WO2007005595A1 - Pregnancy-associated progenitor cells - Google Patents

Abstract

The present invention describes a previously unknown source of fetal stem cells. These fetal stem cells, which have been termed 'pregnancy-associated progenitor cells', are derived from tissue samples obtained from female mammals, including women, that have been pregnant at least once. The present invention provides compositions and methods for using such stem cells and/or progeny or products thereof in applications including research, diagnostic, and transplantation therapies.

Description

Pregnancy-Associated Progenitor Cells

Related Application

[1] The present application claims priority to Provisional Patent Application

No. 60/696,612, filed on July 5, 2005 and entitled "Pregnancy-Associated Progenitor Cells". The Provisional Application is incorporated herein by reference in its entirety.

Government Support

[2] The work described herein was funded by the National Institutes of Health,

Grant No. P30 DK34928 (Center for Digestive Disease Research, GRASP). The United States Government may have certain rights in the invention.

Background of the Invention

[3] Stem cells are the foundation cells for every organ, tissue and cell in the body. They have important characteristics that distinguish them from other types of cells. All stem cells, regardless of their source, have three general properties: they are capable of dividing and renewing themselves for long periods of time; they are undifferentiated or unspecialized (z.e., they do not have a specific function); and, under certain physiological or experimental conditions, they can give rise to multiple specialized cell types. Because of these unique characteristics, stem cells are of wide interest in the research and medical fields.

[4] In particular, due to their ability to regenerate and develop into a large variety of different types of cells in the body, stem cells have great therapeutic potential in regenerative and reparative medicine. Most of the body's specialized cells cannot be replaced by natural processes if they are seriously damaged or diseased. Some conditions or injuries can currently be treated through transplantation of entire healthy organs or tissues, but the need for transplantable organs or tissues far outweighs the available supply. Stem cells, either undifferentiated or directed to mature into specific cell types, can be used to replace damaged or dysfunctional cell populations or to repair diseased or defective tissues. Any disease associated with tissue degeneration can be a potential candidate for stem cell-based therapies, including conditions and disabilities such as Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, heart disease, Type I diabetes, skin and eye wounds or diseases, osteoarthritis, rheumatoid arthritis, muscular dystrophies, and liver diseases. Another approach in regenerative and reparative medicine involves the administration of drugs that coax stem cells already present in the body to promote and/or to participate in the repair of a damaged or diseased tissue or organ.

[5] Study of stem cells behavior in the laboratory can provide insight into the development of both humans and animals. This approach may lead to the isolation of novel precursor cells and/or to the identification of medically important genes. Furthermore, a better understanding of the molecular events involved in the normal mechanisms of development and specialization can help identify the, as yet unknown, processes responsible for abnormal development in utero that result in birth defects, inborn anomalies or other clinical conditions.

[6] The ability of stem cells to produce and supply almost unlimited quantities of healthy differentiated human cells of various tissue types can also have major implications for pharmaceutical research and development. The wide range of cell types that may be derived from human stem cells represents an in vitro biological system that mimics many of the complex interactions of the cells and tissues of the body, and, as such, provides an attractive and valuable research tool. Using these physiologically-relevant cells for drug screening, testing and drug toxicology studies will allow for a more efficient and accurate evaluation of the safety and efficacy of candidate drugs than existing screening assays that mainly rely on animal models and transformed human cells.

[7] However, there are many different cells encompassed by the term stem cell and each type of stem cells has different properties, functions, and living environments. Additionally, different stem cells exist at various time points during an individual's lifespan, from conception to old age. Thus, stem cells are routinely found in embryos, fetuses, and adults. The advantage of embryonic stem cells as a cell source includes virtually indefinite growth and differentiation potential that encompasses many if not all cells and tissues of the body. Embryonic stem cells are isolated from the early embryo stage known as the blastocyst. Since, at the present time, this can only be achieved with the concomitant destruction of the embryo, the derivation of stem cells from embryonic sources has raised ethical and moral issues.

[8] Many types of stem cells can also be isolated after birth from mature organs and tissues (e.g., brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, and liver). Adults stem cells are thought to reside in a specific area of certain tissues where they remain quiescent (i.e., non-dividing) for many years until they are activated by disease or tissue injury. Unlike embryonic stem cells, adult stem cells are generally multipotent and only develop into cells of a specific tissue or organ type. Some new evidence suggests that adult stem cell plasticity may exist, increasing the number of cell types a given adult stem cell can become. However, compared to embryonic stem cells which can be grown in almost unlimited quantities, adults stem cells have the disadvantage of being rare in mature tissues, and methods for expanding their numbers in cell culture have not yet been worked out satisfactorily.

[9] Fetal tissue is also a rich source of stem cells and has several properties that make it superior to mature tissue in particular with regard to cell-based therapies. First, fetal stem cells are capable of proliferating faster and more often than mature, specialized cells, and can often differentiate in response to environment cues around them. It has been found that fetal stem cells are not easily rejected by a recipient due to the low levels of histocompatibility antigens in the fetal tissue. At the same time, fetal stem cells produce angiogenic and trophic factors at high levels, enhancing their ability to grow once they are transplanted. Stem cells isolated from fetal tissue tend to survive excision, dissection, and grafting better than cells from mature tissue because they generally do not have long extensions or strong intercellular connections. Finally, fetal cells can survive at lower oxygen levels than mature cells, which can make them more resistant to the ischemic conditions found during transplantation or in vivo situations. However, the use of fetal stem cells isolated from aborted fetuses carries with it the same controversial issues as the use of embryonic stem cells.

[10] Post-partum tissues have generated interest as an alternative source for human stem cells. For example, methods for recovery of stem cells by perfusion of the placenta or collection from umbilical cord blood have been described. A limitation of stem cell procurement from these methods is often the small volume of cord blood or quantity of cells obtained. Thus, alternative sources of adequate supplies of cells having the ability to differentiate into an array of cell lineages remain in great demand.

Summary of the Invention

[11] The present invention discloses a previously unknown source of fetal stem cells. Unlike stem cells from embryonic and fetal sources, the cells described herein can be isolated without harming or destroying the fetus. More specifically, the present invention encompasses the discovery by the Applicants that fetal cells that are transferred to the mother during pregnancy and persist in the maternal circulation and tissues decades after delivery or termination of pregnancy have multi-lineage potential.

[12] In one aspect, the present invention provides methods for obtaining one or more isolated fetal cells, which comprise steps of: providing a tissue sample harvested from a female mammal, wherein the female mammal has been pregnant with a fetus at least once; and isolating one or more fetal stem cells from the tissue sample. In certain embodiments, the female mammal is a woman.

[13] The fetal stem cells isolated by an inventive method may comprise fetal stem cells that are pluripotent and/or fetal stem cells that are multipotent. In certain embodiments, the isolated fetal stem cells are characterized by (a) the ability to self- renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype.

[14] The tissue sample used in the inventive methods may be harvested from a tissue or organ selected from the group consisting of peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, heart, kidney, lung, and liver. In certain embodiments, isolating one or more fetal stem cells from the tissue sample harvested from the female mammal comprises submitting the tissue sample to a mechanical or enzymatic treatment, or both. In some embodiments, isolating one or more fetal stem cells from the tissue sample comprises centrifuging the treated tissue sample.

[15] The inventive methods for obtaining one or more isolated fetal cells may further comprise one or more of: purifying the fetal stem cells after isolation, expanding the fetal stem cells in an undifferentiated state to obtain undifferentiated fetal stem cells, differentiating the fetal stem cells into cells of a desired cell phenotype to obtain differentiated cells, and differentiating the fetal stem cells into a desired cell type to obtain specialized cells. Differentiating fetal stem cells into cells of a desired cell phenotype may comprise exposing the fetal stem cells to one or more differentiation-inducing agents. A desired cell phenotype may be selected from the group consisting of adipogenic cells, chondrogenic cells, cardiogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, perocardiogenic cells, stromal cells, epithelial cells, neurogenic cells, neurogliagenic cells, pleurigenic cells, hepatogenic cells, pancreogenic cells, and splanchogenic cells. A desired cell type may be selected from the group consisting of red blood cell, B lymphocyte, T lymphocyte, natural killer cell, neutrophil, basophil, eosinophil, monocyte, macrophage, platetet, osteocyte, chondrocyte, adipocyte, neuron, astrocyte, oligodendrocyte, absorptive cell, goblet cell, Paneth cell, enteroendocrine cell, hepatocyte, and keratinocyte.

[16] In certain embodiments, the inventive methods further comprise a step of genetically modifying the fetal stem cells after isolation to obtain genetically modified fetal stem cells. In some embodiments, the inventive methods further comprise a step of cryopreserving the fetal stem cells after isolation under such conditions that at least some of the fetal stem cells are viable upon recovery. The fetal stem cells may be cryopreserved after isolation, purification, differentiation, and/or genetic modification. Cryopreserving the cells may comprise using liquid nitrogen and/or using dimethyl sulfoxide.

[17] In another aspect, the present invention provides an isolated fetal stem cell derived from a tissue sample harvested from a female mammal that has been pregnant with a fetus at least once, wherein the fetal stem cell is characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as it is passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype. In certain embodiments, the female mammal is a woman. The tissue sample may be harvested from a tissue or organ as described above. [18] In still another aspect, the present invention provides a cell population comprising one or more fetal stem cells derived from a tissue sample harvested from a female mammal that has been pregnant at least once, wherein said fetal stem cells are characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype. The cell population may be a substantially homogeneous population of fetal stem cells, or a heterogeneous population comprising the fetal stem cells and at least one other cell type (e.g., a pluripotent stem cell, a multipotent stem cell, an adult stem cell, a progenitor cell, a differentiated cell, and a specialized cell). The fetal stem cells in the inventive cell populations may be expanded in or on a medium in an undifferentiated state, expanded in or on a medium comprising one or more factors which stimulate stem cell differentiation along a desired cell phenotype, or expanded in or on a medium comprising one or more factors which stimulate stem cell differentiation along a desired cell type. The desired cell phenotypes and cell types may be as described above. Alternatively or additionally, the cell population may be cryopreserved under such conditions that at least some of the fetal stem cells are viable upon recovery.

[19] In yet other aspects, the present invention provides a cell lysate prepared from an inventive cell population, an extracellular matrix produced from an inventive cell population, and a conditioned medium prepared from an inventive cell population. Also provided are pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of an inventive cell population, a cell lysate, an extracellular matrix, or a conditioned medium. The pharmaceutical compositions of the invention may further comprise one or more bioactive agents, such as a differentiation-inducing factor, an antϊ- apoptotic agent, an anti-inflammatory agent, an immunosuppressive/immunomodulatory agent, an antiproliferative agent, an antibody, a combinations thereof.

[20] In another aspect, the present invention provides a method of treating a disease or condition in a subject, comprising administering to the subject a therapeutic composition in an amount sufficient to treat the disease or condition, wherein the therapeutic composition comprises one or more fetal stem cells as described above, a cell lysate prepared from fetal stem cells, an extracellular matrix produced by fetal stem cells, or a conditioned medium prepared from fetal stem cells, wherein said fetal stem cells are derived from a tissue sample harvested from a female mammal that has been pregnant with a fetus at least once, and wherein said fetal stem cells are characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype.

[21] A treatment according to the inventive methods comprises at least one of tissue repair, tissue regeneration, tissue augmentation, tissue sealing, tissue function restoration, and therapeutic action.

[22] In certain preferred embodiments, the female mammal is a woman. The subject receiving a treatment according to the present invention may be related to the woman. For example, the subject may be a biological child or grand-child of the woman. Alternatively, the subject is not related to the woman.

[23] In certain embodiments, the therapeutic composition administered to the subject comprises one or more fetal stem cells and the fetal stem cells are induced to differentiate into cells of a desired cell phenotype or cell type in vitro prior to administration. In other embodiments, the therapeutic composition comprises one or more fetal stem cells and the fetal stem cells are induced to differentiate into cells of a desired cell phenotype or cell type in vivo following administration. In still other embodiments, the therapeutic composition comprises one or more fetal stem cells and at least some of said fetal stem cells are genetically modified in vitro prior to administration. The genetic modification may result in the fetal stem cells expressing a gene product that promotes treatment of the disease or condition. In yet other embodiments, the therapeutic composition stimulates adult stem cells present in the subject to divide or differentiate, or both.

[24] Administration of the therapeutic composition may be performed by transplanting, implanting, injecting, fusing, delivering by catheter, or providing as a matrix-cell complex. The methods of treatment of the invention may further comprise administering to the subject at least one other agent selected from the group consisting of a differentiation-inducing factor, an anti-apoptotic agent, an antiinflammatory agent, an immunosuppressive/immunomodulatory agent, an anti- proliferative agent, an antibody or a combination thereof. The other agent may be administered simultaneously with, before, or after, the therapeutic composition.

[25] The therapeutic composition may further comprise cells of at least one other cell type selected from the group consisting of a pluripotent stem cell, a multipotent stem cell, an adult stem cell, a progenitor cell, a differentiated cell, and a specialized cell.

Brief Description of the Drawing

[26] Figure 1 is a set of pictures showing cytokeratin expression in microchimeric cells in thyroid. Photomicrographs show fluorescence in situ hybridization (FISH) analysis using Cy3 labeled X (orange) and fluorescein isothiocyanate conjugated-labeled Y (green) chromosome probes and immunofluorescence staining for cytokeratin using mouse monoclonal AE1/AE3 anticytokeratin antibody and fluorochrome Texas Red (red). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (blue). (A) Male microchimeric cell with 1 Y chromosome (green), 1 X chromosome (orange), and stained with anticytokeratin antibody (red) (patient A; magnification XlOOO). (B) Interphase FISH of thyroid tissue showing a group of microchimeric cells identified by the presence of X and Y chromosomes (orange and green, respectively). The X or Y chromosome may not be observed in each nucleus, as they may not be in the same plane of focus (patient C; magnification x400). This group of cells did not stain positively for cytokeratin. (C) Combined FISH and immunofluorescence staining of a group of microchimeric cells with 1 X and 1 Y chromosome. This group of cells express cytokeratin (red). The X or Y chromosome may not be observed in each cell, as they may not be in the same plane of focus (patient A; magnification X 400).

[27] Figure 2 illustrates CD45 expression in microchimeric cells in liver

(Patient I). The photomicrograph shows a male microchimeric cell with Cy3-labeled X (orange) and fluorescein isothiocyanate conjugated-labeled Y (green) chromosome probes and immunoperoxidase staining for CD45 using mouse monoclonal anti-CD45 antibody and 3-amino-9-ethylcarbazole (AEC) as the chromogen (red). Nuclei are counterstained with 4',6-diamidino-2-ρhenylindole (blue). The microchimeric cell is in a sinusoid area. Surrounding AEC stain (red) reveals CD45 expression (magnification XlOOO).

[28] Figure 3 illustrates heppar-1 (a Hepatocyte Marker) expression in microchimeric cells in liver parenchyma (Patient G). The figure presents photomicrographs of interphase fluorescence in situ hybridization using Cy3-labeled X (orange) and fluorescein isothiocyanate conjugated-labeled Y (green) chromosome probes and immunoperoxidase staining for heppar-1 using mouse monoclonal anti— heppar-1 antibody and 3-amino-9-ethylcarbazole as the chromogen (red). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (blue). (A) Photomicrograph using ultraviolet light of liver parenchyma showing a microchimeric cell with a Y chromosome (green, arrowhead). The morphology is similar to surrounding hepatocytes (magnification XlOOO). (B) Photomicrograph using bright light with immunoperoxidase staining of the microchimeric cell in A showing that the cell is stained with heppar-1 as demonstrated by dark immunoprecipitate (magnification XlOOO).

[29] Figure 4 shows that microchimeric fetal cells in female thyroid express cytokeratin. Photomicrographs show FISH studies using Cy3-labeled X (orange) and FITC -labeled Y (green) chromosome probes, and immunofluorescence staining with anti-cytokeratin antibody, Texas Red (red). Nuclei are counterstained with DAPI (blue). (A) Male microchimeric cell in maternal thyroid with one Y chromosome (small arrow) and one X chromosome; 100Ox magnification. (B) Same cell stained with anti-cytokeratin antibody (large arrow, red), indicating an epithelial cell; lOOOx magnification. (C) Interphase FISH study of female thyroid tissue showing a group of male microchimeric cells; 40Ox magnification. This group of cells did not stain positively for cytokeratin; therefore, they are not epithelial cells. (D) Combined FISH and immunofluorescence staining of a group of male microchimeric cells with one X and one Y (small arrow) chromosome. Note that in this plane of focus not all of the X chromosomes can be seen. This group of cells express cytokeratin (large arrow); 40Ox magnification.

[30] Figure 5 is a set of in vivo imaging pictures of pregnant mice with pups in abdomen. Female wild-type FVB/NJ mice were mated to FVB/NJ males transgenic for the luciferase gene under the control of the CMV (a and b) or the VEGFR2 promoter (c and d). Ventral and dorsal images were captured on day 14 of gestation. The color of the signal represents the intensity of luciferase activity.

[31] Figure 6 shows an in vivo imaging picture of a mouse with cells that express VEGFR.Luc at site of injury (a) and a photograph of the same mouse with inflammation secondary to eye scratch anterior to left eye (b).

[32] Figure 7 is a set of in vivo imaging pictures of (a) a virgin mouse, (b) a pregnant FVB/NJ female with CMViLuc fetuses; (c) a virgin mouse; (d) a pregnant FVB/NJ female with VEGFR2:Luc fetuses; and (e) a pregnant FVB/NJ female with wild-type fetuses. These pictures show that microchimeric fetal cells expressing luciferase migrate specifically to the site of skin biopsy in cases (b) and (d).

[33] Figure 8 is a polynomial graph showing the quadratic relationship between signal intensity ratio and time following skin biopsy in control mice (CT), VEGFR2:Luc bred mice (VL) and CMV:Luc bred mice (CL).

[34] Figure 9 is a set of photomicrographs of liver sections of mice injected with CCl₄ and demonstrating that CCl₄ injury induces liver necrosis followed by fibrosis, steatosis and inflammation. (A) Four weeks after injury, most of the liver is still necrotic. Very few nuclei can be visualized. The general architecture of the liver is lost (10OX magnification). (B) Eight weeks after the injury, the liver parenchyma is organized. There is a mild level of fibrosis after trichrome blue staining (200X magnification). (C) Eight weeks after injury, there is a diffuse microvesicular steatosis (200X magnification). (D) Eight weeks after injury, the liver parenchyma has many inflammatory cells, sometimes organized in aggregates (200X magnification).

