KR101176146B1 - Embryonic-like stem cells derived from post-partum mammalian placenta and uses and methods of treatment using said cells - Google Patents

Abstract

The present invention provides compositions and methods using conventional cord blood compositions or other stem or progenitor cells and embryo-like stem cells derived from the postpartum placenta. Embryonic-like stem cells can be used alone or in combination with other stem cell populations. In accordance with the present invention, embryo-like stem cells may be mixed with other stem cell populations including, but not limited to, umbilical cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells, human stem cells and progenitor cells derived from bone marrow. Can be. Embryonic-like stem cells and mixed groups of embryo-like stem cells and stem cells may be used for various applications, including but not limited to therapeutic uses for transplantation, treatment and prevention of diseases, and diagnostic and research uses. Has application.

Placenta, Embryonic-like Stem Cells, Umbilical Cord Blood, Stem Cells, Progenitor Cells

Description

Embryo-like stem cells derived from post-partum mammalian placenta and uses and methods of treatment using said cells}

The present invention relates to the use of conventional cord blood compositions or other stem or progenitor cells and embryonic-like stem cells derived from the postpartum placenta. Embryonic-like stem cells can be used alone or in combination with other stem cell populations. In accordance with the present invention, embryo-like stem cells include, for example, but not limited to, umbilical cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells, human stem cells and progenitor cells derived from bone marrow, and the like. It can also be mixed with other stem cell populations. Embryonic-like stem cells and mixed groups of embryo-like stem cells and stem cells have a variety of uses and indications, such as, but not limited to, therapeutic, diagnostic and research applications for transplantation. Embryonic-like stem cells and mixed groups also include, for example, but are not limited to, vascular diseases, neurological diseases or disorders, autoimmune diseases or disorders, diseases or disorders including inflammation and diseases such as cancer or disorders associated therewith. Or for the treatment of abnormalities. In particular, embryo-like stem cells or mixtures comprising the same are administered at high doses without HLA typing.

Significant attention is focused on the identification, isolation and development of human stem cells. Human stem cells are omnipotent or pluripotent precursor cells capable of forming a variety of mature human cells. This ability serves as the basis for the cellular differentiation and specialization required for tissue and organ formation.

Recent transplantation success of such stem cells has provided new clinical tools for reconstructing and / or replenishing bone marrow after exposure to disease and toxins and / or radiation. There is further evidence that stem cells, although not all tissues, can be applied to restore many tissues and restore physiological and anatomical function. The application of stem cells in tissue engineering, gene therapy delivery, and cell therapy is also rapidly developing.

Many different types of mammalian stem cells have been characterized. For example, embryonic stem cells, embryonic germ cells, adult stem cells or other committed cells or progenitor cells are known. Certain stem cells have not only been isolated and characterized, but have also been cultured under conditions capable of differentiation to some extent. However, basic problems remain, such as it is almost impossible to obtain a sufficient amount and group of human stem cells capable of differentiating into all cell types. Stem cells are seriously insufficient. This is important for the treatment of various abnormalities such as malignancy, genetic metabolic disorders, hemochromatosis and immunodeficiency. It would be very advantageous to have more sources of embryonic stem cells.

For many reasons, obtaining a sufficient number of human stem cells has been a problem. Firstly, the isolation of a group of stem cells commonly occurring in adult tissues is technically difficult and expensive, in part because of the very limited amount found in blood or tissue. Second, the acquisition of these cells from embryonic or fetal tissue, including the use of abortions, has raised religious and ethical concerns. The widely accepted belief that human embryos and fetuses are independent organisms has triggered government restraint on the use of such sources for all purposes, including medical research. Therefore, alternative sources that do not require the use of cells obtained from embryonic or fetal tissue are necessary to clinically advance the use of stem cells. However, there are few practical alternative sources of stem cells, especially human stem cells, and their supply is also limited. Furthermore, securing sufficient amounts of stem cells from alternative sources generally requires a lot of labor. Such labor, for example, involves the acquisition of cells or tissues from a donor or patient, in vitro culture and / or proliferation, dissection, and the like.

For example, Caplan et al. (US Pat. No. 5,486,359, "Human Mesenchymal Stem Cells," issued Jan. 23, 1996) describe human mesenchymal stems derived from bone marrow that can act as precursors of mesenchymal cell lineage. A human mesenchymal stem cell (hMSC) composition is disclosed. Caplan et al. Disclosed that hMSCs can be identified by specific cell surface markers that are identified as monoclonal antibodies. Homologous hMSC compositions are obtained by positive selection of adherent marrow or periosteal cells without markers associated with hematopoietic cells or differentiated mesenchymal cells. These isolated mesenchymal cell populations exhibit epitopic characteristics associated with mesenchymal stem cells, have the ability to regenerate without differentiation in culture, and are placed in vivo or in vitro at damaged tissue sites. Have the ability to differentiate into specific mesenchymal lineage. However, a drawback of such methods is that they require the securing of bone marrow or periosteal cells from the donor, and the continuous separation of MSCs from them.

Hu et al. (PCT Patent No. 00/73421, "Isolation Methods of Human Amniotic Epithelial Cells, Cold Preservation Methods and Therapeutic Uses," issued Dec. 7, 2000) show that human amnion epithelial cells are obtained from the placenta at delivery. To isolate, incubate, cryopreserved, or induce differentiation for future use. According to Hu et al., The placenta is secured immediately after delivery, and the amniotic membrane is separated from the chorion by incision or the like. Amnion epithelial cells are isolated from amnion by standard cell separation techniques. The disclosed cells can be cultured in a variety of media, expanded to culture, cryopreserved, or induced to differentiate. Hu et al. Disclose that amnion epithelial cells are multipotential (and possibly pluripotential) and can differentiate into epithelial tissues such as corneal surface epithelial cells or vaginal epithelial cells. However, a disadvantage of such methods is that they are labor intensive and the production of stem cells is very low. For example, in order to obtain a sufficient number of stem cells for conventional therapeutic or research purposes, amnion epithelial cells must first be isolated from the amnion by incision and cell separation techniques, then cultured and amplified in vitro .

Umbilical cord blood (cord blood) is a well known alternative source of hematopoietic progenitor stem cells. Stem cells of cord blood are commonly stored cold for the use of hematopoietic reconstitution and are widely used in therapeutic procedures used in bone marrow and other related transplants (Boyse et al., US Pat. No. 5,004,681, "Blood of Blood Preservation of Hematopoietic Stem and Progenitor Cells in Fetuses and Neonates; Boyse et al., US Pat. No. 5,192,553, "Isolation and Preservation of Blood and Fetal and Neonatal Hematopoietic Stem and Progenitor Cells and Therapeutic Uses," March 9, 1993. See publication). Conventional techniques for the recruitment of cord blood are based on the use of a needle or cannula, which uses the aid of gravity to draw cord blood from the placenta, for example bleeding (exsanguinate). (Boyse et al., US Pat. No. 5,192,553, issued March 9, 1993; Boyse et al., US Pat. No. 5,004,681, issued April 2, 1991; Anderson, US Pat. No. 5,372,581, "For Placental Blood Recruitment. Methods and Tools, "issued December 13, 1994; Hessel et al., US Pat. No. 5,415,665," Umbilical Cord Clamping, Cutting, and Blood Recruitment Devices and Methods, issued May 16, 1995). The needle or cannula is usually located in the umbilical vein, and the placenta is gently massaged to help draw cord blood from the placenta, after which the placenta from which the blood has been drawn is considered to be no longer useful and is generally used It was also the main limit it to secure stem cells from cord blood was a sufficient amount of cord blood obtained, which is a cell to be sufficient to reconstitute the bone marrow after transplantation effectively.

Naughton et al. (US Pat. No. 5,962,325, "Three-dimensional Stromal Tissue Culture," issued October 5, 1999) show that fetal cells, including fibroblast-like cells and chondrocyte-progenitors, have umbilical or placental tissue. Or from cord blood.

Kraus et al. (US Pat. No. 6,338,942, "Selective Amplification of Target Cell Populations," issued January 15, 2002) are starting samples of cord blood or peripheral blood to select a predetermined target cell population from other cells in a growth medium. The cells are introduced into the growth medium, induced to divide the cells of the target cell population, and the cells in the growth medium have a specific affinity for the predetermined cell population (such as CD34 cells) (such as monoclonal antibodies to CD34). Disclosed is the fact that it is also possible to selectively amplify a predetermined target cell population by bringing into contact with a selection factor comprising a).

Rodgers et al. (US Pat. No. 6,335,195, "Methods for Promoting Hematopoietic and Mesenchymal Cell Proliferation and Differentiation," published on Jan. 1, 2002) disclose an ex vivo culture method of hematopoietic and mesenchymal stem cells and angiotensinogen ( angiotensinogen), angiotensin I (AI), AI analogues, AI fragments and their analogues, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT ₂ type 2 receptor agonists A method of inducing proliferation and differentiation of lineage-specific cells by culturing alone or in combination with other growth factors and cytokines is disclosed. Stem cells were obtained from bone marrow, peripheral blood or umbilical cord blood. However, a disadvantage of these methods is that such ex vivo methods for inducing proliferation and differentiation of stem cells require a lot of time as described above, and the productivity of stem cells is low.

Because of the limitations on the collection and use of stem cells and the insufficient number of cells generally collected from cord blood, stem cells are decisively in short supply. Stem cells have the potential to be used for the treatment of various abnormalities, including malignant tumors, genetic metabolic disorders, hemochromatosis and immunodeficiency. There has been a decisive need for a source that can readily obtain a large number of human stem cells for various therapeutic and other medically related purposes. The present invention addresses its needs and others.

In addition, there is a need for the treatment of neurological conditions such as amylotrophic lateral sclerosis (ALS). Although recent research using an irradiated mouse model with a less common form of ALS, a family of ALS, suggests that cord blood is useful for the treatment of these diseases, the issue with these sources is one of these options. Make it an ideal. See, for example, Ende et al., Life Sci. 67: 53059 (2000). Thus, there remains a need for larger groups of stem or progenitor cells that can be used in the treatment of diseases, especially when treating diseases such as ALS.

Summary of the Invention

The present invention relates to cord blood compositions or stem or progenitor cells resulting therefrom which are supplemented with or contacted with embryonic-like stem cells derived from post-partum placenta. Embryonic-like stem cells that are the subject of other applications can be used herein as a composition or as a mixture with other stem or progenitor cell populations. According to the present invention, embryonic-like stem cells include other stem or progenitor cell populations including, but not limited to, umbilical cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells, human stem cells and progenitor cells derived from bone marrow It may also be in contact with. Embryonic-like stem cells and mixed groups of embryo-like stem cells and stem or progenitor cells can be used in a variety of ways, including, but not limited to, therapeutic use for transplantation, treatment and prevention of diseases, and diagnostic and research uses. Has use and application.

According to the present invention, a group of stem cells is mixed with a group of embryo-like stem cells to supplement, augment or increase the concentration of pluripotent and multipotent stem cells in the stem cell population. For example, in one embodiment, umbilical cord blood, or stem or progenitor cells derived therefrom, is augmented or contacted with embryonic-like stem cells of the present invention prior to administration to a patient. It should be appreciated that embryonic-like stem cells may be administered simultaneously or sequentially with umbilical cord blood, or cells derived therefrom. However, it is desirable to contact each cell prior to administration.

Embryonic-like stem cells of the invention are the presence of cell surface markers of CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 and ABC-p and cells of CD34, CD38, CD45, SSEA3 and SSEA4. Characterized by the absence of surface markers. In one preferred embodiment, such embryonic-like stem cells can be characterized by the presence of cell surface markers OCT-4 and APC-p. Embryonic-like stem cells derived from the placenta have properties of embryonic stem cells but do not originate from embryos. That is, the present invention includes a mixture of cord blood and embryo-like stem cells isolated from placenta that are OCT-4 + and / or ABC-p +. Such embryo-like stem cells possess a variety of talents (eg pluripotency) like human embryonic stem cells.

According to the present invention, a group of stem cells are mixed with embryo-like stem cells that are either fullypotents or multipotents. Such embryo-like stem cells can be separated from the perfused placenta at different time points, such as, for example, CD34 + / CD38 +, CD34 + / CD38-, and CD34- / CD38-hematopoietic cells. In one embodiment of the invention, such cells may be used to replenish a group 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 containing a mixture of embryonic-like stem cells and stem cells in one bag or container. In one preferred embodiment, the composition is a pharmaceutically acceptable single dose composition. In another embodiment, the present invention provides a composition wherein a group of stem cells and a group of embryo-like stem cells are contained in two separate bags or containers. In certain embodiments, such “two bags” kits may be mixed at or before administration, especially immediately before administration, to a patient in need thereof. In another embodiment, the contents of each bag may be administered separately to the patient, where mixing of the two cell populations occurs in vivo . In another embodiment, the container is sealed, airflow is blocked, and sterilized.

