JP2007525193A - Clinical and commercial stem cells - Google Patents

Abstract

【wrap up】
In accordance with the present invention, in addition to compositions and cells, methods for the accumulation, replenishment and recovery of stem cells derived from body cavity of mature mammals are disclosed. According to another aspect of the present invention, there is provided a method for obtaining a new origin of stem cells by transplanting a foreign substance into a body cavity space of a mammal and collecting stem cells resulting from the transplantation. Furthermore, according to the present invention, after transplanting a foreign body, one or more stem cells of the present invention are required by recovering stem cells in the mammalian body cavity space, manipulating the stem cells, and introducing the stem cells into a subject in need of the stem cells. A method of providing a subject with one or more stem cells of the invention is identified.

Description

This application is supported in part by National Institutes of Health grant number EB-00287. The government has certain rights in the invention.
This application claims priority to US Provisional Patent Application No. 60 / 474,020, filed May 29, 2003.
The present invention relates to the general field of cell physiology, and more particularly to clinically viable stem cells for medical applications such as diagnostics, screening, testing, treatment, and rehabilitation, as well as screening, testing and biotechnology. The present invention relates to compositions and methods for developing and producing cells for commercial use.

Stem cell research has shown that patients suffering from cancer, spinal cord injury, stroke, degenerative diseases, and other symptoms because stem cells are plastic and can replace pathological, damaged or aged tissues and organs Providing new panacea for It has been suggested that by using stem cell transplantation instead of drugs, biologics, and other current therapies, stem cells can provide new therapies for the prevention and / or treatment of various human disorders and symptoms .
For years, scientists have known that stem cells can be derived from embryos, adult bone marrow and other tissues, or fetuses (eg, umbilical cord blood). Due to ethical concerns regarding the recovery of stem cells from embryonic tissue, this particular range of research has recently received less attention. Instead, research focuses on the isolation, proliferation, and tissue / cell transplantation of stem cells derived from adult and fetal tissue. One limitation of using this type of stem cell is that stem cells from the same patient are generally required because stem cells can cause graft rejection. Another limitation is that only a small number of adult or fetal stem cells can be recovered from any one tissue.
This prevented the use of stem cells for extensive or cost-effective clinical treatment. To date, the most abundant origin of adult stem cells is derived from bone marrow. However, currently less than 500,000 adult stem cells are recovered from 15 mL of bone marrow fluid (amount generally recovered from adult humans). In addition, these stem cells require weeks or months of cell culture to produce a suitable number for use such as for transplantation. Furthermore, human liquid bone marrow cultures often fail to produce significant numbers of non-adherent hematopoietic or clonogenic progenitor cells even after 6-8 weeks.
Yet another limitation is that only a few types of adult tissue have been reported to contain stem cells. This includes the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, heart, skin and liver. More importantly, stem cells recovered from adult tissue (also referred to herein as adult stem cells) have limited ability to differentiate. Most often, adult stem cells can only differentiate into the cell type of the original tissue. Most adult stem cells cannot be considered truly pluripotent in the true sense.

With current technology, some drawbacks remain when using adult stem cells. Specifically, adult stem cells are rarely found in mature tissues, and the methods of recovery and expansion in culture are generally inadequate, resulting in very low yields and destructive to the tissue . That is, in the process of obtaining stem cells, some if not all of the original tissue is destroyed. For this reason, a large number of cells are required and / or the use of adult stem cells is difficult in replacement or intensive therapy where the original tissue is essential and cannot be destroyed. Therefore, there is a need to improve current stem cell collection technology to increase stem cell yield without destroying the original tissue. By eliminating tissue destruction, adults can be recipients of cells after donating stem cells. Such a process eliminates rejection by the immune system and reduces or eliminates the need for lifetime use of immunosuppressive drugs.
The present invention addresses not only the many requirements described above, but also other objectives apparent to those skilled in the art.

The present invention solves the problems associated with current stem cell collection and recovery techniques by providing a new and improved method of obtaining stem cells in high yield without destroying the original tissue and methods of using the stem cells. To do. Further, the present invention provides clinical, diagnostic, pharmaceutical and commercial stem cell populations, stem cell lines, and stem cell compositions.
According to one aspect of the present invention, a method is provided for obtaining a new origin of stem cells by introducing a foreign body or graft into a mammalian body cavity space and collecting the stem cells resulting from the transplantation.
Furthermore, according to the present invention, one or more stem cells of the present invention are required by recovering stem cells from the body cavity space of a mammal after transplanting a foreign body, manipulating the stem cells, and introducing the stem cells into a subject in need thereof. A method of providing a subject with one or more stem cells of the invention is identified. Stem cells may be genetically engineered, contacted with another composition, directed to differentiation into a desired cell type, and / or contacted with a three-dimensional scaffold prior to introduction into a subject. May be. Furthermore, the subject may be the same as or different from the origin of the stem cells.

  The advantages of stem cells recovered according to the present invention are that they are inexpensive to recover, produce high yields of cells, are capable of differentiation, proliferation and genetic modification (in vivo and ex vivo), in a physiological manner. Functions (eg, conducts, produces, secretes, regulates, etc. biological compounds), exhibits true pluripotency, and is clinical, diagnostic and commercial (eg, cell / tissue transplant, replacement, Implantation, grafting, genetic or diagnostic screening, product development), and treatment, prevention or other treatment purposes (eg spinal cord injury, damage to other tissues or organs, burns, cirrhosis, hepatitis, Parkinson's disease, Alzheimer, for example) Disease, stroke, muscular dystrophy, diabetes, arthritis, osteoporosis, leukemia, sickle cell disease and other anemias). In addition, the present invention provides for the use of cancer or end-stage illness, an option not available when using most traditional stem cell harvesting methods, such as bone marrow collection that can only be performed in relatively healthy patients. Suitable for use in individuals who may be susceptible to such other treatment options. Furthermore, the present invention is safer than current traditional methods such as bone marrow collection without complications and can be used repeatedly even in the same patient without any known difficulties or side effects.

The present invention addresses current problems regarding graft rejection by the immune system and reduces or eliminates the use of immunosuppressive drugs in transplantation. The present invention provides economical clinical treatment options, researchers and healthcare providers for compositions and methods for recruiting and recovering large numbers of human stem cells for clinical procedures including transplantation, genes, proteins and cell therapy Meet the current demands of the industry. The compositions and methods of the present invention serve as a unique and powerful tool for supplying stem cells, particularly to those in need. Examples of custom designed products of the present invention include compositions for medical, therapeutic, diagnostic, engineering, and biotechnology applications.
Upon reading the following detailed description in conjunction with the drawings, those skilled in the art will further appreciate the above advantages and superior features of the present invention, as well as other important aspects.

In the following, the manufacture and use of the various aspects of the present invention will be described in detail, but it should be recognized that the present invention provides many inventive concepts that can be embodied in a wide variety of aspects. The specific aspects and embodiments described herein are merely illustrative of methods for making and using the invention and do not limit the scope of the invention.
In general, stem cells are pluripotent cells in an unspecialized (eg, undifferentiated) state that can give rise to one or more unspecialized cells. One important feature that identifies a stem cell is its ability to exhibit self-renewal or to generate multiple stem cells themselves; thus, a stem cell is a cell that has the ability to self-maintain. Furthermore, herein, stem cells not only can proliferate (replicate over and over), self-maintain, and produce large numbers of specialized functional progeny, but also have the ability to regenerate damaged tissue. It has unspecialized cells.

  One role of stem cells is to replenish cells lost due to natural cell death, aging, injury or disease. The presence of stem cells in a tissue is usually related to the turnover of cells in the tissue, but stem cells can also exist in tissues lacking a high turnover rate. Unlike specialized cells that do not have the ability to replicate many times (also referred to as “replication” or “proliferation”), adult stem cells will be able to replicate without differentiation for many months. However, current methods recover adult stem cells that have only a limited ability to grow / proliferate (sometimes automatically) before differentiation begins by the cells. In particular, the advantages of the present invention provide for the recovery of stem cells capable of replicating for many months (even more than a year) while maintaining an unspecialized state and thus countless for long-term self-renewal It is to provide a method for producing cells.

