HK1127369A - Production of oligodendrocytes from placenta-derived stem cells - Google Patents

Abstract

The present invention provides methods and compositions for the production of glial cells and oligodendrocytes from placenta stem cells. The invention further provides for the use of these glia and oligodendrocytes in the treatment of, and intervention in, for example, trauma, ischemia and degenerative disorders of the central nervous system (CNS), particularly in the treatment of demyelinating diseases such as multiple sclerosis.

Description

Preparation of oligodendrocytes using placenta-derived stem cells

1. Field of the invention

The present invention provides methods and compositions for preparing glial cells and oligodendrocytes from placenta-derived stem cells (hereinafter referred to as PDSCs). The invention also provides the use of these glia and oligodendrocytes in the treatment and intervention of, for example, trauma, ischemia, Central Nervous System (CNS) degenerative diseases.

2. Background of the invention

Embryonic stem cells capable of producing Central Nervous System (CNS) glia can promote functional recovery after spinal cord trauma, and have the potential to repair demyelinating and dysmyelinating diseases, such as multiple sclerosis. However, the use of embryonic stem cells in clinical therapy poses ethical problems that are not easily solved.

Adult stem cells have also been proposed for therapeutic use. For example, in animal models of cell-replenishment therapy. The therapeutic potential of engrafted stem cells can only be used for clinical purposes if an ethically acceptable, autologous source of stem cells is available and the autologous renewal and fate decisions that allow the programmed maturation of stem cells into specific cell types can be controlled.

The significant morbidity and mortality of neurodegenerative diseases is increasing. Myelin destruction underlies the most common neurological disease in young adults, multiple sclerosis, and it affects repair after traumatic spinal cord injury, preventing not only the regeneration of damaged neuronal axons, but also electrical conduction in the nearest undamaged axons. Therefore, oligodendrocyte replacement is an important clinical goal. While oligodendrocytes can be obtained from neural stem cells, such stem cells are difficult to obtain.

3. Summary of the invention

The present invention provides methods and compositions for preparing oligodendrocytes from placenta-derived stem cells, and methods of using such oligodendrocytes to treat diseases, disorders, or conditions, such as those systemic disorders including trauma, ischemia, or the central nervous system. For example, in one aspect, the invention relates to the use of oligodendrocytes prepared from placenta-derived stem cells for treating a disease, disorder, or condition associated with abnormal myelination. In one embodiment, the present invention provides a method of making oligodendrocytes comprising culturing placenta-derived stem cells under conditions and for a time sufficient for the stem cells to exhibit oligodendrocyte characteristics. In a specific embodiment, the characteristic is the production of a myelin oligodendrocyte-specific protein, or the expression of a gene encoding a myelin oligodendrocyte-specific protein. In another specific embodiment, said culturing comprises contacting said stem cells with Isobutylmethylxanthine (IBMX). In another embodiment, the present invention provides oligodendrocytes prepared by differentiation of placenta-derived stem cells. The invention also provides a method of treating an individual having a disease, disorder or condition associated with abnormal myelination, the method comprising introducing such oligodendrocytes into the individual. In a more specific embodiment, the disease, disorder or condition is multiple sclerosis.

4. Brief description of the drawings

FIG. 1: maturation of oligodendrocyte precursor cells (McMorris & McKinnon, BrainPathology 6: 313-329 (1996)). Transient presentation of antigen indicates progression of migratory "early" (O2A) precursors to non-migratory "late" (O4, pre-OL blasts) and post-mitotic OL. Maturation can be reversibly inhibited (ζ) or reversed (7) by the indicated factors. The monoclonal antibodies and target antigens are listed in table 1.

FIG. 2: human placental stem cells. Left panel: clones of placental stem cells formed in primary culture. Right panel: placental stem cells treated with isobutylmethylxanthine (IBMX, a non-specific phosphodiesterase inhibitor, also having adenosine agonist activity); immunostaining revealed the presence of neural lineage markers including neural stem cell markers (vimentin, GFAP, nestin), and markers of both neuronal (neurofilament protein, neuron-specific enolase) and glial (myelin oligodendrocyte-specific protein (MOSP)) lineage processes.

5. Detailed description of the invention

5.1 preparation of oligodendrocytes

The present invention provides methods and compositions for preparing oligodendrocytes from placenta-derived cells, particularly placental stem cells, also known as placenta-derived stem cells (PDSCs). Stem cells can be obtained from the placenta of a mammal by perfusion (see, e.g., Hariri, U.S. application publication nos. 2002/0123141 and 2003/0032179, which are incorporated herein in their entirety). Stem cells can also be obtained from the placenta by disrupting (e.g., macerating) the placenta or a portion thereof (see, e.g., section 6.2). Cells exhibiting oligodendrocyte characteristics may be obtained from placental-derived stem cells. The cells are useful in the treatment of diseases, disorders or conditions associated with, for example, demyelination or dysmyelination, such as multiple sclerosis.

In one embodiment, differentiable cells, such as stem cells, may be obtained from placenta according to the following method. Primary cultures of mononuclear cells (MNCs) are isolated from placenta, e.g., human placental perfusate. The placenta is obtained after birth of a full term infant under informed consent of the donor. Briefly, the umbilical vessels are inserted into a cannula and then connected to a circuit in which flow is controlled and the placenta is perfused under conditions such as: at a rate of 1 mL/min (at room temperature, up to 24 hours), Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL) containing high concentrations of glucose, 1% heparin and penicillin/streptomycin was used. Placental perfusate (750 ml) was then pooled, centrifuged, and the cell pellet resuspended in PBS containing 1% Fetal Bovine Serum (FBS) and then resuspended with Lymphoprep^TM(Gibco/BRL) by differential gradient density centrifugation. The buffy coat interface containing monocytes containing attached PDSC was recovered, resuspended in DMEM/10% FBS, plated onto fibronectin-coated (Sigma) Falcon plates, and incubated at 37 ℃ with 5% humidified CO₂Culturing in medium. After 24 hours of culture, nonadherent cells were discarded, while adherent cells were maintained and expanded in fresh medium. Single cell clones developed at day 10-18 and were expanded into PDSC lines.

Human Placental Derived Stem Cells (PDSCs) showed a morphology similar to fibroblasts in culture (fig. 2a) and were HLA family I positive. These cells did not express the hematopoietic markers CD34 or CD45 as seen using FACS analysis. However, they express multipotent surface markers: CD10(CALLA), CD29 (integrin beta)₁) CD54(ICAM-1), CD90(Thy-1) and SH2 and SH 3. Under standard growth conditions, the number doubling time of the PDSC is 18 to 36 hours, anAnd the cells maintained this phenotype in vitro in more than 40 colony doublings.

A number of studies describe the neural differentiation of stem cells in vitro and in vivo, including embryonic, hematopoietic and bone marrow stromal cells (Glaser et al, FASEB J.200519 (1): 112-4 (2005); Rogist et al, Cellular Neuroscience 14: 287-300 (1999); Rao and Mayer Proschel, Dev.biol.188: 48-63 (1997); Anderson, Neuron 30: 19-35 (2001); Rao, Stemcells and Development 13: 452-455 (2004); Hermanson et al, Nature 419: 934-939 (2002); John et al, Genes Dev.10: 3129-3140 (1996)).

Agents that elevate intracellular cAMP can be used to promote neural differentiation in vitro. To determine whether human placenta-derived cells are capable of generating the neural line, they were tested in vitro under similar conditions. A monolayer of PDSC (0.25% trypsin, 1mM EDTA) was collected and then re-inoculated (5X 10)³Pieces/ml) to a medium containing 0.5mM IBMX (Sigma company), and after 24 to 72 hours, the morphological change was monitored by phase contrast microscopy, fluorescence immunoassay, and flow cytometry. After 3 days of culture, approximately 50% of the cells had a nerve cell-like morphology with long processes and a pronounced spherical cell body, while the control culture remained undifferentiated. IBMX treated cultures also showed immunoreactivity for markers of a number of neuroepithelial cell lines, including neural precursor cell markers (nestin, vimentin, GFAP), neuronal markers (enolase, neurofilament protein), and cells displaying glial Markers (MOSP) (fig. 2). To determine the status of neural antigen expression, flow cytometry was performed on both treated and untreated PDSCs. After one day treatment with IBMX, there was a significant change in antigen expression for all markers tested, and no change in untreated cells. Thus, the changes observed under these induction conditions reflect the rapid acquisition of antigen markers, consistent with neural differentiation, rather than with selective enrichment or survival.

Placental-derived stem cells can differentiate into oligodendrocytes by culturing in a medium comprising IBMX, a neural stem cell maturation factor (e.g., EGF, FGF), and/or an oligodendrocyte precursor mitogen (e.g., FGF, PDGF). Oligodendrocytes can be prepared from placenta-derived stem cells as described above and maintained or cultured as described in section 6.1. Differentiation of oligodendrocytes can be assessed using immunohistochemistry and PCR as described in section 6.3 and flow cytometry as described in section 6.5. Oligodendrocyte proliferation, migration, and survival can be assessed as described in section 6.4.

5.2 placental stem cells and placental stem cell populations

The immunosuppressive methods of the invention employ placental stem cells, i.e., stem cells obtained from the placenta or a portion thereof, which are (1) attached to a tissue culture medium matrix; (2) (ii) has the ability to differentiate into non-placental cell types; (3) in sufficient quantity, have a detectable ability to suppress immunological effects, e.g. inhibit CD4 in a mixed lymphocyte reaction assay⁺And/or CD8⁺Proliferation of stem cells. Placental stem cells are not derived from blood, such as placental blood or umbilical cord blood. Placental stem cells used in the methods and compositions of the invention have the ability to suppress the immune system of an individual and are screened for this ability.

The placental stem cells can be derived from a fetus or a mother (i.e., can possess a maternal or fetal genotype). The population of placental stem cells or the population of cells comprising placental stem cells may comprise placental stem cells derived from a fetus or a mother alone, or may comprise a mixed population of placental stem cells derived from both a fetus and a mother. Placental stem cells and cell populations comprising placental stem cells can be identified and screened by morphology, labeling, and culture characteristics discussed below.

5.2.1 physical and morphological characteristics

The placental stem cells used in the present invention, when cultured in the original culture medium or cell culture medium, adhere to a tissue culture medium substrate, such as the surface of a tissue culture vessel (e.g., plastic used for tissue culture). In culture, placental stem cells generally exhibit a fibrous, star-like appearance with a number of cytoplasmic processes extending from the central cell body. However, the placental stem cells are morphologically different from fibroblasts cultured under the same conditions, because placental stem cells exhibit such processes in greater numbers than fibroblasts. Morphologically, placental stem cells are also different from hematopoietic stem cells, which generally take on a more rounded or pebbled morphology during culture.

5.2.2 cell surface, molecular and genetic markers

In the methods and compositions of the present invention, suitable placental stem cells and populations of placental stem cells express a variety of markers that can be used to identify and/or isolate the stem cells or populations of cells comprising the stem cells. The placental stem cells and stem cell populations (i.e., two or more placental stem cells) of the present invention include stem cells and cell populations comprising stem cells obtained directly from the placenta or any portion thereof (e.g., the amniotic membrane, chorion, and chorion of the placenta, etc.). The placental stem cell population also includes populations of cultured placental stem cells (i.e., two or more), or populations in a container (e.g., a bag). However, placental stem cells are not trophoblast cells.

Placental stem cells typically express markers CD73, CD105, CD200, HLA-G, and/or OCT-4 markers, and do not express CD34, CD38, or CD 45. Placental stem cells also express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify placental stem cells and distinguish placental stem cells from other stem cell types. Because placental stem cells can express CD73 and CD105, they can have similar characteristics of mesenchymal stem cells. However, because placental stem cells can express CD200 and HLA-G, an embryo-specific marker, they can be distinguished from mesenchymal stem cells that express neither CD200 nor HLA-G (e.g., bone marrow-derived mesenchymal stem cells). Likewise, placental stem cells that do not express CD34, CD38, and/or CD45 can be identified as non-hematopoietic stem cells.

In one embodiment, the invention provides an isolated population of cells, the cell populationThe community contains a large number of CDs 200⁺And HLA-G⁺The immunosuppressive placental stem cells of (a), wherein said plurality of cells detectably inhibits T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment of the isolated population, the stem cells are also CD73⁺And CD105⁺In (1). In another specific embodiment, the stem cell is also CD34^-、CD38^-Or CD45^-In (1). In another specific embodiment, the stem cell is also CD34^-、CD38^-、CD45^-、CD73⁺And CD105⁺In (1). In another embodiment, the isolated population produces one or more embryoid bodies when cultured under conditions that allow the formation of embryoid bodies.

In another embodiment, the invention provides an isolated population of cells comprising a plurality of CD73⁺、CD105⁺And CD200⁺The immunosuppressive placental stem cells of (a), wherein said plurality of cells detectably inhibits T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment of said population, said stem cells are HLA-G⁺In (1). In another specific embodiment, the stem cell is CD34^-、CD38^-Or CD45^-In (1). In another specific embodiment, the stem cell is CD34^-、CD38^-And CD45^-In (1). In a more specific embodiment, the stem cell is CD34^-、CD38^-、CD45^-And HLA-G⁺In (1). In another specific embodiment, the population of cells produces one or more embryoid bodies when cultured under conditions that allow the formation of embryoid bodies.

The invention provides an isolated population of cells comprising a plurality of CD200 s⁺And OCT-4⁺The immunosuppressive placental stem cells of (a), wherein said plurality of cells detectably inhibits T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment, the stem cell is CD73⁺And CD105⁺In (1). At another placeIn a specific embodiment, the stem cell is HLA-G⁺In (1). In another specific embodiment, the stem cell is CD34^-、CD38^-And CD45^-In (1). In a more specific embodiment, the stem cell is CD34^-、CD38^-、CD45^-、CD73⁺、CD105⁺And HLA-G⁺In (1). In another embodiment, the population produces one or more embryoid bodies when cultured under conditions that allow the formation of embryoid bodies.

