EP1636341A2 - Cellules souches a des fins cliniques et commerciales - Google Patents

Cellules souches a des fins cliniques et commerciales

Info

Publication number
EP1636341A2
EP1636341A2 EP04809422A EP04809422A EP1636341A2 EP 1636341 A2 EP1636341 A2 EP 1636341A2 EP 04809422 A EP04809422 A EP 04809422A EP 04809422 A EP04809422 A EP 04809422A EP 1636341 A2 EP1636341 A2 EP 1636341A2
Authority
EP
European Patent Office
Prior art keywords
cells
stem cells
body cavity
cavity space
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04809422A
Other languages
German (de)
English (en)
Other versions
EP1636341A4 (fr
Inventor
Liping Tang
Robert H. Hansen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
Original Assignee
University of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/857,443 external-priority patent/US20050014255A1/en
Application filed by University of Texas System filed Critical University of Texas System
Publication of EP1636341A2 publication Critical patent/EP1636341A2/fr
Publication of EP1636341A4 publication Critical patent/EP1636341A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells

Definitions

  • the present invention relates to the general field of cell physiology, and more particularly to compositions and methods of developing and producing clinically viable stem cells for medical applications, such as diagnosis, screening, testing, therapy, and rehabilitation, as well as cells for use in commercial applications, such as screening, testing, and bioengineering.
  • Stem cell research provides a new panacea for patients with cancer, spinal cord injuries, stroke, degenerative diseases, and other conditions because of their plasticity and potential use to replace diseased, injured or aged tissues and organs. It has been suggested that by using stem cell transplants instead of drugs, biologies, and other current therapies, stem cells can offer new therapies for the prevention and/or treatment of various human disorders and conditions.
  • stem cells can be derived from an embryo, adult bone marrow and other tissue, or a fetus (e.g., umbilical cord blood). Due to ethical concerns regarding the retrieval of stem cells from embryonic tissue, this particular area of research has encountered significantly less attention of late. Research has instead focused on the isolation, proliferation, and tissue/cell transplantation of stem cells derived from adult and fetal tissue.
  • One limitation to the use of these types of stem cells is that because the cells are capable of triggering transplant rejection, stem cells from the same patient are generally required.
  • Another limitation is that only a small number of adult or fetal stem cells may be retrieved from any one tissue. This has hindered the use of stem cells for widespread or cost-effective clinical treatment.
  • stem cells retrieved from adult tissue are often limited in their ability to differentiate. Most often, adult stem cells are only able to differentiate into the cell type of the tissue of origin. As such, most adult stem cells cannot be considered truly pluripotent.
  • adult stem cells are rare in mature tissue and methods for recovery and expansion in culture are generally inefficient, result in a very low yield and are destructive to the tissue— part if not all of the original tissue is destroyed in the process of obtaining stem cells. This makes it difficult to use adult stem cell for replacement or enhancement therapy where large numbers of cells are needed and/or the tissue of origin is essential and cannot be destroyed.
  • current stem cell retrieval techniques in order to increase stem cell yield without destroying the tissue of origin.
  • the present invention solves the problem associated with current stem cell collection and retrieval techniques by providing a new and improved method of obtaining stem cells at a high yield without destroying the tissue of origin as well as methods of use of such stem cells.
  • the present invention provides populations of stem cells, stem cell lines, and compositions of stem cells for clinical, diagnostic, pharmaceutical, and commercial use.
  • a method of obtaining a new source of stem cells is provided by introducing a foreign object or an implant into a body cavity space of a mammal and collecting those stem cells resulting from implantation.
  • a method for providing to a subject in need thereof one or more stem cells of the present invention is identified by retrieving stem cells from a body cavity space of a mammal after implanting a foreign object, manipulating the stem cells and introducing the stem cells into the subject in need thereof.
  • the stem cell may be manipulated by genetics, allowed to contact another composition, directed to specialize into a desired cell type, and/or allowed to contact a three-dimensional scaffold prior to introducing into the subject.
  • the subject may be the same as the source of the stem cells or different.
  • stem cells retrieved in accordance with the present invention are that they are inexpensive to retrieve, produce a high yield of cells, are capable of differentiation, proliferation, and genetic modification (in vivo and ex vivo), function in a physiologic manner (e.g., conduct, produce, secrete, regulate biologic compounds, etc.), exhibit true pluripotency, and are amenable for clinical, diagnostic and commercial uses, such as cell/tissue transplantation, replacement, implantation, grafting, genetic or diagnostic screenings, product development, and for therapeutic, preventative or other treatments purposes (e.g., for spinal cord injuries, other tissue or organ injuries, burns, cirrhosis, hepatitis, Parkinson's Disease, Alzheimer's Disease, stroke, muscular dystrophy, diabetes, arthritis, osteoporosis, leukemia, sickle cell disease and other anemias, as examples).
  • a physiologic manner e.g., conduct, produce, secrete, regulate biologic compounds, etc.
  • a physiologic manner e.g., conduct, produce,
  • the present invention is well suited for use in individuals who may be sensitive to other treatment options such as persons with cancer or those at a late-stage in their disease, an option not available when using most other traditional methods of stem cell retrieval, such as bone marrow harvest which may only be performed in patients considered relatively healthy.
  • the present invention is safer than current traditional methods such as bone marrow harvest with no added complications, and may be used repeatedly even on the same patient without any known difficulties or side effects.
  • the present invention addresses current problems regarding transplant rejection by the immune system and reduce or eliminate the need for use of immunosuppressive drugs with transplantation.
  • the present invention provides an economic clinical treatment option and fulfills the current need of researchers, healthcare providers, and industry for compositions and method of recruiting and retrieving a large number of human stem cells for clinical procedures, including transplantation, gene, protein and cell therapy.
  • the compositions and methods of the present invention serve as unique and powerful tools to supply stem cells, especially to a person in need thereof.
  • Custom designed products of the present invention include compositions for use in medicinal, therapeutic, diagnostic, engineering, and biotechnology applications, as examples.
