JP2010059193A - Embryonic-like stem cell derived from post-partum mammalian placenta and use and method of treatment using the cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition and a method for using embryonic-like stem cells that originate from a post-partum placenta with cord blood compositions or other stem or progenitor cells. <P>SOLUTION: The embryonic-like stem cells can be used alone or in a mixture with other stem cell populations. The embryonic-like stem cells may be mixed with other stem cell populations including umbilical cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells, human stem cells and progenitor cells derived from bone marrow. The embryonic-like stem cells and the mixed populations of embryonic-like stem cells and stem cells have a multitude of uses and applications, including e.g. therapeutic uses for transplantation and treatment and prevention of disease, and diagnostic and research uses. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

1. Introduction The present invention relates to the use of embryonic-like stem cells derived from postpartum placenta with conventional cord blood compositions or other stem or progenitor cells. Embryonic-like stem cells are used by themselves or as a mixture with other stem cell populations. According to the present invention, embryonic-like stem cells can be mixed with other stem cell populations including, but not limited to, cord blood, fetal and neonatal hematopoietic stem and progenitor cells, human Examples include stem cells and bone marrow-derived progenitor cells. Embryo-like stem cells, and mixed populations of embryo-like stem cells and stem cells have a variety of uses, including, but not limited to, therapeutic uses, diagnostic and research uses for transplantation. Embryonic-like stem cells and mixed populations also treat various diseases or disorders, including vascular diseases, neurological diseases or disorders, autoimmune diseases or disorders, diseases or disorders with inflammation, cancers or related disorders Also useful. In particular, embryonic-like stem cells or mixtures comprising them can be administered at high doses without HLA typing.

2. BACKGROUND OF THE INVENTION There is considerable interest in the identification, isolation and production of human stem cells. Human stem cells are totipotent or multipotent progenitor cells capable of generating a variety of mature human cell lines. This ability serves as the basis for cell differentiation and specialization required for organ and tissue development.

  The recent successful transplantation of such stem cells has provided a new clinical tool to reconstitute and / or replenish the bone marrow after exposure to diseased bone marrow resection, toxic chemicals and / or radiation. Furthermore, there is evidence that stem cells can be used to repopulate many, if not all, tissues and restore physiological and anatomical functionality. The application of stem cells to tissue engineering, gene therapy delivery and cell therapy is also rapidly progressing.

  A number of different types of mammalian stem cells have been characterized. For example, embryonic stem cells, embryonic germ cells, adult stem cells, or other committed stem cells or progenitor cells that are destined for terminal differentiation are known. Some stem cells are not only isolated and characterized, but are also cultured under conditions that allow differentiation to a limited extent. However, the fundamental problem remains that it is almost impossible to obtain a sufficient amount and population of human stem cells capable of differentiating into any cell type. The supply of stem cells is extremely scarce. These are important for the treatment of a wide variety of diseases such as malignant diseases, inborn errors of metabolism, abnormal hemoglobinosis, immunodeficiencies. Therefore, it would be very advantageous to have a source of higher amounts of embryonic stem cells.

  Obtaining a sufficient number of human stem cells has been difficult for several reasons. First, the separation of a normal population of stem cells in adult tissue is technically difficult and costly, in part because only a very limited amount of blood or tissue is observed. Take it. Second, the acquisition of these cells from fetal tissue, including embryos or miscarriages, has caused religious and ethical problems. Widespread support for the idea that human embryos and fetuses are independent lives has led to government regulations on the use of these sources for all uses, including medical research. Thus, alternative sources that do not require the use of cells obtained from embryonic or fetal tissue are essential in developing clinical use of stem cells. However, the supply is limited because there are very few possible alternative sources of stem cells, especially human stem cells. In addition, recovering sufficient amounts of stem cells from alternative sources for therapeutic and research purposes includes harvesting cells or tissues from donor subjects or patients, in vitro cell culture and / or proliferation, incisions, etc. Generally, it is complicated.

  For example, Caplan et al. (US Pat. No. 5,486,359 entitled “Human Mesenchymal Stem Cells”, published January 23, 1996), describes a bone marrow-derived human mesenchymal stem cell (hMSC) composition that serves as a precursor to the mesenchymal cell lineage. Is disclosed. Caplan et al. Disclose that hMSCs are identified by specific cell surface markers identified with monoclonal antibodies. Positive selection of adherent bone marrow or periosteal cells that do not contain markers associated with either hematopoietic cells or differentiated mesenchymal cells results in uniform hMSC compositions. These isolated mesenchymal stem cell populations display the characteristics of the epitopes associated with mesenchymal stem cells and are capable of regenerating in culture without differentiation and are therefore induced or damaged in vitro When placed in vivo at a tissue site, it has the ability to differentiate into a specific mesenchymal lineage. However, such methods have the disadvantage that MSCs must be isolated after harvesting bone marrow or periosteal cells from a donor.

  Hu et al. (WO 00/73421 entitled “Isolation, cryopreservation and therapeutic use of human amniotic epithelial cells” published December 7, 2000) was isolated, cultured and used for future use. Disclosed are placenta-derived human amniotic epithelial cells that have been cryopreserved or induced to differentiate. According to Hu et al., The placenta is collected immediately after delivery and the amniotic membrane is separated from the chorion, for example, by incision. The amniotic epithelial cells are then isolated from the amniotic membrane according to standard isolation techniques. The disclosed cells can be cultured in various media, grown in culture, cryopreserved, or induced to differentiate. Hu et al. Disclose that amniotic epithelial cells are pluripotent (possibly pluripotent) and can differentiate into epithelial tissues such as corneal surface epithelium or vaginal epithelium. However, the disadvantages of such methods are that they require a lot of effort and the yield of stem cells is very low. For example, to obtain a sufficient number of stem cells for typical therapeutic or research purposes, the amnion epithelial cells must first be isolated from the amnion by incision and cell separation techniques, and then cultured and expanded in vitro. I must.

  Umbilical cord blood is known as an alternative source of hematopoietic progenitor stem cells. Umbilical cord blood-derived stem cells are usually cryopreserved for use in hematopoietic reconstitution, which is a treatment method widely used in bone marrow and other related transplants (eg, “blood” Isolation and preservation of blood-derived fetal and neonatal hematopoietic stem and progenitor cells, issued on March 9, 1993, Boyse et al., US Pat. No. 5,004,681, entitled “Preservation of fetal and neonatal hematopoietic stem and progenitor cells derived from As well as US Pat. No. 5,192,553 to Boyse et al. The usual technique for collecting umbilical cord blood consists of using a needle or cannula, which is used to drain (ie, phlebotomy) cord blood from the placenta using gravity (March 1993). Boyse et al. US Pat. No. 5,192,553 issued on 9th; Boyse et al. US Pat. No. 5,004,681 issued on 2nd April 1991; “Method and apparatus for placental blood collection” issued 13 December 1994; Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665, issued May 16, 1995, entitled “Umbilical Clamping, Cutting, and Blood Collection Apparatus and Method”. A needle or cannula is usually placed in the umbilical vein and the placenta is gently massaged to drain the cord blood from the placenta. However, after that, the spilled placenta was considered useless and generally discarded. The main limitation in obtaining stem cells from umbilical cord blood is that the amount of umbilical cord blood obtained is often insufficient, resulting in a lack of cells to effectively reconstitute post-transplant bone marrow. Met.

  Naughton et al. (US Pat. No. 5,962,325, entitled “Three-dimensional stromal tissue culture”, issued October 5, 1999), introduces fetal cells such as fibroblast-like cells and chondrocyte progenitor cells into umbilical cord or placental tissue. Or it discloses that it can be obtained from umbilical cord blood.

  Kraus et al. (US Pat. No. 6,338,942, entitled “Selective Proliferation of Target Cell Population” issued on January 15, 2002) states that selective growth of a predetermined target cell population is possible by: Disclosed: After introducing a starting sample of cells from umbilical cord blood or peripheral blood into the growth medium to divide the cells of the target cell population, the cells in the growth medium are allowed to bind to binding molecules (with specific affinity) ( By contacting with a selection element containing a monoclonal antibody (for CD34), cells of a predetermined target population can be selected from cells in the growth medium.

Rodgers et al. (US Pat. No. 6,335,195, entitled “Methods of Promoting Hematopoietic and Mesenchymal Cell Proliferation and Differentiation”, issued January 1, 2002), describes a method for ex vivo culture of hematopoietic and mesenchymal stem cells, and , angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof, or AII presence of AT ₂ 2 type receptor agonists Below, the induction of lineage specific cell growth and differentiation consisting of growing alone or in combination with other growth factors and cytokines is disclosed. The stem cells are derived from bone marrow, peripheral blood or umbilical cord blood. However, the disadvantages of such methods are that the ex vivo methods for stem cell proliferation and differentiation are time consuming and have low stem cell yields as described above.

  Stem cells are extremely in short supply due to the limited collection and use of stem cells and the generally insufficient number of cells recovered from umbilical cord blood. Stem cells have the potential to be used to treat a wide variety of disorders such as malignant diseases, inborn errors of metabolism, abnormal hemoglobinosis, and immunodeficiencies. There is an urgent need for a source that makes a large number of human stem cells readily available for a variety of treatments and other medically relevant purposes. The present invention solves this demand and others.

  In addition, there remains a need to treat neurological symptoms such as amyotrophic lateral sclerosis (ALS). Recent studies using a murine model of familial ALS (a less common form of ALS) suggested that umbilical cord blood was useful in the treatment of this disease, but due to the source issues mentioned earlier This choice is also far from ideal. See Ende et al., Life Sci. 67: 53059 (2000). Thus, there remains a need for a population of stem or progenitor cells that can be used to treat a disease (especially those larger populations when trying to treat a disease such as ALS).

U.S. Pat.No. 5,486,359 WO 00/73421 U.S. Pat.No. 5,004,681 US Patent No. 5,192,553 U.S. Pat.No. 5,372,581 U.S. Pat.No. 5,415,665 US Patent No. 5,962,325 U.S. Pat.No. 6,338,942 U.S. Pat.No. 6,335,195

Ende et al., Life Sci. 67: 53059 (2000)