The present invention provides an isolated population of cells comprising a plurality of CD73⁺、CD105⁺And HLA-G⁺The immunosuppressive placental stem cells of (a), wherein said plurality of cells detectably inhibits T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In one embodiment of the above plurality of cells, the stem cell is also CD34^-、CD38^-Or CD45^-In (1). In another specific embodiment, the stem cell is also CD34^-、CD38^-And CD45^-In (1). In another specific embodiment, the stem cell is also OCT-4⁺In (1). In another specific embodiment, the stem cell is also CD200⁺In (1). In a more specific embodiment, the stem cell is also CD34^-、CD38^-、CD45^-、OCT-4⁺And CD200⁺In (1).

The invention also provides an isolated population of cells comprising a plurality of CD73⁺And CD105⁺The immunosuppressive placental stem cell of (a), wherein said plurality of cells form one or more embryoid bodies under conditions that allow formation of embryoid bodies, and wherein said plurality of cells detectably inhibit T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment, the stem cell is also CD34^-、CD38^-Or CD45^-In (1). In another specific embodiment, the stem cell is also CD34^-、CD38^-And CD45^-In (1). In another embodiment, the dry powderThe cell is also OCT-4⁺In (1). In a more specific embodiment, the stem cell is also OCT-4⁺、CD34^-、CD38^-And CD45^-In (1).

The invention also provides an isolated population of cells comprising a plurality of OCT-4 cells⁺The immunosuppressive placental stem cells of (a), wherein said population forms one or more embryoid bodies when cultured under conditions that allow formation of embryoid bodies, and wherein said plurality of cells detectably inhibit T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In various embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said isolated placental cells are OCT-4⁺The stem cell of (1). In a specific embodiment of the above population, the stem cell is CD73⁺And CD105⁺In (1). In another specific embodiment, the stem cell is CD34^-、CD38^-Or CD45^-In (1). In another specific embodiment, the stem cell is CD200⁺In (1). In a more specific embodiment, the stem cell is CD73⁺、CD105⁺、CD200⁺、CD34^-、CD38^-And CD45^-In (1). In another specific embodiment, the population has been expanded, e.g., passaged at least once, at least three times, at least five times, at least ten times, at least fifteen times, or at least twenty times.

In another embodiment, the invention provides an isolated population of cells comprising a plurality of CD29⁺、CD44⁺、CD73⁺、CD90⁺、CD105⁺、CD200⁺、CD34^-And CD133^-The immunosuppressive placental stem cells of (a).

In a specific embodiment of the above placental stem cells, the placental stem cells constitutively secrete IL-6, IL-8, and monocyte chemotactic protein (MCP-1).

Each of the plurality of placental stem cells referenced above may comprise: placental stem cells obtained and isolated directly from a placenta of a mammal, or placental stem cells cultured and passaged for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 or more passages, or a combination thereof.

The immunosuppressive plurality of placental stem cells described above can comprise about, at least, or no more than 1 x 10⁵、5×10⁵、1×10⁶、5×10⁶、1×10⁷、5×10⁷、1×10⁸、5×10⁸、1×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹Or more placental stem cells.

5.2.3 screening and preparation of placental stem cell populations

In another embodiment, the present invention also provides a method of screening a plurality of immunosuppressive placental stem cells from a plurality of placental cells, the method comprising screening a plurality of placental cells, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said cells are CD200⁺、HLA-G⁺And wherein said placental stem cells detectably inhibit T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In particular embodiments, the screening comprises screening for CD73⁺And CD105⁺The stem cell of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-Or CD45^-The stem cell of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-、CD45^-、CD73⁺And CD105⁺The placental stem cells of (1). In another embodiment, the screening also comprises screening a plurality of placental stem cells that form one or more embryoid bodies when cultured under conditions that allow for embryoid body formation.

In another embodiment, the present invention is a method of treating a subject suffering from a disease or disorderAlso provided are methods of screening a plurality of immunosuppressive placental stem cells from a plurality of placental cells, the method comprising screening a plurality of placental cells, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said cells are CD73⁺、CD105⁺、CD200⁺And wherein said placental stem cells detectably inhibit T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment, the screening comprises screening or HLA-G⁺The stem cell of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-Or CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-And CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-、CD45^-And HLA-G⁺The placental stem cells of (1). In another specific embodiment, the screening additionally comprises screening a population of placental cells that form one or more embryoid bodies when the population is cultured under conditions that allow the formation of embryoid bodies.

In another embodiment, the present invention also provides a method of screening a plurality of immunosuppressive placental stem cells from a plurality of placental cells, the method comprising screening a plurality of placental cells, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said cells are CD200⁺、OCT-4⁺And wherein said placental stem cells detectably inhibit T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment, said screening comprises whether screening or CD73⁺And CD105⁺The placental stem cells of (1). In another specific embodiment, said screening comprises screening or HLA-G⁺The placental stem cells of (1). In another specific embodiment, said screening comprisesScreening is also CD34^-、CD38^-And CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-、CD45^-、CD73⁺、CD105⁺And HLA-G⁺The placental stem cells of (1).

In another embodiment, the present invention also provides a method of screening a plurality of immunosuppressive placental stem cells from a plurality of placental cells, the method comprising screening a plurality of placental cells, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said cells are CD73⁺、CD105⁺And HLA-G⁺And wherein said placental stem cells detectably inhibit T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay. In a specific embodiment, said screening comprises whether screening or CD34^-、CD38^-Or CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-And CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises screening or CD200⁺The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-、CD45^-、OCT-4⁺And CD200⁺The placental stem cells of (1).

In another embodiment, the present invention also provides a method of screening a plurality of immunosuppressive placental stem cells from a plurality of placental cells, the method comprising screening a plurality of placental cells, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said cells are CD73⁺、CD105⁺And wherein said plurality of cells form one or more embryoid bodies under conditions that allow the formation of embryoid bodies. In particular embodiments, the screening comprises whether screening or CD34^-、CD38^-Or CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-And CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises screening or OCT-4⁺The placental stem cells of (1). In a more specific embodiment, the screening comprises whether screening or OCT-4⁺、CD34^-、CD38^-And CD45^-The placental stem cells of (1).

In another embodiment, the present invention also provides a method of screening a plurality of immunosuppressive placental stem cells from a plurality of placental cells, the method comprising screening a plurality of placental cells, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of said isolated placental cells are OCT-4⁺And wherein the plurality of cells form one or more embryoid bodies under conditions that allow the formation of embryoid bodies. In particular embodiments, the screening comprises whether screening or CD73⁺And CD105⁺The placental stem cells of (1). In another specific embodiment, said screening comprises whether screening or CD34^-、CD38^-Or CD45^-The placental stem cells of (1). In another specific embodiment, said screening comprises screening or CD200⁺The placental stem cells of (1). In a more specific embodiment, the screening comprises whether screening or CD73⁺、CD105⁺、CD200⁺、CD34^-、CD38^-And CD45^-The placental stem cells of (1).

The invention also provides methods of preparing an immunosuppressive placental stem cell population or plurality of placental stem cells. For example, the present invention provides a method of preparing a population, the method comprising screening a plurality of placental stem cells, any of which is described above, and isolating the plurality of placental stem cells from other cells (e.g., other placental cells). In a specific embodiment, the present invention provides a method for preparing a population of cells, the method comprising screening placental cells, whereinSaid placental cells (a) are attached to a matrix, (b) express CD200 and HLA-G; or express CD73, CD105, and CD 200; or express CD200 and OCT-4; or express CD73, CD105, and HLA-G; or express CD73 and CD105, and allow the formation of one or more embryoid bodies in a population of placental cells comprising said stem cells and when said population is cultured under conditions that allow the formation of embryoid bodies; or expressing OCT-4, in a population of placental cells comprising stem cells and allowing formation of one or more embryoid bodies when said population is cultured under conditions that allow formation of embryoid bodies; and (c) detectably inhibits CD4 in MLR (mixed lymphocyte reaction)⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells.

In a more specific embodiment, the present invention provides a method for preparing a cell population, the method comprising screening placental stem cells for (a) adherence to a matrix, (b) expression of CD200 and HLA-G, and (c) detectable inhibition of CD4 in MLR (mixed lymphocyte reaction)⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells. In another embodiment, the invention provides a method of preparing a population of cells, the method comprising screening placental stem cells for (a) adherence to a substrate, (b) expression of CD73, CD105, and CD200, and (c) detectable inhibition of CD4 in MLR⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells. In another embodiment, the invention provides a method of preparing a population of cells, the method comprising screening placental stem cells for (a) adherence to a substrate, (b) expression of CD200 and OCT-4, and (c) detectable inhibition of CD4 in MLR⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells. In another embodiment, the invention provides a method of preparing a population of cells, the method comprising screening placental stem cells for (a) adherence to a substrate, (b) expression of CD73 and CD105, (c) formation of embryoid bodies when cultured under conditions that allow embryoid body formation, and (d) detectable inhibition of CD4 in MLR⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells. In another embodiment, the invention provides a method of preparing a population of cells, the method comprising screening placental stem cells for (a) adherence to a substrate, (b) expression of CD73, CD105 and HLA-G, and (c) detectable inhibition of CD4 in MLR⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells. The method of preparing a cell population comprises screening placental stem cells that (a) adhere to a substrate, (b) express OCT-4, (c) form embryoid bodies when cultured under conditions that allow embryoid body formation, and (d) detectably inhibit CD4 in MLR⁺Or CD8⁺T cell proliferation; and isolating said placental cells from other cells to form a population of cells.

In a specific embodiment of the method of making a population of immunosuppressive placental stem cells, said T cells and said placental cells are present in said MLR in a ratio of about 5: 1. The placental cells used in the method may be derived from an intact placenta, or may be derived primarily from the amniotic membrane, or from both the amniotic membrane and the chorion. In another specific embodiment, said placental cells inhibit CD4 in the MLR relative to the number of T cell proliferations in the absence of said placental cells in said MLR⁺Or CD8⁺At least 50%, at least 75%, at least 90%, or at least 95% of T cell proliferation. The methods may additionally include screening, and/or preparing, immunomodulatory placental stem cell populations, e.g., to inhibit the activity of other immune cells, such as Natural Killer (NK) cells.

5.2.4 growth in culture

Growth of placental stem cells as described herein, as with any mammalian cell, depends in part on the particular culture medium chosen for growth. Under optimal conditions, placental stem cells typically multiply in number within 3 to 5 days. During culturing, the placental stem cells of the invention adhere to a substrate of a culture medium, such as the surface of a container for tissue culture (e.g., a plastic tissue culture dish, a fibronectin-coated plastic article, etc.), and form a monolayer.

Isolated placental cell populations comprising the placental stem cells described herein form embryoid bodies, i.e., three-dimensional clusters of cells that grow on top of the adherent stem cell layer, when cultured under appropriate conditions. Cells within embryoid bodies express markers associated with very early stem cells, such as OCT-4, Nanog, SSEA3, and SSEA 4. Cells within the embryoid body do not typically adhere to the media substrate as do placental stem cells described herein, but rather remain attached to the adherent cells during culture. Cells of embryoid bodies survive on top of adherent placental stem cells because embryoid bodies do not form when adherent stem cells are absent. Thus, in a population of placental cells comprising adherent placental stem cells, the adherent placental stem cells promote the growth of one or more embryoid bodies. Without being bound by any theory, it is believed that cells of embryoid bodies grow on adherent placental stem cells, much as embryonic stem cells grow on feeder layer cells. Mesenchymal stem cells (e.g., bone marrow-derived mesenchymal stem cells) do not appear embryoid bodies in culture.

5.2.5 differentiation

Placental stem cells useful in the methods of the invention differentiate into different committed cell lineages. For example, placental stem cells can be differentiated into an adipocyte cell line, a chondrocyte cell line, a neural cell line, or an osteoblast cell line. Such differentiation may be accomplished according to any known differentiation method in the art, such as a method in which bone marrow-derived mesenchymal stem cells are differentiated into similar cell lines.

5.3 method for obtaining placental stem cells

5.3.1 Stem cell Collection compositions

The invention also provides methods for collecting and isolating placental stem cells. Generally, stem cells are obtained from the placenta of a mammal using a physiologically acceptable solution, such as a stem cell collection composition. A Stem cell collection Composition is described in detail in related U.S. provisional application No. 60/754,969 entitled "Improved Composition for Collecting and preserving placental Stem Cells and method of Using the same" filed on 29.12.2005.2005.

Any physiologically acceptable solution suitable for collecting and/or culturing stem cells may be included in the stem cell collection composition, for example, a salt solution (e.g., phosphate buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl, etc.), a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.

The stem cell collection composition may comprise one or more components that tend to protect the placental stem cells, i.e., prevent death of the placental stem cells from the time of collection to the time of culture, or delay death of the placental stem cells, reduce the number of dead placental stem cells in the population of cells, etc. These components may be, for example, apoptosis inhibitors (e.g., cysteine protease inhibitors, or JNK inhibitors); vasodilators (e.g., magnesium sulfate, antihypertensive drugs, Atrial Natriuretic Peptide (ANP), adrenocorticotropic hormone releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin sulfate or magnesium sulfate, phosphodiesterase inhibitors, etc.); necrosis inhibitors (e.g. 2- (1-hydro-indol-3-yl) -3-pentylamine-maleimide, pyrrolidine dithiocarbamate or clonazepam); a TNF-alpha inhibitor; and/or oxygen-carrying perfluorocarbons (e.g., bromoperfluorooctane, bromoperfluorodecane, etc.).