  • FIGURE 1 is an example illustrating the pluripotent nature of stem cells of the present invention
  • FIGURE 2 depicts a light microscopic view of cells of the present invention after retrieval and plating on a substrate
  • FIGURE 3 depicts the relationship between time and stem cell production in accordance with the present invention, depicting cells exposed to an implant (diamond) and not exposed to an implant (square);
  • FIGURE 4 depicts fibrocyte growth over time in accordance with the present invention, depicting those exposed to an implant with or without lavage fluid (diamond or square, respectively) and not exposed to an implant (crosses or triangles);
  • FIGURE 5 shows two views of cells of the present invention (A) induced to form calcium rich-osteoblast aggregates and (B) control or non-differentiated cells depicting a random distribution;
  • FIGURE 6 depicts a view of cells of the present invention after having been induced to become specialized nerve cells
  • FIGURES 7A and 7B depict views of stem cells of the present invention further induced to become specialized nerve cells with typical neuronal morphology
  • FIGURES 8 A and 8B depict views of stem cells of the present invention induced to become specialized nerve cells by displaying typical neuronal morphology
  • FIGURES 9 A, 9B and 9C depict views of stem cells of the present invention induced to become specialized nerve cells after staining positive for NFM and FIGURE 9D show the stem cells not induced to differentiate and showing no positive NFM staining;
  • FIGURE 10 depict views of stem cells of the present invention induced to become specialized muscle cells with muscle striations (10A and 10B), and linear growth patterns (IOC) as well as ability to form myotubes (10D), wherein anchors are striated at the ends of the myotubes after prolonged culturing;
  • FIGURE 11 depicts cell growth as a function of time in accordance with the present invention, depicting exposure to GM-CSF and IL-4 (diamonds) as compared with non growth- inducing signals (squares);
  • FIGURE 12 depicts views of cells of the present invention following the addition of one or more differentiation-inducing signals that promote specialization of the cells into dendritic cells;
  • FIGURE 13 shows several panel views of stem cells of the present invention induced to differentiate into dendritic cells after which immunocytochemistry was performed, wherein arrows in FIGURES A and B show some of the CD1 lc positive cells, a unique marker for dendritic cells, as compared with control cells not induced to differentiate and displaying no CD1 lc (no positive staining); and
  • FIGURE 14 depicts images of cells of the present invention following the addition of one or more differentiation-inducing signals that promote specialization of cells into adipocyte cells (FIGURES 14A, 14B, and 14C), wherein differentiated stem cells are oil red o positive (darkened areas) and cells not induced to differentiate into adipocyte cells are not oil red o positive (no darkened areas, FIGURE 14D).
  • a stem cell is a pluripotent cell in an unspecialized (e.g., undifferentiated) state that may give rise to one or more unspecialized cells.
  • One critical identifying feature of a stem cell is its ability to exhibit self-renewal or to generate more of itself; therefore, a cell with the capacity for self-maintenance.
  • a stem cell is an unspecialized cell capable of proliferation (replication many times over), self-maintenance, and production of a large number of specialized functional progeny, as well as an ability to regenerate tissue after injury.
  • stem cells One role of stem cells is to replace cells that are lost by natural cell death, ageing, injury or disease.
  • the presence of stem cells in a tissue usually correlates with a high turnover of cells in that tissue, but may also exist in tissue lacking a high turnover rate.
  • replication or “proliferation”
  • adult stem cells should be able to replicate without differentiating for many months.
  • current methods retrieve adult stem cells with only a finite ability to grow/proliferate before differentiation is initiated (sometimes automatically) by the cells.
  • an advantage of the present invention is that it provides for the retrieval of stem cells with an ability to replicate for many months (even greater than a year) while remaining unspecialized and, thus, provides a method of yielding millions of cells for long-term self- renewal.
  • Another interesting property of stem cells is that they typically generate the same cell type as the tissue from which they are derived.
  • blood-forming adult stem cell from bone marrow normally gives rise to only blood cells; mesenchymal stem cells (blood marrow stromal cells) normally give rise to specific connective tissue cell types; hematopoietic stem cells normally give rise to all types of blood cells; neural stem cells in the brain normally give rise to nerve cells/neurons, astrocytes, oligodendrocytes; epithelial stem cells from the digestive tract lining normally give rise to digestive-specific cell types; skin stem cells retrieved from the basal layer of the epidermis normally give rise to keratinocytes, while follicular stem cells retrieved from the base of the hair follicle of the epidermis normally give rise to hair follicle cells and epidermal cells.
  • mesenchymal stem cells blood marrow stromal cells
  • hematopoietic stem cells normally give rise to all types of blood cells
  • neural stem cells in the brain normally give rise to nerve cells/neurons, astrocytes, oligodendrocytes
  • the present invention provides stem cells that are able to differentiate into non-derived tissue (e.g., a cell type that differs from the tissue from which they were derived). This ability is termed plasticity.
  • stem cells of the present invention are able function by (a) generating a line of genetically identical cells (e.g., same cells or clones) and (b) differentiating into a number of specialized cell types.
  • Stem cells of the present invention are identified by: (1) labeling cells in a living tissue with molecular markers to determine the specialized cell type; (2) removing the cells from a living tissue (i.e., donor, subject or patient), culturing cells followed by manipulation in vitro (e.g., introduction of one or more nucleic acids sequences or addition of one or more differentiation-inducing substituents) to determine the range of specialized cell types that the cells may become; and (3) removing cells from a living tissue (i.e., donor, subject or patient of the same or different genetic background), labeling cells after they are cultured and then transplanting them back into a host (e.g., donor, different subject or patient of the same or different genetic background) to determine whether the cells repopulate their tissue of origin. Infection of stem cells with a virus that provides a unique identifier to each new stem cell progeny is used to demonstrate that stem cell clones will repopulate one or more injured tissues in a patient in need (e.g., donor or host
  • the present invention provides methods for developing, recruiting, and retrieving pluripotent stem cells from a body cavity space of an adult mammalian host.
  • cells retrieved from the body cavity space of the adult mammalian host are able to (a) create a clonal population of stem cells; (b) proliferate both in vitro or in vivo (e.g., in situ), to generate large numbers of stem cell progeny; (c) differentiate into one or more specialized cell-type upon manipulation in vitro (e.g., demonstrate plasticity) or in vivo.
  • Methods for proliferation, manipulation, and differentiation of the stem cells retrieved from a body cavity space of a mammalian host are also provided.
  • proliferation, manipulation, and differentiation of the stem cells retrieved from the body cavity space of an adult mammalian host are generally performed in suspension or on a substrate to which the cells adhere (e.g., monolayer, three-dimensional network).
  • a substrate or second cell may be used, one that has one or more adherent molecules, chemicals or biologic substances on its surface (e.g., crosslinked or noncrosslinked amino acids, nucleic acids, polymers, polysaccharides, etc).
  • proliferation, manipulation and differentiation of stem cells may be performed within the host prior to or without retrieval.
  • biologic substances are organic compounds that may influence a cell, tissue, or organ.
  • Proliferation and differentiation of stem cells of the present invention are induced, under appropriate conditions, either ex vivo or in vivo as follows: (a) proliferation and differentiation in vitro followed by incorporation into a host; (b) proliferation in vitro followed by incorporation into a host followed by further proliferation and/or differentiation in vivo; (c) proliferation and differentiation after incorporation into a host (i.e., in vivo).
