CN101326280A - Immunomodulation using placental stem cells - Google Patents

Abstract

The present invention provides methods of immunomodulation using placental stem cells and placental stem cell populations. The invention also provides methods for producing and selecting placental cells and cell populations based on immunomodulation and compositions comprising such cells and cell populations.

Description

Immunomodulation using placental stem cells

1. Technical field

The present invention provides methods of modulating the immune system and immune response to antigens using placental stem cells. The present invention also provides compositions comprising placental stem cells for immunoregulation, and methods of transplanting tissues and organs comprising administering placental stem cells to prevent or suppress immune-mediated rejection.

2. Background technology

Human stem cells are totipotent or multipotent precursor cells capable of giving rise to various mature human cell lines. There is evidence that stem cells can be used to repopulate a variety of tissues (if not all) and restore physiological and anatomical function.

Many different types of mammalian stem cells have been characterized. See, eg, Caplan et al., U.S. Patent No. 5,486,359 (human mesenchymal stem cells); Boyse et al., U.S. Patent No. 5,004,681 (fetal and neonatal hematopoietic stem and progenitor cells); Boyse et al., U.S. Patent No. 5,192,553 (identical); Beltrami et al., Cell 114(6):763-766 (2003) (cardiac stem cells); Forbes et al., J. Pathol 197(4):510-518 (2002) (hepatic stem cells). Umbilical cord blood and total nucleated cells derived from umbilical cord blood have been used in transplantation to partially or completely restore hematopoiesis in patients undergoing stripping therapy.

The placenta is a particularly interesting source of stem cells. Because mammalian placentas are abundant and often discarded as medical waste, they represent a unique source of medically useful stem cells. The present invention provides these isolated placental stem cells, populations of placental stem cells and methods of their use.

3. Outline of the invention

The present invention provides a method for immunosuppression using a variety of placental stem cells or umbilical cord stem cells, placental stem cell populations or umbilical cord stem cell populations, and compositions containing and/or prepared from the stem cells. The present invention also provides compositions, including compositions comprising placental stem cells or umbilical cord stem cells having immunosuppressive properties. The invention further provides populations of placental cells or umbilical cord stem cells selected on the basis of their ability to modulate an immune response, and compositions having immunomodulatory properties.

In one aspect, the invention provides a method of suppressing or reducing an immune response comprising contacting a plurality of immune cells with a plurality of placental stem cells for a time sufficient for the placental stem cells to detectably suppress the immune response, wherein the placental stem cells are mixed with T cell proliferation was detectably inhibited in the lymphocyte response (MLR) assay. In a specific embodiment, the placental stem cells: express CD200 and HLA-G; express CD73, CD105 and CD200; express CD200 and OCT-4; express CD73, CD105 and HLA-G; express CD73 and CD105, and when The population promotes the formation of one or more embryoid bodies in a population of placental cells comprising the plurality of placental stem cells when cultured under conditions suitable for embryoid body formation; and/or expresses OCT-4 and when the population is in Formation of one or more embryoid bodies in the population of placental cells comprising the plurality of placental stem cells is promoted when cultured under conditions suitable for embryoid body formation. In another specific embodiment, the plurality of immune cells are T cells or NK (natural killer) cells. In a more specific embodiment, said T cells are CD4 ⁺ T cells. In another more specific embodiment, said T cells are CD8 ⁺ T cells. In another specific embodiment, said contacting is performed in vitro. In another specific embodiment, said contacting is performed in vivo. In a more specific embodiment, said contacting in vivo is performed in a mammalian subject (eg, a human). In another more specific embodiment, said contacting comprises administering said placental cells intravenously, intramuscularly, or into an organ (eg, pancreas) of said individual. The method of suppressing an immune response, particularly in vivo, may further comprise administering to said individual (e.g., to a mammal) an antibody such as an anti-macrophage inflammatory protein (MIP)-1α or an anti-MIP-1β, wherein said antibody Administration is in an amount sufficient to cause a detectable decrease in the amount of MIP-la or anti-MIP-lbeta (eg, in the blood of the subject).

In a more specific embodiment of this method, said placental stem cells expressing CD200 and HLA-G also express CD73 and CD105, ie, are CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the placental cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said plurality of placental stem cells promotes the formation of one or more embryoid bodies in a population of isolated placental cells comprising the placental stem cells when said population is cultured under conditions suitable for embryoid body formation. form.

In another more specific embodiment of this method, said placental stem cells expressing CD73, CD105 and CD200 are also HLA-G ⁺ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ and HLA-G ⁺ . In another specific embodiment, said placental stem cells promote the formation of one or more embryoid bodies in a population of isolated placental cells comprising the placental stem cells when said population is cultured under conditions suitable for the formation of embryoid bodies .

In another more specific embodiment of this method, said placental stem cells expressing CD200 and OCT-4 also express CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said placental stem cells are HLA-G ⁺ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ , CD105 ⁺ and HLA-G ⁺ . In another specific embodiment, said placental stem cells promote the formation of one or more embryoid bodies in a population of isolated placental cells comprising placental stem cells when said population is cultured under conditions suitable for the formation of embryoid bodies.

In another more specific embodiment, the placental stem cells expressing CD73, CD105 and HLA-G are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said placental stem cells are OCT-4 ⁺ . In another specific embodiment, said placental stem cells are CD200 ⁺ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , OCT-4 ⁺ and CD200 ⁺ . In another specific embodiment, said stem cells promote the formation of one or more embryoid bodies in a population of isolated placental cells comprising said placental stem cells when said population is cultured under conditions suitable for the formation of embryoid bodies.

In another more specific embodiment, said one or more of the placental cell populations expressing CD73 and CD105 and promoting the formation of said placental stem cells when said populations are cultured under conditions suitable for embryoid body formation The placental stem cells that form embryoid bodies are also CD34 ^- , CD38 ^- or CD45 ^- . In another specific embodiment, said placental stem cells are OCT-4 ⁺ . In another specific embodiment, said placental stem cells are CD200 ⁺ . In another specific embodiment, the placental stem cells are OCT-4 ⁺ , CD200 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

In another more specific embodiment, said one or more of the placental cell populations that express OCT-4 and promote the formation of embryoid bodies when said populations are cultured under conditions suitable for embryoid body formation include said placental stem cells. The placental stem cells for the formation of embryoid bodies were also CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said placental stem cells are CD200 ⁺ . In another specific embodiment, the placental stem cells are CD73 ⁺ , CD 105 ⁺ , CD200 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

In another specific embodiment of the method of reducing or inhibiting an immune response, said immune response is graft versus host disease. In another specific embodiment, said immune response is an autoimmune disease, eg, diabetes, lupus erythematosus or rheumatoid arthritis.

In another specific embodiment of the method, said plurality of immune cells is also contacted with a plurality of non-placental cells. Such non-placental cells may include, for example, CD34 ⁺ cells. In a more specific embodiment, said CD34 ⁺ cells are peripheral blood hematopoietic progenitor cells, umbilical cord blood hematopoietic progenitor cells or placental blood hematopoietic progenitor cells. In another specific embodiment, said non-placental cells comprise mesenchymal stem cells. In a more specific embodiment, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. In another specific embodiment, said non-placental cells are contained within an allograft.

The methods can use the amount of placental stem cells required to achieve a detectable suppression of the immune response. For example, a population of placental stem cells for exposure to a population of immune cells may comprise 1 x ¹⁰⁵ placental stem cells, 1 x ¹⁰⁶ placental stem cells, 1 x ¹⁰⁷ placental stem cells, or 1 x ¹⁰⁸ placental stem cells or more .

The invention further provides methods of preparing a population of cells comprising placental stem cells selected for their ability to modulate (eg, suppress) an immune response. For example, in one embodiment, the invention provides a method of selecting a population of placental cells comprising (a) analyzing a population of placental cells in a mixed lymphocyte reaction (MLR) assay; and (b) if said population of placental stem cells Detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation in an MLR (mixed lymphocyte reaction), the plurality of placental stem cells are selected, wherein said placental stem cells: (i) adhere to the substrate; and (ii) Express CD200 and HLA-G, or express CD73, CD105 and CD200, or express CD200 and OCT-4, or express CD73, CD105 and HLA-G, or express CD73 and CD105, and when the population is suitable for embryoid bodies promotes the formation of one or more embryoid bodies in a population of placental cells comprising the stem cells when cultured under conditions that form, or expresses OCT-4, and promotes the formation of embryoid bodies when said population is cultured under conditions suitable for embryoid body formation. Formation of one or more embryoid bodies in a population of placental cells comprising the stem cells.

The present invention also provides a method for preparing a population of cells, which comprises selecting from a large number of cells that (a) adhere to a substrate, (b) express CD200 and HLA-G, and (c) are available in an MLR (mixed lymphocyte reaction) placental stem cells that detectably inhibit proliferation of CD4 ⁺ or CD8 ⁺ T cells; and isolating said placental stem cells from other cells to form a cell population. The present invention also provides a method of preparing a population of cells comprising selecting from a large number of cells that (a) adhere to a substrate, (b) express CD73, CD105, and CD200, and (c) detectably inhibit CD4 ⁺ or CD8 ⁺ T cell-proliferated placental stem cells; and isolating said placental stem cells from other cells to form a cell population. The present invention also provides a method of preparing a population of cells comprising selecting for (a) adhesion to a substrate, (b) expression of CD200 and OCT-4, and (c) detectable suppression of CD4 ⁺ or CD8 ⁺ T cells in the MLR proliferating placental stem cells; and isolating said placental stem cells from other cells to form a cell population. The present invention also provides a method of preparing a cell population comprising selecting from a large number of cells that (a) adhere to a substrate, (b) express CD73 and CD105, (c) form when cultured under conditions suitable for embryoid body formation Embryoid bodies, and (d) placental stem cells that detectably inhibit proliferation of CD4 ⁺ or CD8 ⁺ T cells in the MLR; and isolating said placental stem cells from other cells to form a cell population. The present invention also provides a method of preparing a population of cells comprising selecting from a population of cells that (a) adhere to a substrate, (b) express CD73, CD105 and HLA-G, and (c) detectably inhibit CD4 in the MLR ⁺ or CD8 ⁺ T cell-proliferated placental stem cells; and isolating said placental cells from other cells to form a cell population. The present invention also provides a method of preparing a cell population comprising selecting from a large number of cells that (a) adhere to a substrate, (b) express OCT-4, (c) form when cultured under conditions suitable for embryoid body formation Embryoid bodies, and (d) placental stem cells that detectably inhibit proliferation of CD4 ⁺ or CD8 ⁺ T cells in the MLR; and isolating said placental cells from other cells to form a cell population.

In a specific embodiment of any of the embodiments herein, said T cells and said placental stem cells are in the MLR at, for example, about 20:1, 15:1, 10:1, 5:1, 2:2, 1: 1, 1:2, 1:5, 1:10 or 1:20, preferably 5:1 ratio is present.

In another specific embodiment, the method comprises selecting cells expressing ABC-p. In another specific embodiment, the method comprises selecting cells exhibiting at least one particular characteristic of mesenchymal stem cells. In a more specific embodiment, the specific characteristic of the mesenchymal stem cells is the expression of CD29, CD44, CD90 or a combination thereof. In another specific embodiment of the method, said selection is achieved using antibodies. In another specific embodiment, said selection is accomplished using flow cytometry. In another specific embodiment, said selection is achieved through the use of magnetic beads. In another specific embodiment, said selection is achieved by fluorescence activated cell sorting. In another specific embodiment of the above method, said population of cells is expanded.

The placental stem cells used in this method can be derived from whole placenta, or from any part of placenta. For example, in various embodiments, the placental stem cells are derived primarily (or exclusively) from amnion, or amnion and chorion, or from placental perfusate collected during placental perfusion. In a specific embodiment, said placental stem cells can at least inhibit 50%, 70%, 90% or 95% of T cell proliferation in said MLR compared with the amount of T cell proliferation in said MLR lacking said placental stem cells. CD4 ⁺ or CD8 ⁺ T cells proliferate. In another specific embodiment, said placental stem cells also detectably inhibit natural killer (NK) cell activity.

The invention further provides an isolated cell population comprising placental stem cells prepared by any of the methods described herein for selecting an immunomodulatory placental cell population, wherein the population has been identified as detectably inhibiting in the MLR CD4 ⁺ or CD8 ⁺ T cells proliferate. For example, in one embodiment, the invention provides a population of cells comprising isolated placental stem cells that: (a) adhere to a substrate, (b) express CD200 and HLA-G, or express CD73, CD105 and CD200, or express CD200 and OCT-4, or express CD73, CD105 and HLA-G, or express CD73 and CD105, and promote the inclusion of the placental stem cells when said population is cultured under conditions suitable for embryoid body formation Formation of one or more embryoid bodies in a placental cell population of placental cells, or expresses OCT-4, and promotes the formation of one or more embryoid bodies in a population of placental cells comprising the placental stem cells when said population is cultured under conditions suitable for embryoid body formation Formation of one or more embryoid bodies, wherein such populations have been identified in the MLR as detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation.

The present invention also provides an isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD200 and HLA-G, and (c) have been identified as detectably inhibiting in the MLR CD4 ⁺ or CD8 ⁺ T cells proliferate. The present invention also provides an isolated cell population comprising placental stem cells that (a) adhere to the substrate, (b) express CD73, CD105 and CD200, and (c) have been identified as detectably inhibiting in the MLR CD4 ⁺ or CD8 ⁺ T cells proliferate. The present invention also provides an isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD200 and OCT-4, and (c) have been identified as detectably inhibiting in the MLR CD4 ⁺ or CD8 ⁺ T cells proliferate. The present invention also provides an isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD73 and CD105, and (c) form embryoid-like bodies when cultured under conditions suitable for embryoid-like body formation. embryoid bodies, and (d) have been identified in the MLR to detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation. The present invention also provides an isolated cell population comprising placental stem cells that (a) adhere to the substrate, (b) express CD73, CD105 and HLA-G, and (c) have been identified as detectable in the MLR Inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation. The present invention also provides an isolated population of cells comprising placental stem cells that (a) adhere to a substrate, (b) express OCT-4, and (c) form embryoid-like bodies when cultured under conditions suitable for embryoid-like body formation. embryoid bodies, and (d) have been identified in the MLR to detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation.

In a more specific embodiment of the composition, the placental stem cells expressing CD200 and HLA-G also express CD73 and CD105, namely CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said plurality of placental stem cells promotes formation of one or more embryoid bodies in a population of isolated placental cells comprising said placental stem cells when said population is cultured under conditions suitable for embryoid body formation. form.

In a more specific embodiment of said composition, said placental stem cells expressing CD73, CD105 and CD200 are also HLA-G ⁺ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ and HLA-G ⁺ . In another specific embodiment, said placental stem cells promote the formation of one or more embryoid bodies in a population of isolated placental cells comprising said placental stem cells when said population is cultured under conditions suitable for embryoid body formation. form.

In another more specific embodiment of the composition, the placental stem cells expressing CD200 and OCT-4 also express CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said placental stem cells are HLA-G ⁺ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ , CD105 ⁺ and HLA-G ⁺ . In another specific embodiment, said placental stem cells promote the formation of one or more embryoid bodies in a population of isolated placental cells comprising said placental stem cells when said population is cultured under conditions suitable for the formation of embryoid bodies .

In another more specific embodiment of the composition, the placental stem cells expressing CD73, CD105 and HLA-G are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said placental stem cells are OCT-4 ⁺ . In another specific embodiment, said placental stem cells are CD200 ⁺ . In a more specific embodiment, the placental stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , OCT-4 ⁺ and CD200 ⁺ . In another specific embodiment, said stem cells promote the formation of one or more embryoid bodies in a population of isolated placental cells comprising said placental stem cells when said population is cultured under conditions suitable for embryoid body formation.

In another more specific embodiment of said composition, said expresses CD73 and CD105 and promotes a population of placental cells comprising said placental stem cells when said population is cultured under conditions suitable for embryoid body formation The placental stem cells formed by one or more embryoid bodies are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said placental stem cells are OCT-4 ⁺ . In another specific embodiment, said placental stem cells are CD200 ⁺ . In another specific embodiment, the placental stem cells are OCT-4 ⁺ , CD200 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

In another more specific embodiment of said composition, said expresses OCT-4 and promotes a population of placental cells comprising said placental stem cells when said population is cultured under conditions suitable for embryoid body formation One or more embryoid bodies in the forming placental stem cells are likewise CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said placental stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said placental stem cells are CD200 ⁺ . In another specific embodiment, the placental stem cells are CD73 ⁺ , CD105 ⁺ , CD200 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

The present invention further provides immunomodulatory compositions. In one embodiment, the invention provides a composition comprising a culture supernatant of any cell population described herein. In another embodiment, the present invention provides a composition comprising a culture medium of placental stem cells, wherein said placental stem cells: (a) adhere to a substrate; (b) express CD200 and HLA-G, or express CD73 , CD105, and CD200, or express CD200 and OCT-4, or express CD73, CD105, and HLA-G, or express CD73 and CD105, and promote inclusion of the placenta when said population is cultured under conditions suitable for embryoid body formation Formation of one or more embryoid bodies in a placental cell population of stem cells, or express OCT-4, and promote in a placental cell population comprising the placental stem cells when said population is cultured under conditions suitable for embryoid body formation and (c) detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation in MLR (mixed lymphocyte reaction), wherein said placental stem cell culture has been cultured in said Incubate for 24 hours or more. In a specific embodiment, said composition comprises a plurality of said placental stem cells. In another specific embodiment, said composition comprises a plurality of non-placental cells. In a more specific embodiment, said non-placental cells comprise CD34 ⁺ cells. The CD34 ⁺ cells can be, for example, peripheral blood hematopoietic progenitor cells, umbilical cord blood hematopoietic progenitor cells, or placental blood hematopoietic progenitor cells. In another specific embodiment, said non-placental cells comprise mesenchymal stem cells. In a more specific embodiment, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. In another specific embodiment, the composition further comprises an anti-MIP-1α or anti-MIP-1β antibody.

In another specific embodiment, any of the foregoing compositions comprises a matrix. In a more specific embodiment, said matrix is a three-dimensional framework. In another more specific embodiment, said matrix comprises collagen, gelatin, laminin, fibronectin, pectin, ornithine or vitronectin. In another more specific embodiment, the matrix is amnion or amnion derived biological material. In another more specific embodiment, said matrix comprises an outer membrane protein. In another more specific embodiment, said matrix comprises a synthetic compound. In another more specific embodiment, said matrix comprises a biologically active compound. In another more specific embodiment, said biologically active compound is a growth factor, cytokine, antibody, or organic molecule of less than 5,000 Daltons.

The invention further provides a cryopreserved stem cell population, eg, a cell population comprising placental stem cells described herein, wherein the cell population has an immunomodulatory effect. For example, the invention provides a CD200 ⁺ , HLA-G ⁺ placental stem cell population that has been identified in a mixed lymphocyte reaction (MLR) assay to detectably inhibit T cell proliferation, wherein said stem cells have been cryopreserved, and The population described therein is contained in a container. The present invention also provides CD73 ⁺ , CD105 ⁺ , CD200 ⁺ placental stem cell populations, which have been identified as detectably inhibiting T cell proliferation in a mixed lymphocyte reaction (MLR) assay, wherein said stem cells have been cryopreserved, And wherein said group is packed in the container. The present invention also provides a population of CD200 ⁺ , OCT-4 ⁺ placental stem cells which have been identified as detectably inhibiting T cell proliferation in a mixed lymphocyte reaction (MLR) assay, wherein said stem cells have been cryopreserved, and The population described therein is contained in a container. The present invention also provides CD73 ⁺ , CD105 ⁺ placental stem cell populations, which have been identified as detectably inhibiting T cell proliferation in a mixed lymphocyte reaction (MLR) assay, wherein said stem cells have been cryopreserved, and wherein said The population is contained in a container, and when the population of stem cells and placental cells is cultured under conditions suitable for the formation of embryoid bodies, the formation of one or more embryoid bodies can be promoted. The present invention further provides CD73 ⁺ , CD105 ⁺ , HLA-G ⁺ placental stem cell populations which have been identified as detectably inhibiting T cell proliferation in a mixed lymphocyte reaction (MLR) assay, wherein said cells have been cryogenically Preserved, and the group described therein is packed in the container. The present invention also provides a population of OCT-4 ⁺ placental stem cells that has been identified as detectably inhibiting T cell proliferation in a mixed lymphocyte reaction (MLR) assay, wherein said stem cells have been cryopreserved, wherein said population contained in a container, and promote the formation of one or more embryoid bodies when said population of stem cells and placental cells are cultured under conditions suitable for the formation of embryoid bodies.

In any specific embodiment of the aforementioned cryopreservation group, the container is a bag. In various specific embodiments, said population comprises about, at least or at most 1×10 ⁶ said stem cells, 5×10 ⁶ said stem cells, 1×10 ⁷ said stem cells, 5×10 ⁷ Said stem cells, 1×10 ⁸ said stem cells, 5×10 ⁸ said stem cells, 1×10 ⁹ said stem cells, 5×10 ⁹ said stem cells, or 1×10 ¹⁰ said stem cells. In other specific embodiments of any of the foregoing cryopreserved populations, said stem cells have been passaged about, at least, or no more than 5 times, no more than 10 times, no more than 15 times, or no more than 20 times. In another specific embodiment of any of the foregoing cryopreserved populations, said stem cells have been expanded in said container.

3.1 Definition

The term "SH2" as used herein refers to an antibody that binds to an epitope on the marker CD105. Thus, cells called SH2 ⁺ are CD105 ⁺ .

The terms "SH3" and "SH4" as used herein refer to antibodies that bind to an epitope on the marker CD73. Thus, cells referred to as SH3 ⁺ and/or SH4 ⁺ are CD73 ⁺ .