The stem cell collection composition can comprise one or more tissue degrading enzymes, such as a metalloprotease, a serine protease, a neutral protease, a ribonuclease, or a deoxyribonuclease, among others. These enzymes include, but are not limited to: collagenase (e.g., collagenase I, II, III, or IV, collagenase from clostridium histolyticum, etc.); dispase, thermolysin, elastase, trypsin, releasease, hyaluronidase, and the like.

The stem cell collection composition can comprise a bactericidal or bacteria-inhibiting effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide antibiotic (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime, or cefadroxil), clarithromycin, erythromycin, a penicillin (e.g., penicillin V), or a quinolone (e.g., ofloxacin, ciprofloxacin, or norfloxacin), tetracycline, streptomycin, or the like. In particular embodiments, the antibiotic is active against gram positive and/or gram negative bacteria, e.g., pseudomonas aeruginosa, staphylococcus aureus, and the like.

The stem cell collection composition may further comprise one or more of the following compounds: adenosine (about 1mM to about 50 mM); d-glucose (about 20mM to about 100 mM); magnesium ions (about 1mM to about 50 mM); macromolecules having a molecular weight greater than 20000 daltons, and in one embodiment, are present in an amount sufficient to maintain endothelial integrity and cell viability (e.g., synthetic or naturally occurring colloids, polysaccharides such as dextran or polyethylene glycol, from about 25g/l to about 100g/l, or from about 40g/l to about 60 g/l); antioxidants (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C, or vitamin E, about 25 μ M to about 100 μ M); a reducing agent (e.g., N-acetylcysteine, about 0.1mM to about 5 mM); substances that prevent calcium entry into cells (e.g., verapamil, about 2 μ M to about 25 μ M); nitroglycerin (e.g., about 0.05g/l to about 0.2 g/l); an anticoagulant, in one embodiment, is present in an amount sufficient to help prevent residual blood clotting (e.g., heparin or hirudin, about 1000 units/l to about 100,000 units/l); or a compound containing an amiloride group (e.g., amiloride, ethylisopropylamiloride, cyclohexylamiloride, dimethylaminochloropiride, or isobutylamiloride, from about 1.0 μ M to about 5 μ M).

5.3.2 placenta Collection and processing

Typically, human placenta is recovered shortly after the post-partum discharge. In a preferred embodiment, the placenta is recovered from the patient after the patient has informed consent and the patient's complete medical history is recorded and correlated with the placenta. Preferably, the medical history extends until after delivery. Such a history can be used to coordinate subsequent use of the placenta or stem cells harvested therefrom. For example, human placental stem cells can be used as individualized drugs, by medical history, for infants associated with the placenta, or parents, siblings, or other relatives of the infant.

Prior to recovery of placental stem cells, cord blood and placental blood are removed. In certain embodiments, following delivery, cord blood in the placenta is recovered. The placenta is subjected to a conventional cord blood recovery procedure. Typically, the placenta is exsanguinated using a needle or cannula with the aid of gravity (see, e.g., U.S. Pat. No. 5,372,581 to Anderson; U.S. Pat. No. 5,415,665 to Hessel et al). The needle or cannula is typically placed in the umbilical vein and the placenta is gently pressed to help the cord blood drain out of the placenta. Such Cord Blood recovery can be commercially performed, for example, by LifeBank, Cedar Knolls N.J., ViaCord, Cord Blood Registry and Cryocell. Preferably, the placenta is drained by gravity, without further treatment, to reduce tissue damage during cord blood recovery.

Typically, the placenta is transported from a delivery or birth room to another location, such as a laboratory, to recover cord blood and collect stem cells by, for example, perfusion and tissue isolation. The placenta is preferably transported in a sterile, insulated transport facility (maintaining the temperature of the placenta between 20-28℃.), for example, by placing the placenta with the proximal umbilical cord clamped therein in a sterile zip-lock plastic bag, which is then placed in an insulated container. In another embodiment, the placenta is transported using an umbilical cord blood collection kit as described in pending U.S. patent application No. 11/230,760, filed on 9/19/2005. Preferably, the placenta is delivered to the laboratory 4 to 24 hours after delivery. In certain embodiments, the proximal cord is clamped, preferably, inserted 4 to 5cm (centimeters) of the fetal disc surface prior to cord blood recovery. In other embodiments, the proximal umbilical cord is clamped after cord blood recovery and prior to further processing of the placenta.

The placenta can be stored at room temperature or under sterile conditions of 5 to 25 ℃ (celsius) prior to stem cell collection. The placenta may be stored for longer than 48 hours, preferably 4 to 24 hours before perfusing the placenta to remove any residual cord blood. The placenta is preferably stored in an anticoagulant solution at a temperature of 5 to 25 deg.C (Celsius). Suitable anticoagulant solutions are well known in the art. For example, heparin or warfarin sodium solution may be used. In a preferred embodiment, the anticoagulant solution comprises a heparin solution (e.g., 1% w/w in a 1: 1000 solution). The exsanguinated placenta is preferably stored for no more than 36 hours before placental stem cells are collected.

The placenta of the mammal, or a portion thereof, after collection and preparation, generally as described above, may be treated in any manner known in the art, e.g., may be perfused or divided, e.g., digested with one or more tissue-disrupting enzymes, to obtain stem cells.

5.3.3 physical disruption and enzymatic digestion of placental tissue

In one embodiment, the stem cells are collected from the placenta of the mammal by physical disruption of the organ, such as enzymatic digestion. For example, the placenta, or a portion thereof, may be, e.g., crushed, sheared, chopped, diced, chopped, macerated, etc., upon contact with a stem cell collection composition of the invention, followed by digestion of the tissue with one or more enzymes. The placenta or a portion thereof may also be physically disrupted and digested with one or more enzymes, and the resulting material is then immersed or mixed into the stem cell collection composition of the present invention. Any physical disruption method may be used as long as the disruption method allows a large, preferably a majority, preferably at least 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in the organ to survive, as detected by trypan blue exclusion, for example.

The placenta may be divided into components prior to physical disruption and/or enzymatic digestion and stem cell recovery. For example, the placental stem cells can be obtained from the amniotic membrane, chorion, placental chorion, or any combination thereof. Preferably, the placental stem cells are obtained from placental tissue comprising an amniotic membrane and a chorion. Typically, placental stem cells can be obtained by disrupting small pieces of placental tissue, e.g., pieces of placental tissue having a volume of about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 cubic millimeters.

Preferred stem cell collection compositions comprise one or more tissue-disrupting enzymes. Preferably, enzymatic digestion is performed using a combination of enzymes, e.g., a combination of matrix metalloprotease and neutral protease, e.g., a combination of collagenase and dispase. In one embodiment, enzymatic digestion of placental tissue uses a combination of matrix metalloproteinases, neutral proteases, and mucolytic enzymes that digest hyaluronic acid, such as a combination of collagenase, dispase, and hyaluronidase, or a combination of releaser (Boehringer Mannheim, Indianapolis, Ind.) and hyaluronidase. Other enzymes that may be used to destroy placental tissue include papain, deoxyribonuclease, serine proteases such as trypsin, chymotrypsin, or elastase. Serine proteases are inhibited by α 2 microglobulin in serum, so the medium used for digestion is usually serum free. EDTA and dnase are commonly used in enzymatic digestion processes to increase the efficiency of cell resuscitation. The digest is preferably diluted to avoid trapping stem cells in the viscous digest.

Any combination of tissue digesting enzymes may be used. Typical concentrations of tissue digesting enzymes include, for example, collagenase I and collagenase IV: 50-200U/mL, dispase: 1-10U/mL, elastase: 10-100U/mL. The proteases may be used in combination, i.e. two or more proteases in the same digestion reaction, or consecutively to release placental stem cells. For example, in one embodiment, the placenta or portion thereof is first digested with the appropriate amount of collagenase I at 2mg/ml for 30 minutes, followed by digestion with 0.25% trypsin at 37 ℃ for 10 minutes. Serine proteases are preferably used after the use of other enzymes.

In another embodiment, a chelating agent, such as ethylene glycol bis (2-aminoethyl ether) -N, N, N 'N' -tetraacetic acid (EGTA) or Ethylene Diamine Tetraacetic Acid (EDTA), may also be added to the stem cell collection composition comprising stem cells or to the solution that divides and/or digests the tissue prior to isolating the stem cells with the stem cell collection composition.

It will be appreciated that when cells from both the fetus and the mother are contained in an intact placenta or a portion of the placenta (e.g., when the portion of the placenta comprises chorion or placental chorions), the collected placental stem cells will comprise a mixture of placental stem cells derived from both the fetus and the mother. Where a portion of the placenta contains no or negligible amounts of maternal cells (e.g., amnion), the collected placental stem cells will contain nearly all placental stem cells belonging to the fetus.

5.3.4 placental perfusions

Placental stem cells can also be obtained by perfusing a mammalian placenta. Methods for obtaining Stem Cells by perfusing mammalian placenta have been disclosed, for example, in U.S. application publication No. 2002/0123141 to Hariri, and related U.S. provisional application No. 60/754,969 entitled "Improved compositions for Collecting and preserving Placental Stem Cells and Methods of Using the same" (Improved Composition for Collecting and preserving Placental Stem Cells and Methods of Using the same) "filed on 29.12.2005.

Placental stem cells can be collected by perfusion, e.g., via the vasculature of the placenta, such as by perfusion using a stem cell collection composition as a perfusion solution. In one embodiment, the placenta of the mammal is perfused by flowing a perfusion solution through the umbilical artery or umbilical vein or both. The flow of perfusion solution into and through the placenta may be by, for example, gravity. Preferably, a pump, such as a peristaltic pump, is used to force the perfusion solution through the placenta. The umbilical vein may be inserted, for example, with a cannula (e.g., a teflon or plastic cannula) that is connected to a sterile connection device, such as a sterile tube. The sterile connection device is connected to the perfusion manifold.

In preparation for perfusion, the placenta is preferably oriented (e.g., suspended) in such a way that both the umbilical arteries and the umbilical veins are at the highest point of the placenta. The placenta may be perfused through the vasculature of the placenta or the placental vasculature and surrounding tissue by a perfusate, such as the stem cell collection composition of the invention. In one embodiment, the umbilical artery and umbilical vein are simultaneously connected to a pipette that is connected via a deformable connector to a reservoir of perfusion solution. The perfusion solution flows into the umbilical vein and artery. The perfusion solution seeps out and/or through the vessel wall, into the surrounding tissue of the placenta, and is collected in a suitably open container from the surface of the placenta, which is the part of the surface that adheres to the mother's uterus during pregnancy. The perfusion solution may also be introduced through the umbilical cord opening and allowed to drain or filter out of the opening in the placental wall, that portion of the placental wall that interfaces with the maternal uterine wall. In another embodiment, the perfusion solution flows through a cord vein and is collected from a cord artery, or flows through a cord artery and is collected from a cord vein.

In one embodiment, the proximal umbilical cord is clamped during perfusion, preferably, the proximal umbilical cord is inserted 4 to 5cm (centimeters) of the fetal disc surface.

During exsanguination, the perfusate collected from the mammalian placenta for the first time is usually stained by residual red blood cells in cord blood and/or placental blood. As perfusion progresses, the perfusate gradually becomes colorless and residual cord blood cells are washed out of the placenta. At the beginning of the exsanguination of the placenta, typically 30 to 100ml (milliliters) of perfusate is sufficient, but more or less perfusate may be used depending on the observation.

The volume of perfusate used to collect placental stem cells can vary depending on the number of stem cells to be collected, the size of the placenta, the number of collections that can be made from a single placenta, and the like. In various embodiments, the volume of the perfusate may be 50mL to 5000mL, 50mL to 4000mL, 50mL to 3000mL, 100mL to 2000mL, 250mL to 2000mL, 500mL to 2000mL, or 750mL to 2000 mL. Typically, after exsanguination, the placenta is perfused with 700 to 800mL of perfusate.

The placenta may be perfused multiple times over the course of hours or days. When the placenta is to be perfused multiple times, it may be maintained or cultured in a container or other suitable vessel under sterile conditions and the stem cell collection composition is used, or a standard perfusion solution (e.g., a physiological salt solution, such as phosphate buffered saline ("PBS")), with or without an anticoagulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), with or without an antimicrobial (e.g., beta-mercaptoethanol (0.1 mM)), an antibiotic such as streptomycin (e.g., at a concentration of 40-100 μ g/mL), a penicillin (e.g., at a concentration of 40U/mL), amphotericin B (e.g., at a concentration of 0.5 μ g/mL) is perfused 2.3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 days or 3 or more days. The perfused placenta may be maintained for a period of time or more additional time, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and may be re-perfused with, for example, 800mL of perfusion fluid at 700-. The placenta may be perfused 1, 2, 3, 4, 5 or more times, for example once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, the perfusion of the placenta and the collection of the perfusion solution, e.g., the stem cell collection composition, is repeated until the number of nucleated cells recovered falls below 100 cells/ml. The perfusate at different time points may be further processed individually to recover time-dependent cell populations, such as stem cells. The perfusate at different time points can also be collected.

Without wishing to be bound by any theory, after exsanguinating and perfusing the placenta for a sufficient time, the placental stem cells are believed to migrate into the placental exsanguinated and perfused microvascular circulation where they are collected according to the methods of the present invention, preferably by perfusion rinsing into a collection vessel. Perfusing the isolated placenta not only removes residual cord blood, but also provides the placenta with appropriate nutrients, including oxygen. The placenta may be cultured and may be perfused with a similar solution used to remove residual cord blood cells, preferably without the addition of anticoagulant substances.