[35] Figure 10 is a set of two photomicrographs showing that partial hepatectomy induces cell division and inflammation in the regenerating liver. Photomicrographs represent H&E staining of regenerating liver sections after partial hepatectomy. (A) Hepatocyte undergoing cell division (arrow)(400X magnification). (B) One of multiple foci of inflammation present in the parenchyma (200X magnification). [36] Table 1 presents the results of immunolabeling studies performed on microchimeric cells of women (n = 10).

[37] Table 2 shows results of fluorescence in situ hybridization on tissues from control women without a known history of a male pregnancy (n = 11).

[38] Table 3 shows the association between fetal cell microchimerism and maternal autoimmune diseases.

[39] Table 4 lists the signal intensity ratios of luciferase expression at site of skin biopsy over time.

[40] Table 5 shows results (histology, immunofluorescence and PCR) following CCl₄ exposure in Group 1 mice (see Example 4).

[41] Table 6 shows results (histology, immunofluorescence and PCR) following CCl₄ or vegetable oil exposure in Group 2 mice (see Example 4).

[42] Table 7 shows fetal cell microchimerism in the liver before and after regeneration induced by parital hepatectomy.

Definitions

[43] Throughout the specification, several terms are employed that are defined in the following paragraphs.

[44] The term "cell proliferation" refers to an expansion of a population of cells by the continuous division of a single cell into two identical daughter cells.

[45] The term "cell differentiation", as used herein, refers to the elaboration of particular characteristics that are expressed by an end-stage cell type or a cell en route to becoming an end-stage cell (i.e., a specialized cell). The term "directed cell differentiation" refers to a process of manipulating cell culture conditions to induce differentiation into a particular cell type. The term "cell trans-differentiation" refers to the process by which a cell changes from one stage of differentiation into another.

[46] As used herein, the term "stem celF refers to a relatively undifferentiated cell that has the capacity for sustained self-renewal, often throughout the lifetime of an animal or human, as well as the potential to give rise to differentiated progeny (i.e., to different types of specialized cells). An "embryonic stem ceir is a stem cell derived from a group of cells called the inner cell mass, which is part of the early (4 to 5 days old) embryo called the blastocyst. Once removed from the blastocyst, the cells of the inner cell mass can be cultured into embryonic stem cells. In the laboratory, embryonic stem cells can proliferate indefinitely, a property that is not shared by adult stem cells. An "adult stem celF is an undifferentiated cell found in a differentiated (specialized) tissue. Adult stem cells are capable of making identical copies of themselves for the lifetime of the organism. Adult stem cells usually divide to generate progenitor or precursor cells, which then differentiate or develop into "mature" cell types that have characteristic shapes and specialized functions. Sources of adult stem cells include, for example, bone marrow, blood, the cornea and retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, the lining of the gastrointestinal tract, and pancreas. The term "fetal stem celF refers to an undifferentiated cell of fetal origin. Sources of fetal stem cells include fetal tissues (i.e., aborted fetuses and post-partum tissues). The present invention discloses a previously unknown source of fetal stem cells. More specifically, fetal stem cells of the invention, called "pregnancy-associated progenitor cells", can be isolated from tissue samples obtained from female mammals (including humans) that have been pregnant at least once.

[47] The term "pluripotent stem celF refers to a stem cell that has the ability to give rise to types of cells that develop from the three germ layers (mesoderm, endoderm, and ectoderm) from which all the cells of the body arise.

[48] The term "plasticity" refers to the ability of an adult stem cell from one tissue to generate the specialized cell type(s) of another tissue.

[49] The term "progenitor celF or "precursor celF are used herein interchangeably. They refer to a cell that occurs in fetal or adult tissue and is partially specialized; it divides and gives rise to differentiated cells. In vivo, precursor cells belong to a transitory amplifying populations of cells derived from stem cells. Progenitor cells do not have the capacity for sustained, undifferentiated self-renewal.

[50] As used herein, the term "isolated" refers to a cell which has been separated from at least some components of its natural environment. This term includes gross physical separation of the cells from its natural environment {e.g., removal from the donor). Preferably, "isolated" includes alteration of the cell's relationship with the neighboring cells with which it is in direct contact by, for example, dissociation. The term "isolated" does not refer to a cell which is in a tissue section, is cultured as part of a tissue section, or is transplanted in the form of a tissue section.

[51] A cell is "derived from" a subject or sample if the cell is obtained from the subject or sample or if the cell is the progeny or descendant of a cell that was obtained from the subject or sample. A cell that is derived from a cell line is a member of that cell line or is the progeny or descendant of a cell that is a member of that cell line. A cell derived from an organ, tissue, cell line, etc, may be modified in vitro after it is obtained. Such a modified cell is still considered to be derived from the original source.

[52] As used herein, the term "essentially free of indicates that the relevant missing item (e.g., a cell) is undetectable using either a detection procedure described herein or a comparable procedure known to one of ordinary skill in the art.

[53] As used herein, the term "tissue sample" refers to a sample (i.e., whole or part) of a tissue harvested from an organ or tissue of the body. For example, a tissue sample may be harvested from bone marrow, blood, blood vessels, brain, eye, skeletal muscle, tooth, liver, skin, gastrointestinal tract, pancreas, and the like

[54] The term "individual and "subject' are used herein interchangeably.

They refer to a human or another mammal (e.g., primates, dogs, cats, goats, horses, pigs, mice, rabbits, and the like). In certain preferred embodiments, the subject is human.

[55] The term "treatment" is used herein to characterize a method that is aimed at (1) delaying or preventing the onset of a medical condition, disease or disorder; (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the condition; (3) bringing about ameliorations of the symptoms of the condition; and/or (4) curing the condition. The treatment may be administered prior to the onset of the disease, for a prophylactic or preventive action. It may also be administered after initiation of the disease, for a therapeutic action. [56] A "pharmaceutical composition" is herein defined as comprising a pharmaceutically acceptable carrier and an effective amount of at least one of: fetal stem cells as described herein, differentiated cells derived from fetal stem cells, cell populations comprising fetal stem cells and/or differentiated cells derived from fetal stem cells, cell lysates obtained from fetal stem cells or cells derived therefrom, extracellular matrix generated by fetal stem cells or cells derived therefrom, and condition medium produced by fetal stem cells or cells derived therefrom.

[57] As used herein, the term "effective amount' refers to any amount of cells of the invention or products derived therefrom, or pharmaceutical composition thereof, that is sufficient to achieve an intended purpose. For example, the intended purpose may be to delay or prevent the onset of a medical condition, disease or disorder; to slow down or stop the progression, aggravation, or deterioration of the symptoms of the condition, to bring amelioration of the symptoms of the condition, and/or to cure the condition.

[58] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients of a pharmaceutical composition and which is not excessively toxic to the host at the concentrations at which it is administered. The term includes solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see, for example, Remington 's Pharmaceutical Sciences, E. W. Martin, 18^th Ed., 1990, Mack Publishing Co., Easton, PA, which is incorporated herein by reference in its entirety).

[59] As used herein, the term "conditioned medium" refers to a medium in which a specific cell or cell population has been cultured, and optionally removed. While the cells are cultured in the medium, they secrete cellular factors that can provide trophic support to other cells. Such trophic factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, and granules. A conditioned medium according to the present invention contains all or part of the cellular factors released by the cells.

[60] The term "trophic factor" refers to a substance that promotes survival, growth, proliferation, maturation, differentiation, and/or maintenance of a cell, or stimulates increased activity of a cell. The term "trophic support', as used herein, refers to the ability of a substance or mixture of substances to promote survival, growth, maturation, differentiation, and/or maintenance of a cell, or to promote increased activity of a cell.

Detailed Description of Certain Preferred Embodiments

[61] As mentioned above, the present invention provides stem cells of fetal origin that can be retrieved without the ethical controversy associated with obtaining embryonic or fetal material. More specifically, the present invention encompasses the discovery by the Applicants that fetal cells that are transferred to the mother during pregnancy and persist in the maternal circulation and tissues decades after delivery or termination of pregnancy have multi-lineage potential. In addition to possessing the developmental advantage of being fetal in origin, these so-called pregnancy- associated progenitor cells are capable of self-renewal and expansion in culture, and have the ability to differentiate into cells of other phenotypes, which makes them of high interest for clinical and therapeutic applications.

[62] Accordingly, the present invention provides populations comprising such cells, methods for obtaining them, pharmaceutical compositions comprising the cells or components or products thereof, and methods of using the pharmaceutical compositions for therapeutic purposes. Also provided are methods for using these cells for drug testing or screening.

I. Pregnancy-Associated Progenitor Cells

[63] In one aspect, the prevent invention provides isolated fetal stem cells. As mentioned above, isolation of such cells from the mother does not carry with it the same controversial issues as obtaining stem cells from embryonic or fetal sources.

Tissue Samples

[64] Practicing the methods of the present invention involves providing a tissue sample harvested from a suitable female mammal and isolating fetal stem cells from the tissue sample. Preferably, the fetal stem cells are isolated from tissue samples harvested from women. However, the fetal stem cells may be isolated from tissue samples obtained from females of other species. Examples of such species include, but are not limited to, primates, dogs, cats, goats, cattle, horses, pigs, mice, rabbits, and the like.

[65] A suitable female mammal is one that has been pregnant with a fetus at least once. The terms "pregnant with a fetus at least once" and "pregnant at least once" are used herein interchangeably to characterize a female mammal that has given birth at least once and/or has undergone natural or induced pregnancy termination at least once. In the latter case, the female mammal has preferably been pregnant long enough for fetal-maternal cell trafficking to have started. In general, fetal cells cross the placenta early in gestation. However, as will be appreciated by one skilled in the art, the time at which such cell trafficking starts will highly depend on the particular species of the female mammal considered. For example, in humans, feto-maternal cell trafficking starts as early as six weeks of pregnancy (H Ariga et ah, Transfusion, 2001, 41: 1524-1530). In mice, the Applicants have shown that it happens as early as 9 days after conception (K. Khosrotehrani et ah, "Fetal Cells Migrate Specifically to Maternal Skin Wounds and Participate in Maternal Angiogenesis", submitted to the Journal of Clinical Investigation in 2005).

[66] Pregnancy-associated progenitor cells according to the present invention may be isolated from any tissue sample containing fetal stem cells. Accordingly, the term "tissue sample", as used herein, refers to any sample of tissue harvested from a suitable female mammal from which cells of fetal origin can be isolated. Examples of tissues and organs from which tissue samples can be obtained for practicing the present invention include, but are not limited to, peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, kidney, heart, lung, and liver. Methods of harvesting samples from such tissues and organs are known in the art and can be used in the practice of the present invention.

[67] The tissue sample may be harvested from the female mammal and processed on site or it may be transported to a laboratory in a sterile container, preferably at low temperature (e.g., 4⁰C), to be processed. The container may be filled with a solution or medium such as, for example, a salt solution (e.g., Dulbecco's Modified Eagle's Medium (DMEM) or phosphate buffered saline (PBS)), or any solution suitable for transportation of organs used for transplantation. The solution or medium may optionally comprise one or more antibiotics or antimytotic agents (e.g., penicillin, streptomycin, amphotericin B₅ gentamicin, and nystatin).

Isolation of Fetal Stem Cells

[68] Fetal stem cells may be isolated from tissue samples using any suitable method. Isolation of fetal stem cells preferably occurs in an aseptic environment. In embodiments where the tissue sample is solid or semi-solid, blood and debris are removed from the tissue sample prior to isolation of the cells. For example, the tissue sample may be washed with a buffer solution (e.g., buffered saline) optionally comprising antimytotic and/or antibiotic agents.

[69] In certain embodiments, the different cell types present in the tissue sample are fractioned into subpopulations from which the fetal stem cells can be isolated. This may be accomplished using techniques for cell separation including, but not limited to, mechanical treatment (e.g., mincing or shear forces) and/or enzymatic digestion (e.g., using one or more proteolytic enzymes or combination of proteolytic enzymes including, but not limited to, neutral proteases, metalloproteases, serine proteases, mucolytic enzyme activities and deoxyribonucleases, for example, collagenase, trypsin, chymotrypsin, thermolysin, dispase, elastase, hyaluronidase, and pepsin) to dissociate the tissue sample into its component cells, followed by cloning and selection of specific cell types. As well-known in the art, methods of enzymatic digestions are generally performed by incubating the tissue sample at 37⁰C in the presence of one or more enzymes for 30 minutes, 1 hour, 2 hours or longer.

[70] Any suitable clonal selection and cell separation techniques may be used in the practice of the present invention (see, for example, methods described in Freshney, "Culture of Animal Cells: A Manual of Basic Techniques", (3^rd Ed.), Wiley- Liss, Inc: New York). Suitable methods of cell selection and/or separation include, but are not limited to, selection based on morphological and/or biochemical markers, selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation, unit gravity separation, countercurrent distribution, electrophoresis, and flow cytometry (e.g., fluorescence activated cell sorting (FACS)). In certain embodiments, cells of fetal or maternal lineage may be identified by karyotype analysis or in situ hybridization for the X and/or the Y-chromosome. Karyotype analysis can also be used to identify cells of normal karyotype. In animals, fetal cells can be distinguished from maternal cells by the presence or absence of a specific transgene.

[71] Fetal stem cells can also be isolated from blood of suitable female mammals. In certain embodiments, a blood sample is removed from the circulatory system and processed to isolate fetal stem cells. In other embodiments, fetal stem cells are isolated using apheresis, a process in which blood is withdrawn directly from the circulating blood of a suitable female donor, and processed through a cell separator such that cells of interest (i.e., fetal stem cells) are retained and the other blood components (i.e., other cells and plasma) are returned to the body. Since it is performed on a large volume of blood, a single apheresis donation can provide a larger number of fetal stem cells than several whole blood samples. Furthermore, in many circumstances, using apheresis for the isolation of fetal stem cells may be preferred to harvesting bone marrow as apheresis is a relatively cheap and simple process which is not painful for the donor and does not present risks such as those associated with anesthesia, analgesia, blood transfusion, and infection.

[72] Fetal stem cells of the present invention may be isolated from a suitable female mammal any time following delivery or pregnancy termination. For example, fetal stem cells may be isolated immediately following delivery or pregnancy termination. Alternatively or additionally, fetal stem cells may be isolated less than one year after delivery of pregnancy termination (e.g., 1 month, 3 months, 6 months, 9 months, 12 months) or more than one year after delivery or pregnancy termination (e.g., between 1 and 5 years, between 2 and 10 years, more than 10 years, more than 20 years, more than 30 years, more than 40 years, or more than 50 years).

Expansion of Isolated Fetal Stem Cells

[73] Undifferentiated fetal stem cells can be expanded using any suitable culture method. Generally, cells isolated from tissue samples are transferred to a sterile culture vessel at a density that allows cell growth. Culture vessels suitable for use in the practice of the present invention can be uncoated or coated with extracellular matrix, ligands (e.g., laminin, collagen, gelatin, and the like) or extracellular membrane protein (e.g., Matrigel^®).

[74] Expansion of the fetal stem cells of the present invention may be achieved by using any culture medium capable of sustaining growth of the cells. Examples of suitable culture media include, but are not limited to, Dulbecco' modified Eagle's medium (DMEM)₃ mesenchymal stem cell growth medium, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), RPMIl 640, CELL-GRO FREE, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), Ham's FlO medium (FlO), Ham's F12 medium (F 12), DMEM/F12, Iscove's modified Dulbecco's medium, and Eagle's basal medium, RPMI 1640, and advanced DMEM (Gibco), which are commercially available. The culture medium may be supplemented with one or more components including, for example, serum (e.g., fetal calf serum (FCS), fetal bovine serum (FBS), and human serum (HS)); glucose; beta-mercaptoethanol; and antibiotic and/or antimitotic agents (e.g., penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin). Preferably, fetal stem cells are cultured in an atmosphere containing about 0% to about 5% CO₂ in air (v:v) and at a temperature of about 35⁰C to about 39⁰C, more preferably at 37⁰C, for example in an incubator. The medium in the culture vessel may be static or agitated.

[75] Selection of the most appropriate culture medium and culture conditions can be readily performed by one skilled in the art. Cell culture techniques are well known in the art (see, for example, "Cell and Tissue Culture Laboratory Procedures", Doyle et al. (Eds), 1995, John Wiley & Sons: Chichester; "Animal Cell Bioreactors", Ho and Wang (Eds.), 1991, Butterworth-Heinemann: Boston).

Characterization of Fetal Stem Cells

[76] In certain embodiments, the fetal stem cells of the invention (or cell populations comprising fetal stem cells) are characterized before being used in research or clinical applications. Characterization of fetal stem cells may comprise determination of one or more cell features and/or properties such as growth characteristics (e.g., population doubling capacity, doubling time, etc.), karyotype (e.g., presence or absence of chromosomal abnormalities), gene expression profile, protein expression profile, cell-surface marker expression profile, ability to differentiate into cells of different phenotypes, absence of viruses within the cells, and the like.

[77] Any suitable analytical method may be used to characterize the fetal stem cells of the present invention including, but not limited to, histological, morphological, biochemical, immunohistochemical, immunocytochemical, molecular, and genetic methods. Non-limiting examples of suitable characterization methods include flow cytometry (e.g., FACS analysis), gene expression profiling using gene chip arrays and/or polymerase chain reaction (e.g., PCR, reverse transcriptase PCR, and real time PCR), protein arrays, plasma clotting assays, gel electrophoresis, and Enzyme Linked Immunosorbent Assay (ELISA) methods.

[78] Preferably, the fetal stem cells of the invention are characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into cells of at least one phenotype. In certain embodiments, the fetal stem cells are pluripotent. In other embodiments, the fetal stem cells are multipotent. Preferred fetal stem cells do not spontaneously differentiate. Furthermore, preferred fetal stem cells are substantially stable with respect to the cell markers produced on their surface, and with respect to the expression pattern of various genes.

Cryopreservation of Isolated Fetal Stem Cells.