Another interesting property of stem cells is that stem cells usually produce the same cell type as the tissue from which they are derived. For example, bone marrow-derived hematopoietic adult stem cells usually give rise only to blood cells; mesenchymal stem cells (bone marrow stromal cells) usually give rise to unique connective tissue cell types; hematopoietic stem cells usually give all types Give rise to blood cells; neural stem cells in the brain usually give rise to neurons / neurons, astrocytes, oligodendrocytes; epithelial stem cells from the digestive tract wall usually give rise to digestion-specific cell types; epidermis Cutaneous stem cells collected from the basal layer usually produce keratinocytes, and follicular stem cells collected from the base of the hair follicle epidermis usually give rise to hair follicle cells and epidermal cells.
The present invention provides stem cells that can differentiate into non-induced tissues (eg, a cell type different from the tissue from which they were derived). This ability is called plasticity. Thus, the stem cells of the invention can function by (a) generating lineages of genetically identical cells (eg, the same cells or clones) and (b) by differentiating into a number of specialized cell types. .

  The stem cells of the present invention comprise (1) labeling cells in living tissue with molecular markers to determine specialized cell types; (2) cells from living tissue (ie, donor, subject or patient) Determining the range of specialized cell types that the cell can become by in vivo manipulation (eg, introduction of one or more nucleic acid sequences or addition of one or more differentiation-inducing substituents); And (3) removing cells from living tissue (ie, donor, subject or patient of the same or different genetic background), labeling the cells after culturing and then transplanting the cells to the host (eg, the same Or a different genetic background donor, another subject or patient) to determine if the cells will live in the original tissue as before. Infecting stem cells with a virus that provides a unique identifier for each new stem cell progeny, the stem cell clones will still reside in one or more damaged tissues in the patient (eg, donor or host) in need. Prove that.

  As described herein, the present invention provides methods for developing, supplying and recovering pluripotent stem cells from the body cavity space of a mature mammalian host. In accordance with the present invention, cells recovered from the body cavity space of a mature mammalian host can (a) create a clonal population of stem cells; (b) grow in vitro or in vivo (eg, in situ) to generate a large number of stem cell progeny (C) may differentiate into one or more specialized cell types by in vitro manipulation (eg, exhibiting plasticity) or in vivo manipulation. Also provided are methods of growing, manipulating, and differentiating stem cells recovered from the body cavity space of a mammalian host. As used herein, the growth, manipulation, and differentiation of stem cells recovered from the body cavity space of a mature mammalian host are generally in suspension or on a substrate to which the cells can adhere (eg, a monolayer, three-dimensional network structure). ) Done on. Substrates or second cells having one or more attachment molecules, chemicals or biological substances on their surface may be used (eg, cross-linked or non-cross-linked amino acids, nucleic acids, polymers, polysaccharides, etc. ). Alternatively, stem cell proliferation, manipulation and differentiation can be performed in the host before or without recovery. As used herein, a biological substance is an organic compound that can affect cells, tissues, or organs.

  Proliferation and differentiation of the stem cells of the present invention is induced either under the appropriate conditions, ex vivo or in vivo as follows: (a) after in vitro proliferation and differentiation, integration into the host; (b) in After in vivo growth, integration into the host, further in vivo growth and / or differentiation; (c) after integration into the host, growth and differentiation (ie, in vivo). As used herein, in vivo (eg, in a host) growth and / or differentiation is in conjunction with a surgical procedure, an injection procedure, a non-surgical approach (eg, an approach to proliferate stem cells with pharmaceutical manipulation), or a combination of these approaches. It can happen. Proliferation and differentiation are under controlled stimulation where possible. In general, the conditions for in vitro cell culture are close to physiological conditions.

  When culturing stem cells, the culture medium is generally a culture medium used under standard conditions suitable for stem cells or cell specialization. As is known to those skilled in the art, it can be supplemented with growth promoting signals or substituents. As used herein, the terms “growth-promoting signal”, “growth-promoting substituent”, “growth-inducing signal”, “growth-inducing substituent”, or “growth factor” usually refer to growth, proliferation, and Refers to a compound (eg, protein, peptide or other molecule) or stimulant having a nutritional effect. As used herein, “differentiation-inducing signal”, “differentiation-inducing substituent” generally refers to a compound or stimulus that induces differentiation or specialization of a cell. Such compounds are commonly referred to as “signals” or “substituents”. Those skilled in the art recognize that one or more substituents may be used in combination, and combinations that promote growth, proliferation and cell type-specific differentiation are well known in the art, so induction of growth, proliferation, and differentiation is excessive. This experiment is not required. Examples of signals or substituents include cytokines, growth-promoting drugs, growth factors (eg, epidermal growth factor (EGF), amphiregulin, fibroblast growth factor (PDFG), thyroid-stimulating hormone releasing hormone (TRH), transforming Growth factors (TGF), and insulin-like growth factors (IGF), such substituents are usually added to the culture medium at concentrations ranging from about 1 fg / ml to 1 mg / ml. Therefore, a simple titration can be performed to easily determine the optimal substituent concentration, as additional signals include mechanical and / or electrical signals (eg, cell-cell contact, adhesion, migration, electrical Examples include irritation, physical pressure, and strain.

[Cavity space]
The present invention introduces or transplants foreign material into the body cavity space of a mammal (eg, a donor or host) to induce the formation, accumulation and recruitment of stem cells within the body cavity space. Examples include the peritoneal cavity, subcutaneous space, pleural space, lung and / or brain space. These areas are particularly large (biologically) in comparison with protected, space-constrained organs, blood vessels, or bone marrow, often have significant areas surrounding them, and are generally accessible. is there. In this specification, the body cavity space may be referred to as “cavity”, “peritoneal cavity”, “subcutaneous cavity”, “pulmonary cavity”, “pleural cavity”, “brain space”, and “cavity space”.

  As used herein, a “foreign body” includes any object (solid, liquid or gel) that is introduced into the body cavity space and guides it to form stem cells within the body cavity stem cells, where formation is defined as body cavity space Including migration, replenishment and accumulation of stem cells within. Examples include degradable grafts, non-degradable grafts, inflammatory drugs (eg, vaccine adjuvants, dead cell fragments), fibrous materials, pharmaceutical compositions, injectable materials (liquid, gel, or solid), biocompatibility Compositions (proteins, nucleic acids, polymers, dyes, plastics, synthetic or natural drugs, chemical isotopes, metal composites, etc.), pharmaceutical infusion solutions (eg saline, dextran, water) and compositions used in surgical procedures Thing or material. The biocompatible material (biomaterila) may be a single composition or polymer in a layer or mixture, or a composite blend of many materials, and may include substances that are believed to promote biological growth. In one aspect of the invention, the implant of the invention is a biocompatible support that allows for biological fixation of cells. Alternatively, stem cell induction is promoted by inflow of a foreign substance such as a pharmaceutical infusion solution (for example, water, dextran, physiological saline) into the body cavity space.

  Access to the body cavity space is by surgical approach, injection, perfusion, etc. Surgery includes lateral abdominal wall incision, laparoscopic surgery (use of one or more small incisions or instruments to access body cavities), endoscopy (access via flexible endoscope), and the like. . Injection is usually done with a needle and syringe. Those skilled in the art are aware of methods and techniques that allow access to and into the body cavity space.

  In general, the larger the cavity (eg, peritoneal, pulmonary, or pleural cavity), the higher the yield of pluripotent stem cells. In part, the larger the space, the wider the path of cell movement and the greater amount of fluid that can be injected and removed, so that a large amount of cells can be collected from the space. If necessary, stem cells may be continuously recovered from the body cavity space by several rounds of injection and removal. In one aspect, collection from the cell cavity is as simple as loading the fluid into the peritoneum and draining the fluid. The present invention has found that the fluid contains stem cells in a true sense only after introducing a foreign substance into the cavity. For example, a method similar to peritoneal dialysis can be performed, in which the abdomen is filled with up to 3 liters of solution (eg, dialysate) using a catheter and the abdominal wall (ie, peritoneum) when draining from the body. Fluid back into solution with other particles from the cavity (dialyzed).