As used herein, the term "isolated stem cell" refers to a stem cell that is substantially separated from other, non-stem cells in the tissue from which the stem cell was obtained (eg, placenta). For example, if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the non-stem cells naturally associated with the stem cell are removed from the stem cell during harvesting and/or culturing If removed, the stem cells are "isolated."

As used herein, the term "isolated cell population" refers to a cell population that is substantially separated from other cells in the tissue from which the cell population was obtained (eg, placenta). For example, if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells naturally associated with the stem cell population are derived from the stem cell population during collection and/or culture A stem cell population is "isolated" if the population is removed.

The term "placental stem cells" as used herein refers to stem cells or progenitor cells derived from the placenta of mammals, regardless of morphology, cell surface markers, or number of passages after primary culture, which cells are cultured on tissue culture substrates (for example, tissue culture plastic or coated tissue culture plates coated with fibronectin). However, the term "placental stem cells" as used herein is not used to refer to trophoblast cells. A cell is considered a "stem cell" if it retains at least one characteristic of a stem cell, eg, the ability to differentiate into at least one other type of cell.

As used herein, a stem cell is "positive" for a particular marker when it is detectable. For example, a placental stem cell is positive for CD73 because CD73 is detectable on the placental stem cell in an amount detectably greater than background (eg, compared to an isotype control). A cell is also positive for a marker when it can be used to distinguish the cell from at least one other cell type, or can be used to select or isolate the cell when present in or expressed by the cell.

As used herein, "immunomodulation" and "immunomodulatory" refer to causing or having the ability to cause a detectable change in an immune response, as well as the ability to cause a detectable change in an immune response.

As used herein, "immunosuppressive" and "immunosuppressive" refer to causing or having the ability to cause a detectable reduction of an immune response, as well as the ability to cause a detectable suppression of an immune response.

4. Description of drawings

Figure 1: Viability of placental stem cells derived from perfusion (A), amnion (B), chorion (C), or amnion-chorion plates (D), or umbilical cord stem cells (E). Numbers on the x-axis refer to the placenta from which the stem cells were obtained.

Figure 2: HLA ABC ⁻ /CD45 ⁻ /CD34 derived from perfusion (A), amnion (B), chorion (C), or amnion-chorion plates (D), or umbilical cord stem cells (E) as determined by FACSCalibur Percentage of ⁻ /CD133 ⁺ cells. Numbers on the x-axis refer to the placenta from which the stem cells were obtained.

Figure 3: HLA ^ABC- /CD45- ^/ Percentage of CD34 ⁻ /CD133 ⁺ cells. Numbers on the x-axis refer to the placenta from which the stem cells were obtained.

Figure 4: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in stem cells derived from placental perfusate.

FIG. 5 : Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, and CD200 in amnion-derived stem cells.

FIG. 6 : Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, and CD200 in chorion-derived stem cells.

Figure 7: Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200 in stem cells from the amnion-chorion plate.

FIG. 8 : Expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, and CD200 in umbilical cord-derived stem cells.

Figure 9: HLA-G, CD10, CD13, CD33, CD38, Mean expression of CD44, CD90, CD105, CD117, CD200 expression.

Figures 10A and 10B: Mixed lymphocyte reaction (MLR) is a model of an innate immune response and is suppressed by placental stem cells. The percentage of carboxyfluorescein succinimidyl ester (CFSE) ^Low cells can be monitored from gating "live" and CD8 ⁺ and CD4 ⁺ T cells (Figures 10A and 10B, respectively). This percentage increased after six days of MLR (Figures 10C and 10D, MLR curves), and with the addition of placental stem cells (Figures 3C and 3D, PMLR curves), the effect was reversed in both CD8 ⁺ and CD4 ⁺ T cell compartments.

Figure 11 : Placenta-derived stem cells from amnion chorionic plates (AC) and umbilical cord stroma (UC) inhibit allo-MLR. The MLR is performed with CD4 ⁺ T cells, CD8 ⁺ T cells, or an equal number of CD4 ⁺ and CD8 ⁺ T cells. Abscissa: percentage inhibition of proliferation.

Figure 12: Placental stem cells and umbilical cord stem cells inhibit allo-MLR. Six-day assay in wells of a round bottom 96-well plate. Placental cells: T cells: dendritic cells = about 1:10:1. Stem cells are obtained from amnion-chorion (AC), amnion (AM) or umbilical cord (UC). FB = fibroblasts. BM = bone marrow derived mesenchymal stem cells.

Figure 13: Placental stem cells from different donors suppress allo-MLR to varying degrees. This figure compares the suppression of placental stem cells in the MLR from two placental donors designated 61665 and 63450. Stem cells from placenta 63540 showed greater suppression of MLR than stem cells from 61665.

Figure 14: 17-day regression assay and modified placental stem cell regression assay. The X-axis represents the number of placental stem cells added to the experiment. The number of surviving CD23 ⁺ LCLs (lymphoblastoid cell lines, artificially created transformed B cell lines) is measured on the Y axis.

Figure 15: Inhibition of T cell proliferation by placental stem cells in a six-day regression assay. Regression assays were set up using CFSE-stained T cells. Six days later, T cell proliferation was assessed. Relative inhibition of T cell proliferation by stem cells from amnion-chorion (AC), umbilical cord (UC), amnion (AM) or bone marrow (BM) is shown.

Figure 16: Changes in percent inhibition when placental cells were isolated from T cells while allowing medium exchange by introducing a transwell block into the MLR. At 25,000, 50,000, 75,000 or 100,000/reaction, umbilical cord stem cells showed a relatively high level of suppression and a relatively high requirement for cell-to-cell contact in high titers to achieve suppression.

Figure 17: Umbilical cord mesenchymal stem cells added at 12,500 (UC OP/TW 12.5) to 100,000 (UC OP/TW 100) separated by membrane with NLR membrane (TW) or contacted with MLR (OP). Equal amounts of CD4 ⁺ T cells and CD8 ⁺ T cells were used and the percent inhibition of MLR was calculated (%CFSE ^Low = 89%).

Figure 18: The relationship between placental stem cell dose and cell-to-cell contact dependence is not linear. The change in MLR suppression after the introduction of the block was calculated from the values given in Fig. 17.

Figure 19: Differential suppression of T cell responses by placental stem cells and BMSCs. The degree of suppression by PDAC or BMSC was calculated by comparing the percentage of MLR T cells (over 70%) in the CFSE ^Lo gate compared to the MLR of adherent cells. The MLR was either dissociated from adherent cells (transwell) or performed in an open well (open). The X-axis gives the number of adherent cells (in thousands) added to 500,000 T cells and 50,000 DC. The ratio of adherent cells to T cells ranged from 1:5 to 1:40.

Figure 20: Different cell-to-cell contact requirements for immunosuppression of placental stem cells and bone marrow-derived stem cells. From the inhibition data presented in Figure 15, the contact dependence was calculated and shown for the ratio of adherent cells/T (n=3 except UC: n=2).

Figure 21: T regulatory cells are not required for PDAC T cell suppression. Regression assays can be performed using whole PBMCs (red and blue panels) or PBMCs depleted of T regulatory cells (green panels), both stained with CFSE and supplemented with UC PDAC under certain conditions (blue and green panels). N=1.

Figure 22: Proliferation of CFSE ^Hi cells in the secondary MLR. CFSE ^Hi T cells were isolated on a FACS Aria from the PDAC MLR of cells stained with CFSE. The cells were used in the MLR. N=1.

Figure 23: Supernatants of suppressed stem cell MLR do not suppress MLR at 75% replacement. UC (PUC), AC (PAC) and BMSC (PBM) MLR were performed, and the inhibition of MLR was more than 50%. The supernatant from this experiment can be used to replace 10 to 150 μl in 200 μl medium for fresh MLRs. Media from co-cultures of T cells and AC (T/AC) or T cells and bone marrow-derived stem cells (T/BM) were also used as controls in the same manner (N=2).

Figure 24A, 24B: Pre-culture of T cells and adherent cells does not affect MLR inhibition. T cells from 2 donors were used in two independent experiments. Mature DC (A) or CFSE-stained CD3 ^{+ T} cells (B) were cultured with umbilical cord stem cells (UC) or bone marrow-derived stem cells before adding DC (at day 0, A) or CFSE+ CD3 ⁺ T cells (B) as indicated Number of days to start MLR. The adherent cell MLR was then performed as usual for six days. N=2.

Figures 25A, 25B: A. MIP-1α and MIP-1β secretion in MLR and MLR with placental stem cells or bone marrow-derived stem cells, inversely proportional to MLR inhibition. B: T cell and NK cell CFSE data from the same experiment. Supernatants collected from MLRs are shown in Figure 14B and analyzed for MIP-1α and MIP-1β. B: MLR was performed as described above and an average of 55% (T cells) or 83% (NK cells) CFSE ^Low cells were observed. The inhibitory effect of stem cell addition was calculated. N=2 (NK part: N=1).

Figure 26: Measurement of MCP-1 in modified regression assay and MLR supernatants. Placental stem cell suppression and regression assays of the MLR are associated with secretion of the chemoattractant protein MCP-1. AC: Stem cells from amnion-chorion plates. UC: stem cells from the umbilical cord. Light bars: MLR test results. Dark bars: regression test results. Y-axis: pg of MCP-1 in the test solution.

Figure 27: IL-6 measurement in modified MLR and regression assay supernatants. Placental stem cell suppression in MLR and regression assays correlated with IL-6 secretion. AC: Stem cells from amnion-chorion plates. UC: stem cells from the umbilical cord. Light bars: MLR test results. Dark bars: regression test results. Y axis: pg of IL-6 in the test solution.

5. Detailed Description of the Invention

5.1 Immunomodulation using placental stem cells

The invention provides modulation (eg, inhibition) of the activity (eg, proliferation) of an immune cell or population of immune cells by contacting the immune cell with a population of placental stem cells. In one embodiment, the present invention provides a method of suppressing an immune response comprising contacting a plurality of immune cells with a plurality of placental stem cells for a time sufficient for the placental stem cells to detectably suppress the immune response, wherein said Placental stem cells were able to detectably inhibit T cell proliferation in a mixed lymphocyte reaction (MLR) assay.

Placental stem cells are, for example, placental stem cells described elsewhere herein (see Section 5.2). Placental stem cells for immunosuppression can be obtained from a single placenta or from multiple placentas. Placental stem cells for immunosuppression may also be from a single species (eg, the intended recipient species or the species in which immune cell function is to be reduced or suppressed) or may be from multiple species.

"Immune cell" in this method refers to any cell of the immune system, especially T cells and NK (natural killer) cells. Accordingly, in various embodiments of the method, placental stem cells are contacted with a plurality of immune cells, wherein the plurality of immune cells is, or includes, a plurality of T cells (e.g., a plurality of CD3 ⁺ T cells, CD4 ⁺ T cells, and/or or CD8 ⁺ T cells) and/or natural killer cells. An "immune response" in the present methods can be any response of an immune cell to a stimulus normally sensed by an immune cell, eg, a response to the presence of an antigen. In various embodiments, the immune response can be T cells (e.g., CD3 ⁺ T cells, CD4 ⁺ T cells and/or CD8 ⁺ T cells). The immune response may also be the proliferation of T cells contained in the graft. The immune response can also be any activity of natural killer (NK) cells, maturation of dendritic cells, and the like. The immune response can also be a local, tissue or organ specific or systemic effect of the activity of one or more types of immune cells, for example, the immune response can be graft-versus-host disease, inflammation, inflammation-associated scar tissue formation And autoimmune symptoms (for example, rheumatoid arthritis, type I diabetes, lupus erythematosus, etc.) and the like.

"Contacting" herein includes placing placental stem cells and immune cells together in a single container (eg, petri dish, bottle, vial, etc.) or in vivo as one individual (eg, mammal, eg, human). In preferred embodiments, the contacting is for a sufficient length of time, and with sufficient quantities of the placental stem cells and the immune cells, such that changes in the immune function of the immune cells are detectable. More preferably, in various embodiments, the exposure is sufficient to suppress immune function by at least 50%, 60%, 70%, 80%, 90%, or 95% compared to immune function in the absence of placental stem cells (e.g. , proliferation of T cells in response to antigen). Such inhibition in an in vivo setting can be determined by in vitro assays (see below); that is, for a specific number of placental stem cells and a certain number of immune cells in a recipient individual, the degree of inhibition in the in vitro assay can be extrapolated to that in the individual degree of inhibition.

In certain embodiments, the present invention provides methods of using placental stem cells to modulate an immune response, or the activity of a population of one or more types of immune cells, in vitro. Contacting the placental stem cells and the plurality of immune cells may comprise combining the placental stem cells and the immune cells in the same physical space such that at least some of the plurality of placental stem cells interact with at least some of the plurality of immune cells; The common medium is maintained in a separate physical space; or may involve contacting the medium of a culture of placental stem cells or immune cells with another type of cell (e.g., obtaining medium from a placental stem cell culture and placing the isolated Immune cells were resuspended in this medium). In specific embodiments, the contacting is a mixed lymphocyte reaction (MLR).

For example, such contacting can occur in an experimental setting designed to detect the degree of immunomodulation (eg, immunosuppression) of a particular amount of placental stem cells. Such an experimental setting may be, for example, a mixed lymphocyte reaction (MLR) or a regression assay. Methods for performing MLR and regression tests are well known in the art. See, eg, Schwarz, "The Mixed Lymphocyte Reaction: An In Vitro Test for Tolerance," J Exp. Med. 127(5): 879-890 (1968); Lacerda et al., "Human Epstein-Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes Home Preferentially to and Induce Selective Regressions of Autologous EBV-Induced BLymphoproliferations in Xenografted CB-17 Scid/Scid Mice," J Exp. Med. 183: 1215-1228 (1996). In a preferred embodiment, MLR is performed in which a population of placental stem cells is contacted with a population of immune cells (eg, lymphocytes, eg, CD3 ⁺ , CD4 ⁺ and/or CD8 ⁺ T lymphocytes).

MLR can be used to measure the immunosuppressive capacity of large numbers of placental stem cells. For example, a large number of placental stem cells can be tested using an MLR comprising pooling CD4 ⁺ or CD8 ⁺ T cells, dendritic cells (DCs) and placental stem cells in an approximately 10:1:2 ratio, wherein the T cells are stained with a dye such as CFSE staining into progeny cells can be dispensed and wherein the T cells are allowed to proliferate for about 6 days. A large number of placental stem cells are immunosuppressive if T cell proliferation in the presence of placental stem cells is detectably reduced at day 6 when compared to T cell proliferation in the presence of DC and absence of placental stem cells. In this MLR, placental stem cells can be thawed or harvested from culture. Approximately 20,000 placental stem cells were resuspended in 100 μl of medium (RPMI 1640, 1 mM HEPES buffer, antibiotics, and 5% pooled human serum) and allowed to attach to the bottom of the wells for 2 hours. CD4 ⁺ and/or CD8 ⁺ T cells can be isolated from whole peripheral blood mononuclear cells with Miltenyi magnetic beads. Cells were stained with CFSE and a total of 100,000 T cells (CD4 ⁺ T cells alone, CD8 ⁺ T cells alone, or equal amounts of CD4 ⁺ and CD8 ⁺ T cells) were added per well. The volume in the wells was brought up to 200 μl and MLR was performed.

Accordingly, in one embodiment, the present invention provides a method of suppressing an immune response comprising contacting a plurality of immune cells with a plurality of placental stem cells for a time sufficient for said placental stem cells to fail in a mixed lymphocyte reaction (MLR) test. Detectably inhibits T cell proliferation. In one embodiment, said placental stem cells employed in the MLR represent a sample or aliquot of placental stem cells from a larger population of placental stem cells.

Placental stem cell populations obtained from different placentas or different tissues in the same placenta may have different abilities to regulate immune cell activity, eg, have different abilities to inhibit T cell activity or proliferation or NK cell activity. It is therefore ideal to test the immunosuppressive capacity of specific placental stem cell populations prior to use. This capacity can be determined by testing a sample of the placental stem cell population in an MLR or regression assay. In one embodiment, the sample is used for MLR in which the degree of immunosuppression produced by the placental stem cells is measured. This degree of immunosuppression was then attributed to the sampled placental stem cell population. Therefore, MLR can be used as a method to measure the absolute and relative ability of specific placental stem cell populations to suppress immune function. The parameters of the MLR can be altered to provide additional data or to best detect the immunosuppressive capacity of placental stem cell samples. For example, since immunosuppression of placental stem cells appears to rise roughly proportional to the number of placental stem cells in the assay, in one embodiment two or more numbers of placental stem cells may be used for MLR, e.g., in each reaction 1×10 ³ , 3×10 ³ , 1×10 ⁴ and/or 3×10 ⁴ placental stem cells were used. The ratio of the number of placental stem cells to the number of T cells in the assay may also vary. For example, placental stem cells and T cells may be present in assays in any ratio, eg, from about 10:1 to about 1:10, preferably in a ratio of about 1:5, although relatively larger amounts of placental stem cells or T cells may be used.

Regression tests can be used in a similar manner.

The invention also provides methods of using placental stem cells in vivo to modulate an immune response, or the activity of a plurality of one or more types of immune cells. Placental stem cells and immune cells can be contacted, eg, in a plurality of placental stem cell recipient individuals. In one embodiment, when the contacting is in an individual, the contacting is between placental stem cells exogenous to the individual (ie, placental stem cells not derived from the individual) and a plurality of endogenous immune cells. In specific embodiments, the immune cells in the individual are CD3 ⁺ T cells, CD4 ⁺ T cells, CD8 ⁺ T cells and/or NK cells.

Such immunosuppression using placental stem cells may be beneficial in any condition caused or exacerbated by or associated with an inappropriate or undesired immune response. For example, placental stem cell-mediated immunomodulation (eg, immunosuppression) can be used to suppress an inappropriate immune response by an individual's immune system against one or more of its own tissues. Accordingly, in various embodiments, the invention provides methods of suppressing an immune response, wherein the immune response is an autoimmune disease, eg, lupus, diabetes, rheumatoid arthritis, or multiple sclerosis.

Contact between a plurality of placental stem cells and a plurality of one or more types of immune cells may occur in vivo or concomitantly, for example, upon grafting or transplantation of one or more types of tissue into a recipient individual. The tissues can be, for example, bone marrow or blood; organs; specific tissues (eg, skin grafts); composite tissue allografts (ie, grafts comprising two or more different types of tissues), and the like. In this regard, the placental stem cells can be used to suppress one or more immune responses of one or more immune cells contained within the recipient individual, within the transplanted tissue or graft, or both. Such contacting can be performed before, simultaneously with and/or after grafting or transplantation. For example, placental stem cells can be administered at the time of transplantation or engraftment. In addition, placental stem cells can also be administered prior to transplantation or grafting (eg, about 1, 2, 3, 4, 5, 6, or 7 days prior to transplantation or grafting). Alternatively, the placental stem cells can also be administered to the transplant or graft recipient after transplant or graft (eg, about 1, 2, 3, 4, 5, 6, or 7 days after transplant or graft). Preferably, the bulk placenta can be removed before any detectable manifestation or symptom of the immune response of the recipient individual or the transplanted tissue or graft (e.g., a detectable manifestation or symptom of graft-versus-host disease or measurable inflammation) becomes detectable. Stem cells are in contact with a large number of placental stem cells.

In another embodiment, the contacting within the individual is primarily between exogenous placental stem cells and exogenous progenitor or stem cells (eg, exogenous progenitor or stem cells that differentiate into immune cells). For example, an individual undergoing partial or complete immunopurification or bone marrow depletion in conjunction with cancer therapy may receive placental stem cells in combination with one or more other types of stem or progenitor cells. For example, placental stem cells can be used in combination with a large number of CD34 ⁺ cells (eg, CD34 ⁺ hematopoietic stem cells). These CD34 ⁺ cells can be, for example, CD34 ⁺ cells from tissue sources such as peripheral blood, cord blood, placenta, or bone marrow. The CD34 ⁺ cells can be isolated from the tissue source or whole tissue sources (eg, cord blood or bone marrow units) or a partially purified preparation from the tissue source (eg, leukocytes from cord blood) can be combined with placental stem cells. Hariri, US Application Publication No. 2003/0180269 describes the combination of placental stem cells and cord blood or stem cells derived from cord blood.

Preferably, the placental stem cells are administered to the individual in a ratio of about 10:1 to about 1:10, preferably about 1:5, relative to known or expected numbers of immune cells (eg, T cells) in the individual. However, in non-limiting examples, the plurality of placental stem cells may be about 10,000:1, about 1,000:1, about 100:1, about 10:1, about 1:1, about 1:100, about 1:1,000, or about The ratio of 1:10,000 was administered to the individual. Generally, about 1 x ¹⁰⁵ to about 1 x ¹⁰⁸ placental stem cells per kilogram of recipient, preferably about 1 x ¹⁰⁶ to about 1 x ¹⁰⁷ placental stem cells per kilogram of recipient may be administered to achieve immunosuppression. In various embodiments, the amount of placental stem cells administered to an individual or subject comprises at least, about, or no more than 1×10 ⁵ , 3×10 ⁵ , 1×10 ⁶ , 3×10 ⁶ , 1×10 ⁷ , 3×10 ⁷ , 1×10 ⁸ , 3×10 ⁸ , 1×10 ⁹ , 3×10 ⁹ placental stem cells, or more.

The placental stem cells may also be co-administered with one or more other types of stem cells, such as mesenchymal stem cells from bone marrow. Such other stem cells may be administered to the individual in a ratio of, for example, about 1:10 to about 10:1 to placental stem cells.

To facilitate in vivo contact of placental stem cells and immune cells, the placental stem cells can be administered to an individual by any route that allows the placental stem cells and immune cells to contact each other. For example, the placental stem cells can be administered to an individual, eg, intravenously, intramuscularly, intraperitoneally, or directly to an organ (eg, pancreas). For in vivo administration, the placental stem cells can be formulated into a pharmaceutical composition as described in Section 5.6.1 below.