3. Summary of the invention The present invention relates to:

[1] A composition comprising a stem or progenitor cell and an embryonic stem cell.
[2] A composition comprising cord blood cells and embryonic stem cells.
[3] The composition of [1], comprising a population of stem or progenitor cells and a population of embryonic-like stem cells.
[4] The composition according to [1] or [2], which is contained in a container.
[5] The composition according to [4], wherein the container is sealed, airtight, and sterile.
[6] The composition of [1] or [2], wherein the stem or progenitor cell and the embryonic-like stem cell are in contact with each other before or at the time of administration to a patient in need thereof.
[7] The composition of [1] or [2], wherein the stem or progenitor cells and the embryonic-like stem cells are suitable for separate administration to a patient.
[8] The composition according to [1] or [2], which is suitable for bone marrow transplantation.
[9] The composition of [1] or [2], which is suitable for human administration.
[10] The composition of [1] or [2], wherein the stem or progenitor cell is derived from umbilical cord blood or placental blood, fetal or neonatal hematopoietic stem or progenitor cell, human stem cell, adult cell, or bone marrow stem or progenitor cell object.
[11] The composition of [10], wherein the stem or progenitor cell is a fetal or neonatal hematopoietic stem or progenitor cell.
[12] The composition of [11], wherein a plurality of hematopoietic stem or progenitor cells express the cell surface markers CD34 + and CD38-.
[13] The composition of [10], wherein the stem or progenitor cell is cord blood stem cell.
[14] The composition of [13], wherein a plurality of cord blood stem cells express cell surface markers CD34 + and CD38−.
[15] The composition of [13], wherein a plurality of cord blood stem cells express cell surface markers CD34 + and CD38 +.
[16] The embryonic-like stem cell exhibits at least one of the following cell surface markers: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 or ABC-p, or the next cell Surface marker: The composition of [1] or [2], which lacks at least one of CD34, CD45, SSEA3, SSEA4.
[17] The composition of [16], wherein the embryonic stem cells are OCT-4 + and ABC-p +.
[18] The composition of [16], wherein the embryonic stem cells are SSEA3- and SSEA4-.
[19] A kit comprising the composition of [1] or [2], wherein the stem or progenitor cells are in a first container and the embryonic-like stem cells are in a second container.
[20] A method for preparing a composition comprising a plurality of stem or progenitor cells and a plurality of embryonic-like stem cells, comprising isolating and contacting the plurality of stem or progenitor cells and the plurality of embryonic-like stem cells How to
[21] The method according to [20], wherein a plurality of stem or progenitor cells and a plurality of embryonic-like stem cells are stored in separate containers before mixing.
[22] The method of [20], wherein the composition is suitable for human administration.
[23] The method of [20], wherein the stem or progenitor cell is cord blood or placental blood-derived, fetal or neonatal hematopoietic stem or progenitor cell, human stem cell, adult cell, or bone marrow stem or progenitor cell.
[24] The method of [20], wherein the stem or progenitor cell is a fetal or neonatal hematopoietic stem or progenitor cell.
[25] The method of [20], wherein a plurality of hematopoietic stem or progenitor cells express the cell surface markers CD34 + and CD38−.
[26] The method according to [20], wherein a plurality of stem or progenitor cells and a plurality of embryonic-like stem cells are physically mixed.
[27] A method for treating a patient in need thereof, comprising administering a plurality of stem or progenitor cells and a plurality of embryonic-like stem cells.
[28] The method according to [27], wherein a plurality of stem or progenitor cells and a plurality of embryonic-like stem cells are stored in separate containers before mixing.
[29] The method of [27], wherein the plurality of stem or progenitor cells and the plurality of embryonic-like stem cells are in contact with each other before or at the time of administration to a patient in need thereof.
[30] The method of [27], wherein the stem or progenitor cell and the embryonic-like stem cell are suitable for bone marrow transplantation.
[31] The method of [27], wherein the stem or progenitor cell and the embryonic-like stem cell are suitable for human administration.
[32] The method according to [27], wherein the stem or progenitor cell is cord blood or placental blood-derived, fetal or neonatal hematopoietic stem or progenitor cell, human stem cell, adult cell, or bone marrow stem or progenitor cell.
[33] The method of [32], wherein the stem or progenitor cell is a fetal or neonatal hematopoietic stem or progenitor cell.
[34] The method of [33], wherein a plurality of hematopoietic stem or progenitor cells express the cell surface markers CD34 + and CD38-.
[35] The method of [32], wherein the stem or progenitor cell is cord blood stem cell.
[36] The method of [35], wherein a plurality of cord blood stem cells express cell surface markers CD34 + and CD38-.
[37] The method of [35], wherein a plurality of cord blood stem cells express cell surface markers CD34 + and CD38 +.
[38] Embryonic-like stem cells have the following cell surface markers: CD10 +, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT-4 + and ABC-p + The method according to [27], wherein at least one of them is indicated.
[39] The method of [38], wherein the embryonic-like stem cells are OCT-4 + and ABC-p +.
[40] The method of [38], wherein the embryonic-like stem cells are SSEA3- and SSEA4-.
[41] The method of [27], wherein the plurality of stem or progenitor cells and the plurality of embryonic-like stem cells are mixed before or at the time of administration to a patient in need thereof.
[42] The method of [27], wherein a plurality of stem or progenitor cells and a plurality of embryonic-like stem cells are physically mixed.
[43] The method of [27], wherein a plurality of stem or progenitor cells and / or a plurality of embryonic-like stem cells are treated with a growth factor to induce differentiation into a specific cell type.
[44] The method of [27], wherein a plurality of stem or progenitor cells and / or a plurality of embryonic-like stem cells are treated with a growth factor to prevent or suppress differentiation into a specific cell type.
[45] A method for treating a patient in need thereof, comprising administering a plurality of cord blood cells and a plurality of embryonic-like stem cells.
[46] The method according to [45], wherein a plurality of cord blood cells and a plurality of embryonic-like stem cells are stored in separate containers before mixing.
[47] The method of [45], wherein the plurality of cord blood cells and the plurality of embryonic-like stem cells are in contact with each other before or at the time of administration to a patient in need thereof.
[48] The method of [45], wherein a plurality of cord blood cells and a plurality of embryonic-like stem cells are suitable for separately administering to a patient.
[49] The method of [45], wherein the umbilical cord blood cells are fetal or neonatal hematopoietic stem or progenitor cells.
[50] The method of [49], wherein a plurality of hematopoietic stem or progenitor cells express the cell surface markers CD34 + and CD38-.
[51] The method of [45], wherein a plurality of cord blood stem cells express cell surface markers CD34 + and CD38-.
[52] The method of [45], wherein a plurality of cord blood stem cells express cell surface markers CD34 + and CD38 +.
[53] Embryonic-like stem cells have the following cell surface markers: CD10 +, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT-4 + and ABC-p + [45] The method of [45] which shows at least 1 of them.
[54] The method of [53], wherein the embryonic-like stem cells are OCT-4 + and ABC-p +.
[55] The method of [53], wherein the embryonic stem cells are SSEA3- and SSEA4-.
[56] The method of [45], wherein the plurality of cord blood stem cells and the plurality of embryonic-like stem cells are mixed before or at the time of administration to a patient in need thereof.
[57] The method of [45], wherein a plurality of cord blood stem cells and a plurality of embryonic-like stem cells are physically mixed.
[58] The method of [45], wherein a plurality of cord blood stem cells and / or a plurality of embryonic-like stem cells are treated with a growth factor.
[59] The method of [58], wherein a plurality of cord blood stem cells and / or a plurality of embryonic-like stem cells are treated with a growth factor to induce differentiation into a specific cell type.
[60] The method of [58], wherein a plurality of cord blood stem cells and / or a plurality of embryonic-like stem cells are treated with a growth factor to prevent or inhibit differentiation into a specific cell type.
[61] The method of [45], wherein the patient has a disease, disorder or symptom containing an inflammatory component.
[62] The method of [45], wherein the patient has a vascular disease, disorder or symptom.
[63] The method of [62], wherein the disease, disorder, or symptom is atherosclerosis.
[64] The method of [45], wherein the patient has a neurological disease, disorder or symptom.
[65] The method of [64], wherein the disease, disorder or symptom is selected from the group consisting of amyotrophic lateral sclerosis and multiple sclerosis.
[66] The method of [45], wherein the patient has an autoimmune disorder.
[67] The method of [66], wherein the autoimmune disorder is selected from the group consisting of diabetes and amyotrophic lateral sclerosis.
[68] The method of [45], wherein the patient has symptoms caused by trauma or injury, or symptoms associated with trauma or injury.
[69] The method of [68], wherein the trauma or damage is trauma or damage to the central nervous system.
[70] The method of [68], wherein the trauma or damage is trauma or damage to the peripheral nervous system.
[71] A method for treating myelodysplasia, comprising administering cord blood cells or stem cells isolated therefrom and embryonic-like stem cells to a patient in need thereof.
[72] The method of [71], wherein administration of umbilical cord blood cells (or stem cells isolated therefrom) and embryonic stem cells are administered simultaneously.
[73] The method of [71], wherein cord blood cells (or stem cells isolated therefrom) and embryonic-like stem cells are combined prior to administration.
[74] A method of transplanting hematopoietic progenitor cells for treatment or prevention of a disease, comprising administering cord blood cells (or stem cells isolated therefrom) and embryonic stem cells to a patient in need thereof. Comprising a method.
[75] The method of [74], wherein the administration of cord blood cells or stem cells isolated therefrom and the administration of embryonic-like stem cells are the same.
[76] The method of [74], wherein cord blood cells or stem cells isolated therefrom and embryonic-like stem cells are combined prior to administration.
[77] A composition comprising cord blood-derived stem or progenitor cells supplemented with a plurality of embryoid-like stem cells.
[78] A method of treating a patient in need thereof comprising administering to a patient at least 5 × 10 ⁹ nucleated cells comprising embryonic-like stem cells.
[79] The method of [78], wherein the embryonic stem cells are contained in a container.
[80] The method of [78], wherein the embryonic stem cells are suitable for administration to a patient.
[81] The method of [78], wherein the embryonic stem cells are suitable for bone marrow transplantation.
[82] The method of [78], wherein the embryonic stem cells are suitable for human administration.
[83] Embryonic-like stem cells have the following cell surface markers: CD10 +, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT-4 + and ABC-p + [78] The method of [78] which shows at least 1 of them.
[84] The method of [83], wherein the embryonic-like stem cells are OCT-4 + and ABC-p +.
[85] The method of [83], wherein the embryonic-like stem cells are SSEA3- and SSEA4-.
[86] The method of [78], wherein the embryonic-like stem cells are treated with a growth factor.
[87] Growth factor is cytokine, lymphokine, interferon, colony stimulating factor (CSF), interferon, chemokine, interleukin, human hematopoietic growth factor, hematopoietic growth factor ligand, stem cell factor, thrombopoietin (Tpo), granulocyte colony stimulating factor ( G-CSF), leukemia inhibitory factor, basic fibroblast growth factor, placenta-derived growth factor, or epidermal growth factor [86].
[88] The method of [87], wherein the embryonic-like stem cells are treated with a growth factor to induce differentiation into a plurality of cell types.
[89] The method of [87], wherein a plurality of embryonic-like stem cells are treated with a growth factor to prevent or suppress differentiation into a specific cell type.
[90] The method of [78], wherein the treatment comprises administering at least 10 × 10 ⁹ total nucleated cells.
[91] The method of [78], wherein the treatment comprises administering at least 20 × 10 ⁹ total nucleated cells.
[92] The method of [27], wherein at least 30% of the cells in the composition are embryonic-like stem cells.
[93] The method of [27], wherein at least 60% of the cells in the composition are embryonic-like stem cells.
[94] The method of [78], wherein the patient has a disease, disorder or symptom comprising an inflammatory component.
[95] The method of [78], wherein the patient has a vascular disease, disorder or symptom.
[96] The method of [95], wherein the disease, disorder, or symptom is atherosclerosis.
[97] The method of [78], wherein the patient has a neurological disease, disorder, or symptom.
[98] The method of [97], wherein the disease, disorder or symptom is selected from the group consisting of amyotrophic lateral sclerosis and multiple sclerosis.
[99] The method of [78], wherein the patient has an immune-related disorder.
[100] The method of [99], wherein the autoimmune disorder is selected from the group consisting of allergy, diabetes and amyotrophic lateral sclerosis.
[101] The method of [78], wherein the patient has symptoms caused by trauma or injury, or symptoms associated with trauma or injury.
[102] The method of [101], wherein the trauma or damage is trauma or damage to the central nervous system.
[103] The method of [101], wherein the trauma or damage is trauma or damage to the peripheral nervous system.
[104] The method of [78], wherein the at least 5 × 10 ⁹ nucleated cells are collected and pooled from a plurality of donors.
[105] The method of [78], wherein HLA typing is not performed on any of the cells in the at least 5 × 10 ⁹ nucleated cells prior to the administration.
[106] The method of [78], wherein the at least 5 × 10 ⁹ nucleated cells are preconditioned for 18 hours to 21 days prior to the administration.
[107] The method of [78], wherein the at least 5 × 10 ⁹ nucleated cells are preconditioned for 48 hours to 10 days prior to the administration.
[108] The method of [78], wherein the at least 5 × 10 ⁹ nucleated cells are preconditioned for 3 to 5 days prior to the administration.

  The present invention relates to cord blood compositions or stem or progenitor cells derived therefrom supplemented with or in contact with embryonic-like stem cells derived from postpartum placenta. In the present invention, embryonic-like stem cells that are the subject of other patent applications are used as a composition or as a mixture with other stem or progenitor cell populations. According to the invention, embryonic-like stem cells are contacted with other stem or progenitor cell populations, including but not limited to cord blood, fetal and neonatal hematopoietic stem and progenitor cells, human stem cells and bone marrow-derived progenitor cells. Can be made. Embryo-like stem cells, and mixed populations of embryo-like stem cells and stem or progenitor cells can be used in a variety of applications, for example, for therapeutic use, diagnostic and research purposes for transplantation and disease treatment and prevention. The

  According to the present invention, a population of stem cells is mixed with a population of embryonic-like stem cells for the purpose of recruiting, enhancing, and increasing the concentration of multipotent and pluripotent stem cells in the stem cell population. For example, in one embodiment, umbilical cord blood or stem or progenitor cells derived therefrom are augmented with or contacted with embryonic-like stem cells of the invention prior to administration to a patient. Embryo-like stem cells can be administered simultaneously or sequentially with cord blood or cells derived therefrom, but it is preferred to contact each cell prior to administration.

  The embryonic-like stem cells of the present invention have the following cell surface markers: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 and ABC-p, and the following cell surface markers: CD34, CD38 Can be characterized by the absence of CD45, SSEA3 and SSEA4. In preferred embodiments, such embryonic stem cells are characterized by the presence of cell surface markers OCT-4 and APC-p. Embryonic stem cells derived from placenta have the characteristics of embryonic stem cells, but are not derived from embryos. In other words, the present invention encompasses a mixture of umbilical cord blood and embryonic-like stem cells that are OCT-4 + and / or ABC-p + isolated from the placenta. Such embryonic stem cells are as pluripotent (eg pluripotent) as human embryonic stem cells.

  According to the present invention, a population of stem cells is mixed with embryonic-like stem cells that are pluripotent or multipotent. Such embryonic-like stem cells can be isolated from the perfused placenta at various time points, such as CD34 + / CD38 +, CD34 + / CD38-, and CD34- / CD38-hematopoietic cells. In one embodiment, such cells are used to replenish a population of hematopoietic stem cells, such as those found in umbilical cord blood, according to the methods of the invention.

  The present invention also provides a composition in which a mixture of stem cells and embryonic stem cells is contained in a single bag or container. In a preferred embodiment, the composition is a pharmaceutically acceptable unit dose composition. In another embodiment, the present invention provides a composition in which a population of stem cells and a population of embryonic-like stem cells are contained in two separate bags or containers. In certain embodiments, such “two bag” kits are mixed before administration, particularly immediately before or at the time of administration, to a patient in need thereof. In other embodiments, the contents of each bag are administered to the patient separately, in which case mixing of the two cell populations occurs in the body. In other embodiments, the container is sealed, airtight, and sterile.

  The present invention relates to a population of stem cells mixed with embryonic stem cells. According to the present invention, stem cells that can be mixed with embryonic-like stem cells include, but are not limited to, cord blood, fetal or neonatal hematopoietic stem and progenitor cells, human stem cells and bone marrow-derived progenitor cells. . In a preferred embodiment of the invention, the embryonic-like stem cells of the invention are mixed with umbilical cord blood.

  The invention further provides a method for treating a patient in need thereof by administering a population of stem cells supplemented with embryonic-like stem cells. In one embodiment, supplementation of cord blood cell populations with embryonic-like stem cells is accomplished by mixing the stem cells with embryonic-like stem cells prior to administering the combined or “spiked” cell population to the patient. . In another embodiment, supplementation of embryonic-like stem cells to a population of stem cells occurs upon administration of the supplementary population to a patient, eg, by administering umbilical cord blood cells and embryonic-like stem cells simultaneously. In another embodiment, embryonic-like stem cells are supplemented to a population of stem cells after administration of umbilical cord blood cells to the patient, eg, embryonic-like stem cells are administered before or after administration, separately from administration of stem cells.

  According to the present invention, a population of stem cells (eg, umbilical cord blood) supplemented with placenta-derived embryonic-like stem cells has a variety of uses, including prophylactic, therapeutic and diagnostic uses. The replacement stem cell population can be used for transplantation and / or treatment or prevention of disease. In one embodiment of the invention, the supplemental stem cell population is used for tissue or organ repair or regeneration, thereby replacing or repairing the affected tissue, organ or portion thereof. In another embodiment, the supplemented stem cell population can be used as a diagnostic agent to screen for genetic disorders or for a predisposition to a particular disease or disorder.

  In another embodiment, the present invention isolates other embryonic-like stem cells and / or pluripotent or pluripotent stem cells from the extract or perfusate of phlebotomized placenta and uses them according to the methods of the invention A method for replenishing a population of cord blood cells is provided.

  The invention also provides a pharmaceutical composition comprising a population of stem cells (eg, cord blood cells) supplemented with one or more populations of embryonic-like stem cells of the invention.

  The present invention provides an isolated homogeneous population of human placental stem cells capable of differentiating into any cell type. In another embodiment, the population of human placental stem cells is capable of differentiating into one cell type. In yet another embodiment, the population of human placental stem cells has the ability to differentiate into several different cell types. Such cells can be used to replenish a population of stem cells (eg, cord blood) according to the methods of the invention.

  The present invention further includes one or more cells comprising high concentrations (or relatively large populations) of uniform hematopoietic stem cells, including but not limited to CD34 + / CD38- cells, CD34- / CD38- cells and CD133 + cells. A pharmaceutical composition containing a population of hematopoietic stem cells supplemented by the population is included. One or more of these cell populations may be used or mixed with hematopoietic stem cells obtained from umbilical cord blood or other sources (ie CD34 + / CD38 + hematopoietic stem cells) for transplantation and other uses. it can.

  The invention also provides a method of mixing a population of stem cells, progenitor cells or cord blood cells (including cord blood cells stored in a bank or cryopreserved) with a population of embryonic-like stem cells. In one embodiment, the two populations are physically mixed. In another form of this embodiment, the two populations are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another form of this embodiment, stem cells and / or embryonic-like stem cells are treated with growth factors such as cytokines and / or interleukins to induce cell differentiation and then physically mixed. In certain embodiments, the mixed population is treated with growth factors to induce differentiation into various cell types. In another embodiment, the mixed population is treated with a growth factor to induce differentiation into a specific cell type. In another embodiment, the mixed population is treated with a growth factor to prevent or inhibit differentiation into a particular cell type. In certain embodiments, by directing or inducing differentiation to a particular cell type by adjusting the culture conditions, for example by treating the mixed cell population with a special cocktail of cytokines or interleukins.

  In another embodiment, the present invention provides a method of treating a patient in need thereof comprising administering a plurality of cord blood cells and a plurality of embryonic-like stem cells.

  In another embodiment, the present invention provides a method for treating myelodysplasia comprising administering cord blood cells (or stem cells isolated therefrom) and embryonic-like stem cells to a patient in need thereof. provide.

  The present invention also relates to a novel use of human placental stem cells (embryonic stem cells). Also encompassed herein are methods of treating or preventing disease using compositions containing embryonic-like stem cells and other stem or progenitor cells or sources thereof. Also included are methods of administering such compositions. Finally, it should be noted that the compositions of the present invention may include stem or progenitor cell populations from multiple donors. The present invention includes the use of compositions with non-matching HLA in patients as well as compositions with matching HLA. Blood type matching with the patient is preferred, but blood type matching is not required when using compositions that contain both embryonic-like stem cells and stem or progenitor cells.

3.1. Definitions As used herein, the term “bioreactor” refers to growing cells, producing or expressing biological material, or growing cells, tissues, organs, viruses, proteins, polynucleotides and microorganisms. Or means an ex vivo system for culturing.

  As used herein, “cord blood” and “umbilical cord blood” are interchangeable.

  As used herein, the term “embryonic stem cell” refers to a cell that is derived from the inner cell mass of a blastocyst (eg, a human embryo of 4-5 days of life) and is multipotent.

  As used herein, the term “embryonic stem cell” refers to a cell that is not derived from the inner cell mass of a blastocyst. As used herein, the term “embryonic-like stem cell” may also be referred to as “placental stem cell”, with human placental stem cells derived from the perfused placenta postpartum being preferred. Embryonic-like stem cells are preferably pluripotent. However, stem cells obtained from the placenta also include embryonic-like stem cells, pluripotent cells, and committed progenitor cells. According to the methods of the present invention, embryonic-like stem cells derived from placenta can be collected from the isolated placenta once the placenta has been phlebotomized and perfused for a time sufficient to remove residual cells.