[79] Fetal stem cells of the invention, either freshly isolated from a tissue sample or following expansion in culture, can be cryopreserved for future use. Preferably, the fetal stem cells are cryopreserved under such conditions that at least some of the cells are viable upon recovery (i.e., thawing). Preferably, more than 50%, 75%, 80%, or 85% of the cryopreserved cells are viable after recovery. More preferably, more than 90% of the cryopreserved cells are viable after recovery. Even more preferably, more than 95% or 99% of the cryopreserved cells are viable after recovery.

[80] Preferably, the cryopreservation conditions are such that viable fetal stem cells have identical morphological and functional characteristics as the cells prior to cryopreservation. In particular, viable fetal stem cells obtained upon thawing are preferably characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged, and (c) the ability to differentiate into cells of at least one phenotype.

[81] Methods for the cryopreservation of different types of cells are known in the art. Any suitable method of cryopreservation may be used in the practice of the present invention. Typically, the cryopreservation medium contains dimethyl sulfoxide (DMSO). Preferably, the cryopreservation medium contains between about 1% to about 80% of DMSO (v:v). More preferably, the cryopreservation medium contains between about 5% and about 30% of DMSO (v:v). Most preferably, the cryopreservation medium contains between about 8% and about 12% of DMSO (v:v). The cryopreservation medium may further comprise cryopreservation agents such as, for example, methylcellulose. In the cryopreservation medium, fetal stem cells may be suspended at a density of between about 0.5 x 10 to about 10 x 10 cells per milliliter.

[82] Preferably, freezing of the fetal stem cells in a cryopreservation medium is performed at a controlled rate, for example from about -0.1°C/minute to about -1O⁰C/ minute. Fetal stem cells isolated from a tissue sample obtained from a suitable female donor may be stored in a single vial or, alternatively, they may be stored as aliquots in several small vials. Once frozen, the fetal stem cells can be stored indefinitely under liquid nitrogen until needed, as long as care is taken to prevent the possibility of accidental thawing or warming of the frozen cells at any time during their storage period.

[83] Prior to cryopreservation, quality control procedures can be performed (as described above) to check for chromosomal abnormalities, the ability of fetal stem cells to undergo the freeze-thawing processes, the immune compatibility of the fetal stem cells with patients potentially requiring the cells, the presence of viruses within the fetal stem cells that may cause disease, the ability of the fetal stem cells to give rise to a given specialized cell types when required, and the ability of the fetal stem cell numbers to be increased to useful amounts.

[84] When the fetal stem cells are to be used, they can be thawed under controlled conditions, for example by transferring one or more vials containing frozen fetal stem cells to a water bath set to 37⁰C. The thawed contents of the vial(s) are then rapidly transferred under sterile conditions to a culture vessel containing an appropriate medium (e.g., DMEM containing 10% FBS). Preferably, DMSO (which < is present in the cryopreservation medium) is diluted to less than about 1% of the cell culture volume. The thawed samples can then be tested for viability, growth properties, karyotype, and differentiation ability. The thawed fetal stem cells may be grown in an undifferentiated state for as long as desired (as described above) and can then be cultured under certain conditions to allow progression to a differentiated state (as described below).

Differentiation of Fetal Stem Cells

[85] As mentioned above, fetal stem cells of the present invention have multi- lineage potential, i.e., they can be deliberately induced to differentiate into various lineage phenotypes by subjecting them to differentiation-inducing cell culture conditions. Accordingly, the present invention provides differentiated or specialized cells derived from the fetal stem cells described herein. Also provided are populations of cells incubated in the presence of one or more different factors, or under conditions, that stimulate stem cell differentiation along a desired pathway.

[86] Methods relating to stem cell differentiation techniques which can be useful for differentiating the fetal stem cells of the present invention are known in the art and have been reported (see, for example, "Teratocarcinomas and Embryonic Stem Cells: A Practical Approach", J. Robertson (Ed.), 1987, IRL Press Ltd; "Guide to Techniques in Mouse Development, P.M. Wasserman et al, (Eds.)., 1993, Academic Press; M.V. Wiles and G. Keller, Development, 1991, 111: 259-267; R. Wang et al, Development, 1992, 114: 303-316; M.V. Wiles, Meth. Enzymol., 1993, 225: 900-918; G. Keller et al, MoL Cell. Biol., 1993, 13: 473-486; J. Rohwedel et al, Dev. Biol., 1994, 164: 87-101; M. Klug et al, Am. J. Physiol., 1995, 269: H1913- H1921; I.N. Rich, Blood, 1995, 86L 463-472; G. Bain et al, Dev. Biol., 1995, 168: 342-357; K Abe et al, Exp. Cell Res., 1996, 229: 27-34; M.J. Weiss and S.H. Orkin, J. Clin. Invest., 1996, 97: 591-595 and references cited therein; J. Rohwedel et al, Cell Biol. Int., 1996, 20: 579-587; T. Nakano et al, Leukemia, 1997, 3: 496-500; P.D. Rathjen et al, Reprod. Fert, 1998, 10: 31-47; O. Brustle et al, Science, 1999, 285: 754-758; S Liu et al, Proc. Natl. Acad. Sci. USA, 2000, 97: 6126-6131; B.E. Reubinoff et al, Nature BiotechnoL, 2000, 18: 381-382; B. Soria et al, Diabetes, 2000, 49: 157-162; M.J. Shamblott et al, Proc. Natl. Acad. Sci. USA, 2001, 98: 113- 118; A. Suzuki and T. Nakano, Int. J. Hematol., 2001, 73: 1-5; M. Schuldiner et al, Brain Res., 2001, 913: 201-205, each of which is incorporated herein by reference in its entirety). O'Donoghue and coworkers have also described conditions under which male (presumed fetal) stem cells isolated from bone marrow samples of women who had carried sons can differentiate into muscle, nerve, bone and fat cells (K. O'Donoghue et al, Lancet, 2004, 364: 179-182, which is incorporated herein by reference in its entirety).

[87] Agents and conditions that stimulate stem cell differentiation include, but are not limited to, maturation-inducing and differentiation-inducing agents {e.g., growth or trophic factors, peptide hormones, cytokines, ligand receptor complexes, corticosteroids, organic solvents, N-butyrate, demethylating agents, glucocorticoid with cAMP-elevating agents, methyl-isobutylxanthine, indomethacin and the like); culture in a medium conditioned by cells of a particular lineage, and co- culture with cells of a particular lineage.

[88] Maturation and/or differentiation of fetal stem cells into cells of a particular lineage or cells of a particular cell type may be demonstrated by one or more suitable methods including, but not limited to, histological, morphological, biochemical, immunohistochemical, immunocytochemical, molecular and genetic analytic methods. For example, specialized cells derived from fetal stem cells of the invention may be characterized by identification (including both absence and presence) of factors secreted by the differentiated cells or by the presence or absence of specific cell-surface markers.

Modifications of Fetal Stem Cells

[89] In another aspect, the present invention provides fetal stem cells (or cells derived therefrom) and populations of fetal stem cells (or of cells derived therefrom) that are modified.

[90] For example, antigens on the surface of a cell may be altered in such a way that upon transplantation, lysis of the cell is inhibited. Alteration of an antigen can induce immunological non-responsiveness or tolerance, thereby preventing the inducing of the effector phases of an immune response {e.g., cytotoxic T cell generation, antibody production, etc.) which are ultimately responsible for rejection of foreign (i.e., allogeneic or xenogeneic) cells in a normal immune response. Antigens that can be altered to achieve this goal include, for example MHC class I antigens, MHC class II antigens, LFA-3 and ICAM-I. Preferred methods for altering an antigen on a cell to inhibit immune response against the cell have been disclosed in U.S. Pat. No. 6,673,604 and U.S. Pat. Application No. 2003-0113301 (which are incorporated herein by reference in their entirety).

[91] Alternatively or additionally, cells of the invention may be genetically modified. For example, the cells may be modified to express a gene product (i.e., cells may be treated in a manner that results in the production of a gene product by the cell). Alternatively, modification of the cells may result in an increased production of a gene product already expressed by the cells or may result in production of a gene product (e.g., an antisense RNA molecule) which decreases production of another, undesirable gene product normally expressed by the cells.

[92] Examples of methods that can be used to genetically modify the cells of the present invention include, but are not limited to, DNA or RNA gene/sequence insertion of a suitably promoted gene construct, electroporation of said gene, infection by retroviral, lentiviral or other viral vector constructs encoding a gene of interest, mechanical gene introduction or the transfer of specific protein, glycoprotein or phosphoprotein entities. The nucleic acid molecule of interest can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extra-chromosomal molecule, such as a vector or plasmid. Other examples of methods that can be used to genetically modify the cells of the present invention include, but are not limited to, complete inactivation of a gene using the homologous recombinant technique, deletion in part of a gene or deletion of the complete gene, reduction of the target gene activity level using inhibitors of the expression of the target gene (e.g., antisense, small interfering RNA, ribozyme molecules, and triple helix molecules).

[93] Techniques for the genetic modifications of cells are known in the art (see, for example, "Molecular Cloning: A Laboratory Manual", 2^nd Ed., Sambrook et ah, 1989; "Oligonucleotide Synthesis", MJ. Gait (Ed.), 1984; "Animal Cell Culture", R.I. Freshney (Ed.), 1987; "Gene Transfer Vectors for Mammalian Cells", LM. Miller and M.P. Calos (Eds.), 1987; "Current Protocols in Molecular Biology and Short Protocols in Molecular Biology", 3^rd Ed., F.M. Ausubel et al. (Eds.), 1987 and 1995; and "Recombinant DNA Methodology IF, R. Wu et al, (Eds.), Academic Press, 1995; "Basic Methods in Molecular Biology", 2^nd Ed., L.G. Davis et al. (Eds.), Appleton & Lange: Norwalk, CO, 1994, each of which is incorporated herein by reference in its entirety).

[94] The genetically modified cells may be used as bioreactors for generating a specific gene product in vitro, or for producing a tissue in vitro which may then be implanted into a subject, or for delivering a transgene and its product in vivo {e.g., to a mammal subject). For example, cells may be modified to generate gene products that can prevent future disorders {e.g., growth factors such as fibroblast growth factors or transforming growth factors, which encourage blood vessels to invade a diseased or degenerated tissue of the body). Other gene products that can be delivered to a subject via implantation of genetically modified cells include factors which promote survival of the implanted cells, and factors which stimulate stem cells already present in the body to differentiate into cells of a specific cell type. Alternatively or additionally, cells may be altered such that the modification results in suppression of expression of one or more gene products that promote inflammation or rejection at the implant site.

Cell Populations

[95] The present invention also provides cell populations, which preferably comprise fetal stem cells and/or cells derived therefrom (including modified cells).

[96] In certain embodiments, the cell population is heterogeneous and comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 95% of fetal stem cells. The heterogeneous cell populations of the invention may further comprise cells that have been isolated at the same time as the fetal stem cells {i.e., from the same tissue sample). Alternatively or additionally, the heterogeneous cell populations may further comprise cells that have been added to a substantially homogeneous population of fetal stem cells. Cells present in a heterogeneous cell population of the invention may be of maternal or other origin and include, but are not limited to, embryonic stem cells, fetal stem cells (for example isolated from a different suitable female mammal or obtained from post-partum material), adult stem cells, progenitor cells {e.g., adipogenic cells, chondrogenic cells, cardiogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, perocardiogenic cells, stromal cells, epithelial cells, neurogenic cells, neurogliagenic cells, pleurigenic cells, hepatogenic cells, pancreagenic cells, splanchogenic cells, and the like), and specialized cells (e.g., red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, osteocytes, chondrocytes, adipocytes, neurons, astrocytes, oligodendrocytes, absorptive cells, goblet cells, Paneth cells, enteroendocrine cells, hepatocytes, keratinocytes, vascular endothelial cells, myoblasts, myocytes, stromal cells, and the like).

[97] In other embodiments, the cell population is substantially homogeneous and comprises substantially only fetal stem cells (preferably at least about 96%, 97%, 98%, 99% or more fetal stem cells). The homogeneous cell populations of the invention may comprise maternal cells, for example maternal adult stem cells. As described above, homogeneity of a cell population may be achieved by any methods, for example, by cell sorting (e.g., using flow cytometry techniques such as FACS), bead separation, or by clonal expansion.

[98] As will be appreciated by one skilled in the art, fetal stem cells of the invention may be expanded, purified, modified, induced to mature into cells of a given lineage, induced to differentiate into cells of a given cell type, combined with other cells or reagents, and otherwise processed using other methods than those described herein without departing from the true scope and spirit of the present invention.

II. Components and Products Derived from Pregnancy-Associated Progenitor Cells

[99] In another aspect, the present invention provides components and biological products of the fetal stem cells described herein. The fetal stem cells (undifferentiated or induced to differentiate into cells of a desired cell phenotype or cell type) can be cultured in vitro to produce biological products in high yields. Biological products (e.g., growth factors, regulatory factors, trophic factors, peptide hormones, and the like) can be naturally generated by the inventive fetal stem cells; or the fetal stem cells can be genetically modified to produce a particular biological product of interest (as described above).

[100] Conditioned Media. Fetal stem cells of the present invention (undifferentiated or differentiated) may be cultured in vitro to produce conditioned media. Such media may be used to support the in vitro or ex vivo growth and expansion of desired cell types {e.g., stem, progenitor or specialized cells). Alternatively or additionally, such conditioned media may be used in vivo for example to support transplanted homogeneous or heterogeneous cell populations comprising fetal stem cells or cells derived therefrom. Conditioned media, which can promote growth and/or differentiation of cells already present in the body, may also be used in vivo as an alternative to cell transplantation, for example in cases where introducing intact cells could trigger rejection or other immunological responses.

[101] Generally, a medium becomes conditioned upon exposure to cells under conditions sufficient for the cells to condition it. Typically, a culture medium is used to support the growth of fetal stem cells of the invention, which naturally secrete hormones, cell matrix material and other factors in the medium. After a suitable period of time (e.g., one or a few days), the culture medium containing the secreted biological products can be separated from the cells. This process can be repeated several times to obtain large quantities of conditioned medium. Alternatively, the cells can remain in the conditioned medium, for example, to be used in co-cultures with other cells. A conditioned medium prepared from the fetal stem cells of the invention, or cells derived therefrom, may be used as obtained after conditioning, or may be submitted to one or more treatments including concentration (e.g., by ultrafiltration or lyophilization), partial purification, and combination with other reagents including pharmaceutically acceptable carriers and biologically active substances such as proteins, growth factors and/or drugs.

[102] Biological Products and Extracellular Matrix. In other applications one or more biological products of interest secreted by the fetal stem cells in the culture medium may be isolated from the medium. Isolation of such biological products may be performed by any suitable technique such as, for example, differential protein precipitation, electrophoresis, high performance liquid chromatography (HPLC), ion- exchange chromatography, and gel filtration chromatography. As mentioned above, a particular biological product may be isolated from the products naturally secreted in the medium by the fetal stem cells or cells derived therefrom. Alternatively, cells may be cultured under conditions that stimulate the production of that particular biological product, or they can be genetically modified to produce the biological product.

[103] Cell Lysates. Biological products of interest produced by the fetal stem cells of the invention may remain within the cells (instead of being excreted in the culture medium) and thus, their collection may require that the cells be lysed. Cell lysates and fractions thereof may be prepared using homogeneous or heterogeneous cell populations comprising fetal stem cells of the invention or cells derived therefrom. Alternatively, cell lysates may be obtained using fetal stem cells that have been genetically modified or that have been induced to differentiate into cells of a desired cell phenotype or cell type.

[104] Methods of cell lysis are well-known in the art and include techniques based on mechanical disruption, enzymatic disruption, or chemical disruption, or combinations thereof. Cell lysates may be prepared using cells in their culture medium such that they contain secreted growth factors, peptide hormones and the like. Alternatively, cell lysates may be prepared using cells that have been first separated from their culture medium (for example by centrifugation). In certain applications, whole cell lysates are prepared (e.g., by disrupting cells without subsequent separation of cell fractions). In other applications, a cell membrane fraction is separated from a soluble fraction of the cells by centrifugation, filtration, or similar methods. In yet other applications, one or more particular biological products are isolated from a cell lysate or fraction thereof, for example using differential protein precipitation, electrophoresis, or chromatography (e.g., HPLC, ion-exchange chromatography, and gel filtration chromatography).

[105] Cell lysates and fractions thereof prepared from populations of cells of the invention may be used as obtained, or may be concentrated (e.g., by ultrafiltration or lyophilization), purified, and/or combined with other reagents including pharmaceutically acceptable carriers and biologically active substances such as proteins, growth factors, maturation-inducing agents, differentiation-inducing agents, and/or drugs. [106] Cell lysates or fractions thereof may be used in vitro or in vivo, alone or in combination with cell transplantation, as described above for conditioned media.

III. Therapeutic Applications of Pregnancy- Associated Progenitor Cells

[107] In another aspect, the present invention provides methods of using the stem cells described herein, cells derived therefrom (including differentiated cells and modified cells) and products thereof to treat a disease or condition in humans or mammals. Diseases and conditions that can be treated using the present treatment methods are preferably associated with tissue degeneration. Examples of such diseases and conditions include, but are not limited to, neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, heart diseases, Type I diabetes, skin or eye wounds or disorders, osteoarthritis, rheumatoid arthritis, muscular dystrophies, certain types of cancer, genetic blood disorders, and liver diseases.

[108] In certain embodiments, the fetal stem cells (or derivatives or products thereof) are used to treat a subject that is related to the female mammal from which fetal stem cells have originally been obtained (for example, the subject is a biological child or grand-child of a woman who has been pregnant at least once and from which fetal stem cells have been isolated; or the subject is an offspring of a female mammal from which fetal stem cells have been isolated). In other embodiments, the fetal stem cells (or derivatives or products thereof) are used to treat a subject that is not related to the female mammal from which fetal stem cells have originally been obtained. In the latter case, cells and/or cell products used in the treatment methods are preferably allogeneic (rather than xenogeneic) to the subject receiving the treatment.

[109] A treatment according to the methods of the present invention may involve administration (e.g., by injection), to a subject in need of treatment, of fetal stem cells described herein, differentiated cells derived from fetal stem cells, or cell populations comprising fetal stem cells or cells derived therefrom. The cells may be delivered at the site of tissue damage or degeneration (e.g., to the deficient heart of a patient ) or, alternatively, cells may be delivered at a location remote from the tissue in need of repair or regeneration and home to the failing tissue (i.e., migrate by responding to chemical signals). Transplanted fetal stem cells may differentiate in situ and provide trophic support to endogenous cells. Alternatively or additionally, a treatment may involve administration of one or more products or components of fetal stem cells or cell derived therefrom (e.g., cell Iy sates or specific growths or trophic factors) to the subject. Alternatively or additionally, a treatment may involve generation of a desired tissue (e.g., liver tissue, pancreatic tissue, lung tissue, heart tissue, ocular tissue, nerve tissue, brain tissue, muscle tissue, skin, and the like) using cells of the invention (e.g., cultured on three-dimensional substrates) and transplantation of the tissue obtained into the subject in need of treatment.