)或达利珠单抗

)、抗T细胞受体抗体(例如，莫罗单抗-CD3)，咪唑硫嘌呤、皮质类固醇、环孢菌素、他克莫司、霉酚酸酯、西罗莫司、神经钙调蛋白抑制剂等。在具体的实施方式中，该免疫抑制剂是巨噬细胞炎症蛋白(MIP)-1α或MIP-1β的中和抗体。优选地，该抗(MIP)-1α或MIP-1β抗体以足以使所述个体中(MIP)-1α和/或MIP-1β量可检测地下降(例如，在移植时)的量进行施用。The immunosuppressive method may further comprise the addition of one or more immunosuppressants, especially in vivo. In one embodiment, a plurality of placental stem cells is contacted with a plurality of immune cells in the body of an individual, and a composition comprising an immunosuppressant is administered to the individual. Immunosuppressants are well known in the art and include, for example, anti-T cell receptor antibodies (monoclonal or polyclonal antibodies, or antibody fragments or derivatives thereof), anti-IL-2 receptor antibodies (such as basiliximab (

) or daclizumab

), anti-T cell receptor antibodies (eg, murozumab-CD3), azathioprine, corticosteroids, cyclosporine, tacrolimus, mycophenolate mofetil, sirolimus, calmodulin Inhibitors etc. In a specific embodiment, the immunosuppressant is a neutralizing antibody to macrophage inflammatory protein (MIP)-1α or MIP-1β. Preferably, the anti-(MIP)-1α or MIP-1β antibody is administered in an amount sufficient to detectably decrease the amount of (MIP)-1α and/or MIP-1β in said individual (eg, upon transplantation).

5.2 Placental stem cells and placental stem cell populations

The immunosuppressive method of the present invention employs placental stem cells, i.e., stem cells obtained from the placenta or a portion thereof that (1) adhere to a tissue culture substrate; (2) have the ability to differentiate into non-placental cell types; (3) The ability to detectably suppress immune function (eg, proliferation of CD4 ⁺ and/or CD8 ⁺ stem cells in a mixed lymphoid reaction assay) in sufficient numbers. Placental stem cells do not come from blood (for example, placental blood or umbilical cord blood). The placental stem cells used in the methods and compositions of the invention are selected for their ability to suppress the immune system of an individual.

Placental stem cells can be of either fetal or maternal origin (ie, can have a maternal or fetal genotype). A population of placental stem cells or a population of cells comprising placental stem cells may comprise placental stem cells derived only from the fetus or the mother, or may comprise a mixed population of placental stem cells derived from both the fetus and the mother. Placental stem cells and cell populations comprising placental stem cells can be identified and selected by the morphological, marker and culture characteristics discussed below.

5.2.1 Physical and Morphological Characteristics

When the placental stem cells of the present invention are cultured in primary culture or cell culture, they will adsorb to the tissue culture substrate, for example, the surface of the tissue culture vessel (eg, tissue culture plastic). Placental stem cells in culture typically have a fibroblast-like, star-shaped appearance with numerous cytoplasmic processes extending from a central cell body. However, since placental stem cells have more such protrusions than fibroblasts, placental stem cells are morphologically distinguishable from fibroblasts cultured under the same conditions. Morphologically, placental stem cells can also be differentiated from hematopoietic stem cells, which typically have a more rounded or cobblestone morphology in culture.

5.2.2 Cell surface, molecular and genetic markers

Placental stem cells and populations of placental stem cells useful in the methods and compositions of the invention express a number of markers that can be used to identify and/or isolate the stem cells or populations of cells comprising stem cells. Placental stem cells and stem cell populations (ie, two or more types of placental stem cells) of the invention include stem cells and stem cell-containing cell populations obtained directly from the placenta or any part thereof (eg, amnion, chorion, placental villi leaves, etc.). A population of placental stem cells also includes a population (ie, two or more) of placental stem cells in culture, or a population of cells in a container (eg, a bag). However, placental stem cells are not trophoblasts.

Placental stem cells typically express the markers CD73, CD105, CD200, HLA-G and/or OCT-4, and do not express CD34, CD38 or CD45. Placental stem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify and differentiate placental stem cells from other stem cell types. Because placental stem cells can express CD73 and CD105, they have the characteristics of mesenchymal stem cells. However, since placental stem cells can express CD200 and HLA-G (fetal-specific markers), they can be differentiated from mesenchymal stem cells that express neither CD200 nor HLA-G (eg, bone marrow-derived mesenchymal stem cells) come out. Similarly, placental stem cells can be identified as non-hematopoietic stem cells by loss of CD34, CD38 and/or CD45 expression.

In one embodiment, the present invention provides an isolated cell population comprising a plurality of CD200 ⁺ , HLA-G ⁺ immunosuppressive placental stem cells, wherein said plurality of cells detectably inhibits T in a mixed lymphocyte reaction (MLR) assay. Cell Proliferation. In a specific embodiment of the isolate, said stem cells are also CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the stem cells are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In a more specific embodiment, the stem cells are also CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ and CD105 ⁺ . In another embodiment, the isolate produces one or more embryoid bodies when cultured under conditions suitable for embryoid body formation.

In another embodiment, the present invention provides an isolated cell population comprising a large amount of CD73 ⁺ , CD105 ⁺ , CD200 ⁺ immunosuppressive placental stem cells, wherein said large number of cells detectably detectable in a mixed lymphocyte reaction (MLR) assay Inhibits T cell proliferation. In a specific embodiment of the population, the stem cells are HLA-G ⁺ . In another specific embodiment, the stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In a more specific embodiment, said stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ and HLA-G ⁺ . In another specific embodiment, said population of cells produces one or more embryoid bodies when cultured under conditions suitable for embryoid body formation.

The present invention also provides an isolated cell population comprising a plurality of CD200 ⁺ , OCT-4 ⁺ immunosuppressive placental stem cells, wherein said plurality of cells detectably inhibits T cell proliferation in a mixed lymphocyte reaction (MLR) assay. In a specific embodiment, the stem cells are CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said stem cells are HLA-G ⁺ . In another specific embodiment, the stem cells are CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In a more specific embodiment, the stem cells are CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ , CD105 ⁺ and HLA-G ⁺ . In another specific embodiment, the population produces one or more embryoid bodies when cultured under conditions suitable for embryoid body formation.

The present invention also provides an isolated population of immunosuppressive placental stem cells comprising a plurality of CD73 ⁺ , CD105 ⁺ and HLA-G ⁺ , wherein said plurality of cells detectably inhibits T cell proliferation in a mixed lymphocyte reaction (MLR) assay . In the specific embodiment of the above-mentioned large number of cells, the stem cells are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the stem cells are also CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said stem cells are also OCT-4 ⁺ . In another specific embodiment, said stem cells are also CD200 ⁺ . In a more specific embodiment, said stem cells are also CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , OCT-4 ⁺ and CD200 ⁺ .

The present invention also provides an isolated cell population comprising a large amount of immunosuppressive placental stem cells, the stem cells are CD73 ⁺ , CD105 ⁺ stem cells, wherein said large number of cells form one or more when cultured under conditions suitable for embryoid body formation The embryoid body, wherein said plurality of cells detectably inhibits T cell proliferation in a mixed lymphocyte reaction (MLR) assay. In a specific embodiment, the stem cells are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the stem cells are also CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said stem cells are OCT-4 ⁺ . In a more specific embodiment, said stem cells are also OCT-4 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

The present invention also provides an isolated cell population comprising a plurality of immunosuppressive placental stem cells that are OCT-4 ⁺ stem cells, wherein said plurality of cells form one or more embryoid-like bodies when cultured under conditions suitable for embryoid body formation. The embryoid body, wherein said plurality of cells detectably inhibits T cell proliferation in a mixed lymphocyte reaction (MLR) assay. In various embodiments, said isolated placental cells are 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% for OCT-4 ⁺ stem cells. In a specific embodiment of the above population, the stem cells are CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the stem cells are CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said stem cells are CD200 ⁺ . In a more specific embodiment, said stem cells are CD73 ⁺ , CD105 ⁺ , CD200 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said population has been expanded, eg, passaged at least 1 time, at least 3 times, at least 5 times, at least 10 times, at least 15 times, or at least 20 times.

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

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

Each of the above-mentioned large number of placental stem cells may comprise placental stem cells directly obtained or isolated from mammalian placenta, or cultured and passed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 , 16, 18, 20, 25, 30 or more times placental stem cells, or a combination thereof.

The above-mentioned large amount of immunosuppressive placental stem cells may comprise about, at least, or no more than 1×10 ⁵ , 5×10 ⁵ , 1×10 ⁶ , 5×10 ⁶ , 1×10 7 , 5×10 ⁷ , 1×10 7 , 1× ^{10 7} 10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 5×10 ⁹ , 1×10 ¹⁰ , 5×10 ¹⁰ , 1×10 ¹¹ or more placental stem cells.

5.2.3 Selection and preparation of placental stem cell populations

In another embodiment, the present invention also provides a method of selecting a large number of immunosuppressive placental stem cells from a large number of placental cells, which comprises selecting a population of placental cells, wherein at least 10%, at least 20%, at least 30% of said cells , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% are CD200 ⁺ , HLA-G ⁺ placental stem cells, and wherein said placental stem cells are in the mixed lymphocyte T cell proliferation was detectably inhibited in the MLR assay. In a specific embodiment, said selecting comprises selecting stem cells that are also CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said selecting comprises selecting stem cells that are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, the selection includes selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ or CD105 ⁺ . In another specific embodiment, said selecting further comprises selecting a plurality of placental stem cells capable of generating one or more embryoid bodies when cultured under conditions suitable for embryoid body formation.

In another embodiment, the present invention also provides a method for selecting a large number of immunosuppressive placental stem cells from a large number of placental cells, comprising selecting a large number of placental cells, wherein at least 10%, at least 20%, at least 30% of said cells , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% are CD73 ⁺ , CD105 ⁺ , CD200 ⁺ placental stem cells, and wherein said placental stem cells are in mixed lymphoid T cell proliferation was detectably inhibited in a cellular response (MLR) assay. In a specific embodiment, said selecting comprises selecting stem cells that are also HLA-G ⁺ . In another specific embodiment, the selection comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said selection comprises selection of placental stem cells that are also CD34 ⁻ , CD38 ⁻ , CD45 ⁻ and HLA-G ⁺ . In another specific embodiment, said selecting further comprises selecting a population of placental cells capable of generating one or more embryoid bodies when cultured under conditions suitable for embryoid body formation.

In another embodiment, the present invention also provides a method for selecting a large number of immunosuppressive placental stem cells from a large number of placental cells, comprising selecting a large number of placental cells, wherein at least 10%, at least 20%, at least 30% of said cells , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% are CD200 ⁺ , OCT-4 ⁺ placental stem cells, and wherein said placental stem cells are in mixed lymphocytes T cell proliferation was detectably inhibited in the MLR assay. In a specific embodiment, said selecting comprises selecting placental stem cells that are also CD73 ⁺ and CD105 ⁺ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also HLA-G ⁺ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, the selection includes selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , CD73 ⁺ , CD105 ⁺ and HLA-G ⁺ .

In another embodiment, the present invention also provides a method for selecting a large number of immunosuppressive placental stem cells from a large number of placental cells, comprising selecting a large number of placental cells, wherein at least 10%, at least 20%, at least 30% of said cells , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% are CD73 ⁺ , CD105 ⁺ and HLA-G ⁺ placental stem cells, and wherein said placental stem cells are in Detectably inhibits T cell proliferation in the mixed lymphocyte reaction (MLR) assay. In a specific embodiment, said selecting comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also CD200 ⁺ . In another specific embodiment, the selection includes selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ , CD45 ⁻ , OCT-4 ⁺ and CD200 ⁺ .

In another embodiment, the present invention also provides a method for selecting a large number of immunosuppressive placental stem cells from a large number of placental cells, comprising selecting a large number of placental cells, wherein at least 10%, at least 20%, at least 30% of said cells , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% are CD73 ⁺ , CD105 ⁺ placental stem cells, and wherein said large number of cells are suitable for embryoid bodies Formation conditions form one or more embryoid bodies. In a specific embodiment, said selecting comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ and CD45 ⁻ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also OCT-4 ⁺ . In a more specific embodiment, said selecting comprises selecting placental stem cells that are also OCT-4 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

In another embodiment, the present invention also provides a method for selecting a large number of immunosuppressive placental stem cells from a large number of placental cells, which comprises selecting a large number 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% are OCT4 ⁺ stem cells, and wherein said large number of cells are in a state suitable for embryoid body formation One or more embryoid bodies are formed under certain conditions. In a specific embodiment, said selecting comprises selecting placental stem cells that are also CD73 ⁺ and CD105 ⁺ . In another specific embodiment, the selection comprises selecting placental stem cells that are also CD34 ⁻ , CD38 ⁻ or CD45 ⁻ . In another specific embodiment, said selecting comprises selecting placental stem cells that are also CD200 ⁺ . In a more specific embodiment, said selecting comprises selecting placental stem cells that are also CD73 ⁺ , CD105 ⁺ , CD200 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ .

The present invention also provides a method for preparing immunosuppressive placental stem cells or a large amount of immunosuppressive placental stem cells. For example, the present invention provides a method of preparing a population of cells comprising selecting any of the plurality of placental stem cells described above and isolating the plurality of placental stem cells from other cells (eg, other placental cells). In a specific embodiment, the present invention provides a method for preparing a population of cells, comprising selecting placental cells, wherein the placental cells: (a) adhere to a substrate; (b) express CD200 and HLA-G, or express CD73, CD105 and CD200, or express CD200 and OCT-4, or express CD73, CD105 and HLA-G, or express CD73 and CD105, and promote the formation of stem cells comprising the stem cells when the population is cultured under conditions suitable for embryoid body formation. Formation of one or more embryoid bodies in a population of placental cells, or expressing OCT-4, and promoting one or more of a population of placental cells comprising the stem cells when the population is cultured under conditions suitable for embryoid body formation and (c) detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation in an MLR (mixed lymphocyte reaction); and isolating said placental cells from other cells to form a cell population.

In a more specific embodiment, the present invention provides a method of preparing a population of cells comprising selecting placental stem cells that (a) adhere to a substrate, (b) express CD200 and HLA-G, and (c) in the MLR detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation in a (mixed lymphocyte reaction); and isolating said placental stem cells from other cells to form a cell population. In another specific embodiment, the present invention provides a method of preparing a population of cells comprising selecting placental stem cells that (a) adhere to a substrate, (b) express CD73, CD105, and CD200, and (c) in the MLR detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation; and isolating said placental stem cells from other cells to form a cell population. In another specific embodiment, the present invention provides a method of preparing a population of cells comprising selecting placental stem cells that (a) adhere to a substrate, (b) express CD200 and OCT-4, and (c) in the MLR detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation; and isolating said placental stem cells from other cells to form a cell population. In another specific embodiment, the present invention provides a method for preparing a population of cells, comprising selecting placental stem cells that (a) adhere to a substrate, (b) express CD73 and CD105, (c) forming embryoid bodies when cultured under conditions for embryo body formation, and (d) detectably inhibiting proliferation of CD4 ⁺ or CD8 ⁺ T cells in the MLR; and isolating said placental stem cells from other cells to form a cell population. In another specific embodiment, the invention provides a method of preparing a population of cells comprising selecting placental stem cells that (a) adhere to a substrate, (b) express CD73, CD105, and HLA-G, and (c) detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation in the MLR; and isolating said placental stem cells from other cells to form a cell population. A method of preparing a population of cells comprising selecting placental stem cells that (a) adhere to a substrate, (b) express OCT-4, (c) form embryoid bodies when cultured under conditions suitable for embryoid body formation, and (d) detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation in the MLR; and isolating said placental stem cells from other cells to form a cell population.

In a specific embodiment of the preparation method of the immunosuppressive placental stem cell population, the T cells and the placental cells are present in the MLR at a ratio of about 5:1. The placental cells used in the present invention may be derived from whole placenta, or mainly from amnion, or amnion and chorion. In another specific embodiment, compared with the amount of T cell proliferation in the MLR when the placental cells are absent, the placental cells can be suppressed by at least 50%, at least 75%, or at least 90% in the MLR Or at least 95% proliferation of CD4 ⁺ or CD8 ⁺ T cells. The method can further comprise selecting and/or preparing a population of placental stem cells capable of immunomodulating (eg, inhibiting the activity of) other immune cells (eg, the activity of natural killer (NK) cells).

5.2.4 Growth in culture

Growth of placental stem cells described herein, as with any mammalian cell, depends in part on the particular growth medium chosen. Under optimal conditions, the number of placental stem cells usually doubles within 3-5 days. During the culture process, the placental stem cells of the present invention adhere to the culture substrate, for example, the surface of a tissue culture vessel (eg, plastic tissue culture dish, fibronectin-coated plastic, etc.), and form a monolayer.

When cultured under appropriate conditions, isolated placental cell populations comprising placental stem cells of the invention form embryoid bodies, ie, grow three-dimensional clusters of cells on top of a layer of adherent stem cells. Cells within embryoid bodies express markers associated with very early stem cells (eg, OCT-4, Nanog, SSEA3 and SSEA4). Similar to placental stem cells described here, cells within embryoid bodies generally do not adhere to the culture substrate, but remain attached to adherent cells in culture. Since embryoid bodies do not form in the absence of adherent stem cells, survival of embryoid body cells depends on adherent placental stem cells. The adherent placental stem cells thus promote the growth of one or more embryoid bodies in a population of placental cells comprising the adherent placental stem cells. Without wishing to be bound by theory, it is believed that the cells of the embryoid body grow on the adherent placental stem cells as embryonic stem cells grow on the feeder cell layer. Mesenchymal stem cells (eg, bone marrow-derived mesenchymal stem cells) do not form embryoid bodies in culture.

5.2.5 Differentiation

Placental stem cells useful in the methods of the invention can be differentiated into different committed cell lines. For example, the placental stem cells can be differentiated into adipogenic, chondrogenic, neurogenic or osteogenic cell lines. Such differentiation can be achieved by any differentiation method known in the art, for example, a method of differentiating bone marrow-derived mesenchymal stem cells into similar cell lines.

5.3 Methods of obtaining placental stem cells

5.3.1 Stem cell collection composition

The present invention further provides methods for collecting and isolating placental stem cells. In general, stem cells can be harvested from a mammalian placenta by using a physiologically acceptable solution, such as a stem cell harvesting composition. A detailed description of the stem cell collection composition can be found in the paper entitled "Improved Composition for Collecting and Preserving Placental Stem Cells and Methods of Using the Composition" submitted on December 29, 2005. )” U.S. Provisional Application No. 60/754,969.

Stem cell collection compositions include any physiologically acceptable solution suitable for collecting and/or culturing stem cells, for example, a saline solution (e.g., phosphate buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCl, etc.), Medium (for example, DMEM, H.DMEM, etc.) and the like.

The stem cell collection composition may comprise one or more components that tend to preserve the placental stem cells, i.e., prevent the death of the placental stem cells from the time of collection to the time of culture, or delay the death of the placental stem cells, reduce the number of dead placental stem cells in the cell population, etc. components. Such components can be, for example, apoptosis inhibitors (eg, caspase inhibitors or JNK inhibitors); vasodilators (eg, magnesium sulfate, antihypertensive drugs, atrial natriuretic peptide (ANP), corticotropin , corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, phosphodiesterase inhibitors, etc.); necrosis inhibitors (eg, 2-(1H-indole -3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); TNF-α inhibitors; and/or oxygen-carrying perfluorocarbons (e.g., perfluorocarbons fluorooctyl bromide, perfluorodecyl bromide, etc.).

The stem cell collection composition may comprise one or more tissue degrading enzymes, for example, metalloproteases, serine proteases, neutral proteases, ribonucleases or deoxyribonucleases, and the like. 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 , release enzyme, hyaluronidase, etc.

The stem cell collection composition may include a bactericidally or bacteriostatically effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide (eg, tobramycin), cephalosporin (eg, cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefaclor oxime or cefadroxil), clarithromycin, erythromycin, penicillin (eg, penicillin V) or quinolones (eg, ofloxacin, ciprofloxacin, or norfloxacin), tetracycline, streptomycin, and the like. In particular embodiments, the antibiotic is active against Gram (+) and/or Gram (-) bacteria, eg, Pseudomonas aeruginosa, Staphylococcus aureus, and the like.

The stem cell collection composition may also include one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50 mM); molecular weight greater than 20,000 Macromolecules of urton, which, in one embodiment, are present in amounts sufficient to maintain endothelial integrity and cell viability (e.g., from about 25 g/l to about 100 g/l, or from about 40 g/l to about 60 g/l synthetic or naturally occurring colloids, polysaccharides such as dextran or polyethylene glycol); antioxidants (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, present in amounts from about 25 μM to about 100 μM glycine, vitamin C, or vitamin E); reducing agents (e.g., N-acetylcysteine present in an amount from about 0.1 mM to about 5 mM); agents that prevent calcium from entering cells (e.g., verapamil present in an amount); nitroglycerin (e.g., from about 0.05 g/L to about 0.20 g/L); an anticoagulant, which, in one embodiment, is present in an amount sufficient to help prevent residual blood clots (for example, heparin or hirudin present in a concentration of about 1000 units/l to about 100,000 units/l); or amiloride-containing compounds (for example, amiloride in an amount of about 1.0 μM to about 5 μM amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride).