[Stem cell recovery]
Generally, the stem cells of the present invention are recovered from the cavity of a mammalian donor. Any animal capable of producing body space and pluripotent stem cells can be used as a stem cell donor, including insects, fish, reptiles, birds, amphibians, and mammals. In accordance with the present invention, stem cells collected from donors can be used in many applications, including genetic manipulation, diagnosis, phenotyping, screening, and / or heterologous, autologous, or xenogeneic. Examples include transplantation to a host.

  Stem cells obtained from the donor cavity are generally recovered by collecting the fluid used to wash the body cavity space. The cells can also be collected from within and around the implant material, such as a three-dimensional matrix with pores. As used herein, lavage includes gastric lavage, dialysis, drug instillation and other lavage, perfusion, disruption or separation methods to collect cells from the body cavity space. The cells may also be collected by separation (before or after washing) from any extracellular matrix tissue or graft in a cavity and generally under aseptic procedures. If separation and / or disruption is necessary, routine methods well known in the art such as treatment with enzymes (eg trypsin, collagenase) or physical methods (eg using blunting) are performed. The fluid used for washing / separation is a typical or modified pH such as water, saline, pharmaceutical infusion, dextran, or tissue culture media (eg, HEH, DMEM, PRMI, F-12 used alone or in combination) An equilibrium (physiological) solution may be used. In one embodiment, the cells are centrifuged at low speed (eg, 200-2000 rpm) and then resuspended in fresh culture medium before being cultured on a fixed material or in suspension. Alternatively, the cells are collected with the fluid. Fluids used for washing / separation are, for example, those necessary for growth and cell metabolism (eg, growth factors, amino acids, vitamins, minerals, and / or proteins) and those that prevent infection by yeast, bacteria, fungi, etc. (eg, , Antibacterial agents, antibiotics, antifungal agents, antiviral agents) may contain one or more signals, substituents and / or supplements. Wash and separation fluids and fresh media are used with or without serum (eg, from cows, horses, chickens, etc.). Routine culture media used to collect cells and induce cell growth, differentiation, dedifferentiation, expansion, immortalization, specialization, and for transplantation are well known in the art. No undue experimentation is necessary to obtain a fluid suitable for cleaning. Furthermore, methods for optimizing cell collection are routine to those skilled in the art. When the collected cells are transferred for growth, differentiation, or other manipulation, the appropriate medium is replaced or perfused continuously or periodically as needed. Standard culture methods can be used to further enrich the collected / collected cells to any desired amount (eg, for cell expansion). This enrichment can be achieved using another known method (eg, negative selection method or positive selection method). Stem cells and progenitor cells can be enriched to any desired degree by this procedure or other art known procedures. An alternative is to use a packing cell line infected with retrovirus, or when the supernatant obtained from the culture of such a packing cell line is carried out according to the invention and with an enriched stem cell pool ( Addition to stem cells recovered (even in the presence of further differentiation or growth inducing substituents) provides a very efficient means for obtaining in vitro infection of stem cells.

  Examples of stem cell applications for recovery include proliferation induction, cell expansion, differentiation into one or more specialized cell types, genetic modification, short or long term storage (eg, cryopreservation or any method known in the art) Therapeutic measures such as screening, diagnostic exploration, phenotyping, and transplantation, chemotherapy, disease treatment, disease prevention, and replacement or enhancement of cells, organs or tissues.

  The addition of one or more recruitment-derived substituents to the donor cavity can increase the actual number of stem cells recovered from the cavity. As used herein, “recruitment-inducing substituent” refers to a substituent that stimulates cell recruitment and / or proliferation, and examples include anti-miotics, anti-differentiation substituents, or proliferation-inducing signals. This ability to enhance the recruitment and proliferation of stem cells recovered from subjects and donors reduces the time between recovery and treatment, increases the efficiency of the present invention, and is a form of cell enhancement.

[Proliferation]
In order to induce proliferation, cell division is generally performed by at least one growth-inducing substituent or substance (ie, a molecule that binds to a receptor on the surface of the cell and exerts a trophic or growth-inducing effect). Including normal nutrient chemical or biological factors). Examples of growth-inducing growth factors include EGF, amphiregulin, FGFs, and TGFs, which are used alone or in combination. Additional substituents can be added to the culture medium, particularly lineage specific substituents such as vitamins, NGF, PDGF, TRH, TGF, BMP, GM-CSF, or IGF.

  The proliferation of the cells of the present invention may occur before or after collection from the body cavity space, and similar proliferation-inducing signals can be used. The growth of cells collected from the cavity can begin as early as several hours or can take several days. In general, when cells collected from a body cavity space are placed on a substrate, the cells become attached within a few hours. After growth, new unspecialized cells appear in culture. These proliferating cells are generally clonal (ie, progeny of a single stem cell). If a proliferation-inducing signal persists, the total number of cells in the culture increases. Furthermore, the original cells recovered from the cavity increase in size. Providing a suitable culture medium as described above continues to grow proliferating cells in suspension. Proliferation cells in culture may be passaged to resume growth. Importantly, successive passages of growth and resumption (eg, weekly) result in a logarithmic increase in cell number after each passage. After growth and / or passage, stem cells can be genetically modified in vitro using techniques such as those described below.

[Differentiation]
Any method that activates a cascade of biological events leading to growth can induce differentiation of the cells of the invention, such as free inositol triphosphate (ITP), intracellular Ca ²⁺ , diacylglycerol And / or activation of protein kinase C (PKC) and other cellular kinases. Differentiation is controlled by external signals such as chemical secretion by other cells, physical contact with neighboring cells, and other specific molecules in the environment (eg, molecular substituents). Other examples of methods of inducing differentiation include treatment with phorbol esters, differentiation-inducing growth factors and other chemical signals alone, or in time series or in combination with other signals. In addition, place the cells on a fixed substrate (eg, a flask, culture plate, or a coverslip that may be covered with an ionically charged surface such as poly-L-lysine and poly-L-ornithine). It can also be applied to induce differentiation. Other substrates that can induce differentiation include those resembling an extracellular matrix such as collagen, fibronectin, laminin, and can be used alone or in combination. Differentiation can also be induced in suspensions in which growth-inducing substituents are present.

  The differentiation of the cells of the present invention occurs in a short time, such as 2 hours, or as long as at least a week, depending on the selected pedigree. When an appropriate differentiation inducer is added to the cells collected by the method of the present invention, many cells differentiate. The differentiation and detection of a specific cell type or pedigree can be determined by morphological, immunocytochemical and / or immunohistochemical methods, or by expression of cell type specific RNA or DNA. For example, cell type specific antibodies, specific gene expression, or specific histochemical assays can be used to distinguish cell characteristics or specialized cell phenotypic characteristics. Those skilled in the art will know the methodologies that characterize the best specific cell types. A cell can be specialized to any one cell type depending on the substituents (or time series) added to the cell culture medium. Examples of cell types include nerve cells (eg, glia, dendrites), non-neuronal cells (astrocytes, oligodendrocytes), epithelial cells, hematopoietic cells, hepatocytes, heart cells, endothelial cells, muscle cells (Smooth and skeletal), epidermal cells, osteoblasts, chondrocytes, stromal cells, adipocytes.

[Gene modification and manipulation]
Stem cells recovered from the peritoneum or other such cavity spaces are non-transformed cells, but they have the characteristics of a continuous cell line. In the unspecialized state, cells continually divide in the presence of growth-inducing substituents, making them excellent targets for genetic modification. As used herein, the term “genetic modification” or “genetic manipulation” refers to the stable or transient alteration of the genotype of cells recovered from the animal cavity by the intentional introduction of a heterologous nucleic acid. The nucleic acid may be synthetically derived or naturally derived and may contain a gene, part of a gene, or other useful nucleic acid sequence.

  Viral vectors (eg, retrovirus, degenerative hepatitis virus, adenovirus, adeno-associated virus, cytomegalovirus) or mammalian cells that can be used to direct the expression of one or more genes encoding the desired protein and promote differentiation Heterologous nucleic acid can be introduced into the stem cells of the invention by specific promoter or transfection (eg, lipofection, calcium phosphate transfection, DEAE-dextran, electroporation). As used herein, a protein can be any protein or combination of proteins in question and can be linked to a selectable marker for detection. In addition, the vector may contain a drug selection marker (see, eg, methods known in the art, Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harabor, N.Y.).