The placental stem cells harvested by perfusion according to the methods of the invention are significantly greater than the number obtained from a mammalian placenta without perfusion with the solution, and without other treatment to obtain stem cells (e.g., by tissue disruption, such as enzymatic digestion). In this context, "significantly more" means at least 10% more. The number of placental stem cells prepared by perfusion according to the methods of the invention is significantly greater than, for example, the number of placental stem cells obtained from a culture by culturing a placenta or a portion thereof.

Stem cells may be isolated from placenta by perfusion with a solution comprising one or more proteases or other tissue-disrupting enzymes. In particular embodiments, the placenta or portion thereof (e.g., the amniotic membrane, amniotic membrane and chorion, placental leaflets or chorion, or a combination of any of the foregoing) is placed at 25-37 ℃ and co-cultured with one or more tissue disrupting enzymes for 30 minutes in 200 milliliters of culture medium. The cells in the perfusate were collected, placed at 4 ℃, and washed with a cold inhibitor mix containing 5mM edta, 2mM dithiothreitol, and 2mM β -mercaptoethanol. After a few minutes, the stem cells are washed with a cold (e.g., 4 ℃) stem cell collection composition of the present invention.

It will be appreciated that perfusion using the pan method (pan method) according to the present invention results in a mixture of fetal and maternal cells, as the perfusate is collected after it has been shed from the maternal side of the placenta. Thus, the cells collected by this method comprise a mixed population of placental stem cells derived from both the fetus and the mother. In contrast, the placental stem cell population collected solely by perfusion of the placental vasculature is almost exclusively derived from the fetus, as the perfusate flow is collected through one or two placental vessels and only through the remaining vessels.

5.3.5 isolation, Classification and characterization of placental stem cells

Stem cells from mammalian placenta, whether obtained by perfusion or enzymatic digestion, can be first purified (i.e., isolated) from other cells by Ficoll gradient centrifugation. Such centrifugation may follow standard protocols for any centrifugation speed, and the like. For example, in one embodiment, cells collected from the placenta are recovered from the perfusate by centrifugation at 5000 × g for 15 minutes at room temperature to separate the cells from, for example, contaminating debris and platelets. In another embodiment, placental perfusate is concentrated to about 200 ml, gently layered on Ficoll, centrifuged at 22 ℃ at about 1100 Xg for 20 minutes, and the low density of middle layer cells are collected for further processing.

The cell pellet can be resuspended in a fresh stem cell collection composition, or in a medium suitable for stem cell maintenance, for example, IMDM serum-free medium containing 2U/ml heparin and 2mM EDTA (GibcoBRL, N.Y.). The total monocyte fraction can be isolated, for example, using Lymphoprep (Nycomed Pharma, Oslo, Norway) following the manufacturer's recommended protocol.

As used herein, "isolated" placental stem cells refers to the removal of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells that are normally associated with stem cells within an intact mammalian placenta. Stem cells from an intact organ are "isolated" when they exist in a population of cells that contains less than 50% of the cells normally associated with stem cells in the organ.

Placental cells obtained by perfusion or digestion may be isolated, for example, further or initially by differential trypsinization using, for example, a 0.05% trypsin solution (Sigma, st louis, missouri) with 0.2% EDTA added. The differential approach of trypsin digestion is feasible because placental stem cells are usually detached from plastic surfaces within about 5 minutes, whereas other attached colonies usually require more than 20-30 minutes of culture. Separated from each otherPlacental stem cells can be harvested after trypsinization and trypsin neutralization using, for example, Trypsin Neutralization Solution (TNS) (Cambrex corporation). In embodiments where adherent cells are isolated, aliquots are used, e.g., 5-10X 10⁶The individual cells are placed in each of several T-75 flasks, preferably fibronectin-coated T-75 flasks. In this embodiment, the cells may be cultured in commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex) and placed in a tissue culture incubator (37 ℃, 5% CO)₂). After 10 to 15 days, nonadherent cells were removed from the flask by PBS rinsing. PBS was then replaced with MSCGM. The flasks are preferably checked daily for the presence of various adherent cell types, in particular for the presence and expansion of fibroblast-like cell clusters.

The number and type of cells collected from the mammalian placenta can be monitored, for example, by detecting morphological changes and cell surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., tissue-specific or cell marker-specific antibody staining), Fluorescence Activated Cell Sorting (FACS), Magnetic Activated Cell Sorting (MACS); monitoring by examining the morphology of the cells using optical microscopy or confocal microscopy; and/or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling. These techniques can also be used to identify cells that are positive for one or more specific markers. For example, using the CD34 antibody, it can be detected whether the cell contains a detectable amount of CD34 using the above techniques; if so, the cell is CD34⁺In (1). Similarly, if a cell produces enough OCT-4 RNA to be detected by RT-PCR, or significantly more OCT-4 RNA than an adult cell, then the cell is OCT-4⁺In (1). Cell surface labeled antibodies (e.g., CD markers such as CD34) and sequences of stem cell specific genes such as OCT-4 are well known in the art.

Placental cells, particularly cells isolated by Ficoll isolation, different attachment capacities, or a combination of both, can be sorted using Fluorescence Activated Cell Sorting (FACS). Fluorescence Activated Cell Sorting (FACS) is a well-known method for separating particles, including cells, based on their fluorescent properties (Kamarch, 1987, Methods Enzymol, 151: 150-. Laser excitation of the fluorescent moieties in the individual particles results in tiny charges, which allows electromagnetic separation of positive and negative particles in a mixture. In one embodiment, the cell surface marker specific antibody or ligand is labeled with a different fluorescent label. The cells are processed through a cell sorter, which separates the cells based on their binding capacity to the antibody used. FACS sorted microparticles can be stored directly into individual wells of a 96-well or 384-well plate for ease of isolation and cloning.

In one sorting protocol, stem cells from placenta are sorted based on the expression of markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4, and/or HLA-G. This can be done in conjunction with a stem cell screening procedure based on its adherence properties in culture. For example, adherent selection of stem cells can be performed before or after sorting based on expression of the marker. In one embodiment, for example, cells are first sorted based on their expression of CD34, CD34^-Are preserved, and those CD200 s⁺HLA-G⁺Is derived from all other CD34^-Is isolated from the cells of (1). In another embodiment, cells from the placenta are based on their expression of the markers CD200 and/or HLA-G; for example, cells displaying either of these two markers are isolated for further use. Cells expressing e.g. CD200 and/or HLA-G may in a particular embodiment be further sorted based on their expression of CD73 and/or CD105, or based on epitopes recognized by antibodies SH2, SH3 or SH4, or based on lack of expression of CD34, CD38 or CD 45. For example, in one embodiment, the placental cells are sorted by expression or absence of CD200, HLA-G, CD73, CD105, CD34, CD38, and CD45, and CD200 is⁺、HLA-G⁺、CD73⁺、CD105⁺、CD34^-、CD38^-And CD45^-The placental cells of (a) are isolated from other placental cells for further use.

In another embodiment, magnetic beads may be used to isolate cells. Cells can be sorted using Magnetic Activated Cell Sorting (MACS) technique, a method that separates particles based on their ability to bind magnetic beads (0.5-100 μm in diameter). Various advantageous modifications may be made to the magnetic microspheres, including the covalent addition of antibodies that specifically recognize particular cell surface molecules or haptens. The beads and cells are then mixed for binding. The cells are then passed through a magnetic field to isolate cells having specific cell surface markers. In one embodiment, the cells may then be separated and remixed with magnetic beads that have additional cell surface labeled antibodies bound thereto. The cells are again passed through a magnetic field to isolate cells that have bound both antibodies. Such cells are then diluted into separate culture dishes, for example microwell culture dishes for isolation of clones.

Placental stem cells can also be identified and/or sorted based on cell morphology and growth characteristics. For example, placental stem cells can be characterized as having, and/or screened based on, a fibroid appearance, e.g., in culture. Placental stem cells can also be characterized as having the ability to form embryoid bodies and/or screened for their ability to form embryoid bodies. In one embodiment, for example, placental cells that are fibroblast-like in shape, express CD73 and CD105, and form one or more embryoid bodies in culture are isolated from other placental cells. In another embodiment, OCT-4 will form one or more embryoid bodies in culture⁺The placental cells of (a) are separated from other placental cells.

In another embodiment, placental stem cells can be identified and characterized by a clonogenic unit assay. The clone forming unit assay is well known in the art, e.g., Mesen Cult^TMCulture medium (Stem Cell Technologies, Vancouver, British Columbia).

The viability, proliferative potential and longevity of placental stem cells can be assessed using standard techniques known in the art, such as trypan blue exclusion test, fluorescein diacetate uptake test, propidium iodide uptake test (to assess viability), and thymidine uptake test, MTT cell proliferation test (to assess proliferative capacity). Longevity can be determined by methods known in the art, for example by measuring the maximum number of colony doublings in a long-term culture.

Placental stem cells can also be isolated from other placental cells using other techniques known in the art, such as selective growth of desired cells (positive selection), selective elimination of undesired cells (negative selection); segregation based on differences in cell agglutination in mixed populations, for example with soybean agglutinin; a freeze-thaw procedure; filtering; conventional centrifugation and zonal centrifugation; centrifugal elution (reverse flow centrifugation); unit gravity sorting; counter-current distribution; electrophoresis, and the like.

5.4 culture of placental Stem cells

5.4.1 culture Medium

The isolated placental stem cells or population of placental stem cells or placental tissue from which the placental stem cells have grown can be used to initiate or seed the cell culture medium. The cells are typically transferred to a sterile tissue culture vessel, coated or uncoated with extracellular matrix or ligands such as laminin, collagen (e.g., natural or denatured), gelatin, fibronectin, ornithine, vitronectin, and extracellular membrane proteins (e.g., MATRIGEL (BDDiscovery Labware, bedford, ma)).

Placental stem cells can be cultured under any conditions and in any medium acceptable for stem cell culture as recognized in the art. Preferably, the culture medium comprises serum. Placental stem cells can be cultured in, for example, DMEM-LG (Dulbecco's modified essential Medium, Low sugar)/MCDB 201 (Chicken fibroblast basal Medium) comprising ITS (insulin ferroselenoprotein), LA + BSA (linoleic acid-bovine serum Albumin), glucose, L-ascorbic acid, PDGF, EGF, IGF-1 and penicillin/streptomycin; DMEM-HG (high glucose) containing 10% Fetal Bovine Serum (FBS); DMEM-HG containing 15% FBS(ii) a IMDM (Iscove's modified Dulbecco's medium) comprising 10% FBS, 10% horse serum and hydrocortisone; m199, comprising 10% FBS, EGF and heparin; alpha-MEM (minimum essential Medium) containing 10% FBS, GlutaMAX^TMAnd gentamicin; DMEM containing 10% FBS, GlutaMAX^TMAnd gentamicin, and the like. Preferred medium is DMEM-LG/MCDB-201 containing 2% FBS, ITS, LA + BSA, glucose, L-ascorbic acid, PDGF, EGF and penicillin/streptomycin.

Other media that can be used to culture placental stem CELLs include DMEM (high or low sugar), Eagle's basal medium, Ham's F10 medium (F10), Ham's F12 medium (F12), Iscove's modified Dulbecco's medium, Mesenchymal Stem CELL Growth Medium (MSCGM), Liebovitz' sL-15 medium, MCDB, DMIEM/F12, RP1640 MI, advanced DMEM (Gibco), DMEM/MCDB 201 (Sigma), and CELL-GRO FREE.

One or more of the following components may be supplemented into the culture medium, including, for example, serum (e.g., Fetal Bovine Serum (FBS), preferably about 2-15% (v/v); horse serum (ES); Human Serum (HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one or more growth factors, such as Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), Leukemia Inhibitor (LIF), Vascular Endothelial Growth Factor (VEGF), and Erythropoietin (EPO); amino acids, including L-valine; and one or more antibiotic and/or antifungal substances for controlling microbial contamination, such as penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin, either alone or in combination.

5.4.2 expansion and proliferation of placental stem cells

Once the placental stem cells or stem cell population is isolated (e.g., stem cells or stem cell population are separated from at least 50% of the placental cells that are normally associated with the stem cells or stem cell population in vivo), the stem cells or stem cell population can be expanded and expanded in vitro. For example, a population of placental stem cells can be cultured in a tissue culture container, e.g., a petri dish, flask, multi-well plate, etc., for a sufficient time to allow the stem cells to proliferate to 70-90% confluence, i.e., the stem cells and progeny thereof occupy 70-90% of the culture surface area of the tissue culture container.

Placental stem cells can be seeded into a culture vessel at a density that allows for cell growth. For example, the cells can be at low density (e.g., about 1000 to about 5000 cells/cm)²) To high densities (e.g., about 50,000 or more cells/cm)²) And (4) inoculating. In a preferred embodiment, the cells are cultured in CO₂Air in an amount of about 0% to about 5% by volume. In some preferred embodiments, the cells are cultured in O₂Air in an amount of about 2% to about 25%, preferably, O₂Air in an amount of about 5% to about 20%. The cells are preferably cultured at about 25 ℃ to about 40 ℃, preferably 37 ℃. The cells are preferably cultured in an incubator. The culture medium may be static or agitated, for example, using a bioreactor. Placental stem cells are preferably grown under conditions of low oxidative stress (e.g., addition of glutathione, ascorbic acid, catalase, vitamin E, N-acetylcysteine, etc.).

Once a 70% to 90% confluence is achieved, the cells can be passaged. For example, the cells may be enzymatically treated, e.g., trypsinized, to detach them from the tissue culture surface using techniques well known in the art. After removing the cells with a pipette and counting, approximately 20,000 to 100,000 stem cells, preferably approximately 50,000 stem cells, are passaged onto fresh medium in a new culture vessel. Typically, the new medium and the medium prior to cell transfer are the same type of medium. The invention encompasses placental stem cell populations that have been passaged at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 or more times.