  As used herein, the terms “contaminated” or “contained” when used with respect to the placenta means the removal and / or drainage of substantially all cord blood from the placenta. According to the present invention, placental phlebotomy can be achieved by, for example, but not limited to, drainage, gravity-induced outflow, massage, squeezing, pumping, and the like. In a preferred embodiment, placental phlebotomy can be achieved by perfusing, rinsing, or flushing the placenta with a fluid (with or without an agent such as an anticoagulant) that aids in placental phlebotomy. .

  As used herein, the term “mixing” is generated by combining or blending into a lump or mixture, bringing together the components so that the components are more or less homogeneous, and combining the ingredients. Or forming, adding, augmenting, forming by replenishment or mixing, or adding a component or element to another component or element, or vice versa.

  As used herein, the term “perfuse” or “perfusion” refers to the act of injecting or passing fluid over or through an organ or tissue, preferably any residual cells from the organ or tissue, such as non-adherent cells. By passing liquid through the organ or tissue with sufficient force or pressure to be removed. As used herein, the term “perfusate” refers to fluid that is collected after passage through an organ or tissue. In a preferred embodiment, the perfusate contains one or more anticoagulants.

  As used herein, the term “exogenous cell” refers to “foreign” cells or heterologous cells (ie, “non-self” cells derived from a source other than a placental donor), or self from an organ or tissue other than the placenta. By derived cell (ie, “self” cell from a placental donor) is meant.

  As used herein, the term “organoid” refers to the assembly of one or more cell types into an actual structure or superficial appearance as an organ or gland of a mammalian body (particularly the human body). Means an agglomerate.

  As used herein, the term “pluripotent cell” refers to a cell that has the ability to grow into any subset of about 260 cell types of the mammalian body. Unlike pluripotent cells, pluripotent cells are not capable of forming all cell types.

  As used herein, the term “pluripotent cell” refers to a cell that has full differentiation potential, ie, the ability to grow into any of the approximately 260 cell types of the mammalian body. Pluripotent cells are capable of self-renewal and can remain dormant or quiescent within the tissue. Unlike totipotent cells (eg, fertilized diploid eggs), embryonic stem cells usually cannot form new blastocysts.

  As used herein, the term “progenitor cell” means a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue.

  As used herein, the term “stem cell” refers to a master cell that can regenerate indefinitely to form specialized cells of tissues and organs. Stem cells are pluripotent or pluripotent cells in development. The stem cells divide to produce two daughter stem cells, or one daughter cell and one progenitor (“transit”) cell, which later proliferates with mature fully formed tissue cells. Become. As used herein, “stem cell” includes “progenitor cells” unless otherwise specified.

  As used herein, the term “totipotent cell” refers to a cell that can form a complete embryo (eg, a blastocyst).

4). Detailed Description of the Invention The present invention is, in part, pluripotent so that embryonic-like stem cells obtained from phlebotomy, perfusion and / or cultured placenta can be easily differentiated into any desired cell type. It is based on the unexpected discovery that it is a sex stem cell. These embryonic-like stem cells are used to recruit, augment or augment a population of stem cells including, but not limited to, cord blood, fetal and neonatal hematopoietic stem and progenitor cells, human stem cells and bone marrow derived progenitor cells Can do. According to the present invention, the stem cell population and the embryonic-like stem cell population are mixed in order to recruit, enhance, and increase the concentration of multipotent and pluripotent stem cells in the stem cell population. According to the present invention, a population of stem cells mixed with a population of embryonic-like stem cells can be used for a variety of applications, for example, for transplantation, therapeutic use for disease treatment and prevention, diagnostic and research use. Is done.

  The present invention also provides a composition in which a mixture of stem cells and embryonic stem cells is contained in a single bag or container. In another embodiment, the present invention provides a composition in which a population of stem cells and a population of embryonic-like stem cells are contained in two separate bags or containers. In certain embodiments, such “two-bag” compositions are mixed prior to administration, particularly immediately before, or upon administration to a patient in need thereof. In other embodiments, the contents of each bag are administered to the patient separately, in which case mixing of the two cell populations occurs in the body.

  The invention also provides a method of mixing a population of stem cells or progenitor cells or cord blood (including cord blood stored in a bank or cryopreserved) with a population of embryonic-like stem cells. In one embodiment, the two populations are physically mixed. In another form of this embodiment, the two populations are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another form of this embodiment, stem cells and / or embryonic-like stem cells are treated with growth factors such as cytokines and / or interleukins to induce cell differentiation and then physically mixed.

  The invention also provides a population of committed cells destined for terminal differentiation, such as nerve cells, muscle cells, hematopoietic cells, vascular cells, adipocytes, chondrocytes, bone cells, hepatocytes, pancreatic cells, or Methods are provided for mixing a population of cells destined to differentiate into heart cells with a population of embryonic-like stem cells. In one embodiment, the two populations are physically mixed. In another form of this embodiment, the two populations are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another form of this embodiment, cells and / or embryonic stem cells destined for terminal differentiation are treated with growth factors such as cytokines and / or interleukins to induce cell differentiation and then physically mixed To do.

  According to the method of the present invention, embryonic-like stem cells are extracted from the drained placenta by a perfusion technique using either or both of the umbilical artery and umbilical vein. The placenta is preferably drained by phlebotomy and collection of residual blood (eg, residual umbilical cord blood). The drained placenta is then treated to establish an ex vivo natural bioreactor environment in which embryonic stem cells resident in the parenchyma and extravascular space are mobilized. Embryonic-like stem cells migrate into the drained empty microcirculation, where they are collected according to the method of the invention, preferably by flushing into a collection vessel by perfusion.

  As mentioned above, several different pluripotent or pluripotent stem cells can be isolated from the perfused placenta at various times during perfusion, such as CD34 + / CD38 +, CD34 + / CD38-, and CD34- / CD38-hematopoietic cells. In one embodiment, such cells are used to replenish a population of stem cells (eg, cord blood cells) according to the methods of the invention.

  The present invention further provides an isolated homogeneous population of human placental stem cells capable of differentiating into any cell type. In another embodiment, the population of human placental stem cells is capable of differentiating into one cell type. In yet another embodiment, the population of human placental stem cells has the ability to differentiate into a number of different cell types. Such cells can be used to replenish a population of stem cells (eg, cord blood cells) according to the methods of the invention.

  The invention also provides a method of mixing a population of stem cells with a population of embryonic stem cells. In one embodiment, the two populations are physically mixed. In another form of this embodiment, the two populations are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another form of this embodiment, stem cells and / or embryonic-like stem cells are treated with growth factors such as cytokines and / or interleukins to induce cell differentiation and then physically mixed. In certain embodiments, the mixed population is treated with growth factors to induce differentiation into various cell types. In another embodiment, the mixed population is treated with a growth factor to induce differentiation into a specific cell type. In another embodiment, the mixed population is treated with a growth factor to prevent or inhibit differentiation into a particular cell type. In certain embodiments, differentiation to a particular cell type can be directed or induced by adjusting the culture conditions, eg, by treating the mixed cell population with a special cocktail of cytokines or interleukins.

  The present invention provides a pharmaceutical composition comprising a population of stem cells (eg, cord blood cells) supplemented with one or more populations of embryonic-like stem cells of the present invention.

  The invention further replenishes with one or more cell populations containing high concentrations (or relatively large populations) of homogeneous hematopoietic stem cells, including but not limited to CD34 + / CD38- cells and CD34- / CD38- cells. Pharmaceutical compositions containing a population of treated stem cells (eg, cord blood cells). One or more of these cell populations can be used with or mixed with hematopoietic cells derived from umbilical cord blood (ie, CD34 + / CD38 + hematopoietic cells) for transplantation and other uses.

  According to the present invention, populations of stem cells (eg, umbilical cord blood) supplemented with placenta-derived embryonic-like stem cells have a variety of uses, including therapeutic and diagnostic uses. The replacement stem cell population can be used for transplantation and / or treatment or prevention of disease. In one embodiment of the invention, the supplemental stem cell population is used for tissue or organ repair or regeneration, thereby replacing or repairing the affected tissue, organ or portion thereof. In another embodiment, the supplemented stem cell population can be used as a diagnostic agent to screen for genetic disorders or for a predisposition to a particular disease or disorder.

  The invention further provides a method for treating a patient in need thereof by administering a population of stem cells supplemented with embryonic-like stem cells. In one embodiment, supplementing embryonic-like stem cells to a population of cord blood cells is accomplished by mixing the stem cells and embryonic-like stem cells prior to administering the supplemented population to a patient. In another embodiment, supplementation of embryonic-like stem cells to a population of stem cells occurs upon administration of the supplementary population to a patient, eg, by administering umbilical cord blood cells and embryonic-like stem cells simultaneously. In another embodiment, embryonic-like stem cells are supplemented to a population of stem cells after administration of umbilical cord blood cells to the patient, eg, embryonic-like stem cells are administered before or after administration, separately from administration of stem cells.

4.1. Placenta isolation and culture methods
4.1.1. Pretreatment of the placenta According to the method of the present invention, the human placenta immediately after delivery and after delivery is collected, and in a particular embodiment, cord blood in the placenta is collected. In certain embodiments, the placenta is subjected to normal umbilical cord blood collection methods. Such cord blood collection is commercially available from, for example, LifeBank Inc., Cedar Knolls, N, J., ViaCord, Cord Blood Registry and Cryocell. Umbilical cord blood can be drained immediately after delivery of the placenta.

  In another embodiment, the placenta is pretreated according to the method disclosed in co-pending patent application 10 / 076,180 filed February 13, 2002, which is hereby incorporated by reference in its entirety.

4.1.2. Placental hemoptysis and removal of residual cells
Activate if the placenta is properly treated after delivery, as disclosed in PCT Publication WO 02/064755 published August 22, 2002, which is incorporated herein by reference in its entirety. Containing resting cells capable of For example, after delivery from the uterus, the placenta is bled as soon as possible to prevent or minimize apoptosis. Subsequently, the placenta is perfused as soon as possible after phlebotomy to remove all blood, residual cells, proteins, factors, and other substances present in this organ. Material debris can also be removed from the placenta. Perfusion usually continues for at least 2 hours but less than 24 hours with the appropriate perfusate. Thus, the placenta can be used immediately as an abundant source of embryonic-like stem cells, and these embryonic-like stem cells are used in research, including drug discovery, disease treatment and prevention (especially transplant surgery or therapy), And used to form cells, tissues and organs destined to terminal differentiation.

  Moreover, surprisingly, human placental stem cells obtained from phlebotomy, perfusion and / or cultured placenta are pluripotent stem cells that can easily differentiate into any desired cell type.

  According to the present invention, stem or progenitor cells, including but not limited to embryonic-like stem cells, are phlebotomized, ie, a placenta that has completely drained cord blood remaining after labor and / or normal cord blood recovery Can be recovered from. According to the method of the present invention, the placental phlebotomy method and residual cell removal method are known in the art, such as PCT publication WO 02/064755 published Aug. 22, 2002 (in its entirety by reference). Are incorporated herein by reference).

4.1.3. Culture of placenta Following placental hemoptysis and perfusion for a sufficient amount of time, it is observed that embryonic-like stem cells migrate to the microcirculatory system of the hemoptysis and perfused placenta. According to the method of the invention, the embryonic-like stem cells are collected, preferably by perfusion and washing into a collection vessel. In another embodiment, the placenta is cultured according to the method described in PCT Publication WO 02/064755 published Aug. 22, 2002, which is incorporated herein by reference in its entirety, and proliferated cells are monitored. , Screen and / or characterize.

4.2. Collection of cells from the placenta After phlebotomy and perfusion of the placenta, embryonic-like stem cells migrate to the empty microcirculatory system where the placenta has been drained, and in this microcirculation system, preferably the effluent perfusate is placed in a collection vessel. By collecting, embryonic-like stem cells are collected according to the present invention.

  In preferred embodiments, culture in the placenta using techniques known to those skilled in the art, such as density gradient centrifugation, magnetic cell separation, flow cytometry, or other cell separation and sorting methods well known in the art. The cells are isolated from the effluent perfusate and sorted.

  In certain embodiments, embryonic-like stem cells are collected from the placenta and according to the methods described in PCT Publication WO 02/064755 published Aug. 22, 2002, which is hereby incorporated by reference in its entirety. save.

4.3. Embryo-like stem cells Embryo-like stem cells obtained according to the method of the present invention are pluripotent cells, that is, have full differentiation ability, self-renewal ability, and are dormant or quiescent in the tissue. Includes cells that can remain. Stem cells that can be obtained from the placenta include embryonic-like stem cells, pluripotent cells, progenitor cells destined for terminal differentiation, and fibroblast-like cells.

  The first blood collected from the placenta is called umbilical cord blood, which mainly contains CD34 + and CD38 + hematopoietic progenitor cells. High concentrations of CD34 + and CD38− hematopoietic progenitor cells can be isolated from the placenta along with high concentrations of CD34− and CD38 + hematopoietic progenitor cells within the first 24 hours after delivery. After about 24 hours of perfusion, along with the cells, high concentrations of CD34− and CD38− cells can be isolated from the placenta. The isolated and perfused placenta of the present invention provides a large source of stem cells enriched for CD34 + and CD38− stem cells and CD34− and CD38 + stem cells. Isolated placenta perfused for more than 24 hours provides a large source of stem cells enriched for CD34- and CD38- stem cells.

  In a preferred embodiment, the embryonic-like stem cells obtained by the method of the present invention are viable, quiescent, multipotent stem cells that are present in full term human placenta and have good birth and It can be recovered after placental delivery, resulting in the recovery of as many as 1 billion nucleated cells resulting in 50-100 million pluripotent and multipotent stem cells.

  The human placental stem cells provided by the placenta are surprisingly embryonic-like cells, for example, the presence of the following cell surface markers in these cells: SSEA3-, SSEA4-, OCT-4 + And ABC-p +. Preferably, the embryonic-like stem cells of the invention are characterized by the presence of OCT-4 + and ABC-p + cell surface markers. Thus, the present invention encompasses stem cells that are not isolated or obtained from embryonic sources but can be identified by the following markers: SSEA3-, SSAE4-, OCT-4 + and ABC-p +. In one embodiment, the human placental stem cells do not express MHC class 2 antigen.

  Stem cells isolated from the placenta are uniform and sterile. Furthermore, the stem cells are readily obtained in a form suitable for human administration, i.e. they are of pharmaceutical grade.

  Preferred embryonic-like stem cells obtained by the methods of the invention can be identified by the presence of OCT-4 + and ABC-p + cell surface markers. Furthermore, the present invention also includes embryonic stem cells having the following markers: CD10 +, CD38-, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT -4+ and ABC-p +. Such cell surface markers are routinely examined by methods known to those skilled in the art, for example, flow cytometry, followed by washing and staining with anti-cell surface marker antibodies. For example, to test for the presence of CD34 or CD38, cells are washed in PBS and then double stained with anti-CD34-phycoerythrin and anti-CD38-fluorescein isothiocyanate (Becton Dickinson, Mountain View, CA).

In another embodiment, cells cultured in a placental bioreactor are identified and characterized by a colony forming unit assay. This assay is generally known in the art and includes Mesen Cult ^™ medium (stem cell Technologies, Inc. Vancouver, British Columbia).