  An alternative approach is to deliberately immortalize the stem cells by introducing carcinogens that alter the genetic makeup of the cells to induce the cells to grow indefinitely. In addition, stem cells to eliminate cell death by administering Bcl-2 or genetically modifying cells with the bcl-2 gene (the product is known to interfere with programmed cell death (apoptosis)), In particular, differentiation-induced stem cells can be genetically modified.

  The genetically modified cells of the present invention have the added advantage of having the ability to fully specialize to become cell type specific cells. Specialization is reproducible and can be manipulated by any of a number of well-known differentiation protocols. In one aspect, for cell / gene therapy, the genetically modified stem cells recovered from the cavity are transferred to one or more heterologous and / or autologous hosts that require one or more bioactive molecules produced by the genetically modified stem cells. Transplant. The transplantation will be described in detail later. Alternatively, the genetically modified stem cells may be subjected to one or more differentiation protocols before transplantation. Once the cells are specialized, the cells can be isolated and transplanted into one or more hosts in need of proteins or biologically active molecules expressed by the genetically modified cells.

[Stem cell transplantation, rehabilitation and therapeutic use]
In one aspect of the invention, insufficient function has been obtained by transplanting with one or more stem cells and / or compositions of the invention to treat or prevent one or more disorders, illnesses or degenerative conditions, or have been damaged. Or repair or replace dysfunctional tissues / organs or increase the function of cells, tissues, or organs. Such organ or tissue damage (also referred to as affected) may be from mechanical, chemical, molecular or electrolytic seizures, changes or abnormalities. Transplantation with the compositions of the present invention may be performed with immunosuppression as deemed necessary by those skilled in the art. In some cases, stem cells that have been genetically modified (including gene replacement by homologous recombination or gene knockout) can be used. For example, excision of the major histocompatibility complex (MHC) gene is well known in the art (Zheng et al., 1991, PNAS, 88: 8067-8071). Stem cells lacking MHC expression allow the cells of the present invention to be used across allogeneic and xenogeneic histocompatibility barriers without the need for immunosuppression. Furthermore, transplanted stem cells can be recovered from the cavities of transgenic animals with altered or deleted MHC antigens.

  By transplantation, the stem cells and compositions of the present invention are delivered to the entire nerve lesion or to one or more specific sites deemed appropriate to those skilled in the art. Cells are administered by any method known to maintain organ or tissue integrity. In one embodiment of the present invention, the transplanted stem cells or unique components of the cells are genetically modified to include one or more tracers (eg, rhodamine- or fluorescein-labeled microspheres, or fast blue bis). Benzamide, or histochemical markers into which retroviruses have been introduced, or isotope compounds, or dyes or markers such as light-variable chemicals or green fluorescent protein). Here, stem cells are used as diagnostic markers, for exploration, for visualization of tissue or organ remodeling changes, for one or more stimuli (for example, mechanical or chemical such as electric fields, proteins, chemicals, drugs, etc. Can be used for markers of response to other substances) or for other therapeutic purposes.

  The stem cells of the present invention can also be used for biotechnological purposes. In one aspect, stem cells are collected within a three-dimensional matrix (eg, skeleton) that represents the tissue in question. At the time of collection, stem cells may be induced to differentiate or may not be induced. Stem cells may remain in the skeleton, where they may be genetically modified or induced to differentiate, or the stem cells may be removed from the skeleton and further manipulated before use. Importantly, stem cells can be harvested and induced in vivo at the site of interest and differentiated to give rise to new organs and / or tissues. Alternatively, the skeleton is collected and transplanted elsewhere or provided to the subject.

[Screening, diagnosis, testing, and exploration with stem cells]
Stem cells of the present invention (stem cells that have been induced or not differentiated) recovered from the cavity are essential for screening for toxins in potentially therapeutic compositions and pharmaceutical formulations. As is well known in the art, the composition is applied to cells in vitro at varying dosages, the time of proliferation and / or differentiation is varied, and the cellular response is monitored over time. Morphological (physical), genetic, secretory, conductive (eg, ion channels or neurotransmission) and other such responses to one or more methods well known in the art (eg, western as a general example) Blot, Southern blot, Northern blot gene screening, immunohistochemistry, protein, receptor and enzyme assay, enzyme-linked immunosorbent assay (ELISA), electrophoresis analysis, HPLC, radioimmunoassay, electrophysiological measurement) . Similarly, cell type-specific or proliferative stem cells of the present invention can be grown on a feeder layer (acting as a substrate) or in a three-dimensional network structure. Therefore, the stem cells may already have undergone differentiation before screening.

  Similar to in vitro screening and testing, the stem cells of the invention (with or without differentiation induction) transplanted in the absence and presence of one or more unique compositions or formulations are observed for their efficiency and safety ( For example, host viability, pharmacology, biochemistry and immunological effects). In addition, the stem cells or type-specific cells of the present invention are used to measure the effect of an implant or another graft on the cells or host.

  The term “therapeutic composition” or “pharmaceutical formulation” includes, for example, chemicals, polymers, radioactive materials, viruses, proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, nucleotides, drugs, prodrugs, implants , And any material such as a device. In the present invention, cells that have been induced and specialized before screening are also screened.

Screening with stem cells of the present invention (in vitro or in vivo, with and without further induction for specialization) is an economical method for testing and monitoring industrial and biological drugs and compounds I will provide a. In one aspect, the cells are utilized for rapid identification (eg, by high-throughput screening methods) of substances involved in proliferation or differentiation and survival of host cells in vitro. Furthermore, a cDNA library can be constructed from the stem cells or pedigree-specific cells of the present invention using methods well known in the art. As such, it analyzes nucleic acids or factors involved in cell regulation, dysfunction, repair, remodeling, etc., and designs industrial compositions and / or pharmaceutical formulations to improve positive cell characteristics and negative Neutralizes cell characteristics. Diagnostic probes are also developed, particularly those that identify one or more genetic disorders or dysfunctions. In addition, the cells of the invention are studied for their ability to secrete or produce potential therapeutic or industrial compositions.
Examples are provided below to fully illustrate the various aspects of the present invention. However, the examples should in no way be construed as limiting the scope of the invention as defined in the appended claims.

[Examples of accumulation and induction of pluripotent cell formation]
In the present invention, an inflammatory reaction occurs by supplementation or recovery of stem cells in the body cavity space of the present invention. Inflammatory responses are generally induced (surgically or otherwise) by introducing foreign bodies into the body cavity space. In one embodiment, an implant (including a polyethylene terephthalate (PET) disk) is surgically placed in the peritoneal cavity of a mouse and the results are compared to the results of a mouse undergoing sham surgery. Induction of pluripotent cells (eg, stem cells) in the cavity occurs as early as at least about 2 hours after introduction of the implant into the peritoneal space (data not shown). The implant continues to induce the formation of pluripotent cells for at least about 14 days (data not shown). Cells present and cells recruited to the cavity were collected by washing the cavity. Collected cells were placed in culture and allowed to adhere to a substrate (eg, culture plate). In culture, the attached cells were found to increase at an exponential rate. Initially looking at adherent cells, several different morphological features were observed as shown in FIG. After several days, weeks and / or months of cell growth in culture, stem cells are induced to induce different cell types (osteoblasts, smooth muscle cells, fibroblasts, neurons, and dendrites, as described below). Cell).
As is apparent, replenishment and recovery (eg, recovery from a lavage fluid) of stem cells derived from the peritoneal cavity requires an inflammatory reaction and a foreign body. The foreign body itself is necessary for stem cells to appear in the lavage fluid and to grow in culture (data not shown).