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

The invention also provides a method of treating a patient in need thereof by administering a group of stem cells supplemented with embryo-like stem cells. In one embodiment, replenishing a group of cord blood cells with embryo-like stem cells occurs by mixing the embryo-like stem cells with the stem cells prior to administration of the combined or "spiked" population to the patient. . In another embodiment, supplementing a group of stem cells with embryo-like stem cells occurs by administering a supplemented group to the patient, such as simultaneous administration of embryo-like stem cells and cord blood cells. In another embodiment, supplementing a group of stem cells with embryonic-like stem cells is performed by administering cord blood cells to a patient, such as administering embryo-like stem cells isolated from stem cells before or after administration of the stem cells. Occur after administration.

According to the present invention, a group of stem cells supplemented with embryo-like stem cells, such as placenta, for example umbilical cord blood, have a variety of uses, including prophylactic, therapeutic and diagnostic uses. A supplemented group of stem cells can be used to prevent or treat transplantation and / or tinnitus. In one embodiment of the invention, a supplemented group of cells can be used to recover and reconstruct tissues and organs, thereby replacing or repairing diseased tissues, organs or portions thereof. In another embodiment, the supplemented group of stem cells can be used as a diagnostic for testing predisposition or genetic abnormality for a particular disease or condition.

In another embodiment, the present invention provides a method for separating other embryo-like and / or multipotent or fullypotent stem cells from an extract or perfusate of the placenta from which blood has been removed and according to the present invention. Provides a way to use it to replenish cord blood cells.

The invention also provides a pharmaceutical composition comprising several stem cell populations, such as umbilical cord blood cell populations, supplemented with one or more embryo-like stem cell populations of the invention.

The present invention provides an isolated homogeneous human placental stem cell population with the potential to differentiate into all cell types. In another embodiment, a group of human placental stem cells has the potential to differentiate into one cell type. In another embodiment, a group of human placental stem cells has the potential to differentiate into a variety of different cell types. Such cells may be used to replenish stem cell populations such as umbilical cord blood according to the methods of the present invention.

The invention also encompasses pharmaceutical compositions comprising hematopoietic stem cell populations supplemented with one or more cell populations having a high concentration (or larger cell population) of uniform hematopoietic stem cells, wherein the uniform hematopoietic stem cells CD34 + / CD38- cells; CD34- / CD38- cells, and CD133 + cells are included, but are not limited to these. One or more such populations of cells may be used or mixed with hematopoietic stem cells, such as CD34 + / CD38 + hematopoietic cells obtained from umbilical cord blood or other sources for transplantation and other uses.

The invention also provides a method of mixing a group of stem, progenitor or cord blood cells with a group of embryo-like stem cells, including stored or cryostored cord blood cells. In one embodiment, the two groups are physically mixed. In another aspect of this example, the two groups are physically mixed and then treated with growth factors such as cytokines and / or all interleukins to induce cell differentiation. In another aspect of this embodiment, stem cells and / or embryo-like stem cells are treated with growth factors such as cytokines and / or interleukins to induce cell differentiation and then physically mixed. In one embodiment, the mixed group is treated with growth factors to induce differentiation into various cell types. In another embodiment the mixed group is treated with growth factors to induce differentiation into one particular cell type. In another embodiment the mixed group is treated with growth factors to prevent or inhibit differentiation into one particular cell type. In certain embodiments, the culture state can be controlled, for example, a mixed group of cells may be treated with a specific cytokine or interleukin mixture to induce or link differentiation to a particular cell type.

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

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

The invention also relates to new uses of human placental stem cells (embryonic-like stem cells). Also included in the present invention are methods for treating or preventing a disease with compositions comprising embryonic-like stem cells and other stem or progenitor cells or sources thereof. Similarly, methods of administering such compositions are included. Finally, it should be appreciated that the compositions of the present invention may comprise stem or progenitor cell populations derived from several donors. The present invention encompasses the use of non-HLA matched compositions as well as HLA-matched compositions in a patient. Blood types that match certain patients are preferred. However, it is not necessarily required when compositions comprising embryonic-like stem cells and stem or progenitor cells are used.

Definition of Terms

The term "bioreactor" as used herein refers to proliferating cells, producing or expressing biological substances, or culturing or maturing cell tissues, organoids, viruses, proteins, polynucleotides and microorganisms. Ex vivo system that can be used.

As used herein, the terms "cord blood" or "umbilical cord blood" may be used interchangeably.

As used herein, the term “embryonic stem cell” refers to pluripotent cells derived from the inner cell mass of a blastocyst, such as, for example, 4-5 day mature human embryos.

As used herein, the term “embryo-like stem cell” refers to a cell that is not derived from the inner cell mass of the blastocyst. The term "embryonic-like stem cells" as used herein may be expressed as "placental stem cells" and preferably refers to human placental stem cells derived from postpartum perfused placenta. Embryonic-like stem cells are preferably pluripotent. However, stem cells that may be obtained from the placenta include embryonic-like stem cells, multipotent cells, and committed progenitor cells. According to the method of the present invention, embryo-like stem cells derived from the placenta may be collected from isolated placenta perfused for a time sufficient to remove blood and remove residual cells.

As used herein, the term "bleeded" or "exsanquinated or exsanguination" refers to the removal and / or drainage of all cord blood from the placenta when used in connection with the placenta. In accordance with the present invention, blood removal of the placenta is, for example, but not limited to draining, gravity induced efflux, massaging, squeezing, pumping, etc. ) And the like. In a preferred embodiment, deplacement of the placenta may be performed by perfusing, rinsing or flushing the placenta with a constant solution. The solution may or may not contain ingredients such as anticoagulants to aid in blood removal of the placenta.

As used herein, the term "mix" refers to combining or blending into one mass or mixture; Placing together in a mass so that the components are somewhat homogeneous; Combining or producing components; Made by admixture, supplementation, or commingling; Or addition of one component or part to another component or part.

As used herein, the term “perfuse” or “perfusion” refers to the act of drawing or passing a solution through or over an organ or tissue, preferably with non-adherent cells, for example. By passing the solution through the organ or tissue with sufficient pressure or force to remove the same residual cells from the organ or tissue. As used herein, the term “perfusate” refers to the fluid collected after passing through an organ or tissue. In a preferred embodiment, the perfusate comprises one or more anticoagulants.

The term "exogenous cells" as used herein refers to "foreign" cells. That is, heterogeneous cells ("non-autologous" cells derived from sources other than placenta donors) or cells of own tissue ("autologous" cells derived from placental donors) from tissues or organs other than the placenta.

As used herein, the term "organoid" refers to an aggregate of one or more cell types that are apparently or actually structurally gathered, such as any organ or gland of the mammalian body, preferably the human body. .

As used herein, the term "multipotent" refers to a cell having the ability to grow to any number of cell types of about 260 cell types of the mammalian body. Unlike pluripotent cells, redundant cells do not have the ability to form all cell types.

As used herein, the term "pluripotent" refers to the ability to fully differentiate cells. That is, it refers to a cell having the ability to grow to about 260 cell types of the mammalian body. Pluripotent cells are self-neoplastic and may exist at rest or in silence (silent) in tissue. Unlike pluripotent cells (such as diploid embryos, for example), embryonic stem cells are generally unable to form new blastocysts.

As used herein, the term “progenitor cell” refers to a cell designated to differentiate into a specific type of cell or to form a specific type of tissue.

As used herein, the term “stem cells” refers to master cells that can be infinitely repeated to form specialized cells of tissues and organs. Stem cells are developmentally pluripotent or redundant cells. Stem cells can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor (“transit”) cell. One progenitor cell then proliferates into mature, fully formed cells of the tissue. As used herein, “stem cells” include progenitor cells unless otherwise indicated.

As used herein, the term "totipotent" refers to a cell capable of forming a complete embryo, such as a pouch embryo.

Detailed description of the invention

The present invention is due in part to the unexpected discovery that embryo-like stem cells formed by perfused and / or cultured placenta are bloodipotent stem cells that can readily differentiate into the desired cell type. Such embryo-like stem cells complement, augment or promote a group of stem cells, including but not limited to umbilical cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells, human stem cells and progenitor cells derived from bone marrow Can be used to According to the present invention, a group of stem cells is mixed with a group of embryo-like stem cells to supplement, augment or promote the concentrations of fullypotent and multipotent stem cells in the stem cell population. According to the present invention, a group of stem cells includes a group of embryo-like stems having various uses and applications, including but not limited to therapeutic uses for transplantation, treatment and prevention of diseases, and diagnostic and research uses. Mixed with cells.

The invention also provides a composition wherein a mixture of stem cells and embryo-like stem cells is contained in one bag or container. In another embodiment, the present invention provides a composition wherein a group of stem cells and a group of embryo-like stem cells are contained in two separate bags or containers. In certain embodiments, such “two bags” compositions may be mixed upon or prior to administration, preferably just prior to administration, to a patient in need thereof. In another embodiment, the contents of each bag may be administered separately to the patient, in which case the two cell populations are used as accessory in vivo .

The present invention also provides a method of mixing cord blood comprising a group of stem or progenitor cells or stored or cryopreserved cord blood with a group of embryo-like stem cells. In one embodiment, the two groups are physically mixed. In another aspect of this example, the two groups are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another aspect of this embodiment, stem cells and / or embryo-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 refers to a group designated to differentiate into cells such as, for example, nerves, muscle cells, hematopoietic cells, vascular cells, adipocytes, chondrocytes, osteoblasts, hepatocytes, pancreas, or heart cells. It provides a method of mixing the cells of the group with a group of embryo-like stem cells. In one embodiment, the two groups are physically mixed. In another aspect of this example, the two groups are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another aspect of this embodiment, the designated cells and / or embryo-like stem cells are treated with growth factors such as cytokines and / or interleukins, and then physically mixed to induce cell differentiation.

According to the method of the present invention, embryo-like stem cells are extracted from the drained placenta by perfusion techniques using either or both umbilical artery and umbilical vein. The placenta is preferably drained by desorption and collection of residual blood such as residual umbilical cord blood. The drained placenta is then processed in a manner that establishes an ex vivo , natural bioreactor environment in which embryonic-like stem cells residing in parenchyma and extravascular space are recruited. Embryonic-like stem cells migrate to drained, empty microcirculation, where they are collected according to the method of the present invention, preferably as washed by the recruitment vessel by perfusion.

As described above, a large number of other pluripotent or redundant stem cells can be separated from the perfused placenta at different points during perfusion, such as CD34 + / CD38 +, CD34 + / CD38−, and CD34− / CD38− hematopoietic cells. In one embodiment, such cells may be used to supplement a group of stem cells, such as cord blood cells, in accordance with the methods of the present invention.

The invention also provides isolated homogeneous human placental stem cell populations with the potential to differentiate into all cell types. In another embodiment, a group of human placental stem cells has the potential to differentiate into one cell type. In another embodiment, a group of human placental stem cells have the potential to differentiate into a variety of different cell types. Such cells may be used to replenish a group of stem cells, such as cord blood, in accordance with the methods of the present invention.

The invention also provides a method of mixing a group of stem cells with a group of embryo-like stem cells. In one embodiment, the two groups are physically mixed. In another aspect of this example, the two groups are physically mixed and then treated with growth factors such as cytokines and / or interleukins to induce cell differentiation. In another aspect of this embodiment, stem cells and / or embryo-like stem cells are treated with growth factors such as cytokines and / or interleukins to induce cell differentiation and then physically mixed. In one embodiment, the mixed groups are treated with growth factors to induce differentiation into various cell types. In another embodiment, the mixed groups are treated with growth factors to induce differentiation into specific cell types. In other embodiments, the mixed groups are treated with growth factors to prevent or inhibit differentiation to specific cell types. In certain embodiments, the culture state can be controlled, for example, a mixed group of cells may be treated with a specific cytokine or interleukin mixture to induce or link differentiation to a particular cell type.

The present invention provides a pharmaceutical composition comprising a group of stem cells, such as, for example, cord blood cells, supplemented with one or more embryo-like stem cell populations of the present invention.

The invention also encompasses pharmaceutical compositions comprising a population of stem cells, such as a group of umbilical cord blood cells, supplemented with one or more cell populations having a high concentration (or greater number of cells) of homogeneous hematopoietic stem cells, wherein Qualitative hematopoietic stem cells include CD34 + / CD38-cells; And CD34- / CD38- cells. One or more such cell populations may be used or mixed with cord blood hematopoietic cells, ie, CD34 + / CD38 + hematopoietic cells for transplantation and other uses.

According to the present invention, a group of stem cells, such as, for example, cord blood, supplemented with embryo-like stem cells derived from the placenta, have a variety of uses, including therapeutic and diagnostic uses. The supplemented group of stem cells can be used for transplantation or for the treatment or prevention of a disease. In one embodiment of the invention, a supplemented group of cells can be used to repair and reposition tissues and organs, thereby relocating or restoring the diseased tissues, organs or portions thereof. In another embodiment, the supplemented group of stem cells may be used for diagnostic purposes to test for genetic abnormalities or as predisposition for certain diseases or disorders.