  Embryonic stem cells obtained by the method of the present invention are induced to differentiate along specific cell lineages such as adipogenesis, cartilage formation, bone formation, hematopoiesis, myogenesis, angiogenesis, neurogenesis, and liver development. be able to. In certain embodiments, embryonic-like stem cells obtained according to the methods of the invention are induced to differentiate for use in transplantation and ex vivo treatment protocols. In certain embodiments, the embryonic-like stem cells obtained by the methods of the present invention are induced to differentiate into a specific cell type and then engineered to provide a therapeutic gene product. In certain embodiments, embryonic-like stem cells obtained by the methods of the invention are incubated in vitro with a compound that differentiates them, and then the differentiated cells are transplanted directly into the subject. Accordingly, the present invention encompasses a method for differentiating human placental stem cells using standard media. Furthermore, the present invention also includes hematopoietic cells, neuronal cells, fibroblasts, strand cells, mesenchymal cells and hepatocytes.

  After collection from the placenta, the embryonic-like stem cells are derived from the same placenta as the embryonic-like stem cells or from another human or non-human source (irradiated fibers) for the purpose of obtaining a continuous long-term culture thereof. The cells may be further cultured using methods known to those skilled in the art by culturing on blast cells, etc.) or in a conditioned medium obtained from a culture of such feeder cells. Embryonic-like stem cells may also be expanded in the placenta prior to collection from the placenta bioreactor or in vitro after collection from the placenta. In certain embodiments, the embryonic stem cells to be expanded are exposed to or cultured in the presence of an agent that inhibits cell differentiation. Such agents are known to those of skill in the art and include, but are not limited to, human Delta-1 and human Serrate-1 polypeptides (Sakano, entitled “Differentiation Inhibiting Polypeptides” published January 8, 2002). US Pat. No. 6,337,387), leukemia inhibitory factor (LIF) and stem cell factor. Methods for expanding cell populations are also known to those skilled in the art (see, for example, “Ex vivo replication of stem cells, optimization of hematopoietic progenitor cell cultures, human stromal cell metabolism, GM-CSF, published December 4, 2001). US Pat. No. 6,326,198 to Emerson et al. Entitled “Selective Expansion of Target Cell Population”, entitled “Methods and Compositions for Increased Secretion and / or IL-6 Secretion”; Kraus et al., US Pat. No. 6,338,942).

  Embryonic-like stem cells can be assessed for viability, proliferative capacity, and longevity using standard techniques known to those skilled in the art, such as trypan blue exclusion assay, fluorescein diacetate Uptake assays, propidium iodide uptake assays (to assess viability); and thymidine uptake assays, MTT cell proliferation assays (to assess proliferation). Lifespan can be determined by methods known to those skilled in the art, such as determining the maximum number of population doublings in an expanded culture.

  In certain embodiments, one or more administrations inhibit the differentiation of cells collected from the placenta in modulating the differentiation of stem or progenitor cells cultured in phlebotomy, perfusion and / or cultured placenta. A drug or pharmaceutical composition is used that contains a dose effective to effect sufficient to modulate, and / or modulate.

Agents capable of inducing stem or progenitor cell differentiation are known to those of skill in the art and include, but are not limited to: Ca ²⁺ , EGF, αFGF, βFGF, PDGF, keratinocyte proliferation Factor (KGF), TGF-β, cytokine (eg, IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormone (eg, androgen, estrogen, insulin, prolactin, triiodothyronine, Hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix elements (eg, collagen, laminin, heparan sulfate, Matrigel ™) or combinations thereof.

  Agents that inhibit cell differentiation are also known to those of skill in the art and include, but are not limited to: human Delta-1 and human Serrate-1 polypeptides (issued January 8, 2002) Sakano et al., US Pat. No. 6,337,387, entitled “Differentiation Inhibiting Polypeptides”), leukemia inhibitory factor (LIF) and stem cell factor.

  By introducing an agent used to modulate differentiation into a placental bioreactor, differentiation of cells cultured in the placenta can be induced. Alternatively, the agent can be used to modulate in vitro differentiation after collecting or removing cells from the placenta.

  To determine whether a stem cell has differentiated into a particular cell type, methods known to those skilled in the art, such as flow cytometry or immunocytochemistry (eg, staining cells with tissue specific or cell marker specific antibodies), etc. Measure the changes in morphology and cell surface markers using techniques, examine cell morphology using optical or confocal microscopy, or use known techniques such as PCR and gene expression profiling Changes in expression are measured.

4.4. Supplementing Stem Cell Populations with Embryonic Stem Cells The present invention relates to a population of stem cells mixed with embryonic stem cells. According to the present invention, stem cells that can be mixed with embryonic-like stem cells include, but are not limited to, cord blood, fetal and neonatal hematopoietic stem and progenitor cells, human stem cells and bone marrow-derived progenitor cells. In a preferred embodiment of the invention, the embryonic-like stem cells of the invention are mixed with umbilical cord blood.

  The present invention provides an isolated homogeneous population of human placental stem cells (embryonic-like stem cells) capable of differentiating into any cell type. Such cells can be used to replenish a population of stem cells (eg, cord blood cells) according to the methods of the invention.

  The invention also provides a population of cord blood cells supplemented with a population of embryonic-like stem cells derived from the placenta (ie, mixed, combined or enhanced).

  The supplemented population includes a population of cells that are totipotent or pluripotent stem cells (eg, cells exhibiting a CD34 + / CD38 +, CD34 + / CD38-, or CD34- / CD38- phenotype), Has a wide variety of uses.

  According to the present invention, the population of supplemented stem cells of the present invention comprises embryonic-like stem cells and other stem or progenitor cells of 100,000,000: 1, 50,000,000: 1, 20,000,000: 1, 10,000,000: 1, 5,000,000: 1, 2,000,000: 1, 1,000,000: 1, 500,000: 1, 200,000: 1, 100,000: 1, 50,000: 1, 20,000: 1, 10,000: 1, 5,000: 1, 2,000: 1, 1,000: 1, 500: 1, 200: 1, 100: 1, 50: 1, 20: 1, 10: 1, 5: 1, 2: 1, 1: 1, 1: 2, 1: 5, 1:10, 1: 100, 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, Contain at a ratio of 1: 20,000,000, 1: 50,000,000 or 1: 100,000,000 (compare the total number of nucleated cells in each population).

  In another embodiment, the present invention converts stem cells (eg, umbilical cord blood) with a composition of the invention (eg, a population of pure embryonic placental stem cells or a population of cells enriched for embryonic placental stem cells). Methods for replenishing, mixing, combining or enhancing are provided. In certain embodiments, an aliquot (or population) of placental embryonic-like stem cells is added to an aliquot of cord blood and then delivered to a patient in need thereof.

  The invention also provides a method of supplementing a population of cord blood cells with a population of embryonic-like stem cells. In one embodiment, the two populations are physically mixed. In another aspect of this embodiment, the two populations are physically mixed and then treated with growth factors (eg, cytokines and / or interleukins) to induce cell differentiation. In another aspect of this embodiment, cord blood cells and / or embryonic-like stem cells are treated with growth factors (eg, cytokines and / or interleukins) and then physically mixed.

  The present invention also provides a method of treating a patient in need thereof by administering a population of cord blood cells supplemented with embryonic-like stem cells. In one embodiment, supplementation of a cord blood cell population with embryonic stem cells is performed by mixing the cord blood cells and embryonic stem cells prior to administering the supplemented population to a patient. In another embodiment, supplementation of a cord blood cell population with embryonic-like stem cells occurs when the supplemented population is administered to a patient, eg, by administering cord blood cells and embryonic-like stem cells simultaneously. In another embodiment, replenishment of the cord blood cell population with embryonic-like stem cells is performed after administering the cord blood cells to the patient, e.g., administering the embryonic-like stem cells before or after administration, separately from administration of the cord blood cells. To do.

  In one embodiment, the present invention provides a method of replenishing cord blood cells with embryonic-like stem cells, in which case the mixture is contained in a single bag. In another embodiment, the present invention provides a method of supplementing cord blood cells with embryonic-like stem cells, wherein the cord blood cells and embryonic-like stem cells are each contained in separate bags. Such “two-bag” compositions can be mixed before or when administered to a patient.

  In another embodiment, an aliquot (or population) of placental embryonic-like stem cells is conditioned before being added to and mixed with an aliquot of cord blood. For example, in one aspect of this embodiment, the population of embryonic placental stem cells can be added to a particular cell line (eg, hematopoietic cells, as described above in Section 4.3) before being added to and mixed with an aliquot of cord blood. Neural cells, adipogenic cells, chondrogenic cells, osteogenic cells, liver-derived cells, pancreatic cells, or myogenic cell lines). For example, embryonic placental stem cell populations can be expressed by cytokines (eg, IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormones (eg, androgens, estrogens, insulin, prolactin, avians). Exposure to iodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix elements (eg, collagen, laminin, heparan sulfate, Matrigel ™) or combinations thereof.

  In another aspect of this embodiment, agents that inhibit differentiation (eg, human Delta-1 and Serrate-1 polypeptides, before adding a placental embryonic-like stem cell population to an aliquot of cord blood before mixing) Or a combination of these).

  In another embodiment, an aliquot (or population) of unconditioned embryonic-like placental stem cells and an aliquot of cord blood are mixed and the mixed population of cells is conditioned prior to delivery to a patient in need thereof. In certain embodiments, a mixed population of embryonic placental stem cells and cord blood cells is conditioned with an agent that induces or inhibits cell differentiation as described above.

  In certain embodiments, a population of embryonic-like stem cells of the invention is added to or mixed with a cord blood cell population prior to administration to a patient in need thereof. In another specific embodiment, the population of embryonic-like stem cells of the invention is added or mixed during or simultaneously with administration of the cord blood cell population to a patient in need thereof. In another specific embodiment, the population of embryonic-like stem cells of the invention and the population of cord blood cells are administered sequentially to a patient in need thereof. In one embodiment, a population of embryonic-like stem cells is administered first, followed by administration of a cord blood cell population. In another embodiment, the cord blood cell population is administered first, followed by the placental embryonic-like stem cell population.

  A population of cord blood cells spiked with embryonic-like stem cells can be cultured under various conditions, induced to proliferate and / or induced to differentiate, including, for example, nutrients, vitamins Including, but not limited to, treating spiked populations by introducing growth factors, or combinations thereof, into the medium. Serum and other growth factors may be added to the medium. Growth factors are usually proteins, including but not limited to cytokines, lymphokines, interferons, colony stimulating factor (CSF), chemokines, and interleukins. Other growth factors that can be used include ligand, stem cell factor, thrombopoietin (Tpo), granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor, basic fibroblast growth factor, placenta-derived growth factor, and epithelial growth Recombinant human hematopoietic growth factors, including factors, are included. In one embodiment, the supplemented population is treated with growth factors that induce differentiation into various cell types. In another embodiment, the spiked population is treated with a growth factor that induces differentiation into a particular cell type. In another embodiment, the supplemented population is treated with a growth factor that prevents or inhibits differentiation to a particular cell type.

  In certain embodiments of the invention, a method of replenishing a population of cord blood cells comprises (a) inducing differentiation of embryonic-like stem cells, (b) mixing the embryonic-like stem cells with a population of cord blood cells, and (c ) Including administering the mixture to a patient in need thereof.

  In another embodiment of the invention, the method of replenishing a population of umbilical cord blood cells comprises: (a) mixing embryonic-like stem cells with a population of umbilical cord blood cells; and (b) differentiation of a mixture of umbilical cord blood cells and spiked populations of embryonic-like stem cells. And (c) administering the mixture to a patient in need thereof.

  In another embodiment of the invention, the method of replenishing a population of cord blood cells comprises: (a) administering a mixture of cord blood cells supplemented with embryonic-like stem cells to a patient in need thereof; and (b) the Inducing differentiation of the mixture and (c) administering the mixture to a patient in need thereof.

In certain embodiments, stem or progenitor cells are induced to differentiate into specific cell types by exposure to growth factors by methods well known in the art. In certain embodiments, the growth factor is GM-CSF, IL-4, Flt3L, CD40L, IFN-α, TNF-α, IFN-γ, IL-2, IL-6, retinoic acid, basic fibroblast proliferation Factor, TGF-β-1, TGF-β-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensin II (AII), AII AT ₂ type 2 A receptor agonist, or an analog or fragment thereof.

  In one embodiment, by exposing stem or progenitor cells to β-mercaptoethanol or DMSO / butylated hydroxyanisole according to methods well known in the art, for example, as described in Section 5.4.1, It induces to differentiate into nerve cells.

  In another embodiment, stem or progenitor cells are differentiated into adipocytes by exposure to dexamethasone, indomethacin, insulin and IBMX according to methods well known in the art, for example, by the method described in Section 5.4.2. Guide you to.

  In another embodiment, stem or progenitor cells are differentiated into chondrocytes by exposure to TGF-β-3 according to methods well known in the art, for example, by the method described in Section 5.4.3. To guide.

  In another embodiment, stem or progenitor cells are exposed to dexamethasone, ascorbic acid-2-phosphate and β-glycerophosphate according to methods well known in the art, for example by the method described in Section 5.4.4. To induce differentiation into bone cells.

  In another embodiment, hepatocytes are obtained by exposing stem or progenitor cells to IL-6 +/- IL-15 according to methods well known in the art, for example, as described in Section 5.4.5. Induces to differentiate into.

  In another embodiment, stem or progenitor cells are converted to basic fibroblast growth factor and transforming growth factor β-1 according to methods well known in the art, for example, as described in Section 5.4.6. Induction is induced to differentiate into pancreatic cells.

  In another embodiment, stem or progenitor cells are obtained according to methods well known in the art, for example, retinoic acid, basic fibroblast growth factor, TGF-β-1 by the method described in Section 5.4.7. And induced to differentiate into pancreatic cells by exposure to epidermal growth factor, exposure to cardiotropin-1, or exposure to human myocardium extract.

  In another embodiment, embryonic-like stem cells are stimulated to produce biologically active molecules such as immunoglobulins, hormones, enzymes.

  In another embodiment, for example, erythropoietin, cytokine, lymphokine, interferon, colony stimulating factor (CSF), chemokine, interleukin; ligand, stem cell factor, thrombopoietin (Tpo), interleukin, and granulocyte colony stimulating factor (G- Embryonic-like stem cells are stimulated to proliferate by administration of recombinant human hematopoietic growth factors such as CSF) or other growth factors.

  In another embodiment, either before or after collection from the placenta, eg, using a viral vector such as an adenovirus or retroviral vector, or a machine such as liposome or chemical mediated DNA uptake Engineered embryonic-like stem cells by genetic means.

  Methods known to those skilled in the art, such as transfection, transformation, transduction, electroporation, infection, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, liposomes, LPOFECTIN ™, lysosomal fusion, synthetic cationic lipids Using a gene gun or a DNA vector carrier, a vector containing the transgene is introduced into the cell of interest, whereby the transgene is introduced into daughter cells (eg, daughter embryo-like cells produced by the division of embryonic-like stem cells). Stem cells or progenitor cells). For various techniques for transformation or transfection of mammalian cells, see Keown et al., 1990, Methods Enzymol. 185: 527-37; Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor laboratory Press, See NY.

  Preferably, the transgene may be introduced by any technique as long as it does not damage the cell nuclear membrane or other existing cells or genetic structures. In certain embodiments, the transgene is introduced into the nuclear genetic material by microinjection. Microinjection of cells and cell structures is generally known and practiced in the art.

  For stable transfection of cultured mammalian cells (eg, embryonic-like stem cells), only a small percentage of cells can incorporate foreign DNA into their genome. The efficiency of integration depends on the vector and transfection technique used. In order to identify and select integrants, a gene that encodes a selectable marker (eg, resistance to antibiotics) is generally introduced into the host embryonic stem cells along with the gene sequence of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated a selectable marker gene survive while other cells die). Such a method is particularly useful in methods for performing homologous recombination on mammalian cells (eg, embryonic-like stem cells) prior to introducing or transplanting the recombinant cells into a subject or patient.