[Example of stem cell recovery]
In one embodiment, cells were replenished by transplanting a 1.2 cm diameter PET disc into the peritoneum of Swiss Webster mice through a midline incision. After implantation of two PET discs (one disc on either side of the peritoneal cavity), the wound was closed with a steel wound clip. Mice were sacrificed at various times after introduction of the implant and cells in the peritoneum were collected by lavage. Washing was performed by injecting at least about 4-5 mL of DMEM medium into the peritoneum of mice and carefully collecting the wash with a moving pipette after various time points. Next, at least about 10% fetal bovine serum (FBS) and 1-5% antibiotics (eg penicillin and streptomycin) to protect against possible bacteria from dying macrophages or to protect the intestines ) And the recovery medium (washing solution) were placed in a tissue flask or cell culture plate. The cells that adhered were pluripotent stem cells. Most importantly, approximately 500,000 stem cells were collected per gram of mouse after the first wash. Stem cell recovery was continued for the same subject by repeating the same procedure several times. For example, a subject undergoing peritoneal dialysis collects stem cells after each dialysis treatment as long as there is a foreign substance that induces an inflammatory reaction in the peritoneal cavity.
The adherent stem cells were then passaged and maintained in cultures that induced or did not induce proliferation or differentiation. In the presence of normal culture media, antibiotics and minimal serum supplementation, stem cells continued to grow for at least about 1 year (eg, dividing into a population of progenitor cells) (data not shown).

[Example of stem cell growth]
After adding a washing solution (used to wash the peritoneal cavity) to the culture surface, usually spindle-like adherent cells were easily observed. After a sufficient application time, usually at least about 24 hours, non-adherent cells were removed and fresh media was introduced into the cells. The cells were monitored by counting adherent spindle cells over time in random areas of the culture surface. FIG. 3 shows that adherent cells grew at an exponential rate (diamonds). On the other hand, cells recovered from the peritoneal cavity of mice in the absence of implants (eg, do not introduce foreign material into the cavity prior to lavage) and attached in the same manner as described above do not grow and eventually Usually died within at least a week. In general, only stem cells remained in culture after the first passage. To ensure a pure pluripotent population, the population can be further “purified” using α-fetoprotein as a marker.

[Example of stem cell supplementation]
As described above, the induction of inflammation or a similar response (eg, fiber or wound healing response) induces stem cells to accumulate and / or migrate into the body cavity space. This occurs with the foreign material because in the absence of the foreign material, the fluid and its inflammatory components cannot recover the stem cells. In one embodiment, cells were collected from the peritoneum of 16 mice (8 animals received implants and 8 died). After gastric lavage, the cells were recovered from the cavity by a further step of separating the lavage fluid from the extracted cells by simple centrifugation. After storing the washing solution after centrifugation, the groups were exchanged between 2 groups (4 animals that received the implant, 4 animals that did not receive the implant). The remaining 8 animals (4 with implants and 4 without implants) retained their own lavage fluid. Cells from all groups were cultured and spindle cells were counted over several days.
FIG. 4 shows the growth curves of these cultured cells. As can be seen from the number of cells counted over 2 weeks, cells from mice that received implants grew at an exponential rate (diamonds and squares), while cells from mice without implants died (triangles and triangles). X). Cells did not grow even when cells collected from mice without implants were introduced with a lavage fluid from mice with implants (containing many inflammatory proteins and chemokines). Furthermore, FIG. 4 shows that it is not the washing solution itself but the introduction of foreign matter that induces the formation, infiltration and recovery of stem cells. This is because the cells introduced into the implant grew in the absence (squares) or presence (diamonds) of the washing solution.

[Examples of stem cell plasticity]
Cells collected from the peritoneal cavity of mice after introduction of the foreign body were cultured for several weeks and months, left as stem cells, and some were induced to differentiate. For ordinary stem cells, when the stem cells of the present invention are cultured for several days or weeks to reach confluence, not only differentiation but also production of extracellular matrix can be promoted. Differentiation was also induced by adding culture media additives and / or changing culture conditions. Similarly, extracellular matrix formation was induced under appropriate conditions. Both behaviors demonstrate the plasticity of the stem cells of the present invention. Further examples are provided below.

[Example of specialization to bone cells]
In addition to incorporating culture conditions well known in the art to induce bone cell formation, bone cell differentiation by the cells of the present invention can occur as follows. In one aspect, in the presence of DMEM, fetal bovine serum (FBS) and antibiotics, cell aggregation and formation of cultured bone ossicle cells occurs during long-term cell growth, and can be dense (e.g., near confluence or Confluence). In addition, when cells are induced as follows, they become bone cells. After washing, the cells of the invention are collected from the mouse with the implant. Cells are plated on 24-well culture plates using DMEM, 5% FBS and 2% antibiotics. After 3 hours, fresh media was added to each group (to remove non-adherent cells and wash solution). Cells are grown in the same medium to an approximate density of at least about 5 × 10 ⁴ cells / cm ² . Next, differentiation induction additives (substituents) such as the following are added to the cells: (a) DMEM, 5% FBS, and 1000 ng / mL bone morphogenetic protein (BMP) -2; (b) DMEM, 5% FBS and 300 ng / mL BMP-2; (c) DMEM, 5% FBS, 1% antibiotics and 2.5 ng / mL TGF-β1; or (d) DMEM, 5% FBS, 1% antibiotic (control). After at least about 1 week, the medium is replaced with the same additives. After at least about 2 weeks, all groups are given fresh media containing DMEM, 5% FBS, 1% PNC, and 4 mmol NaHPO ₄ (to promote extracellular matrix production). In addition, after at least about 3 weeks with a medium containing NaHPO ₄ and 2% alizarin red S mixed in deionized water, the bone for bone mineralization with standard alizarin red S protocol as known in the art. Stain. FIG. 5A shows an example in which BMP-2 is used to induce bone cells. Note the calcium-rich matrix of the cells treated with BMP-2 and the surrounding nodules, as well as the many individual cells in the surrounding that are also stained red. FIG. 5B shows that stem cells without BMP pretreatment do not show positive red staining (dark area on image).

[Example of specialization for nerve cells]
In addition to incorporating culture conditions well known in the art to induce neuronal cell formation, neuronal differentiation by the cells of the present invention can occur as follows. As shown in FIG. 6, after incubation with β-mercaptoethanol, the cells can differentiate into neuron-like cells with long, branched processes from the cell body center. In one aspect, the protrusions can grow to a length that spans half of the culture plate. The cells of the invention form protrusions after an extended period of time in culture (eg, at least one month in the presence of serum and antibiotics).
In another embodiment, after culturing with DMEM and 10% FBS, stem cells collected by washing are induced as follows to become neurons. After the cells are grown to at least about 50% confluence, 10 mM β-mercaptoethanol (BME) is added to the medium. At least about 24 hours after the introduction of BME, the cell morphology begins to change; the cell body contracts to complete the process. At least about 48 hours after the addition of BME, the cells display a specialized multipolar morphology. FIGS. 7A and 7B are two examples of neuronal cells differentiated at least about 24 hours after the introduction of differentiation-inducing substituents, and the morphology is not observed before the addition of differentiation-inducing substituents. The cells were identified as neurons by epithelial histochemical staining for H & E staining (FIGS. 8A and 8B) and neuron-specific enolase (NSE) and neurofilament-M (NFM) (FIGS. 9A, 9B, and 9C). Only neurons that have been induced to differentiate with nerve-specific inducing substituents stain positively for both NSE and NFM.

[Example of specialization to skeletal muscle cells]
As is well known in the art, differentiation of skeletal muscle cells by the cells of the present invention can occur by carrying out the culture conditions for inducing skeletal muscle cell formation and the following conditions. At least about several weeks after introduction of stem cells collected from the peritoneal cavity by lavage into the culture surface, the morphology of skeletal muscle cells begins to change in the presence of DMEM, FBS, antibiotics and 5-azacytidine. An example of how these cells look morphologically is shown in FIG. Note that the skeletal striated muscle clearly visible in FIGS. 10A and 10B and after further culture, myotubes are formed and look like FIGS. 10C and 10D.

[Example of specialization to smooth muscle cells]
The cells of the present invention can be induced to differentiate into smooth muscle cells by carrying out culture conditions that are well known in the art for inducing smooth muscle cell formation and the following conditions. Cells are collected from the peritoneal cavity and cultured in DMEM, FBS and antibiotics for at least about 2 weeks, sometimes after two subcultures, RNA extracted from stem cells preferentially or specifically to proliferating smooth muscle cells It exhibits similar morphology and expression as the unique protein present. For example, after at least about 2 weeks in culture, both smooth muscle cells and stem cells of the invention express type III collagen and smooth muscle actin heavy chain-5 ′ (data not shown).