The invention also provides a method of treating a patient in need thereof by administering a group of stem cells supplemented with embryo-like stem cells. In one embodiment, supplementing a group of cord blood cells with embryo-like stem cells occurs by mixing the embryo-like stem cells with the stem cells before administering the supplemented group to the patient. In another embodiment, supplementing a group of cord blood cells with embryo-like stem cells occurs by administering a supplemented group to a patient, such as, for example, simultaneous administration of cord blood cells and embryo-like stem cells. In another embodiment, supplementing a group of cord blood cells with embryo-like stem cells is performed to a patient, such as, for example, by administering embryo-like stem cells before or after, separate from stem cell administration. Code occurs after administration of blood cells.

How to separate and incubate the placenta

Pretreatment of the placenta

According to the method of the present invention, a human placenta is recovered immediately after its release after birth, and in some embodiments cord blood in the placenta is recovered. In some embodiments, the placenta is used in conventional cord blood recovery procedures. Such cord blood recoveries can be obtained commercially, for example, from LifeBank Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. The cord blood can be discharged immediately after the compression release of the placenta.

In another embodiment, the placenta is pretreated according to co-pending US application Ser. No. 10 / 076,180, filed Feb. 13, 2002, whereby the citation is hereby incorporated by reference in its entirety. do.

Blood removal of the placenta and removal of residual cells

As disclosed in PCT Patent No. 02/064755 (published Aug. 22, 2002), the citation is hereby incorporated by reference in its entirety, whereby the placenta after birth is activated if the placenta is properly manipulated after birth. Contains stationary cells that may be present. For example, after release of pressure from the uterus, blood is removed from the placenta as soon as possible to minimize or prevent apoptosis. Then, as soon as possible after blood removal, the placenta is perfused to remove blood, residual cells, proteins, factors and other substances present in the organs. Material debris may also be removed from the placenta. Perfusion generally continues with a suitable perfusate for at least 2 hours and at least 24 hours. The placenta can therefore be used as a rich source of embryo-like stem cells, which studies include drug development, treatment and prevention of disease, in particular transplant surgery or treatment, and production of committed cells, tissues and organoids. Can be used for

In addition, surprisingly and unexpectedly, human placental stem cells produced by blood-removed, perfused and / or cultured placenta are pluripotent stem cells that can readily differentiate into several desirable cell types.

According to the method of the present invention, stem or progenitor cells, including but not limited to embryonic-like stem cells, have complete removal of cord blood, that is, remaining cord blood after birth and / or conventional cord blood recovery processes. It may be recovered from the released placenta. According to the method of the present invention, the placental ocular blood removal method and residual cell removal method are any conventionally known methods, such as those disclosed in PCT Patent No. 02/064755, issued August 22, 2002. It can be easily done by means of which, the citation is hereby incorporated by reference in its entirety.

Placental culture

After blood is removed from the placenta and perfused for a sufficient time, embryo-like stem cells are observed to migrate to the microcirculatory system of the blood removed and perfused placenta, where embryo-like stem cells are collected according to the method of the present invention. Lose. Preferably by washing into the recruitment vessel by perfusion. In another embodiment, according to the method disclosed in PCT Patent No. 02/064755, issued August 22, 2002, the placenta is cultured and the proliferated cells are observed, sorted and / or characterized, thereby citing the above references. Is incorporated by reference in its entirety herein.

Collection of cells from the placenta

After blood is removed from the placenta and perfused for a sufficient time, embryo-like stem cells migrate into the empty placenta's microcirculatory system, where the embryo-like stem cells are collected according to the method of the present invention. Preferably it is collected by collecting perfusion liquid which flows out into the collection vessel.

In a preferred embodiment, cells cultured in the placenta are well known to those of ordinary skill in the art, such as density gradient centrifugation, magnet cell separation, flow cytometry. , Or are separated from the effluent perfusate using other cell separation or sorting methods.

In certain embodiments, embryo-like stem cells are collected from the placenta and, in certain embodiments, are preserved according to the method described in PCT Patent No. 02/064755, issued August 22, 2002, whereby the citations The entirety of which is incorporated herein by reference.

Embryonic-like Stem Cells

Embryonic-like stem cells obtained according to the methods of the present invention may also include pluripotent cells, ie cells with complete differentiation variability, which are self-renewing and can remain dormant or stationary in tissue. Stem cells that may be obtained from the placenta include embryo-like stem cells, redundant cells, designated progenitor cells, and fibroblatoid cells.

The first blood collected from the placenta is referred to as cord blood containing hematopoietic progenitor cells, mainly CD34 + and CD38 +. Within the first 24 hours of postpartum perfusion, high concentrations of CD34 + and CD38− hematopoietic progenitors and high concentrations of CD34− and CD38 + hematopoietic progenitors may be isolated. After 24 hours of perfusion, high concentrations of CD34- and CD38- cells can be separated from the placenta along with the aforementioned cells. The isolated perfused placenta of the present invention provides a large amount of stem cell source rich in CD34 + and CD38− stem cells and CD34− and CD38 + stem cells. Separated placenta perfused for 24 hours or more provides a source of large amounts of stem cells, rich in CD34- and CD38- stem cells.

In a preferred embodiment, the embryo-like stem cells obtained by the methods of the invention are viable, stationary, and pluripotent stem cells. These stem cells are present in the full-term human placenta and recovered with successful delivery and placental compression release to recover 1 billion nucleated cells, which are approximately 50 million to 100 million redundant cells and pluripotency. Produce stem cells.

Human placental stem cells provided by the placenta are surprisingly embryo-like, for example the presence of the following cell surface markers (SSEA3-, SSEA4-, OCT-4 + and ABC-p +) for the identification of these cells Confirmed. Preferably, embryonic-like stem cells of the invention are characterized by the presence of OCT-4 + and ABC-p + cell surface markers. The present invention thus encompasses stem cells which have not been isolated or otherwise obtained from embryonic sources but which can be identified by the following markers: SSAE3-, SSAE4-, OCT-4 + and ABC-p +. In one embodiment, human placental stem cells do not express MHC class 2 antigen.

Stem cells isolated from the placenta are homogeneous and sterile. In addition, stem cells are readily obtained in a form suitable for human administration, ie, pharmaceutical grade. Preferred embryo-like stem cells obtained by the methods of the invention may be identified by the presence of the following cell surface markers: OCT-4 + and ABC-pt. Furthermore, the present invention provides the following markers: CD10 +, CD38-, CD29 +, CD34-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4-, OCT-4 +, and ABC-p +. Includes embryonic stem cells. Such cell surface markers are generally determined according to methods well known in the art, for example flow cytometry, after washing and staining with anti-cell surface marker antibodies. For example, to determine the presence of CD-34 or CD-38, cells are washed with PBS and then anti-CD34 phycoerythrin and anti-CD38 fluorescein isothiocyanate ( isothiocyanate) (Becton Dickinson, Mountain View, Calif.).

In another embodiment, cells cultured in placental bioreactors are identified and characterized by colony forming unit assays. Such methods are well known in the art, such as Mesen Cult ^™ medium (stem cell Technologies, Inc., Vancouver British Columbia).

Embryonic-like stem cells obtained according to the method of the present invention are adipogenic, chondrogenic, osteogenic, hematopoietic, myogenic, angiogenic. It may also be induced to differentiate into particularly cell lineages, including specific cell lineages of vasogenic, neurogenic, and hepatocellular. In certain embodiments, embryo-like stem cells obtained according to the methods of the present invention are induced to differentiate for use in transplantation and for ex vivo treatment protocols. In certain embodiments, embryonic-like stem cells obtained according to the methods of the present invention are induced to differentiate into specific cell types and genetically engineered to provide a therapeutic gene product. In certain embodiments, embryo-like stem cells obtained according to the methods of the invention are cultured in vitro with a compound that induces differentiation, followed by direct transplantation of the differentiated cells into certain subjects. Accordingly, the present invention encompasses methods for differentiating human placental stem cells using standard culture media. In addition, the present invention includes hematopoietic cells, nerve cells, fibroblasts, strand cells, mesenchymal cells and hepatocytes.

Embryonic-like stem cells may also be further cultured after being collected from the placenta using methods well known in the art. For example, cultured on feeder cells, such as irradiated fibroblasts obtained from the same placenta or other human or non-human sources as embryonic-like stem cells, to obtain continued long-term culture of embryo-like stem cells, or such feeders It can be further cultured by culturing in an engineered medium obtained from the culture of the cells. Embryonic-like stem cells may also be expanded in vitro prior to collection from placental bioreactor or after recovery from placenta. In certain embodiments, embryo-like stem cells to be expanded are exposed to or cultured in the presence of a component that inhibits cell differentiation. Such ingredients are well known in the art and include human delta-1 and human serrate-1 polypeptides (Sakano et al. US Pat. No. 6,337,387, "Differentiation-Inhibiting Polypeptides", Jan. 8, 2002). One publication), leukemia inhibitory factor (LIF) and stem cell factor. Methods for the proliferation of cell populations are well known in the art (see, eg, Emerson et al., US Pat. No. 6,326,198, “For ex vivo replication of stem cells, for optimization of hematopoietic progenitor cells, and metabolism, Methods and compositions for increasing GM-CSF secretion and / or IL-6 secretion of human stromal cells ", issued December 4, 2001; Kraus et al., US Pat. No. 6,338,942," Selection of target cell populations. " Multiplication ", published January 15, 2002).

Embryonic-like stem cells may be assessed using standard techniques whose viability, proliferation potential, and longevity are well known in the art. Such standard techniques include trypan blue exclusion assays, fluorescein diacetate uptake assays, propidium iodide uptake assays and proliferation to assess viability. Thymidine uptake assays, MTT cell proliferation assays, and the like. Lifespan can be measured by methods well known in the art, such as assessing the maximum number of group doublings in extended culture.

In certain embodiments, differentiation of stem cells or progenitor cells cultured in a blood-removed, perfused and / or cultured placenta has sufficient effect to inhibit, modulate and / or regulate the differentiation of cells collected from the placenta. To be exerted, the dosage is controlled using a pharmaceutical composition or ingredient comprising a single and / or repeated dose effective in a single or repeated dose.

Components that induce stem or progenitor cell differentiation are well known in the art. Examples include Ca ²⁺ , EGF, α-FGF, β-FGF, PDGF, keratinocyte growth factor (KGF), TGF-β, cytokines (eg, IL-1α, IL-1β, IFN- γ, TFN), retinoic acid, transferrin, hormones (e.g. androgen, estrogen, insulin, prolactin, triiodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix elements (eg, collagen, heparan sulfate, Matrigel ^™ ), or mixtures thereof, but are not limited thereto.

Ingredients that inhibit cell differentiation are also well known in the art. Examples include human delta-1 and human serrate-1 polypeptides (Sakano et al., US Pat. No. 6,337,387, "Differentiation-inhibiting polypeptide", issued January 8, 2002), leukemia inhibitory factor (LIF), and stem Cellular factors, but are not limited thereto.

Components used to control differentiation can be introduced into placental bioreactors to induce differentiation of cells in culture in the placenta. Alternatively, the component can be used to control differentiation in vitro after cells have been collected or removed from the placenta.

Determination of whether the stem cells have differentiated into specific cell types is performed by methods well known in the art. Flow cytometry or immunocytochemistry, for example by investigating the morphology of cells using light or focal sharing microscopy or by measuring changes in gene expression using well known techniques such as PCR and gene-expression profiling. This may be accomplished by measuring changes in morphology and cell surface markers using techniques such as immunocytochemistry (e.g., staining cells with tissue-specific or cell-marker specific antibodies).

Group of Stem Cells Supplemented with Embryonic-like Stem Cells

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

The present invention provides isolated homogeneous human placental stem cell populations (embryo-like stem cells) with the potential to differentiate into all cell types. Such cells can be used to replenish a group of stem cells, for example cord blood cells, according to the methods of the present invention.

The present invention also provides a group of cord blood cells supplemented (ie mixed, bound or augmented) with a group of embryo-like stem cells derived from the placenta.

The supplemented groups are very viable and include a group of cells that are pluripotent or redundant stem cells, such as, for example, cells displaying a CD34 + / CD38 +, CD34 + / CD38- or CD34- / CD38- phenotype. .

According to the present invention, a supplemented group of stem cells of the present invention is 100,000,000: 1, 50,000,000: 1, 20,000,000: 1, 10,000,000: 1, 5,000,000: 1, 2,000,000: when compared based on the total number of nucleated cells in each group. 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; 1: 20,000,000; 1: 50,000,000; Or embryonic-like stem cells and other stem or progenitor cells in a ratio of 1: 100,000,000. In one embodiment, a number (or group) of embryo-like placental stem cells are added to a number of umbilical cord blood prior to delivery to the patient in need thereof.