5.4.3 placental stem cell populations

The present invention provides placental stem cell populations. The population of placental stem cells can be isolated directly from one or more placentas, i.e., the population of placental stem cells can be a population of placental cells comprising placental stem cells, obtained from or contained in a perfusate; or obtained or contained in a digest (i.e., a collection of cells obtained by enzymatic digestion of the placenta or a portion thereof). The isolated placental stem cells of the invention can also be cultured and expanded to produce a population of placental stem cells. A population of placental cells comprising placental stem cells can also be cultured and expanded to produce a population of placental stem cells.

The population of placental stem cells of the invention comprises placental stem cells, e.g., placental stem cells as described above. In various embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in the isolated population of placental stem cells are placental stem cells. That is, the population of placental stem cells can comprise, for example, up to 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of non-stem cells.

The present invention provides methods for preparing isolated populations of placental stem cells, whether from enzymatic digestion or perfusion, that express specific markers and/or specific culture properties or morphological characteristics, by, for example, screening for embryonic stem cells. In one embodiment, for example, the invention provides a method of preparing a population of cells, the method comprising: screening placental cells for (a) adherence to a substrate, and (b) expression of CD200 and HLA-G; and separating the cells from other cells to form a population of cells. In another embodiment, a method of preparing a population of cells comprises: screening for placental cells that (a) are attached to a substrate, and (b) express CD73, CD105, and CD 200; and separating the cells from other cells to form a population of cells. In another embodiment, a method of preparing a population of cells comprises: screening for placental cells that (a) adhere to a substrate, and (b) express CD200 and OCT-4; and separating the cells from other cells to form a population of cells. In another embodiment, a method of preparing a population of cells comprises: screening placental cells that (a) adhere to a substrate, (b) express CD73 and CD105, and (c) promote the formation of one or more embryoid bodies in a population of placental cells comprising said stem cells when said population is cultured under conditions that allow the formation of embryoid bodies; and separating the cells from other cells to form a population of cells. In another embodiment, a method of preparing a population of cells comprises: screening placental cells for (a) adherence to a matrix, and (b) expression of CD73, CD105 and HLA-G; and separating the cells from other cells to form a population of cells. In another embodiment, a method of preparing a population of cells comprises: screening placental cells that (a) adhere to a substrate, (b) express OCT-4, and (c) promote the formation of one or more embryoid bodies in a population of placental cells comprising said stem cells when said population is cultured under conditions that allow the formation of embryoid bodies; and separating the cells from other cells to form a population of cells. In any of the above embodiments, the method may additionally comprise screening for placental cells that express ABC-p (a placenta-specific ABC transporter; see, e.g., Allikmets et al, Cancer Res.58 (23): 5337-9 (1998)). The method may also include screening for cells that exhibit at least one characteristic, e.g., that is characteristic of mesenchymal stem cells, such as cells that express CD29, CD44, CD90, or any combination of the foregoing.

In the above embodiments, the substrate may be any surface upon which culturing and/or screening of cells (e.g., placental stem cells) may be accomplished. Representative substrates are plastic articles such as plastic tissue culture dishes or plastic multiwell plates. Plastic articles used for tissue culture may be coated with a layer of biomolecules such as laminin or fibronectin.

Cells may be screened for placental stem cell populations by any method known in the art of cell screening, such as placental stem cells. For example, one or more cell surface labeled antibodies can be used to screen cells, for example, in flow cytometry or FACS. Screening can be performed using antibody-bound magnetic beads. Antibodies specific for certain stem cell-related markers are well known in the art. For example, an antibody to OCT-4 (Abcam, Cambridge, Mass.), an antibody to CD200 (Abcam), an antibody to HLA-G (Abcam), an antibody to CD73 (BD Biosciences Pharmingen, san Diego, Calif.), an antibody to CD105 (Abcam; BioDesign International, Saco, Maine), and the like. Other labeled antibodies are also commercially available, for example, antibodies to CD34, CD38, and CD45 are available from, for example, StemCell technologies, Inc. or Biodesign International, Inc.

The isolated population of placental stem cells can comprise placental cells that are not stem cells, or cells that are not placental cells.

The isolated population of placental stem cells can be combined with one or more populations of non-stem cells or populations of non-placental cells. For example, the isolated placental stem cell population can be combined with blood (e.g., placental blood or umbilical cord blood), blood-derived stem cells (e.g., stem cells derived from placental blood or umbilical cord blood), a population of blood-derived nucleated cells, bone marrow-derived mesenchymal cells, a population of bone-derived stem cells, native bone marrow, adult (adult) stem cells, a population of stem cells contained in a tissue, cultured stem cells, a population of fully differentiated cells (e.g., chondrocytes, fibroblasts, amniotic cells, osteoblasts, muscle cells, cardiomyocytes, etc.), and the like. The cells in the isolated placental stem cell population may be combined with a plurality of cells of another type, the total number of nucleated cells in each population being compared at a ratio of about 100,000,000: 1, 50,000,000: 1, 20,000,000: 1, 10,000,000: 1, 5,000,000: 1, 2,000,000: 1, 1,000,000: 1, 500,000: 1, 200,000: 1, 100,000: 1, 50,000: 1, 20,000: 1, 10,000: 1, 5,000: 1, 2,000: 1, 1,000: 1, 500: 1, 200: 1, 100: 1, 50: 1, 20: 1, 10: 1, 5: 1, 1: 2, 1: 5, 1: 10, 1: 100, 1: 200, 1: 500, 1: 1,000, 1: 2,000, 1: 1,000, 1,000: 1, 1,000: 1,000, 1,000: 1, 1,000. Likewise, cells in an isolated placental stem cell population can be combined with a large number of cells of a variety of cell types.

In one embodiment, the isolated population of placental stem cells is combined with a plurality of hematopoietic stem cells. These hematopoietic stem cells may be, for example, contained in raw placenta, umbilical cord blood or peripheral blood; in all nucleated cells in placental blood, umbilical cord blood, or peripheral blood; isolated CD34 in blood from placenta, umbilical cord or periphery⁺In a population of cells; in unprocessed bone marrow; in all nucleated cells in the bone marrow; isolated CD34 in bone marrow⁺Cell populations, etc.

5.5 preservation of placental Stem cells

Placental stem cells can be stored, i.e., placed under conditions that allow long-term storage, or that inhibit cell death (e.g., apoptosis or necrosis).

Placental Stem Cells can be preserved, for example, with a composition comprising an apoptosis inhibitor, a necrosis inhibitor, and/or an oxygen-carrying perfluorocarbon, as described in related U.S. provisional application No. 60/754,969 entitled "Improved composition for Collecting and Preserving Placental Stem Cells and method of Using the same" (2005-12-25 application). In one embodiment, the present invention provides a method of preserving a population of placental stem cells, the method comprising contacting said population of stem cells with a stem cell collection composition comprising an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon, wherein said inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of stem cells relative to a population of stem cells not contacted with the inhibitor of apoptosis. In a specific embodiment, the apoptosis inhibitor is a cysteine protease inhibitor. In another specific embodiment, the apoptosis inhibitor is a JNK inhibitor. In a more specific embodiment, the JNK inhibitor does not modulate the differentiation or proliferation of the stem cell. In another embodiment, said stem cell collection composition comprises said apoptosis inhibitor and said oxygen-carrying perfluorocarbon in separate phases. In another embodiment, the stem cell collection composition comprises the apoptosis inhibitor and the oxygen-carrying perfluorocarbon in an emulsion. In another embodiment, the stem cell collection composition additionally comprises an emulsifier, such as lecithin. In another embodiment, the apoptosis inhibitor and the perfluorocarbon when contacted with the stem cells is at a temperature of about 0 ℃ and about 25 ℃. In another more specific embodiment, the apoptosis inhibitor and the perfluorocarbon are at a temperature of about 2 ℃ and 10 ℃, or at about 2 ℃ and about 5 ℃ when contacted with stem cells. In another more specific embodiment, said contacting is performed during trafficking of said stem cell population. In another more specific embodiment, said contacting is performed during freezing and thawing of said stem cell population.

In another embodiment, the invention provides a method of preserving a population of placental stem cells, the method comprising contacting said population of stem cells with an apoptosis-inhibitor and an organ preservation compound, wherein said apoptosis-inhibitor is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of stem cells relative to a population of stem cells not contacted with the apoptosis-inhibitor. In one embodiment, the organ preservation compound is a UW solution (described in U.S. Pat. No. 4,798,824; also known as ViaPasan; see also Southard et al, Transplantation 49 (2): 251-257(1990)) or a solution described in U.S. Pat. No. 5,552,267 to Stern et al. In another embodiment, the organ preservation compound is hydroxyethyl starch, lactobionic acid, raffinose, or a combination thereof. In another embodiment, the stem cell collection composition additionally comprises an oxygen-carrying perfluorocarbon, whether separated into two phases or in the form of an emulsion.

In another embodiment of the method, during perfusion, the placental stem cells are contacted with a stem cell collection composition comprising an apoptosis inhibitor and oxygen-carrying perfluorocarbon, an organ preservation compound, or a combination thereof. In another embodiment, the stem cells are contacted during tissue disruption, such as enzymatic digestion. In another embodiment, the placental stem cells are contacted with the stem cell collection composition after collection by perfusion, or after collection by tissue disruption, e.g., enzymatic digestion.

Typically, during collection, enrichment, and isolation of placental cells, it is preferable to minimize or eliminate cellular stress caused by hypoxia and mechanical stress. Thus, in another embodiment of the method, the stem cells or stem cell populations are exposed to hypoxic conditions, i.e., oxygen concentrations below the usual blood oxygen concentrations, for less than six hours during collection, enrichment or isolation during said storage. In a more specific embodiment, said population of stem cells is exposed to said hypoxic conditions for less than two hours during said preservation. In another more specific embodiment, the population of stem cells is exposed to the hypoxic conditions for less than one hour, or less than thirty minutes, or is not exposed to hypoxic conditions during the collection, enrichment or isolation. In another specific embodiment, the population of stem cells is not exposed to shear stress during collection, enrichment, or isolation.

The placental stem cells of the invention can be cryopreserved, for example, in cryopreservation media in a small vessel such as an ampoule. Suitable cryopreservation media include, but are not limited to, culture media including, for example, growth media or cell freezing media, such as commercially available cell freezing media such as C2695, C2639 or C6039 (Sigma). The cryopreservation medium preferably comprises DMSO (dimethyl sulfoxide) at a concentration of, for example, about 10% (v/v). The cryopreservation media may comprise other reagents, such as methylcellulose and/or glycerol. During cryopreservation, the preferred cooling rate of placental stem cells is about 1 ℃/minute. Preferred cryopreservation temperatures are from about-80 ℃ to about-180 ℃, preferably from about-125 ℃ to about-140 ℃. The cryopreserved cells were transferred into liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoules reach about-90 ℃, they are transferred to a liquid nitrogen storage area. The cryopreserved cells are preferably thawed at about 25 ℃ to about 40 ℃, preferably at about 37 ℃.

5.6 uses of placental stem cells

5.6.1 compositions comprising placental stem cells

The method of suppressing an immune response of the present invention may use a composition comprising placental stem cells, or a biomolecule derived therefrom. Likewise, the plurality and population of placental stem cells of the present invention can be used in conjunction with any physiologically or medically acceptable compound, composition, or device, for example, in research or therapy.

5.6.1.1 frozen placental stem cells

The immunosuppressive placental stem cell population of the present invention can be stored, e.g., cryopreserved, for later use. Methods for cryopreserving cells, such as stem cells, are well known in the art. The population of placental stem cells can be prepared in a manner that facilitates administration to an individual. For example, the present invention provides a population of placental stem cells contained within a container suitable for medical use. These containers may be, for example, sterile plastic bags, bottles, jars, or other containers from which the placental stem cell population can be readily dispensed. For example, the container may be a blood bag or other plastic, medically acceptable bag suitable for intravenous injection of a liquid into a recipient. The container is preferably one that allows cryopreservation of the combined stem cell populations.

The cryopreserved population of immunosuppressive placental stem cells can comprise placental stem cells derived from a single donor or from multiple donors. The population of placental stem cells can be completely HLA-matched, or partially HLA-mismatched, or completely HLA-mismatched to the intended recipient.

Accordingly, in one embodiment, the present invention provides a composition comprising a population of immunosuppressive placental stem cells contained in a container. In particular embodiments, the population of stem cells is cryopreserved. In another embodiment, the container is a bag, bottle or jar. In a more specific embodiment, the bag is a sterile plastic bag. In a more specific embodiment, said bag is adapted to, allow for, or facilitate intravenous administration of said population of placental stem cells. The pouch may contain a plurality of interconnected cavities or compartments to allow the placental stem cells to be mixed with one or more other solutions, such as a drug, prior to or during administration. In another embodiment, the composition comprises one or more compounds that facilitate cryopreservation of the combined stem cell population. In another specific embodiment, said population of placental stem cells is comprised within a physiologically acceptable aqueous solution. In a more specific embodiment, the physiologically acceptable aqueous solution is a 0.9% NaCl solution. In another specific embodiment, said population of placental stem cells comprises placental cells that are HLA-matched to a recipient of said population of stem cells. In another specific embodiment, said combined stem cell population comprises placental cells that are at least partially HLA-mismatched to a recipient of said stem cell population. In another specific embodiment, said placental stem cells are derived from a plurality of donors.