[110] Cells of the invention may be implanted alone or in combination with other cells (e.g., as cell populations such as those described above) and/or in combination with other biologically active factors or reagents, and/or drugs (see below). As will be appreciated by those skilled in the art, these other cells, biologically active factors, reagents, and drugs may be administered simultaneously or sequentially with the cells and/or products of the invention. Cells, products and compositions according to the present invention may be administered to a subject in need of treatment using any suitable method. For example, they may be surgically implanted, injected, or delivered using a catheter or syringe. Administration may be intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, oral, or nasal administration.

[Ill] In certain embodiments, a treatment according to the present invention further comprises pharmacologically irnmunosuppressing the subject prior to initiating the cell-based treatment. Methods for the systemic or local immunosuppression of a patient are well known in the art. Alternatively, fetal stem cells or their derivatives may be modified to reduce their immunogenicity, as described above. In other embodiments, the treatment further comprises assessing the effects of the treatment. Physicians and artisans skilled in the art can readily determine the best methods to evaluate the effects of a treatment based on the disease or condition treated.

[112] Other methods of treatment provided by the present invention involve treating a disease or condition affecting a female mammal that has been pregnant at least once, by activating or stimulating fetal stem cells present in her system. IV. Pharmaceutical Compositions

[113] The cells and products thereof disclosed herein may be administered per se or may be administered as pharmaceutical compositions. Accordingly, the present invention provides pharmaceutical compositions comprising homogeneous or heterogeneous populations of differentiated and/or undifferentiated fetal stem cells, cultures thereof, cell lysates thereof, extracellular matrix generated thereby, or conditioned medium produced therefrom and at least one pharmaceutically acceptable carrier. Preferably, the cells and related products {i.e., extracellular matrix, cell lysate and conditioned medium) are of human origin {i.e., derived from tissue samples harvested from suitable women). However, pharmaceutical compositions may also be prepared using cells and related products derived from female mammals of other species, as described above.

[114] The specific formulation of an inventive pharmaceutical composition will depend upon several factors including the nature of the active component {e.g., cells, cell lysate, extracellular matrix or conditioned medium), the specific purpose of the composition {e.g., tissue repair, tissue regeneration, tissue augmentation, tissue sealing, tissue function restoration, stimulation of differentiation of stem cells present in the body, and the like) and the route of administration selected.

[115] Pharmaceutically acceptable carriers and diluents include any suitable organic or inorganic carrier substances which do not deleteriously react with the active component(s) of the pharmaceutical composition. The use of such carriers and diluents is well known in the art. Similarly, carriers and diluents are known and have been described, for example in "Remington 's Pharmaceutical Sciences", E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA). Examples of suitable pharmaceutically acceptable carriers include water, salt solution (e.g., Ringer's solution), alcohols, oils, gelatins, can carbohydrates {e.g., lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, and polyvinyl pyroline. Other auxiliary agents may be added to the pharmaceutical compositions such as lubricants, preservatives, stabilizers, wetting agents, emulsifϊers, osmostic pressure enhancing agents (e.g., salts), viscosity enhancing agents, and buffers. Pharmaceutical compositions comprising cellular components or products are preferably formulated as liquids. Pharmaceutical compositions comprising cells and cell populations may be formulated as liquids, semi-solids (e.g., gels) or solids (e.g., matrix, lattices, scaffolds, and the like). If desired, the pharmaceutical composition may be sterilized.

[116] In certain embodiments, the pharmaceutical composition may further comprise other biologically active substances or bioactive factors such as antiinflammatory agents, anti-apoptotic agents, immunosuppressive or immunomodulatory agents, antioxidants, growth factors, and drugs. Examples of anti-apoptotic agents include erythropoietin (EPO), EPO mimetibody, thrombopoietin, insulin-like growth factor (IGF-II or IGF-II)₅ hepatocyte growth factor (HGF), caspase inhibitors. Examples of anti-inflammatory agents include p38 MAP kinase inhibitors, TGF-β inhibitors, statins, and interleukin (IL)-6 and IL-I inhibitors, and non-steroidal anti-inflammatory drugs (e.g., Flurbiprofen, Indomethacin, Naproxen, Sulindac, and Tenoxicam). Examples of immmunosuppressive/immunomodulatory agents include calcineurin inhibitors (e.g., cyclosporine, tacrolimus), mTOR inhibitors (e.g., sirolimus), antiproliferative (e.g., azathioprine, mycophenolate mofetil), corticosteroids (e.g., prednisoline, hydrocortisone) and various antibodies such as monoclonal anti-IL-2Rα receptor antibodies (e.g., basiliximab, daclizumab). Examples of antioxidants include vitamins C and E, co-enzyme Q-IO, glutathione, sodium sulfite, sodium meta-bisulfite, L- cysteine, N-acetyl cysteine, and β-mercaptoethylamine.

[117] Effective dosages and administration regimens can be readily determined by good medical practice based on the clinical condition of the individual patient, and will depend on a number of factors including, but not limited to, the extent of the symptoms of the condition, the nature of the active component(s) of the pharmaceutical composition (i.e., differentiated or undifferentiated fetal stem cells, cell lysate, extracellular matrix or conditioned medium), and characteristics of the patient (e.g., age, body weight, gender, general health, and the like).

V. Use of Pregnancy- Associated Progenitor Cells for IM Vitro Drug Screening

[118] The fetal stem cells (or cells derived therefrom) of the present invention may be used in cell-based assays to identify, characterize, screen and/or test biologically active agents. Biologically active agents include factors or entities (e.g., compounds, molecules, drugs and the like) as well as environmental conditions (such as culture conditions or manipulations) that affect one or more characteristics of the cells used in the assay.

[119] Generally, the inventive assays include incubating fetal stem cells (undifferentiated or differentiated) with at least one candidate agent under conditions and for a time sufficient to allow contact between the candidate agent and the cells; and determining the effect of the candidate agent on a cell characteristic before and after incubation in the presence of the candidate agent. Incubation can be performed in vitro or in vivo.

[120] For in vitro assays, fetal stem cells or cells derived therefrom may be cultured according to standard cell culture techniques. For example, cells are often grown in a suitable vessel in a sterile environment at 37⁰C in an incubator containing a humidified 95% air-5% CO₂ atmosphere. Vessels may contain stirred or stationary cultures. Various cell culture media may be used. Cell culture techniques are well- known in the art and established protocols are available for the culture of diverse cell types including stem cells (see, for example, R.I. Freshney, "Culture of Animal Cells: A Manual of Basic Technique", 2^nd Edition, 1987, Alan R. Liss, Inc.). In some applications, the assays may be performed using cells contained in a plurality of wells of a multi-well assay plate. Such assay plates are commercially available, for example, from Stratagene Corp. (La Jolla, CA) and Corning Inc. (Acton, MA) and include, for example, 48-well, 96-well, 384-well, and 1536-well plates.

[121] Biologically active agents may be tested or screened for their ability to affect or modulate (e.g., change or maintain; inhibit or stimulate) proliferation, lineage-commitment, differentiation, survival, phenotype, or function of the cells used in the assay. Depending on the assay, the read-out may be gene expression, expression of one or more markers (e.g., molecular markers and/or non-molecular markers), production of growth factors, response to growth factors, morphology, metabolic activity, DNA synthesis or repair, modification of cell membrane permeability, cell viability, survival, and the like. For example, cytotoxicity can be assessed by using vital staining techniques. The number and/or robustness of the cultured cells after incubation with the candidate agent as compared with cells not exposed to the agent can be analyzed using standard cytological and/or histological techniques, including the use of immunocytochemical techniques employing antibodies that define type-specific cellular antigens.

[122] In some applications, the cell-based assays provided herein may be used to identify, characterize, screen and/or test agents that promote proliferation and maintenance of cells of a desired phenotype in a long-term culture; agents that promote commitment of cells to a particular lineage or cell type; agents that promote progressive differentiation (i.e., maturation to a more committed/differentiated state) of a cell to a desired differentiated cell type; or agents that promote terminal differentiation of a cell to a desired differentiated cell type. Such agents could be used in the development of improved differentiation and culture methods for the fetal stem and progenitor cells provided herein. Alternatively or additionally, such agents could find applications in therapeutic transplantations involving these cells, for example to facilitate or promote their proliferation and/or commitment to a particular lineage or cell type in vivo.

[123] The fetal stem cells described herein and cells derived therefrom may be particularly useful for drug screening and testing, drug toxicology studies as well as for new drug target identification. In order to improve their research and development productivity, companies in the pharmaceutical and biotechnology industry are more and more frequently adopting cell-based assays in the early phases of the drug discovery process. The use of cell-based assays is expected to reduce the late-stage failure rates of compounds in the pipeline by allowing improved, early selection of drug candidates with higher probability of success in pre-clinical and clinical trials (O.E. Beeske and S. Goldbard, Drug Discov. Today, 2002, 7: S131-S135). However, cells currently used in these screening assays are human cell lines that have usually been maintained in vitro for long periods of time and as such often have different characteristics than cells in vivo. These differences can make it difficult to predict the action of a drug in vivo based on the response of human cell lines in vitro. Differentiated cells of a variety of cell types may be derived from the fetal stem cells of the present invention and prepared in virtually unlimited quantities using standardized conditions. By conducting drug screening assays in these physiologically-relevant, unaltered cells, agents identified using these assays are more likely to behave similarly in other physiological contexts, such as in vivo conditions. [124] As will be appreciated by those of ordinary skill in the art, any kind of compounds, factors or agents can be tested using these screening assays. A candidate agent may be a synthetic or natural compound; it may be a single molecule, or it may be a mixture or complex of different molecules. Exemplary agents include, but are not limited to, nucleic acids, peptides, polypeptides (including fusion proteins), polyketides, peptidomimetics, antibodies (including fragments or variants thereof), antisense RNAs, RNAi constructs (including siRNAs), ribozymes, and chemical compounds (including small organic molecules).

[125] Screening assays according to the present invention may be performed for testing one or a few compounds. Alternatively, screening assays may be used for screening collections or libraries of compounds. Agents identified in the assays described herein can be further evaluated, detected, cloned, sequenced, chemically modified, and the like using techniques well known in the art.

VL Kits

[126] The fetal stem cells of the present invention, as well as cells derived therefrom and products thereof can conveniently be employed as part of a kit, for example, for cell culture or implantation.

[127] Accordingly, a kit is provided herein that comprises cells of the invention and one or more other components, such as cell culture media (in liquid or powered form), cell culture containers (e.g., culture dishes, multi-well plates, vials, etc.), antibiotics, antimitotic agents, hormones, matrix or support, maturation-inducing agents, differentiation-inducing agents, hydrating agents (e.g., physiologically- compatible saline solutions), pharmaceutically acceptable carriers (in liquid, semisolid or solid form), means for implanting or injecting the fetal stem cells (e.g., a syringe or a catheter), and the like. Preferably, the kit contains all the components necessary for its intended use (e.g., all the components necessary for culturing the fetal stem cells in vitro, and/or all the components necessary for differentiating the fetal stem cells into cells of a desired cell phenotype or cell type, and/or all the components necessary for implanting the fetal stem cells in vivo). In certain embodiments, the fetal stem cells comprised in the kit are cryopreserved under such conditions that at least some of the fetal stem cells are viable upon recovery. In certain embodiments, the conditions of cryopreservation allow for storage and/or shipping of the kit. Other additional components of the kit include information about the fetal stem cells comprised in the kit (e.g., immunological, biochemical and genetic properties of the stem cells) and instructions describing how to use the kit to culture, differentiate, and/or implant the fetal stem cells.

[128] Other kits are provided by the present invention that utilize cell populations comprising fetal stem cells, differentiated cells derived from fetal stem cells, specialized cells derived from fetal stem cells, and/or products generated from or by fetal stem cells. As described above, these kits preferably further comprise other components necessary for their intended purpose. Kits for in vitro screening assays as disclosed herein may contain, in addition to the fetal stem cells or derivatives, reagents for practicing the screening assay, and instructions for conducting the assay.

Examples

[129] The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained.

[130] Most of the results presented below have been reported by the present Applicants in recent scientific publications (K. Khosrotehrani and D.W. Bianchi "Multi-lineage Potential of Fetal Cells in Maternal Tissue: A Legacy in Reverse", J. Cell Science, 2005, 118: 1559-1563; K. Khosrotehrani et al, "Transfer of Fetal Cells with Multi-Lineage Potential to Maternal Tissue", J. Am. Med. Assoc, 2004, 292: 75- 80; K. Khosrotehrani et al, "Fetal Cells Migrate Specifically to Maternal Skin Wounds and Participate in Maternal Angiogenesis", submitted to the Journal of Clinical Investigation in 2005; K. Khosrotehrani et al., "Fetal Cells Participate Over Time in the Response to Specific Types of Murine Maternal Hepatic Injury", submitted to Human Reproduction in June 2006; and D.W. Bianchi and N.M. Fisk, "Gender Matters: Fetomaternal Cell Trafficking and the Stem Cell Debate", submitted to Nature in April 2006), each of which is incorporated herein by reference in its entirety.

Example 1: Transfer of Fetal Cells With Multi-Lineage Potential To Maternal

Tissue

1 - Introduction

[131] Fetal cells from both male and female fetuses enter the maternal circulation during all pregnancies (H. Ariga et at, Transfusion, 2001, 41: 1524-1530; K. Krabchi et at, Clin. Genet., 2001, 60: 145-150). They can persist in maternal blood or tissues for decades, creating a state of physiologic microchimerism in the parous woman (D.W. Bianchi et at, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708). Recent studies detected male cells of presumed fetal origin in 30% to 50% of healthy women who had prior male pregnancies (N.C. Lambert et at, Blood, 2002, 100: 2845-2851). The long-term consequences of fetal cell microchimerism for maternal health are only beginning to be appreciated. Fetal microchimeric cells are present in higher numbers in women with some autoimmune diseases, such as systemic sclerosis, than in control groups (J.L. Nelson et at, Lancet, 1998, 351: 559-562; CM. Artlett et at, New Engl. J. Med., 1998, 338: 1186-1191). The Applicants have also observed fetal cells in the tissues of women with non-autoimmune disorders, such as hepatitis C (K.L. Johnson et at, Hepatology, 2002, 36: 1295-1297) and cervical cancer (D. Cha et at, Obstet. Gynecol., 2003, 102: 774-781). Thus, the Applicants have developed an alternate hypothesis in which fetal cells were associated with the maternal response to injury as opposed to causing disease.

[132] During pregnancy, the fetal cells that enter the maternal circulation are predominantly of hematopoietic origin, such as nucleated red blood cells, lymphocytes, or hematopoietic stem cells (D.W. Bianchi et at, Br. J. Haematol., 1999, 105: 574-583; H. Osada et at, Transfusion, 2001, 41: 499-503). Trophoblasts and mesenchymal stem cells also circulate within maternal blood (LJ. can Wijk et at, Am. J. Obstet. Gynecol., 1996, 174: 871-878; K. O'Donoghue et al, MoI. Hum. Reprod., 2003, 9: 497-502). Following pregnancy, male fetal cells have been demonstrated in the CD34⁺ compartment (D.W. Bianchi et at, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708; K.M. Adams et at, Blood, 2003, 102: 3845-3847; E. Guetta et at, Blood Cells MoI. Dis., 2003, 30: 13-21). They have also been found in various sorted subsets of maternal peripheral mononuclear blood cells, such as T-, B-, and natural killer cells, or cells that express the CD4 or CD8 antigens (P.C. Evans et al, Blood, 1999, 93: 2033-2037; CM. Artlett et al, Clin. Immunol., 2002, 103: 303-308), suggesting that fetal microchimeric cells may be capable of engraftment and differentiation along the hematopoietic pathway. Little information is available on the phenotype of fetal microchimeric cells in non-hematopoietic tissues and most published studies suggest that fetal cells express hematopoietic markers (P.A. Fanning et al, J. Hepatol., 2000, 33: 690-695; C. Scaletti et al, Arthritis Rheum., 2002, 46: 445-450). In contrast, the Applicants have previously reported that the male cells of presumably fetal origin observed in the thyroid of a woman affected with a multinodular goiter had a follicular morphology (B. Srivatsa et al, Lancet, 2001, 358: 2034-2038). The hypothesis was therefore tested by examining tissue specimens from women, affected with a variety of diseases, who had male offspring to determine the morphology, cell surface, and intracellular phenotype of fetal cells within maternal organs.

2 - Methods

[133] Study Design. Individual male (XY+) microchimeric cells were evaluated for their cell surface and intracellular phenotype. Tissue samples from 10 women that were previously studied by the Applicants' laboratory group were selected that had significant and easily detectable male cell microchimerism (K.L. Johnson et al, Hepatology, 2002, 36: 1295-1297; D. Cha et al, Obstet. Gynecol., 2003, 102: 774- 781; B. Srivatsa et al, Lancet, 2001, 358: 2034-2038; KX. Johnson et al, Arthritis Rheum., 2001, 44: 2017-2111; KX. Johnson et al, Arthritis Rheum., 2001, 44: 1848- 1854). All of the initial studies documenting the presence of microchimerism included appropriate control patients, including women who had no prior male pregnancies. In the present study, the Applicants contemporaneously analyzed skin and cervical tissue from 11 women with no known history of a male pregnancy. Approval from the institutional review board and written informed consent from all patients who underwent surgery or biopsies were obtained. To the extent possible, complete pregnancy histories from study participants were obtained, including the number of sons, daughters, and abortions (spontaneous and elective), as well as the possibility of other sources of microchimeric cells. None of the women had a twin brother or had received an organ transplant at the time of tissue collection. One woman had a history of blood transfusion from a donor of unknown sex.