The invention also provides a method of supplementing a group of cord blood cells with a group of embryo-like stem cells. In one embodiment, the two groups are physically mixed. In another aspect of this example, the two groups are physically mixed and then treated with growth factors such as cytokines and / or all interleukins to induce cell differentiation. In another aspect of this embodiment, cord blood cells and / or embryo-like stem cells are treated with growth factors such as cytokines and / or interleukins, and then physically mixed to induce cell differentiation.

The invention also provides a method of treating a patient in need thereof by administering a group of cord blood cells supplemented with embryo-like stem cells. In one embodiment, replenishing a group of cord blood cells with embryo-like stem cells occurs by mixing cord blood cells with embryo-like stem cells prior to administering the supplemented group to a patient. In another embodiment, replenishing a group of cord blood cells with embryo-like stem cells occurs upon administration of the supplemented group to a patient, such as for example simultaneously administering cord blood cells and embryo-like stem cells. do. In another embodiment, replenishing a group of cord blood cells with embryo-like stem cells may, for example, be administered with embryo-like stem cells, such as before or after administration, separated from administration of cord blood cells. Occurs after administration of blood.

In one embodiment, the present invention provides a method for supplementing cord blood cells with embryo-like stem cells, characterized in that the mixture is contained in one bag. In another embodiment, the present invention provides a method for supplementing cord blood cells with embryo-like stem cells, wherein cord blood cells and embryo-like stem cells are contained in different bags, respectively. Such “two bags” compositions may be mixed upon or prior to administration to a patient in need thereof.

In another embodiment, a certain number (or group) of embryo-like placental stem cells are adjusted before they are added to a certain number of cord blood before delivery to a patient in need and before mixing. For example, in one aspect of this embodiment, a group of embryo-like placental stem cells are added to a certain number of umbilical cord blood and mixed with cytokines (eg, IL-1α, IL-1β, IFN-γ, TFN), retinoic acid, transferrin, hormones (e.g. androgen, estrogen, insulin, prolactin, triiodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA, By exposure to DMSO, NMF, DMF, matrix elements (e.g. collagen, heparan sulfate, Matrigel ^™ ), or mixtures thereof, adipogenic, chondrogenic, osteogenic ( It may also be induced to differentiate into particularly cell lineages such as osteogenic, hematopoietic, myogenic, vasogenic, neurogenic, and hepatocellular specific cell lineages.

In another aspect of this embodiment, a group of embryo-like placental stem cells are added to, and mixed with, a certain number of cord blood, for example human delta-1 and human serrate-1 polypeptides, or mixtures thereof It can be adjusted by exposure to a component that suppresses differentiation, such as.

In another embodiment, a number (or group) of non-regulated embryo-like placental stem cells and a number of umbilical cord blood are mixed and the mixed cell population is adjusted before delivery to the patient in need thereof. In certain embodiments, the group of mixed embryo-like placental stem cells and umbilical cord blood cells are adjusted with components to induce or inhibit cell differentiation as described above.

In certain embodiments, a group of embryo-like stem cells of the invention is added to or mixed with a group of umbilical cord blood cells prior to administration to a patient in need thereof. In another specific embodiment, a group of embryo-like stem cells of the invention is added to or mixed with a group of umbilical cord blood cells during or concurrent with administration to a patient in need thereof. In another specific embodiment, a group of embryo-like stem cells and a group of umbilical cord blood cells of the present invention are administered continuously to a patient in need thereof. In one embodiment, a group of embryo-like stem cells is administered first, and a group of umbilical cord blood cells is administered second. In another embodiment, a group of umbilical cord blood cells is administered first, and a group of embryo-like placental stem cells is administered second.

A group of cord blood cells spiked into embryonic-like stem cells includes, but is not limited to, processing the spiked group by introducing nutrients, hormones, vitamins, growth factors, or mixtures thereof into the culture medium. , May be cultured under a variety of conditions, induced to proliferate, and / or induced to differentiate. Serum and other growth factors can be added to the culture medium. Growth factors are generally proteins and include, but are not limited to, cytokines, lymphokines, interferons, colony stimulating factors (CSFs), interferons, chemokines, and interleukins. Other growth factors that can be used include recombinant human hematopoietic growth factor, stem cell factor, thrombopoietin (Tpo), granulocyte boney-stimulating factor (G-CSF), leukemia inhibitory factor, basal fibroblast growth factor, including ligands. , Placental growth factor and epidermal growth factor, but is not limited thereto. In one embodiment, the supplemented group is treated with growth factors to induce differentiation into various cell types. In another embodiment, the spiked group is treated with growth factors to induce differentiation into specific cell types. In another embodiment, the supplemented group is treated with growth factors such that differentiation to specific cell types is inhibited or prevented.

In certain embodiments of the invention, a method of replenishing a group of cord blood comprises (a) inducing differentiation of embryo-like stem cells, (b) mixing a group of cord blood cells with an embryo-like stem cell, and (c) Administration of the mixture to a patient in need thereof.

In another embodiment of the present invention, a method of replenishing a group of cord blood comprises (a) mixing a group of cord blood cells with an embryo-like stem cell; (b) inducing differentiation of a spiked group of cord blood cells and an embryo-like stem cell mixture and (c) administering the mixture to a patient in need thereof.

In another embodiment of the invention, a method of supplementing a group of cord blood comprises (a) administering a mixture of cord blood cells supplemented with embryo-like stem cells to a patient in need thereof, and (b) inducing differentiation of the mixture and ( c) administration of 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 according to methods well known in the art. In certain embodiments, growth factors are: GM-CSF, IL-4, Flt3L, CD40L, IFN-alpha, TNF-alpha, IFN-gamma, IL-2, IL-6, Retinoic acid, basal fibroblast growth factor , TGF-beta-1, TGF-beta-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensin II (AII), AII AT ₂ type 2 receptor agonists, or analogs or fragments thereof.

In one embodiment, the stem or progenitor cells are well known in the art to which the present invention pertains, such as, for example, exposure to β-mercaptoethanol or DMSO / butyrated hydroxyanisole according to the methods described below. Thus induced to differentiate into nerves.

In another embodiment, the stem or progenitor cells are adipocytes according to methods well known in the art, such as, for example, exposure to dexamethasone, indomethacin, insulin and IBMX according to the methods described below. Is induced to differentiate.

In another embodiment, stem or progenitor cells are induced to differentiate into chondrocytes according to methods well known in the art, such as, for example, exposing to TGF-beta-3 according to the methods described below.

In other embodiments, stem or progenitor cells are well known in the art, for example, by exposing to dexamethasone, ascorbic acid-2-phosphate and beta-glycerophosphate according to the methods described below. Thus induced to differentiate into osteocytes.

In another embodiment, the stem or progenitor cells are induced to differentiate into hepatocytes according to methods well known in the art, such as, for example, exposing to IL-6 +/- IL-15 according to the methods described below. do.

In another embodiment, the stem or progenitor cells are, for example, basic fibroblast growth factor and transforming growth factor beta-1, TGF-beta-1 according to the method described below. Is induced to differentiate into pancreatic cells according to methods well known in the art.

In another embodiment, the stem or progenitor cells are exposed to cardiotropin-1, for example, to basal fibroblast growth factor, TGF-beta-1 and epidermal growth factor, according to the methods described below. Is induced to differentiate into heart cells according to methods well known in the art, such as exposing to human myocardial extracts.

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

In another embodiment, embryonic-like stem cells are, for example, erythropoietin, cytokines, lymphokines, interferons, colony stimulating factors, interferons, including interferons, chemokines, ligands, recombinant human hematopoietic growth Stimulated to proliferate by administration of factors, stem cell factors, thrombopoietin (Tpo), interleukin, and granulocyte colony-stimulating factor (G-CSF) or other growth factors.

In another embodiment, embryo-like stem cells are collected from the placenta, for example by using viral vectors such as adenovirus or retroviral vectors or by mechanical means such as liposomes or chemically mediated DNA uptake. Genetically engineered before or after collection.

Vectors containing transgenes are for example transfection, transformation, transduction, electroporation, infection, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, liposomes It may be introduced into cells of interest by methods well known in the art, such as, LIPOFECTIN ^™ , lysosomal fusion, use of synthetic cationic lipids, gene guns or DNA vector transporters. The transgene is thus delivered to daughter cells, such as, for example, daughter embryo-like stem cells or progenitor cells formed by division of embryo-like stem cells. For various techniques for the transformation or transfection of mammalian cells, see Keown et al., 1990, Methods Enzymol. 185: 527-37; See Sambrook et al., Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY.

Preferably, the transgene can be introduced using any technique as long as it does not destroy the nuclear membrane or other existing cellular or genetic structure of the cell. In certain embodiments, the transgene is inserted into nuclear genetic material by microinjection. Microinjection and cellular structure of cells are well known in the art and generally performed.

For stable transfection of cultured mammalian cells, such as embryonic-like stem cells, only a small number of cells need to integrate foreign DNA into their genomes. The efficiency of the integration depends on the vector and the transfection technology used. To identify and select integrators, genes encoding selective markers (eg antibiotic resistance) are introduced into host embryo-like stem cells along with the gene sequence of interest. Preferred selective markers include those that confer resistance to drugs such as G418, hygromycin, and mesotrexate. Cells stably transfected with the nucleic acid to be introduced can be identified by drug selection (eg cells containing the selective marker gene survive while other cells die). Such methods are particularly useful in methods involving homologous recombination into mammalian cells (eg embryonic-like stem cells) prior to introduction or transplantation of recombinant cells into a patient or subject.

Many selection systems may be used to select modified host embryo-like cells. In particular, the vector may comprise a detectable or selectable marker. Other methods of selection are herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypersantin-guanine phosphoribosyltransferase (applied to tk-, hgprt- or aprt-cells, respectively) Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and selections for other markers including adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes. However, it is not limited thereto. In addition, antimetabolite resistance can be used as a basis for selection for the following genes: dhfr (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77) which confers resistance to mesotrexate: 3567; O'Hare et al., 1981, Proe. Natl. Acad. Sci. USA 78: 1527); Gpt conferring resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); Neo (Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1) conferring resistance to aminoglycoside G418; And hygro conferring resistance to hygromycin (Santerre et al., 1984, Gene 30: 147).

The transgene can be integrated into the genome of the cell of interest, with random integration being preferred. In other embodiments, the transgene can be, for example, directed homologous recombination (ie, "knock-in" or "knock-out" of the gene of interest into the cell of interest), Chappel, U.S. Pat. 5,272,071; And PCT Patent No. 91/06667, issued May 16, 1991; US Patent No. 5,464,764, Capecchi et al., Issued November 7, 1995; US Patent No. 5,627,059, Capecchi et al., Issued May 6, 1997; Such as US Pat. No. 5,487,992, Capecchi et al., Issued Jan. 30, 1996).

Methods for producing cells subjected to target genetic engineering through homologous recombination are well known in the art. The construct will contain at least a portion of the gene of interest that has undergone the desired genetic engineering and will include a homologous portion of the target genetic locus, an internal copy of the target gene in the host genome. In contrast to those used for homologous recombination, DNA constructs for partial integration do not need to include homologous regions to coordinate recombination. The marker can be included into a target construct or random construct to perform positive and negative selections for insertion of the transgene.

In order to make homologous recombinant cells such as, for example, homologous recombinant embryo-like stem cells, internal placental cells or exogenous cells cultured in the placenta, homologous recombinant vectors may be carried by the vector and the genes of interest within the genome of the target cell and the gene of interest. Genes of interest at the 5- and 3-terminals are flanked with gene sequences internally present in the genome of the target cell for homologous recombination that occurs between. Additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with internal genes in the genome of the target cell. Generally several tens of kilobases (kb) of flanking DNA (5- and 3- termini) are included in the vector. Methods of making homologous recombinant animals from homologous recombinant vectors and recombinant stem cells are well known in the art (eg Thomas and Capecchi, 1987, Cell 51: 503; Bradley, 1991, Curr. Opin. Bio / Technol 2: 823-29; and PCT Patent Nos. 90/11354, 91/01140, and 93/04169.

In one embodiment, the genome of the foreign cell cultured in the placenta according to the method of the present invention is the target of gene targeting via homologous recombination or random integration.

In certain embodiments, Bonadio et al. (US Pat. No. 5,942,496, methods and compositions for delivering various genes to bone marrow cells, issued August 24, 1999; and PCT Patent No. 95/22611, for stimulating bone marrow cells Methods and Compositions, issued August 24, 1995, were used to introduce nucleic acids into cells of interest, such as foreign cells cultured in the placenta, for example stem cells, progenitor cells or bone marrow precursor cells. .

Embryonic-like Stem Cells and Use of a Group of Stem Cells Replenished

Embryonic-like stem cells can be obtained from perfused placenta according to the method described in co-pending US patent application Ser. No. 01 / 076,180, issued February 13, 2002.