5.6.1.2 pharmaceutical composition

The population of immunosuppressive placental stem cells or the population of cells comprising placental stem cells can be formulated as a pharmaceutical composition for in vivo use. Such pharmaceutical compositions comprise a population of placental stem cells, or a population of cells comprising placental stem cells, in a pharmaceutically acceptable carrier, such as a salt solution or other physiologically acceptable solution for in vivo administration. The pharmaceutical compositions of the invention may comprise any of the placental stem cell populations or placental stem cell types described herein. The pharmaceutical composition may comprise fetal, maternal, or both fetal and maternal placental stem cells. In addition, the pharmaceutical composition of the present invention may comprise placental stem cells obtained from a single individual or placenta, or placental stem cells obtained from a plurality of individuals or placentas.

The medicine of the present inventionThe compositions of matter may comprise any immunosuppressive amount of placental stem cells. For example, a single unit dose of placental stem cells can comprise about, at least, or no more than 1 x 10 placental stem cells in various embodiments⁵、5×10⁵、1×10⁶、5×10⁶、1×10⁷、5×10⁷、1×10⁸、5×10⁸、1×10⁹、5×10⁹、1×10¹⁰、5×10¹⁰、1×10¹¹One or more placental stem cells.

The pharmaceutical compositions of the invention comprise a population of cells that includes 50% or more viable cells (i.e., at least 50% of the cells in the population are viable or functional). Preferably, at least 60% of the cells in the population are viable cells. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the population in the pharmaceutical composition are viable.

The pharmaceutical compositions of the invention may comprise one or more compounds that, for example, facilitate implantation (e.g., anti-T cell receptor antibodies, immunosuppressive agents, etc.), stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.

5.6.1.3 placenta stem cell conditioned medium

The placental stem cells of the invention can be used to prepare an immunosuppressive conditioned medium, i.e., a medium comprising one or more biomolecules secreted or excreted by the stem cells, which biomolecules have a detectable immunosuppressive effect on a large number of immune cells of one or more types. In various embodiments, the conditioned medium comprises a medium in which the placental stem cells have been grown for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the conditioned medium comprises a medium in which the placental stem cells are grown to a confluency of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% confluency. Such conditioned medium may be used to support the culture of separate placental stem cell populations or other types of stem cells. In another embodiment, the conditioned medium comprises a medium in which the placental stem cells differentiate into an adult cell type. In another embodiment, the conditioned medium of the invention comprises a medium in which placental stem cells and non-placental stem cells are cultured.

Accordingly, in one embodiment, the present invention provides a composition comprising a culture medium from a culture of cultured placental stem cells, wherein said placental stem cells (a) are attached to a substrate; (b) express CD200 and HLA-G; or express CD73, CD105, and CD 200; or express CD200 and OCT-4; or express CD73, CD105, and HLA-G; or express CD73 and CD105, and promote the formation of one or more embryoid bodies in a population of placental cells comprising the placental stem cells when the population is cultured under conditions that allow the formation of embryoid bodies; or expressing OCT-4 and promoting the formation of one or more embryoid bodies in a population of placental cells comprising the placental stem cells when the population is cultured under conditions that allow the formation of embryoid bodies; and (c) detectably inhibits CD4 in MLR (mixed lymphocyte reaction)⁺Or CD8⁺T cell proliferation, wherein said culture of placental stem cells is cultured in said culture medium for 24 hours or more. In particular embodiments, the composition further comprises a plurality of said placental stem cells. In another embodiment, the composition comprises a plurality of non-placental cells. In a more specific embodiment, said non-placental cells comprise CD34⁺Cells, e.g., hematopoietic progenitor cells, such as peripheral blood hematopoietic progenitor cells, umbilical cord blood hematopoietic progenitor cells, or placental blood hematopoietic progenitor cells. The non-placental cells may also comprise other stem cells, for example mesenchymal stem cells, such as bone marrow-derived mesenchymal stem cells. The non-placental cells can also be one or more types of adult cells or cell lines. In another embodiment, the composition comprises an anti-proliferative substance, such as an anti-MIP-1 α or anti-MIP-1 β antibody.

5.6.1.4 matrix comprising placental stem cells

The invention also encompasses matrices, hydrogels, scaffolds, and the like comprising the immunosuppressive placental stem cell population.

The placental stem cells of the invention can be seeded onto a natural substrate, for example, a placental biological material, such as an amniotic membrane material. These amniotic membrane materials may be, for example, amniotic membranes which are divided directly from mammalian placentas; fixed or heat treated amniotic membrane, substantially dry (i.e., less than 20% moisture content) amniotic membrane, chorion, substantially dry amniotic membrane, chorion, and the like. Preferred placental biomaterials that can be seeded with placental stem cells are described in U.S. application publication No. 2004/0048796 to Hariri.

The placental stem cells of the invention can be suspended in a hydrogel solution for, e.g., injection. Hydrogels suitable for these compositions include self-assembling polypeptides, such as RAD 16. In one embodiment, the hydrogel solution containing the cells may be allowed to harden, for example in a mold to form a matrix in which the cells are dispersed for implantation. Placental stem cells in this matrix can also be cultured such that the cells are mitotically expanded prior to implantation. Hydrogels are, for example, organic polymers (natural or synthetic) which are cross-linked by covalent, ionic or hydrogen bonds to form a three-dimensional open lattice structure which entraps water molecules to form a gel. Hydrogel-forming materials include ionically crosslinked polysaccharides such as alginic acid and its salts, polypeptides, polyphosphazines, and polyacrylic acids, or block copolymers such as polyethylene oxide-polypropylene glycol block copolymers, which are crosslinked by temperature or pH, respectively. In some embodiments, the hydrogel or matrix of the invention is biodegradable.

In some embodiments of the invention, the compositions comprise in situ polymerizable gels (see, e.g., U.S. patent application publication No. 2002/0022676; Anseth et al, J.control Release, 78 (1-3): 199-209 (2002); Wang et al, Biomaterials, 24 (22): 3969-80 (2003)).

In some embodiments, the polymer is at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous ethanol solutions, having charged pendant groups or monovalent ion salts thereof. Examples of polymers having acidic side groups that can react with cations are poly (phosphazenes), poly (acrylic acids), poly (methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate), and sulfonated polymers such as sulfonated polystyrene. Copolymers having acidic side groups formed by the reaction of acrylic or methacrylic acid with vinyl ether monomers or polymers may also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) ethanol groups, phenolic OH groups and acidic OH groups.

The placental stem cells of the invention or their co-cultures can be seeded onto a three-dimensional framework or scaffold and implanted into the body. Such a framework may be implanted in combination with any one or more growth factors, cells, drugs or other components that stimulate tissue formation or otherwise enhance or improve the practice of the invention.

Examples of scaffolds that can be used in the present invention include nonwoven mats, porous foams, or self-assembling polypeptides. The nonwoven felt may be formed using fibers including synthetic absorbable glycolic acid and lactic acid copolymers (e.g., PGA/PLA) (VICRYL, Ethicon, samevill, new jersey). Foams formed by procedures such as freeze-drying, or lyophilization (see, e.g., U.S. Pat. No. 6,355,699) composed of, for example, poly (epsilon-caprolactone/poly (glycolic acid) (PCL/PGA) copolymers can also be used as scaffolds.

The placental stem cells of the present invention can also be seeded onto or contacted with physiologically acceptable ceramic materials, including, but not limited to: mono-, di-, tri-, alpha-, beta-and tetra-calcium phosphates, hydroxyapatite, fluorapatite, calcium sulphate, calcium fluoride, calcium oxide, calcium carbonate, magnesium salts of calcium phosphates, bioactive glasses such asAnd mixtures thereof. The porous biocompatible ceramic materials currently commercially available include(Canmedica corporation, Canada),(Merck Biomaterial France, France),(Mathys, AG, Bettlach, Switzerland) and mineralized collagen-based bone graft products such as HEALOS^TM(DePuy, Raynham, Mass.) andRHAKOSS^TMand, and(Orthovita, Marvin, Pa.). The framework may be a mixture, blend or composite of natural and/or synthetic materials.

In another embodiment, placental stem cells can be seeded onto or contacted with a felt, which can be made, for example, from multifilament yarns made using bioabsorbable materials such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.

The placental stem cells of the invention can, in another embodiment, be seeded onto a foam scaffold of a composite structure. These foam scaffolds can be molded into useful shapes, such as the shape of a portion of a particular structure of the body that requires repair, replacement, or augmentation. In some embodiments, the frame is treated, for example with 0.1M acetic acid, then cultured in polylysine, PBS, and/or collagen, followed by seeding with cells of the invention to enhance cell attachment. The outer surface of the matrix may be modified to improve cell attachment or growth and tissue differentiation, such as by coating the matrix with plasma, or by adding one or more proteins (e.g., collagen, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), cell matrices, and/or other materials such as, but not limited to, gelatin, alginate, agar, agarose, vegetable gums, and the like.

In some embodiments, the scaffold comprises or is treated with a material that renders the scaffold non-thrombogenic. These treatments and materials may also promote and sustain endothelial growth, transplantation, and extracellular matrix deposition. Examples of such materials and treatments include, but are not limited to: natural materials such as basement membrane proteins, e.g. laminin and type IV collagen, synthetic materials such as EPTFE, and blocked polyurethaneurea silicones, e.g. PURSPAN^TM(The Polymer Technology Group company, Burkely, Calif.). The stent may also contain an anti-thrombotic agent, such as heparin; scaffolds may also be treated to alter surface charge (coated with plasma) prior to seeding with placental stem cells.

5.6.2 immortalized placental stem cell lines

Mammalian placental cells can be conditionally immortalized by transfection with any suitable vector comprising growth-promoting genes, genes encoding the following proteins: which under appropriate conditions promote growth of the transfected cells such that the production, and/or activity, of the growth promoting proteins can be regulated by external factors. In a preferred embodiment, the growth-promoting gene is an oncogene, such as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T antigen, polyoma virus large T antigen, E1a adenovirus, or human papilloma virus E7 protein.

External regulation of the growth-promoting protein may be achieved by placing the growth-promoting gene under the control of an externally controllable promoter, e.g., a promoter whose activity may be controlled, e.g., by altering the temperature of the transfected cell or the composition of the medium contacting the cell. In one embodiment, a tetracycline (tet) controlled gene expression system may be used (see, Gossen et al, Proc. Natl. Acad. Sci. USA 89: 554)7-5551, 1992; hoshimaru et al, proc.natl.acad.sci.usa93: 1518-1523, 1996). Tet in this vector controls the strong activation of the transcriptional activator (tTA) ph in the absence of tet_CMV*-1Transcription of the minimal promoter from human cytomegalovirus fused to the tet operator sequence. tTA is a fusion protein of the repressor protein (tetR) of the transposon-10-derived anti-tet operon of E.coli and the acidic domain of herpes simplex virus VP 16. Low, non-toxic concentrations of tet (e.g., 0.01-1.0. mu.g/mL) almost completely abolished transcriptional activation of tTA.

In one embodiment, the vector further comprises a gene encoding a selectable marker, such as a protein that renders the drug resistant. Bacterial neomycin resistance gene (neo)^R) Is such a marker that may be used in the present invention. With neo^RLabeled cells may be selected by methods well known in the art, such as by adding, for example, 100-.

Transfection may be performed by any of a variety of methods known in the art, including but not limited to retroviral infection. Typically, cell cultures can be transfected by co-culture with a mixture of conditioned media collected from the vector-producing cell line and DMEM/F12 media containing N2 supplement. For example, placental cell cultures prepared as described above can be infected in vitro after five days, e.g., by culturing in one volume of conditioned medium and two volumes of DMEM/F12 containing N2 supplement for about 20 hours. Transfected cells with the selectable marker are then selected as described above.

Following transfection, the medium is passaged onto a surface that allows proliferation, e.g., allowing at least 30% cell doubling over a 24 hour period. Preferably, the substrate is a polyornithine/laminin substrate, consisting of a tissue culture plastic coated with polyornithine (10 μ g/mL) and/or laminin (10 μ g/mL); polylysine/laminin matrix or surfaces treated with fibronectin. The cultures are then fed every three to four days with growth medium, which may or may not be supplemented with one or more proliferation-enhancing factors. The proliferation-enhancing factor may be added to the growth medium when the confluency of the culture is less than 50%.

When the degree of confluence reaches 80-95%, the conditionally-immortalized placental stem cell lines can be passaged using standard techniques, e.g. by trypsinization. In some embodiments, it is advantageous to maintain the screen until about the twentieth passage (by, for example, adding G418 to cells containing the anti-neomycin gene). Cells may also be frozen in liquid nitrogen for long term storage.

Clonal cell lines can be isolated from conditionally-immortalized human placental stem cell lines prepared as described above. Typically, such clonal cell lines can be isolated and expanded using standard techniques, for example by limiting dilution methods or using clonal loop isolation. Clonal cell lines can generally be fed and passaged as described above.

Conditionally-immortalized human placental stem cell lines, which may, but need not, be clonal, can generally be induced to differentiate by inhibiting the production and/or activity of growth-promoting proteins in culture conditions that promote differentiation. For example, if the gene encoding a growth-promoting protein is under the control of an externally controllable promoter, transcription of the growth-promoting gene can be inhibited by changing conditions, such as temperature or media composition. For the tetracycline-controlled gene expression systems discussed above, differentiation can be achieved by adding tetracycline to inhibit transcription of the growth-promoting gene. Typically, 1. mu.g/mL tetracycline for four to five days is sufficient to initiate differentiation. To promote further differentiation, other agents may be included in the growth medium.

5.6.3 test

Embryonic stem cells of the present invention can be used in assays to detect the effect of culture conditions, environmental factors, molecules (e.g., biomolecules, small inorganic molecules, etc.), and the like, on the proliferation, expansion, and/or differentiation of stem cells relative to placental stem cells not exposed to such conditions.