  A number of selection systems can be used to select for transformed host embryo-like cells. In particular, the vector may contain a detectable or selectable marker. Other selection methods include, but are not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and selection of alternative markers such as the adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) gene, which are used in tk-, hgprt- or aprt-cells, respectively. be able to. Antimetabolite resistance can also be used as a selection criterion for the following genes: dhfr conferring resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt conferring resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); aminoglycoside G-418 Neo (Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); and hygro (Santerre et al., 1984, Gene 30: 147) conferring resistance to hygromycin.

  Preferably, the transgene is integrated into the genome of the target cell by random integration. In another embodiment, the transgene is directed in a directed manner, such as directed homologous recombination (ie, “knock-in” or “knock-out” of the gene of interest in the genome of the cell of interest) (Chappel, US Pat. No. 5,272,071). And PCT publication number WO 91/06667 published May 16, 1991; US Pat. No. 5,464,764 issued November 7, 1995 to Capecchi et al., US Pat. No. 5,627,059 issued May 6, 1997; No .; US Pat. No. 5,487,992 issued by Capecchi et al. Issued January 30, 1996).

  Methods for producing cells containing a target gene modification by homologous recombination are known to those skilled in the art. Such a construct includes at least a portion of the gene of interest having the desired genetic modification and also includes a region homologous to the target locus (ie, an endogenous copy of the target gene in the host's genome). In contrast to those used for homologous recombination, DNA constructs for random integration may not contain regions of homology to mediate recombination. By including the marker in the targeting construct or random construct, positive and negative selections for the insertion of the transgene can be performed.

  In order to produce homologous recombinant cells, for example homologous recombinant embryonic stem cells cultured in placenta, endogenous placental cells or exogenous cells, the homologous recombination vector (the gene of interest is 5 ′ and Between the target gene carried by the vector and the endogenous gene in the genome of the target cell. Allow recombination to occur. The additional flanking nucleic acid sequence is of sufficient length to successfully achieve homologous recombination with an endogenous gene in the genome of the target cell. Typically, several kilobases of flanking DNA (both at the 5 'and 3' ends) are included in the vector. Methods for producing homologous recombinant animals from homologous recombinant vectors and recombinant stem cells are known to those skilled in the art (see, eg, Thomas and Capecchi, 1987, Cell 51: 503; Bradley, 1991, Curr. Opin. Bio / Technol. 2: 823-29; and PCT publication numbers WO 90/11354, WO 91/01140 and WO 93/04169).

  In one embodiment, the genome of exogenous cells cultured in the placenta according to the method of the invention is targeted for gene targeting by homologous recombination or random integration.

  In certain embodiments, the method of Bonadio et al. (US Pat. No. 5,942,496 entitled “Methods and Compositions for Multiple Gene Introduction into Bone Cells” issued August 24, 1999; published August 24, 1995) PCT WO95 / 22611) entitled “Methods and Compositions for Stimulating Bone Cells” of No. 1, which introduces nucleic acid into target cells such as stem cells, progenitor cells or exogenous cells cultured in placenta, eg, osteoprogenitor cells .

4.5 Uses of Embryonic- like Stem Cells and Supplemented Stem Cell Populations Embryonic-like stem cells can be obtained from perfused placenta according to the method described in pending US Patent Application No. 01 / 076,180, filed February 13, 2002 .

  Placental stem cells (embryonic-like stem cells) can be induced to differentiate into specific cell types, either ex vivo or in vivo. For example, pluripotent embryonic stem cells can be injected into damaged organs to achieve organogenesis and injury repair in vivo. Such injuries may be due to conditions and disorders including, but not limited to: myocardial infarction, stroke disorder, multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia, inflammation, addiction Cognitive decline with age, radiation damage, cerebral palsy, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Lee's disease, AIDS dementia, memory loss, amyotrophic lateral sclerosis, ischemic kidney disease, brain or spinal cord trauma Cardiopulmonary bypass, glaucoma, retinal ischemia, or retinal trauma.

  In certain embodiments, embryonic-like stem cells isolated from placenta are used alone or in combination with stem or progenitor cell populations (ie, cell compositions of the invention) in autologous or heterologous enzyme replacement therapy, Diseases or conditions can be treated including, but not limited to, lysosomal storage diseases such as Tay-Sachs, Neiman-Pick, Fabry, Gaucher, Hunter, and Fuller's syndrome, as well as other gangliosides Cis, mucopolysaccharidosis, and glycogenosis.

  In other embodiments, embryonic-like stem cells, alone or in combination with stem or progenitor cell populations, are used as autologous or heterologous transgene carriers in gene therapy, resulting in inborn errors of metabolism, adrenal brain leukodystrophy, cystic Fibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia, Pearson's syndrome, Pomp disease, phenylketonuria (PKU), porphyria, maple syrup urine disease, homocystinuria, mucopolysaccharidosis, chronic granulomatous The disease, tyrosinemia and Tay-Sachs disease can be ameliorated, or cancer, tumors or other medical conditions can be treated.

  In other embodiments, the cellular composition can be used in autologous or heterogeneous tissue regeneration / replacement therapy or protocols including, but not limited to, treatment of corneal epithelial defects, cartilage repair, facial skin abrasions Methods, mucous membranes, tympanic membrane, intestinal lining, nerve structures (eg, retina, auditory neurons in the basement membrane, olfactory neurons in the olfactory epithelium), skin burns and wound repair, or other damaged or affected organ or tissue reconstruction .

The large number of embryonic-like stem cells and / or progenitor cells obtained using the methods of the present invention can reduce the need for providing a large amount of bone marrow in certain embodiments. For bone marrow transplantation, 1 × 10 ⁸ to 2 × 10 ⁸ bone marrow mononuclear cells must be infused per kilogram of patient weight (ie, about 70 ml of bone marrow for a 70 kg donor). Obtaining 70 ml requires intensive bone marrow donation, and a significant amount of blood is lost during the donation process. In certain embodiments, cells from a small amount of bone marrow donation (eg, 7-10 ml) are grown in a placental bioreactor and then injected into the recipient.

  In addition, a small number of stem and progenitor cells usually circulate in the bloodstream. In another embodiment, such exogenous stem cells or exogenous progenitor cells are collected by pheresis. Ferresis is a method in which blood is collected, one or more components are selectively removed, and the remaining blood is reinfused into the donor. Exogenous cells recovered by pheresis can be expanded by growth in a placental bioreactor, thus completely eliminating the need for bone marrow donation.

  Blood cells regenerate while chemotherapy treatment is stopped, but during that time the cancer grows and proliferates, and naturally becomes more resistant to chemotherapeutic drugs due to natural selection. Therefore, the longer the treatment period by chemotherapy and the shorter the treatment stop period, the higher the chance of successfully killing the cancer. In order to shorten the period of cessation of chemotherapy treatment, embryonic-like stem cells or progenitor cells collected according to the method of the present invention may be introduced into a patient alone or in combination with other populations of stem cells or progenitor cells. it can. Such treatment shortens the time that the patient presents with a low blood cell count, and thus chemotherapy treatment can be resumed earlier than before.

  Embryonic-like stem cells, progenitor cells, foreign cells, or genetically engineered cells obtained from the placenta according to the method of the present invention can be used to produce tissues or organs in vivo, either alone or in combination with other populations of stem or progenitor cells. Can be used. The method of the present invention differentiates cells by inoculating a matrix with cells obtained from placenta (eg, embryonic-like stem cells, progenitor cells, exogenous stem or progenitor cells) and culturing under appropriate conditions. , Including grouping into a matrix. The tissues and organs obtained by the method of the present invention can be used for various purposes such as research and therapeutic purposes.

  The population of embryonic-like stem cells and supplemented stem cells of the present invention can be used in a wide variety of prophylactic or therapeutic protocols, in which the desired cell population, such as a stem cell or progenitor cell population, is engrafted. Transplantation or injection augments, repairs or replaces body tissues or organs. The population of embryonic-like stem cells and supplemented stem cells of the present invention can be used to replace or augment existing tissue, introduce new or modified tissue, or join biological tissues or structures together can do. Also, the population of embryonic-like stem cells and supplemented stem cells of the present invention can be used in place of embryonic stem cells in the treatment protocols described herein that generally use embryonic stem cells.

  In preferred embodiments of the invention, embryonic-like stem cells and supplemented stem cell populations can be used as autologous and allogeneic (including matched and mismatched HLA-type hematopoietic grafts). When using embryonic-like stem cells as allogeneic hematopoietic transplants, US Pat. No. 5,800,539 issued September 1, 1998; and US Pat. No. 5,806,529 issued September 15, 1998 (both for reference) The host may have to be treated to suppress immune rejection of the donor cells, as described in US Pat.

  For example, the embryonic stem cells and supplemented stem cell populations of the present invention are used in therapeutic transplant protocols, for example, liver, pancreas, kidney, lung, nervous system, muscular system, bone, bone marrow, thymus, spleen, mucous tissue, gonadal , Or hair stem cells or progenitor cells can be augmented or replaced.

  Embryo-like stem cells and supplemented stem cell populations are treatments where progenitor cells are commonly used instead of certain classes of progenitor cells (eg, chondrocytes, hepatocytes, hematopoietic cells, pancreatic parenchymal cells, neuroblasts, muscle progenitor cells, etc.) Or it can be used in research protocols.

  The embryonic-like stem cells and supplemented stem cell populations of the present invention can be used for cartilage, tendon or ligament augmentation, repair or replacement. For example, in certain embodiments, a prosthesis (eg, a hip prosthesis) is coated with a replacement cartilage tissue construct grown from embryonic-like stem cells of the invention. In another embodiment, a joint (eg, knee) is reconstituted with cartilage tissue grown from embryonic-like stem cells. Cartilage tissue constructs can be used for major reconstructive surgery of various joints (for protocols see, for example, Resnick, D., and Niwayama, G., 1988, Diagnosis of Bone and Joint Disorders, 2nd edition , See WB Saunders Co.).

  The embryonic-like stem cells and supplemented stem cell populations of the present invention can be used to repair tissue and organ damage due to trauma, metabolic disorders or diseases. In such embodiments, the patient is allowed to regenerate or restore tissue or organs damaged as a result of disease by administering embryonic-like stem cells alone or in combination with other stem or progenitor cell populations, e.g., chemical The immune system after receiving therapy or radiation can be strengthened, or heart tissue after myocardial infarction can be repaired.

  The embryonic-like stem cells and supplemented stem cell populations of the present invention can be used to augment or replace bone marrow cells in bone marrow transplantation. Human autologous and allogeneic bone marrow transplantation is currently used as a treatment for diseases such as leukemia, lymphoma, and other life-threatening disorders. However, the disadvantage of these methods is that a large amount of donor bone marrow must be collected to ensure sufficient cells for transplantation.

  The embryonic-like stem cells and supplemented stem cell populations of the present invention can provide stem cells and progenitor cells, thereby reducing the need for large amounts of bone marrow donation. Also, according to the method of the present invention, after obtaining a small amount of bone marrow, the number of stem cells and progenitor cells is increased by culturing and growing in the placenta before being injected or transplanted into the recipient.

  In certain embodiments, embryonic-like stem cells and supplemented stem cell populations can be used for autologous or heterologous enzyme replacement therapy to treat specific diseases or conditions including, but not limited to, lysosomal accumulation Diseases such as Tay-Sachs, Niemann-Pick, Fabry, Gaucher, Hunter, and Fuller's syndrome, as well as other gangliosidosis, mucopolysaccharidosis, and glycogenosis.

  In other embodiments, the cells are used in gene therapy as an autologous or heterologous transgene carrier, resulting in adrenal white matter dystrophy, cystic fibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia, Pearson syndrome, Improve inborn errors of metabolism such as Pomp disease, phenylketonuria (PKU), and Tay-Sachs disease, porphyria, maple syrup urine disease, homocystinuria, mucopolysaccharidosis, chronic granulomatous disease and tyrosineemia Or treating cancer, tumors or other pathological neoplastic conditions.

  In other embodiments, it can be used in autologous or xenogeneic tissue regeneration or replacement therapy or protocols including, but not limited to, treatment of corneal epithelial defects, cartilage repair, facial skin abrasion, mucosa, Tympanic membrane, intestinal lining, neural structures (eg, retina, auditory neurons in the basement membrane, olfactory neurons in the olfactory epithelium), skin burns and wound repair, scalp (hair) transplantation, or other damaged or affected organs or tissues Reconstruction.

The large number of embryonic-like stem cells and / or progenitor cells obtained using the methods of the present invention can reduce the need for providing a large amount of bone marrow in certain embodiments. For bone marrow transplantation, 1 × 10 ⁸ to 2 × 10 ⁸ bone marrow mononuclear cells must be infused per kilogram of patient weight (ie, about 70 ml of bone marrow for a 70 kg donor). To obtain 70 ml requires intensive delivery and a significant amount of blood is lost during the delivery process. In certain embodiments, cells from a small amount of bone marrow donation (eg, 7-10 ml) are grown in a placental bioreactor and then injected into the recipient.

  In another embodiment, the embryonic-like stem cells of the invention and the supplemented stem cell population are used as a supplemental treatment in addition to chemotherapy. Most chemotherapeutic drugs used to target and destroy cancer cells work by killing all proliferating cells (ie, cells that are dividing). Because bone marrow is one of the most actively proliferating tissues in the body, hematopoietic stem cells are often damaged or destroyed by chemotherapeutic drugs, resulting in decreased or stopped blood cell production. End up. Chemotherapy must be stopped after a certain period of time and resumed after the patient's hematopoietic system has replenished the blood cell supply. Previously quiescent stem cells proliferate, increase white blood cell counts to an acceptable level, and it may take more than a month before chemotherapy can be resumed. Will be destroyed again).

  While the chemotherapy treatment is stopped, blood cells regenerate, but during that time the cancer grows, and natural selection increases resistance to chemotherapeutic drugs. Therefore, the longer the period of treatment with chemotherapy and the shorter the period of stopping treatment, the higher the chance of reliably killing the cancer. To shorten the interval of chemotherapy treatment, embryonic-like stem cells or progenitor cells collected according to the method of the present invention can be introduced into a patient. Such treatment reduces the time it takes for the patient to present a low blood cell count, so that chemotherapy treatment can be resumed earlier.

  In another embodiment, human placental stem cells can be used to treat or prevent genetic diseases such as chronic granulomatous disease.

4.6. Pharmaceutical Compositions The present invention includes pharmaceutical compositions containing the embryonic-like stem cells of the present invention and a supplemented stem cell population. The present invention relates to a cell line with one or more placenta-derived human pluripotent and pluripotent progenitor cells that are administered one or more times before or after transplantation of conditioned or not conditioned human progenitor stem cells, such as Inhibits and modulates differentiation into mesoderm, adipocytes, chondrocytes, bone cells, muscle cells, vascular cells, neurons, endothelial cells, hepatocytes, kidney cells, pancreatic cells and / or hematopoietic lineage cells; And / or pharmaceutical compositions comprising effective single doses and / or multiple doses that can have a sufficient effect to modulate.

  According to this embodiment, the embryonic-like stem cells and supplemented stem cell populations of the present invention can be formulated as injection solutions (eg, PCT WO96 / 39101, which is incorporated herein by reference in its entirety). In another embodiment, the cells and tissues of the invention are polymerized as described in US Pat. Nos. 5,709,854; 5,516,532; 5,654,381, each incorporated herein by reference in its entirety. Possible or crosslinkable hydrogels can be used to formulate. Embryonic stem cells may be administered as obtained from the placenta, or spiked into umbilical cord blood and administered as a mixed cell composition. Alternatively, it may be placed in a physiologically acceptable buffer or liquid for administration to an individual.

  The invention also encompasses pharmaceutical compositions comprising high concentrations (or larger populations) of homogeneous embryoid-like stem cells, in which case one or more of these cell populations are used for transplantation and other uses. Use with other stem or progenitor cells or as a mixture with other stem or progenitor cells. Other stem or progenitor cells include, but are not limited to, adipogenesis, chondrogenesis, osteogenesis, hematopoiesis, myogenic, vasogenic, neurogenic, and liver derived Stem cells; mesenchymal stem cells, stromal cells, endothelial cells, hepatocytes, keratinocytes, and certain cell types, tissues or organs (nerve cells, myelin, muscle, blood, bone marrow, skin, heart, connective tissue, lung, kidney, liver) And stem or progenitor cells, including but not limited to pancreas, such as islet cells.