[Example of specialization to dendritic cells]
The cells of the present invention can be induced to differentiate into dendritic cells by performing culture conditions well known in the art for inducing dendritic cell formation and by performing the following conditions. As described above, the cells of the present invention recovered from the peritoneal cavity of a mouse with an implant are collected by washing. Suspend cells in DMEM with at least about 10% FBS and 1% antibiotic. Next, after culturing the cells on a 24-well plate for at least about 3 hours, the non-adherent cells are discarded and fresh medium (as described above) is added to the cells. Divide cells into control and differentiation-inducing groups. In one aspect, the differentiation-inducing substituent comprises GM-CSF (20 ng / mL) and IL-4 (20 ng / mL). After the addition of such substituents, morphological changes occur across the culture plate, usually uniformly, including elongation of each cell (eg, a longer morphology than the control) and faster growth as shown in FIG. FIG. 11 shows the growth profiles of control cells (squares) and cells that received GM-CSF and IL-4 differentiation-inducing substituents (diamonds) when observed for 6 days. After at least about 1 week in culture, fresh medium was added. Control cells received the same medium (DMEM, FBS and antibiotics) as described above, and highly proliferating cells were added to GM-CSF (20 ng / mL) and IL-4 (20 ng / mL). The same medium was received to induce dendritic cell maturation (see FIG. 12A for examples of dendritic cells in culture). Next, the cells were cultured for at least about 24 hours and then fixed with 10% formalin, and immunostaining was performed for the presence of dendritic cell markers (see FIGS. 12B and 12C). As is known in the art, dendritic cells express MHC II, CD86, and CD40, among other markers. In addition, recent studies have shown that dendrites can be selected by CD11c expression. Thus, cells that are immunostained with an antibody against CD11c (eg, antibody-bound magnetic beads) are considered dendritic cells. FIGS. 13A and 13B show that cells induced to differentiate into dendritic cells as described above are also positive for CD11c (as shown using anti-CD11-FIT-C conjugated antibodies). ; See arrow). Positive fluorescence is most prominent around the cell. On the other hand, as shown in FIGS. 13C and 13D, the CD11c antibody is not detected on the surface of the control stem cell (without differentiation-inducing signal).

[Example of specialization for fat cells]
The cells of the present invention can be induced to differentiate into adipocytes by performing culture conditions well known in the art for inducing adipocyte and fat cell formation, and by performing the following conditions. Cells recovered from the peritoneal cavity by lavage were cultured in DMEM with 20% FBS in DMEM and grown to over 90% confluence. Next, the culture medium was α-MEM, 10% FBS, 10% rabbit serum, 10% dexamethasone, 5 μg / mL insulin, and 50 μM 5,8,11,14-eicosatetraynoic acid. Was replaced. After at least about 2 days in culture, the cells were cultured in the same medium excluding dexamethasone. Next, it can be seen that when cells are maintained for at least one week, induction of adipocytes usually begins during that time. If the cell is large, round, and shows a very large yellow sac in the cell membrane, it indicates that the cell is an adipocyte. As shown in FIGS. 14A and 14B, cells were identified as adipocytes by staining with Oil Red O (a dye that is more soluble in the lipid reservoir than the remainder of the cell matrix or cell body).
Stem cells not induced to differentiate did not stain oil red O positive (FIG. 14D).

Examples of some protocols used to induce differentiation / cell specialization
Nerve cell specialization protocol:
1. Increased to 70% confluency with 20% FBS. Induced with 1 mM β-mercaptoethanol, 20% FBS in EMEM for at least about 24 hours. Fresh medium with 10 mM β-mercaptoethanol, 20% FBS in EMEM Alternative protocol for neuronal cell specialization:
1. 1. Co-culture with neuronal cells, glial cells, Schwann cells, or astrocyte cell line Grow in conditioned media from neuronal, glial, Schwann or astrocyte cell lines or introduce at least one extracellular matrix protein Adipocyte Specialization Protocol:
1. Increase to 90% or more confluence with DMEM and 20% FBS. 2. Replace with medium containing α-MEM, 10% FBS, 10% rabbit serum, 10-8 dexamethasone, 5 μg / mL insulin, and 50 μM 5,8,11,14-eicosatetraic acid. After at least 2 days, grow in medium similar to 2 but without dexamethasone. Differentiation begins as early as a few days or may take at least about a week Dendritic cell specialization protocol:
1. Incubate with DMEM, serum and GM-CSF in combination with IL-4 and / or SCF (stem cell factor) for at least about 9 days or up to 9 days 2.3. 3. Continue with one dose of GM-CSF and TNF-α for at least 24 hours Myocyte specialization protocol (including myoblasts, myotubes, and cardiomyocytes)
1. Grow cells in DMEM with 10-20% FBS (optionally and 5% horse serum) and do not allow cells to reach confluence of at least about 90%. 3. Re-apply cells to achieve at least about 80% confluency 3. Grow in DMEM containing 5-azacytidine or 5-aza-2'-deoxycytidine 4. After 24 hours, 5-azacytidine or 5-aza 4. Remove the medium with -2'-deoxycytidine and grow again in the same medium without additives Myotubes usually appear after at least about 7 days with continued culture. Alternative protocol for myocyte specialization:
1. 1. Co-culture with myoblasts or cardiomyocyte cell line Growing on myoblast conditioned medium or extracellular matrix protein Chondrocyte specialization protocol (including chondrocyte and chondroblast)
1. 1. Incubate cells with DMEM, serum and antibiotics Add medium containing FGF and / or IGF-I and / or TGF-β ₁ Alternative protocol for chondrocyte specialization:
1. 1. Incubate cells with DMEM, serum and antibiotics After at least one week, introduce the cells into a three-dimensional matrix (eg, biomaterial-based, polymer-based, or including hyaluronic acid, proteoglycan, collagen, and / or dechlorinated bone as examples) 3. Optionally: Introduce cell line gradients into culture conditions (using protocols well known in the art) to promote extracellular matrix production Bone cell specialization protocols (including osteoblasts and osteoocytes)
1. 1. Grow cells in DMEM with serum and antibiotics to at least about 90% confluency 2. Replace with the same medium containing recombinant BMP-2 (or another BMP) Optionally: After at least about 12-24 hours, add another dose of recombinant BMP-2. Small bones are visible to the naked eye when cultured. Alternative protocol for bone cell specialization:
1. 1. Incubate cells with DMEM, serum and antibiotics After at least one week, introduce cells into any three-dimensional matrix (eg, biocompatible material-based, polymer-based, or including, for example, hyaluronic acid, proteoglycan, collagen, and / or dechlorinated bone)

  The present invention provides a means for generating a large number of unspecialized stem cells and many types of specialized cells by implanting a foreign body into a mammalian body cavity space and accumulating stem cells in the body cavity space. As such, the method of the present invention can recover more than 200 times the number of stem cells as compared to methods currently used for stem cell recovery (eg, from bone marrow). The accumulated stem cells (specialized and / or unspecialized) of the present invention can be applied for in vivo or ex vivo applications. Thus, the stem cells of the present invention accumulate in the host mammal and remain in the host as stem cells, or can be used for another subject in need thereof after induction or recovery from the specialized cell type. Can (eg for biotechnological purposes). Similarly, the stem cells of the present invention can be used as diagnostic tools (eg, biological markers) for cell and product screening, such as for the prevention and treatment of one or more conditions associated with cellular dysfunction or abnormality. It can be used for many commercial applications and can be used to create established cell lines, cDNA libraries, to manufacture therapeutics, and to be used in genetic engineering. As such, the cells, cell lines, and compositions of the present invention can be used in many applications, including transplantation, engraftment, rehabilitation, cell replacement, diagnostics, compound and drug screening, biotechnology Etc.

Unlike stem cells collected by previously established methods, the stem cells of the present invention can be induced to differentiate into bone marrow derived hematopoietic stem cells, bone marrow derived mesenchymal stem cells, and brain and spinal cord neurons, Shows true plasticity.
According to another aspect of the present invention, there is provided a method for inducing the formation of pluripotent cells having differentiation and proliferation ability by transplanting a foreign substance into a body cavity space of a mammal and accumulating stem cells. Furthermore, another aspect is a method for recovering pluripotent cells having proliferation and differentiation ability from a mammalian body cavity space by washing the body cavity space with a physiological solution after introducing a foreign substance into the body cavity space. Importantly, physiological solutions include, for example, peptides, cytokines, antibiotics, anti-inflammatory and / or pro-inflammatory agents, antioxidants, other nutrients, growth-inducing signals, proliferation-inducing signals, differentiation-inducing signals, And / or may include a differentiation blocking signal.
According to yet another aspect of the present invention, a method is provided for creating an environment for the accumulation of stem cells with differentiation and proliferation potential by introducing foreign matter into a mammalian body cavity space for at least about 2 hours.