Placental stem cells (embryonic-like stem cells) can be induced to differentiate into specific cell types, ex vivo or in vivo . For example, fluripotent embryo-like stem cells can be injected in vivo into damaged organs for long-term angiogenesis and damage repair. Such injuries are mainly myocardial infarction, seizure abnormalities, multiple sclerosis, fainting, hypotension, cardiac arrest, ischemia, inflammation, impaired cognitive impairment, radiation damage, cerebral palsy, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, Leigh disease, AIDS It may be due to conditions and abnormalities, including but not limited to dementia, memory impairment, muscular atrophic lateral sclerosis, ischemic kidney disease, brain or spinal cord cord trauma, heart-lung bypass, glaucoma, retinal ischemia, or retinal trauma. .

Embryonic-like stem cells isolated from the placenta, alone or in combination with stem or progenitor cell populations (ie, the cell compositions of the present invention), in certain embodiments, are Tay-Sachs, Niemann-Pick , Lysosomes such as Fabry's, Gaucher's, Hunter's, and Hurler's syndrome and other gangliosidosis, mucopolysaccharidosis, and glycogenosis It can be used in enzyme replacement therapy of autologous or heterologous to treat certain diseases or conditions, including, but not limited to, accumulation diseases.

In other embodiments, embryonic-like stem cells, alone or in combination with stem or progenitor cell populations, may be a genetic metabolic disorder, adreoleukodystrophy, cystic fibrosis, glycogen accumulation disease, hyperthyroidism, Sickle cell anemia, Pearson syndrome, Pompe disease, phenylketonuria, porphyrias, maple syrupuria, homocystinuria, mucoplysaccharidosis, It can be used as a transgene carrier of autologous or heterologous to treat chronic granulomatous disease and tyrosineemia and Tay-Sachs disease or to treat cancer, tumor or other pathological conditions.

In other embodiments, the cell composition may be used for corneal epithelial injury, cartilage tissue treatment, facial dermabrasion, mucosa, tympanic membrane, intestinal lining, neurological structures (e.g., retina, auditory nerve of basement membrane, olfactory nerve of olfactory epithelium). Of autologous or heterologous origin, including but not limited to treatment, repair of bums and wounds for traumatic damage of skin, or reconstitution of damaged or diseased organs or tissues It can be used in tissue regeneration or relocation treatments or protocols.

In some embodiments, the large number of embryo-like stem cells and / or progenitor cells obtained using the method according to the present invention reduces the need for a large number of bone marrow donations. Bone marrow transplantation requires injection of about 1 × 10 ⁸ to 2 × 10 ⁸ bone marrow mononuclear cells per kg body weight of the patient (ie about 70 ml of bone marrow for a 70 kg donor). Obtaining 70 ml requires excessive donation and severe loss of blood during the donation process. In certain embodiments, cells resulting from small amounts of bone marrow donation (about 7-10 ml) can be amplified in placental bioreactors before being injected into the recipient.

In addition, small amounts of stem cells and progenitor cells generally circulate in the bloodstream. In other embodiments, such exogenous stem cells or exogenous progenitor cells are collected by apheresis where blood is drawn, one or more components are selectively removed, and injected into the donor, the remainder of the blood. The extraneous sheaths recovered by the biopsy are propagated in placental bioreactors, thus completely eliminating the need for bone marrow donation.

While blood cells regenerate between chemotherapy treatments, the cancer has time to grow and become resistant to chemotherapy drugs by natural selection. Therefore, the longer the application of chemotherapy and the shorter the time between treatments, the greater the chance of successfully killing the cancer. To reduce the time between chemotherapy treatments, embryo-like stem cells or progenitor cells collected according to the present invention may be introduced to a patient alone or in combination with other stem cells or progenitor cell populations. Such treatment will reduce the period of time for which patients have low blood cell levels and therefore help to speed up chemotherapy treatment.

Embryonic-like stem cells, progenitor cells, foreign cells, or engineered cells obtained from the placenta according to the present invention can be used alone or in combination with other stem or progenitor cell populations for in vivo manufacture of tissues or organs. The method of the invention uses cells obtained from the placenta, such as, for example, embryonic-like stem cells, progenitor cells, or foreign cells or progenitor cells, to inoculate the matrix and incubate under appropriate conditions to allow the cells to differentiate and reside in the matrix. It involves doing. Tissues and organs obtained in accordance with the methods of the present invention may be used for a variety of purposes, including research and therapeutic purposes.

A group of stem cells supplemented with embryonic-like stem cells of the present invention may be used for various prophylactic or reinforcement, repair or repair of tissues or organs of the body by implantation, transfection or infusion of a preferred cell population such as stem or progenitor cell population. It can be used in therapeutic protocols. Embryonic-like stem cells of the present invention and a group of supplemented stem cells can be used to augment or supplement existing tissue, to introduce new or altered tissue, or to connect biological tissue or structures. A group of stem cells supplemented with embryonic-like stem cells of the invention may also be used in place of embryonic stem cells in a treatment protocol that typically involves the use of embryonic stem cells.

In a preferred embodiment of the present invention, embryonic-like stem cells and supplemented stem cell populations can be used for autologous and allogenic uses, including hematopoietic transplantation which is not compatible with HLA type. Depending on the use of embryo-like stem cells with allogeneic hematopoietic transplantation, it may be necessary to treat the host to reduce the immune rejection response of the donor cells. This is US Pat. No. 5,800,539, issued September 1, 1998; And U.S. Patent No. 5,806,529, issued September 15, 1998, which is hereby incorporated by reference in its entirety.

For example, embryonic-like stem cells and supplemented stem cell populations of the invention may be, for example, liver, pancreas, kidney, lung, nervous system, muscular system, bone, bone marrow, thymus, spleen, muscle tissue, gonads, or stems of hair. Or in therapeutic transplant protocols to supplement or augment progenitor cells.

Embryonic-like stem cells and supplemented stem cell populations may be used to identify specific classes of progenitor cells (eg chondrocytes, hepatocytes, hematopoietic cells, pancreatic parenchymal cells, neuroblasts, muscle) in therapeutic or research protocols in which progenitor cells are commonly used. Progenitor cells, etc.).

Embryonic-like stem cells and supplemented stem cell populations of the present invention can be used for augmentation, repair or supplementation of cartilage, tendons, or ligaments. For example, in some embodiments, prostheses (eg hip prostheses) are coated with alternate cartilage tissue constructs grown from embryonic-like stem cells of the present invention. In another embodiment, the joint (eg knee) is reconstituted with cartilage tissue constructs grown from embryonic-like stem cells. Cartilage tissue structures can also be applied to reconstructive surgery for joints of various other types. (For protocols on this, see Resnick, D. and Niwayama, G. et al., 1988, Diagnosis of Bone and Joint Disorders, 2d ed., W.B. Saunders Co.).

Embryonic-like stem cells and supplemented stem cell populations of the invention can be used to repair tissue and organ damage resulting from trauma, metabolic abnormalities, or disease. In one such embodiment, the patient has an embryo-like stem to regenerate or recover tissue or organs damaged as a result of the disease, such as, for example, palpation of the immune system after chemotherapy or irradiation, recovery of heart tissue after myocardial infarction. The cells can be administered alone or in combination with other stem or progenitor cell populations.

Embryonic-like stem cells and supplemented stem cell populations of the invention can be used to augment or replace bone marrow cells in bone marrow transplantation. Human autologous and allogeneic bone marrow transplantation is commonly used as a treatment for diseases such as leukemia, lymphoma and other life-threatening disorders. However, a disadvantage of this means is that a large amount of donor bone marrow must be removed to ensure that there are enough cells for transplantation.

Embryonic-like stem cells and supplemented stem cell populations of the present invention provide stem and progenitor cells that can reduce the need for many bone marrow donations. According to the method of the present invention, a small amount of bone marrow donation is obtained and the number of stem and progenitor cells is increased by culturing and propagating in the placenta prior to injection or transplantation into the recipient.

In certain embodiments, embryonic-like stem cells supplemented with stem cells of the present invention may comprise Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's, Hunter. (Hunter's) and lysosomal accumulations such as Hurler's syndrome and other gangliosidoses, mucopolysaccharidoses, and glycogenogenes Can be used in enzyme replacement therapy of autologous or heterologous tissue to treat a particular disease or condition.

In another embodiment, the cells are adrenoleukodystrophy, cyst fibrosis, glycogen accumulation disease, hyperthyroidism, sickle cell anemia, Pearson syndrome, Pompe disease, phenylkenonyria (PKU), porphyrias, maple syrup urine disease, homocystinuria, mucoplysaccharide nosis, chronic granulomatous disease and tyrosineemia and tei-sa It can be used as a transgene carrier of autologous or heterologous to treat genetic metabolic disorders such as Tay-Sachs disease or to treat cancer, tumors or other pathological conditions.

In other embodiments, the cells may be of corneal epithelial injury, cartilage tissue treatment, facial dermabrasion, mucosa, tympanic membrane, intestinal lining, neurological structures (e.g., retina, auditory nerve of basement membrane, olfactory nerve of olfactory epithelium). Of autologous tissue, including, but not limited to, treatment, burns and wound repair for traumatic damage of the skin, hair transplantation or reconstruction of damaged or diseased organs or tissues It may be used in tissue regeneration or relocation treatments or protocols derived from heterologous.

In some embodiments, the large number of embryo-like stem cells and / or progenitor cells obtained using the method according to the present invention reduces the need for a large number of bone marrow donations. Bone marrow transplantation requires injection of about 1 × 10 ⁸ to 2 × 10 ⁸ bone marrow mononuclear cells per kg body weight of the patient (ie about 70 ml of bone marrow for a 70 kg donor). Obtaining 70 ml requires excessive donation and severe loss of blood during the donation process. In certain embodiments, cells resulting from small amounts of bone marrow donation (about 7-10 ml) can be amplified in placental bioreactors before being injected into the recipient.

In another embodiment, embryonic-like stem cells and supplemented stem cell populations of the invention may be used for supplemental therapeutic use in addition to chemotherapy. Most chemotherapeutic agents used to target and destroy cancer cells work by killing all proliferating cells, all cells that divide the cell. Because bone marrow is one of the most actively proliferating tissues in the body, hematopoietic stem cells are frequently damaged or destroyed by chemotherapeutic agents, resulting in reduced or interrupted blood cell production. Before restarting chemotherapy, chemotherapy should be discontinued at regular intervals to ensure that the patient's hematopoietic system provides sufficient blood cell supply. Prior to that, it took a month or more to allow the stem cells at rest to multiply and increase white blood cell counts to an acceptable level to resume chemotherapy treatment (when resumed, bone marrow stem cells are destroyed). do).

While blood cells regenerate between chemotherapy treatments, cancer has time to grow and become resistant to chemotherapy drugs by natural selection. Therefore, the longer the application of chemotherapy and the shorter the time between treatments, the greater the chance of successfully killing the cancer. To reduce the time between chemotherapy treatments, embryo-like stem cells or progenitor cells collected according to the present invention can be introduced into a patient. Such treatment will reduce the period of time that patients have low blood cell counts and therefore help to speed up chemotherapy treatment.

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

Pharmaceutical composition

The present invention includes a pharmaceutical composition comprising an embryo-like stem cell of the present invention and a population of stem cells supplemented. The invention relates to one or more cells, such as, for example, mesoderm, fat, chondrocytes, osteocytes, myocytes, vascular, neuronal, epithelial, liver, kidney, pancreatic, and / or hematopoietic lineage cells. Before or after transplantation of engineered or unengineered human progenitor stem cells capable of exerting sufficient effects to inhibit, regulate and / or modulate differentiation of placental derived human fluripotent and multipotent progenitor stem cells into the lineage. Pharmaceutical compositions comprising doses and / or repeated doses effective for single or repeated administrations are included.

According to this embodiment, embryonic-like stem cells and supplemented stem cell populations of the invention can be formulated with injections (see, eg, PCT Patent No. 96/39101, whereby the above references are incorporated by reference in their entirety). Is included herein). In an alternative embodiment, the cells and tissues of the present invention are described in U.S. Patent Nos. 5,709,854; 5,516,532; 5,516,532; It may be formulated using polymerized or cross linking hydrogels as described in US Pat. No. 5,654,381, such that the references are hereby incorporated by reference in their entirety. Embryonic-like stem cells may be administered in a state obtained from the placenta, or spiked into umbilical cord blood and administered as a mixed cell composition, or may be placed in a physiologically acceptable buffer or solution for administration to an individual. It may be.

The invention also encompasses pharmaceutical compositions having high concentrations (or large groups) of homogeneous embryo-like stem cells, wherein one or more such populations are combined with other stem or progenitor cells for transplantation and other uses. Or as a mixture mixed with other stem or progenitor cells. Other stem or progenitor cells include adipogenic, chondrogenic osteocytic, hematopoietic, muscular, angiogenic, neurogenic, and hepatic stem cells; Mesenchymal stem cells, stromal cells, epithelial cells, hepatocytes, keratinocytes, and nerves, maelin, muscle, blood, bone marrow, skin, heart, connective tissue, lungs, kidneys, liver, and pancreas (eg Pancreatic islet cells), including but not limited to, stem or progenitor cells of a particular cell type, tissue or organ.