5.3.2 Collection and processing of placenta

In general, human placentas are recovered immediately after delivery. In a preferred embodiment, the placenta is recovered from the patient with the patient's informed consent and after obtaining the patient's complete medical history related to the placenta. Preferably, the medical history continues after delivery. This medical history can be used to adjust the subsequent use of the placenta or stem cells harvested therefrom. For example, based on the medical history, human placental stem cells can be used as individualized medicine for the infant associated with the placenta, or the infant's parents, siblings, or other relatives.

Cord blood and placental blood are removed prior to placental stem cell recovery. In certain embodiments, the cord blood in the placenta is recovered after delivery. The placenta can be subjected to routine cord blood salvage processing. Blood is usually drawn from the placenta with the aid of gravity using a needle or cannula (see, eg, Anderson, US Patent No. 5,372,581; Hessel et al., US Patent No. 5,415,665). This needle or cannula is usually inserted into the umbilical cord vein and gently massages the placenta to help it drain the cord blood. The cord blood salvage can also be done commercially, such as LifeBank Corporation, Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. Preferably, the placenta is gravity drained and no other manipulation is performed to minimize tissue disruption during cord blood recovery.

The placenta is usually shipped from the delivery room to another location (eg, a laboratory) to recover the cord blood and harvest the stem cells by methods such as perfusion or tissue separation. Ship the placenta preferably in a sterile, thermally insulated transport device (maintaining the placenta temperature at 20-28°C), eg, place the placenta clamping the proximal umbilical cord in a sterile zip-lock plastic bag and place the bag in the Insulated container. In another embodiment, the placenta is shipped in a cord blood collection kit substantially as described in pending US Patent Application Serial No. 11/230,760, filed September 19, 2005. Preferably, the placenta is sent to the laboratory within 4 to 24 hours after delivery. In certain embodiments, the proximal umbilical cord is clamped within preferably 4-5 cm (centimeters) of insertion into the placenta prior to cord blood recovery. In another embodiment, the proximal cord is clamped after cord blood recovery but prior to further processing of the placenta.

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

Once harvested and prepared substantially as described above, the mammalian placenta or portion thereof may be processed (e.g., perfused or disrupted, e.g., digested with one or more tissue disrupting enzymes) by any method known in the art ) to obtain stem cells.

5.3.3 Physical disruption and enzymatic digestion of placental tissue

In one embodiment, stem cells can be harvested from a mammalian placenta by physically disrupting (eg, enzymatically digesting) the organ. For example, the placenta or portion thereof can be crushed, sheared, minced, diced, minced, macerated, etc. while contacting the stem cell collection composition of the invention, followed by digestion of the tissue with one or more enzymes. The placenta or portion thereof may also be physically disrupted and digested with one or more enzymes, and the resulting material immersed or mixed in the stem cell collection composition of the invention. Any method of physical disruption is suitable, as long as a large amount, more preferably most, more preferably at least 60%, 70%, 80%, 90%, 95%, 98% of the organ can be determined by e.g. trypan blue exclusion method or 99% of the cells survived.

The placenta can be sectioned prior to physical disruption and/or enzymatic digestion and stem cell recovery. For example, placental stem cells can be obtained from amnion, chorion, placental villi, or any combination thereof. Preferably, placental stem cells are obtained from placental tissue comprising amnion and chorion. Placental stem cells can typically be obtained by disrupting small pieces of placental tissue, e.g., volumes 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 1000 cubic millimeters of placental tissue.

Preferred stem cell collection compositions comprise one or more tissue disrupting enzymes. Enzymatic digestion is preferably performed using a combination of enzymes (eg, a combination of matrix metalloprotease and dispase, eg, a combination of collagenase and dispase). In one embodiment, enzymatic digestion of placental tissue uses a combination of matrix metalloproteinases, neutral proteases, and mucopolysaccharases for digestion of hyaluronic acid, such as collagenase, a combination of dispase and hyaluronidase, or a release enzyme (Boehringer Mannheim Company, Indianapolis, Ind.) and hyaluronidase. Other enzymes that can be used to disrupt tissue include papain, deoxyribonuclease, serine proteases such as trypsin, chymotrypsin or elastase. Serine proteases are inhibited by α2 microglobulin in serum, so the matrix used for digestion is usually serum-free. EDTA and DNase are commonly used during enzymatic digestion to increase the efficiency of cell recovery. It is preferable to dilute the digest to avoid entrapment of stem cells in the viscous digest.

Any combination of tissue-digesting enzymes can be used. Common concentrations of tissue-digesting enzymes include, for example, 50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100 U/mL for elastase. Proteases can be used in combination, ie, two or more proteases are employed in the same digestion reaction, or can be used sequentially to release placental stem cells. For example, in one embodiment, the placenta or a portion thereof is first digested with an appropriate amount of about 2 mg/ml collagenase I at 37° C. for 30 minutes, followed by digestion with 0.25% trypsin at 37° C. for 10 minutes. Preferably the serine protease is used immediately after the other enzyme.

In another embodiment, a chelating agent, such as ethylene glycol bis(ethylene glycol), can be added to the stem cell harvesting composition containing the stem cells, or to the solution in which the tissue is disrupted and/or digested prior to using the stem cell harvesting composition to isolate the stem cells. (2-Aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA) to further disrupt the tissue.

It will be appreciated that when an intact placenta or a portion of a placenta contains both fetal and maternal cells (e.g., when the placental portion contains chorion or chorionic lobes), the placental stem cells harvested will contain placental stem cells from both fetal and maternal sources. mixture. When the placental portion contains no or negligible amounts of maternal cells (eg, amnion), the harvested placental stem cells comprise almost exclusively fetal placental stem cells.

5.3.4 Placental perfusion

Placental stem cells can also be obtained by perfusion of mammalian placenta. Methods of perfusing a mammalian placenta to obtain stem cells are disclosed, for example, in Hariri, U.S. Application Publication No. 2002/0123141, and in "Improved Compositions for Harvesting and Preserving Embryonic Stem Cells and Methods of Such Compositions," filed December 29, 2005. U.S. Provisional Application No. 60/754,969 for "Improved Composition for Collecting and Preserving Placental StemCells and Methods of Using the Composition."

或塑料套管)插入脐静脉。该无菌连接装置与灌注集流腔相连。Placental stem cells can be harvested by perfusing, for example, placental vasculature using, for example, a stem cell harvesting composition as a perfusion solution. In one embodiment, a mammalian placenta may be perfused by passing a perfusion solution through either or both of the umbilical artery and umbilical vein. The perfusion solution may flow through the placenta by gravity flow or the like. Preferably, the perfusion solution is forced through the placenta by a pump (eg, a peristaltic pump). A cannula (e.g.,

or plastic cannula) into the umbilical vein. The sterile connection is connected to the perfusion manifold.

In preparation for perfusion, the placenta is preferably oriented (eg, suspended) in such a way that the umbilical artery and umbilical vein are positioned at the apex of the placenta. The placenta is perfused by passing a perfusate (eg, a stem cell harvesting composition of the invention) through the placental vasculature or through the placental vasculature and surrounding tissue. In one embodiment, the umbilical artery and umbilical vein are connected simultaneously to a suction tube connected to a perfusion solution reservoir via a flexible connector. The perfusion solution flows into the umbilical vein and artery. The perfusion solution flows from and/or through the walls of the blood vessels into the surrounding tissue of the placenta and is collected from the surface of the placenta in connection with the mother's uterus during pregnancy into a suitable open container. The perfusion solution may also be directed through the opening in the umbilical cord and out or filtered out of the opening in the placental wall joined to the maternal uterine wall. In another embodiment, the perfusion solution is passed through the umbilical vein and collected from the umbilical artery, or passed through the umbilical artery and collected from the umbilical vein.

In one embodiment, the proximal umbilical cord may be clamped during perfusion, more preferably within 4-5 cm (centimeters) of the insertion of the umbilical cord into the placenta.

Perfusate first collected from a mammalian placenta during exsanguination is often colored due to residual red blood cells in the cord blood and/or placental blood. This perfusate will become shallower as perfusion progresses and residual cord blood is washed out of the placenta. Usually 30 to 100 ml (milliliters) of perfusate is sufficient for initial deblood removal of the placenta, but more or less perfusate can be used depending on observations.

The volume of perfusate used to harvest placental stem cells can vary depending on the number of stem cells to be harvested, the size of the placenta, the number of harvests from a single placenta, and other factors. In various embodiments, the volume of perfusate may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Perfusion is usually performed with 700-800 mL of perfusate after phlebotomy.

The placenta can be perfused multiple times over the course of hours or days. When multiple perfusions of the placenta are required, the placenta can be stored or cultured under sterile conditions in containers or other suitable vessels with or without anticoagulants (e.g., heparin, warfarin sodium, coumadin, etc.) dihydroxycoumarin) and/or with or without antibacterial agents (e.g., β-mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., 40-100 μg/ml), penicillin (e.g., 40 U/ml ml), amphotericin B (eg, 0.5 μg/ml)), or a standard perfusion solution (eg, conventional saline solution, such as phosphate-buffered saline) for perfusion. In one embodiment, the isolated placenta is preserved or cultured for a period of time without harvesting the perfusate, for example preserving or culturing the placenta 1, 2, 3, 4, 5, 6, 7, 8, prior to perfusion and perfusate harvesting. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or 2 or 3 or more days. The perfused placenta may be preserved for an additional period of time, e.g., 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 perfuse again with eg 700-800 mL of perfusate. The placenta may be perfused 1, 2, 3, 4, 5 or more times, for example every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, perfusion of the placenta and harvesting of the perfusion solution (eg, stem cell harvesting composition) can be repeated until the number of recovered nucleated cells falls below 100 cells/ml. Perfusates at different time points can be further processed individually to recover time-dependent cell populations, such as stem cells. Perfusates from different time points can also be pooled.

Without wishing to be bound by any theory, after exsanguination of the placenta and perfusion of the placenta for a sufficient period of time, placental stem cells are believed to transfer into the bloodless and perfused placental microcirculation, wherein as described herein, preferably by perfusion into the collection container for They collect. Perfusing the isolated placenta not only helps to remove residual cord blood but also provides proper nutrients, including oxygen, to the placenta. The placenta is incubated and perfused using a solution similar to that used to remove residual cord blood cells (preferably without added anticoagulant).

Placental stem cells in the harvest obtained from perfusion according to the methods of the invention are significantly greater than those obtained from mammalian placenta not perfused with said solution and without other treatments to obtain stem cells (e.g., by tissue disruption, e.g., enzymatic digestion) number of placental stem cells. "Significantly more" in this context means at least 10% more. Perfusion performed according to the method of the invention results in significantly greater numbers of placental stem cells than, for example, placental stem cells obtained from culture medium in which the placenta or parts thereof are cultured.

Stem cells can be isolated from the placenta by perfusion with a solution comprising one or more proteases or other tissue disrupting enzymes. In specific embodiments, the placenta or portion thereof (e.g., amnion, amnion and chorion, placental lobules or cotyledons, or any combination of the foregoing) may be conditioned at 25-37°C prior to disruption with one or more tissues Enzymes were incubated in 200 mL of medium for 30 minutes. Cells were harvested from the perfusate, temperature adjusted to 4°C, and washed with a cold inhibitor cocktail containing 5 mM EDTA, 2 mM dithiothreitol, and 2 mM β-mercaptoethanol. After several minutes, the stem cells are washed with the cold (eg, 4° C.) stem cell collection composition of the invention.

Perfusion by the present pan method is understood to be the collection of perfusate from the maternal side of the placenta to obtain a mixture of fetal and maternal cells. Thus, cells harvested by this method comprise a mixture of placental stem cell populations of both fetal and maternal origin. In contrast, perfusion through the placental vasculature alone (where the perfusate is passed through one or both placental vessels and harvested only through the remaining vessels) results in a collection of placental stem cell populations that are almost exclusively of fetal origin.

5.3.5 Isolation, sorting and characterization of placental stem cells

Stem cells obtained from mammalian placenta, whether by perfusion or enzymatic digestion, can first be purified (ie, isolated) from other cells by Ficoll gradient centrifugation. This centrifugation can refer to any standard protocol for centrifugation speed. For example, in one embodiment, cells harvested from the placenta can be recovered from the perfusate by centrifugation at 5000 xg for 15 minutes at room temperature, which can separate the cells from contaminating debris or platelets, for example. In another embodiment, the placental perfusate is concentrated to about 200 ml, gently layered on Ficoll, and centrifuged at about 1100 xg for 20 minutes at 22°C to harvest the low-density cell medial layer for further processing.

The cell pellet was resuspended in fresh stem cell collection composition or a medium suitable for stem cell storage, such as IMDM serum-free medium (GibcoBRL, NY) containing 2 U/ml heparin and 2 mM EDTA. Total monocyte fractions can be isolated using, for example, Lymphoprep (Nycomed Pharma, Oslo, Norway) following the manufacturer's recommendations.

"Isolated" placental stem cells as used herein refers to removal of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells normally present in an intact mammalian placenta. Cells related to the stem cell. A stem cell of an organ is "isolated" when the population of cells in which the stem cell is located contains less than 50% of the cells normally associated with the stem cell in an intact organ.

Placental cells obtained by perfusion or digestion can be further or first isolated by differential trypsinization using, for example, 0.05% trypsin solution containing 0.2% EDTA (Sigma, St. Louis MO). Differential trypsinization is possible because placental stem cells typically detach from plastic surfaces within approximately 5 minutes, whereas other adherent populations typically require more than 20-30 minutes of incubation. Detached placental stem cells can be harvested after trypsinization and trypsinization using eg Trypsin Neutralization Solution (TNS, Cambrex). In one embodiment for isolating adherent cells, aliquots (e.g., about 5-10 x 10 ⁶ ) of cells are each placed into one of a plurality of T-75 flasks, preferably into fibronectin-coated T75 flasks. bottle. In this embodiment, the cells can be cultured in a tissue culture incubator (37°C, 5% _CO2 ) with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex). After 10 to 15 days, unattached cells were washed from the flasks with PBS. Then replace PBS with MSCGM. Flasks are preferably checked daily for the presence of various adherent cell types, particularly for identification and expansion of fibroblast clusters.

The number and type of cells collected from mammalian placenta can be monitored, for example, by using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., by tissue-specific or cell marker-specific detection of changes in morphology and cell surface markers, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), etc., by using optical or confocal microscopy to detect cell morphology, and/or by methods known in the art Techniques such as PCR and gene expression profiling detect changes in gene expression. These techniques can also be used to identify cells that are positive for one or more specific markers. For example, one can determine whether a cell contains measurable CD34 by the techniques described above using an antibody to CD34; if so, the cell is CD34 ⁺ . Likewise, a cell is OCT-4+ if it produces enough OCT-4 RNA to be detected by RT-PCR, or produces significantly more OCT- ⁴ RNA than mature cells. Antibodies against cell surface markers (eg, 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 separation, differential adhesion, or a combination of the two, 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: 150-165). Laser excitation of fluorescent moieties in individual particles creates a tiny charge that electromagnetically separates the positive and negative particles in the mixture. In one embodiment, cell surface marker-specific antibodies or ligands can be labeled with a unique fluorescent label. Cells are processed through a cell sorter to separate cells based on their ability to bind the antibodies used. FACS-sorted particles can be placed directly into individual wells of 96- or 384-well plates to facilitate isolation and cloning.

In one sorting protocol, stem cells from placenta are sorted for expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4 and/or HLA-G. This can be achieved in conjunction with a step of selecting stem cells based on their adherent properties in culture. For example, adherent selection of stem cells can be accomplished before or after sorting based on marker expression. For example, in one embodiment, cells may first be sorted based on their CD34 expression; ^CD34- cells are retained and CD200 ⁺ HLA-G ⁺ cells are separated from all other ^CD34- cells. In another embodiment, cells from the placenta can be sorted for expression of the markers CD200 and/or HLA-G; for example, cells displaying either marker can be isolated for further use. In a specific embodiment, cells expressing eg CD200 and/or HLA-G can be based on their expression of CD73 and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, or CD34, CD38 or CD45 The deletions were further sorted. For example, in one embodiment, placental cells can be sorted based on the expression or absence of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and CD200 ⁺ , HLA- G ⁺ , CD73 ⁺ , CD105 ⁺ , CD34 ⁻ , CD38 ⁻ and CD45 ⁻ placental cells were used for further use.

In another embodiment, magnetic beads can be used to isolate cells. Cells can be sorted using Magnetic Activated Cell Sorting (MACS), a method of separating particles based on their ability to bind to magnetic beads (0.5-100 μm in diameter). A variety of useful modifications can be made to magnetic microspheres, including the covalent addition of antibodies that specifically recognize specific cell surface molecules or haptens. The beads are then mixed with the cells for binding. The cells are then passed through a magnetic field to isolate cells with specific cell surface markers. In one embodiment, cells can be subjected to such separation and remixed with magnetic beads bound to antibodies to other cell surface markers. The cells are again passed through the magnetic field to separate cells that bind both antibodies. Such cells are then diluted into individual culture dishes, such as microtiter dishes, for clonal isolation.

Placental stem cells can also be characterized and/or sorted based on cell morphology and growth characteristics. For example, placental stem cells can be characterized, eg, have a fibroblastic appearance in culture, and/or placental stem cells can be selected based on, eg, a fibroblastic appearance in culture. Placental stem cells can also be characterized and/or selected for the ability to form embryoid bodies. In one embodiment, for example, placental cells that are fibroblast-like in culture, express CD73 and CD105, and generate one or more embryoid bodies are isolated from other placental cells. In another embodiment, OCT-4 ⁺ placental cells that generate one or more embryoid bodies in culture are isolated from other placental cells.

In another embodiment, placental stem cells can be identified and characterized by a colony forming unit assay. Colony forming unit assays are known in the art, for example Mesen Cult medium (Stem Cell Technologies, Vancouver British Columbia).

Viability, proliferative potential, and longevity of placental stem cells can be assessed by standard techniques known in the art, such as trypan blue exclusion test, fluorescent diacetic acid uptake test, propidium iodide uptake test (to assess viability); and thymidine Uptake test, MTT cell proliferation test (assessment of proliferation). Lifespan can be determined by methods known in the art, eg, determining the maximum number of doubling populations in expanded culture.

Placental stem cells can also be isolated from other placental cells by other techniques known in the art, for example, selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection); For example, soybean lectin is used in separation based on differential cell agglutination; freeze-thaw method; filtration; conventional and zonal centrifugation; centrifugal elutriation (countercurrent centrifugation); unit gravity separation; countercurrent distribution;

5.4 Culture of placental stem cells

5.4.1 Medium

Isolated placental stem cells, or populations of placental stem cells, or cells growing placental stem cells or placental tissue can be used to initiate or seed the cell culture. Cells are typically transferred into uncoated or coated extracellular matrix or ligands such as laminin, collagen (eg, native or denatured collagen), gelatin, fibronectin, ornithine, vitronectin, and extracellular Sterile tissue culture vessels for membrane proteins (e.g., MATRIGEL (BD Discovery Labware, Bedford, Mass.)).

Placental stem cells can be cultured in any medium and under any conditions recognized in the art for culturing stem cells. Preferably, the medium contains serum. For example, placental stem cells can be cultured in a medium containing ITS (insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serum albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1 and penicillin DMEM-LG (Dulbecco's modified essential medium, low sugar)/streptomycin/MCDB 201 (chicken fibroblast basal medium); DMEM-HG (high sugar) containing 10% fetal bovine serum (FBS); containing 15 DMEM-HG with %FBS; IMDM (Iscove's modified Dulbecco's medium) containing 10% FBS, 10% horse serum and hydrocortisone; M199 containing 10% FBS, EGF and heparin; containing 10% FBS, GlutaMAX ^TM and α-MEM (minimum essential medium) of gentamicin; DMEM containing 10% FBS, GlutaMAX ^™ and gentamicin, etc. A preferred medium is DMEM-LG/MCDB-201 containing 2% FBS, ITS, LA+BSA, dextrose, L-ascorbic acid, PDGF, EGF and penicillin/streptomycin.

Other media that can be used to culture placental stem cells include DMEM (high or low glucose), Eagle's Basal Medium, Ham's F10 Medium (F10), Ham's F-12 Medium (F12), Iscove's Modified Dulbecco's Medium, Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 Medium, MCDB, DMIEM/F12, RPMI 1640, Advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO FREE.

The medium may be supplemented with one or more components 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, e.g., platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fiber protocellular growth factor (bFGF), insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO); amino acids, including L-valericin and one or more antibiotics and/or antimycotics to control microbial contamination, for example, one or a combination of penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and nystatin.

5.4.2 Expansion and proliferation of placental stem cells

Once the placental stem cell or population of stem cells is isolated (e.g., from at least 50% of the placental cells with which the stem cell or population of stem cells are normally associated in vivo), the stem cell or population of stem cells can be propagated or expanded in vitro. For example, a population of placental stem cells can be cultured in a tissue culture vessel (e.g., dish, bottle, multiwell plate, etc.) for a sufficient time to allow the stem cells to proliferate to 70-90% confluency, i.e., until the stem cells and their progeny occupy the tissue culture vessel 70-90% of the culture surface area.

Placental stem cells can be seeded into a culture vessel at a density that allows for cell growth. For example, the cells can be seeded at a low density (eg, about 1,000 to about 5,000 cells/cm ² ) to a high density (eg, about 50,000 or more cells/cm ² ). In a preferred embodiment, the cells are cultured in an atmosphere containing about 0 to about 5% _CO2 by volume. In some preferred embodiments, the cells may be cultured in an atmosphere of about 2% to about 25% _O2 , preferably about 5% to about 20% _O2 in air. Cells are preferably cultured at about 25°C to about 40°C (preferably 37°C). Cells are preferably grown in an incubator. The medium can be static or shaken by a bioreactor or the like. Placental stem cells are preferably grown under low oxidative stress (eg, supplementation of glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, etc.).