Yet another aspect of the present invention is to introduce a foreign substance into a mammalian body cavity space, collect stem cells accumulated in the mammalian body cavity space, and introduce the collected cells into a patient in need thereof. A method for treating a host disease caused by stem cells collected from a body cavity space. In general, the stem cells can be expanded (eg, expanded) before the stem cells are introduced into the patient. Stem cells are generally used to treat diseases with cellular abnormalities (including genetic diseases, aging and age-related disorders, acquired diseases, tumors (eg, cancer)), and tissue damage (eg, injury or inflammation); Repair again. In yet another aspect, one or more stem cells proliferate and undergo genetic manipulation (eg, one or more viruses, pharmaceuticals, or biologicals such as proteins, genes, peptides, labels, cells or chemical protectants) Substance) and / or induced to differentiate.
Another aspect of the present invention is a method for producing a clonal population of stem cells. As such, stem cells can be induced to differentiate, proliferate, and / or be introduced into the subject (typically cells of the desired cell type are present in the subject). Furthermore, in addition to the method of providing one or more pluripotent stem cells to a subject, pluripotent stem cells are provided. Still further, a cDNA library, a method for constructing the library, and one or more diagnostic probes obtained from the cDNA library are provided. As such, it analyzes nucleic acids or factors involved in cell regulation, dysfunction, repair, remodeling, etc., and designs compositions and / or pharmaceutical formulations to enhance positive cell characteristics and negative Neutralizes cell characteristics.

According to yet another aspect of the present invention, the foreign substance is introduced into the body cavity space, the cells accumulated in the body cavity space are collected and cultured, and then the immortalizing gene is introduced into the cell under conditions acceptable for the immortalization gene incorporation. A method is provided for immortalizing cells recovered from the body cavity space of a mammal by allowing the cells to self-renew and specialize.
Yet another aspect of the present invention is a stem cell (or population thereof) recovered from a mammalian body cavity space and immortalized with one or more genes capable of producing a protein, wherein the stem cell stimulates the production of said protein. The protein can be expressed when induced with a substance that does so.
Yet another aspect of the invention includes a stem cell recovered from a mammalian body cavity space that is induced to express at least one property of a non-cavity-derived cell and a pharmaceutically acceptable carrier. And (a) at least one non-endogenous nucleic acid sequence is introduced into the cell, (b) at least one non-endogenous peptide is introduced into the cell, and (C) A composition is provided wherein at least one monoclonal antibody is introduced into the cell. Examples of the characteristics include nerve cells, astrocytes, dendritic cells, hematopoietic cells, hepatocytes, heart cells, skeletal cells, epithelial cells, adipocytes, alveolar cells, ocular cells, endothelial cells, osteoblasts, Properties defined as chondrocytes, epidermis cells, pancreatic cells, kidney cells, tendon cells, and germ cells. These stem cells may have cell function (eg, liver cell, kidney cell function), physical structure (bone or adipocyte), or treatment of diseases such as diabetes, liver failure or kidney failure, plastic surgery Essential proteins or biological materials (eg, insulin, growth factors) for the like.

In yet another aspect of the present invention, stem cells recovered from the body cavity space of the present invention, which can be used for identification of substances involved in the growth of in vitro or host cells, are provided in addition to methods for testing such substances. The Examples of such substances include approved and investigational pharmaceuticals, agricultural chemicals, foods, chemical products used for industrial purposes, and known or suspected toxins.
The present invention is a method for producing cells that can be used in cell-based therapy, wherein cells recovered from a mammalian body cavity space are directed to differentiate into one or more specialized cell types and treat human diseases Renewable source of replacement cells that are introduced into the patient to survive (for example, withstand transplantation into damaged tissue or organ or pathological tissue or organ, live as before in the organ or tissue, and integrate into the surrounding tissue after transplantation) A useful method is provided.
Upon reading the foregoing detailed description, further objects, advantages and novel features of the invention as set forth herein will be apparent to those skilled in the art or may be ascertained from practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out herein.

It is an example which shows the pluripotency of the stem cell of this invention. The optical microscope photograph after collection | recovery of the cell of this invention and application | coating to a board | substrate is shown. The relationship between time and stem cell production of the present invention is shown, showing cells exposed to the implant (diamonds) and cells not exposed to the implants (squares). FIG. 5 shows the growth of fibrocytes over time according to the present invention, showing cells exposed to the implant with a washing solution (diamonds), cells exposed to the implant without washing solution (squares) and cells not exposed to the implants (x marks or triangles). 1 shows an overview of the cells of the invention induced to form calcium-rich osteoblast aggregates. An overview of control cells or undifferentiated cells showing a random distribution is shown. 2 shows an overview of the cells of the present invention after being induced to become specialized neuronal cells. Figure 2 shows an overview of the stem cells of the present invention further induced to become specialized neuronal cells with typical neuronal morphology. To become a specialized neuron with typical neuron morphology Fig. 2 shows an overview of the stem cells of the present invention induced to become specialized neurons by displaying typical neuronal morphology. Fig. 2 shows an overview of the stem cells of the present invention induced to become specialized neurons by displaying typical neuronal morphology. An overview of the stem cells of the invention induced to become specialized neurons after staining positive for NFM is shown. An overview of the stem cells of the invention induced to become specialized neurons after staining positive for NFM is shown. An overview of the stem cells of the invention induced to become specialized neurons after staining positive for NFM is shown. Stem cells that do not induce differentiation and do not show NFM positive staining are shown. 2 shows an overview of the stem cells of the present invention induced to become specialized muscle cells with striated muscle. 2 shows an overview of the stem cells of the present invention induced to become specialized muscle cells with striated muscle. 2 shows an overview of stem cells of the present invention induced to become specialized muscle cells with a linear growth pattern. 2 shows an overview of the stem cells of the present invention induced to become specialized myocytes with myotube formation ability (after long-term culture, the anchor is grooved at the end of the myotube). FIG. 4 shows cell growth as a function of time according to the present invention, showing exposure to GM-CSF and IL-4 (diamonds) compared to no growth induction signal (squares). FIG. 4 shows cell growth as a function of time according to the present invention, showing exposure to GM-CSF and IL-4 (diamonds) compared to no growth induction signal (squares). FIG. 2 shows an overview of the cells of the invention after the addition of one or more differentiation-inducing signals that promote specialization of the cells to dendritic cells. After inducing differentiation into dendritic cells, shows an overview of the stem cell panel of the present invention in which immunocytochemistry was achieved, the arrows show some CD11c positive cells compared to control cells that do not display CD11c (non-positive staining), That is, it shows a unique marker of dendritic cells. After inducing differentiation into dendritic cells, shows an overview of the stem cell panel of the present invention in which immunocytochemistry was achieved, the arrows show some CD11c positive cells compared to control cells that do not display CD11c (non-positive staining), That is, it shows a unique marker of dendritic cells. Figure 3 shows an overview of the stem cell panel of the present invention where immunocytochemistry was achieved after inducing differentiation into dendritic cells. Figure 3 shows an overview of the stem cell panel of the present invention where immunocytochemistry was achieved after inducing differentiation into dendritic cells. The image of the stem cell of the present invention after addition of one or more differentiation-inducing signals that promote cell specialization to adipocytes is shown, the differentiated stem cells are oil red o positive (dark part), and differentiate into adipocytes Cells that do not induce NO are not oil red o positive (non-dark). The image of the stem cell of the present invention after addition of one or more differentiation-inducing signals that promote cell specialization to adipocytes is shown, the differentiated stem cells are oil red o positive (dark part), and differentiate into adipocytes Cells that do not induce NO are not oil red o positive (non-dark). The image of the stem cell of the present invention after addition of one or more differentiation-inducing signals that promote cell specialization to adipocytes is shown, the differentiated stem cells are oil red o positive (dark part), and differentiate into adipocytes Cells that do not induce NO are not oil red o positive (non-dark). The image of the stem cell of the present invention after addition of one or more differentiation-inducing signals that promote cell specialization to adipocytes is shown, the differentiated stem cells are oil red o positive (dark part), and differentiate into adipocytes Cells that do not induce NO are not oil red o positive (non-dark).