In one embodiment, the present invention provides a high concentration (or large group) of CD34 + / CD38-cells; And homogeneous hematopoietic stem cells, including but not limited to, CD34- / CD38-cells. One or more such cell populations are used in combination with other stem cells or in a mixture with other stem cells for transplantation and other uses. In certain embodiments, the pharmaceutical composition comprises embryonic-like placental stem cells of the invention and cord blood hematopoietic cells such as, for example, CD34 + / CD38 + hematopoietic cells.

One or more such cell populations are used with cord blood hematopoietic cells, such as CD34 + / CD38 + hematopoietic cells, or in admixture with cord blood hematopoietic cells for transplantation and other uses.

In one embodiment, the invention provides a heterogeneous group of nucleated cells comprising embryo-like placental stem cells. In certain embodiments, heterologous groups of nucleated cells (other than the pure group of CD34 + cell embryo-like placental stem cells) are preferred.

In another embodiment, the present invention provides a mixed group of cells (eg cord blood cells and embryo-like placental stem cells). The group of mixed cells may or may not be frozen. Such mixed groups may be stored and / or used in one container, such as for example one bag or one syringe.

In another embodiment, the present invention provides two or more separate or distinct groups of different cell types (eg cord blood cells and embryo-like placental stem cells). Each separate group is a separate container containing one type of cell or cell group, for example one bag (e.g., a blood storage bag from Baxter, Becton-Dickinson, Medcep, National Hospital Products or Terumo) or one It may be used and / or stored in separate containers, such as dog syringes. In some aspects of these examples, the present invention provides separate containers of different cell types to be mixed prior to administration. Such cells may or may not be frozen.

In one particular embodiment, cord blood cells are contained in one bag and embryo-like placental stem cells are contained in a second bag.

In another embodiment, the present invention provides embryonic-like placental stem cells "conditioned" prior to freezing.

In another embodiment, a group of cells, including but not limited to embryonic-like placental stem cells, can be adjusted to remove erythrocytes and / or granulocytes according to standard methods. This leaves a group of nucleated cells rich in embryo-like placental stem cells. Such a rich group of embryo-like placental stem cells may be used unfrozen, or frozen for later use. If a group of cells are frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife ^™ Cell Freezing Medium (Cascade Biologics) are added to the rich group of cells before freezing).

In another embodiment, a group of cells, including but not limited to embryonic-like placental stem cells, may be adjusted to remove red blood cells and / or granulocytes after being frozen and thawed.

According to the invention, components that induce cell differentiation can be used to modulate a group of embryo-like stem cells. In certain embodiments, components that induce cell differentiation can be added to a group of cells in a container. Its components include Ca ²⁺ , EGF, α-FGF, β-FGF, PDGF, keratinocyte growth factor (KGF), TGF-β, cytokines (e.g. IL-1α, IL-1β, IFN γ, TFN), retinoic acid, transferrin, hormones (e.g. androgen, estrogen, insulin, prolactin, triiodothyronine, hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO , NMF, DMF, matrix elements (eg, collagen, heparan sulfate, Matrigel ^™ ), or mixtures thereof, but are not limited thereto.

In other embodiments, components that inhibit cell differentiation can be added to a group of embryo-like stem cells. In certain embodiments, components that inhibit differentiation can be added to a group of cells in a container. Components include human delta-1 and human serrate-1 polypeptides (Sakano et al., US Pat. No. 6,337,387, "Differentiation-Inhibiting Polypeptides", issued January 8, 2002), leukemia inhibitory factor (LIF), and Stem cell factors, but are not limited thereto.

In certain embodiments, one or more groups of embryo-like stem cells are delivered to a patient in need thereof. In certain embodiments, two or more groups of fresh (unfrozen) cells are delivered from one container or a single delivery system.

In another embodiment, two or more groups of frozen and thawed cells are delivered in one container or a single delivery system.

In another embodiment, fresh (unfrozen) cells of each of the two or more groups are transferred to and delivered from one container or a single delivery system. In another aspect of this embodiment, each group is delivered from a different IV infusion bag (eg, a bag of Baxter, Becton-Dickinson, Medcep, National Hospital Products or Terumo). The contents of each container (eg IV infusion bag) may be delivered through a separate delivery system, and each container may be "piggybacked" so that their contents are combined prior to delivery from one delivery system. Or mixed. For example, two or more groups of cells are fed and / or mixed into a common flow tube (eg a tube), or they are fed and / or mixed into one common container (eg a chamber or a bag).

According to the invention, cells of two or more groups can be combined prior to, during or at the time of administration or simultaneously with delivery.

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

In one embodiment, the present invention provides a method for treating or preventing a disease or condition of a subject comprising administering to the subject. Such treatment or prevention here requires a therapeutically effective amount of the embryo-like stem cells of the invention, or a supplemented cell population.

In another embodiment, the present invention provides a method of treating or preventing a disease or condition of a subject comprising administering to the subject. Such treatment or prevention here requires a therapeutically effective amount of the embryo-like stem cells of the invention.

Embryonic-like stem cells of the present invention are expected to have an anti-inflammatory effect when administered to individual empirical inflammations. In a preferred embodiment, embryonic-like stem cells or supplemented cell populations of the invention can be used to treat any disease, condition or condition resulting from or associated with inflammation. Inflammation can include, for example, the brain, spinal cord and peripheral nervous system; Vascular tissues including cardiac tissues; Pancreas; Other organs of the small intestine or digestive tract; lungs; kidney; liver; Reproductive organs; Epithelial tissue; Or muscles including endoderm tissue; It may be present in any organ or tissue, such as the nervous system.

Embryonic-like stem cells or supplemented cell populations of the invention can also be used to treat autoimmune or immune system-related abnormalities, including those associated with inflammation. Thus, in some embodiments, the present invention includes a method of treating an individual with an autoimmune disease or condition comprising administering to the subject a therapeutically effective amount of a cell or population of cells of the invention. Such disease or condition may be, but is not limited to, diabetes mellitus, muscular atrophic lateral sclerosis, myasthenia gravis, diabetic neuropathy or lupus. In related embodiments, embryonic-like stem cells or supplemented cell populations of the invention can be used to treat immune-related abnormalities such as chronic or acute allergies.

In certain embodiments, the disease or condition is all diseases referred to herein and dysplastic anemia, myelodysplastic disease, myocardial infarction, seizure abnormalities, multiple sclerosis, fainting, hypotension, cardiac arrest, ischemia, inflammation, age-related cognitive function. Damage, radiation injury, cerebral palsy, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, Leigh disease, AIDS, dementia, memory loss, muscular dystrophy (ALS), ischemic kidney injury, brain or spinal cord trauma, heart-lung bipolar Pass, Glaucoma, Retinal Ischemia, Retinal Trauma, Lysosomal Accumulation, Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's, Hunter's, and Hurler's Disease , Other gangliosidosis, mucopolysaccharidosis, glycogenosis, genetic metabolic abnormalities, adrenoleukodystrophy, cyst fibrosis, glycogen accumulation disease, hyperthyroidism Hypothyroidism, sickle cell anemia, Pearson syndrome, Pompe disease, phenylketonuria, porphyrias, maple syrup urine, homocystinuria, mucus Polysaccharides, chronic granulomatous diseases and tyrosinemia and Tay-Sachs disease, cancer, tumors or other pathological or oncological conditions.

In another embodiment, the cells can be used to treat any kind of injury due to trauma, including trauma, particularly inflammation. Examples of such trauma-related conditions include central nervous system (CNS) injury, injury of the peripheral nervous system (PNS), including tissues surrounded by the brain, spinal cord, or CNS; Or injury to any part of the body. Such trauma may be caused by an accident or may be caused by normal or abnormal consequences of a surgical procedure such as surgery or angioplasty. Trauma may be associated with rupture or blockage of blood vessels such as fainting or phlebitis, for example. In certain embodiments, the cells may be of corneal epithelial injury, cartilage tissue treatment, facial dermabrasion, mucosa, tympanic membrane, intestinal lining, neurological structure (e.g., retina, auditory nerve of basement membrane, olfactory nerve of olfactory epithelium). Regeneration of autologous or heterologous tissue, including, but not limited to, treatment, repair of burns and wounds for traumatic damage of the skin, or reconstitution of damaged or diseased organs or tissues, or It can be used in relocation treatments or protocols.

In certain embodiments, the disease or condition is aplastic anemia, myelodysplasia, leukemia, bone marrow disorder or hematopoietic disease or condition. In another particular embodiment, the object is a human.

In another embodiment, the present invention provides a method of treating a subject having a disease, condition or condition arising from or associated with inflammation. In another embodiment, the present invention provides a method of treating a subject having a neuropathic disease, disorder or condition. In a more specific embodiment, the neuropathic disease is ALS. In another specific embodiment, said neuropathic disease is Parkinson's disease. In another specific embodiment, the disease is a vascular or cardiovascular disease. In another specific embodiment, the disease is arteriosclerosis. In another specific embodiment, the disease is diabetes.

In certain embodiments, the pharmaceutical compositions of the present invention comprise a number of umbilical cord blood to which embryo-like placental stem cells have been added, as described above.

A large number of embryo-like stem cells, or supplemented cell populations, once administered, form long-term "colonies" and can be planted into the host. This results in a host that is essentially chimeric. Because chimeras are generally more energetic and resilient in other genetic contexts, such chimerism is expected to promote host health and well-being. As such, embryo-like stem cells may not only be administered to an individual suffering from a particular disease, condition or condition, but may also be administered to an individual for the purpose of increasing the overall health and well-being of the individual.

Embryonic-like stem cells or supplemented cell populations of the invention can be treated with compounds that modulate TNF-α activity prior to administration to a subject. Such compounds are well described in co-pending U.S. Provisional Application No. 60 / 372,348, issued April 12, 2002, which is hereby incorporated by reference in its entirety. Preferred compounds are IMiDs and SelCids, and more preferred compounds are those available under the trade names Actimid ^™ and Revimid ^™ .

A particularly useful aspect of the embryo-like stem cells of the present invention is that, in some embodiments, there is no need to test the HLA-type of the cells prior to administration. That is, embryo-like stem cells can be obtained from a heterologous donor, or from a plurality of heterologous donors, which are transplanted into a subject in need, and the transplanted cells remain indefinitely in the host. The elimination of the need for HLA typing greatly facilitates both the transplantation procedure itself and the identification of the donor for transplantation. However, embryo-like stem cells or supplemented cell populations containing them may have HLA match between donor and recipient prior to administration.

The inventors have found that loading these cells increases the efficiency of treatment of individuals with embryonic-like stem cells or supplemented cell populations. Preconditioning involves storing the cells in a gas-pass container at −5-23 ° C., 0-10 ° C., or preferably 4-5 ° C. for a period of time. The period is 18 hours to 21 days, 48 hours to 10 days, preferably 3-5 days. The cells may be cryopreserved prior to shipment, or preferably immediately prior to administration.

Thus, in one embodiment, the present invention provides a method for treating a subject comprising administering to said subject embryo-like stem cells collected from at least one donor. The term "donor" as used herein refers to adults, children. Infant, or preferably placenta. In another preferred embodiment, the method comprises administering to said individual embryo-like stem cells obtained and collected from a plurality of donors. In certain embodiments the embryo-like stem cells are stem cells obtained from a plurality of donors. When collected from a plurality of donors, the dosage unit, wherein the “dosage unit” is a collection obtained from one donor, may be collected before administration, may be administered continuously, or alternatively may be administered. . In another embodiment of the invention, the embryo-like stem cells are mixed with umbilical cord blood or spiked into umbilical cord blood. The mixture is then administered to the subject. In a more specific embodiment of the method, the ratio of embryo-like stem cells to 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.

Administration of Stem Cells: Dosage

A particularly useful aspect of the present invention is the administration of high doses of stem cells to an individual; Such numbers of cells are much more effective than the materials from which they are derived (eg bone marrow or cord blood). In this context, “high dose” refers to 5, 10, 15, or 20 or more multiples of total nucleated cells than administered, for example, in bone marrow transplants. Nucleated cells here include stem cells, in particular embryonic-like stem cells. Generally, patients receiving stem cell infusions, for example for bone marrow transplantation, receive one unit of cells. One unit here is about 1 × ^{10 9} nucleated cells, which correspond to 1-2 × ^{10 8} stem cells. For high dose treatment, therefore, the patient may be 3 billion, 5 billion, 10 billion, 15 billion, 20 billion, 30 billion, 40 billion, 50 billion or more, or alternatively 3, 5, 10, 20, 30 , 40, or 50 units or more of total nucleated cells, ie embryonic-like stem cells alone, or other stem or progenitor cell populations spiked with embryo-like stem cells (ie, umbilical cord blood spiked with embryo-like stem cells) ).