  • proliferation and/or differentiation in vivo e.g., in a host
  • proliferation and/or differentiation in vivo may occur in combination with a surgical procedure, injection procedure, a non-surgical approach (e.g., one that coaxes stem cells proliferation using pharmaceutical manipulation), or a combination of these approaches.
  • Proliferation and differentiation are under controlled stimulation when available. In general, conditions for in vitro cell culturing are near physiologic conditions.
  • culture medium When culturing stem cells, culture medium is generally one used under standard conditions appropriate for stem cells or for cell specialization. Supplementation may occur with a growth-promoting signal or substituent, as known to one of ordinary skill in the art.
  • growth-promoting signal As used herein, the terms “growth-promoting signal,” “growth-promoting substituent,” “proliferation- inducing signal,” “proliferation-inducing substituent,” or “growth factor” generally refer to a compound (e.g., protein, peptide or other molecule) or stimulus having a growth, proliferative, and/or trophic effect on a cell.
  • differentiation-inducing signal As used herein, “differentiation-inducing substituent,” generally refer to a compound or stimulus that induces cell differentiation or specialization.
  • signals Such compounds may be referred to generally as “signals” or “substituents.”
  • signals or substituents include cytokines, growth-promoting pharmaceutical agents, growth factors such as epidermal growth factor (EGF), amphiregulin, fibroblast growth factor (FGF, acidic or basic), nerve growth factor (NGF), platelet-derived growth factor (PDGF), thyrotropin releasing hormone (TRH), transforming growth factor (TGF), and insulin-like growth factor (IGF), as examples.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TRH thyrotropin releasing hormone
  • TGF insulin-like growth factor
  • IGF insulin-like growth factor
  • substituents are usually added to the culture medium at concentrations ranging between about 1 fg/ml to 1 mg/ml. To optimize culture conditions, simple titrations can be performed easily to determine optimal substituent concentrations. Additional signals include mechanical and/or electrical signals (e.g., cell-cell contact, adhesion, movement, electrical stimulation, physical pressure, distortion, etc.).
  • the present invention uses the introduction or implantation of a foreign object in a body cavity space of a mammal (e.g., donor or host) to induce the formation, accumulation and recruitment of stem cells into the body cavity space.
  • a mammal e.g., donor or host
  • stem cells include the peritoneal cavity, subcutaneous space, pleural space, lung and/or brain space. These regions are generally larger (biologically), often with significant circumferential area and generally afford easy access, especially as compared with protected and space-limited organs, blood vessels, or bone marrow.
  • body cavity space may also be referred to as "cavity,” "peritoneal cavity,” “subcutaneous cavity,” “lung cavity,” “pleural cavity,” “brain space,” and “cavity space.”
  • foreign object includes any object (solid, liquid or gel) that is introduced into and induce stem cells to form in the body cavity space, wherein formation includes the migration, recruitment and accumulation of stem cells in the body cavity space.
  • a degradable implant non-degradable implant, inflammatory agent (e.g., vaccine adjuvants, dead bacterial fragments), fibrotic agent, pharmaceutical composition, injectable substance (liquid, gel, or solid), biocompatible composition (protein, nucleic acid, polymer, dye, plastic, synthetic or natural drug, chemical isotope, metal composite, etc.), medical instillation solution (e.g., saline, dextran, water) as well as compositions or materials used in a surgical procedure.
  • inflammatory agent e.g., vaccine adjuvants, dead bacterial fragments
  • fibrotic agent e.g., fibrotic agent
  • pharmaceutical composition injectable substance (liquid, gel, or solid), biocompatible composition (protein, nucleic acid, polymer, dye, plastic, synthetic or natural drug, chemical iso
  • the biomaterial may be of a single composition or a polymer or composite blend of many materials either in a layer or in a mixture and may include a substance thought to promote biologic growth.
  • the implant of the invention is a biocompatible support making possible the biological anchoring of cells.
  • stem cell initiation is promoted by entry of a foreign object substance such as a medical instillation (e.g., water, dextran, saline) into the body cavity space.
  • a medical instillation e.g., water, dextran, saline
  • Surgical techniques include trans-abdominal wall incision, laparoscopic surgical technique (one or more small incisions or using microsurgical instruments to access the cavity), endoscopy (access with a flexible endoscope), etc.
  • Injection is generally with a needle and syringe.
  • One skilled in the art is acquainted with methods and techniques that allow body cavity space access or entry into the body cavity space.
  • the larger the cavity e.g., peritoneal cavity, lung cavity, or pleural cavity
  • several rounds of infusion and withdrawal may be performed to continuously retrieve stem cells from the body cavity space.
  • gathering cells from the cavity may be as simple as loading the peritoneum with fluid and draining the fluid out. As such, the present invention has found that the fluid will contain stem cells only after a foreign object is introduced into the cavity.
  • a process similar to that of peritoneal dialysis may be performed in which a catheter is used to fill the abdomen with up to 3 liters of a solution (e.g., dialysis solution) and from the walls of the abdominal cavity (i.e., the peritoneum) the fluid along with other particulates from within the cavity are returned (dialyzed) as a solution when drained from the body.
  • a solution e.g., dialysis solution
  • the peritoneum the fluid along with other particulates from within the cavity are returned (dialyzed) as a solution when drained from the body.
  • Stem cells of the present invention are generally retrieved from the cavity of a donor that is a mammal. Any animal with a body cavity space and the ability to generate pluripotent cells may be used as the stem cell donor, including insects, fish, reptiles, birds, amphibians, and mammals, as examples.
  • stem cells retrieved from the donor are available for a number of applications, including genetic manipulation, diagnostics, phenotyping, screening, and/or transplantation into a heterologous, autologous, or xenogeneic host.
  • Stem cells from the cavity of a donor are generally retrieved by collecting fluid used to wash the body cavity space. They can also be collected from within and around an implanted material, such as one that includes a three-dimensional matrix with pores.
  • washing may include lavage, dialysis, medical instillation and other methods of washing, perfusion, disruption or dissociation in order to collect cells from the body cavity space.
  • Cells may also be collected by dissociation (before or after washing) from any extracellular matrix tissue or implant in the cavity and generally under sterile procedures. Where dissociation and/or disruption is required, routine methods well known in the art are implemented, such as treatment with enzymes (trypsin, collagenase, as examples) or by physical methods (e.g., using a blunt instrument).
  • Fluid used for washing/dissociation may be a regular or modified pH balanced (physiologic) solution such as water, saline, medical instillation solution, dextran, or tissue culture medium (e.g., HEH, DMEM, RPMI, F-12, as examples, used alone or in combination).
  • cells may be centrifuged at low speed (e.g., 200 to 2000 rpm) and then resuspended in fresh culture medium prior to culturing which may be on a fixed substrate or in suspension. Alternatively, cells are collected with fluid.