Claims (39)

A method for collecting pluripotent stem cells, comprising the following steps:
Introducing a foreign substance into the body cavity space of a mammal;
Washing the body cavity space with a physiological solution at least once; and collecting the physiological solution to recover pluripotent stem cells;
Including methods.   The method of claim 1, wherein the body cavity space is one or more of the group consisting of a peritoneal cavity, a subcutaneous space, a pleural space, a lung space, and a brain space.   The method of claim 1, wherein the physiological solution is selected from the group consisting of culture media, dextran, saline, serum, antibiotics, pharmaceutical infusions, and combinations thereof.   The method of claim 1, wherein the washing step is performed at least about 2 hours after introduction of the foreign substance into the body cavity space, and optionally repeated at least about every 2 hours.   The method of claim 1, wherein the lavage step is selected from the group consisting of gastric lavage, dialysis, and pharmaceutical infusion.   The method of claim 1, further comprising genetically engineering the pluripotent stem cells.   2. The method of claim 1, wherein the pluripotent stem cells are reintroduced into a patient in need.   Induced pluripotent stem cells by incubating in a medium further containing serum and GM-CSF, and optionally reapplying dendritic cells in a medium containing serum and TNF-α The method of claim 1, wherein the method is differentiated into dendritic cells. Serum by incubation in a medium further comprising a bone-specific growth factor selected from the group consisting of bone morphogenic proteins and TGF-beta _1, differentiated into bone cells by inducing pluripotent stem cells, The method of claim 1.   The method according to claim 1, wherein pluripotent stem cells are induced to differentiate into nerve cells by incubating in a medium further containing serum and β-mercaptoethanol.   The pluripotent stem cells are induced to differentiate into muscle cells by incubating in a medium further containing serum and antibiotics and allowing the cells to grow for several weeks without reaching full confluency. Method.   The method of claim 1, wherein pluripotent stem cells are induced to differentiate into adipocytes by incubating in a medium further comprising serum, dexamethasone, insulin, and 5,8,11,14-eicosatetraic acid. Serum, FGF, IGF-1, and by incubating at further comprises medium and one or more growth factors selected from the group consisting of TGF-beta _1, into chondrocytes induced pluripotent stem cells 2. The method of claim 1, wherein the method is differentiated. A method for treating a disease in a patient in need of treatment comprising the following steps:
Collecting pluripotent stem cells from the body cavity space of the subject;
Culturing the pluripotent stem cells; and introducing the pluripotent stem cells into a patient in need of the pluripotent stem cells;
Including methods.   15. The method of claim 14, further comprising the step of inducing and differentiating pluripotent stem cells in the presence of a differentiation induction signal.   15. The method of claim 14, wherein pluripotent stem cells are collected by washing the body cavity space at least about 2 hours after introducing a foreign body into the body cavity space.   15. The method of claim 14, wherein the subject is a patient.   By introducing one or more selected from the group consisting of nucleic acid sequences, amino acids, antibodies, differentiation-inducing signals, proliferation-inducing signals, biological compounds, chemicals, selectable markers, three-dimensional scaffolds, and combinations thereof, 15. The method of claim 14, further comprising the step of manipulating pluripotent stem cells.   15. The method of claim 14, wherein the pluripotent stem cell is manipulated in vivo. The following steps:
Introducing a foreign substance into the body cavity space of a mammal;
Washing the body cavity space with a physiological solution at least once; and collecting the physiological solution to recover a cell population;
A cell population obtained by the method of claim 1 comprising:   A stem cell that is recovered from the body cavity space at least about 2 hours after introduction of a foreign substance into the body cavity space of a mammal and can create a clonal population of stem cells.   One or more selected from the group consisting of genetic manipulation, screening, diagnosis, phenotyping, xenotransplantation, autotransplantation, transplantation, cell replacement, tissue replacement, cell strengthening, tissue strengthening, treatment of cell abnormalities, and combinations thereof 21. The stem cell of claim 20, wherein the stem cell is used in a use.   A method for producing a stem cell comprising the step of transplanting a three-dimensional object into a body cavity space of a subject, wherein the three-dimensional object acts as a skeleton for stem cell accumulation and growth.   24. The method of claim 23, wherein the stem cells are manipulated in vivo by selecting from the group consisting of genetic modification, induced recruitment, induced differentiation, induced growth, induced proliferation, and combinations thereof.   18. The method of claim 17, wherein the stem cell is used to treat a condition selected from the group consisting of tumor, disease, cancer, tissue damage, cell death.   A cell population recovered from a body cavity space of a subject that has been induced to express at least one characteristic of a non-body cavity derived cell and suitable for transplantation into a host, wherein the cell population comprises the body cavity The cell population recovered at least about 2 hours after introduction of the three-dimensional foreign material into the space.   An isolated stem cell recovered from the body cavity space of a subject that is immortalized with at least one gene capable of producing the compound, wherein the isolated stem cell is induced by induction of a signal that stimulates production of the compound. And the isolated stem cell is recovered at least about 2 hours after introduction of the foreign substance into the body cavity space.   A composition comprising stem cells recovered from a body cavity space of a subject, induced to express at least one characteristic of non-body cavity derived cells, and a pharmaceutically acceptable carrier, The composition wherein the stem cells are collected at least about 2 hours after introduction of the foreign substance into the body cavity space.   The characteristics are nerve cells, astrocytes, dendritic cells, hematopoietic cells, hepatocytes, heart cells, skeletal cells, epithelial cells, adipocytes, alveolar cells, ocular cells, endothelial cells, osteoblasts, chondrocytes 30. The composition of claim 28, selected from the group consisting of: a pancreatic cell, a lymphocyte, an epidermal cell, a renal cell, a tendon cell, and a germ cell characteristic.   A composition comprising stem cells recovered from a body cavity space of a subject, induced to express at least one characteristic of non-body cavity derived cells, and a pharmaceutically acceptable carrier, The composition, wherein at least one non-endogenous nucleic acid sequence has been introduced into the stem cell, and the stem cell is recovered at least about 2 hours after introduction of the foreign substance into the body cavity space.   A composition comprising stem cells recovered from a body cavity space of a subject, induced to express at least one characteristic of non-body cavity derived cells, and a pharmaceutically acceptable carrier, The composition wherein at least one non-endogenous amino acid sequence has been introduced into the stem cells and the stem cells are collected at least about 2 hours after introduction of the foreign substance into the body cavity space.   A composition comprising stem cells recovered from a body cavity space of a subject, induced to express at least one characteristic of non-body cavity derived cells, and a pharmaceutically acceptable carrier, The composition, wherein at least one monoclonal antibody is introduced into the stem cells, and the stem cells are collected at least about 2 hours after introduction of the foreign substance into the body cavity space.   Stem cells recovered from a mammalian body cavity space that can be used for identification of substances involved in growth in vitro or host cells, wherein the stem cells are recovered at least about 2 hours after introduction of a foreign substance into the body cavity space. The stem cell. A method for testing the safety of a composition prepared on the basis of stem cells collected from a body cavity space of a mammal, comprising the following steps:
Collecting stem cells recovered from the mammalian body cavity space by washing the body cavity space with a physiological solution at least about 2 hours after introduction of the foreign substance into the body cavity space; and one or more preparation compositions Incubating said stem cells in a suitable medium in the presence of
Including said method.   35. The method of claim 34, further comprising examining stem cells at various times after incubation to assess cell changes.   A stem cell line obtained by the method of claim 1 and used to construct a cDNA library.   37. The stem cell line of claim 36, wherein one or more diagnostic probes are obtained from the cDNA library.   38. The stem cell line of claim 36, wherein the stem cells produce one or more therapeutic compositions. A method for collecting pluripotent stem cells comprising the following steps:
Introducing a foreign substance into the body cavity space of a mammal; and recovering pluripotent stem cells from the body cavity space;
Including methods.

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