  In one embodiment, the present invention provides a pharmaceutical composition comprising a high concentration (or larger population) of homogeneous hematopoietic stem cells, including but not limited to CD34 + / CD38− cells and CD34− / CD38− cells. I will provide a. One or more of these cell populations can be used with or as a mixture with other stem cells for use in transplantation and other applications. In certain embodiments, the pharmaceutical composition comprises placental embryonic-like stem cells of the invention and cord blood hematopoietic cells (ie, CD34 + / CD38 + hematopoietic cells).

  One or more of these cell populations can be used with umbilical cord hematopoietic cells (ie, CD34 + / CD38 + hematopoietic cells) or as a mixture with the hematopoietic cells for use in transplantation and other applications.

  In one embodiment, the present invention provides a heterogeneous population of nucleated cells comprising placental embryonic-like stem cells. In certain embodiments, heterogeneous populations of nucleated cells (CD34 + cells and placental embryonic stem cells that are not pure populations) are preferred.

  In another embodiment, the invention provides a mixed population of cells (eg, cord blood cells and placental embryonic stem cells). The mixed cell population may or may not be frozen. Such a mixed population can be stored and / or used in one container (eg, one bag or syringe).

  In another embodiment, the present invention provides two or more distinct populations of different cell types (eg, cord blood cells and placental embryonic stem cells). Each separate population is in a separate container containing one type of cell or cell population, such as one bag (eg, a blood storage bag available from Baxter, Becton-Dickinson, Medcep, National Hospital Products or Terumo) or syringe. Can be stored and / or used. In a particular aspect of this embodiment, the present invention provides separate containers for different cell types that are mixed prior to administration. Such cells may or may not be frozen.

  In certain embodiments, cord blood cells are contained in one bag and placental embryonic stem cells are contained in another bag.

  In another embodiment, the present invention provides placental embryonic-like stem cells that are “conditioned” prior to freezing.

In another embodiment, a population of cells, including but not limited to placental embryonic stem cells, is conditioned by removing erythrocytes and / or granulocytes according to standard methods, resulting in placental embryoid-like A population of nucleated cells enriched in stem cells remains. Such enriched populations of embryonic placental stem cells may be used without freezing or may be frozen for later use. When freezing a population of cells, standard cryopreservation agents (eg, DMSO, glycerol, Epilife ™ cell freezing medium (Cascade Biologics)) are added to the enriched cell population prior to freezing.
In another embodiment, a population of cells, including but not limited to embryonic placental stem cells, may be conditioned by freezing and thawing and then removing red blood cells and / or granulocytes.

According to the present invention, embryonic-like stem cell populations can be conditioned using agents that induce cell differentiation. In certain embodiments, an agent that induces differentiation is added to a population of cells in a container, such agents include: Ca ²⁺ , EGF, αFGF, βFGF, PDGF, keratinocytes Growth factors (KGF), TGF-β, cytokines (eg, IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormones (eg, androgen, estrogen, insulin, prolactin, triiodothyronine) , Hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix elements (eg, collagen, laminin, heparan sulfate, Matrigel ™) or combinations thereof.

  In another embodiment, an agent that inhibits cell differentiation can be added to a population of embryonic-like stem cells. In certain embodiments, an agent that inhibits differentiation is added to a population of cells in a container, such agents include the following: human Delta-1 or human Serrate-1 polypeptide (2002) Sakano et al., US Pat. No. 6,337,387 entitled “Differentiation Inhibiting Polypeptides” issued on Jan. 8), leukemia inhibitory factor (LIF), stem cell factor and combinations thereof

  In certain embodiments, one or more populations of embryonic-like stem cells are delivered to a patient in need thereof. In certain embodiments, two or more populations of fresh (never frozen) cells are delivered from one container or one delivery system.

  In another embodiment, two or more populations of frozen and thawed cells are delivered from one container or one delivery system.

  In another embodiment, each of two or more populations of fresh (never frozen) cells are transferred to and delivered from one container or one delivery system. In another embodiment, each of two or more populations of frozen and thawed cells is transferred to and delivered from one container or one delivery system. In another aspect of these embodiments, each population is delivered from a different intravenous bag (eg, available from Baxter, Becton-Dickinson, Medcep, National Hospital Products or Terumo). The contents of each container (eg, IV bag) can be delivered from a separate delivery system, or each container can be piggybacked and mixed together before mixing in one delivery. It may be delivered by the system. For example, two or more cell populations are fed into a common flow line (eg, tubes) and / or mixed therein, or they are fed into a common container (eg, a chamber or bag) and / or Or they can be mixed in.

  According to the present invention, two or more cell populations can be combined prior to, during or at the time of administration, and can be delivered simultaneously.

In one embodiment, at least 1.7 × 10 ⁷ nucleated cells per kg are delivered to a patient in need thereof. Preferably, at least 2.5 × 10 ⁷ nucleated cells per kg are delivered to a patient in need thereof.

  In one embodiment, the present invention provides a method of treating or preventing a disease or disorder in a subject, the method comprising subjecting a subject wishing to such treatment or prevention to a therapeutically effective amount of the present invention. Administering embryonic-like stem cells or supplemented cell populations.

  In another embodiment, the present invention provides a method of treating or preventing a disease or disorder in a subject, the method comprising subjecting a subject wishing to such treatment or prevention to a therapeutically effective amount of the present invention. Administering embryonic-like stem cells.

  The embryonic-like stem cells of the present invention are expected to exhibit an anti-inflammatory effect when administered to an individual experiencing inflammation. In a preferred embodiment, embryonic-like stem cells or supplemented cell populations of the invention can be used to treat diseases, conditions or disorders caused by or associated with inflammation. Inflammation may be present in any organ or tissue, such as muscle; nervous system including brain, spinal cord, peripheral nervous system; vascular tissue including heart tissue; pancreas; intestine or digestive tract Other organs; lung; kidney; liver; reproductive organ; endothelial tissue, or endoderm tissue.

  The embryonic stem cells or supplemented cell populations of the present invention can also be used to treat autoimmune disorders or immune system related disorders (including those involving inflammation). Accordingly, in certain embodiments, the present invention provides a method of treating an individual having an autoimmune disease or condition, wherein the method includes treating such an individual with a therapeutically effective amount of an embryonic-like stem cell of the present invention. Or administering a supplemented cell population. Such a disease or disorder can be, but is not limited to, diabetes, amyotrophic lateral sclerosis, myasthenia gravis, diabetic neuropathy or lupus. In related embodiments, the embryonic-like stem cells or supplemented cell populations of the present invention can be used to treat immune related disorders such as chronic or acute allergies.

  In certain embodiments, the disease or disorder includes, but is not limited to, a disease or disorder described herein, for example, aplastic anemia, spinal dysplasia, myocardial infarction, convulsive disorder, multiple Systemic sclerosis, stroke, hypotension, cardiac arrest, ischemia, inflammation, age-related cognitive decline, radiation damage, cerebral palsy, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Leigh disease AIDS dementia, memory loss, amyotrophic lateral sclerosis (ALS), ischemic kidney disease, brain or spinal cord trauma, cardiopulmonary bypass, glaucoma, retinal ischemia, retinal trauma, lysosomal storage disease, -Saxophone, Neiman-Pick, Fabry, Gaucher, Hunter, and Fuller syndrome, and other gangliosidosis, mucopolysaccharidosis, glycogenosis, inborn errors of metabolism, adrenal brain white matter Trophy, cystic fibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia, Pearson syndrome, Pamp's disease, phenylketonuria (PKU), porphyria, maple syrup urine disease, homocystinuria, mucopolysaccharidosis , Chronic granulomatous disease, tyrosinemia, Tay-Sachs disease, cancer, tumor, or other pathological or neoplastic symptoms.

  In other embodiments, the cells can be used to treat any type of injury caused by trauma (especially trauma with inflammation). Examples of such trauma-related symptoms include central nervous system (CNS) damage, such as damage to the brain, spinal cord or tissues surrounding the CNS, peripheral nervous system (PNS) damage, or other parts of the body There is damage. Such trauma can be caused by an accident or can be the normal or abnormal result of medical procedures such as surgery or angioplasty. Trauma can be associated with rupture or occlusion of blood vessels, such as in stroke and phlebitis. In certain embodiments, the cells can be used for autologous or xenogeneic tissue regeneration or replacement therapy, including but not limited to treatment of corneal epithelial defects, cartilage repair, facial skin abrasion, Mucosa, tympanic membrane, intestinal lining, neural structures (eg, retina, auditory neurons in the basement plate, olfactory neurons in the olfactory epithelium), skin burns and wound repair, or other reorganization of damaged or affected organs or tissues included.

  In certain embodiments, the disease or disorder is aplastic anemia, spinal dysplasia, leukemia, bone marrow disorder, or hematopoietic disease or disorder. In another embodiment, the subject is a human.

  In another embodiment, the present invention provides a method of treating an individual having a disease, disorder or condition associated with or caused by inflammation. In another embodiment, the present invention provides a method for treating an individual having a neurological disease, disorder or condition. In a more specific embodiment, said neurological disorder is ALS. In another more specific embodiment, the neurological disorder is Parkinson's disease. In another specific embodiment, the disease is a vascular or cardiovascular disease. In a more specific embodiment, the disease is atherosclerosis. In another specific embodiment, the disease is diabetes.

  In certain embodiments, the pharmaceutical composition of the invention contains an aliquot of cord blood to which embryonic placental stem cells have been added as described in Section 4.4.

  Many embryonic-like stem cells or supplemented cell populations, once administered, can settle into the host and form long-term “colony”. As a result, this results in a host that is essentially chimeric. Such chimerism is expected to increase the health and well-being of the host as chimeras are generally strong and resilient in other genetic backgrounds. Thus, embryonic-like stem cells can be administered to an individual not only to suffer from a particular disease, disorder or condition, but also to improve overall health and well-being.

The embryonic-like stem cells or supplemented cell populations of the invention can be treated with a compound that modulates the activity of TNF-α prior to administration to an individual. Such compounds are described in detail in co-pending US Provisional Application No. 60 / 372,348, dated April 12, 2002, the disclosure of which is incorporated herein in its entirety. Preferred compounds are referred to as IMiD and SelCid, and particularly preferred compounds are available under the trade names Actimid ^™ and Revimid ^™ .

  A particularly useful aspect of the embryonic-like stem cells of the present invention is that in certain embodiments, there is no need for HLA typing of those cells prior to administration. In other words, embryonic-like stem cells can be taken from one or more xenogeneic donors and transplanted into an individual in need of such cells, and the transplanted cells will remain in the host indefinitely. By eliminating the need for HLA typing, both the transplant operation itself and the search for transplant donors become much easier. However, embryonic-like stem cells or supplemental cell populations containing them can be matched to HLA (donor vs. recipient) prior to administration.

  The inventors have found that the efficacy in treating individuals with embryonic-like stem cells or supplemental cell populations is enhanced when preconditioning these cells. Preconditioning involves storing the cells in a gas permeable container for a period of time at about -5-23 ° C, 0-10 ° C, preferably 4-5 ° C. The period can be 18 hours to 21 days, 48 hours to 10 days, preferably 3 to 5 days. Prior to preconditioning, the cells may be cryopreserved and are preferably preconditioned immediately prior to administration.

  Accordingly, in one embodiment, the present invention provides a method of treating an individual comprising administering to the individual embryonic-like stem cells collected from at least one donor. As used herein, “donor” means an adult, child, infant, preferably placenta. In another preferred embodiment, the method comprises administering to the individual embryonic-like stem cells collected and pooled from multiple donors. In certain embodiments, the embryonic-like stem cells are stem cells taken from multiple donors. If collected from a large number of donors, the dosage unit (where “dose unit” is a collection from a single donor) is pooled prior to administration and administered sequentially or selectively. Can be. In another embodiment of the method, embryonic-like stem cells are mixed with umbilical cord blood or “spiked” into umbilical cord blood and the mixture is administered to an individual. In a more specific embodiment of the method, the ratio of embryonic-like stem cells to umbilical cord blood is at least 20:80, 30:70, 40:60, 50:50, 60:40, 70 based on the total number of nucleated cells. : 30 or 80:20.

4.7 Administration of Stem Cells: Dosage A particularly useful embodiment of the present invention is the administration of high doses of stem cells to an individual. Such numbers of cells are significantly more effective than their source material (eg, bone marrow or umbilical cord blood). As used herein, a “high dose” is 5, 10, 15 or 20 times or more of the number of total nucleated cells, including stem cells (especially embryonic-like stem cells) compared to the number administered, for example, in bone marrow transplantation. Indicates. Typically, for example, for bone marrow transplantation, a patient who receives an infusion of stem cells receives 1 unit of cells. Here, one unit is about 1 × 10 ⁹ nucleated cells (corresponding to 1 to 2 × 10 ⁸ stem cells). Thus, for high-dose treatment, patients will have 3 billion, 5 billion, 10 billion, 15 billion, 20 billion, 30 billion, 40 billion, 50 billion or more total nucleated cells, or 3, 5, 10 , 20, 30, 40 or 50 units of total nucleated cells (embryo-like stem cells alone or embryo-like stem cells spiked into another stem or progenitor cell population (eg embryo-like stem cells spiked into cord blood)) Will be administered. In one preferred embodiment, for example, an individual is administered 15 units of spiked umbilical cord blood, wherein the unit comprises about 750 million cord blood cells and 500 million embryonic-like stem cells. Thus, in one embodiment, the number of nucleated cells administered to an individual is at least 5 times the number of cells normally administered in bone marrow replacement. In another specific embodiment of the method, the number of nucleated cells administered to the individual is at least 10 times the number of cells normally administered in bone marrow replacement. In another particular embodiment of the method, the number of nucleated cells administered to the individual is at least 15 times the number of cells normally administered in bone marrow replacement. In another embodiment of the method, the total number of nucleated cells (including stem cells) administered to the individual is 1-100 × 10 ⁸ per kg body weight. In another embodiment, the number of total nucleated cells administered is at least 5 billion cells. In another embodiment, the total number of nucleated cells administered is at least 15 billion cells.

  In another embodiment of the method, embryonic-like stem cells and umbilical cord blood are mixed just prior to administration to the individual (ie, within 5 minutes). In another embodiment, the embryonic-like stem cells and umbilical cord blood are mixed at a time point of 5 minutes or more prior to administration to the individual. In another embodiment of the method, embryonic-like stem cells are cryopreserved and thawed prior to administration to an individual. In another embodiment, embryonic-like stem cells and umbilical cord blood are mixed at a time point no less than 24 hours prior to administration to the individual to form a replacement cell population. The replacement cell population is cryopreserved and thawed prior to administration. In another embodiment, the embryonic-like stem cell and / or replacement cell population can be administered more than once. In another embodiment, embryonic-like stem cells and / or replacement cell populations are preconditioned by storage for 18 hours to 21 days prior to administration. In a more specific embodiment, the cells are preconditioned for 48 hours to 10 days prior to administration. In a preferred specific embodiment, the cells are preconditioned for 3-5 days prior to transplantation. In a preferred embodiment of any of the methods, embryonic-like stem cells are not HLA typed prior to administration to an individual.

In another specific embodiment of the method, the embryonic-like stem cells are predominantly (ie,> 50%) CD34 ⁺ cells. In a more specific embodiment of the method, the embryonic stem cells are predominantly CD34 ⁺ 33 ⁺ stem cells.