  • Fluid used for washing/dissociation may also contain one or more signals, substituents and/or supplements, such as those required for growth, cellular metabolism (e.g., growth factors, amino acids, vitamins, minerals, and/or proteins, as examples) and those that prevent infection or contamination by yeast, bacteria, fungi, etc. (e.g., antimicrobial, antibiotic, antifungal, antiviral).
  • Washing and dissociation fluid as well as fresh culture medium are used with or without serum (e.g., from bovine, equine, chicken, as examples). Routine culture medium used for cell harvesting, inducing growth, differentiation, dedifferentiation, cell expansion, immortalization, specialization and for transplantation are well known in the art.
  • the appropriate medium is replaced or perfused, either continuously or periodically as needed.
  • the retrieved/collected cells may be further enriched by any desired amount (e.g., for cell expansion). Different known methods may be used to achieve this enrichment, (e.g., negative selection method or positive selection method). Via this or other procedures known in the art, stem cells and progenitor cells may be concentrated to any degree desired.
  • An alternative means includes using a packing cell line infected with a retroviras, or a supernatant obtained from such a packaging cell line culture, is added to the stem cells retrieved in accordance with the present invention and when practiced together with an enriched stem cell pool (even in the presence of additional differentiation or proliferation-inducing substituents) provides a very effective means for obtaining stem cell infection in vitro.
  • stem cells upon retrieval may include: induction of proliferation, cell expansion, differentiation into one or more specialized cell types, genetic modification, short- or long-term storage (e.g., cryopreservation or any method known in the art), screening, diagnostic probing, phenotyping, and therapeutic interventions such as for transplantation, chemotherapy, disease treatment, disease prevention, and cell, organ or tissue replacement or enhancement, as examples.
  • Adding one or more recruitment-inducing substituents into the cavity of the donor may increase the actual number of stem cells retrieved from the cavity.
  • "recruitment- inducing substituents” refer to substituents that stimulate cell recruitment and/or proliferation, examples of which include anti-mitotic agents, anti-differentiating substituents, or proliferation- inducing signals. This ability to enhance the recruitment and proliferation of stem cells retrieved from a subject or donor reduces the time between retrieval and therapeutic intervention and improves the efficiency of the present invention and is one form of cell enrichment.
  • the culture medium may be generally supplemented with at least one proliferation-inducing substituent or substance (i.e., a chemical or biologic factor, generally trophic, that includes cell division, such as molecules that binds to a receptor on the surface of the cell and exert trophic or growth-inducing effects).
  • proliferation-inducing growth factors include EGF, amphiregulin, FGFs, TGFs, as examples, used alone or in combination.
  • Additional substituents may be added to the culture medium, especially those that are lineage specific substituents such as vitamins, NGF, PDGF, TRH, TGF, BMP, GM-CSF, or IGF, as examples.
  • Proliferation of cells of the present invention may occur prior to or after collection from the body cavity space, where similar proliferation-inducing signals maybe used. Proliferation of the cells retrieved from the cavity may begin as early as a few hours or may take several days. Generally, cells retrieved from the body cavity space, when placed on a substrate, become adherent within a few hours. After proliferation, new unspecialized cells will appear in culture. These proliferative cells are generally clonal (i.e., are progeny of a single stem cell). In the continued presence of a proliferation-inducing signal, the number of total cells in culture will increase. In addition, the original cells retrieved from the cavity may increase in size.
  • Proliferating cells will continue to proliferate in suspension if continually offered the appropriate culture medium as described above.
  • the proliferating cells in culture may also be passaged and proliferation reinitiated.
  • passaging and reinitiating proliferation may be continuously repeated (e.g., weekly) resulting in a logarithmic increase in the number of cells after each passage.
  • the stem cells may be genetically modified in vitro using techniques as described below.
  • Differentiation of cells of the present invention may be induced by any method that activates the cascade of biological events leading to growth, including such things as liberating inositol triphosphate (ITP), intracellular Ca 2+ , liberation of diacyl glycerol and/or the activation of protein kinase C (PKC) and other cellular kinases, as examples.
  • ITP inositol triphosphate
  • Ca 2+ intracellular Ca 2+
  • PLC protein kinase C
  • PLC protein kinase C
  • Differentiation is controlled by external signals, such as chemical secretions by other cells, physical contact with neighboring cells, and certain other molecules in the environment (e.g., molecular substituents).
  • Other examples of methods that induce differentiation include treatment with phorbol esters, differentiation-inducing growth factors and other chemical signals, alone, in a temporal sequence or in combination with other signals.
  • plating the cells on a fixed substrate may also induce differentiation.
  • a fixed substrate e.g., flask, culture plate, or coverslip that may also be coated with an ionically charged surface such as poly-L-lysine and poly-L-omithine, as examples
  • Other substrates that may induce differentiation include those that resemble the extracellular matrix such as collagen, fibronectin, laminin, as examples and may be used alone or in combination. Differentiation may also be induced in a suspension in the presence of a proliferation-inducing substituent.
  • Differentiation of cells of the present invention occurs in as little as two hours or as long as at least a week, depending on the chosen lineage.
  • many of the cells will differentiate.
  • Differentiation and detection of a specific cell-type or lineage may be determined by morphology, immunocytochemistry and/or immunohistochemical methods or by expression of cell-type specific RNA or DNA.
  • cell-type specific antibodies, expression of specific genes, or specific histochemical assays maybe used to distinguish cellular characteristics or phenotypic properties of the specialized cells. Those skilled in the art will be able to recognize the methodologies that best characterize a specific cell type.
  • Cells may specialize into one of any cell type depending on the substituent (or temporal sequence thereof) that is added to the cell culture medium.
  • Examples of cell types include neural (e.g., glia, dendrites, etc.), non-neural (astocytes, oligodendrocytes), epithelial, hematopoietic, hepatic, cardiac, endothelial, muscular (smooth and skeletal), epidermal, osteoblastic, osteoclastic, chondrocytic, stromal, adipocytic, as examples.
  • neural e.g., glia, dendrites, etc.
  • non-neural astocytes, oligodendrocytes
  • epithelial hematopoietic
  • hepatic hepatic
  • cardiac endothelial
  • muscular smooth and skeletal
  • epidermal e.g., osteoblastic, osteoclastic, chondrocytic
  • stem cells retrieved from the peritoneum or other such cavity spaces are non-transformed cells, they possess features of a continuous cell line. In the unspecialized state, in the presence of a proliferation-inducing substituent, cells continuously divide and are, therefore, excellent targets for genetic modification.
  • the term "genetic modification” or “genetic manipulation,” as used herein, refers to the stable or transient alteration of the genotype of a cell retrieved from the cavity of an animal by intentional introduction of exogenous nucleic acid.