[134] Fluorescence in Situ Hybridization and Immunolabeling. Fluorescence in situ hybridization (FISH) analysis of the tissue sections was performed as previously described (K.L. Johnson et al, Arthritis Rheum., 2001, 44: 1848-1854; K.L. Johnson et al, Biotechniques, 2000, 29: 1220-1224) with simultaneous immunolabeling (K. Khosrotehrani et al, Biotechniques, 2003, 34: 242-244). Three different mouse monoclonal IgGl antibodies were tested: AE1/AE3 anticytokeratin (Chemicon International, Temecula, Calif ) was used to identify epithelial cells, anti- CD45 (Dako, Carpintera, Calif) to identify leukocytes, and heppar-1 (Dako) to identify hepatocytes. In all experiments, a mouse IgGl (BD Bioscience, San Diego, Calif) was used as an isotypic control.

[135] Scoring. Following hybridization and immunostaining, tissue sections were included for subsequent analysis if the following criteria were met: FISH, immunostaining, and morphologic.

[136] FISH Criteria. During the hybridization procedure, there was minimal loss of cells and more than 75% of nuclei contained FISH signals. Male cells had 2 differently-colored FISH signals, representing both the X and Y chromosomes, and an intact nuclear border. The coordinates of microchimeric cells were recorded, allowing retrieval of 701 (97.9%) of 716 cells on the slide. The total number of nuclei was estimated in each section by counting them in 10 fields at 40Ox magnification and counting the number of fields to cover the whole tissue section. The frequency of male cells among a million maternal cells was then extrapolated for each tissue section.

[137] Immunostaining Criteria. The immunostaining results were considered to be positive if target areas were stained and non-target areas were not stained. For CD45, the target areas were defined as nucleated cells inside blood vessels and non- target areas were defined as any epithelial tissue. For heppar-1 and cytokeratin, the target area was defined as liver parenchyma or epithelial area, respectively, and non- target areas were defined as cells inside blood vessels. In addition, to further prove the specificity of the antibodies used, immunostaining was performed with the anticytokeratin antibody on liver, lymph node, and spleen tissue, and with heppar-1 on skin, spleen, heart, and thyroid tissue. Two series of immunostaining experiments were also performed on a cord blood sample obtained during a full-term cesarean delivery with all the antibodies described above to determine if circulating fetal cells express hepatocyte or epithelial cell markers.

[138] Morphologic Criteria. After evaluating both FISH and immunostaining results, tissue sections were stained with hematoxylin and eosin. Microchimeric male cells were then relocated based on their slide coordinates and their morphology and relative location were assessed within a section using a light microscope. Cells that were not part of the section were excluded. Morphology and immunostaining were independently evaluated by 2 investigators.

[139] Statistical Analyses. Each microchimeric cell received a score of 0 if hematopoietic or 1 if epithelial or hepatocyte. The concordance between the morphology and immunohistochemical assessments were compared for all cells that had both criteria scored by estimating the K value. In thyroid specimens, the cells were also evaluated as being inside or outside the diseased area of the tissue section. Thyroid samples were the only specimens in which the pathologic area (adenomatous tissue) could be clearly distinguished from healthy surrounding tissue. All other specimens contained exclusively diseased tissue. The frequencies of cytokeratin positive microchimeric cells inside and outside the diseased area were compared by using the Kruskal-Wallis test (M. Hollander and D.A. Wolfe, "Nonparametric Statistical Methods", 1999 (2^nd Ed), John Wiley & Sons: Hoboken, NJ, pp. 190-191).

3 - Results

[140] FISH analyses were performed and a total number of 701 XY+ cells (mean [SD], 227 [128] XY+ microchimeric per million maternal cells) were identified in archived paraffin-embedded tissue section specimens from 10 women (mean age, 51.7 years; range, 34-74 years) who had male offspring. The cell surface and intracellular phenotype of the XY+ cells were subsequently evaluated by immunolabeling, morphology, and relative location within the sample (Table 1). FISH analysis was also performed on tissue biopsies from women who had no history of a male pregnancy (n = 11) and found no XY+ cells (Table 2). [141] Anticytokeratin did not stain hematopoietic tissues, such as lymph node or spleen, but did stain biliary epithelium as expected. Antihepatocyte antibody (heppar- 1) was specific for liver and did not stain any of the additional tissues tested (skin, heart, thyroid, and spleen). In addition, unlike anti-CD45, anticytokeratin and heppar- 1 antibodies did not stain cord blood cells. In 90% of cases in which there was positive immunochemical staining of a cell, independent histological assessment of that cell after hematoxylin and eosin staining was substantially concordant with regard to morphology (κ=0.72). A mean (SD) frequency of 190 (157) XY+ microchimeric cells per million maternal cells was found among 3 women with multinodular goiters who underwent partial thyroidectomy. In each of the 3 women, 14% to 60% of the XY+ cells stained positively with cytokeratin, a marker of epithelial differentiation (Figure 1). In one case, some of the XY+ fetal cells that expressed cytokeratin were integrated into a thyroid follicle. In 2 of 3 thyroid specimens, none of the microchimeric cells expressed CD45, a common leukocyte antigen. A large inflammatory infiltrate was observed in the third woman's thyroid; 67% of the XY+ cells expressed CD45.

[142] The differentiation pattern of XY+ cells was also analyzed according to their physical location within a pathologic or healthy area. The 3 thyroid specimens studied included a macroscopically visible adenoma surrounded by healthy thyroid tissue. Histological examination of these 3 specimens revealed that most of the microchimeric cells (114 of 150 cells successfully relocated) were not part of the adenomatous tissue but were in the surrounding healthy thyroid tissue. Interestingly, fetal cells inside the adenoma (36 of 150) had a significantly higher percentage of cytokeratin expression than cells outside the adenoma (92% vs. 17%, respectively; P < 001). The reverse situation was found for CD45: XY+ cells outside the adenoma more frequently expressed CD45 than cells inside the adenoma (32% vs. 3%, respectively; P < 001).

[143] Other epithelial tissues, such as cervical epithelium specimens from 3 women, and digestive epithelial (gallbladder, intestine) tissues from 2 women were also analyzed. A comparable pattern of differentiation was found: 20% to 56% of the XY+ cells expressed cytokeratin and 30% to 55% expressed CD45. In hematopoietic tissues, such as lymph nodes and spleen from 2 women, 90% of the XY+ cells expressed CD45. None of the cells expressed cytokeratin. Double staining (CD45 and cytokeratin) was performed in most tissues; microchimeric cells never stained positively with both antibodies. In liver specimens of two women (patients G and I), most of the XY+ cells expressed CD45 (Figure 2). In one woman, 4% of the fetal microchimeric cells stained with the hepatocyte marker heppar-1. These cells had a morphology compatible with that of hepatocytes (Figure 3).

4 - Comments

[144] The use of stem cells as a novel treatment for repair of diseased organs in the human is an area of intense interest for the worldwide scientific community, as well as the lay public and many governments. In this study, the Applicants have shown that XY+ microchimeric cells in maternal tissues, acquired most likely through pregnancy, express leukocyte, hepatocyte, and epithelial markers. These data suggest that pregnancy may result in the physiologic acquisition of a fetal cell population with the capacity for multi-lineage differentiation. The Applicants have coined the term "pregnancy-associated progenitor cells" to describe this population.

[145] The present study was based on a small number of patients already selected for having high numbers of microchimeric cells. It is recognized that there is an inherent selection bias in the studied women, but to perform the study, adequate numbers of fetal microchimeric cells must be present in the tissue to be further analyzed. Therefore, the conclusions drawn from the present study may only apply to women with high numbers of microchimeric cells.

[146] Most of the women did not have any additional sources of microchimerism, such as solid organ transplantation. One of the 10 patients had a history of blood transfusion. Transfusion-associated microchimerism is highly unlikely to develop unless large quantities of blood are transfused in the setting of trauma (T.H. Lee et al, Blood, 1999, 93: 3127-3139). Therefore, it is most likely that the XY+ cells in this study are fetal in origin.

[147] In almost all tissues, XY+ cells bearing CD45, the common leukocyte antigen, were observed at variable frequencies. These results are consistent with previous findings that suggest that fetal microchimeric cells are originally blood cells, including hematopoietic progenitor cells (D. W. Bianchi et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708; H. Osada et al, Transfusion, 2001, 41: 499-503; E. Guetta et al, Blood Cells MoI. Dis., 2003, 30: 13-21; P.C. Evans et al, Blood, 1999, 93: 2033-2037).

[148] XY+ microchimeric cells that expressed cytokeratin, a marker of epithelial cell differentiation, were never observed in hematopoietic tissues {e.g., lymph node). The concordance of morphological and immunohistochemical findings supports the idea that some fetal cells may have an epithelial phenotype. In one woman, in whom higher numbers of microchimeric cells were present, cells with evidence of a hepatocyte marker were also detected. Fetal cord blood cells were also shown not to express epithelial or hepatocyte markers, suggesting that the microchimeric fetal cells acquire these markers in the environment of maternal tissues.

[149] The present study did not determine the type of fetal progenitor cells originally transferred during the pregnancies of the women. Fetal blood contains a variety of stem cell types, including mesenchymal stem cells and hematopoietic stem cells (C. Campagnoli et al, Blood, 2001, 98: 2396-2402). During pregnancy, fetal hematopoietic and mesenchymal progenitor cells circulate within maternal blood and can be cultured in maternal peripheral blood for up to 6 months after delivery (E. Guetta et al, Blood Cells MoI. Dis., 2003, 30: 13-21; K. O'Donoghue et al, MoI. Hum. Reprod., 2003, 9: 497-502; P.C. Evans et al, Blood, 1999, 93: 2033-2037).

[150] Feto-maternal transfusion may be even higher after an elective termination of pregnancy (D.W. Bianchi et al, Am. J. Obstet. Gynecol., 2001, 184: 703-706). The Applicants have shown previously by meta-analysis that a reproductive history that includes an elective termination or an early fetal loss is associated with a higher incidence of microchimerism in maternal tissues (K. Khosrotehrani et al, Arthritis Rheum., 2003, 48: 3237-3241). The CD34⁺ fetal cells are present in maternal blood for decades after delivery in 75% of women studied (D.W. Bianchi et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708) as well as in the CD34⁺-enriched cell fraction of women undergoing granulocyte colony-stimulating factor bone marrow stimulation (K.M. Adams et al, Blood, 2003, 102: 3845-3847). The present results imply but do not prove that fetal CD34⁺ hematopoietic stem cells that persist post partum may have multi-lineage capacity. Another possibility is that pregnancy results in the acquisition of a different type of circulating stem cell, perhaps from the placenta, which has epithelial characteristics. [151] The non-hematopoietic morphology and phenotypes of the fetal cells that were observed may result from different mechanisms. Fetal progenitor cells could transdifferentiate into the hematopoietic, hepatic, or epithelial cells. They could also adopt the host tissue phenotype by fusing with hepatocytes or epithelial cells (X. Wang et al, Nature, 2003, 422: 897-901). In the identification of microchimeric XY+ cells based on X and Y chromosome FISH signals, an XY+ cell with an interphase karyotype suggestive of a fused nucleus (XXXY) or having 2 separate nuclei was never detected. However, one cannot exclude the possibility that some fetal and maternal cells fuse their cytoplasm, especially in dense tissues, such as liver, in which the outer limits of each cell are hard to distinguish. Whatever the mechanism involved, the Applicants believe that the idea of fetal cells expressing non- hematopoietic markers is novel and may have important long-term health implications for the woman who has undergone pregnancy by providing her with a younger population of cells that may have different capabilities in the response to tissue injury.

[152] In conclusion, the present study has shown that fetal cells, in a variety of maternal tissues, have morphologic and protein expression characteristics of not only hematopoietic but also epithelial and hepatic cells. These data suggest that, at least in some women after pregnancy, fetal cells transferred during pregnancy develop multi- lineage capacity either by cell fusion or transdifferentiation. Further study of naturally occurring fetal cell microchimerism may be useful in determining the characteristics of the specific progenitor cell population and the exact mechanisms involved in its apparent differentiation.

Example 2: Multi-Lineage Potential of Fetal Cells in Maternal Tissue: A Legacy in Reverse

1 - Introduction

[153] The recent discovery of the long-term persistence of fetal cells in maternal blood and tissues decades after pregnancy has opened up a new field of investigation (D.W. Bianchi et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708; JX. Nelson et al, Lancet, 1998, 351: 559-562; K. O'Donoghue et al, Lancet, 2004, 364: 179-182). Fetal cell microchimerism, originally described in mice, is defined as the persistence of fetal cells in maternal organs and circulation without any apparent graft-vensro-host reaction or graft rejection (A. Liegeois et al, Transplant. Proc, 1977, 9: 273-276). It has become apparent that fetal cell microchimerism is a widespread phenomenon, and it is now known that fetal cells are transferred to the maternal circulation during all human pregnancies and after delivery.

[154] The findings in humans have led to the hypothesis that autoimmune diseases that are found predominantly in women may result from an immune reaction between the mother and the fetal cells that remain post-partum (J.L. Nelson, Arthritis Rheum., 1996, 39: 191-194). Persisting fetal cells are also found in a wide range of tissues from women affected with a variety of non-immune diseases, such as hepatitis C and cervical cancer. Here recent findings are discussed that suggest that, in all pregnancies, fetal cells that have stem-cell-like properties are transferred into maternal blood. The Applicants hypothesize that these cells, which they term pregnancy-associated progenitor cells (PAPCs), persist after delivery in a maternal stem cell niche and, in the case of tissue injury, home to the damaged organ and differentiate as part of the maternal repair response.

2 - Fetal Cells Circulate During Pregnancy

[155] In all human pregnancies, fetal cells can be detected in the maternal circulation (H. Ariga et al, Transfusion, 2001, 41 : 1524-1530). Feto-maternal cell trafficking starts as early as six weeks of gestation (H. Ariga et al, Transfusion, 2001, 41: 1524-1530). The frequency with which fetal cells can be detected in blood from pregnant women increases with gestational age. hi normal second-trimester pregnancies, the number of fetal cells in the maternal circulation is estimated to be 1-6 cells/mL of maternal venous blood (D. W. Bianchi et al, Am. J. Obstet. Gynecol, 2001, 184: 703-706; K. Krabchi et al, Clin. Genet, 2001, 60: 145-150; D.W. Bianchi et al, Am. J. Hum. Genet., 1997, 61: 822-829). At 36 weeks of gestation, 100% of pregnant women have fetal cells in their circulation (H. Ariga et al, Transfusion, 2001, 41 : 1524-1530). After delivery, this fraction rapidly decreases. Sensitive PCR techniques indicate that 30-50% of healthy women have detectable fetal cells in their blood from four weeks to decades after delivery (H. Ariga et al, Transfusion, 2001, 41: 1524-1530; CM. Artlett et al, Clin. Immunol., 2002, 103: 303-308; N.C. Lambert et al, Blood, 2002, 100: 2845-2851). By examining specific peripheral blood mononuclear cell sub-populations, one can detect microchimerism in as many as 90% of healthy postpartum women (P.C. Evans et al, Blood, 1999, 93: 2033-2037). Fetal cell microchimerism is thus probably a widespread phenomenon, although difficult to detect.

3 - Factors that Influence the Transfer of Fetal Cells during and after Pregnancy

[156] The amount of fetal cell transfer to the maternal circulation during pregnancy may be influenced by feto-maternal histocompatibility. In animal models, female mice with a syngenic fetus (one with identical histocompatibility alleles at the H-2 locus) have higher numbers of microchimeric cells in their hematopoietic tissues, such as blood, lymph node and spleen, compared with female mice with allogenic fetuses (those that have different histocompatibility alleles at the H-2 locus) (E.A. Bonney and P. Matzinger, J. Immunol., 1997, 158: 40-47). However, in humans, the same trend is not observed between feto-maternal histocompatibility and the persistence of fetal cell microchimerism (P.C. Evans et al, Blood, 1999, 93: 2033- 2037).

[157] Although certain maternal HLA alleles, such as HLA-DQ Al*0501, appear to be more frequently associated with fetal cell microchimerism (N.C. Lambert et al, J. Immunol., 2000, 164: 5545-5548; JX. Nelson et al, Lancet, 1998, 351: 559- 562), this finding is controversial (CM. Artlett et al, Arthritis Rheum., 2003, 48: 2567-2572). The number of fetal cells in the maternal circulation is affected by fetal and placental abnormalities. There is increased fetomaternal cell transfer in cases of fetal aneuploidy (D. W. Bianchi et al, Am. J. Hum. Genet., 1997, 61: 822-829), maternal preeclampsia (W. Holzgreve et al, Obstet. Gynecol., 1998, 9: 669-672) or following terminations of pregnancy (D. W. Bianchi et al, Am. J. Obstet. Gynecol., 2001, 184: 703-706). In the latter case, in the second trimester, the number of fetal cells in the maternal circulation before and after a termination increases from 19 to 1500/16 mL of maternal whole blood.

[158] A woman's reproductive history is also important. By systematically analyzing all published cases of microchimerism that described the study subjects' individual reproductive histories, the present Applicants observed that a prior history of fetal loss (either miscarriage or termination) significantly increases the chance that fetal cells can be detected in that woman's organs (K. Khosrotehrani et al, Arthritis Rheum., 2003, 48: 3237-3241). Women who have a history of fetal loss are 2.4 times more likely to exhibit fetal cell microchimerism than are women with no history of fetal loss. Unfortunately, this meta-analysis cannot distinguish between natural and voluntary pregnancy loss in the published literature. There may be significant differences in the incidence of microchimerism between these scenarios. Other variables, such as the number of pregnancies, do not appear to influence the persistence of fetal cells significantly. The increased microchimerism following fetal loss is probably due either to increased transfusion of fetal cells at the time of loss or to the transfer of a cell type that is at an earlier developmental stage and thus more likely to engraft in the mother. Another factor that might influence the presence of microchimerism is the length of time that has elapsed since completion of pregnancy. Several studies have suggested that fetal cells are not detectable in women with younger sons (D. W. Bianchi et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708; M. A. Filho et al, Transplant. Proc, 2002, 34: 2951-2952).