Once 70%-90% confluent is achieved, the cells can be passaged. For example, cells can be detached from tissue culture surfaces by enzymatic treatment (eg, trypsinization) using techniques known in the art. After removing the cells with a pipette and counting the cells, about 20,000-100,000 stem cells, preferably about 50,000 stem cells, are passaged to a new culture vessel containing fresh medium. Typically, the new medium is the same type of medium from which the stem cells were removed. The invention encompasses populations of placental stem cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more.

5.4.3 Placental stem cell population

The present invention provides a population of placental stem cells. A population of placental stem cells may be isolated directly from one or more placentas; i.e., the population of placental stem cells may be a population of placental cells including those obtained from or contained in perfusate, or obtained from digestive fluid (i.e., for Placental stem cells obtained from a cell collection obtained by enzymatic digestion of a placenta or a part thereof) or contained in the digested solution. The isolated placental stem cells of the invention can also be cultured and expanded to produce a population of placental stem cells. Placental stem cell populations can also be prepared by culturing and expanding placental cell populations comprising placental stem cells.

Placental stem cell populations of the invention comprise placental stem cells, eg, placental stem cells described herein. 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 placental stem cell population can comprise, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% non-stem cells.

The present invention provides methods for preparing isolated populations of placental stem cells by, for example, selecting for placental stem cells (whether obtained from enzymatic digestion or perfusion) that express specific markers and/or specific cultural or morphological characteristics. For example, in one embodiment, the invention provides a method of preparing a population of cells comprising selecting placental cells that (a) adhere to a substrate and (b) express CD200 and HLA-G; and from other cells The cells are isolated to form cell populations. In another embodiment, a method of preparing a population of cells comprises selecting placental cells that (a) adhere to a substrate and (b) express CD73, CD105, and CD200; and isolating said cells from other cells to form cells group. In another embodiment, a method of preparing a population of cells comprises selecting placental cells that (a) adhere to a substrate and (b) express CD200 and OCT-4; and isolating said cells from other cells to form cells group. In another embodiment, a method of preparing a population of cells comprises selecting placental cells that (a) adhere to a substrate, (b) express CD73 and CD105, and (c) when said population is in a culture suitable for embryoid body formation promoting the formation of one or more embryoid bodies in a population of placental cells comprising said stem cells when cultured under conditions; and isolating said cells from other cells to form a population of cells. In another embodiment, a method of preparing a population of cells comprises selecting placental cells that (a) adhere to a substrate and (b) express CD73, CD105, and HLA-G; and isolating said cells from other cells to form cell clusters. In another embodiment, a method of preparing a population of cells comprises selecting placental cells that (a) adhere to a substrate, (b) express OCT-4, and (c) when said population is in a state suitable for embryoid body formation promoting the formation of one or more embryoid bodies in a population of placental cells comprising said stem cells when cultured under conditions; and isolating said cells from other cells to form a population of cells. In any of the above embodiments, the method may further comprise selecting placental cells expressing ABC-p (placenta-specific ABC transporter; e.g., see Allikmets et al., Cancer Res. 58(23):5337-9 (1998)) . The method may also include selecting cells exhibiting at least one characteristic characteristic of, eg, mesenchymal stem cells (eg, expression of CD29, expression of CD44, expression of CD90, or expression of a combination of the foregoing).

In the embodiments described above, the substrate can be any surface on which cell (eg, placental stem cells) culture and/or selection can be performed. The substrate is usually plastic, eg, a tissue culture dish or a plastic multiwell plate. Tissue culture dishes can be coated with biomolecules such as laminin or fibronectin.

Cells, such as placental stem cells, can be selected for use in a placental stem cell population by any cell selection method known in the art. For example, cells can be selected by flow cytometry or FACS using antibodies to one or more cell surface markers. Selection can be performed using antibody-conjugated magnetic beads. Antibodies specific for certain stem cell-related markers are known in the art. For example, for OCT-4 (Abeam, Cambridge, MA), CD200 (Abeam), HLA-G (Abeam), CD73 (BD BiosciencesPharmingen, San Diego, CA), CD 105 (Abeam; BioDesign International, Saco, ME) etc. antibodies. Antibodies against other markers are also commercially available, for example CD34, CD38 and CD45 from companies such as StemCell Technologies or BioDesign International.

The isolated population of 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 or non-placental cells. For example, an isolated population of placental stem cells can be combined with blood (e.g., placental blood or umbilical cord blood), blood-derived stem cells (e.g., stem cells from placental blood or umbilical cord blood), blood-derived nucleated cell populations, bone marrow-derived mesenchymal cells, Bone-derived stem cell populations, native bone marrow, adult (body) stem cells, stem cell populations contained in tissues, cultured stem cells, fully differentiated cell populations (e.g., chondrocytes, fibroblasts, amnion cells, osteoblasts, muscle cells, cardiomyocytes, etc.) and other combinations. Cells in an isolated population of placental stem cells may be combined with a number of other cell types in ratios of about 100,000,000:1, 50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1 relative to the total number of nucleated cells in each population 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, 2:1, 1:1, 1:2, 1:5, 1:10, 1:100, 1:1 200, 1:500, 1:1,000, 1:2,000, 1:5,000, 1:10,000, 1:20,000, 1:50,000, 1:100,000, 1:500,000, 1:1,000,000, 1:2,000,000, 1:5,000,000, 1:10,000,000, 1:20,000,000, 1:50,000,000 or about 1:100,000,000. Cells in an isolated population of placental stem cells may also be combined with a plurality of cells of a plurality of cell types.

In one embodiment, an isolated population of placental stem cells is combined with a plurality of hematopoietic stem cells. For example, such hematopoietic stem cells may be contained within unprocessed placenta, cord blood, or peripheral blood; within total nucleated cells from placental blood, cord blood, or peripheral blood; in isolated in ^{untreated bone marrow; in total nucleated cells from bone marrow; in isolated CD34+ cell} populations from bone marrow, etc.

5.5 Preservation of placental stem cells

Placental stem cells can be preserved, that is, placed under conditions that allow long-term storage, or under conditions that inhibit cell death due to apoptosis, necrosis, or the like.

U.S. Provisional Application No. entitled "Improved Composition for Collecting and Preserving Placental Stem Cells and Methods of Using the Composition" filed on December 25, 2005 60/754,969, placental stem cells can be preserved using, for example, a composition comprising an inhibitor of apoptosis, an inhibitor of necrosis, and/or an oxygen-carrying perfluorocarbon. In one embodiment, the present invention provides a method of preserving a population of stem cells comprising contacting said population of stem cells with a stem cell harvesting composition comprising an apoptosis inhibitor and an oxygen-carrying perfluorocarbon, wherein the stem cell population not contacted with the apoptosis inhibitor The inhibitor of apoptosis is present in an amount and for a time sufficient to reduce and prevent apoptosis in the stem cell population compared to the stem cell population of the agent. In a specific embodiment, said inhibitor of apoptosis is a caspase inhibitor. In another specific embodiment, said inhibitor of apoptosis is a JNK inhibitor. In a more specific embodiment, said JHK inhibitor does not modulate differentiation or proliferation of said stem cells. In another embodiment, said stem cell collection composition comprises said inhibitor of apoptosis 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 further includes an emulsifier, such as lecithin. In another embodiment, said inhibitor of apoptosis and said perfluorocarbon are at a temperature between about 0°C and about 25°C when contacting the stem cells. In another more specific embodiment, said inhibitor of apoptosis and said perfluorocarbon are contacted with stem cells at a temperature between about 2°C and about 10°C, or between about 2°C and about 5°C. In another more specific embodiment, said contacting takes place during transport of said population of stem cells. In another more specific embodiment, said contacting is performed during freezing and thawing of said population of stem cells.

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

In another embodiment of the method, placental stem cells are contacted with a stem cell harvesting composition comprising an apoptosis inhibitor and an oxygen-carrying perfluorocarbon, an organ preservation compound, or a combination thereof during perfusion. In another embodiment, the stem cells are contacted in a tissue disruption (eg, enzymatic digestion) treatment. In another embodiment, the placental stem cells are contacted with the stem cell harvesting compound after harvesting by perfusion, or harvesting by tissue disruption (eg, enzymatic digestion).

During placental cell harvesting, enrichment and isolation, it is generally preferred to minimize or eliminate cellular stress such as hypoxia and mechanical stress. Thus, in another embodiment of the method, during said preservation, during collection, enrichment or isolation, the stem cells or population of stem cells are exposed to hypoxic conditions for less than six hours, wherein hypoxic conditions are hypoxic conditions below normoxia Concentration of oxygen concentration. In a more specific embodiment, during said preserving, said population of stem cells is exposed to said hypoxic condition for less than two hours. In another more specific embodiment, said stem cell population is exposed to said hypoxic condition for less than one hour, or less than 30 minutes, or is not exposed to said hypoxic condition during collection, enrichment or isolation. In another specific embodiment, said population of stem cells is not exposed to shear stress during harvesting, enrichment or isolation.

The placental stem cells of the present invention can be cryopreserved, for example, in a cryopreservation medium in a small container such as an ampoule. Suitable cryopreservation media include, but are not limited to, media, including growth media or cell freezing media, such as commercially available cell freezing media such as C2695, C2639 or C6039 (Sigma). The cryopreservation medium preferably contains DMSO (dimethyl sulfoxide) at a concentration of, for example, about 10% (v/v). Cryopreservation media may contain other agents, eg, methylcellulose and/or glycerol. Placental stem cells are preferably cooled at a rate of about 1 °C/min during cryopreservation. The preferred cryopreservation temperature is from about -80°C to about -180°C, preferably from about -125°C to about -140°C. Cryopreserved cells can be transferred to liquid nitrogen before thawing for use. For example, in some embodiments, once the ampoule reaches about -90°C, it is transferred to a liquid nitrogen storage area. Cryopreserved cells are preferably thawed at a temperature of from about 25°C to about 40°C, preferably at about 37°C.

5.6 Use of placental stem cells

5.6.1 Compositions comprising placental stem cells

The immunosuppressive methods of the invention may employ compositions comprising placental stem cells or biomolecules obtained therefrom. Similarly, the plurality of placental stem cells and populations of placental stem cells of the invention may be combined with any physiologically acceptable or medically acceptable compound, composition or device for use, eg, in research or therapy.

5.6.1.1 Placental stem cells cryopreserved

Populations of immunosuppressive placental stem cells of the invention can be preserved (eg, cryopreserved) for later use. Methods for cryopreserving cells, such as stem cells, are well known in the art. Placental stem cell populations can be prepared in a form that is readily administered to an individual. For example, the invention provides a population of placental stem cells contained in a container suitable for medical administration. For example, the container can be a sterile plastic bag, bottle, jar, or other container that facilitates administration of the placental stem cell population. For example, the container may be a blood bag or other plastic, medically acceptable bag suitable for intravenous administration of fluids to a recipient. The container is preferably a container capable of cryopreserving pooled populations of stem cells.

The cryopreserved immunosuppressive placental stem cell population may comprise placental stem cells from a single donor or from multiple donors. The placental stem cell population may be fully HLA-matched, or partially or fully HLA-mismatched, with the intended recipient.

Accordingly, in one embodiment, the present invention provides a composition comprising a population of immunosuppressive placental stem cells in a container. In a specific embodiment, the stem cell population is cryopreserved. In another embodiment, the container may be 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 support or facilitate intravenous administration of said population of placental stem cells. The bag may comprise a plurality of interconnected chambers or chambers to allow mixing of the placental stem cells or one or more other solutions (eg, drugs) prior to or at the time of administration. In another specific embodiment, the composition comprises one or more compounds that facilitate cryopreservation of a pooled population of stem cells. In another specific embodiment, said population of placental stem cells is contained in a physiologically acceptable aqueous solution. In a more specific embodiment, said 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 pooled stem cell population comprises placental cells that are at least partially HLA-mismatched to the recipient of said stem cell population. In another specific embodiment, said placental stem cells are from multiple donors.

5.6.1.2 Pharmaceutical composition

Immunosuppressive placental stem cell populations or cell populations comprising placental stem cells can be formulated into pharmaceutical compositions for in vivo use. These 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 saline solution or other physiologically acceptable solution suitable for in vivo administration. The pharmaceutical compositions of the invention may comprise any of the placental stem cell populations or types of placental stem cells described herein. The pharmaceutical composition may comprise fetal, maternal or both fetal and maternal placental stem cells. The pharmaceutical composition of the present invention may further comprise placental stem cells obtained from a single individual or placenta or from a plurality of individuals or placentas.

The pharmaceutical composition of the invention may comprise any immunosuppressive amount of placental stem cells. For example, in various embodiments, a single unit dose of placental stem cells may comprise about, at least or no more than 1 x 10 ⁵ , 5 x 10 ⁵ , 1 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 5×10 ⁹ , 1×10 ¹⁰ , 5×10 ¹⁰ , 1×10 ¹¹ or more placental stem cells.

Pharmaceutical compositions of the invention comprise cell populations that contain 50% or more viable cells (ie, at least 50% of the cells in the population are functional or viable). Preferably, at least 60% of the cells in the population survive. More preferably, at least 70%, 80%, 90%, 95% or 99% of the cells in the population of the pharmaceutical composition are alive.

The pharmaceutical composition of the present invention may comprise one or more compounds such as to promote implantation (for example, anti-T cell receptor antibody, immunosuppressant, etc.); stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl ether base starch etc.

5.6.1.3 Placental stem cell conditioned medium

The placental stem cells of the present invention can be used to prepare immunosuppressive conditioned medium, that is, containing one or more biological substances secreted by said stem cells that have a detectable immunosuppressive effect on one or more types of immune cells. Molecular media. In various embodiments, the conditioned medium comprises placental stem cells grown therein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days culture medium. In another embodiment, the conditioned medium comprises a medium in which placental stem cells have been grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100% confluency . These conditioned media can be used to support the culture of another population of placental stem cells or populations of other types of stem cells. In another embodiment, the conditioned medium comprises a medium in which placental stem cells have differentiated into mature cell types. In another embodiment, the conditioned medium of the invention comprises a medium in which placental stem cells and non-placental stem cells have been cultured.

Accordingly, in one embodiment, the invention provides a composition comprising a culture medium derived from a culture of placental stem cells, wherein said placental stem cells: (a) adhere to a substrate; (b) express CD200 and HLA- G, or express CD73, CD105 and CD200, or express CD200 and OCT-4, or express CD73, CD105 and HLA-G, or express CD73 and CD105 and when the population is cultured under conditions suitable for embryoid body formation promoting the formation of one or more embryoid bodies in a population of placental cells comprising the placental stem cells, or expressing OCT-4 and promoting the formation of a placenta comprising the placental stem cells when said population is cultured under conditions suitable for the formation of embryoid bodies the formation of one or more embryoid bodies in the cell population; and (c) detectably inhibit CD4 ⁺ or CD8 ⁺ T cell expansion in an MLR (mixed lymphocyte reaction), wherein said placental stem cell culture has Culture in the medium for 24 hours or more. In a specific embodiment, the composition further comprises a plurality of said placental stem cells. In another specific embodiment, the composition comprises a plurality of non-placental cells. In a more specific embodiment, said non-placental cells comprise CD34 ⁺ cells, eg, 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, eg mesenchymal stem cells, eg bone marrow derived mesenchymal stem cells. The non-placental cells can also be one or more types of mature cells or cell lines. In another specific embodiment, the composition further comprises an anti-proliferative agent, eg, an anti-MIP-1α or anti-MIP-1β antibody.

5.6.1.4 Matrix containing placental stem cells

The invention further includes matrices, hydrogels, scaffolds and the like comprising immunosuppressive placental stem cell populations.

The placental stem cells of the present invention may be seeded onto a natural substrate, eg, placental biomaterial such as amniotic membrane material. Such amnion material can be, for example, amnion cut directly from mammalian placenta; fixed or heat-treated amnion, well-dried (i.e., <20% _H20 ) amnion, chorion, well-dried chorion, well-dried amnion and chorion etc. Preferred placental biomaterials on which to seed placental stem cells can be found in Hariri, US Application Publication No. 2004/0048796.

The placental stem cells of the present invention can be suspended in, for example, a hydrogel solution suitable for injection. Hydrogels suitable for use in these compositions include self-assembling peptides such as RAD16. In one embodiment, the cell-containing hydrogel solution can harden in a mold to form a matrix for implantation in which the cells are dispersed. Placental stem cells in such a matrix can also be cultured for mitotic expansion prior to implantation. For example, the hydrogel may be an organic polymer (natural or synthetic) that is cross-linked by covalent, ionic, or hydrogen bonding to form a three-dimensional open lattice structure that entraps water molecules to form a gel. Hydrogel-forming materials include polysaccharides (such as alginic acid and its salts), peptides, polyphosphazenes, and ionically cross-linked polyacrylic acids, block polymers (such as polyethylene oxide-polypropylene glycol cross-linked by temperature or pH, respectively). block copolymer). In some embodiments, the hydrogels or matrices of the invention are biodegradable.

In some embodiments of the invention, the formulation comprises an in situ polymerizable gel (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 an aqueous solution, such as water, buffered saline solution, or aqueous alcoholic solution, the polymer has charged pendant groups or a monovalent ionic salt thereof. Examples of polymers having acidic side groups reactive with cations are poly(phosphorazo), poly(acrylic acid), poly(methacrylic acid), 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 and vinyl ether monomers or polymers may also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

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

Examples of scaffolds that can be used in the present invention include nonwoven felts, porous foams, or self-assembling peptides. Nonwoven mats can be formed using fibers comprising synthetic acetic and lactic acid absorbable copolymers (eg, PGA/PLA) (VICRYL, Ethicon Corporation, Somerville, N.J.). Foams composed of, for example, poly(ε-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymers formed by processes such as freeze-drying or lyophilization (see, for example, U.S. Pat. No. 6,355,699) are also useful as bracket use.

及其混合物。目前商业上可获得的多孔生物相容性陶瓷材料包括

(CanMedica公司，加拿大)、

(Merck BiomaterialFrance，法国)、

(Mathys，AG，Bettlach，瑞士)，以及矿化胶原质骨嫁接产品例如HEALOS^TM(DePuy公司，Raynham，MA)和

RHAKOSS^TM以及

(Orthovita，Malvern，Pa.)。该框架可以是天然和/或合成材料的混合物、掺杂物或复合物。The placental stem cells of the present invention can also be seeded onto or contacted with physiologically acceptable ceramic materials, which include, but are not limited to, mono, di, tri, α-tri, β-tri, and calcium tetraphosphate, hydroxyapatite, fluorine Apatite, calcium sulfate, calcium fluoride, calcium oxide, calcium carbonate, calcium magnesium phosphate, bioactive glass such as

and mixtures thereof. Currently commercially available porous biocompatible ceramic materials include

(CanMedica, Canada),

(Merck Biomaterial France, France),

(Mathys, AG, Bettlach, Switzerland), and mineralized collagen bone graft products such as HEALOS ^™ (DePuy Corporation, Raynham, MA) and

RHAKOSS ^TM and

(Orthovita, Malvern, Pa.). The framework may be a mixture, dopant or composite of natural and/or synthetic materials.

In another embodiment, placental stem cells may be seeded on or contacted with a mat, which may consist of, for example, multifilaments obtained from bioabsorbable materials such as PGA, PLA, PCL copolymers or dopants or hyaluronic acid. be made of.

In another embodiment, the placental stem cells of the present invention can be seeded onto a foam scaffold, which can be a composite structure. Such foam scaffolds can be molded into useful shapes, such as the shape of specific structural parts in the body to be repaired, replaced or augmented. In some embodiments, the framework can be treated with, for example, 0.1 M acetic acid before seeding the cells of the invention, and then placed in polylysine, PBS and/or collagen to enhance cell attachment. The outer surface of the matrix can be modified to enhance cell attachment or growth and tissue differentiation, for example, by coating the matrix with plasma, or adding one or more proteins (e.g., collagen, elastic fibers, reticular fibers), glycoproteins , mucopolysaccharides (e.g., heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), cell matrix and/or other materials such as, but not limited to, gelatin, alginic acid , agar, agarose and vegetable gum.

In some embodiments, the scaffold comprises or is treated with a material that renders it non-thrombogenic. Such treatments and materials can also promote and maintain endothelial growth, migration, and extracellular substrate deposition. Examples of such materials and treatments include, but are not limited to, natural materials such as basement membrane proteins such as laminin and collagen type IV, synthetic materials such as EPTFE, and segmented polyurethane urea silicones such as PURSPAN ^™ (The Polymer Technology Group Inc., Berkeley, Calif). Scaffolds may also contain antithrombotic agents such as heparin; the scaffolds may also be treated to alter surface charge (eg, coating with plasma) prior to inoculation 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 containing a growth-promoting gene, i.e., a gene encoding a protein that promotes the growth of the transfected cell under appropriate conditions such that the growth Production and/or activity of facilitative 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 E7 protein of papillomavirus.

External regulation of a growth-promoting protein can be achieved by placing the growth-promoting gene in the presence of an externally regulatable promoter (e.g., a promoter whose activity can be controlled by changing the temperature of the transfected cell or the composition of the medium in contact with the cell). come under control. In one embodiment, a tetracycline (tet) controlled gene expression system can be used (see Gossen et al., Proc. Natl Acad. Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. . USA 93:1518-1523, 1996). A tet-controlled transactivator (tTA) within this vector strongly activates transcription from ph _CMV*-1 (human minicytomegalovirus minimal promoter fused to the tet operator sequence) when tet is absent. tTA is a fusion protein of the repressor (tetR) of the Escherichia coli transposon-10-derived tet resistance operon and the acidic domain of VP16 of the herpes simplex virus. Nontoxic low concentrations of tet (eg, 0.01-1.0 μg/mL) almost completely abolished the transcriptional activation of tTA.