  • the nucleic acid may be synthetic or naturally derived, and may contain genes, portions of genes, or other useful nucleic acid sequences.
  • Exogenous nucleic acid may be introduced to a stem cell of the present invention by viral vectors (retrovirus, modified herpes viral, herpes- viral, adenovirus, adeno-associated virus, cytomegalovirus as examples) or mammalian cell-specific promoter or transfection (lipofection, calcium phosphate transfection, DEAE-dextran, electroporation, as examples) that direct the expression of one or more genes encoding a desired protein and may be used to promote differentiation.
  • the protein may be any protein or protein combination of interest and may be linked to a selectable marker for detection.
  • the vectors may include a drug selection marker. (See Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. for examples of techniques known in the art.)
  • stem cells especially those induced to differentiate can be genetically modified to cease cell death by administering Bcl-2 or by genetically modifying the cells with the bcl-2 gene, whose product is known to prevent programmed cell death (apoptosis).
  • the genetically modified cells of the present invention possess the added advantage of having the capacity to fully specialize to become cell-type specific cells. Specialization is reproducible and may be operable by using any of a number of well-known differentiation protocols.
  • the genetically modified stem cells retrieved from the cavity are implanted for cell/gene therapy into one or more heterologous and/or autologous hosts in need of one or more biologically active molecules produced by the genetically modified cells. Transplantation techniques are detailed below.
  • the genetically modified stem cells may be subjected to one or more differentiation protocols prior to implantation. Once the cells have specialized, they maybe isolated and implanted into one or more hosts in need of the protein or biologically active molecule that is expressed by the genetically modified cell.
  • transplantation with one or more stem cells and/or compositions of the present invention is performed in order to treat or prevent one or more disorders, diseases, or degenerative conditions, to repair or replace a damaged, poorly functioning or malfunctioning tissue/organ, or to enhance cell, tissue, or organ function.
  • organ or tissue injuries also referred to as affected areas
  • Transplantation with compositions of the present invention may be accompanied by immunosuppression as deemed necessary by one of ordinary skill in the art.
  • stem cells with genetic modification that includes gene replacement or gene knockout using homologous recombination may be employed.
  • MHC major histocompatibility complex
  • stem cells and compositions of the present invention are delivered throughout the affected neural area or to one or more specific sites as deemed appropriate to one of ordinary skill in the art.
  • the cells are administered using any method known to maintain the integrity of organ or tissue.
  • transplanted stem cells or specific components of the cells have been genetically modified to include one or more tracers
  • stem cells can be used as diagnostic markers, for probing, for visualization of tissue or organ remodeling changes, for markers of response to one or more stimuli (e.g., mechanical or chemical, such as electric fields, proteins, chemicals, drugs, etc.) or other therapeutic purposes.
  • stimuli e.g., mechanical or chemical, such as electric fields, proteins, chemicals, drugs, etc.
  • Stem cells of the present invention are also used for bioengineering purposes.
  • stem cells are recruited in a three-dimensional matrix (e.g., scaffold) that represent the tissue of interest.
  • a three-dimensional matrix e.g., scaffold
  • stem cells may or may not be induced to differentiate.
  • Stem cells may remain in the scaffold, where they are genetically modified or induced to differentiate or may be removed from the scaffold where they undergo further manipulation prior use.
  • stem cells may be recruited and induced to differentiate in vivo at the site of interest in order to create a new organ and/or tissue.
  • the scaffold is retrieved and implanted elsewhere or donated to a recipient.
  • Stem cells retrieved from the cavity of the present invention will be essential for screening of toxins and of potential therapeutic compositions and pharmaceutical preparations.
  • compositions are applied to cells at differing dosages, varying times during proliferation and or differentiation and cell response is likewise monitored over time as is well known in the art.
  • Morphologic (physical), genetic, secretory, conductivity (e.g., ion channels or nerve conduction) and other such responses are analyzed by one or more methods well known in the art (e.g., Western blot, Southern blot, Northern blot gene screening, immunohistochemistry, protein, receptor and enzyme assays, enzyme-linked immunosorbant assays (ELISA), electrophoresis analysis, HPLC, radioimmune assays, electrophysiologic measures, as general examples).
  • cell type-specific or proliferating stem cells of the present invention may be grown on a feeder layer (acting as a substrate) or in a three-dimensional network.
  • stem cells, prior to screening may have already undergone differentiation.
  • transplanted stem cells of the present invention (with or without the induction to differentiate) in the absence and presence of one or more specific compositions or preparations are observed for their efficacy and safety (e.g., host survival, pharmacologic, biochemical and immunologic effects, etc.).
  • the stem cells or type-specific cells of the present invention are used to measure the effect of an implant or another transplant on cells or a host.
  • compositions refer to any agent, such as a chemical, polymer, radioactive substance, virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro-drug, implant, and device, as examples.
  • agent such as a chemical, polymer, radioactive substance, virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro-drug, implant, and device, as examples.
  • stem cells of the present invention provides an economic way to test and monitor industrial or biologic chemicals and compounds.
  • cells are use for rapid identification of substances (e.g., via high throughput screening methods) involved in the proliferation, differentiation and survival of in vitro or host cells (including an organ or tissue).
  • cDNA libraries may be constructed from stem cell or lineage-specific cells of the present invention using techniques known in the art. As such, nucleic acids or their factors involved in cell regulation, dysfunction, repair, remodeling, etc. are analyzed and industrial compositions and/or pharmaceutical preparations are designed to promote positive cell features and counteract negative ones. Diagnostic probes are also developed, especially those that identify one or more genetic disorders or dysfunction.
  • cells of the present invention are investigated for their ability to secrete or produce potential therapeutic or industrial compositions.
  • an inflammatory response occurs with the recruitment or retrieval of stem cells in the body cavity space of a mammal.
  • the inflammatory response is generally induced by introducing (surgically or otherwise) a foreign object into the body cavity space.
  • an implant is surgically placed into the peritoneal cavity of a mouse, wherein the implant includes Polyethylene Terephthalate (PET) disks, and results are compared to those from mice undergoing sham surgery.
  • PET Polyethylene Terephthalate
  • the induction of pluripotent (e.g., stem) cells in the cavity occurs as early as at least about two hours after the introduction of the implant into the peritoneal space (data not shown).
  • the implant will continue to induce the formation of pluripotent cells for at least about 14 days and longer (data not shown).
  • Cells residing and recruited to the cavity were collected by lavage of the cavity. Collected cells were placed in culture and allowed to adhere to a substrate (e.g., culture plate). In culture, adherent cells were found to expand at an exponential rate. Initially, when viewing adherent cells, several different morphologic features were observed as illustrated in FIGURE 2. After days, weeks and/or months of cell growth in culture, stem cells are induced to differentiate into different cell types, including osteoblast, smooth muscle, fibroblast, neurons, and dendritic cells as will be discussed below.