In a more preferred embodiment, for example, the subject is given about 15 units of spiked cord blood, which contains about 7.5 billion cord blood cells and 500 million embryo-like stem cells. Thus, in one embodiment, the number of nucleated cells administered to an individual is at least five times greater than the number of cells generally administered in bone marrow replacement. In another particular embodiment of the invention, the number of nucleated cells administered to the individual is at least 10 times greater than the number of cells generally administered in bone marrow replacement. In another specific embodiment of the present invention, the number of nucleated cells administered to the individual is at least 15 times greater than the number of cells generally administered in bone marrow replacement. In another embodiment of the invention, the total number of nucleated cells comprising stem cells administered to the subject is 1-100 × ^{10 8} per kg body weight. In another embodiment, the total number of nucleated cells administered is at least 5 billion. In another embodiment, the total number of nucleated cells administered is at least 15 billion.

In another embodiment of the invention, the embryo-like stem cells and the cord blood are mixed immediately (ie within 5 minutes of administration) prior to administration to the subject. In another embodiment, the embryo-like stem cells and the cord blood are mixed at a time point of at least 5 minutes prior to administration to the subject. In another embodiment of the method, the embryo-like stem cells are cryopreserved and thawed prior to administration to the subject. In another embodiment, the embryo-like stem cells and the cord blood are mixed to form a supplemented cell population at a time point of at least 24 hours prior to administration to the subject. Wherein said supplemented cell population is cryopreserved and thawed prior to said administration. In other embodiments, the embryo-like stem cells and / or supplemented cell populations may be administered multiple times. In another embodiment, the embryo-like stem cells and / or supplemented cell populations are stored and preconditioned for 18 hours to 21 days prior to administration. In another specific embodiment, the cells are stored and preconditioned for 48 hours to 10 days prior to administration. In certain preferred embodiments, the cells are preconditioned for 3-5 days prior to transplantation. In a preferred embodiment of any of the methods herein, the embryo-like stem cells are not examined for HLA type prior to administration to the subject.

In another specific embodiment of the method, said embryo-like stem cells are predominantly (ie at least 50%) CD34 + cells. In another specific embodiment of the method, said embryo-like stem cells are predominantly CD34 + 33 + stem cells.

Therapeutic or prophylactic treatment of individuals with embryonic-like stem cells or supplemented cell populations comprising them may be considered effective if the disease, condition or condition is measurably enhanced in any way. Such improvement may be indicated by various indicators. Measurable indicators include, for example, detectable changes in a physiological state or series of physiological states associated with a particular disease, condition or condition (such changes include blood pressure, heart rate, respiratory rate, number of various blood cell types, blood of certain proteins Controlled expression of internal levels, carbohydrates, lipids or cytokines or genetic markers associated with a disease, condition or condition). Normal values for the various indicators may be set by normal ranges well known in the art, or may be set by comparison of such values with controls. In medical science, treatment efficiency is also often characterized by the subjective feeling of the individual's feelings and the individual's condition of health. Therefore, improvements that appear after administration of stem cells or supplemented cell populations of the invention are also characterized by subjective indicators such as the subject's feeling of subjective improvement, increased well-being, increased health status, improved energy levels, or the like. Cleared.

Embryonic-like stem cells and supplemented cell populations of the invention can be administered to a patient in any pharmaceutically or medically acceptable manner, including methods by injection or transfusion. The cell or supplemented cell population may comprise or be included in the pharmaceutically acceptable carrier described below. Embryonic-like stem cells or supplemented cell populations may be transported, stored in, or transported in any pharmaceutically or medically acceptable container such as, for example, a blood bag, transfer bag, plastic tube or vial.

Kit

The 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 invention. Optionally related to such containers are: an instrument for cell culture, a cell culture medium or one or more containers filled with components of one or more cell culture media, such as a tool for intravenous injection of a composition of the invention. Tools for use in the delivery of the compositions of the present invention, and / or in a form defined by a governmental body regulating the manufacture, use or sale of a medicament or biological product, the knots being manufactured for human administration, Means permission by mechanism of use or sale. In certain embodiments, the kit includes one or more other containers filled with stem cells, such as umbilical cord blood, as described above, and one or more containers filled with embryo-like stem cells of the present invention.

In another embodiment, the kit comprises a mixture of stem cells supplemented with embryo-like stem cells, such as, for example, cord blood cells, contained within one bag or container. In another embodiment, the kit comprises a group of cord blood cells and a group of embryo-like stem cells contained in two separate bags or containers. In certain embodiments, the kit comprises a "two bags" composition in which a bag containing cord blood cells and a bag containing embryonic-like stem cells are mixed at the time of administration, prior to administration to a patient in need thereof. In another embodiment, the kit contains two separate bags or containers containing a group of cord blood cells and a group of embryo-like stem cells, which are administered to a patient separately (eg, simultaneously or sequentially). The mixing of the two cell populations takes place in vivo .

In another embodiment, the kit provides a group of cord blood cells and a group of embryo-like stem cells that are physically mixed prior to administration. In another aspect of this embodiment, the kit is for example GM-CSF, IL-4, Flt3L, CD40L, IFN-alpha, TNF-alpha, IFN-gamma, IL-2, IL-6, Retinoic acid, basal fiber Blast growth factor, TGF-beta-l, TGF-beta-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensin II (AII), AII AT Growth factors such as ₂ type 2 receptor agonists, or analogs or fragments thereof. In another aspect of this embodiment, the two groups are physically mixed prior to administration to the patient and then treated with growth factors included in the kit to induce cell differentiation. In another aspect of this embodiment, the cord blood cells and / or embryo-like stem cells are treated with growth factors contained in the kit and then physically mixed to induce cell differentiation prior to administration to the patient.

The following examples are provided by way of illustration for further explanation and should not be construed as limiting the scope of the invention.

Example 1

Analysis of cell types recovered from perfusate of drained placenta

This example describes the analysis of cell types recovered from eluted perfusate of placenta cultured according to the method of the present invention. 20 ml of phosphate buffered saline (PBS) was added to the perfusion solution and the 10 ml portions were collected and centrifuged for 25 minutes at 3000 rpm (rpm). The eluate was divided into four tubes and placed in an ice bath. A 1% fetal calf serum (FCS) solution in 2.5 ml PBS was added to the tube and the tube was centrifuged at 140 min x 10 g (acceleration due to gravity).

The pellet was resuspended in 5 ml of 1% FCS and the two tubes were mixed. Total mononuclear cells (mononucleocytes) were calculated by adding total lymphocytes and total multinucleated cells and then multiplying the result by the total cell suspension volume.

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

WBC Lym% MID% GRA% Total Volume Cell Count

1000 / ml

Umbilical cord blood (CB) 10.5 43.2 8 48.8 60 ml 6.3 X 10 ⁸

Placental perfusate (at room temperature) 12.0 62.9 18.2 18.9 15 ml 1.8 X 10 ⁸

Placental perfusate (37 ° C) 11.7 56.0 19.2 24.8 30 ml 3.5 X 10 ⁸

In the table, the sample of placental perfusate is after Ficoll, the total cell number of placental perfusate after Ficoll is 5.3 X 10 ⁸ , and the cell number of cord blood before processing is 6.3 X 10 ⁸ . Lym% represents the percentage of lymphocytes; MID% represents the percentage of the midrange of leukocytes; GRA% represents the percentage of granulocytes.

Example 2

Analysis of cells obtained by placenta culture and perfusion

The following example describes the analysis of cells obtained by culture and perfusion of the placenta according to the method of the present invention.

1. Materials and Methods

Pregnant women enrolled in the Private Umbilical Cord Blood Banking Program were provided with informed consent to recruit placenta donors and recognize the use of the placenta without any blood following the recovery of umbilical cord blood for research purposes. Donor data will be confidential.

The donors also recognized the use of the blinded data produced from steady state processing of their cord blood samples for cryopreservation.

This permitted comparison between the eluted perfusate and the collected umbilical cord blood composition recovered using this experimental method is described below.

After bleeding of umbilical cord blood from the umbilical cord and placenta, it was stored at room temperature and delivered to the laboratory within 4 to 24 hours, according to the method described above, the placenta was sterilized at room temperature, placed in an isolated container and delivered to the laboratory within 4 hours of birth.

Investigation revealed that the placenta was discarded if the placenta had a physical injury, such as a cut in the trachea or a wound in the umbilical vessel.

Placenta were kept at room temperature (23 ± 2 ° C.) or refrigerated (4 ° C.) in sterile containers for 2 to 20 hours. At regular intervals, the placenta was immersed in sterile saline at 25 ± 3 ° C and washed to remove any type of surface blood or debris. The umbilical cord was incised approximately 5 cm laterally from its insertion into the placenta, and the umbilical conduit cannulates with TEFLON ® or polypropylene catheters connected with a sterile liquid pathway that allows bilateral perfusion and recovery of the eluate from the placenta. ).

The method described above allowed placental conditioning, perfusion and elution collection to be performed under controlled ambient pressure conditions as well as real-time monitoring of intravascular pressure and flow rates, core and perfusate temperatures and recovered eluate volumes.

The range of conditioned protocols was assessed for 24 hours postpartum period and cell composition of the eluate was analyzed by flow cytometry, light microscopy and colony forming unit analysis.

2. Placental Conditioning

Donor placenta were treated at room temperature within 12 to 24 hours after delivery.                 

Prior to treatment, the membranes were removed and the mother site was washed away with residual blood. The umbilical conduit was cannulated with catheters made with the use of a 20 gauge buffer needle for blood sample collection.

Donor placentals were allowed to simulate and maintain a physiologically compatible environment for recruitment and proliferation of residual embryonic-like stem cells in a range of 5-37 ° C., 5% CO ₂ , pH 7.2 to 7.5, preferably pH 7.45. It was maintained under various conditions such as

The cannula was flushed with IMDM serum free medium (GibcoBRL, NY) containing 2U / ml heparin (Elkins-Sinn, NJ).

Perfusion of the placenta was continued at a rate of 50 ml per minute until approximately 150 ml of perfusate had collected. The vessel of this perfusate was labeled "initial fraction."

Perfusion of the placenta was continued at the same rate and a second fraction of about 150 ml was collected and labeled "late fraction". The placenta was gently massaged during the course of the experiment to aid in the recovery of cellular material and the perfusion process. Eluate was collected from the perfusion ash through gravity drainage and aspiration through arterial cannula.

The placenta was then perfused for 10 minutes at a rate of 15 ml / min with heparinized (2 U / ml) Dulbecco's modified Eagle's medium (H. DMEM), and the perfusate was collected from the mother site within 1 hour and nucleated. The number of cells was counted.

Perfusion and collection were repeated one or two times until the number of nucleated cells was lowered below 100 / ml.

Perfusions were collected and centrifuged lightly to remove platelets, cell debris and unnucleated cell membranes.

Nucleated cells were then separated by Ficoll-Hypaque density centrifugation and resuspended in Dulbecco's modified Eagle's medium after washing.

To isolate adherent cells, 5-10 x 10 ⁶ cell aliquots were placed in each of several T-75 flasks and a commercially available Mesenchymal Stem Cell Growth Medium obtained from BioWhittaker. ; MSCGM) and placed in a tissue culture incubator (37 ° C., 5% CO ₂ ).

After 10 to 15 days, unattached cells were removed by washing with PBS and replaced by MSCGM.

Flasks were examined daily for the presence of various attached cell types and in particular for the identification and expansion of a group of fibroblastoid cells.

3. Cell recovery and isolation

Cells were recovered from the perfusate by centrifugation at 5000 × g for 15 minutes at room temperature.

This process acts to separate cells from contaminated cell debris and platelets.

Cell pellets were resuspended in IMDM serum overlapping medium containing 2U / ml heparin and 2 mM EDTA (GibcoBRL, NY).

The whole mononuclear cell fraction was isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) following the procedure recommended by the manufacturer, and the single nuclear cell fraction was resuspended. Cells were counted using a hematocytometer.

Survival numbers were assessed by trypan blue exclusion.

Isolation of mesenchymal cells was achieved through "differential trypsinization" using a 0.05% trypsin solution with 0.2% EDTA (Sigma, St. Louis MO).

Differential trypsinization is possible because fibroblast-like cells fall off the plastic surface in about 5 minutes and other attached groups require 20 to 30 minutes or more of culture.

The fallen fibroblast-like cells were collected after trypsinization and trypsin neutralized using trypsin neutralization solution (TNS, BioWhittaker).

Cells were washed with H.DMEM and resuspended in MSCGM.

Flow cytometry was performed using the Becton-Dickinson FACSCalibur instrument and FITC and PE labeled monoclonal antibodies (mAb) selected based on known markers for bone marrow derived MSCs (mesenchymal cells) were selected from B.D. And Caltag Laboratories (South San Francisco, CA.) and the reactivity of mAbs in their cultured supernatants was detected by FITC or PE labeled F (ab) 'dichlorine anti-mouse antibodies and produced SH2, SH3 and SH4 antibodies. Got hybridomas.