In one embodiment, the vector further comprises a gene encoding a selectable marker (eg, a protein that confers drug resistance). Bacterial neomycin resistance gene (neo ^R ) is one such marker that can be used in the present invention. Cells carrying neo ^R can be selected by methods known to those of ordinary skill in the art, such as adding 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of methods known to those of ordinary skill in the art, including, but not limited to, retroviral infection. In general, cell cultures can be transfected by co-cultivation with a mixture of conditioned medium taken from the vector-producing cell line and DMEM/F12 with N2 supplementation. For example, placental cell cultures prepared as described above can be infected in vitro five days later by culturing for about 20 hours in one volume of conditioned medium and two volumes of DMEM/F12 with N2 supplementation. Transfected cells carrying a selectable marker can then be selected for as described above.

Following transfection, the culture is passaged to a surface that allows proliferation (eg, allowing at least 30% of the cells to double over a period of 24 hours). Preferably, the substrate is a polyornithine/laminin substrate consisting of a tissue culture plastic coated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), polylysine Acid/laminin substrate or fibronectin-treated surface. The cultures are then fed growth medium supplemented or not with one or more proliferation promoting factors every 3-4 days. Proliferation promoting factors can be added to the growth medium when the culture is less than 50% confluent.

The conditionally immortalized placental stem cell line can be passaged using standard techniques, such as trypsinization at 80-95% confluency. In some embodiments, it is advantageous to maintain selection (eg, by adding G418 to cells containing the neomycin resistance gene) until about the twentieth passage. Cells can also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from the conditionally immortalized human placental stem cell lines prepared as above. Generally, such clonal cell lines can be isolated and expanded by standard techniques such as limiting dilution or using cloning rings. Clonal cell lines can generally be fed and passaged as described above.

Differentiation of a conditionally immortalized human placental stem cell line, which may, but need not be, is typically clonal, can generally be induced by inhibiting the production and/or activity of growth-promoting proteins under differentiation-promoting culture conditions. For example, if a gene encoding a growth-promoting protein is under the control of an externally regulatable promoter, conditions (eg, temperature or composition of the medium) can be altered to inhibit transcription of the growth-promoting gene. For the tetracycline-controlled gene expression system described above, differentiation can be achieved by adding tetracycline to inhibit the transcription of growth-promoting genes. Generally, 1 μg/mL tetracycline for 4-5 days is sufficient to induce differentiation. To facilitate further differentiation, other agents can be added to the growth medium.

5.6.3 Test

Placental stem cells of the invention can be used in assays to determine the effect of culture conditions, environmental factors, molecules (e.g., biomolecules, small inorganic molecules, etc.) and/or differentiation effects.

In a preferred embodiment, changes in proliferation, expansion or differentiation of the placental stem cells of the present invention after exposure to a certain molecule are measured. For example, in one embodiment, the present invention provides a method of identifying a compound that modulates the proliferation of a population of placental stem cells comprising contacting said population of stem cells with said compound under conditions that permit proliferation, wherein if said compound is not contacted with said A compound is identified as a compound that modulates placental stem cell proliferation if the compound causes a detectable change in the proliferation of the stem cell population compared to the compound. In a specific embodiment, the compound is identified as an inhibitor of proliferation. In another specific embodiment, said compound is identified as an enhancer of proliferation.

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

In another embodiment, the present invention provides a method of identifying a compound capable of modulating the differentiation of placental stem cells comprising contacting said stem cells with said compound under conditions that permit differentiation, wherein if the stem cells not contacted with said compound If, in contrast, the compound causes a detectable change in the differentiation of the stem cells, the compound is identified as a compound that modulates placental stem cell proliferation. In a specific embodiment, the compound is identified as an inhibitor of differentiation. In another specific embodiment, said compound is identified as an enhancer of differentiation.

5.6.4 Placental Stem Cell Bank

Stem cells from postpartum placenta can be cultured in a number of different ways to obtain a batch of placental stem cells (eg, a set of doses that can be administered individually). For example, such batches can be obtained from stem cells in placental perfusion or enzymatically digested placental tissue. Groups of placental stem cell batches obtained from a large number of placentas can be arranged in a placental stem cell bank for, eg, long-term storage. Adherent stem cells are typically obtained from primary cultures of placental material to form seed cultures, which are expanded under controlled conditions to form cell populations approximately equal to the doubling number. Batches are preferably obtained from tissue of a single placenta, but may also be obtained from tissue of a large number of placenta.

In one embodiment, a stem cell batch is obtained as follows. For example, placental tissue is first disrupted by mincing, digested with an appropriate enzyme such as collagenase (see section 5.2.3 above). The placental tissue preferably comprises, for example, intact amnion, intact chorion, or both from a single placenta, but may also comprise only a portion of amnion or chorion. The digested tissue can be cultured for about 1-3 weeks, preferably about 2 weeks. After removal of non-adherent cells, high-density colonies formed were harvested by trypsinization, etc. These cells were harvested and resuspended in a certain volume of medium, which were defined as passage 0 cells.

The expanded culture was then inoculated with passage 0 cells. Expansion culture can be performed by any setup of a stand-alone cell culture device, for example, NUNC ^™ 's Cell Factory. Passage ⁰ cell ^{cultures can} be ^subdivided to ^any degree ^{to e.g.} 8×10 ³ , 9×10 ³ , 1×10 ⁴ , 1×10 ⁴ , 2×10 ⁴ , 3×10 ⁴ , 4×10 ⁴ , 5×10 ⁴ , 6×10 ⁴ , 7×10 ⁴ , 8 x 10 ⁴ , 9 x 10 ⁴ or 10 x 10 ⁴ stem cells were inoculated into the expanded culture. Preferably, about 2 x ¹⁰⁴ to about 3 x ¹⁰⁴ passage 0 cells are used to inoculate each expansion culture. The number of expanded cultures can depend on the number of passage 0 cells and can be larger or smaller depending on the particular placenta from which the stem cells were obtained.

Expansion cultures can be grown until the cell density in culture reaches a certain value, eg, about 1 x ¹⁰⁵ cells/ ^cm2 . At this point the cells can be harvested or cryopreserved, or passaged into new expansion cultures as described above. Cells may be passaged eg 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times prior to use. It is preferred to keep records of the cumulative number of population doublings in expansion cultures. Cells from passage 0 cultures can be expanded to 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 multipliers, or up to 60 multipliers. However, the number of population doublings before dividing the cell population into individual doses is preferably between about 15 and about 30 doublings, preferably about 20 doublings. The cells can be cultured continuously throughout the expansion process, or frozen at one or more points during expansion.

Cells to be used in individual doses can be frozen (eg, cryopreserved) for later use. Individual doses may contain, for example, from about 1 million to about 100 million cells/ml, and can contain from about ¹⁰⁶ to about ¹⁰⁹ cells in total.

In a specific embodiment of the method, the cells at passage 0 are cultured to about 4 doublings and then frozen in the first cell bank. Cells from the first cell bank were frozen and used to inoculate the second cell bank, which was expanded an additional eight times. Cells at this stage were harvested and frozen and used to inoculate a new expansion culture, which was allowed to undergo another eight doublings, bringing the cumulative number of cell doublings to approximately 20. Cells at the midpoint of passaging may be frozen in units of about 100,000 to about 10 million cells/ml, preferably about 1 million cells/ml for subsequent expansion in culture. Cells at about 20 doublings may be frozen as individual doses of about 1 million to about 100 million cells/ml for administration or for use in the preparation of stem cell-containing compositions.

In a preferred embodiment, the donor (eg parent) from which the placenta was obtained is tested for at least one pathogen. If the mother tested positive for the test pathogen, the entire batch obtained from that placenta was discarded. This test can be performed at any time during the preparation of the placental stem cell batch, including before or after the establishment of passage 0 cells, or during the expansion culture process. Pathogens for which the presence can be tested include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, human immunodeficiency virus (types I and II), cytomegalovirus, herpes virus, and the like.

5.6.5 Treatment of multiple sclerosis

In another aspect, the invention provides methods of treating an individual suffering from multiple sclerosis or multiple sclerosis-related symptoms comprising administering to the individual in an amount and for a duration sufficient to detectably modulate (e.g., suppress) an immune response in the individual. Individuals are administered large quantities of placental stem cells.

Multiple sclerosis (MS) is a chronic, relapsing inflammatory disease of the central nervous system. The disease can result in damage to the myelin sheath around CNS and PNS axons, oligodendrocytes, and the nerve cells themselves. The disease is mediated by autoreactive T cells, especially CD4 ⁺ T cells, that proliferate under the influence of cell adhesion molecules and proinflammatory cytokines, cross the blood-brain barrier and enter the CNS. Symptoms of MS include sensory disturbances in the extremities, optic nerve disturbances, pyramidal tract disturbances, bladder dysfunction, bowel dysfunction, sexual dysfunction, ataxia, and diplopia.

Four distinct MS types or clinical courses have been identified. First, relapsing/remitting MS (RRMS) is characterized by acute self-limiting attacks of neurological deficits over a period of days to weeks, followed by a recovery period of several months, sometimes not completely. The second type, secondary progressive MS (SPMS), is initially RRMS but subsequently changes so that its clinical course is characterized by a continuous deterioration of function unrelated to the acute episode. A third type, primary progressive MS (PPMS), is characterized by a sustained decline in function from onset without acute attacks. A fourth type, Progressive/Relapsing MS (PRMS), also begins progressively, with occasional episodes amid progressive decline in function.

Motor skills assessment, optionally combined with MRI, is often used to evaluate people with MS. For example, a motor skills assessment (Extended Disability Status Scale) scores infected individuals on the following level of ability:

0.0 Neurological examination is normal

1.0 No disability, minimal signal in one FS

1.5 No disability, minimal signal in multiple FS

2.0 with minimal disability in a FS

2.5 Mild disability in one FS or minimal disability in two FS

3.0 Moderate disability in one FS, or mild disability in three or four FSs. fully mobile

3.5 Fully ambulatory, but moderately disabled in one FS and more than minimally disabled in several other systems

4.0 Fully ambulatory, self-care without assistance, up and about 12 hours a day despite considerable disability; able to walk about 500 meters without assistance or rest

4.5 Fully ambulatory without assistance, up and about most of the day, able to work all day, full mobility may have some limitations or require minimal assistance; characterized by fairly severe disability; able to walk approximately 300 m without assistance or rest

5.0 Walks approximately 200 meters without assistance or rest; disability severe enough to impair full day's daily activities (full day's work without special equipment)

5.5 Walks approximately 100 m without assistance or rest; disability severe enough to prevent full day's daily activities

6.0 Walk about 100 meters with intermittent assistance or continuous assistance on one side (bamboo stick, crutches, braces), with or without rest

6.5 Continuously walk about 20 meters with bilateral assistance (bamboo sticks, crutches, brackets) without rest

7.0 Cannot walk more than about 5 meters even with assistance, basically confined to wheelchair; self-moves into standard wheelchair and moves alone; gets up and walks about 12 hours a day in wheelchair

7.5 Walks no more than a few steps; confined to wheelchair; may need assistance with locomotion; moves wheelchair on its own, but cannot stay in standard wheelchair all day; may require automated wheelchair

8.0 Basically limited to bed or chair or ambulatory in wheelchair, but can leave bed most of the day; retains self-care functions; generally has effective use of arms

8.5 Most of the time of the day is basically confined to the bed; some effective use of the arms, retaining some self-care functions

9.0 Confined to bed; still able to communicate and eat

9.5 Totally helpless bedridden patient; unable to communicate effectively or eat/swallow

Death caused by 10.0MS

In the scoring system above, "FS" refers to the eight functional systems measured, including the pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral systems, and others.

Other similar scoring systems are known, including the Scripps Neurological Scale, the Walking Index, and the Multiple Sclerosis Functional Composite (MSFC).

MS progression can also be assessed by measuring incidence.

MS progression can also be assessed with magnetic resonance imaging, which detects neurological lesions associated with MS (eg, new lesions, enhancing lesions, or combined uniquely active lesions).

Accordingly, in one embodiment, the invention provides a method of treating an individual with MS (eg, an individual diagnosed with MS) comprising administering to the individual an amount of placental stem cells sufficient to suppress an immune response in the individual. In a specific embodiment, the administering measurably improves one or more symptoms of MS in the individual. In a more specific embodiment, the symptom is, for example, one or more sensory disturbances of a limb, optic nerve dysfunction, pyramidal tract dysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction, ataxia, and Diplopia. In another specific embodiment, said administering results in an increase of at least half a point in the EDSS score. In another specific embodiment, said administering results in an increase in the EDSS score of at least one point. In another specific embodiment, said administering results in an increase of at least two points in the EDSS score. In other specific embodiments, said administering results in a measurable improvement in multiple sclerosis score or MRI.

MS has been treated with other therapeutic agents such as immunomodulators or immunosuppressants such as interferon beta (IFNβ), including IFNβ-1a and IFNβ-1b; glatiramer acetate (Copaxone); cyclophosphamide; methotrexate ; Azathioprine (Imuran); Clardribine (Leustatin); Cyclosporine; Mitoxantrone, etc. MS has also been treated with anti-inflammatory therapeutics, such as glucocorticoids, including adrenocorticotropic hormone (ACTH), methylprednisolone, dexamethasone, and the like. MS has also been treated with other types of therapeutic agents, such as intravenous immunoglobulin, plasma exchange, or sulfasalazine.

Accordingly, the present invention further provides methods of treating an individual with MS (eg, an individual diagnosed with MS) comprising administering to the individual a quantity of placental stem cells sufficient to suppress an immune response in the individual (wherein the administration detectably improves one or more symptoms of MS in an individual) and one or more therapeutic agents. In one embodiment, the therapeutic agent is a glucocorticoid. In a specific embodiment, the glucocorticoid is adrenocorticotropic hormone (ACTH), methylprednisolone or dexamethasone. In another embodiment, the therapeutic agent is an immunomodulator or immunosuppressant. In different specific embodiments, the immunomodulator or immunosuppressant is IFNβ-1a and IFNβ-1b, glatiramer acetate (Copaxone), cyclophosphamide, methotrexate, azathioprine, cladribine, cyclic mitoxantrone or mitoxantrone. In other embodiments, the therapeutic agent is intravenous immunoglobulin, plasma exchange, or sulfasalazine. In another embodiment, the individual is administered any combination of the foregoing therapeutic agents.

Individuals with MS (eg, individuals diagnosed with MS) can be treated at any time during the progression of the disease using a plurality of placental stem cells in combination with one or more therapeutic agents. For example, the individual can be treated immediately after diagnosis, or within 1, 2, 3, 4, 5, 6 days after diagnosis, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, Within 10, 15, 20, 25, 30, 35, 40, 45, 50 or more weeks, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after diagnosis for treatment. The individual can be treated single or multiple times during the clinical course of the disease. Individuals may be treated during acute episodes, remission, or during chronic regression, as appropriate. In another embodiment, the placental stem cells are administered to a woman with MS postpartum to maintain a state of remission or reduction of relapses experienced during pregnancy.

In one embodiment, the individual is administered a dose of about 300 million placental stem cells. However, the dose can vary according to the individual's physical characteristics (such as body weight), and can vary between 1 million and 10 billion placental stem cells per dose, preferably between 10 million and 1 billion per dose, or between 100 million to 50 million placental stem cells. Administration is preferably intravenous, but any art-accepted route of administration of living cells may be used. In one embodiment, the placental stem cells are from a cell bank.

6. Example

6.1 Example 1: Culture of placental stem cells

Placental stem cells are obtained from postpartum mammalian placenta by perfusion or physical disruption, such as enzymatic digestion. The cells were cultured in medium containing 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma), 2% fetal calf serum (FCS) (Hyclone Laboratories), 1× insulin-transferrin - selenium (ITS), 1 × linoleic acid-bovine serum albumin (LA-BSA), 10 ^-9 M dexamethasone (Sigma), 10 ^-4 M ascorbic acid-2-phosphate (Sigma), epidermal growth factor ( EGF) 10ng/ml (R&D Systems), platelet-derived growth factor (PDGF-BB) 10ng/ml (R&D Systems), and 100U penicillin/1000U streptomycin.

Culture flasks for culturing cells were prepared as follows. T75 flasks were coated with fibronectin (FN) by adding 5 ng/ml human FN (SigmaF0895) in 5 ml PBS to the flask. Place the bottle containing the FN solution at 37 °C for 30 min. The FN solution was then removed prior to cell culture. No need to dry the bottle after handling. Alternatively, leave the bottle in contact with the FN solution at 4°C overnight or longer, then warm the bottle and remove the FN solution before use.

Placental stem cells isolated by perfusion

Placental stem cell cultures from perfused placenta were prepared as follows. Cells from the Ficoll gradient method were inoculated at a concentration of 50–100 × ¹⁰ cells/flask into 15 ml of medium in FN-coated T75 flasks prepared as above. Typically 5 to 10 bottles are inoculated. Incubate the bottle at 37°C for 12-18 hours to allow adherent cells to attach. Add 10 ml of warm PBS to each bottle to remove suspended cells and mix gently. Then 15 ml of medium was removed and replaced with 15 ml of fresh medium. All medium was changed 3-4 days after culture initiation. For subsequent medium replacements, 50% or 7.5 ml of medium was removed.

From about day 12, the cultures were examined microscopically for the growth of adherent cell colonies. Adherent cells were harvested by trypsinization, typically between day 13 and day 18 after initiation of culture, when the cell culture reached approximately 80% confluency. Cells harvested from primary cultures are referred to as passage 0 (zero).

Placental stem cells isolated by physical disruption and enzymatic digestion

Placental stem cell cultures from digested placental tissue were prepared as follows. Place the perfused placenta maternal side up on sterile paper. A razor blade was used to scrape about 0.5 cm of the superficial layer on the mother side of the placenta, and the blade was used to remove chunks of placental tissue, measuring about 1 x 2 x 1 cm. The placental tissue was then cut into approximately 1 ^mm3 fragments. These fragments were placed in 50ml Falcon tubes and digested with collagenase IA (2 mg/ml, Sigma) for 30 minutes and then with trypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes in a water bath at 37°C. The resulting solution was centrifuged at 400 g for 10 min at room temperature, and the digestate was removed. The pellet was resuspended in approximately 10 volumes of PBS (eg, 5 ml of pellet was resuspended in 45 ml of PBS), and the tube was centrifuged at 400 g for 10 minutes at room temperature. Resuspend the tissue/cell pellet in 130 mL of culture medium, then inoculate 13 ml of cells in each fibronectin-coated T75 flask. Cells were cultured at 37 °C in a humidified atmosphere containing 5% _CO2 . Placental stem cells can be selectively cryopreserved at this stage.

Subculture and Expansion of Placental Stem Cells

Thaw cryopreserved cells rapidly in a 37°C water bath. Placental stem cells were quickly removed from the cryovial with 10 ml of warm medium and transferred to a 15 ml sterile tube. Cells were centrifuged at 400g for 10 minutes at room temperature. Cells were gently resuspended in 10 ml of warm medium by pipetting, and the number of viable cells was counted by trypan blue exclusion. Cells were seeded at a density of approximately 6000-7000 cells/ ^cm2 in RN-coated flasks prepared as above (approximately 5 x ¹⁰⁵ cells per T-75 flask). Cells were incubated at 37°C, 5% CO ₂ and 90% humidity. When the cells reached 75-85% confluency, aseptically remove all spent medium from the bottle and discard. Add 3 ml of 0.25% trypsin/EDTA (w/v) solution to cover the cell layer, and then incubate the cells at 37°C, 5% CO ₂ and 90% humidity for 5 minutes. Tap the bottle once or twice to speed up cell detachment. When >95% of the cells were rounded and detached, 7 ml of warm medium was added to each T-75 flask, and the solution was dispersed by pipetting multiple times on the surface of the cell layer.

After the cells were counted and assayed for viability as described above, the cells were centrifuged at 1000 RPM for 5 minutes at room temperature. Cell passage was performed by gently resuspending the cell pellet in the T-75 flask with medium and spreading evenly across two FN-coated T-75 flasks.

Using the method described above, a population of adherent placental stem cells expressing the markers CD105, CD117, CD33, CD73, D29, CD44, CD10, CD90 and CD133 was identified. This cell population does not express CD34 or CD45. Some, but not all, of these placental stem cell cultures express HLA-ABC and/or HLA-DR.

6.2 Example 2: Isolation of placental stem cells from placental structures

6.2.1 Materials and methods

6.2.1.1 Isolation of target phenotypes

Five different placental cell populations were obtained from normal, term-pregnant placentas. All donors provided full written consent for the use of their placenta for research purposes. Five cell populations were examined: placental cells from (1) placental perfusate (from perfusion of placental vasculature); and (2) enzymatically digested amnion, (3) enzymatically digested chorion , (4) Enzymatically digested placental cells from amnion-chorion plates and (5) Enzymatically digested umbilical cord cells. Different tissues were cleaned with sterile PBS (Gibco-Invitrogen Corporation, Carlsbad, CA) and placed on separate sterile Petri dishes. Each tissue was dissected with a sterile surgical scalpel and placed in a 50 mL Falcon conical tube. Minced tissue was digested with 1× collagenase (Sigma-Aldrich, St.Louis, MO) in a 37°C water bath for 20 minutes, centrifuged, and then treated with 0.25% trypsin-EDTA (Gibco-Invitrogen) in a 37°C water bath Digest for 10 minutes. After digestion the tissues were centrifuged and washed once with sterile PBS (Gibco-Invitrogen). The reconstituted cells were filtered twice (once with a 100 μm cell strainer and once with a 30 μm separation filter) to remove any residual extracellular matrix or cellular debris.

6.2.1.2 Cell Viability Assessment and Cell Counting

Cell numbers were counted and cell viability was assessed by manual trypan blue exclusion after digestion. Cells were mixed 1:1 with trypan blue dye (Sigma-Aldrich) and cells were read on a hemocytometer.