  • a substrate e.g., culture plate
  • adherent cells were found to expand at an exponential rate. Initially, when viewing adherent cells, several different morphologic features were observed as illustrated in FIGURE 2. After days, weeks and/or months of cell growth in culture, stem cells are induced to differentiate into different cell types, including osteoblast, smooth muscle, fibroblast, neurons, and dendritic cells as will be discussed below.
  • an inflammatory response as well as a foreign object are necessary for the recruitment and retrieval of stem cell from the peritoneal cavity (e.g., retrieval from lavage fluid).
  • a foreign object is required for stem cells to appear in the lavage fluid and to proliferate in culture (data not shown).
  • cells were recruited by implantation of 1.2 cm diameter PET disks into the peritoneum of Swiss Webster mice by midline incision. Two PET disks — one disk on either side of the peritoneal cavity — were implanted after which the wound was closed with steel wound clips. After introduction of the implant for various time periods, mice were sacrificed, and cells within the peritoneum gathered by lavage. Lavage was performed by injection of at least about 4-5 mL of DMEM media into the peritoneum of the mouse and careful withdrawal of the lavage fluid with a transfer pipette after different time points.
  • the withdrawn medium (lavage fluid) was then placed into a tissue culture flask or cell culture plate with at least about 10% fetal bovine serum (FBS) and 1-5% antibiotics (e.g., penicillin and streptomycin) to protect against possible bacteria from dying macrophages or the gut.
  • FBS fetal bovine serum
  • antibiotics e.g., penicillin and streptomycin
  • Cells that adhere were pluripotent stem cells.
  • approximately 500,000 stem cells per gram of mouse were retrieved.
  • the same procedure may be repeated on the same subject several more time to continue stem cell recovery. For example, a subject undergoing peritoneal dialysis will retrieve stem cells after every dialysis treatment as long as there is a foreign object in the peritoneal cavity that induces an inflammatory response.
  • Adherent stem cells were then passaged to maintain them in culture with or without inducing proliferation or differentiation. In the presence of normal culture medium, an antibiotic and with minimal serum supplementation, stem cells continued to grow (e.g., divide into a population of progenitor cells) for at least about a year (data not shown).
  • Adherent cells generally with spindle shape morphology were readily recognizable shortly after lavage fluid (used to lavage the peritoneal cavity) was added to a culture surface. Nonadherent cells were removed and fresh media introduced to the cells after sufficient plating time, generally at least about 24 hours. Growth of the adherent spindle shaped cells was monitored by counting them over time in random areas of the culture surface (generally observed under a 40X microscope). FIGURE 3 shows that adherent cells grew at an exponential rate (diamonds).
  • mice recovered from the peritoneal cavity of mice in the absence of an implant (i.e., no foreign object introduced into the cavity prior to lavage) and allowed to adhere in the exact same manner as described above did not grow and eventually died out, generally within at least about one week.
  • an implant i.e., no foreign object introduced into the cavity prior to lavage
  • stem cells remained in culture.
  • alpha-fetoprotein may be used as a marker to further 'purify' the population.
  • induction of an inflammatory or similar such response induces stem cells to accumulate and /or migrate into the body cavity space.
  • stem cells e.g., fibrotic or wound healing response
  • cells were gathered from the peritoneum of 16 mice, of which eight animals received an implant and eight did not. Cells were retrieved from the cavity by lavage followed by an additional step to separate lavage fluid from the extracted cells by a brief centrifugation. Lavage fluid was saved following centrifugation and then swapped between the two groups of mice, 4 having received the implant, 4 having not received the implant.
  • FIGURE 4 illustrates the growth curves of these cultured cells. As can be seen from the number of cells counted for more than two weeks, cells from mice that received an implant grew at an exponential rate (diamonds and squares), while cells from mice without implant died (triangles and cross-hatches). Even with the introduction of lavage fluid from mice with implant (containing a multitude of inflammatory proteins and chemokines) to cells retrieved from mice without implant, cells did not grow.
  • FIGURE 4 shows that it is the introduction of the foreign object not the lavage fluid itself that induces stem cell formation, infiltration, and retrieval, because cells introduced to an implant grew in the absence (squares) or presence (diamonds) of the lavaged fluid.
  • bone cell differentiation by cells of the present invention may occur as follows.
  • cell aggregation and formation of cultured bone ossicles occurs in cells grown for an extended time and to high density prior to passaging (e.g., near or at confluence) in the presence of DMEM, fetal bovine serum (FBS) and an antibiotic.
  • FBS fetal bovine serum
  • Cells are also induced to become bone cells as follows.
  • Cells of the present invention are collected from mice with an implant following lavage. Cells are plated in 24-well culture plates using DMEM, 5% FBS and 2% antibiotic. After three hours, fresh media is added to each group (to remove non-adherent cells and lavage fluid).
  • Cells are grown in same media until reaching an approximate density of at least about 5 x 10 4 cells/cm 2 .
  • Differentiation-inducing additives substituted with DMEM, 5% FBS, and 1000 ng/mL bone morphogenetic protein (BMP)-2;
  • BMP bone morphogenetic protein
  • DMEM 5% FBS, and 300 ng/mL BMP-2;
  • DMEM 5% FBS, 1% antibiotic and 2.5 ng/mL TGF- ⁇ l; or
  • Media is replaced with identical additives after at least about one week.
  • FIGURE 5 A shows an example of the induction into bone cells using BMP-2. Note the calcium rich matrix and surrounding nodules of cells treated with BMP-2, as well as many individual cells on the periphery that also stain red.
  • FIGURE 5B shows that stem cells without BMP pretreatment do not show a positive red stain (dark area on image).
  • neural cell differentiation by cells of the present invention may occur as follows. As shown in FIGURE 6, following incubation with ⁇ -mercaptoethanol, cells are capable of differentiation into neural-like cells with a long, branched protrusions from a central cell body. In one embodiment, protrusions may grow to lengths that span over half of the culture plate. Cells of the present invention will form protrusions after a long time in culture (e.g., at least about one month with serum and antibiotics present).
  • stem cells retrieved by lavage are induced to become neural cells after culturing with DMEM, 10% FBS as follows.
  • Cells are grown to at least about 50% confluence after which lOmM ⁇ -mercaptoethanol (BME) is added into the medium.
  • BME lOmM ⁇ -mercaptoethanol
  • morphology of cells will began to change; cell bodies retract and processes elaborate.
  • At least about 48 hours after the addition of BME cells show a specialized multipolar morphology.