  A therapeutic or prophylactic treatment of an individual with embryonic-like stem cells or a population of supplemented cells containing them can be considered effective if the disease, disorder or condition is measurablely improved in any way . Such an improvement is indicated by several indicators. Measurable indicators include, for example, one or more physiological conditions associated with a particular disease, disorder or symptom (blood pressure, heart rate, respiratory rate, counts of various blood cell types, specific proteins, carbohydrates, Detectable changes in lipid levels or cytokine levels, including but not limited to the expression of genetic markers associated with a disease, disorder or condition. When any one of such indicators responds to such treatment by changing to a value that is within normal values or more closely approximates normal values, the stem cell or supplemented cell population of the present invention Individual treatment would be considered effective. Normal values can be established by normal ranges known in the art for various indicators or by comparison with such control values. In the field of medical science, the therapeutic effect is often judged by the individual's impression and the subjective feeling of the individual's health. Therefore, the determination of improvement is a subjective indicator such as a feeling of subjective improvement of the individual after administration of the stem cell or supplemental cell population of the present invention, increased happiness, improved health status, increased energy level. Can also be done.

  The embryonic stem cells or replacement cell populations of the present invention can be administered to a patient by pharmaceutically or medically acceptable methods (eg, injection or infusion). Such cells or supplemented cell populations can include or be included in a pharmaceutically acceptable carrier (see Section 4.8). Embryo-like stem cells or supplemented cell populations can be transported, stored and transported in pharmaceutically or medically acceptable containers such as blood bags, transfer bags, plastic tubes or vials.

4.8 Kits The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Optionally, such a container may be accompanied by the following: a device for cell culture, one or more containers containing cell culture media or components of the media, a device for delivering a composition of the invention ( For example, a device for the intravenous injection of the composition of the present invention) and / or a notification form in the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product (this notification form is a human Reflects the approval by the government of manufacture, use or sale to administer. In certain embodiments, the kit comprises one or more containers filled with embryonic-like stem cells of the invention and one or more different containers filled with stem cells (eg, umbilical cord blood) as described above.

  In one embodiment, the kit comprises a mixture of stem cells (eg, cord blood cells) supplemented with embryonic-like stem cells contained in a single bag or container. In another embodiment, the kit comprises a population of umbilical cord blood cells and a population of embryoid-like stem cells housed in two separate bags or containers. In certain embodiments, the kit comprises a “two-bag” composition, wherein the bag containing cord blood cells and the bag containing embryonic-like stem cells are mixed before or when administered to a patient in need thereof. Is done. In other embodiments, the kit is housed in two separate bags or containers and is administered to a patient separately (eg, simultaneously or sequentially) a population of cord blood cells and a population of embryonic-like stem cells Comprising. In this case, the mixing of the two cell populations occurs in the body.

In another embodiment, the kit provides a population of cord blood cells and a population of embryoid-like stem cells that are physically mixed prior to administration. In another aspect of this embodiment, the kit is a growth factor such as GM-CSF, IL-4, Flt3L, CD40L, IFN-α, TNF-α, IFN-γ, IL-2, IL-6, retinoic acid , Basic fibroblast growth factor, TGF-β-1, TGF-β-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensin II (AII) ), An AII AT ₂ type 2 receptor agonist, or an analog or fragment thereof. In another aspect of this embodiment, the two populations are physically mixed and then treated with growth factors included in the kit to induce cell differentiation and then administered to the patient. In another aspect of this embodiment, cord blood cells and / or embryonic-like stem cells are treated with growth factors included in the kit to induce cell differentiation, and then they are physically mixed and then administered to the patient. To do.

  The following experimental examples are for explaining the present invention and are not intended to limit the present invention.

5. Examples
5.1. Example 1: Analysis of cell types recovered from drained placental perfusate This example describes the analysis of cell types recovered from placental outflow perfusate cultured by the method of the present invention.

  20 ml of phosphate buffer solution (PBS) was added to the perfusate and a 10 ml portion was collected and centrifuged at 3,000 rpm (rotations per minute) for 25 minutes. The effluent was divided into four tubes and placed in an ice bath. 2.5 ml of 1% fetal calf serum (FCS) solution in PBS was added and the tube was centrifuged (140 minutes × 10 g (acceleration by gravity)). After resuspending the pellet in 5 ml of 1% FCS solution, the two tubes were combined together. The total number of mononuclear cells was calculated by multiplying the sum of the total lymphocyte count and total monocyte count by the total cell suspension volume.

The table below shows the cell types obtained by perfusion of the cultured placenta according to the method described above.

The PP sample was after Ficoll.
The total number of PP cells after Ficoll was 5.3 × 10 ⁸ , and the number of CB before treatment was 6.3 × 10 ⁸ .
Lym% is the percentage of lymphocytes; MID% is the percentage of central leukocytes; GRA% is the percentage of granulocytes.

5.2. Example 2: Analysis of Cells Obtained by Placenta Perfusion and Incubation The following example describes the analysis of cells obtained by placenta perfusion and incubation according to the method of the present invention.

5.2.1. Materials and Methods Placental donors were recruited from pregnant mothers who registered with a private umbilical cord blood bank program and provided informed consent to allow the use of a placenta that had been bled after collection of umbilical cord blood for research purposes. . Donor data is confidential. These donors also accepted the use of data obtained from routine processing of cord blood samples for cryopreservation. Thereby, the composition of the collected umbilical cord blood and the collected outflow perfusate could be compared using the experimental method described below.

  In order to store the placenta at room temperature within 4-24 hours after umbilical cord blood is bled from the umbilical cord and transfer it to the laboratory, place the placenta in a sterile insulated container at room temperature according to the method described above and experiment within 4 hours after delivery. Moved to the room. When the placenta was examined and physical damage such as organ fragmentation or umbilical cord laceration was evident, the placenta was discarded. The placenta was placed in a sterile container and maintained at room temperature (23 ± 2 ° C.) for 2-20 hours or stored refrigerated (4 ° C.). Periodically, the placenta was immersed in 25 ± 3 ° C. sterile saline and washed to remove all surface blood and debris visible to the naked eye.

  The umbilical cord was cut at a point of about 5 cm from the insertion part into the placenta, and a TEFLON (registered) or polypropylene catheter was inserted into the umbilical cord blood vessel. The catheter is connected to a sterile fluid pathway that allows for bidirectional perfusion of the placenta and recovery of the effluent. With the above method, placental conditioning, perfusion and effluent recovery in all situations under controlled ambient atmospheric conditions, while at the same time intravascular pressure, flow rate, core and perfusate temperature, and recovered effluent volume Could be monitored in real time. The range of conditioning protocol was evaluated over 24 hours postpartum and the cellular composition of the effluent was analyzed by flow cytometry, light microscopy and colony forming unit assays.

5.2.2. Placenta conditioning The donor placenta was treated at room temperature within 12-24 hours of delivery. Prior to treatment, the membrane was removed and the maternal site was washed away to remove residual blood. A catheter consisting of a 20 gauge butterfly needle used for blood sample collection was inserted into the umbilical cord blood vessel.

Change conditions such as maintaining at 5-37 ° C, 5% CO ₂ , pH 7.2-7.5 to mimic and maintain a physiologically compatible environment for the growth and mobilization of residual embryonic stem cells While maintaining the donor placenta. The cannula was flushed with IMDM serum-free medium (GibcoBRL, NY) containing 2 U / ml heparin (Elkins-Sinn, NJ). Placental perfusion continued at a flow rate of 50 ml per minute until approximately 150 ml of perfusate was collected. This amount of perfusate was labeled “Initial fraction”. By continuing the perfusion at the same flow rate, a second fraction of approximately 150 ml was collected and labeled “late fraction”. During the course of this procedure, the placenta was gently massaged to facilitate the perfusion process and helped with the recovery of cellular material. The effluent was collected from the perfusion circuit by gravity drainage and aspiration through the arterial cannula.

Next, perfuse the placenta with heparinized (2U / ml) Dulbecco's Modified Eagle Medium (H.DMEM) at a flow rate of 15ml / min for 10 minutes, and then collect perfusate from the mother's site within 1 hour. And nucleated cells were counted. The perfusion and collection procedure was repeated once or twice until the number of nucleated cells recovered was below 100 cells / ml. After pooling the perfusate, platelets, debris and enucleated cell membranes were removed by light centrifugation. The nucleated cells were then isolated by Ficoll-Hypaque density gradient centrifugation, washed and resuspended in H.DMEM. To isolate adherent cells, an aliquot of 5-10 x 10 ⁶ cells was placed in each of several T-75 flasks and placed in commercial mesenchymal stem cell growth medium (MSCGM) obtained from Bio Whittaker. The cells were cultured and placed in a tissue culture incubator (37 ° C., 5% CO ₂ ). After 10-15 days, non-adherent cells were removed by washing with PBS, then MSCGM was used instead of PBS. The flasks were examined daily for the presence of various adherent cell types, particularly for confirmation of fibroblastoid cells and cluster growth.

5.2.3. Cell recovery and isolation Cells were recovered from the perfusate by centrifugation at 5,000 xg for 15 minutes at room temperature. This procedure helped to separate the cells from contaminating debris and platelets. The cell pellet was resuspended in IMDM serum-free medium containing 2 U / ml heparin and 2 mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction was isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) following the manufacturer's recommended procedure, and then the mononuclear cell fraction was resuspended. Cells were counted using a hemocytometer. Viability was assessed by trypan blue exclusion. Isolation of mesenchymal cells was achieved by “differential trypsinization” using 0.05% trypsin solution (Sigma, St. Louis, MO) containing 0.2% EDTA. The differential trypsinization was possible because fibroblast-like cells detached from the plastic surface within about 5 minutes while other adherent populations required 20-30 minutes incubation. . The detached fibroblast-like cells were recovered after trypsin treatment and trypsin neutralization using a trypsin neutralization solution (TNS, Bio Whittaker). Cells were washed in H.DMEM and resuspended in MSCGM.

  Flow cytometry was performed using a Becton-Dickinson FACS Calibur instrument. FITC- and PE-labeled monoclonal antibodies (mAbs) selected based on known markers of bone marrow-derived MSCs (Mesenchymal Stem Cells) are purchased from BD and Caltag Laboratory (South San Francisco, CA) and produce SH2, SH3 and SH4 antibodies Hybridomas were obtained and mAb reactivity in their culture supernatants was detected with FITC or PE labeled F (ab) '2 goat anti-mouse antibody. Cell lineage differentiation was performed using commercially available induction and maintenance media (Bio Whittaker) according to the manufacturer's instructions.

5.2.4. Isolation of placental embryonic-like stem cells Microscopic examination of adherent cells in culture flasks revealed different cell types. Spindle-shaped cells, round cells with large nuclei and many perinuclear vesicles, and star-shaped cells with several protrusions (one of which was attached to the flask) The cells were observed to adhere to the flask. No further characterization of these adherent cells was attempted, but similar cells were found in bone marrow, umbilical cord and peripheral blood cultures and were considered non-stem cell-like. The fibroblast-like cells that finally appeared as clusters were candidates for becoming MSCs (mesenchymal stem cells), were isolated by differential trypticin treatment, and subcultured in secondary flasks. After trypsin treatment, phase contrast microscopy of round cells showed that the cells were highly granulated, which was indistinguishable from bone marrow-derived MSCs made in the laboratory or purchased from Bio Whittaker. When subcultured, placenta-derived embryonic stem cells attached within a few hours, in contrast to their initial phase, have a unique fibroblast-like shape and the same growth pattern as the standard bone marrow-derived MSC. Formed. During subculture and replenishment, if loosely attached mononuclear cells are washed away, the culture remains homogeneous and any contaminants other than fibroblast-like cells are visible to the naked eye. There wasn't.

5.2.5. Results Expression of CD-34, CD-38, and other stem cell-related surface markers on mononuclear cells purified from early and late fractions was assessed by flow cytometry. Harvested and sorted cells were washed in PBS and then double stained with anti-CD-34 phycoerythrin and anti-CD38 fluorescein isothiocyanate (Becton Dickinson, Mountain View, CA).

  For example, cell isolation was achieved using magnetic cell separation such as Auto Macs (Miltenyi). Preferably, CD34 + cell isolation is performed first.

5.3. Example 3: Perfusion Medium The following example shows a preferred perfusate formulation for culturing isolated placenta.

  The composition is a perfusate that can be used at various temperatures to perfuse the placenta. It should also be noted that additional components such as antibiotics, anticoagulants and other growth factors can be used in the perfusate or medium.

5.4 Example 4: Induction of differentiation into specific cell types Umbilical cord blood cells and / or embryonic-like stem cells were induced to differentiate into specific cell types by exposure to growth factors. Growth factors used to induce differentiation include, but are not limited to, GM-CSF, IL-4, Flt3L, CD40L, IFN-α, TNF-α, IFN-γ, IL-2, IL- 6, retinoic acid, basic fibroblast growth factor, TGF-β-1, TGF-β-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin I (AI), Angiotensin II (AII), AII AT ₂ type 2 receptor agonist, or analogs or fragments thereof are included.

5.4.1 Induction of differentiation into nerve cells This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into nerve cells. The following protocol was employed to induce differentiation into neurons.

1. Placental stem cells are grown for 24 hours in preinduction medium consisting of DMEM / 20% FBS and 1 mM β-mercaptoethanol.
2. Remove preinduction medium and wash cells with PBS.
3. Add neuronal cell induction medium consisting of DMEM and 1-10 mM β-mercaptoethanol. Alternatively, the efficiency of neural cell differentiation may be increased using an induction medium consisting of DMEM / 2% DMSO / 200 μM butylated hydroxyanisole.
4. In certain embodiments, morphological and molecular changes may appear as early as 60 minutes after exposure to serum-free medium and β-mercaptoethanol (Woodbury et al., J. Neurosci. Res., 61: 364 -370). RT / PCR can be used, for example, to evaluate expression of nerve growth factor receptor and neurofilament heavy chain genes.

5.4.2 Induction of differentiation into adipocytes This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into adipocytes. The following protocol was employed to induce differentiation into adipocytes.

1. Placental stem cells are grown in DMEM or MSCGM (Bio Whittaker) supplemented with 15% umbilical cord serum.
2. Perform 3 cycles of induction / maintenance. In each cycle, placental stem cells were fed with Adipogenesis Induction Medium (Bio Whittaker), and the cells were cultured for 3 days (37 ° C, 5% CO ₂ ), and then Adipogenesis Maintenance Medium (Adipogenesis Maintenance Medium). Bio Whittaker) for 1 to 3 days. Use induction medium containing 1 μM dexamethasone, 0.2 mM indomethacin, 0.01 mg / ml insulin, 0.5 mM IBMX, DMEM-high glucose, FBS and antibiotics.
3. After 3 cycles of induction / maintenance, the cells are cultured in an adipogenic maintenance medium for an additional 7 days. In the meantime, the medium is changed every 2-3 days.
4. Adipogenesis can be assessed by the development of numerous cytoplasmic lipid vesicles that can be easily observed using the lipophilic dye Oil Red O. RT / PCR assays are used to examine lipase and fatty acid binding protein gene expression.

5.4.3 Induction of differentiation into chondrocytes This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into chondrocytes. The following protocol was employed to induce differentiation into chondrocytes.

1. Placental stem cells are maintained in DMEM or MSCGM (Bio Whittaker) supplemented with 15% umbilical cord serum.
2. Dispense placental stem cells into sterile polypropylene tubes. The cells are centrifuged (150 xg for 5 minutes) and washed twice with Incomplete Chondrogenesis Medium (Bio Whittaker).
3. After the final wash, resuspend the cells in Complete Chondrogenesis Medium (Bio Whittaker) containing 0.01 μg / ml TGF-β-3 at a concentration of 5 x 10 (5) cells / ml .
4. Dispense 0.5 ml of cells into a 15 ml polypropylene culture tube. Cells are pelleted at 150 xg for 5 minutes. This pellet is left in the medium.
5. Incubate loosely capped tubes at 37 ° C, 5% CO ₂ for 24 hours.
6. The cell pellet is supplied with freshly prepared complete chondrogenic medium every 2-3 days.
7. Maintain and suspend the pellet in the medium by daily stirring using a low speed vortex.
8. Collect the chondrogenic cell pellet after 14-28 days in culture.
9. Chondrogenesis can be characterized, for example, by observing the production of eosinophilic base material, assessing cell morphology and / or examining collagen 2 and collagen 9 gene expression by RT / PCR .

