBS, resuspended in 25 ⁇ l 1% BSA/PBS and 100 ⁇ l 3.5% formaldehyde, and stored at 4°C in the dark until ready for cytofluorography.
  • Cells were analyzed with a Becton Dickinson FACS 440 and the Profile 2 by Coulter.
  • the cells were to be double stained, they were incubated as above, substituting directly-conjugated antibodies of different fluorescence colors (i.e., FITC and rhodamine or Texas Red) .
  • the cells were washed three times in 1% BSA/PBS, resuspended at a concentration of 5xlO s /ml in 25 ⁇ l 1% BSA/PBS and 100 ⁇ l 3.5% formaldehyde, and stored at 4°C in the dark until ready for sorting. If the cells were to be cultured after the sort, they were collected into DMEM/20% FBS/0.1% gentamicin and plated.
  • REAGENTS The majority of the antibodies used in the studies described herein were purchased from Becton Dickinson (San Jose, CA) . The exceptions are: anti-fibronectin, anti-desmin, anti-smooth muscle ⁇ -actin and anti-laminin, (Sigma, St. Louis, MO) , anti-vimentin (Labsystems, Raleigh, NC) , anti-collagen (Chemicon, Temecula, CA) and anti-von Willebrands factor (Accurate) .
  • MIGRATION ASSAY Wound chambers were implanted into subcutaneous pockets in the flanks of mice.
  • the wound chambers consisted of a perforated 3 cm length of silastic tubing (Dow Corning) that contained a piece of polyvinyl alcohol sponge (Unipoint, NC) that had been sterilized by autoclaving. Incisions were closed with wound clips and the mice were monitored for infection. Once weekly post implantation, the wound fluid was percutaneously aspirated using a lcc syringe with a 25g needle.
  • the cells obtained were cultured in DMEM/20% FBS/0.1% gentamicin. Cells were analyzed by morphology and fluorescence staining techniques for fibroblast-specific markers.
  • Immunofluorescence analysis for selected cell surface markers was performed by direct visualization under fluorescence microscopy (spot immunofluorescence) and by cytofluorography. The two distinct cell types were further separated, purified and characterized by fluorescence-activated cell sorting (FACs) .
  • FACs fluorescence-activated cell sorting
  • the large "spindle-shaped" cell was identified by antibody staining to be a mesenchymal cell type that displayed typical fibroblast markers; i.e., collagen I, III, vimentin, and fibronectin.
  • MHC class II T cell receptor ( ⁇ and 5)
  • Blood-borne mesenchymal cells could be expanded in vitro by addition of granulocyte- acrophage colony stimulating factor (GM-CSF) at a concentration of 50 U/ml (FIG. 2) .
  • GM-CSF granulocyte- acrophage colony stimulating factor
  • mice were experimentally implanted with wound chambers in their back. The migration of a blood-borne murine cell population corresponding morphologically to the human blood-borne mesenchymal cells into the chambers was observed.
  • the present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

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Abstract

La présente invention se rapporte à une population de cellules mammaliennes à diffusion hématogène qui expriment un profil unique de marqueurs de surface, dont certains marqueurs typiques des fibroblastes du tissu conjonctif, et sont appelées ici 'cellules mésenchymateuses à diffusion hématogène'. Cette invention se rapporte notamment à l'isolation, la caractérisation et les utilisations de ces cellules mésenchymateuses à diffusion hématogène. Les cellules de la présente invention peuvent se distinguer des leucocytes du sang périphérique par leur taille distincte, leur morphologie, le phénotype de surface cellulaire et les activités biologiques, et peuvent se distinguer en outre des fibroblastes du tissu conjonctif par d'autres marqueurs phénotypiques de surface. Ces cellules prolifèrent en culture, et in vivo, tel que mis en évidence dans des modèles animaux, et peuvent migrer dans des sites lésionnels à partir du sang. Ces cellules mésenchymateuses à diffusion hématogène peuvent avoir par conséquent une large plage d'applications, notamment, mais non exclusivement, l'activation de la cicatrisation, le remodelage tissulaire et en thérapie génique.
PCT/US1994/002850 1994-03-16 1994-03-16 Cellules mesenchymateuses a diffusion hematogene Ceased WO1995025164A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
KR1019960705117A KR100352057B1 (ko) 1994-03-16 1994-03-16 혈액-상재간엽세포
NZ265435A NZ265435A (en) 1994-03-16 1994-03-16 Blood-borne mesenchymal cells (fibroblasts) for wound healing and tissue remodelling
EP94913910A EP0759070A4 (fr) 1994-03-16 1994-03-16 Cellules mesenchymateuses a diffusion hematogene
PCT/US1994/002850 WO1995025164A1 (fr) 1994-03-16 1994-03-16 Cellules mesenchymateuses a diffusion hematogene
JP7523995A JPH09510348A (ja) 1994-03-16 1994-03-16 血行性間葉細胞
AU65884/94A AU693441B2 (en) 1994-03-16 1994-03-16 Blood-borne mesenchymal cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1994/002850 WO1995025164A1 (fr) 1994-03-16 1994-03-16 Cellules mesenchymateuses a diffusion hematogene

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WO1995025164A1 true WO1995025164A1 (fr) 1995-09-21

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EP (1) EP0759070A4 (fr)
JP (1) JPH09510348A (fr)
KR (1) KR100352057B1 (fr)
AU (1) AU693441B2 (fr)
WO (1) WO1995025164A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1099754A1 (fr) * 1999-11-10 2001-05-16 Universiteit Leiden Cellules souches mesenchymateuses et/ou cellules progénitrices, leur isolation et leur utilisation
WO2011022045A3 (fr) * 2009-08-17 2011-04-14 Singh Ashok K Appareil et procédé pour générer et récolter des cellules souches adultes et du fluide associé à celles-ci provenant de la peau et de l'épiploon pour une utilisation médicale, cosmétique et vétérinaire
CN115287260A (zh) * 2022-06-30 2022-11-04 江苏汇先医药技术有限公司 一种t细胞的富集方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0759070A4 *
THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY (FASEB) JOURNAL, Volume 7, Number 3, issued 19 February 1993, BUCALA et al., "Identification of a Circulating Mesenchymal Cell Population in Peripheral Blood: Possible Role in Wound Healing and Tissue Remodeling", page A139, Abstract 805. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1099754A1 (fr) * 1999-11-10 2001-05-16 Universiteit Leiden Cellules souches mesenchymateuses et/ou cellules progénitrices, leur isolation et leur utilisation
WO2001034775A1 (fr) * 1999-11-10 2001-05-17 Universiteit Leiden Cellules souches mesenchymenteuses et/ou progenitrices, leur isolation et utilisation
WO2011022045A3 (fr) * 2009-08-17 2011-04-14 Singh Ashok K Appareil et procédé pour générer et récolter des cellules souches adultes et du fluide associé à celles-ci provenant de la peau et de l'épiploon pour une utilisation médicale, cosmétique et vétérinaire
US9173903B2 (en) 2009-08-17 2015-11-03 Ashok K. Singh Fluid associated with adult stem cells for medical, cosmetic, and veterinary use
CN115287260A (zh) * 2022-06-30 2022-11-04 江苏汇先医药技术有限公司 一种t细胞的富集方法

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JPH09510348A (ja) 1997-10-21
AU693441B2 (en) 1998-07-02
AU6588494A (en) 1995-10-03
KR970701777A (ko) 1997-04-12
EP0759070A1 (fr) 1997-02-26
EP0759070A4 (fr) 1998-03-11
KR100352057B1 (ko) 2003-01-24

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