4 - Fetal Stem Cells are Transferred During Pregnancy From the Fetus to the Mother

[159] In all human pregnancies, fetal progenitor cells that express CD34 are transferred into the maternal circulation (E. Guetta et al, Blood Cells MoI. Dis., 2003, 30: 13-21); they can be isolated by culturing maternal blood during pregnancy and up to six months after delivery (H. Osada et al, Transfusion, 2001, 41: 499-503). The number of fetal progenitor cells circulating in the blood of pregnant women has been estimated to be 0-2/niL (E. Guetta et al, Blood Cells MoI. Dis., 2003, 30: 13-21). Decades after delivery, male fetal CD34+ (hematopoietic stem cells; HSCs) and CD34+CD38+ cells (which are committed to early B- and T-cell development) have been identified in 75% of women with sons (D. W. Bianchi et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708). In addition, fetal cells have been detected in the CD34+-enriched fraction obtained by apheresis (a procedure in which blood is drawn and separated into its components by dialysis; CD34+ cells are retained and the rest are returned to the donor) after growth factor-induced mobilization of HSCs in 50% of the women studied (K.M. Adams et al, Blood, 102: 3845-3847).

[160] During the first trimester of pregnancy, fetal blood also contains mesenchymal stem cells (MSCs; C. Campagnoli et al, Blood, 2001, 98: 2396-2402), which were initially described in adult bone marrow. Microchimeric fetal MSCs have been isolated from the peripheral blood of an adult woman following termination of pregnancy (K. O'Donoghue et al, MoI. Hum. Reprod., 2003, 9: 497-502). Fetal stem cells thus seem to enter the maternal circulation during pregnancy and persist in niches such as bone marrow.

5 - Do Fetal Cells cause Autoimmune Disease?

[161] The long-term presence of fetal cells in the semi-allogenic maternal body raises the possibility of an immune reaction between maternal and fetal cells that results in maternal disease. Nelson has hypothesized that some autoimmune diseases that preferentially occur in middle-aged women, and that have clinical and pathological similarities with graft-veraws-host reaction disease, may in fact be allo- immune diseases (Table 3) (JX. Nelson, Arthritis Rheum., 1996, 39: 191-194).

[162] Systemic sclerosis (SS) is such a disease. The number of fetal cells present in blood and other tissues of women affected with SS is significantly higher than in controls (CM. Artlett et al, New Engl. J. Med., 1998, 338: 1186-1191; JX. Nelson et al, Lancet, 1998, 351: 559-562; K. Khosrotehrani and D.W. Bianchi, Curr. Opin. Obstet. Gynecol., 2003, 15: 195-199). The present Applicants studied autopsy specimens from multiple tissues from women affected with SS and showed that male cells of putative fetal origin were most frequently observed in spleen (KX. Johnson et al, Arthritis Rheum., 2001, 44: 1848-1854). They also reported that, in a woman with systemic lupus erythematosus (SLE) who died of severe intestinal vasculitis, affected tissues had more fetal cells present than did healthy tissues (KX. Johnson et al, Arthritis Rheum., 2001, 44: 2107-2111).

[163] Similar techniques do not reveal any microchimeric fetal cells in skin samples from a group of women affected with mild SLE (K. Khosrotehrani et al, "Natural History of Fetal Cell Microchimerism during and following Murine Pregnancy", J. Reprod. Immunol., 2005, in press). Filho et al detected evidence of Y-chromosome sequences in the blood of 68% of SLE patients compared with 33% in controls (M. A. Filho et al, Transplant. Proc, 2002, 34: 2951-2952). Patient disease severity was not described in this study. In SLE, fetal cell microchimerism might be more likely to be found in affected tissues of severe cases (for example with nephritis) rather than benign ones (M. Mosca et al, Ann. Rheum. Dis., 2003, 62: 651-654). Fetal cells thus probably do not trigger the disease but instead home to the affected maternal tissue if the damage reaches a particular 'threshold'.

6 - Does the fetus 'treat' its mother?

[164] What if the fetal cells are found in the clinically affected organs because they are attempting to combat the disease? The present Applicants have analyzed a liver biopsy specimen from a woman with hepatitis C (K.L. Johnson et at, Hepatology, 2002, 36: 1295-1297) who stopped treatment (against medical advice) but, despite this, did well clinically and her disease abated. Her liver specimen contained thousands of male cells detected by dual-color fluorescence in situ hybridization (FISH) studies using probes for the X and Y chromosomes. She had never received a blood transfusion and was not a twin. Follow-up studies using DNA polymorphism analyses indicated that the probable source of the male cells in her liver was a pregnancy that she had terminated 17-19 years earlier (K.L. Johnson et at, Hepatology, 2002, 36: 1295-1297). In this case, the male cells in the liver were morphologically indistinguishable from surrounding liver tissue, which suggests that they were hepatocytes.

[165] Similarly, a study of biopsy material from 29 women with thyroid disorders revealed fetal microchimeric cells in women with Hashimoto's disease as well as other non-immune thyroid disorders (B. Srivatsa et al., Lancet, 2001, 358: 2034-2038). An unexpected result was the detection of large numbers of male fetal cells in an otherwise healthy woman who had a benign thyroid adenoma. DNA probes that map to the X and Y chromosomes showed that mature follicles from the woman's thyroid were partly male and partly female. She had no other potential sources of microchimeric cells: she had never been transfused, had never had an organ transplant, and was not a twin.

[166] More systematic studies have examined the phenotypes of fetal microchimeric cells from patients who have high numbers of such cells by combining in situ hybridization to detect the fetal cells and immunolabeling to identify their phenotype (K. Khosrotehrani et at, Biotechniques, 2003, 34: 242-244). In epithelial tissues such as thyroid, cervix, gallbladder or intestine, 14-60% of the fetal cells express epithelial markers such as cytokeratin (Figure 4). In the liver, 4% of the fetal microchimeric cells have a hepatocytic phenotype (K. Khosrotehrani et at, JAMA, 2004, 292: 75-80). Most of the other fetal microchimeric cells in these tissues express CD45, the common leukocyte antigen, indicating a likely hematopoietic origin. Similarly, 90% of the fetal cells detected in maternal hematopoietic tissues, such as spleen or lymph node, express CD45. In all cases, the morphology of the fetal cells suggests that they have differentiated. In addition, in sections containing diseased and healthy thyroid tissue, the fetal cells more frequently express cytokeratin if they are in the diseased area of the thyroid. These results suggest that fetal cells, possibly hematopoietic in origin, home to the site of injury and adopt the maternal local tissue phenotype. Whether the fetal cells actually differentiate or fuse with the damaged host cells remains an open question. However, using chromosome specific probes and FISH analysis, the Applicants have never observed the tetraploid signals that would be consistent with such fusion. It is therefore concluded that, among the fetal cells transferred to the mother during pregnancy, some have multi-lineage capacity. The Applicants term these cells, Pregnancy-Associated Progenitor Cells or PAPCs.

7 - Cellular Origin of the PAPCs

[167] Fetal CD34+ and CD34+CD38+ cells circulate in maternal blood for decades after delivery (D.W. Bianchi et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 705-708). The Applicants believe that, to persist long-term, the fetal microchimeric cell population must contain stem cells that can proliferate, as initially proposed for bone marrow cell microchimerism (A. Liegeois et al, Transplant. Proc, 1977, 9: 273- 276). It is hard to imagine how fully differentiated fetal cells that have a short half- life and no self-renewal capacity could regularly appear in maternal blood and tissue decades after delivery. Thus, the Applicants hypothesize that they have stem-cell-like properties.

[168] Both fetal blood and the placenta contain various types of stem cells. HSCs have been identified, isolated and cultured from the placenta. In fact, the placenta contains 2-4 times more HSCs than other fetal hematopoietic tissues, such as the liver or yolk sac (M. Alvarez- Silva et al, Development, 2003, 130: 5437-5444). In addition, placental HSCs have a higher proliferation potential than fetal liver progenitors, which are at a later developmental stage. Another possible origin of the PAPCs could be MSCs. Recently, O'Donoghue and coworkers found male (presumed fetal) MSCs in 100% of bone marrow samples obtained at thoracotomy from women with sons who ranged in age from 13 to 51 years (K. O'Donoghue et al, Lancet, 2004, 364: 179-182). They characterized these cells phenotypically, as well as functionally, following culture. Under appropriate culture conditions, the cells differentiate into muscle, nerve, bone and fat.

8 - The Need for Animal Models

[169] Studies of human fetal microchimerism are limited by the amount of appropriate tissue available, the difficulty of obtaining accurate pregnancy histories, and the impossibility of analyzing tissue from healthy individuals. Recently, several animal models have been used in studies of fetal cell microchimerism. Jimenez and Tarantal used rhesus monkeys (Macaca mulatto) to study feto-maternal trafficking (D .F. Jimenez and A.F. Tarantal, Pediatr. Res., 2003, 53: 18-23) and demonstrated long-term persistence of male CD34+ cells in one or more maternal tissues (D.F. Jimenez et al, Transplantation, 2005, 79: 142-146). Fetal cell microchimerism has also been examined in mice (A. Liegeois et al., Transplant. Proc, 1981, 13: 1250- 1252; E.A. Bonney and P. Matzinger, J. Immunol., 1997, 158: 40-47; P. J. Christner et al, Arthritis Rheum., 2000, 43: 2598-2605) and, more recently, in rats (Y. Wang et al, Biochem. Biophys. Res. Commun., 2004, 325: 961-967).

[170] Most studies of murine fetal cell microchimerism rely on the fetus and the mother being of different sex, or on the presence of a marker chromosome (T6) in the fetus. More recently, the present Applicants have used male transgenic mice carrying unique paternal reporter transgenes to identify and track the fetal cells. The transgenic fetal cells can be easily detected in the wild-type maternal tissues. For example, when enhanced green fluorescent protein (GFP) under the control of the chicken β-actin promoter and the cytomegalovirus (CMV) enhancer is used as the reporter (M. Okabe et al, FEBS Lett., 1997, 407: 313-319), cells from transgenic fetuses can be easily detected in wild-type females by fluorescence microscopy or immunohistochemistry. Furthermore, quantifying the number of gfp sequences by real-time PCR amplification of genomic DNA allows detection of the equivalent of one fetal cell in 105 maternal cells (K. Khosrotehrani et al, Hum. Reprod., 2004, 19: 2460-2464). These methods allow fetal cells to be monitored in maternal blood and tissue during and after normal murine pregnancies (K. Khosrotehrani et al, "Natural History of Fetal Cell Microchimerism during and following Murine Pregnancy", J. Reprod. Immunol., 2005, in press). In addition, the present Applicants are currently developing injury models to assess the capabilities of fetal cells to home to maternal injured tissues and to differentiate. Animal models show great promise for determination of which types of maternal injury or disease are most likely to recruit fetal cells from their niche. Furthermore, new bioluminescent imaging techniques will allow study of the behavior of these cells in the living mouse (P.R. Contag et at, Nat. Med., 1998, 4: 245-247).

9 - Conclusions/Perspectives

[171] Fetal cell microchimerism is a new field of investigation. It is a widespread phenomenon that potentially affects every woman who has been pregnant. The discovery of the long-term persistence of fetal cells in maternal tissues, with their evidence of multi-lineage capacity, strongly suggests the presence of a novel population of cells that are acquired physiologically.

[172] The work discussed here provides strong evidence that all adult stem cells are not alike. In the future, consideration must be given to whether adult stem cells originate from a male or female and whether that female has been pregnant, since tissues from adult parous females appear to contain a mixed population of adult and fetal cells.

[173] Pregnancy results in the acquisition of cells that may have clinical applications and therapeutic potential. Whether the PAPCs are HSCs or MSCs, or a new population of stem cells, is an unresolved issue. It is also unknown whether PAPCs respond to all types of maternal injury or only those injuries that recruit stem cells. It is possible that these cells, since they are fetal in origin, have a higher proliferative capacity or more plasticity than their equivalent adult (maternal) cells. In the current debate over the use of embryonic stem cells for treatment of disease, the discovery of a population of fetal stem cells that apparently differentiate in the adult woman and can be acquired without harming the fetus may be significant. Future research will focus on animal models to determine the contribution of the fetal PAPCs to the repair of maternal injury. Example 3: Fetal Cells Migrate Specifically to Maternal Skin Wounds and Participate in Maternal Wounds

1 - Background and Goal of the Study

[174] Fetal cells circulate in the blood of all pregnant women (H. Ariga et ah, Transfusion, 2001, 41: 1524-1530) and approximately half of post-partum women (N.C. Lambert et ah, Blood, 2002, 100: 2845-2851). Some of the fetal cells are stem cells. For example, fetal hematopoietic stem cells that express CD34 enter the maternal circulation and persist for decades post-partum (D. W. Bianchi et ah, Proc. Natl. Acad. Sci. USA, 196, 93: 705-708; E. Guetta et ah, Blood Cells MoI. Dis., 2003, 30: 13-21; K.M. Adams et ah, Blood, 2003, 102: 3845-3847). Fetal mesenchymal stem cells have been successfully cultured from maternal bone marrow as long as 51 years following pregnancy (O'Donoghue et ah, Lancet, 2004, 364: 179-182). These studies have led to the development of the concept that following pregnancy, a woman becomes a chimera.

[175] The long-term health consequences of fetal cell microchimerism for a parous woman are currently the subject of active research and debate. Higher numbers of circulating fetal microchimeric cells are found in the blood of post-partum women with autoimmune diseases, such as systemic sclerosis, than in healthy women (J.L. Nelson et ah, Lancet, 1998, 351: 559-562; K. Khosrotehrani and D. W. Bianchi, Curr. Opin. Obstet. Gynecol., 2003, 15: 195-199). In women with autoimmune conditions, higher numbers of fetal cells are found in spleen and clinically affected organs (K.L. Johnson et ah, Arthritis Rheum., 2001, 44: 1848-1854; K.L. Johnson et ah, Arthritis Rheum., 2001, 44: 2107-2111); their presence or persistence may be affected by maternal-fetal class II human leukocyte antigens (N.C. Lambert et ah, J. Immunol, 2000, 164: 5545-5548; CM. Artlett et ah, Arthritis Rheum, 2003, 48: 2567-2572). In addition, fetal cells have been detected in maternal tissue in nonimmune diseases such as cervical cancer (D. Cha et ah, Obstet. Gynecol, 2003, 102: 774-781), hepatitis C (K.L. Johnson et ah, Hepatology, 2002, 36: 1295-1297), and benign thyroid adenoma (B. Srivatsa et ah, Lancet, 2001, 358: 2034-2038). In the latter two studies, histological analysis of the fetal cells suggested that they had the morphology of fully mature and differentiated liver or thyroid tissue. Using simultaneous immunolabeling techniques and genetic analysis (K. Khosrotehrani et ah, Biotechniques, 2003, 34: 242-244), the Applicants have recently shown that fetal microchimeric cells express markers of hepatocytic, epithelial, or leukocyte differentiation in diseased maternal liver, epithelial or hematopoietic tissue decades after delivery (K. Khosrotehrani et al, JAMA, 2004, 292: 75-80). This implies that the fetus participates in the response to tissue injury in its mother.

[176] Human studies are complicated by lack of accurate reproductive histories, difficulty in retrieving pathologic specimens, and an inability to test mechanisms of disease. The objective of the study presented below was, therefore, to determine, using transgenic mouse models, whether fetal cells participate in the response to an induced maternal skin injury. To do this the Applicants took advantage of relatively recently described whole animal in vivo imaging systems that permit real-time and longitudinal assessment of fetal cell migration and gene expression (P. R. Contag et al, Nature Med., 1998, 4: 245-247). Zhang and coworkers (Blood, 2004, 103: 617- 626) have described the development of a transgenic mouse, VEGFR2:Luc, in which a luciferase reporter gene is under the control of the murine vascular endothelial growth factor receptor-2 (VEGFR2) promoter. VEGFR2 is developmentally regulated in the perinatal and neonatal period and transcriptionally regulated in adult life during angiogenesis. hi an adult murine model of cutaneous wound healing, luciferase expression under the control of a VEGFR2 promoter was induced in wounded skin and suppressed with the administration of dexamethasone (N. Zhang et al, Blood, 2004, 103: 617-626 ). In this study, peak induction of luciferase expression in adult animals occurred between 7 and 10 days after skin biopsy.

[177] In the present study, the Applicants bred wild-type female mice to VEGFR2:Luc males or males that expressed the luciferase reporter gene under the control of a cytomegalovirus (ubiquitous) promoter (CMV: Luc). The dominant transgenes are inherited, on average, by half of the pups. Thus, the timing and location of fetal cell migration following skin injury can be tracked in the mother's body and monitored for VEGFR2 expression to determine if fetal cells participate in 'maternal wound healing.

2 - Methods

[178] Murine Breeding Pairs. The Institutional Animal Care and Use Committee (IACUC) of the Tufts University School of Medicine Division of Laboratory Animal Medicine approved the following protocol. Eight week old wild- type FVB/NJ female mice (Taconic Laboratories, Germantown, NY) were mated to congenic adult males transgenic for the luciferase gene under the control of the CMV (FVB/N-Tg(CW.-Zwc)Xen) (n = 2) or the VEGFR2 (FVB/N-T g(VEGFR2:Luc)Xen) promoter (n = 11). The Xenogen Corporation (Alameda, CA) kindly provided the transgenic males. In addition, two female mice mated to wild type FVB/NJ males and two virgin female mice served as controls. For each animal the beginning day of gestation was retrospectively identified as the delivery date minus 20 days (average duration of a pregnancy in a mouse).

[179] Cutaneous Wounding Procedure. Twelve pregnant, three post-partum, and two virgin mice underwent a punch skin biopsy. The biopsies on the female mice mated to the VEGFR2:Luc males were performed at a mean of 9.5 days of gestation (range 4-12 days). The biopsies on the post-partum mice were performed on either day 13 or 14 following delivery. The female mice mated to the CMV: Luc males were originally supposed to receive their biopsies post-partum, but they became pregnant for a second time immediately after delivery of their first group of pups. Retrospectively, it was determined that they were pregnant at the time of biopsy. Animals were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7 mg/kg). The fur on the back of each mouse was shaved. A full- thickness wound was created by excising the skin using a standard 6mm punch device.