  • FIGURES 7A and 7B are two examples of differentiated neural cells at least about 24 hours after introduction of the differentiation- inducing substituents, a morphology that is not observed before addition of the differentiation- inducing substituents.
  • FIGURES 9A, 9B, and 9C Only neural cells induced to differentiate with neural-specific induction substituents stain positive for both NSE and NFM. Example of specialization into skeletal muscle cells.
  • Skeletal muscle cell differentiation by the cells of the present invention may occur by implementing culture conditions that induce skeletal muscle cell formation as well known in the art, and by implementing the following conditions. At least about several weeks after stem cells retrieved from the peritoneal cavity by lavage are introduced to a culture surface, skeletal muscle cell morphology will begin in the presence of DMEM, FBS an antibiotic, and 5-azacytidine. Examples of how these cells look morphologically are shown in FIGURE 10. Note the skeletal muscle striations seen clearly in both FIGURES 10A and 10B and after more culturing, myotubes are formed and apparent as in FIGURES IOC and 10D.
  • Cells of the present invention may be induced to differentiate into smooth muscle cells by implementing culture conditions well known in the art that induce smooth muscle cell formation and by implementing the following conditions. After retrieving cells from the peritoneal cavity and culturing for at least about two weeks, sometimes with two subcultures, in DMEM, FBS and an antibiotic, RNA extracted from the stem cells demonstrate similar morphology and expression of specific proteins that exist predominantly or specifically in proliferative smooth muscle cells. For example, after at least about two weeks in culture, both smooth muscle cells and stem cells of the present invention express collagen type III and smooth muscle actin heavy chain-5'. (Data not shown)
  • Cells of the present invention may be induced to differentiate into dendritic cells by implementing culture conditions well known in the art that induce dendritic cell formation and by implementing the following conditions.
  • Cells of the present invention retrieved from the peritoneal cavity of a mouse with an implant are gathered by lavage as previously described.
  • Cells are suspended in DMEM, with at least about 10% FBS and 1% antibiotic. Cells are then cultured onto a 24 well plate for at least about 3 hours, after which non-adherent cells are discarded and fresh media (as above) is added to cells. Cells are divided into a control group and one induced to differentiate.
  • the differentiation-inducing substituents include GM-CSF (20ng/mL) and IL-4 (20 ng/mL). After addition of such substituents, morphological changes occur, generally uniformly, across the culture plate that included the expansion of each cells (e.g., longer morphology than control) and a higher rate of growth as shown in FIGURE 11.
  • FIGURE 11 shows the growth profile of control cells (squares) and those receiving the differentiation-inducing substituents of GM-CSF and IL-4 (diamonds) as observed for six days. After at least about one week in culture, fresh media was added. Here control cells received the same media as above (DMEM, FBS and an antibiotic), while the highly proliferating received the same media in addition to GM-CSF (20 ng/mL) and TNF- ⁇ (20 ng/mL) to induce dendritic cell maturation (see FIGURE 12A for an example of dendritic cells in culture).
  • FIGURES 12B and 12C show that dendritic cells express MHC II, CD86, and CD40, among other markers.
  • the dendrites may be selected by their expression of CD1 lc.
  • FIGURE 13A and 13B show that cells induced to differentiate into dendritic cells as described above are also positive for CD lie (as shown using an anti-CDl 1-FIT-C linked antibody, see arrows). Positive fluorescence is most noticeable around the cell periphery.
  • the CD1 lc antibody is not detected on the surface of control stem cells (without the differentiation-inducing signal) as shown in FIGURES 13C and 13D.
  • Cells of the present invention may be induced to differentiate into adipocyte and fat cells by implementing culture conditions well known in the art that induce adipocyte and fat cell formation and by implementing the following conditions.
  • Cells retrieved from the peritonpal cavity by lavage were cultured in DMEM with 20% FBS in DMEM and grown to > 90% confluence. Culture media was then replaced with ⁇ -MEM, 10% FBS, 10% rabbit serum, 10% dexamethasone, 5 ⁇ g/mL insulin, and 50 ⁇ M 5,8,11,14-eicosatetraynoic acid. After at least about two days in culture, cells were cultured in the same media except without dexamethasone.
  • adipocytes were then maintained for at least one week during which the induction of adipocytes generally is found to begin.
  • the cells are indicative of adipocytes as they are larger, rounder and display very large yellow sacs inside the cell membrane.
  • Cells were identified as adipocytes by staining with Oil Red O (a stain more soluble in lipid reservoirs than the rest of the cell matrix or body) as shown in FIGURES 14A and 14B.
  • Oil Red O a stain more soluble in lipid reservoirs than the rest of the cell matrix or body
  • FIGURES 14A and 14B Stem cells not induced to differentiate did not stain positive for Oil Red O (FIGURE 14D). Examples of several protocols used to induce differentiation/cell specialization. Nerve cell specialization protocol:
  • Co-culture with an neuronal, glial, Schwann, or astrocyte cell line 2. Feed with conditioned media obtained from neuronal, glial, Schwann, or astrocyte cell line, or introduce at least one extra cellular matrix protein
  • Adipocyte cell specialization protocol :
  • Muscle cell specialization protocol including myoblasts, myotubes, and cardiomyocytes
  • Co-culture with a myoblast or cardiomyoblast cell line 2. Feed with a myoblast-conditioned medium or with an extracellular matrix protein
  • Cartilage cell specialization protocol including chondrocytes and chondroblasts:
  • Bone cell specialization protocol including osteoblast and osteocyte
  • any 3-dimensional matrix e.g., biomaterial- based, polymer-based, or containing hyaluronic acid, proteoglycan, collagen, and/or demineralized bone, as example
  • the present invention provides a means for generating large numbers of unspecialized stem cells as well as multiple types of specialized cells by implanting a foreign object in a body cavity space of a mammal and allowing stem cells to accumulate in the body cavity space. As such, greater that 200-fold the number of stem cells may be retrieved by methods of the present invention, as compared to methods currently used for the retrieval of stem cells (e.g., from the bone marrow). Accumulated stem cells of the present invention (specialized and/or unspecialized) are amenable to in vivo or ex vivo use.
  • stem cells of the present invention may accumulate in the host mammal and remain within that host either as stem cells or following induction to a specialized cell type or be retrieved from the host and used for another subject in need thereof (e.g., for bioengineering purposes).
  • stem cells of the present invention are amenable to multiple commercial uses such as for cell and product screenings, as a diagnostic tool (e.g., biologic marker), for prophylactic and therapeutic treatment of one or more conditions involving a cellular dysfunction or abnormality, and can be used to create established stem cell lines, cDNA libraries, to produce therapeutic agents, and for genetic engineering.
  • cells, cell lines, and compositions of the present invention may be used for numerous applications, including for transplantation, engraftment, rehabilitation, cell replacement, diagnosis, compound and drug screenings, bioengineering, and the like.