5.4.4 Induction of differentiation into bone cells This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into bone cells. The following protocol was employed to induce differentiation into bone cells.

1. Culture placental stem cell adherent cultures in DMEM or MSCGM (Bio Whittaker) supplemented with 15% umbilical cord serum.
2. Rest culture for 24 hours in tissue culture flask.
3. Replace MSCGM with Osteogenic Induction Medium (Bio Whittaker) containing 0.1 μM dexamethasone, 0.05 mM ascorbic acid-2-phosphate and 10 mM β-glycerophosphate to induce differentiation into osteogenic cells To do.
4. Supply cells with osteogenesis induction medium every 3-4 days for 2-3 weeks.
5. Assess differentiation using calcium-specific staining and RT / PCR to examine alkaline phosphatase and osteopontin gene expression.

5.4.5 Induction of differentiation into hepatocytes This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into hepatocytes. The following protocol was employed to induce hepatocyte differentiation.

1. Placental stem cells are cultured in DMEM / 20% CBS supplemented with 20 ng / ml hepatocyte growth factor and 100 ng / ml epidermal growth factor. Knockout serum replacement may be used instead of CBS.
2. Add 50 ng / ml IL-6 to the induction flask.

5.4.6 Induction of differentiation into pancreatic cells This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into pancreatic cells. The following protocol was employed to induce differentiation into pancreatic cells.

1. Placental stem cells are cultured in DMEM / 20% CBS supplemented with 10 ng / ml basic fibroblast growth factor and 2 ng / ml transforming growth factor β-1. Knockout serum replacement may be used instead of CBS.
2. Add conditioned medium from nestin-positive neuronal cultures to the medium at a 50/50 concentration.
3. Culture cells for 14-28 days, refeed medium every 3-4 days.
4. Characterize differentiation by assaying insulin gene expression by insulin protein or RT / PCR.

5.4.7 Induction of differentiation into heart cells This example describes the induction of differentiation of cord blood cells and / or embryonic-like stem cells into heart cells. The following protocol was employed to induce differentiation into heart cells.

1. Placental stem cells in DMEM / 20% CBS supplemented with 1 μM retinoic acid, basic fibroblast growth factor 10 ng / ml, transforming growth factor β-1 2 ng / ml, and epidermal growth factor 100 ng / ml Incubate at Knockout serum replacement may be used instead of CBS.
2. Alternatively, placental stem cells are cultured in DMEM / 20% CBS supplemented with 50 ng / ml cardiotropin-1 for 24 hours.
3. Alternatively, placental stem cells are maintained in protein-free medium for 5-7 days and then stimulated with human myocardial extract (incremental dose analysis). To obtain myocardial extracts, 1 gram of human myocardium is homogenized in 1% HEPES buffer supplemented with 1% umbilical cord serum. This suspension is incubated for 60 minutes and then centrifuged to collect the supernatant.
4. Cultivate cells for 10-14 days, refeed medium every 3-4 days.
5. Assess differentiation using the RT / PCR gene expression assay for cardiac actin.

5.4.8 Characterization of cord blood cells and / or embryonic-like stem cells before and / or after differentiation A population of cord blood cells spiked with pre-differentiated and / or differentiated embryonic-like stem cells, cord blood cells and / or embryonic-like stem cells To characterize, changes in morphology and cell surface markers are measured using techniques such as flow cytometry and immunocytochemistry, and changes in gene expression are measured using techniques such as PCR. Cells exposed to and / or differentiated from growth factors are characterized by the presence or absence of the following cell surface markers: CD10 +, CD29 +, CD34-, CD38-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 + , SSEA3-, SSEA4-, OCT-4 +, and ABC-p +. Preferably, pre-differentiation embryonic-like stem cells are characterized by the presence of cell surface markers OCT-4 +, APC-p +, CD34− and CD38−. Stem cells carrying these markers are universal (eg, multipotent), similar to human embryonic stem cells. Umbilical cord blood cells before differentiation are characterized by the presence of cell surface markers CD34 + and CD38 +. Differentiated cells derived from a population of embryonic-like stem cells, cord blood cells and / or cord blood cells spiked with embryonic-like stem cells preferably do not express these markers.

5.5 Example 5: Treatment of individuals with amyotrophic lateral sclerosis using embryonic stem cells Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a cortex, It is a fatal neurodegenerative disease that affects motor neurons in the brainstem and spinal cord. As many as 20,000 Americans have ALS, and 5,000 new cases occur every year in the United States. The majority of ALS cases are sporadic (S-ALS), but approximately 5-10% are hereditary (familial F-ALS). ALS develops when certain neurons in the brain and spinal cord that control spontaneous movement gradually degenerate. The main feature of ALS is the loss of spinal motor neurons, which weakens and weakens the muscles under their control, causing paralysis. The way ALS develops depends on which muscles are weakened first. ALS appears in middle age, and men are 1.5 times more likely than women to develop the disease. ALS usually dies within 5 years after diagnosis.
There are both familial and sporadic forms of ALS, but familial forms are now linked to several distinct loci. Only about 5-10% of ALS cases are familial. Of these, 15-20% are due to mutations in the gene encoding Cu / Zn superoxide dismutase 1 (SOD1). These appear to be “gain-of-function” mutations that confer toxicity on this enzyme. The discovery of SOD mutations as the cause of ALS has paved the way for some understanding of the disease. A model animal for this disease is now available and hypotheses are being established and tested for molecular events leading to cell death.

  An example of a method for treating an individual suffering from ALS using embryonic stem cells derived from placenta is shown below. This method involves intravenous infusion through a temporary peripheral vascular catheter.

First, a standard laboratory analysis is performed to assess individuals with ALS. Such analyzes can include measurement of metabolic profiles, CDC with differentiation, lipid profiles, fibrinogen levels, blood ABO rH typing, liver function tests, and BUN / creatine levels. Individuals are instructed to take the following drugs the day before transplantation: diphenhydramine (Benadryl ^™ ), 25 mg tid and prednisone 10 mg.

  Embryonic stem cells (alone or spiked into cord blood) are removed from cryopreserved stock, thawed, and maintained at a temperature of about 5 ° C. for about 2 days prior to transplantation.

  The individual is transplanted at an outpatient clinical center with all necessary equipment for intravenous infusion, physiological monitoring, and physical observation.

Approximately 1 hour prior to transplantation, the individual is administered diphenhydramine (Benadryl ^™ ) 25 mg × 1 PO and prednisone 10 mg × 1 PO. This is for precaution and to reduce the possibility of an acute allergic reaction. At the time of injection, an 18 G indwelling peripheral venous line is placed in one of the individual's four limbs and maintained open by injecting D5 1/2 saline + 20 mEq KC1 at a TKO rate. Individuals are examined prior to transplantation, but pay particular attention to heart rate, respiratory rate, and body temperature. Other monitoring such as electrocardiogram and blood pressure measurement may be performed.

The embryonic stem cells are then injected at a rate of 1 unit / hour with a total delivery volume of 60 ml. In this case, one unit corresponds to about 1-2 × 10 ⁹ total nucleated cells. Alternatively, the units of embryonic stem cells are delivered in cord blood with a total volume of 60 ml. In this case, the ratio of the number of embryonic-like stem cells to the number of stem cells in cord blood is at least 2: 1. The unit to be administered may be only cord blood. Based on data from preclinical studies in mice, a total of 2.0-2.5 × 10 ⁸ cells / kg body weight should be administered. For example, a 70 kg individual will receive about 14-18 × 10 ⁹ total nucleated cells. Individuals need to be monitored for signs of allergic reaction or hypersensitivity. These are signs of immediate discontinuation of the infusion.

  After injection, the individual should be monitored while lying down for at least 60 minutes, when he or she may resume normal activity.

5.6 Example 6: Treatment of individuals with atherosclerosis using embryonic-like stem cells Using the injection protocol described in Example 5, embryonic-like stem cells (alone or spiked into cord blood) are treated with atherosclerosis. Administer to patients with illness. Embryo-like stem cells or replacement cell populations can be administered to individuals who have no subjective symptoms, individuals who are candidates for angioplasty, or patients who have recently undergone cardiac surgery (within a week).

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

  All references cited in this text are all to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The full text of which is incorporated herein by reference for purposes.

  The citation of any publication is because it was disclosed prior to the filing date and should not be construed as an admission that the present invention is not entitled to precede such publications because of prior patents.

Claims (58)

  A composition comprising stem or progenitor cells and placental stem cells.   A composition comprising umbilical cord blood cells and placental stem cells.   2. The composition of claim 1 comprising a population of stem or progenitor cells and a population of placental stem cells.   The composition according to claim 1 or 2, which is contained in a container.   5. A composition according to claim 4, wherein the container is sealed, airtight and sterile.   The composition according to claim 1 or 2, wherein the stem or progenitor cells and placental stem cells are in contact with each other before or at the time of administration to a patient in need thereof.   The composition according to claim 1 or 2, wherein the stem or progenitor cells and placental stem cells are suitable for separate administration to a patient.   The composition according to claim 1 or 2, which is suitable for bone marrow transplantation.   The composition according to claim 1 or 2, which is suitable for human administration.   The composition according to claim 1 or 2, wherein the stem or progenitor cell is cord blood or placental blood-derived, fetal or neonatal hematopoietic stem or progenitor cell, adult cell, or bone marrow stem or progenitor cell.   11. The composition of claim 10, wherein the stem or progenitor cell is a fetal or neonatal hematopoietic stem or progenitor cell.   12. The composition of claim 11, wherein the plurality of hematopoietic stem or progenitor cells are CD34 + and CD38- with respect to cell surface markers.   The composition according to claim 10, wherein the stem or progenitor cell is cord blood stem cell.   14. The composition of claim 13, wherein the plurality of cord blood stem cells are CD34 + and CD38- with respect to cell surface markers.   14. The composition of claim 13, wherein the plurality of cord blood stem cells are CD34 + and CD38 + with respect to cell surface markers.   Placental stem cells exhibit at least one of the following cell surface markers: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 or ABC-p, or the following cell surface marker: CD34 The composition according to claim 1 or 2, which lacks at least one of CD45, SSEA3, SSEA4.   The composition according to claim 16, wherein the placental stem cells are OCT-4 + and ABC-p +.   The composition according to claim 16, wherein the placental stem cells are SSEA3- and SSEA4-.   A kit comprising the composition of claim 1 or 2, wherein the stem or progenitor cells are in a first container and the placental stem cells are in a second container.   A method for preparing a composition comprising a plurality of stem or progenitor cells and a plurality of placental stem cells, the method comprising isolating and contacting the plurality of stem or progenitor cells and the plurality of placental stem cells.   21. The method of claim 20, wherein the plurality of stem or progenitor cells and the plurality of placental stem cells are each contained in separate containers prior to mixing.   21. The method of claim 20, wherein the composition is suitable for human administration.   21. The method of claim 20, wherein the stem or progenitor cells are cord blood or placental blood derived, fetal or neonatal hematopoietic stem or progenitor cells, adult cells, or bone marrow stem or progenitor cells.   21. The method of claim 20, wherein the stem or progenitor cell is a fetal or neonatal hematopoietic stem or progenitor cell.   21. The method of claim 20, wherein the plurality of hematopoietic stem or progenitor cells are CD34 + and CD38- with respect to cell surface markers.   21. The method of claim 20, wherein the plurality of stem or progenitor cells and the plurality of placental stem cells are physically mixed.   Use of a plurality of stem or progenitor cells and a plurality of placental stem cells in the manufacture of a pharmaceutical composition for the treatment of a patient in need thereof.   28. Use according to claim 27, wherein the stem or progenitor cells and placental stem cells are suitable for administration to humans.   28. Use according to claim 27, wherein the stem or progenitor cells are cord blood or placental blood-derived, fetal or neonatal hematopoietic stem or progenitor cells, adult cells, or bone marrow stem or progenitor cells.   30. Use according to claim 29, wherein the stem or progenitor cells are fetal or neonatal hematopoietic stem or progenitor cells.   32. Use according to claim 30, wherein the plurality of hematopoietic stem or progenitor cells are CD34 + and CD38- with respect to cell surface markers.   30. Use according to claim 29, wherein the stem or progenitor cells are cord blood stem cells.   Placental stem cells with respect to cell surface markers, at least one of the following: CD10 +, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT-4 + and ABC-p + 28. The use according to claim 27, wherein   34. Use according to claim 33, wherein the placental stem cells are OCT-4 + and ABC-p +.   34. Use according to claim 33, wherein the placental stem cells are SSEA3- and SSEA4-.   28. Use according to claim 27, wherein a plurality of stem or progenitor cells and / or a plurality of placental stem cells are treated with a growth factor to induce differentiation into a specific cell type.   28. Use according to claim 27, wherein a plurality of stem or progenitor cells and / or a plurality of placental stem cells are treated with a growth factor to prevent or inhibit differentiation into a specific cell type.   Use of multiple cord blood cells and multiple placental stem cells in the manufacture of a pharmaceutical composition for the treatment of a patient in need thereof.   39. Use according to claim 38, wherein the cord blood cells are fetal or neonatal hematopoietic stem or progenitor cells.   40. Use according to claim 39, wherein the plurality of hematopoietic stem or progenitor cells are CD34 + and CD38- with respect to cell surface markers.   39. Use according to claim 38, wherein the plurality of cord blood stem cells are CD34 + and CD38- with respect to cell surface markers.   39. Use according to claim 38, wherein the plurality of cord blood stem cells are CD34 + and CD38 + with respect to cell surface markers.   Placental stem cells with respect to cell surface markers, at least one of the following: CD10 +, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT-4 + and ABC-p + 40. The use according to claim 38, wherein   44. Use according to claim 43, wherein the placental stem cells are OCT-4 + and ABC-p +.   44. Use according to claim 43, wherein the placental stem cells are SSEA3- and SSEA4-.   40. Use according to claim 38, wherein the patient has a vascular disease, disorder or condition.   47. Use according to claim 46, wherein the disease, disorder or condition is atherosclerosis.   40. Use according to claim 38, wherein the patient has a neurological disease, disorder or condition.   40. Use according to claim 38, wherein the patient has an autoimmune disorder.   50. Use according to claim 49, wherein the autoimmune disorder is selected from the group consisting of diabetes and amyotrophic lateral sclerosis.   40. The use of claim 38, wherein the patient has symptoms caused by trauma or injury, or symptoms associated with trauma or injury.   52. Use according to claim 51, wherein the trauma or damage is trauma or damage to the central nervous system.   52. Use according to claim 51, wherein the trauma or damage is trauma or damage to the peripheral nervous system.   Use of cord blood cells or stem cells isolated therefrom and placental stem cells in the manufacture of a pharmaceutical composition for the treatment of myelodysplasia.   Use of cord blood cells or stem cells isolated therefrom and placental stem cells in the manufacture of a pharmaceutical composition for transplanting hematopoietic progenitor cells for the treatment or prevention of disease.   A composition comprising umbilical cord blood-derived stem or progenitor cells supplemented with a plurality of placental stem cells. Use of at least 5 × 10 ⁹ nucleated cells in the manufacture of a pharmaceutical composition for the treatment of a patient in need thereof, the treatment comprising administering the cells to the patient; The use as described above, wherein the nucleated cells comprise placental stem cells and the patient has a disease, disorder or symptom comprising an inflammatory component. Use of at least 5 × 10 ⁹ nucleated cells in the manufacture of a pharmaceutical composition for the treatment of a patient in need thereof, the treatment comprising administering the cells to the patient; The use as described above, wherein the nucleated cells comprise placental stem cells and the patient has an immune related disorder.