In a preferred embodiment, the placental stem cells of the invention are tested for changes in proliferation, expansion or differentiation upon exposure to a molecule. In one embodiment, for example, the invention provides a method of identifying a compound that modulates proliferation of a plurality of placental stem cells, the method comprising contacting said plurality of stem cells with said compound under conditions that allow proliferation, wherein said compound is identified as a compound that modulates proliferation of placental stem cells if said compound causes a detectable change in proliferation of said plurality of stem cells relative to a plurality of stem cells not contacted with said compound. In particular embodiments, the compound is identified as a proliferation inhibitor. In another specific embodiment, the compound is identified as a proliferation promoter.

In another embodiment, the present invention provides a method of identifying a compound that modulates the expansion of a plurality of placental stem cells, the method comprising contacting said plurality of stem cells with said compound under conditions that allow expansion, wherein said compound is identified as a compound that modulates the expansion of placental stem cells if said compound causes a detectable change in the expansion of said plurality of stem cells relative to a plurality of stem cells not contacted with said compound. In particular embodiments, the compound is identified as an amplification inhibitor. In another specific embodiment, the compound is identified as an amplification enhancer.

In another embodiment, the present invention provides a method of identifying a compound that modulates differentiation of placental stem cells, the method comprising contacting said plurality of stem cells with said compound under conditions that allow differentiation, wherein said compound is identified as a compound that modulates differentiation of placental stem cells if said compound causes a detectable change in differentiation of said plurality of stem cells relative to a plurality of stem cells not contacted with said compound. In a specific embodiment, the compound is identified as a differentiation inhibitor. In another specific embodiment, the compound is identified as a differentiation promoter.

5.6.4 placental Stem cell Bank

Stem cells from a postpartum placenta can be cultured in a number of different ways to prepare a series of placental stem cell sets, e.g., a series of doses for individual administration. These groups may, for example, be obtained from stem cells derived from placental perfusate or enzymatically digested placental tissue. A panel of placental stem cells obtained from a large number of placentas may be arranged in a placental stem cell bank for, e.g., long-term storage. Generally, adherent stem cells can be obtained from an initial culture of placental material to form a seed culture that is multiplied approximately a significant number of times under controlled conditions to form a population of cells. Groups are preferably derived from tissue of a single placenta, but may be derived from tissue of multiple placentas.

In one embodiment, the stem cell group is obtained according to the following method. The placental tissue is first disrupted, for example by mincing, and digestion with a suitable enzyme, for example collagenase (see section 5.2.3 above). The placental tissue preferably comprises, e.g., intact amniotic membrane, intact chorion, or both, from a single placenta, and may also comprise a portion of the amniotic membrane or chorion. The digested tissue is cultured, for example for about one to three weeks, preferably about two weeks. After removal of the unattached cells, the high density of colonies formed are collected, for example by trypsinization. These cells were collected and resuspended in an appropriate volume of medium and defined as passage 0 cells.

The 0 th generation cells were then used to inoculate the expansion medium. The expansion culture may be any combination of separate cell culture devices, e.g., NUNC^TMThe cell factory of (1). The cells in the 0 th generation culture may be subdivided to any degree, for example by 1X 10³、2×10³、3×10³、4×10³、5×10³、6×10³、7×10³、8×10³、9×10³、1×10⁴、1×10⁴、2×10⁴、3×10⁴、4×10⁴、5×10⁴、6×10⁴、7×10⁴、8×10⁴、9×10⁴Or 10X 10⁴And inoculating the stem cells with an amplification medium. Preferably, approximately 2X 10 is used per amplification medium⁴To about 3 x 10⁴And 0 th generation cells. The number of expansion cultures may depend on the number of cells at passage 0, and may be greater or lesser in number depending on the particular placenta from which the stem cells are obtained.

The expanded culture is grown until the cell density in the culture medium reaches a certain value, for example about 1X 10⁵One cell per square centimeter. At this point the cells are collected and either cryopreserved or passaged to fresh expansion medium as described above. Prior to use, the cells may be passaged, for example, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. The cumulative number of colony doublings is preferably maintained during the amplification culture. Cells from the 0 th generation culture can be expanded 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 times, or up to 60 doublings. Preferably, however, the number of colony doublings before dividing the cell colony into individual doses is between about 15 and about 30, preferably about 20 doublings. During the expansion process, the cells may be continuously cultured, or may be frozen at one or more points during expansion.

Cells for a single dose may be frozen, e.g., cryopreserved for later use. A single dose may contain, for example, from about one million to about one hundred million cells per milliliter, and may contain a total of about 10⁶From about to about 10⁹Cells in between.

In a specific embodiment of the method, passage 0 cells are cultured to about 4 doublings and then frozen in a first cell bank. The cells in the first cell bank are frozen and used to seed a second cell bank in which the cells are expanded for approximately another 8 doublings. Cells at this stage were collected and frozen and used to inoculate media that allowed approximately 8 additional doublings to be made, resulting in a cumulative number of cell doublings of approximately 20. Cells at the midpoint of passage may be frozen in units of about 100,000 to about ten million cells per ml, preferably about one million cells per ml, for use in subsequent expansion cultures. Cells that are multiplied by about 20 times may be frozen in a single dose of about one million to about one hundred million cells per milliliter for administration or use in the form of a composition comprising stem cells.

In a preferred embodiment, a donor (e.g., a mother) from which the placenta is obtained is tested for at least one pathogen. If the maternal was positive for the pathogen tested, all groups from the placenta were discarded. Such testing can be performed at any time during the preparation of the placental stem cell population, including before or after the establishment of passage 0 cells, or during expansion culture. Pathogens whose presence is being tested for may include, but are not limited to: hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human immunodeficiency virus (type I and type II), cytomegalovirus, herpes virus, and the like.

5.6.5 treatment of multiple sclerosis

In another aspect, the invention provides a method of treating an individual having multiple sclerosis or a symptom associated with multiple sclerosis, the method comprising administering to the individual a plurality of embryonic stem cells in an amount and for a time sufficient to detectably modulate, e.g., inhibit, an immune response in the individual.

Multiple Sclerosis (MS) is a chronic, recurrent disease of the central nervous system. The disease results in damage to the myelin sheaths surrounding the axons of the CNS and PNS, to oligodendrocytes and to the nerve cells themselves. The disease is caused by autoreactive T cells, particularly CD4⁺T cell mediation, which cells proliferate, cross the blood brain barrier, and enter the CNS under the influence of cell adhesion molecules and pro-inflammatory cytokines. Symptoms of multiple sclerosis include sensory disturbances in the limbs, optic nerve dysfunction, pyramidal tract dysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction, ataxia, and diplopia.

Four different types or clinical courses of MS have been identified. First, relapsing/remitting multiple sclerosis (RRMS) is characterized by an autogenous restricted episode of neurological dysfunction that has a severe episode lasting from days to weeks, followed by a period of recovery, sometimes incomplete, lasting months. The second type, Secondary Progressive Multiple Sclerosis (SPMS), begins as RRMS, but changes so that the clinical course becomes characterized by a stable functional deterioration, not involving a sharp attack. Third, Primary Progressive Multiple Sclerosis (PPMS), which is characterized by a stable decline in function from onset, without a sharp onset. The fourth type, progressive/relapsing multiple sclerosis (PRMS), also begins with a progressive process with occasional attacks of progressive decline in function.

Persons with MS are typically evaluated by motor function assessment, optionally together with MRI. For example, one motor function assessment method, an expanded disability status scale, scores the ability of individuals affected by disease as follows:

0.0 neurological check Normal

1.0 No disability, minimal signal in one FS

1.5 No disability, minimal signal among multiple FSs

2.0 minimal disability in one FS

2.5 mild disability in one FS or minimal disability in both FSs

3.0 with moderate disability in one FS, or mild disability in three or four FS. Completely movable

3.5 completely ambulatory, but with moderate disability in one FS and over-minimal disability in several other systems

4.0 completely ambulatory, without assistance, self-care of life, and about 12 hours of getting up to walk every day despite of considerable disability; can walk about 500 meters without assistance or rest

4.5 can completely walk without assistance, the patient can get up and walk for most of time every day, the patient can work all day, and the complete activity can have some limitations or needs minimum help; characterized by a rather severe disability; can walk about 300 meters without assistance or rest

5.0 walk about 200 meters without assistance or rest; disability severe enough to hurt daily activities throughout the day (working throughout the day without special equipment)

5.5 walk about 100 meters without assistance or rest; disability is severe enough to prevent daily activities throughout the day

6.0 walk about 100m with intermittent assistance or with one-side continuous assistance (bamboo cane, crutch, stand), with or without rest

6.5 continuous double-side assistant (bamboo cane, crutch, bracket) walks about 20 meters without rest

7.0 cannot walk more than about 5 meters even with assistance, being substantially confined to wheelchairs; the wheelchair is moved into a standard wheelchair and is moved independently; getting up and walking on the wheelchair for about 12 hours a day

7.5 walk no more than a few steps; restrained on a wheelchair; assistance may be required when moving; the wheelchair is moved by self, but cannot stay on a standard wheelchair all day long; may require an automatic wheelchair

8.0 basically confined to walking on a bed or chair or on a wheelchair, but can leave the bed most of the time each day; the self-care function is kept; there is generally effective use of the arms

8.5 most of each day is substantially confined to bed; has some effective use of arms and partial self-care function

9.0 is confined to the bed; can still communicate and eat

9.5 completely helpless bedridden patients; inability to effectively communicate or feed/swallow

10.0 MS-induced death

In the above scoring system, "FS" refers to eight functional systems measured, including pyramidal, cerebellum, brainstem, sensory, intestinal and bladder, visual, cerebral and other systems.

In addition, similar scoring systems are known, including the Scripps neurological grade scale, walking ability index, and multiple sclerosis functional composite score (MSFC).

The progression of MS was also assessed by determination of the grade of onset.

The progression of MS is also assessed by magnetic resonance imaging, which can detect MS-associated damage to the nervous system (e.g., new damage, enhanced damage, or combined unique active damage).

Accordingly, in one embodiment, the invention provides a method of treating an individual having MS, e.g., an individual diagnosed with MS, comprising administering to the individual a plurality of placental stem cells, wherein said placental stem cells are capable of differentiating into oligodendrocytes, e.g., into oligodendrocytes in the individual. In a specific embodiment, the administering detectably ameliorates one or more symptoms of MS in the individual. In more specific embodiments, the symptom is, for example, one or more of a sensory disorder of a limb, an optic nerve dysfunction, a pyramidal tract dysfunction, a bladder dysfunction, an intestinal dysfunction, a sexual dysfunction, ataxia, or double vision. In another specific embodiment, the administration results in at least a half-score improvement in the EDSS scale. In another specific embodiment, the administration results in at least a one point improvement in the EDSS scale. In another specific embodiment, the administration results in at least two separate improvements in the EDSS scale. In other specific embodiments, the administration results in a detectable improvement in a multiple sclerosis assessment scale or in an MRI.

MS has been treated with other therapeutic agents, such as immunomodulators or immunosuppressants, e.g., interferon beta (IFN β), including IFN-1 a and IFN-1 b; glatiramer acetate (Copaxone corporation); cyclophosphamide; methotrexate; azathioprine (Imuran corporation); cladribine (Leustin Corp.); cyclosporine; mitoxantrone; and so on. MS is also treated with anti-inflammatory therapeutic agents, such as glucocorticoids of the adrenal gland, including corticotropin (ATCH), methylprednisolone, dexamethasone, and the like. MS is also treated with other types of therapeutic agents, such as intravenous immunoglobulin, plasmapheresis, or sulfasalazine.

Accordingly, the invention also provides a method of treating an individual having MS, e.g., an individual who has been diagnosed with MS, comprising administering to the individual a plurality of placental stem cells and one or more therapeutic agents, wherein said administering detectably improves one or more symptoms of MS in the individual, and wherein the placental stem cells are capable of differentiating into oligodendrocytes, e.g., oligodendrocytes within the individual. In one embodiment, the therapeutic agent is a glucocorticoid. In particular embodiments, the glucocorticoid is adrenocorticotropic hormone (ATCH), methylprednisolone, or dexamethasone. In another embodiment, the therapeutic agent is an immunomodulatory agent or an immunosuppressive agent. In various embodiments, the immunomodulator or immunosuppressant is IFN beta-1 a, IFN-1b, glatiramer acetate, cyclophosphamide, methotrexate, azathioprine, cladribine, cyclosporine, or mitoxantrone. In other embodiments, the therapeutic agent is an intravenous immunoglobulin, plasmapheresis, or sulfasalazine. In another embodiment, the individual is administered any combination of the foregoing therapeutic agents.

Individuals with MS, e.g., individuals diagnosed with MS, may be treated with a plurality of placental stem cells, and optionally one or more therapeutic agents, at any time during the course of the disease. For example, the individual may be treated immediately after diagnosis, or within 1, 2, 3, 4, 5,6 days of diagnosis, or within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more weeks of diagnosis, or within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more years of diagnosis. The individual may be treated one or more times during the clinical course of the disease. The individual may be suitably treated during a severe episode, during remission, or during a chronic degenerative stage. In another embodiment, the placental stem cells are administered to a prenatal woman with MS to maintain a state of remission or reduced recurrence experienced during pregnancy. In one embodiment, the individual is administered a dose of about three hundred million placental stem cells. However, the dosage may vary, depending on the physical characteristics of the individual, such as body weight, and may range from one million to one hundred million placental stem cells, preferably from ten million to one billion placental stem cells, or from one hundred million to fifty million placental stem cells per dose. The administration is preferably intravenous, but may be by any route accepted in the art for administration of living cells. In one embodiment, the placental stem cells are from a cell bank.