6.2.1.3 Characterization of cell surface markers

HLA ^ABC- / ^CD45- / ^CD34- /CD133 ⁺ cells were selected for characterization. Cells with this phenotype were identified, quantified and characterized by both Becton-Dickinson flow cytometry, FACSCalibur and FACSAria (Becton-Dickinson, San Jose, CA, USA). Various placental cells were stained at a rate of approximately 10 μL of antibody per 1 million cells for 30 min at room temperature on a shaker. The following anti-human antibodies can be used: fluorescein isothiocyanate (FITC)-conjugated against HLA-G (Serotec, Raleigh, NC), CD10 (BD Immunocytometry Systems, San Jose, CA), CD44 (BD Biosciences Pharmingen, San Jose, CA) and CD105 (R&D Systems, Minneapolis, MN); monoclonal antibodies against CD44, CD200, CD117, and CD13 (BD Biosciences Pharmingen) conjugated to phycoerythrin (PE); Phycoerythrin-Cy5 (PE Cy5)-coupled streptavidin and monoclonal antibodies against CD117 (BD Biosciences Pharmingen); ) monoclonal antibody; allophycocyanin (APC)-conjugated streptavidin and monoclonal antibody against CD38 (BD Biosciences Pharmingen); and biotinylated CD90 (BD Biosciences Pharmingen). After incubation, the cells were washed once to remove unbound antibody and fixed overnight at 4°C with 4% paraformaldehyde (USB, Cleveland, OH). The next day, cells were washed twice, filtered through a 30 μm separation filter, and loaded into a flow cytometer.

Samples stained with an anti-mouse IgG antibody (BD Biosciences Pharmingen) served as a negative control and were used to condition photomultiplier tubes (PMTs). A sample stained with an anti-human antibody alone can be used as a positive control and to adjust spectral overlap/compensation.

6.2.1.4 Cell Sorting and Culture

A panel of placental cells (from perfusate, amnion, chorion) was stained with 7-amino-actinomycin D (7AAD; BD Biosciences Pharmingen) and monoclonal antibodies specific for the phenotype of interest. Cells were stained at a rate of approximately 10 μL of antibody per 1 million cells and incubated on a shaker at room temperature for 30 minutes. Live cells expressing the phenotype of interest were then positively sorted on the BD FACS Aria and plated in culture. Both sorted (target population) and "all" (unsorted) placental cell populations were plated for comparison. Cells were plated on 96-well plates coated with fibronectin (Sigma-Aldrich) at the cell densities listed in Table 1 (cells/cm ² ). Determine cell density and whether to plate that cell type in duplicates or triplicates based on the number of cells expressing that phenotype of interest.

Table 1: Cell plating density

Add complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201 (Sigma); 2% fetal bovine serum (Hyclone Labs.); 1× insulin-transferrin- Selenium (ITS); 1× linoleic acid-bovine serum albumin (LA-BSA); 10 ^-9 M dexamethasone (Sigma); 10 ^-4 M ascorbic acid-2-phosphate (Sigma); epidermal growth factor 10ng/ mL (R&D Systems); and platelet-derived growth factor (PDGF-BB) 10 ng/mL (R&D Systems)) and place the well plate in a 5% CO ₂ /37°C incubator. On day 7, 100 μL of complete medium was added to each well. The 96-well plate was continuously monitored for approximately 2 weeks with a final assessment of the culture on day 12.

6.2.1.5 Data analysis

FACSCalibur data were analyzed in FlowJo (Treestar Corporation) using standard gating techniques. BD FACS Aria data were analyzed using FACSDiva software (Becton-Dickinson). FACS Aria data were analyzed using doublet discrimination gating and standard gating techniques to minimize doublets. All results were compiled in Microsoft Excel and all values here are expressed as mean ± standard deviation (number, standard error of the mean).

6.2.2 Results

6.2.2.1 Cell viability

The activity after digestion can be evaluated by artificial trypan blue exclusion method (Figure 1). The average viability of cells obtained from mostly digested tissue (from amnion, chorion or amnion-chorion plates) was approximately 70%. The mean viability of cells from amnion was 74.35%±10.31% (n=6, SEM=4.21), the mean viability of cells from chorion was 78.18%±12.65% (n=4, SEM=6.32), from amnion- The average viability of cells from the chorionic plate was 69.05%±10.80% (n=4, SEM=5.40) and that of cells from the umbilical cord was 63.30%±20.13% (n=4, SEM=10.06). Undigested cells from perfusion retained the highest mean viability of 89.98±6.39% (n=5, SEM=2.86).

6.2.2.2 Cell quantification

Five different placenta-derived cell populations were analyzed to determine the number of HLA ABC ⁻ /CD45 ⁻ /CD34 ⁻ /CD133 ⁺ cells. From the data analysis of BD FACSCalibur, it can be seen that the amniotic membrane, perfusate and chorion contain the most total cells of this type, which are 30.72±21.80 cells (n=4, SEM=10.90), 26.92±22.56 cells (n= 3, SEM=13.02), and 18.39±6.44 cells (n=2, SEM=4.55) (data not shown). Amnion-chorionic plates and umbilical cords contained the least total number of cells expressing the target phenotype, 4.72±4.16 cells (n=3, SEM=2.40) and 3.94±2.58 cells (n=3, SEM=1.49), respectively (data not shown).

Similarly, when analyzing the percentage of total cells expressing the phenotype of interest, it can be seen that amniotic membrane and placental perfusate contain the highest percentage of cells expressing this phenotype, respectively 0.0319%±0.0202% (n=4, SEM= 0.0101) and 0.0269%±0.0226% (n=3, SEM=0.0130) (Figure 2). Although the umbilical cord contained a relatively small number of cells expressing the phenotype of interest (Fig. 2), it contained the third highest percentage of cells expressing the phenotype of interest 0.020 ± 0.0226% (n = 3, SEM = 0.0131) (Fig. 2 ). Chorionic and amnion-chorionic plates contained the lowest percentages of cells expressing the phenotype of interest, 0.0184±0.0064% (n=2, SEM=0.0046) and 0.0177±0.0173% (n=3, SEM=0.010), respectively ( figure 2).

Consistent with the results of the BD FACSCalibur analysis, the BD FACS Aria data also demonstrated that amnion, perfusate, and chorion provided higher amounts of HLAABC ⁻ /CD45 ⁻ /CD34 ⁻ /CD133 ⁺ compared to other sources. The average total number of cells expressing the target phenotype in amnion, perfusate and chorion were 126.47±55.61 cells (n=15, SEM=14.36), 81.65±34.64 cells (n=20, SEM=7.75) and 51.47± 32.41 cells (n=15, SEM=8.37) (data not shown). Amnion-chorionic plates and umbilical cords contained the least total number of cells expressing the target phenotype, 44.89±37.43 cells (n=9, SEM=12.48) and 11.00±4.03 cells (n=9, SEM=1.34), respectively (data not shown).

BD FACS Aria data showed that B and A cell sources contained the highest percentage of HLAABC ⁻ /CD45 ⁻ /CD34 ⁻ /CD133 ⁺ cells, which were 0.1523±0.0227% (n=15, SEM=0.0059) and 0.0929±0.0419% (n =20, SEM=0.0094) (Figure 3). The D cell source contained the third highest percentage of cells expressing the phenotype of interest, 0.0632±0.0333% (n=9, SEM=0.0111) (Figure 3). The C and E cell sources contained the lowest percentages of cells expressing the phenotype of interest, 0.0623±0.0249% (n=15, SEM=0.0064) and 0.0457±0.0055% (n=9, SEM=0.0018), respectively (Fig. 3) .

After identifying and quantifying the HLA ABC ^- /CD45 ^- /CD34 ^- /CD133 ⁺ cells derived from each cell, the cell surface markers HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117 were further tested , CD200 and CD105 expression were analyzed and characterized.

6.2.2.3 Placental perfusate-derived cells

Perfusate-derived cells were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (Figure 4). The average expression of each marker in perfusate-derived cells was as follows: 37.15%±38.55% (n=4, SEM=19.28) of cells expressed HLA-G; 36.37%±21.98% (n=7, SEM=8.31) of cells expressed CD33; 39.39%±39.91% (n=4, SEM=19.96) cells express CD117; 54.97%±33.08% (n=4, SEM=16.54) cells express CD10; 36.79%±11.42% (n=4, SEM=5.71) cells express CD44; 41.83%±19.42% (n=3, SEM=11.21) cells express CD200; 74.25%±26.74% (n=3, SEM=15.44) cells express CD90; 35.10%± 23.10% (n=3, SEM=13.34) of cells express CD38; 22.87%±6.87% (n=3, SEM=3.97) of cells express CD105; and 25.49%±9.84% (n=3, SEM=5.68) cells express CD13.

6.2.2.4 Amnion-derived cells

Amnion-derived cells were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (Figure 5). The average expression of each marker in amnion-derived cells is as follows: 57.27%±41.11% (n=3, SEM=23.73) of cells express HLA-G; 16.23%±15.81% (n=6, SEM=6.46) of cells express CD33 62.32%±37.89% (n=3, SEM=21.87) of the cells express CD117; 9.71%±13.73% (n=3, SEM=7.92) of the cells express CD10; 27.03%±22.65% (n=3, SEM =13.08) cells express CD44; 6.42%±0.88% (n=2, SEM=0.62) cells express CD200; 57.61%±22.10% (n=2, SEM=15.63) cells express CD90; 63.76%±4.40 % (n=2, SEM=3.11) of cells expressed CD38; 20.27%±5.88% (n=2, SEM=4.16) of cells expressed CD105; and 54.37%±13.29% (n=2, SEM=9.40) of Cells express CD13.

6.2.2.5 Chorion-derived cells

Chorionic villi-derived cells were consistently positive for HLA-G, CD117, CD10, CD44, CD200, CD90, CD38, and CD13, whereas expression of CD33 and CD105 varied (Fig. 6). The average expression of each marker in chorionic cells is as follows: 53.25%±32.87% (n=3, SEM=18.98) of cells express HLA-G; 15.44%±11.17% (n=6, SEM=4.56) of cells express CD33 ; 70.76%±11.87% (n=3, SEM=6.86) of cells express CD117; 35.84%±25.96% (n=3, SEM=14.99) of cells express CD10; 28.76%±6.09% (n=3, SEM =3.52) cells express CD44; 29.20%±9.47% (n=2, SEM=6.70) cells express CD200; 54.88%±0.17% (n=2, SEM=0.12) cells express CD90; 68.63%±44.37 % (n=2, SEM=31.37) of cells expressed CD38; 23.81%±33.67% (n=2, SEM=23.81) of cells expressed CD105; and 53.16%±62.70% (n=2, SEM=44.34) of Cells express CD13.

6.2.2.6 Amnion-chorionic plate placental cells

Cells from the amnion-chorion plate were consistently positive for HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105 and CD13 (Figure 7). The average expression of each marker in amnion-chorion plate-derived cells is as follows: 78.52%±13.13% (n=2, SEM=9.29) of cells express HLA-G; 38.33%±15.74% (n=5, SEM=7.04) 69.56%±26.41% (n=2, SEM=18.67) of cells expressed CD117; 42.44%±53.12% (n=2, SEM=37.56) of cells expressed CD10; 32.47%±31.78% (n =2, SEM=22.47) cells express CD44; 5.56% (n=1) cells express CD200; 83.33% (n=1) cells express CD90; 83.52% (n=1) cells express CD38; 7.25% (n=1) cells expressed CD105; and 81.16% (n=1) of cells expressed CD13.

6.2.2.7 Umbilical cord-derived cells

Umbilical cord-derived cells were consistently positive for HLA-G, CD33, CD90, CD38, CD105, and CD13, whereas expression of CD117, CD10, CD44, and CD200 varied (Figure 8). The average expression of each marker in the umbilical cord-derived cells was as follows: 62.50%±53.03% (n=2, SEM=37.50) of the cells expressed HLA-G; 25.67%±11.28% (n=5, SEM=5.04) of the cells expressed CD33 44.45%±62.85% (n=2, SEM=44.45) cells express CD117; 8.33%±11.79% (n=2, SEM=8.33) cells express CD10; 21.43%±30.30% (n=2, SEM =21.43) cells express CD44; 0.0% (n=1) cells express CD200; 81.25% (n=1) cells express CD90; 64.29% (n=1) cells express CD38; 6.25% (n=1) cells express CD38; ) of cells express CD105; and 50.0% (n=1) of cells express CD13.

A summary of the mean values of expression of all markers is shown in FIG. 9 .

6.2.2.8BD FACS Aria sorting report

Three different placental cell populations (cells from perfusate, amnion and chorion) expressing the highest percentages of HLA ABC, CD45, CD34 and CD133 were stained with 7AAD and antibodies against these markers. Live cells expressing the phenotype of interest were positively sorted for the three populations. The results of BD FACS Aria sorting are shown in Table 2.

Table 2:

Three different positively sorted cell populations ("sorted") and their corresponding unsorted cells were plated and culture results analyzed at day 12 (Table 3). Sorted perfusate-derived cells plated at a cell density of 40,600/ ^cm2 yielded tiny round non-adherent cells. Two of the three groups of unsorted perfusate-derived cells each plated at a cell density of 40,600/cm ² yielded almost all tiny round non-adherent cells with a few adherent cells at the periphery of the wells. Unsorted perfusate-derived cells plated at a cell density of 93,800/ ^cm2 yielded almost all tiny round non-adherent cells with a few adherent cells at the periphery of the well.

Sorted amnion-derived cells plated at a cell density of 6,300/ ^cm2 yielded tiny round non-adherent cells. Unsorted amnion-derived cells plated at a cell density of 6,300/cm ² yielded tiny round non-adherent cells. Unsorted amnion-derived cells plated at a cell density of 62,500/cm ² yielded tiny round non-adherent cells.

Sorted chorion-derived cells plated at a cell density of 6,300/ ^cm2 yielded tiny round non-adherent cells. Unsorted chorion-derived cells plated at a cell density of 6,300/ ^cm2 yielded tiny round non-adherent cells. Unsorted chorion-derived cells plated at a cell density of 62,500/ ^cm2 yielded tiny round non-adherent cells.

6.3 Example 3: Differentiation of placental stem cells

Adherent placental stem cells differentiate into a variety of different cell lines. Adherent placental stem cells can be isolated from placenta by physically disrupting tissues from anatomical parts of the placenta (including amniotic membrane, chorion, placental cotyledon or any combination thereof), while umbilical cord stem cells can be obtained by physically disrupting umbilical cord tissue.

Placental stem cells and umbilical cord stem cells were cultured in media containing low concentrations of fetal bovine serum and limited growth factors. Flow cytometric analysis revealed that typically ≥70% of placental stem cells had a CD200 ⁺ CD105 ⁺ CD73 ^{+ CD34- CD45-} phenotype. Placental stem cells were found to differentiate into adipocytes, chondrocytes and bone cell lineages.

In induction medium containing IBMX, insulin, dexamethasone, and indomethacin, placental stem cells transformed into fat-laden adipocytes within 3 to 5 weeks. Under osteoblast-inducing culture conditions, placental stem cells were found to form bone nodules and deposit calcium in their extracellular matrix. Chondrogenic differentiation of PDAC was carried out in microspheres and was demonstrated by the formation of mucopolysaccharides in tissue aggregates.

6.4 Example 4: Immunomodulation Using Placental Stem Cells

Placental stem cells have immunomodulatory functions, including inhibition of T cell and natural killer cell proliferation. The following experiments demonstrate the ability of placental stem cells to modulate the stimulatory response of T cells in two assays, a mixed lymphocyte reaction assay and a regression assay.

6.4.1 Mixed lymphocyte reaction test

The MLR measures the response of the effector population to the target population. The effectors may be lymphocytes or purified subpopulations such as CD8 ⁺ T cells or NK cells. The target population could be allogeneic irradiated PBMC, or the same mature DC as in this study. The responder population consisted of approximately 20% of the total T cells allospecific cells. The modified placental stem cell MLR uses placental stem cells in this reaction.

Placental stem cells were plated in wells of a 96-well plate, and effector groups were added. Placental stem cells were pre-incubated with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) stained effectors for 24 hours prior to the addition of target cell-mature DCs. Six days later, the supernatant and non-adherent cells were collected. Supernatants were analyzed by Luminex bead assay and cells were analyzed by flow cytometry.

Classically, MLRs generate proliferative responses in both CD8 ⁺ and CD4 ⁺ T cell compartments. Since the two allogeneic donors had never been exposed before, this response was a naive T cell response. Both CD4 ⁺ T cells and CD8 ⁺ T cells proliferated abundantly in standard MLRs. CD4 and CD8 T cell proliferation, as measured by the percentage of CFSE ^Low responding cells, was inhibited when placental stem cells were added to the MLR.

The effect of adding placental stem cells (PMLR) to the MLR can be seen in FIGS. 3A and 3B (PMLR traces) and FIG. 4 . Similar results were obtained whether only CD4 ⁺ or CD8 ⁺ T cells were used alone, or equal amounts of CD4 ⁺ T cells and CD8 ⁺ T cells were used simultaneously. Placental stem cells obtained from amnion-chorion or umbilical cord stroma suppressed MLR to a similar extent, with no difference in suppression observed between CD4 ⁺ T cells and CD8 ⁺ T cells. The same holds true for bulk T cell responses.

MLR alone was performed using CD4 ⁺ T cells, CD8 ⁺ T cells, or both CD4 ⁺ T cells and CD8 ⁺ T cells and allogeneic dendritic cells (DC). Placental stem cells were added to the MLR, and the MLR without placental stem cells was used as a control to evaluate the degree of proliferation of T cells.

CD4 ⁺ and CD8 ⁺ T cells and CD14 ⁺ monocytes were isolated from the buffy coat using Miltenyi MACS columns and beads according to the manufacturer's instructions. By culturing monocytes in RPMI 1640 supplemented with 1% donor plasma, IL-4, and GM-CSF for six days, and in RPMI 1640 supplemented with IL-1β, TNF-α, and IL-6 for two days, day to obtain dendritic cells. Allogeneic T cells and DCs were incubated at a T:Dc ratio of 10:1 to form a classic 6-day MLR. T cell proliferation was assessed by staining T cells with CFSE (carboxyfluorescein diacetate succinimidyl ester) before addition to the assay. CSFE can be used to assess the extent of proliferation by measuring the dilution of the dye in the subpopulation of cells.

Placental stem cells (PSCs) were added to the assay at a T:DC:PSC ratio of 10:1:2. Reactions were performed in 96-well plates in a final volume of 200 μL in RPMI 1640 supplemented with 5% pooled human serum (R5). Six days later, non-adherent cells were briefly resuspended and transferred to RPMI-washed 5 mL tubes and stained with CD4 and CD8 antibodies. Proliferation of CD4 and CD8 compartments can be assessed on a BD FACS Calibur.

placental stem cells. Placental stem cells can be obtained as described in Examples 1 and 2 above. Placental stem cells can be obtained from the following placental tissues: amnion (AM), or amnion/chorion (AC). Umbilical cord stem cells were obtained from digests of umbilical cord (UC). Fibroblasts (FB) and bone marrow-derived mesenchymal stem cells (MSCs) were added as controls.

result. When placental stem cells were added to the MLR, T cell proliferation was inhibited (Figure 10). The placental stem cells used in the experiments reflected in Figure 10 were derived from one placenta, designated 61665. For all placental stem cells tested, the CD4 ⁺ compartment was more suppressed than the CD8 ⁺ compartment when either CD4 ⁺ and CD8 ⁺ T cells were used but not both (Fig. 10A). The inhibition of CD4 ⁺ activation by AM and UC placental stem cells was roughly comparable to that mediated by MSCs, with an inhibition rate of approximately 60%-75%. When MLR was performed using both CD4 ⁺ and CD8 ⁺ T cells, placental stem cells inhibited the proliferation of the CD4 ⁺ compartment to a much greater extent than the CD8 ⁺ compartment (Fig. 10B). In particular, AM placental stem cells inhibited CD4 ⁺ T cell proliferation by nearly 90%, exceeding that shown by MSCs. The difference in inhibition between these two compartments was most pronounced for AM and AC placental stem cells.

Placental stem cells from different donors inhibited T cell proliferation to varying degrees in the MLR (Fig. 11). Placental stem cells from a different placenta (designated 65450) proliferated CD4 ⁺ and CD8 ⁺ T cells in the MLR differently than placental stem cells from placenta 61665. Remarkably, AC and UC PSCs from placenta 65450 inhibited T cell proliferation by 80% to 95%, exceeding that of MSCs in this assay. However, AC placental stem cells from placenta 65450 failed to suppress T cell proliferation to an appreciable degree (compared to AM placental stem cell suppression in Figure 10A).

Placental stem cells also suppress the activity of natural killer (NK) cells in the MLR.

6.4.2 Regression test

Placental stem cells were shown in a regression assay to suppress T cell responses to B cell lines expressing Epstein-Barr virus (EBV) antigens. The regression test is a recall test (recallassay) that measures the mechanism of effector T cells brought about by the presentation of EBV antigen peptides of MHC class I and class II molecules of EBV-transformed B cells. The assay was performed by mixing T cells with an artificially generated transformed B cell line, a lymphoblastoid cell line (LCL) from the same donor. The LCL expresses nine Epstein-Barr virus antigens that elicit a range of adaptive T and B cell responses in relation to each other, although only T cell effector mechanisms were measured in classical regression assays. This regression assay provides a simple assay of cytotoxicity against targets infected with naturally occurring pathogens in which LCLs express the activated B cell marker CD23. Therefore, the number of cells expressing CD23 is a measure of the number of surviving LCLs in this assay.