  • stem cells of the present invention exhibit true plasticity, because they may be induced to differentiate into bone marrow- derived hematopoietic stem cells, bone marrow-derived mesenchymal stem cells, and neuronal cells of the brain and spinal chord.
  • a method for inducing the fo ⁇ nation of pluripotent cells with differentiation and proliferation capabilities is provided by implanting a foreign object in a body cavity space of a mammal a foreign object and allowing stem cells to accumulate. Further, another aspect is a method of retrieving pluripotent cells with proliferation and differentiation capabilities from a body cavity space of a mammal by washing the body cavity space with physiologic solution following the introduction of a foreign body into the body cavity space.
  • the physiologic solution may include additional substituents such as peptides, cytokines, antibiotics, anti- and/or pro-inflammatory agents, anti-oxidants, other nutrients, growth-inducing signals, proliferation-inducing signals, differentiation-inducing signals, and/or differentiation-blocking signals as examples.
  • additional substituents such as peptides, cytokines, antibiotics, anti- and/or pro-inflammatory agents, anti-oxidants, other nutrients, growth-inducing signals, proliferation-inducing signals, differentiation-inducing signals, and/or differentiation-blocking signals as examples.
  • a method of creating an environment for the accumulation of stem cells with differentiation and proliferation capabilities is provided by introducing a foreign object into a body cavity space of an mammal for at least about two hours.
  • Yet another aspect of the present invention is a method of treating a disease in a host with stem cells retrieved from a body cavity space of a mammal by retrieving stem cells that accumulate in the body cavity space of the mammal following the introduction of a foreign object into the body cavity space followed by introducing the retrieved cells into a patient in need thereof.
  • Stem cells are generally allowed to proliferate (e.g., expand) and may be induced to differentiate before they are introduced into the patient.
  • Stem cells are generally used to treat any disease for which there is a cellular abnormality, including a genetic disease, aging and age- related disorders, an acquired disease, tumor (e.g., cancer), as well as for tissue injury (e.g., damage or inflammation) and repair.
  • a genetic disease e.g., aging and age- related disorders
  • an acquired disease e.g., tumor (e.g., cancer)
  • tissue injury e.g., damage or inflammation
  • one or more stem cells proliferate, undergo genetic manipulation (e.g., addition of one or more viral, pharmaceutical, or biologic substances, such as proteins, genes, peptides, labels, cell or chemoprotectants, etc.), and/or induced to differentiate.
  • Another aspect of the present invention is a method of creating a clonal population of stem cells.
  • stem cells may be induced to differentiate, proliferate, and/or introduced into a subject, wherein cells of the desired cell type are typically present in the subject.
  • a pluripotent stem cell is provided in addition to a method for providing to a subject one or more pluripotent stem cells.
  • a cDNA library, methods of constructing such library, diagnostic probes obtained from such a cDNA libraries are provided. As such, nucleic acids or their factors involved in cell regulation, dysfunction, repair, remodeling, etc. are analyzed and compositions and/or pharmaceutical preparations are designed to promote positive cell features and counteract negative ones.
  • a method for immortalizing cells retrieved from a body cavity space of an mammal by introducing a foreign object in a body cavity space, retrieving cells that accumulate in the body cavity space and after culturing, introducing an immortalization gene to the cells under conditions permissive for the uptake of the immortalization gene and allowing cells to self-renew and specialize.
  • Still another aspect of the present invention is a stem cell (or population thereof) retrieved from a body cavity space of an mammal and immortalized with one or more genes capable of producing a protein, wherein the stem cell is capable of expressing the protein upon induction by agents that stimulate production of the protein.
  • compositions that includes a stem cell retrieved from a body cavity space of a mammal that has been induced to express at least one characteristic of a non-cavity derived cell and a pharmaceutically acceptable carrier, and (a) wherein at least one non-endogenous nucleic acid sequence has been introduced into the cell, (b) wherein at least one non-endogenous peptide has been introduced into the cell, and/or (c) wherein at least one monoclonal antibody has been introduced into the cell.
  • the characteristic includes those defined as neuronal, astroglial, dendritic, hematopoietic, hepatic, cardiac, skeletal, epithelial, adipocytic, alveolar, ocular, endothelial, osteoblastic, chondrocytic, epidermal, pancreatic, renal, tenocytic, and reproductive, as examples.
  • These stem cells may possess cell functions (e.g. the function of liver cells, kidney cells), physical structure (bone or fat cells) or produce essential proteins or biological substances (eg. insulin, growth factors) for the treatment of diseases such as diabetes, liver or kidney failure, plastic surgery, and the like.
  • a stem cell retrieved from a body cavity space of a mammal operable for identifying substances involved in the growth of in vitro or host cells is provided, in addition to a method of testing such substance.
  • the substances may include approved and investigational pharmaceutical products, agricultural agents, food products, chemical products used for industrial purposes, and known or suspected toxins, as examples.
  • the present invention also provides a composition and a method for generating cells to be used for cell-based therapies, wherein cells retrieved from a body cavity space of an mammal are directed to differentiate into one or more specialized cell type and serve as a renewable source of replacement cells introduced into a patient to treat a human disease (e.g., transplant into a damaged or disease tissue or organ, repopulate an organ or tissue, integrate into surrounding tissue after transplantation). Cells may be introduced and allowed to integrate as a primary or secondary cell source as needed.
  • a human disease e.g., transplant into a damaged or disease tissue or organ, repopulate an organ or tissue, integrate into surrounding tissue after transplantation.
  • Cells may be introduced and allowed to integrate as a primary or secondary cell source as needed.

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Abstract

L'invention concerne des procédés d'accumulation, de recrutement et de récupération de cellules souches provenant de l'espace d'une cavité corporelle d'un mammifère adulte ainsi que les compositions et les cellules se conformant à cette invention. Selon un autre aspect de cette invention, on prévoit un procédé d'obtention d'une nouvelle source de cellules souches par l'implantation d'un corps étranger dans l'espace d'une cavité corporelle d'un mammifère et de collecte de ces cellules souches provenant de l'implantation. Selon cette invention, on prévoit un procédé administrant au patient une ou plusieurs cellules souches consistant à récupérer des cellules souches pour un espace de cavité corporelle d'un mammifère après implantation d'un corps étranger, à manipuler les cellules souches et à introduire celles-ci dans le patient.
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WO2005033268A9 (fr) 2005-09-15
JP2007525193A (ja) 2007-09-06
WO2005033268A8 (fr) 2005-06-02
CA2527700A1 (fr) 2005-04-14
EP1636341A4 (fr) 2007-08-01
WO2005033268A3 (fr) 2006-03-23
WO2005033268A2 (fr) 2005-04-14

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