[180] In Vivo Imaging of Luciferase Activity. In vivo imaging was performed as previously described (P .R. Contag et at, Nature Med., 1998, 4: 245-247) using an IVIS^® Imaging System 100 Series (Xenogen). Female mice were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (7 mg/kg), and injected with 1 mg of D-Luciferin (Biotium, Hayward, CA). Five minutes after luciferin injection, mice were dorsally imaged for 5 minutes while prone and another 5 minutes ventrally while supine. The signals emitted from specific regions of the maternal body indicate fetal luciferase activity and were quantified as counts/pixel using Living Image^® software (Xenogen). For the skin biopsy model, the intensity of the signal was measured in the following way: the smallest possible circle that included wounded skin only (minimizing fur) was drawn and signal intensity was measured. Next, the signal intensity on an uninjured adjacent area with fur was measured using the same circle. And the measurement was repeated for the black background. This was done because the fur has a low-grade fluorescent signal. A ratio was developed by dividing signal intensity by number of pixels in the circle to permit comparison across mice. Mice were imaged immediately following biopsy (day 1) and at varying intervals following injury up until re-epithelialization of the wound (i.e. mice were imaged at either days 4, 8 and 11 or days 5, 9 and 12 following biopsy, due to weekend scheduling restraints for use of the in vivo imaging system).

[181] Genotyping of Pups. Since on average, only half of the pups would be expected to inherit the paternal transgene, the presence of luciferase positive fetuses resulting from each mating needed to be confirmed. When possible, phenotyping was performed by in vivo imaging of each pregnant mouse. However, because it is difficult to count the number of transgenic pups due to their close proximity to each other in the uterus, postnatal genotyping using polymerase chain reaction (PCR) amplification of the luciferase transgene sequence

(http://www.ncbi.nlm.nih.gov/BLAST) was also performed on liver obtained from each pup following sacrifice. The forward primer was

5'-TGGATTCTAAAACGGATTACCAGGG-S' and the reverse primer was 5'-CCAAAACAACAACGGCGGC-S'. Conventional PCR amplification was performed for 35 cycles. The first cycle consisted of denaturation at 95⁰C for 10 minutes, followed by 95°C for 45 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds. In pups that inherited the paternal transgene the amplification product was seen under ultraviolet light at 1083 base pairs on a 0.5% agarose gel using ethidium bromide staining.

[182] Statistical Analyses. For each mouse the luciferase signal was measured in counts/pixel on the injury day (day 1) and at intervals until the wound appeared fully healed. Signal intensity ratios within and between mice were compared. Medians and 25th and 75th percentile ranges were generated for all studied outcomes. Signal intensity ratios were logarithmically transformed prior to the statistical analyses. Changes in the skin injury signal over time of healing, as well as potential predictors such as gestational age, fur background fluorescence, transgenic type, and number of transgenic pups, were assessed using repeated measures regression analysis. All statistical analyses were performed using SAS/STAT software (SAS Institute, Inc., Cary, NC).

3 - Results

[183] Detection of Fetal Cells That Express Luciferase in the Pregnant Mouse.

Preliminary experiments were performed on six non-biopsied pregnant mice in gestational weeks 1, 2, and 3 with serial in vivo imaging to determine the earliest point in gestation in which a luciferase signal could be detected coming from the transgenic fetuses and placenta. Wild type FVB/NJ females were mated to CMV:Luc or VEGFR2:Luc transgenic males. Luciferase signal appeared for both types of transgenic fetuses on day 10-12 of gestation in maternal ventral images (Figure 5). Median signal intensity ratios of black background were consistent across all measurements (0.029 [25^th percentile: 0.017; 75^th percentile: 0.040]). Median signal intensity ratios of fur background across all measurements were not significantly different between control mice (0.310 [0.254; 0.358]) and transgenic-bred mice (0.275 [0.246; 0.322]) (P=0.70).

[184] "Experiment of Nature" - Early Fetal Response to Eye Injury, In the experiment above, with the exception of the abdomen, other areas of the pregnant mice bearing luciferase transgenic fetuses were devoid of any signal in ventral as well as dorsal images. During the first week of gestation, no luciferase signal was detectable in ventral or dorsal images at various exposure times. However, in one wild-type female mouse a strong and specific luciferase signal was detected anterior to her left eye nine days after she was placed in a cage with a VEGFR2:Luc male (Figure 6(A)). This signal was reproducible on several consecutive days. Upon closer observation it was apparent that this mouse had a scratch and clinical evidence of inflammation in the left eye only (Figure 6(B)). Upon dissection of the uterus, nine fetuses were visualized and five were confirmed by PCR to have inherited the VEGFR2:Luc transgene. Because she was sacrificed while pregnant it can only be estimated that she was no more than 9 days pregnant when the signals first appeared.

[185] Imaging Results Following Skin Biopsy. Table 4 shows the relative change in signal intensity ratios over time in all mice following skin biopsy. On day 1, immediately after skin biopsy was performed, the signal obtained at wound site was considered to be the baseline. On day 3 after biopsy all mice mated to transgenic males, with the exception of VLlO, had detectable fetal signals. Fetal cells that expressed the luciferase transgene were detected only at the site of skin wound and in the uterus (Figure 7). Compared to baseline, maximum fetal cell signal in the wound was obtained at day 4 (P=0.0002) or day 5 (P<0.0001) following biopsy. The difference in peak signal intensity (day 4 or 5) between control mice and transgenic- bred mice was statistically significant (P=O.018). Mouse VLlO underwent skin biopsy on day 4 of gestation and exhibited a delay in fetal cell migration (peak signal observed at day 8 instead of day 4-5 after biopsy). Luciferase signal persisted in most mice until re-epithelialization occurred (day 11 or 12). Neither VEGFR2:luc+ or CMV:luc+ signals were detected above baseline in the wound after the completion of re-epithelialization.

[186] The change in signal intensity ratios over time of healing followed a parabolic curve (P<0.0001) (Figure 8). Non-specific fluorescence in control animals at the wound site followed the same quadratic trend but was significantly lower than in the transgenic-bred mice. No difference in the parabolic curve was seen between the female mice bred to the VEGFR2:Luc males and those bred to CMV: Luc males.

[187] In the eight pregnant mice mated to VEGFR2:Luc transgenic males (VL4 through VLI l), there was a statistically significant interaction between gestational age and signal measurement. When the biopsy occurred early in gestation, wound fluorescence had a single sharp peak, but when the biopsy was performed later in gestation peak wound fluorescence had a longer plateau (P=0.022). Remarkably, the post-partum mice (VLl to VL3) had a similar fetal cell homing response to the wound that was observed in the pregnant mice (P=0.61), indicating that fetal cells not only persisted in the mother following delivery but they retained their capability of responding to maternal injury.

[188] Relationship Between Number of Transgenic Pups and Imaging Signals.

In the ten females (VL3, VL5 through VLI l, CLl and CL2) representing twelve pregnancies mated to transgenic males in which living pups could be studied postpartum, three females had their litters phenotyped by in vivo imaging (VLlO, CLl and CL2) and the other seven females had their pups genotyped by PCR amplification of the luciferase transgene. Since the level of microchimerism may be dependent on the number of pregnancies, those mice that were pregnant twice were excluded from this analysis (CLl and CL2). Day 4 or 5 signal was analyzed as a function of the number of transgenic fetuses present in the eight mice that had a known number of transgenic pups (VL3 and VL5 through VLl 1). No correlation was observed between the number of pups and day 4 or 5 signal intensity (P=O.93).

4 - Discussion

[189] Using unique, paternally inherited transgenes to track fetal cells in a pregnant or post-partum mouse model, the Applicants have shown in the present study that fetal cells migrate specifically to maternal wounds. This occurs in both natural (eye scratch) and induced (punch biopsy) skin injuries. Fetal cells are able to cross the placenta as early as 9 days after a female is put in a cage with a male. Significantly, this is either before mouse circulation begins or very early in the development of the circulation (S.H. Orkin and L.I. Zon, Nat. Immunol., 2002, 3: 323-328). The results presented here demonstrate a specific homing of the fetal cells to the wound site and expression of VEGFR2 by fetal cells at the site of injury. Whether the cells that home to the site of injury already express VEGFR2 or whether the wound environment turns on that promoter is currently unknown.

[190] The rise and fall of VEGFR2 expression by the fetal cells is similar to what has been demonstrated in adult transgenic cells during wound healing (N. Zhang et al, Blood, 2004, 103: 617-626), although the time course is more rapid. The quadratic relationship between luciferase signal and time following skin biopsy was observed in both females mated to transgenic males and in controls (i.e. virgin females and females mated to wild type males). Although there were significant differences in signal intensity ratios between transgenic-mated females and controls, the observation of background signal in controls must be assessed in future experiments. This background may be due to non-specific signal resulting from blood flow or another component of the healing process at the wound site. Nevertheless, fetal cells appear to be active participants in the response to inflammation and in the development of new blood vessels, which are important steps in wound healing. Remarkably, fetal cells are capable of responding to maternal injury both during and following pregnancy. Because only mice that were 2 weeks postpartum were tested, it is not possible to comment upon how long post-partum fetal cells might be capable of responding to a maternal wound. In the human, however, fetal cells persist for decades in the maternal blood and organs (D. W. Bianchi et al, Proc. Natl. Acad. Sci. USA₅ 1996, 93: 705-708; K. O'Donoghue et al, Lancet, 2004, 364: 179-182; K. Khosrotehrani et al, JAMA, 2004, 292: 75-80) , 6, 17).

[191] The fetal cellular response to maternal injury is affected by gestational age of the pregnancy. In animals that were injured early in gestation (such as mouse VLlO) the fetal cells could not be detected in the wound before 6-9 days of gestation. CL2 was biopsied on day 1 but this mouse had a prior pregnancy and fetal cells from the earlier pregnancy could contribute to wound healing. The inability to detect fetal cells in the wound prior to gestational days 6-9 may be due to the fact that the fetal microchinieric cells present in the maternal circulation before that gestational age do not have the capacity to home and/or to express VEGFR2. One could speculate that later in gestation the fetal hematopoietic system is more mature or that there is a larger volume of fetal cells in the maternal circulation capable of response to maternal injury.

[192] As reported in Example 2, the Applicants have shown that fetal cells transferred during pregnancy adopt the phenotype of different maternal tissues such as thyroid, liver, intestine or cervix and concluded that some fetal cells that they named the pregnancy associated progenitor cells transferred during pregnancy had multi- lineage capacity (K. Khosrotehrani et al, JAMA, 2004, 292: 75-80). The specific murine fetal cell type involved in the trafficking and maternal repair described here is currently unknown. In all likelihood that cell is formed quite early during gestation. One candidate cell type is the hemangioblast, since it is formed early during gestation, expresses CD34 and VEGFR2, and gives rise to both endothelial and hematopoietic stem cells (E. Pelosi et al, Blood, 2002, 100: 3203-3208). Another candidate is a bone marrow-derived endothelial or epithelial cell. Using similar skin wounding procedures, a number of other investigators have shown that neovascularization and skin repair occurs in conjunction with cells derived from bone marrow precursors (T. Asahara et al, Cir. Res., 1999, 85: 221-228; E. V. Badiavas et al, J. Cell Physiol., 2003, 196: 245-250). With a gender-mismatched bone marrow transplant model, Borue et al (Am. J. Pathol, 2004, 165: 1161-1112) demonstrated that bone marrow- derived keratinocytes engrafted in wounded skin at significantly higher levels than in uninjured skin and that this was not due to cell fusion. They hypothesized that the cells were recruited nonspecifically via the inflammatory response. In agreement with the results presented here, Fathke et al. (Stem Cells, 2004, 22: 812-822) showed that the bone marrow derived endothelial progenitor cells initially involved in wound healing do not persist after re-epithelialization is complete.

[193] In summary, the present data demonstrate that during pregnancy, female mice acquire a population of cells that are capable of crossing the placenta very early in gestation, and express VEGFR2 specifically at the site of natural and induced skin injury. The present data imply that pregnancy results in the acquisition of cells that play a role in the response to maternal tissue injury. Future work will be directed towards determination of whether the fetal cells make a positive contribution to maternal wound healing, as fetal wound healing is reportedly "scarless" (G.P. Yang et al, Wound Repair Regen., 2003, 11: 411-418).

Example 4: Fetal Cells Participate over time in the Response to Specific Types of

Murine Maternal Hepatic Injury

1 - Introduction

[194] Using immunolabeling techniques, the Applicants have previously shown that decades after delivery fetal microchimeric cells express markers of hepatocytic, epithelial, or leukocyte differentiation in maternal liver, epithelial or hematopoietic organs, respectively (K. Khosrotehrani et al. J. Am. Med. Assoc, 2004, 292: 75-80). This observation led them to hypothesize that fetal microchimeric stem cells may home to maternal injured tissue as part of the maternal repair response to tissue injury. They may therefore be "helpful" and not "harmful" as previously proposed.

[195] The Applicants have also reported a case in which a large number of male fetal cells repopulated the liver of a woman with hepatitis C (K.L. Johnson et al, Hepatology, 2002, 36: 1295-1297). This observation led them to ask whether microchimeric fetal cells could participate in the regeneration of maternal liver. Human studies are often limited by the number of subjects and the availability of healthy and diseased tissues. To better address this question, we developed a murine model of fetal cell microchimerism (K. Khosrotehrani et al, J. Reprod. Immunol., 2005, 66: 1-12; K. Khosrotehrani et al, Hum. Reprod., 2004, 19: 2460-2464). Wild- type female mice were bred to congenic males transgenic for the reporter gene green fluorescent protein. Fetal cells from pups that inherit the transgene are easily detectable in maternal wild-type tissues. Carbon tetrachloride (CCl₄) and partial hepatectomy are well-established injury models to study liver regeneration. CCl₄ induces an acute injury in which the regeneration process involves hepatocyte cell division and oval cell activation (X. Wang et al., Proc. Natl. Acad. Sci. USA, 2003, 100 Suppl., 1 : 11881-11888; N.D. Theise and D.S. Krause, Dev. Biol., 2002, 13: 411- 417). On the other hand, partial hepatectomy induces mitotic activity among remaining hepatocytes and does not recruit oval cells (L. Libbrecht and T. Roskams, Semin. Cell Dev. Biol., 2002, 13: 389-396). The present study aimed to determine whether fetal cells migrate to areas of maternal injury and if so, is the response dependent on the type of injury?

2 - Materials and Methods

[196] Mice. The Institutional Animal Care and Use Committee (IACUC) of the Tufts University School of Medicine Division of Laboratory Animal Medicine approved the present protocol. The enhanced green fluorescent protein (GFP+) transgenic mouse (Jackson Laboratories stock # 03291, Bar Harbor, ME) has a C57BL/6J genetic background with the gfp transgene under the control of a chicken beta-actin promoter and a cytomegalovirus (CMV) enhancer (M. Okabe et al., FEBS Lett., 1997, 407: 313-319). C57BL/6J (wild-type) female retired breeders (Jackson Laboratories) were purchased that were bred to male GFP+ mice and gave birth to an average of three to four litters. Eight (8) week old C57BL/6J virgin female mice were also bred to GFP+ males. Female mice that did not deliver a litter were excluded to avoid confounding results due to microchimerism as a result of spontaneous abortion or resorption. After delivery, the total and transgenic number of pups for each mouse were recorded by using UV excitation to detect green fluorescence.

[197] Liver Injury Models. Chemical Injury. Mice were injected once with 0.1 mL of 20% CCl₄ in vegetable oil intraperitoneally, as previously described (L.G. Koniaris et al, J. Immunol., 2001, 167: 399-406; R.O. Recknagel, Life Sci., 1983, 33: 401-408). Retired breeders (n=16) were studied in the first group of experiments (group 1). Animals were observed for four (n=5) or eight weeks (n=l l) following injection and then sacrificed. Liver and in some cases spleen tissues were studied with histology, immunofluorescence, and real-time quantitative polymerase chain reaction (PCR) amplification. Because PCR results in group 1 suggested a trend towards increasing fetomaternal microchimerism at the later observation point, a second group of experiments (group 2) was performed with 8 week old C57BL/6J virgin females mated to GFP+ males (n=18). In group 2 cases (10/18) were injected once with CCl₄ and controls (8/18) were injected once with vegetable oil only. Each group was injected 5-6 weeks following delivery and observed for 4 (n=9) or 8 (n=9) weeks after injection.

[198] Surgical Injury. Partial hepatectomy was performed 3 to 10 weeks after delivery on virgin female C57BL/6J mice (n=7) that had been bred once to GFP+ male mice. Briefly, animals were anesthetized using isoflurane inhalation and maintained under deep anesthesia throughout the surgical procedure with anesthetic doses titrated as needed. A midline incision was made from xyphoid to mid abdomen and the liver was exposed. The anterior and medial segments of the liver were retracted into the wound and encircled with a 2-0 vicryl ligature and excised sharply. The incision was closed with a running 3-0 vicryl suture.

[199] Tissue Collection. Mice were sacrificed using carbon dioxide inhalation. Liver, and in some cases spleen, was collected. Tissues were either fixed in 4% formaldehyde and 30% sucrose, in formalin, or immediately frozen in liquid nitrogen.

[200] Histology. Paraffin-embedded sections of liver specimens were obtained and stained with hematoxylin-eosin (H&E) or trichrome blue to detect fibrosis. Sections were then assessed histologically, focusing on general structure, number of mitoses, amount of inflammation and presence of steatosis or necrosis.

[201] DNA Extraction and Real-time PCR Amplification. Genomic DNA extraction was performed on all samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Real-time PCR was performed as previously described using an ABI 7700 Sequence Detection System with the SDS vl.9 software (K. Khosrotehrani et al, Hum. Reprod., 2004, 19: 2460- 2464). All experiments were performed in triplicate, and a tissue sample was considered to be positive for fetal cell microchimerism if the amount of gfp transgene detected was equivalent to at least one genome in a background of 100,000-200,000 maternal genome equivalents (GE). Results were then normalized to fetal GE per 1 million maternal GE. [202] Detection of GFP Fluorescence in Maternal Tissues. Anti-GFP rabbit polyclonal antibodies (Chemicon International, Temecula, CA) were used on frozen sections to detect fetal GFP+ cells. Goat anti-rabbit IgG labeled with fluorescein isothiocyanate (FITC)(Jackson Immunoresearch, West Grove, PA, USA) was used as a secondary antibody. Briefly, after rehydration, sections were blocked using 20% normal goat serum. Primary antibody was used at a dilution of 1:100 and incubated overnight at 4⁰C. After washes, secondary antibody was used at a dilution of 1 :50 and incubated for 40 minutes. Slides were then washed, counterstained with 0.3 μg/mL 4,6-diamidino-2-phenylindole (DAPI), and observed with a fluorescence microscope (Zeiss Axioskop).