The classic 17-day regression test yielded results similar to those of the first set of bars in Figure 5. Since all CD23+ cells had been killed by CD4 ⁺ and CD8 ⁺ T cells, no CD23 ⁺ cells were detected. With the addition of placental stem cells, an increase in the survival of CD23 ⁺ cells was observed in the latter two sets of columns. Without wishing to be bound by theory, two explanations can be given for the observed effect. Either the death of T cells allows the free expansion of LCLs, or the placental stem cells mainly increase the lifespan of LCLs and have relatively little effect on T cells.

In separate regression experiments, T cells and dendritic cells were obtained from laboratory donors. The Epstein-Barr virus transformed B cell line, LCL, can be obtained by incubating peripheral blood mononuclear cells (PBMC) with supernatant from a cytolytic EBV line, B95.8 and cyclosporine A for two weeks. LCL expresses nine EBV antigens. The grown LCL lines were maintained in RPMI 1640 containing 10% fetal bovine serum. Regression assays were performed by mixing CD4 ⁺ or CD8 ⁺ T cells with autologous LCL at a T:LCL ratio of 10:1. The assay was performed in 96-well plates in 200 [mu]L of RPMI 1640 supplemented with 5% pooled human serum (R5). Placental stem cells were added to the assay at a T:LCL:PSC ratio of 10:1:2. The trial will be done for 6, 10 or 17 days.

A 6-day regression test can be performed using CSFE-labeled T cells. Placental stem cells from placenta 65450 inhibited T cell proliferation by about 65% to about 97% in the regression assay, a result corresponding to the results for PSCs in the MLR (Figure 12). In addition, UC and AC lines derived from placenta 65450 significantly inhibited T cell proliferation, whereas 65450AM PSCs did not inhibit proliferation.

In separate assays, it was determined that natural killer cells were also inhibited in MLR and regression assays. In MLR or regression assays containing 50 U/ml IL-2, the inhibitory effect on NK cells was about 45% (from about 40% to about 65%, SEM 5%).

Placental stem cells are not immunogenic. In any event, no more than 5% of background T cell proliferation was observed for placental stem cells from any donor or any anatomical site of the placenta.

Cell-cell contact requirements. Cytotoxic effects in regression assays, and allo-recognition in MLRs are dependent on TCR (T cell receptor):MHC interactions between target and effector cells. The requirement of cell-cell contact in placental stem cell-mediated suppression can be assessed by transwell assay. MLR can be performed in this assay, where T cells and placental stem cells are separated by a membrane. As shown in Figure 13, the higher the number of placental stem cells in the MLR, the greater the reduction in inhibition, showing that particularly at higher densities, placental stem cells (UC) require significant contact with T cells to inhibit T cell proliferation.

Separate experiments demonstrated that placental stem cell immunosuppression of T cells appears to involve, at least in part, soluble factors. To determine whether the placental stem cell-mediated immunosuppression was dependent on cell-cell contact, transwell assays were performed in which placental stem cells were placed in an inlay with a membrane at the bottom that allows only soluble factors to pass through. At the bottom of the well, there are only MLR or T cells, separated from the placental stem cells. To determine whether the observable effect was dependent on the relative dosage of placental stem cells, stem cells were added at different relative densities relative to T cells and DC. This inhibitory effect was partially abolished when umbilical cord placental stem cells were separated from the MLR. This MLR inhibition reduced CD4 ⁺ T cells by 75% and CD8 ⁺ T cells by 85% when placental stem cells were used at densities similar to those used in Figure 4 (Figure 7, Figure 8). The inhibitory effect was still 66% when only a quarter of the dose of placental stem cells was used (UC OP 25), which decreased to background levels when 12,500 UC placental stem cells were added. No change in inhibition was observed when block separation was used (Figure 7). For 25,000 placental stem cells, a minimal relative decrease in inhibition by block introduction was observed, although strong inhibition was still present (Fig. 8).

6.5 Example 5: The contact dependence of immunosuppression in placental stem cells differs from that in bone marrow-derived mesenchymal stem cells

In experiments examining the extent of contact dependence of immune regulation, umbilical cord stem cells displayed a significantly different requirement for immunoregulatory cell-cell contacts than bone marrow-derived stem cells. In particular, placental stem cells are more dependent on cell-cell contacts for immune regulation, especially when the number of placental or mesenchymal stem cells is higher.

Bone marrow-derived stem cells (BMSCs) and umbilical cord stem cells (UCs) have different requirements for cell-cell contact according to the ratio of adherent cells to T cells in the mixed lymphocyte reaction assay (MLR). In transwell assays in which placental stem cells were separated from T cells and dendritic cells (DCs) in the MLR, inhibition differed between the two types of adherent cells. Figure 15 shows the open hole and transwell results side by side. Similar inhibition was observed when using approximately 100,000 or 75,000 UC or BMSC in an open-pore format. However, in the transwell format, UC suppressed the MLR to a lesser degree than BMSC, showing greater contact dependence at higher placental stem cell/T cell ratios. Placental stem cells were more suppressive to cells when a lower ratio of placental cells to T cells was used.

The degree of exposure dependence was calculated from this inhibition data. Figure 16 shows the contact dependence of UC and BMSC MLR. Bone marrow-derived cells were less contact-dependent than UC at higher BM/T cell ratios. In other words, UC placental stem cells and BMSCs behave differently with respect to an important mechanistic parameter, the requirement for cell-cell contacts.

Regulatory T cells (Treg) are essential in BMSC-mediated T cell suppression. See Aggarwal and Pittenger, "Human Mesenchymal Stem Cells Modulate Allogeneic Immune Cell Responses," Blood 105(4):1815-1822 (2004). CD4 ⁺ CD25 ⁺ Treg were depleted from healthy donor peripheral blood mononuclear cells (PBMCs) and regression assays were performed using autologous EBV (Epstein-Barr virus) transformed cells. In some cases join UC. As shown in Figure 17, there was no difference in placental stem cell-mediated suppression of T cell responses in the regression assay regardless of the presence of Tregs. Thus, although T regulatory T cells have been reported to be essential for BMSC-mediated T cell suppression, T regulatory cells have not shown a role in placental stem cell-mediated immunosuppression.

MLR was performed in which T cells were taken from an MLR suppressed by placental stem cells and fresh dendritic cells were added. T cells are stained with CFSE, which distributes evenly to progeny cells as they proliferate. CFSE ^Hi cells are non-proliferating T cells (eg, the leftmost peak in Figure 17). This population was obtained by sorting stained T cells on a FACS Aria. These cells can be used in secondary MLRs with fresh dendritic cells. As shown in Figure 18, sustained suppression was not observed as previously suppressed cells proliferated well relative to DCs. CFSE ^Lo cells (ie, daughter cells) are unlikely to be involved in the inhibition, as these cells themselves subsequently proliferate. The CFSE ^Hi population consists of non-allospecific cells that do not proliferate relative to this DC donor and T cells suppressed by placental stem cells. Once the placental stem cells are removed, the suppressor cells proliferate.

MLR was inhibited by BMSCs when approximately 10% of the supernatant was replaced with supernatant from BMSC MLR. In stark contrast, no change in T cell proliferation was observed when the supernatant was replaced with that from the MLR containing placental stem cells, even when 75% of the medium was replaced (Figure 19).

Different times of incubation with placental stem cells before initiation of MLR may affect DC or resting T cells. This was tested by incubating placental stem cells or BMSCs with T cells (Fig. 20A) or DCs (Fig. 20B) for different lengths of time before starting the assay. Preincubation of T cells with placental stem cells did not significantly alter the suppressive phenotype (Fig. 20A). However, BMSC T cell suppression varied with the length of DC/PDAC pre-incubation. As shown in Figure 20B, the suppression by BMSCs was strongest when DCs were added one day after T cells. However, much lower suppression was observed when DCs were added simultaneously with T cells. Incubation of DCs with BMSCs for longer periods of time reversed this loss of inhibition. This suppression was close to expected when DCs were added one day after T cells (+1 day) in the two-day pre-culture. A similar trend was not observed in placental stem cell-mediated suppression.

6.6 Example 6: Cytokine Status of Placental Stem Cells and Umbilical Cord Stem Cells in MLR and Regression Tests

The secretion of certain cytokines into MLR medium by umbilical cord stem cells (UC) and placental stem cells from amnion chorionic plates (AC) was determined.

In some assays, cytokine and chemokine levels in supernatants were measured using cytokine arrays. Several factors were found to be secreted into the supernatant, the most relevant for MLR and regression assays were macrophage inflammatory protein (MIP)-1α and MIP-1β. Both chemoattractant proteins attract T cells and are secreted by CD8 ⁺ T cells in response to human immunodeficiency virus (HIV) infection. When measured in the MLR, the secretion of these chemoattractant proteins was inversely correlated with the inhibition of the MLR by placental stem cells and MSCs (Figure 14). Neither placental stem cells nor MSCs secrete MIP-1α and MIP-1β.

In another study, a correlation was found for the secretion of MCP-1 and IL-6, both important immunomodulators (Figures 9 and 10; compare with Figure 4). Although placental stem cells did not secrete IL-6 or MCP-1 alone, UC and AC lines that inhibited MLR and T cell proliferation in regression assays (Fig. 4) secreted IL-6 or MCP-1 (Fig. 9 and Fig. 10). Although IL-6 is primarily associated with proinflammatory effects (see, for example, Kishimoto et al., Annu. Rev. Immunol. 23:1-21 (2005)), it also has other functions, such as a protective role in liver injury in mice (See, eg, Klein et al., J. Clin. Invest. 115:860-869 (2005)).

In a separate study, analysis of cytokine secretion was performed on the MLR or the AC used in the regression assay. Cytokines can be detected in supernatants from 6-day stem cell cultures, stem cell MLR, or stem cell regression assays on the Luminex system. MLR includes stem cells, dendritic cells (DC) and T cells in a ratio of 2/1/10. Epstein-Barr virus (EBV) regression assays included stem cells, EBV tumor cells (T) and T cells at a TS:stem cell:T ratio of 2:1:10.

IL-6 (11 ng/ml) and IL-8 (16 ng/ml) levels were found to remain constant in stem cell culture alone, MLR and regression assays. It was detected that the concentration of MCP-1 in stem cell culture alone and non-inhibitory control adherent cell MLR and regression assay was about 2ng/ml, but rose to about 10ng/ml in suppressive stem cell MLR and stem cell regression assay. These values pertain to serum levels of MCP-1.

Interleukin-2 (IL-2) is both a T cell survival factor and an obligate factor for CD4 ⁺ CD25 ⁺ T regulatory cells. This T cell subset was not required for T cell suppression by AC stem cells, but IL-2 levels were consistently reduced during MLR suppression by AC stem cells. AC stem cell depleted MLR supernatants contained approximately 35 pg/ml IL-2, whereas AC stem cell containing MLRs contained up to 440 pg/ml IL-2.

IL-2 concentration correlates with inhibition. For example, a CD4 ⁺ T cell MLR showing 85% suppression contained 330 pg/ml IL-2, while a CD8 ⁺ T cell MLR using AC stem cells showing 85% suppression contained 66 pg/ml IL-2. These results show that IL-2 and MCP-1, traditionally regarded as immune response stimulators, have an important role in immunosuppression.

Equivalents:

The present invention is not intended to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such adjustments are intended to fall within the scope of the claims.

Various publications, patents, and patent applications are cited herein, the disclosures of which are incorporated by reference in their entirety.

Claims (58)

1. A method of suppressing an immune response comprising contacting a plurality of immune cells with a plurality of placental stem cells for a time sufficient for said placental stem cells to detectably suppress an immune response, wherein said placental stem cells are tested in a mixed lymphocyte reaction (MLR) assay detectably inhibited T cell proliferation. 2. The method of claim 1, wherein said large number of placental stem cells: Express CD200 and HLA-G; Expresses CD73, CD105 and CD200; Express CD200 and OCT-4; Express CD73, CD105 and HLA-G; express CD73 and CD105, and promote the formation of one or more embryoid bodies in a population of placental cells comprising the stem cells when said population is cultured under conditions suitable for embryoid body formation; and/or OCT-4 is expressed and promotes the formation of one or more embryoid bodies in a population of placental cells comprising the stem cells when the population is cultured under conditions suitable for embryoid body formation. 3. The method of claim 1, wherein said plurality of immune cells are T cells or NK (natural killer) cells. 4. The method of claim 3, wherein said T cells are CD4 ⁺ T cells. 5. The method of claim 3, wherein said T cells are CD8 ⁺ T cells. 6. The method of claim 1, wherein said contacting is performed in vitro. 7. The method of claim 1, wherein said contacting is performed in vivo. 8. The method of claim 7, wherein said contacting is performed in a mammalian subject. 9. The method of claim 8, wherein said mammalian subject is a human. 10. The method of claim 7, wherein said contacting comprises administering said placental cells intravenously. 11. The method of claim 7, wherein said contacting comprises intramuscularly administering said placental cells. 12. The method of claim 7, wherein said contacting comprises administering said placental cells into an organ of said individual. 13. The method of claim 12, wherein said organ is the pancreas. 14. The method of claim 8, further comprising administering to the individual an anti-macrophage inflammatory protein (MIP)-1α or anti-MIP-1β antibody, wherein the antibody is present in an amount sufficient to elicit MIP in the individual's blood -1α or anti-MIP-1β was administered in an amount that resulted in a detectable decrease in the amount. 15. The method of claim 1, wherein said immune response is graft versus host disease. 16. The method of claim 15, wherein said immune response is an autoimmune disease. 17. The method of claim 16, wherein the autoimmune disease is diabetes, lupus erythematosus, rheumatoid arthritis or multiple sclerosis. 18. The method of claim 1, wherein said plurality of immune cells is also contacted with non-placental cells. 19. The method of claim 18, wherein said non-placental cells comprise CD34 ⁺ cells. 20. The method of claim 19, wherein said CD34 ⁺ cells are peripheral blood hematopoietic progenitor cells, umbilical cord blood hematopoietic progenitor cells or placental blood hematopoietic progenitor cells. 21. The method of claim 18, wherein said non-placental cells comprise mesenchymal stem cells. 22. The method of claim 21, wherein said mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. 23. The method of claim 16, wherein said non-placental cells are contained in an allograft. 24. The method of claim 1, wherein said plurality of placental stem cells comprises 1 x ¹⁰⁵ placental stem cells. 25. The method of claim 1, wherein said plurality of placental stem cells comprises 1 x ¹⁰⁶ placental stem cells. 26. The method of claim 1, wherein said plurality of placental stem cells comprises 1 x ¹⁰⁷ placental stem cells. 27. The method of claim 1, wherein said plurality of placental stem cells comprises 1 x ¹⁰⁸ placental stem cells. 28. A method of selecting a population of placental cells comprising (a) analysis of a large number of placental cells in a mixed lymphocyte reaction (MLR) assay; and (b) selecting said plurality of placental stem cells if said plurality of placental stem cells detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation in an MLR (mixed lymphocyte reaction), wherein said placental stem cells: (i) adheres to the substrate; and (ii) express CD200 and HLA-G, or expresses CD73, CD105, and CD200, or Express CD200 and OCT-4, or Express CD73, CD105, and HLA-G, or expressing CD73 and CD105 and promoting the formation of one or more embryoid bodies in a population of placental cells comprising the stem cells when said population is cultured under conditions suitable for embryoid body formation, or OCT-4 is expressed and promotes the formation of one or more embryoid bodies in a population of placental cells comprising the stem cells when the population is cultured under conditions suitable for embryoid body formation. 29. A method for selecting a cell population comprising: selecting from a large number of cells that (a) adhere to a substrate, (b) express CD200 and HLA-G, and (c) detectably in an MLR (mixed lymphocyte reaction) placental stem cells that inhibit proliferation of CD4 ⁺ or CD8 ⁺ T cells; and isolating said placental stem cells from other cells to form a cell population. 30. A method of preparing a population of cells comprising: selecting from a plurality of cells that (a) adhere to a substrate, (b) express CD73, CD105, and CD200, and (c) detectably inhibit CD4 ⁺ or CD8 ⁺ in MLRs T cell-proliferated placental stem cells; and isolating said placental stem cells from other cells to form a cell population. 31. A method of preparing a population of cells comprising: selecting from a population of cells that (a) adhere to a substrate, (b) express CD200 and OCT-4, and (c) detectably inhibit CD4 ⁺ or CD8 ⁺ in MLRs T cell-proliferated placental stem cells; and isolating said placental stem cells from other cells to form a cell population. 32. A method of preparing a population of cells comprising: selecting from a plurality of cells that (a) adhere to a substrate, (b) express CD73 and CD105, (c) form embryoids when cultured under conditions suitable for embryoid body formation and (d) placental stem cells that detectably inhibit proliferation of CD4 ⁺ or CD8 ⁺ T cells in the MLR; and isolating said placental stem cells from other cells to form a cell population. 33. A method of preparing a population of cells comprising: selecting from a plurality of cells that (a) adhere to a substrate, (b) express CD73, CD105, and HLA-G, and (c) detectably inhibit CD4 ⁺ or CD8 ⁺ T cell-proliferated placental stem cells; and isolating said placental cells from other cells to form a cell population. 34. A method for preparing a population of cells comprising: selecting from a large number of placental stem cells that (a) adhere to a substrate, (b) express OCT-4, (c) form embryoid-like bodies when cultured under conditions suitable for embryoid-like body formation embryoid bodies, and (d) cells that detectably inhibit proliferation of CD4 ⁺ or CD8 ⁺ T cells in the MLR; and isolating said placental stem cells from other cells to form a cell population. 35. The method of any one of claims 28-34, wherein said T cells and said placental stem cells are present in said MLR in a ratio of about 5:1. 36. The method according to any one of claims 28-34, wherein said placental stem cells are derived from amnion, amnion and chorion. 37. The method of any one of claims 28-34, wherein said placental stem cells are suppressed by at least 50% in said MLR compared to the amount of proliferation of T cells in said MLR lacking said placental stem cells Proliferation of CD4 ⁺ or CD8 ⁺ T cells. 38. The method of any one of claims 28-34, wherein said placental stem cells inhibit at least 75% in said MLR compared to the proliferation of T cells in said MLR lacking said placental stem cells % of CD4 ⁺ or CD8 ⁺ T cells proliferating. 39. The method of any one of claims 28-34, wherein said placental stem cells inhibit at least 90% in said MLR compared to the proliferation of T cells in said MLR lacking said placental stem cells % of CD4 ⁺ or CD8 ⁺ T cells proliferated. 40. The method of any one of claims 28-34, wherein said placental stem cells in said MLR can inhibit at least 95% of CD4 ⁺ or CD8 ⁺ T cells proliferated. 41. The method of any one of claims 28-34, wherein said placental stem cells also detectably inhibit natural killer (NK) cell activity. 42. An isolated population of placental stem cells prepared according to the method of any one of claims 28-34. 43. An isolated population of placental stem cells, wherein said placental stem cells: (a) adheres to the substrate; (b) express CD200 and HLA-G, or expresses CD73, CD105, and CD200, or Express CD200 and OCT-4, or Express CD73, CD105, and HLA-G, or expressing CD73 and CD105, and promoting the formation of one or more embryoid bodies in a population of placental cells comprising the placental stem cells when said population is cultured under conditions suitable for embryoid body formation, 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 said population is cultured under conditions suitable for embryoid body formation; and (c) have been identified in the MLR to detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation. 44. An isolated cell population comprising placental stem cells that (a) adhere to the substrate, (b) express CD200 and HLA-G, and (c) have been identified in an MLR (mixed lymphocyte reaction) as Detectably inhibits CD4 ⁺ or CD8 ⁺ T cell proliferation. 45. An isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD73, CD105, and CD200, and (c) have been identified as detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation. 46. An isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD200 and OCT-4, and (c) have been identified as detectably inhibiting CD4 ⁺ or CD8 ⁺ T cell proliferation. 47. An isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD73 and CD105, (c) form embryoid bodies when cultured under conditions suitable for embryoid body formation, And (d) have been identified in the MLR to detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation. 48. An isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express CD73, CD 105, and HLA-G, and (c) have been identified as detectably inhibiting in the MLR CD4 ⁺ or CD8 ⁺ T cells proliferate. 49. An isolated cell population comprising placental stem cells that (a) adhere to a substrate, (b) express OCT-4, (c) form embryoid bodies when cultured under conditions suitable for embryoid body formation, And (d) have been identified in the MLR to detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation. 50. A composition comprising a supernatant of a culture of a cell population according to any one of claims 43-49. 51. A composition comprising a culture medium of placental stem cells, wherein said placental stem cells: (a) adheres to the substrate; (b) express CD200 and HLA-G, or expresses CD73, CD105, and CD200, or Express CD200 and OCT-4, or Express CD73, CD105, and HLA-G, or expressing CD73 and CD105, and promoting the formation of one or more embryoid bodies in a population of placental cells comprising the placental stem cells when said population is cultured under conditions suitable for embryoid body formation, 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 said population is cultured under conditions suitable for embryoid body formation; and (c) is identifiable in the MLR to detectably inhibit CD4 ⁺ or CD8 ⁺ T cell proliferation, Wherein said placental stem cell culture has been cultured in said culture medium for 24 hours or longer. 52. The composition of claim 51, further comprising a plurality of said placental stem cells. 53. The composition of claim 51, further comprising a plurality of non-placental cells. 54. The composition of claim 53, wherein said non-placental cells comprise CD34 ⁺ cells. 55. The composition of claim 54, wherein said CD34 ⁺ cells are peripheral blood hematopoietic progenitor cells, umbilical cord blood hematopoietic progenitor cells or placental blood hematopoietic progenitor cells. 56. The composition of claim 53, wherein said non-placental cells comprise mesenchymal stem cells. 57. The composition of claim 56, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. 58. The composition of claim 51, further comprising an anti-MIP-la or anti-MIP-lbeta antibody.

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