6. Examples of the embodiments

6.1 example 1: oligodendrocyte maintenance medium

Typical culture media for oligodendrocytes are as follows: preferred media are serum-free formulations, optimal for the maintenance of rodent OL lineage cells. Basal medium (R1236) contained high glucose DMEM (Sigma) supplemented with 1mM sodium pyruvate, antibiotics (penicillin-streptomycin), 0.05. mu.g/ml insulin to stimulate glucose transporters, 100. mu.g/ml transferrin (iron uptake), 30nM selenium (metabolic cofactor), 10. mu.M forskolin (forskolin) (cAMP), 60. mu.g/ml N-acetylcysteine (Redox, Life) and 5. mu.g/ml bovine serum albumin (transporter). Rodent oligodendrocyte precursor cells were maintained using R1236, supplemented with mitogen to promote proliferation and autoreaction (10ng/mL PDGF-AA +5ng/mL FGF2 or 20% v: vB104 conditioned medium). To promote oligodendrocyte differentiation, the mitogen-containing medium was replaced with R1236 containing 10 μ g/ml bovine insulin +5 μ g/ml T3 (triiodothyronine), both bovine insulin and T3 being survival and maturation factors for oligodendrocytes in rodents. All growth factors contained in the medium were recombinant (human) polypeptides (R & D) and B104-cm was prepared from neuroblastoma cells (available from ATCC) cultured to 50% confluence and then exposed to R123648 for hours. This conditioned medium was then filtered and stored in aliquots at-30 ℃ until use. B104 is one of many nervous system cell lines established by Dave Schubert of Salk's college (Schubert et al, Nature 249: 224-227(1974)), and secretes factors that support the survival and self-renewal of rodent oligodendrocyte precursor cells.

6.2 example 2: obtaining stem cells from placenta by enzymatic digestion

An exemplary protocol for obtaining stem cells from placental tissue by enzymatic digestion is as follows: frozen placenta tissues (about 1X 0.5cm each in length, width and height) were obtained. The tissue is umbilical cord, maternal surface of placenta or amnion. The digestive enzymes used included trypsin-EDTA (0.25%, GIBCO BRL Co.); collagenase IA (Sigma), collagenase I (Worthington), collagenase IA (Sigma) + trypsin-EDTA, collagenase I (Worthington) + trypsin-EDTA, or elastase + collagenase I + collagenase IV + dispase (Worthington). Placental tissue was digested as follows: the tissue was minced in the presence of enzyme (1g in 10 ml of enzyme in 50ml tube), the tube was placed at 45 ℃ and shaken at 37 ℃ for 1 hour at 250 rpm (C25 culture shaker, New Brunswick scientific, Edison, N.J., USA). The supernatant was then discarded. The pellet was washed three times with 20 ml Hank's + 5% FCS and resuspended in 12 ml of medium. The resulting 3ml of suspension was aliquoted into T-75 flasks (four for each digestion) each containing 10 ml of medium. Alternatively, 10 ml trypsin/EDTA was added, shaken at 37 ℃ at 250 rpm for 30 minutes, recentrifuged and washed once more with 10 ml Hank's + 5% FCS. Cells were seeded and cultured, and adherent cells were selected.

6.3 example 3: oligodendrocyte precursor lineage test

The appearance, maturation and differentiation of oligodendrocyte precursor cells can be detected by immunochemistry and transcriptional expression. For immunochemistry, cells grown on glass coverslips are cultured in a medium containing a concentration of growth factors. Coverslips were removed after 1-7 days, fixed with 4% paraformaldehyde, and then characterized using lineage specific antibodies (table 1). The staining was detected using a secondary antibody (Alexa Fluors, Molecular Probes) linked to a fluorescent label and observed by fluorescence microscopy. As a negative control, the secondary antibody alone was used. The proportion of immunoreactive cells was determined by counting up to 200 cells per cover slip.

Table 1. reagents for immunohistochemistry:

stage (2): antibody-specific target site source literature

nSC：Nestin Ms IgG axial filament DSHB (Johe et al, 1996)

NRP：e-NCAM Ms IgG axial filament DSHB

GRP：A2B5 Ms IgM ganglioside conditioned medium (Eisenbarth et al,

(ATCC) 1979)

OPC：olig2 Rabbit IgG bHLH factor (H.Yakoo, (Sun et al, 2001)

JP)

NG2 rabbit IgG proteoglycans, Chemicon (Nishiyama et al,

1996)

pdgfra goat IgG PDGF receptor R & D Inc. (Matsui et al, 1989)

Unc5b goat IgG Netrin receptor R & D Inc. (Lu et al, 2004)

O4 Ms IgM thioester CM (Bansal et al, 1989)

OL：O1 Ms IgM GalC CM (Raff et al, 1978)

CNPase Ms IgG myelin Sigma (Pfeiffer et al, 1993)

CNPase

MBP Rabbit IgG myelin basic Chemicon (Pfeiffer et al, 1993)

Inc

Nerve cellNF-H neurofilament protein Virginia Lee

Star gumGFAP Rabbit IgG glial microglia (Pfeiffer et al, 1993)

Cells Silk

CM-conditioned Medium

Immunohistochemical studies were extended by analyzing transcriptional expression under specific culture conditions. RNA analysis was performed using Northern blotting (McKinnon et al, 1990) and RT-PCR (McKinnon et al, 1993 b). Cells grown in 60mm plates were recovered and RNA was harvested using TRIzol reagent (Gibco Co.). For RT-PCR, analysis was performed by reverse transcription of 1. mu.g RNA into cDNA (MoMuLV reverse transcriptase; cDNA yield 1: 1). Then, 50-100ng of cDNA was used as a template for PCR amplification, and synthesized primers selected using Taq polymerase and the primer selection procedure described in the literature (see, e.g., McKinnon et al, Glia 7: 245-. The primers are constructed to hybridize to transcripts encoding lineage specific oligodendrocytes and oligodendrocyte precursor proteins. For the new primer pairs, a gradient (+ -10 ℃) was used to establish the optimal amplification parameters. The PCR fragments were resolved by electrophoresis, visualized by EtBr staining, and their identity determined by automated DNA sequence analysis (DNAcore facility).

6.4 example 4: proliferation, migration and survival assays

Proliferation test: quantification of the ability of oligodendrocyte precursors to produce mitogenic responses to specific ligands³H-Thymidine incorporation test. Cells were exposed to growth factors at doses that elicited maximal responses of FGF and PDGF to test half maximal response values. The response ranged from 500-1000cpm (no growth factor) to 10,000cpm (recombinant PDGF-AA) in the background, and the test was sensitive enough to accurately detect a partial mitogenic response. All cell proliferation assays are optionally performed at least 3 times independently. For qualitative testing, cells were exposed to mitogens in the presence of 50. mu.M BrdU (Sigma) for the last four hours and DNA synthesis was monitored by a dual fluorescent immunoassay for BrdU (Osterhout et al, J. Neurosci.17: 9122-9132(1997)) and a second lineage marker.

Subjecting the cells to a proliferation assay, the cells being exposed toMitogens had been removed 24 hours prior to recombinant growth factors to reduce background levels of DNA synthesis. The proliferation assay described herein passes through increased DNA synthesis and incorporation during the last 4 hours of this assay depending on exposure to growth factors³H-thymine, and thus the response to mitogens was measured. Cells in 96-well plates (2,000 cells/well) were cultured in R1236 medium lacking growth factors for 24 hours, and then in the presence of specific concentrations of factors for 24 hours at 0.5. mu. Ci/ml³The culture was carried out in the presence of H-thymine (Amersham) for the last 4 hours. The nucleic acids were recovered using an automated harvester (Brandel Co.) and the incorporated radioactivity was measured by scintillation counting. Three sets (three wells) of tests were performed for each growth factor concentration.

Migration test: the migratory capacity of oligodendrocyte precursor cells (chemotaxis) and their directional response in response to growth factors are measured by cinematography. Quantitative testing a modified Boyden cell test was used (Armstrong et al, 1990). In this test, PDGF-AA mediated chemotaxis (4,000 cells/mm) can be distinguished from background migration (1,000 cells/mm) and chemomotility (voluntary movements) by adding an inducer to both the upper and lower wells of the chamber (preventing chemotaxis, but not chemomotility) (see Armstrong et al, J.Neurosci. Res.27: 400-407 (1990)).

Cells were cultured for 16 hours in mitogen-deficient medium, defined as medium separated from the lower chamber containing attractant medium by a polycarbonate filter, and then transferred to the top wells of the microtomograph chamber (20,000 cells/well). Three wells were added for each concentration of growth factor, and cells were cultured at 37 ℃ for 16 hours. The number of cells migrating per square millimeter of the lower layer of the filter was determined by counting GFP-labeled cells, and the total number of migrations was determined after staining the membrane using Dip-Quik (American Scientific).

Survival test: the viability of oligodendrocyte precursor cells alone was determined using the MTT test (Mosmann, 1983) and using chromatin fragments (nucleosome ladder bands) as described (Yasuda et al, J.Neurosci.Res.40: 306-317 (1995)) using a modified TUNEL test (Gavrili et al, J.cell.biol.119: 493-501 (1992)). Cells were cultured in bFGF, PDGF-AA or growth factor-free medium for 24 hours, and the number of MTT-incorporated cells or the level of notch end markers was compared between mutant and wild-type OL cultures. Wortmannin, an inhibitor of PI3K, was used as a positive control for cell death (Ebner et al, j. neurosci. res.62: 336-345 (2000)).

MTT assays were performed on cells grown in 96-well plates and the proportion of labeled cells was determined by counting stained cells as described previously (Barres et al, Cell 70: 31-46 (1992)). The ability of bFGF and PDGF-AA to prevent DNA fragmentation was detected by analysis of chromatin DNA from cells grown in the presence or absence of increasing concentrations of these factors. For quantitative analysis, cells grown in 96-well plates were incubated at 37 ℃ in a medium containing 4 units of terminal transferase (Promega Biotech, Inc.), 2. mu. Ci [ a-³²P]Dideoxy ATP (Amersham), and 0.3% Triton X-100 in PBS for 60 minutes, then Cell lysates were harvested on Whatman GF/C filters (Brandel Cell Harvester), and the 3' -end of the DNA was detected by liquid scintillation counting³²And (4) doping P. The assay was enzyme dependent and produced a background of 10,000cpm, and 100,000cpm was incorporated into cells cultured in the presence of 1 μ M staurosporine (Ebner, 2000) for 72 hours. For qualitative analysis of nucleosome ladder bands, DNA was isolated from cells grown in 35mm dishes using terminal transferase and³²the ends were labeled in vitro with P-ddATP, nucleosome-sized fragments were resolved by size in an agarose gel, and incorporated radioactivity was detected by densitometry.

6.5 example 5: flow cytometry

For intracellular staining for flow cytometry, approximately 5X 10 were paired with 0.5 ml of Beckman Coulter IntraPrep reagent⁵Cell membranes of individual PDSCs were permeabilized for 15 minutes. After washing with PBS, cells were co-incubated with primary antibody (1g) on ice for 30 minutes, followed by washing twice. Cells were resuspended in 1: 100 secondary antibody and incubated for 30 minutes. After staining, cells were washed twice and immediately analyzed on a Beckmann Coulter XL-MCL flow cytometer. To assess protein expression in untreated cells and IBMX-induced cells, 1.5X 10 using FL1(FITC) and FL2(CY3) signals were collected⁴And (4) cells. Dead cells and debris are excluded by using a highly forward and orthogonal light scattering window or by Propidium Iodide (PI) exclusion.

Primary antibodies and final dilutions for mice, rabbits and donkeys were as follows: rabbit anti-nestin, 1: 100 (BDPharMingen); mouse anti-neuron specific enolase, 1: 100(Chemicon Corp.); mouse anti-myelin/oligodendrocyte-specific protein, 1: 100(DAKO Corp.); mouse anti-neurofilament protein-L, 1: 100(DAKO Corp.); rabbit anti-glial fibrillary acidic protein, 1: 200 (DAKO); mouse anti-vimentin, 1: 100(BD PharMingen). The following antibodies used in the flow cytometry experiments were obtained from Becton Dickinson and were used at a dilution of 1: 10: anti-CD 45, anti-CD 34, anti-CD 29, anti-CD 10, anti-HLA-1, anti-CD 54, anti-CD 90, anti-SH 2, and anti-SH 3.

Cells were cultured on polyornithine-coated glass coverslips (Sigma) in DMEM containing 10% FCS. Cells were fixed in PBS with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. The cells and primary antibody were then co-incubated at 37 ℃ for 30 minutes. Cells were then washed three times with PBS and co-incubated with Fluorescein (FITC) -conjugated donkey anti-mouse IgG (Jackson laboratories) or Cy 3-conjugated goat anti-rabbit IgG (Jackson laboratories), both incubated at 37 ℃ for 30 minutes in the dark at a dilution of 1: 50. The labeled cells were washed and mounted with Vectashield mounting medium (Vector laboratories).

Claims (6)

1. A method of producing oligodendrocytes, the method comprising culturing placenta-derived stem cells under conditions and for a time sufficient for said stem cells to exhibit oligodendrocyte characteristics.

2. The method of claim 1, wherein the characteristic is production of myelin oligodendrocyte-specific protein, or expression of a gene encoding myelin oligodendrocyte-specific protein.

3. The method of claim 1, wherein said culturing comprises contacting said stem cells with Isobutylmethylxanthine (IBMX).

4. An oligodendrocyte produced by the method of claim 1.

5. A method of treating a subject having a disease, disorder or condition associated with abnormal myelination, the method comprising introducing the oligodendrocyte of claim 4 into the subject.

6. The method of claim 5, wherein the disease, disorder or condition is multiple sclerosis.