[203] Statistical Analyses. Each mouse was analyzed for the presence of microchimerism, defined by the presence of transgenic cells by microscopy or the number of microchimeric GE by quantitative PCR in the liver or spleen. Fisher's exact test was used to assess the proportion of samples with transgenic cells among cases and controls. The number of microchimeric GE in cases and controls as well as between the two time points (4 and 8 weeks) in the tissues was analyzed using the non-parametric Wilcoxon rank sum test. AU statistical analyses were performed using SAS/STAT software (SAS Institute, Inc., Gary, NC). A p value of less than 0.05 was considered significant.

3 - Results

[204] Histology. Chemical injury. In group 1 mice, CCl₄ injection induced massive (60-90%) liver necrosis at 4 weeks after the injection (Figure 9(A), Table 5). At 8 weeks after the injection, all mice showed histologic evidence of recovery from their liver injury. The livers were slightly fibrotic with excess microvesicular steatosis (Figure 9(B), Figure 9(C)). The injured livers also had more inflammatory cells (Figure 9(D)), sometimes organized in aggregates. In addition, 10 of 16 exposed mice had gross evidence of splenic enlargement; spleens were not available for histology.

[205] Surgical Injury. Post-mortem analysis of livers from the 4 surviving animals in group 1 that were electively sacrificed showed healthy regenerating liver tissue, with mitotic activity present (Figure 10). [206] Real-Time PCR Amplification of Fetal Transgenes. Chemical Injury. Results of real-time PCR amplification for both groups are shown in Tables 5 and 6. For group 2, a comparison of the median number of fetal GE in maternal liver at 4 weeks for CCl₄ exposed (n=5) vs. vegetable oil control (n=4) was not significant in either liver or spleen (p=0.44 and p=1.0, respectively). At 8 weeks, the comparison of exposed (n=5) to control (n=4) was significant for spleen (p=0.016) but not liver (p=0.29), likely due to the small sample size. The effect of time in all CCl₄ exposed mice (groups 1 and 2) was also studied for both the median number of fetal GE and the relative frequency of organs with detectable fetal cell microchimerism. Both were highly significant. In the comparison of 4 weeks (n=10) vs. 8 weeks (n=16), both liver and spleen had more fetal cells present at 8 weeks (p=0.006 and p=0.0006, respectively, by Wilcoxon rank sum test). The frequencies in liver were 2/10 (4 weeks) vs. 11/16 (8 weeks) (p=0.04, by Fisher's exact test). Similar results were observed in spleen: 1/10 (4 weeks) vs. 12/16 (8 weeks) (p=0.004, by Fisher's exact test).

[207] Surgical Injury. For PCR experiments, 3 -lobe hepatectomies were performed on 7 mice, 4 of which were electively sacrificed 7 days following surgery, and 3 of which died 1-4 days following surgery. AU mice that were electively sacrificed had evidence of mitotic activity present, indicating active liver regeneration (Table 7). No GFP+ cells were detected in any of the post-hepatectomy livers nor in their corresponding baseline liver lobes removed, which served as the internal control for each mouse. Six of seven spleens had no GFP+ cells detected. In one spleen, 1 of the triplicate experiments had 4.32 GFP GE per 1 x 10⁶ maternal GE. The other 2 reactions had 0.

[208] Effect of Number of Transgenic Pups. PCR results from the livers and spleens of CCl₄ exposed animals at 8 weeks (n=5) were used to determine if the number of transgenic pups delivered affected the results. Although liver (r²=0.22, p=0.72) and spleen (r²=0.45, p=0.45) fetal cell numbers moderately correlated with the number of transgenic pups, the results did not reach statistical significance.

[209] Immunofluorescence. Immunofluorescence analysis of liver sections allowed us to detect GFP+ cells in 2 of 11 CCl₄ exposed livers when studied at 8 weeks (Table 5). No fetal GFP+ cells were detected in regenerated livers following partial hepatectomy.

4 - Discussion

[210] The persistence of fetal cells in maternal tissues after human pregnancy is well known. The present Applicants have recently proposed that fetal cells, once in the maternal circulation, may participate in the maternal response to tissue injury for years post-partum (K. Khosrotehrani and D. W. Bianchi, Curr. Opin. Obstet. Gynecol., 2003, 15: 195-199). They have also shown that fetal cell microchimerism occurs during all pregnancies in mice; however, by three weeks after delivery no fetal cells can be detected in maternal tissues (K. Khosrotehrani et al, J. Reprod. Immunol., 2005, 66: 1-12). In the present Example, the Applicants demonstrate that fetal GFP+ cells are found in the chemically injured liver and spleen in the post-partum female mouse. Exposure to CCl₄ results in an increase in the frequency and number of fetal cells. This may be due to a migration of fetal cells to the injured organs or to amplification of the existing microchimeric fetal cell population. The results presented herein support both hypotheses. Although the experiments were designed with liver toxicity in mind, CCU is also known to affect the spleen (LN. Alexeyeva et al, Cent. Eur. J. Public Health, 1994, 2: 16-18). The mice of the present study had gross evidence of splenic enlargement, so it is not surprising that fetal cells were found in this injured tissue.

[211] The results presented herein also demonstrate that there is a significant effect of time on fetal cell migration to liver and spleen. An increase in the median number of fetal cells present in the maternal livers occurred some time between four to eight weeks after the chemical injury. According to the histological findings, this corresponds to the period when most of the liver regeneration took place. Another injury model, partial hepatectomy, was used in which the regeneration takes place in the first few days, but no increase in the number of microchimeric cells was observed in the regenerating liver by PCR or by immunostaining. It is possible that the mice were not allowed to survive long enough following surgical injury to detect fetal cell microchimerism, although the histologic results suggest that active repair had already taken place. Liver regeneration after CCl₄ injection involves hematopoietic stem cells and hepatic oval cells (X Wang et al, Proc. Natl. Acad. Sci. USA, 2003, 1: 11881- 11888) whereas the regeneration after partial hepatectomy is based on hepatocyte cell division (L. Libbrecht et al, Semin. Cell Dev. Biol., 2002, 13: 389-396). The present results suggest that specific types of liver injury may elicit different fetal cell responses in the mother, which may be due to the different cell types involved in recovery from chemical versus surgical injury.

[212] The data presented herein agree with previous studies using rodent models that demonstrate the presence of fetal cells in maternal tissue following injury. Christner et al (Arthritis Rheum., 2000, 43: 2598-2605) described a higher number of fetal cells in the circulation of mice injected with vinyl chloride to induce skin fibrosis. Imaizumi et al. (Endocrinology, 2002, 143: 247-253) induced thyroiditis in the mouse and noted an increase in the number of microchimeric cells in the thyroid. Wang et al. (Biophys. Res. Commun., 2004, 325: 961-967) exposed post partum female rats to ethanol and gentamicin; fetal GFP+ cells were detected in liver and kidney. The previous studies, however, did not have controls matched for reproductive history. Furthermore, they did not study animals at different time points in the recovery period.

[213] In humans, the present Applicants have shown that fetal cells can be found in the livers of women with hepatitis C and autoimmune hepatitis (K.L. Johnson et al., Hepatology, 2002, 36: 1295-1297; K. Khosrotehrani et al., JAMA, 2004, 292: 75-80). Some of the microchimeric cells have the morphology and immunohistochemical characteristics of hepatocytes. Most fetal cells express hematopoietic markers such as CD45. In the present study, the fetal GFP+ cells that were detected in the livers after CCl₄ injury were mononuclear. Some of them were isolated, and some were grouped in aggregates. In liver exposed to CCl₄, fewer fetal cells were detected by immunohistochemistry than by real-time PCR. It has been previously suggested that detection of gene sequences by in situ hybridization and PCR is more sensitive than detection of protein markers by immunohistochemistry (E. Mezey et al., Science, 2003, 299: 1184). The present results support this suggestion; thus, the number of GFP+ cells with the morphology of hepatocytes may possibly be higher than what the present immunohistochemical studies can demonstrate.

[214] In conclusion, the Applicants have demonstrated here that in a mouse model fetal microchimeric cells are transferred during pregnancy and can be detected in the maternal liver and spleen as a result of chemical but not surgical injury. The statistically significant differences in fetal cell microchimerism observed at different time points suggest that these cells play a dynamic role in the repair process. Further studies are needed to address whether the fetal cells involved in this process have a well-defined phenotype and whether their presence ultimately improves maternal liver and splenic function.

Other Embodiments

[215] Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

Claims

ClaimsWhat is claimed is:

1. A method for obtaining one or more isolated fetal stem cells, the method comprising steps of: providing a tissue sample harvested from a female mammal, wherein the female mammal has been pregnant with a fetus at least once; and isolating one or more fetal stem cells from the tissue sample.

2. The method of claim 1, wherein the isolated fetal stem cells comprise fetal cells that are pluripotent.

3. The method of clam 1, wherein the isolated fetal stem cells comprise fetal cells that are multipotent.

4. The method of claim I₃ wherein the isolated fetal stem cells are characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype.

5. The method of claim 1, wherein the tissue sample is harvested from a tissue or organ selected from the group consisting of peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, heart, kidney, lung, and liver.

6. The method of claim 1, wherein the female mammal is a woman.

7. The method of claim I₅ wherein isolating one or more fetal stem cells from the tissue sample harvested from the female mammal comprises submitting the tissue sample to a mechanical or enzymatic treatment, or both.

8. The method of claim 7, wherein isolating one or more fetal stem cells further comprises centrifuging the treated tissue sample.

9. The method of claim 1 further comprising purifying said fetal stem cells after isolation.

10. The method of claim 1 further comprising expanding said fetal stem cells in or on a medium after isolation.

11. The method of claim 10, wherein the fetal stem cells are expanded in an undifferentiated state to obtain undifferentiated fetal stem cells.

12. The method of claim 1 further comprising expanding said fetal stem cells in or on a medium after isolation; and differentiating the fetal tern cells into cells of a desired cell phenotype to obtain differentiated cells.

13. The method of claim 12, wherein the desired cell phenotype is selected from the group consisting of adipogenic cells, chondrogenic cells, cardiogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, perocardiogenic cells, stromal cells, eptihelial cells, neurogenic cells, neurogliagenic cells, pleurigenic cells, hepatogenic cells, pancreogenic cells, and splanchogenic cells.

14. The method of claim 1 further comprising expanding said fetal stem cells in or on a medium after isolation; and differentiated the fetal stem cells into cells of a desired cell type to obtain specialized cells.

15. The method of claim 14, wherein the cell type is selected from the group consisting of red blood cell, B lymphocyte, T lymphocyte, natural killer cell, neutrophil, basophil, eosinophil, monocyte, macrophage, platetet, osteocyte, chondrocyte, adipocyte, neuron, astrocyte, oligodendrocyte, absorptive cell, goblet cell, Paneth cell, enteroendocrine cell, hepatocyte, and keratinocyte.

16. The method of claim 12 or 14, wherein differentiating the fetal stem cells comprises exposing said fetal stem cells to at least one differentiation-inducing agent.

17. The method of claim 1 further comprising genetically modifying the fetal stem cells after isolation to obtain genetically modified fetal stem cells.

18. The method of claim 1 further comprising cryopreserving the fetal stem cells after isolation under such conditions that at least some of the fetal stem cells are viable upon recovery.

19. The method of claim 11 further comprising cryopreserving the undifferentiated fetal stem cells under such conditions that at least some of said undifferentiated fetal stem cells are viable upon recovery.

20. The method of claim 12 further comprising cryopreserving the differentiated cells under such conditions that at least some of said differentiated cells are viable upon recovery.

21. The method of claim 14 further comprising cryopreserving the specialized cells under such conditions that at least some of said specialized cells are viable upon recovery.

22. The method of claim 17 further comprising cryopreserving the genetically modified fetal stem cells under such conditions that at least some of said genetically modified fetal stem cells are viable upon recovery.

23. The method of any of claims 18-22, wherein cryopreserving comprises using liquid nitrogen.

24. The method of any of claim 18-22, wherein cryopreserving comprises using dimethyl sulfoxide.

25. An isolated fetal stem cell derived from a tissue sample harvested from a female mammal that has been pregnant with a fetus at least once, wherein said fetal stem cell is characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as it is passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype.

26. The isolated fetal stem cell of claim 25, wherein the tissue sample is harvested from a tissue or organ selected from the group consisting of peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, heart, kidney, lung, and liver.

27. The isolated fetal stem cell of claim 25, wherein the female mammal is a woman.

28. A cell population comprising one or more fetal stem cell isolated from a tissue sample harvested from a female mammal that has been pregnant at least once, wherein said fetal stem cells are characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype.

29. The cell population of claim 28, wherein the fetal stem cells are isolated from a tissue sample harvested from a tissue or organ selected from the group consisting of peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, heart, kidney, lung, and liver.

30. The cell population of claim 28, wherein the female mammal is a woman.

31. The cell population of claim 28 characterized in that it is a substantially homogeneous population of the fetal stem cells.

32. The cell population of claim 28 characterized in that it is a heterogeneous population comprising said fetal stem cells and at least one other cell type.

33. The cell population of claim 32, wherein the other cell type is selected from the group consisting of a pluripotent stem cell, a multipotent stem cell, an adult stem cell, a progenitor cell, a differentiated cell, and a specialized cell.

34. The cell population of claim 28, wherein the fetal stem cells are expanded in or on a medium in an undifferentiated state.

35. The cell population of claim 28, wherein the fetal stem cells are expanded in or on a medium comprising one or more factors which stimulate stem cell differentiation along a desired cell phenotype.

36. The cell population of claim 35, wherein the desired cell phenotype is selected from the group consisting of adipogenic cells, chondrogenic cells, cardiogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, perocardiogenic cells, stromal cells, eptihelial cells, neurogenic cells, neurogliagenic cells, pleurigenic cells, hepatogenic cells, pancreogenic cells, and splanchogenic cells.

37. The cell population of claim 28, wherein the fetal stem cells are expanded in or on a medium comprising one or more factors which stimulate stem cell differentiation along a desired cell type.

38. The cell population of claim 37, wherein the desired cell type is selected from the group consisting of red blood cell, B lymphocyte, T lymphocyt, natural killer cell, neutrophil, basophil, eosinophil, monocyte, macrophage, platetet, osteocyte, chondrocyte, adipocyte, neuron, astrocyte, oligodendrocyte, absorptive cell, goblet cell, Paneth cell, enteroendocrine cell, hepatocyte, and keratinocyte.

39. The cell population of claim 28, wherein the fetal stem cells are cryopreserved under such conditions that at least some of said fetal stem cells are viable upon recovery.

40. A cell lysate prepared from the cell population of claim 28.

41. An extracellular matrix produced from the cell population of claim 28.

42. A conditioned medium prepared from the cell population of claim 28.

43. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of the cell population of claim 28.

44. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of the cell lysate of claim 40.

45. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of the extracellular matrix of claim 41.

46. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of the conditioned medium of claim 42.

47. The pharmaceutical composition of any of claims 43-46, wherein the fetal stem cells are isolated from a tissue sample harvested from a tissue or organ selected from the group consisting of peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, heart, kidney, lung, and liver.

48. The pharmaceutical composition any of claims 43-46, wherein the female mammal is a woman.

49. The pharmaceutical composition of any of claim 48 further comprising one or more bioactive agents.

50. The pharmaceutical composition of claim 49, wherein the bioactive agent is selected from the group consisting of a differentiation-inducing factor, an anti- apoptotic agent, an anti-inflammatory agent, an immunosuppressive immunomodulatory agent, an anti-proliferative agent, and an antibody.

51. A method of treating a disease or condition in a subject, the method comprising a step of administering to the subject a therapeutic composition in an amount sufficient to treat the disease or condition, wherein the therapeutic composition comprises one or more fetal stem cells, a cell lysate prepared from fetal stem cells, an extracellular matrix produced by fetal stem cells, or a conditioned medium prepared from fetal stem cells, wherein said fetal stem cells are isolated from a tissue sample harvested from a female mammal that has been pregnant with a fetus at least once, and wherein said fetal stem cells are characterized by (a) the ability to self-renew and expand in culture; (b) a normal karyotype and the ability to maintain that karyotype as they are passaged; and (c) the ability to differentiate into one or more cells of at least one phenotype.

52. The method of claim 51, wherein the female mammal is a woman.

53. The method of claim 52, wherein the subject is related to the woman.

54. The method of claim 53, wherein the subject is a biological child or grandchild of the woman.

55. The method of claim 52, wherein the subject is not related to the woman.

56. The method of claim 51, wherein the fetal stem cells are isolated from a tissue sample harvested from a tissue or organ selected from the group consisting of peripheral blood, blood vessels, bone marrow, skeletal muscle, brain, skin, heart, kidney, lung, and liver.

57. The method of claim 51, wherein the therapeutic composition comprises one or more fetal stem cells and the fetal stem cells are induced to differentiate into cells of a desired cell phenotype or cell type in vitro prior to administration.

58. The method of claim 51, wherein the therapeutic composition comprises one or more fetal stem cells and the fetal stem cells are induced to differentiate into cells of a desired cell phenotype or cell type in vivo following administration.

59. The method of claim 51, wherein the therapeutic composition comprises one or more fetal stem cells and at least some of said fetal stem cells are genetically modified in vitro prior to administration, wherein the genetic modification results in the fetal stem cells expressing a gene product that promotes treatment of the disease or condition.

60. The method of claim 51, wherein the therapeutic composition stimulates adult stem cells present in the subject to divide or differentiate, or both.

61. The method of claim 51, wherein administering is by transplanting, implanting, injecting, fusing, delivering by catheter, or providing as a matrix- cell complex.

62. The method of claim 51 further comprising co-administering to the subject at least one other agent selected from the group consisting of a differentiation- inducing factor, an anti-apoptotic agent, an anti-inflammatory agent, an antiproliferative agent, an immunosuppressive/immunomodulatory agent, and an antibody.

63. The method of claim 62, wherein the other agent is administered simultaneously with the therapeutic composition.

64. The method of claim 62, wherein the other agent is administered sequentially with the therapeutic composition.

65. The method of claim 51, wherein the therapeutic composition further comprises cells of at least one other cell type selected from the group consisting of a pluripotent stem cell, a multipotent stem cell, an adult stem cell, a progenitor cell, a differentiated cell, and a specialized cell.

66. The method of claim 51, wherein said treatment comprises at least one of: tissue repair, tissue regeneration, tissue augmentation, tissue sealing, tissue function restoration, and therapeutic action.