Lineage differentiation was performed using commercially available induction, and maintenance culture medium (BioWhittaker) was used as directed by the manufacturer.

4. Isolation of Placental Embryonic-like Stem Cells

Microscopic examination of the cells attached to the culture flasks showed morphologically different cell types.

Spinal cells, circular cells with large nuclei and small vacuoles around several nuclei, and star-shaped cells with multiple protrusions (fixed to the flask through one of the star-shaped cells) Attached to the field and observed.

Although no attempt was made to further characterize these attached cells, similar cells were observed in the culture, cord and peripheral blood of the bone marrow and therefore considered natural non-stem cell-like.

Fibroblastoid cells persisting and appearing as a group were candidates for being MSCs (mesenchymal cells), isolated by differential trypsinization, and subcultured in second flasks.

Phase microscopy of the round cells after trypsin showed that these cells were highly granulated and indistinguishable from bone marrow-derived MSCs made in the laboratory or purchased from BioWhittaker.

When subcultured, placental-derived embryo-like stem cells adhered within a few hours in contrast to their early stages, exhibited a unique fibroblast-like appearance and showed the same growth pattern as control bone marrow-derived MSCs.

In addition, loosely bound single nuclear cells were washed out during subculture and refeeding, and the culture remained uniform and free of any type of non-fibroblast-like cell contaminants.

5 .Result

Expression of CD-34, CD-38, and other stem cell related cell surface markers on early and late fraction purified mononuclear cells was assessed by flow cytometry.

The recovered and isolated cells were washed with PBS and then double stained with anti-CD34 phycoerythrin and anti-CD38 fluorescein isothiocyanate (Becton Dickinson, Mountain View, CA).

Cell separation was performed using magnetic cell separation such as, for example, automatic Mac (Miltenyi). Preferably CD 34+ cell isolation is performed first.

Example 3-

Perfusion Medium (PERFUSION MEDIUM)

The following example provides the composition of the preferred perfusate solution for the culture of the separated placenta.

compound Source Stock concentration Final concentration 500 ml DMEM-LG Jibco BRL 11885-084 300 ml MCDB201 SigmaM-6770 Soluble in water pH 7.2 filter 200 ml FCS Hyclone 100% 2% 10 ml ITS Sigma I-3146 or Jibco BRL 41400-045 100x 1x 5ml Pen & Strep Jibco BRL 15140-122 100x 1x 5ml LA + BSA Sigma + Jibco BRL
BSA 100x (1 μg / ml LA) 10ng / ml LA 5ml Dexamethasone Sigma D-2915 0.25 mM (in water) 0.05 μM 100 μl L-ascorbic acid Sigma A-8960 1000x (100mM) 1x (0.1mM) 500 μl PDGF (50 μg) R & D 10 μg / ml in 4 mM HC1 + 0.1% BSA 10 ng / ml 500 μl EGF (200 μg) Sigma E-9644 10 μg / ml in 10 mM HAc + 0.1% BSA 10 ng / ml 500 μl

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

Example 4

Induction of differentiation into specific cell types

Cord blood cells and / or embryonic-like stem cells were exposed to growth factors to induce differentiation into specific cell types.

Growth factors used to induce induction include GM-CSF, IL-4, Flt3L, CD40L, IFN-alpha, TNF alpha, IFN-gamma, IL-2, IL-6, retinoic acid, basic fibroblast growth factor, TGF-beta-1, TGF-beta-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensin II (AII), including but not limited to, AII AT ₂ type 2 receptor agonists or analogs or fragments thereof.

Induce differentiation into 1 neuron

This example describes the differentiation of cord blood cells and / or embryo-like stem cells into neurons.

The following protocol is used to induce neuronal differentiation.

Placental stem cells were grown for 24 hours in preinduction medium consisting of DMEM / 20% FBS and 1 mM beta-mercaptoethanol.

2. The preinduction medium was removed and the cells washed with PBS.

3. Neuronal induction medium consisting of DMEM and 1-10 mM beta-mercaptoethanol was added. Alternatively, induction media consisting of DMEM / 2% DMSO / 200 μM butylated hydroxyanisole can be used to increase neuronal differentiation efficiency.

4. In certain instances, morphological and molecular level changes may occur as early as 60 minutes after exposure to serum-deficient media and beta-mercaptoethanol (Woodbury et al., J. Neurosci. Res., 61: 364-). 370). For example, RT / PCR can be used to measure expression of nerve growth factor receptor and neurofibrillary heavy chain genes.

2. Induction of differentiation into adipocytes

This example describes inducing differentiation of cord blood cells and / or embryo-like stem cells into adipocytes. The following protocol is used to induce adipocyte differentiation.

Placental stem cells grow in DMEM fed with MSCGM (Bio Whittaker) or 15% cord serum.

2. Three cycles of induction / maintenance are used.

Each cycle was fed placental stem cells to adipose cell differentiation induction medium (Bio Whittaker) and incubated cells for 3 days at 37 ° C., 5% CO ₂ , and 1 to 3 days in adipose cell differentiation maintenance medium (Bio Whittaker) Incubated. Induction medium was used 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 complete cycles of induction / maintenance, cells were cultured in adipocyte differentiation maintenance medium for an additional 7 days with medium change every 2 to 3 days.

4. Adipocyte differentiation can be measured by the development of a number of intracytoplasmic lipid vesicles that can be easily observed using oil red O, a fat soluble stain.

RT / PCR analysis is used to investigate the expression of lipase and fatty acid binding protein genes.

3. Induction of Differentiation into Chondroitin

This example describes inducing differentiation of cord blood cells and / or embryo-like stem cells into chondrocytes.

The following protocol is used to cause chondrocyte differentiation: 1.                 

1. Placental stem cells were maintained in DMEM supplemented with MSCGM (Bio Whittaker) or 15% cord serum.

2. Placental stem cells were aliquoted into silver sterilized polypropylene tubes.

Cells were washed twice with incomplete Chondrogenesis medium (Bio Whittaker) by centrifugation at 150 x g for 5 minutes.

3. After the final washes, cells were resuspended in a complete Chondrogenesis medium (Bio Whittaker) containing 0.01 μg / ml TGF-beta-3 at a concentration of 5 × 10 (5) cells / ml.

4. 0.5 ml of cells were aliquoted into 15 ml polypropylene culture tubes.

The cells were pelleted at 150 × g for 5 minutes and the pellets left intact in the medium.

5. Loosely capped tubes were incubated at 37 ° C., 5% CO ₂ for 24 hours.

6. Cell pellets were fed freshly prepared complete Chondrogenesis medium every two to three days.

7. The pellets were suspended in the medium with daily stirring using a low speed vortex.

8. Chondrocyte pellets were harvested after 14 to 28 days of culture.

9. Chondrogenesis is elucidated by RT / PCR, for example, to observe the production of an eoinophilic ground substance, to observe cell morphology and / or to observe collagen 2 and collagen 9 gene expression. Can be.

4. Induce differentiation into osteoblasts

This example describes the induction of cord blood cells and / or embryo-like stem cells into osteocytes.

The following protocol is used to cause osteogenic differentiation.

1. Attachment Cultures of Placental Stem Cells are Cultured in DMEM Supplemented with Bio Whittaker (MSCGM) or 15% Code Serum.

2. Cultures are rested for 24 hours in tissue culture flasks.

3. Osteogenic differentiation is induced by replacing MSCGM with osteogenic induction medium (Bio Whittaker) containing 0.1 μM dexamethasone, 0.05 mM ascorbic acid-2-phosphate, 10 mM beta glycerophosphate.

4. Cells were fed osteogenic induction medium every 3 to 4 days daily for 2-3 weeks.

5. Differentiation was analyzed using RT / PCR for calcium specific staining and alkaline phosphatase and osteopontin gene expression.

5. Induce differentiation into Hepatocytes

This example describes the induction of differentiation of cord blood cells and embryonic-like stem cells into hepatocytes.

The following protocol is used to cause hepatogenic differentiation. Placental stem cells are cultured in DMEM / 20% CBS supplemented with hepatocyte growth factor, 20 ng / ml and epidermal growth factor (100 ng / ml). Knockout serum replacement can be used in place of FBS.

2. Add 50 ng / ml IL-6 to the induction flasks.

6. Induce differentiation into pancreatic cells

This example describes the differentiation of cord blood cells and / or pancreatic cells of embryonic-like stem cells.

The following protocol is used to cause pancreatic differentiation.

Placental stem cells were cultured in DMEM / 20% CBS supplemented with basic fibroblast growth factor (10 ng / ml) and transforming growth factor beta-1 (2 ng / ml). Knockout serum substitutes may be used in place of CBS.

2. Conditioned medium from nestin-positive neuronal cell cultures was added to the medium at a 50/50 concentration.

3. Cells were incubated with refeeding every 3 to 4 days for 14-28 days.

4. Differentiation was characterized by analyzing insulin protein or insulin gene expression by RT / PCR.

7. Induce differentiation into heart cells

This example illustrates the induction of differentiation of cord blood cells and / or embryo-like stem cells into heart cells.

The following protocol is used to cause myogenic differentiation.                 

1. Placental stem cells were retinoic acid (1 μM), basic fibroblast growth factor (10 ng / ml); And in DMEM / 20% CBS supplemented with transforming growth factor beta-1 (2 ng / ml) and epidermal growth factor (100 ng / ml). Knockout serum substitutes may be used in place of CBS.

2. Alternatively placental stem cells are incubated for 24 hours in DMEM / 20% CBS supplemented with 50ng / ml Cardiotropin-1.

3. Alternatively, placental stem cells are maintained in protein deficient medium for 5 to 7 days and then stimulated with human myocardial extract (increase dose assay).

Myocardial extract is obtained by homogenizing 1 gram human myocardium in 1% HEPES buffer supplemented with 1% cord serum. The suspension is incubated for 60 minutes, then centrifuged and the supernatant collected.

4. Cells are re-incubated every 3 to 4 days for 10-14 days.

5. Differentiation was measured using cardiac actin RT / PCR gene expression analysis.

8. Characterization of cord blood cells and / or embryo-like stem cells before and / or after differentiation

Embryonic-like stem cells, cord blood cells, and / or embryo-like stem cells and a group of spiked cord blood cells may be used to measure changes in gene expression using techniques such as PCR before and / or after differentiation, flow cytometry and Techniques such as immune cell chemistry are used to measure and characterize changes in cell surface markers and morphological changes.

Cells and / or differentiated cells that have been exposed to growth factors are those cell surface markers: CD10 +, CD29 +, CD34-, CD38-, CD44 +, CD45-, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3-, SSEA4 -Is characterized by the presence and absence of OCT-4 +, and ABC-p +.                 

Preferably embryonic-like stem cells are characterized by the presence of cell surface markers OCT-4 +, APC-p +, CD34- and CD38- prior to differentiation. Stem cells with these markers are as versatile (eg pluripotent) as human stem cells.

Cord blood cells are characterized by the presence of CD34 + and CD38 + prior to differentiation. Differentiated cells derived from embryonic-like stem cells, cord blood cells and / or a group of stem cells and spiked cord blood cells preferably do not express these markers.

Example 5-

Treatment of Patients with Muscular Atrophic Lateral Sclerosis with Embryonic-like Stem Cells

Amyotrophic Lateral Sclerosis (ALS), also called Lou Gehrig's disease, is a fatal degenerative neuropathy that affects motor neurons, brain stem, and spinal cord in the cortex. ALS suffers from about 20,000 Americans with 5,000 new cases in the United States each year. The majority of ALS patients are sudden (S-ALS), but about 5-10% are genetic (F-ALS). ALS occurs when certain neurons in the brain and spinal cord that control spontaneous movement gradually degenerate.

A key feature of ALS is the loss of spinal motor neurons that weaken and weaken the muscles under their control, leading to paralysis.

ALS appears in a variety of ways, depending on which muscle is weakened for the first time.

ALS develops about one and a half times better in men than in middle-aged women. ALS is usually fatal within 5 years after diagnosis.

ALS has both genetic or unexpected forms, which are currently associated with several unique genetic locations. Only about 5 to 10% of ALS patients are genetic. 15 to 20% of these are due to mutations in the gene encoding copper / zinc superoxide dismutase 1 (SOD1). They represent "gain-of-function" mutations that impart toxic properties to enzymes.

The discovery of SOD mutations as a cause of ALS has opened the way for some progress in understanding the disease; Animal models of the disease are currently available, hypotheses have been developed, and experiments are ongoing on the molecular work that leads to cell death.

An example of how to treat a patient with ALS with embryo-like stem cells derived from the placenta is described below.

The method includes intravenous infusion via a peripheral and transient angiocatheter.

Patients with ALS are first examined by performing standard laboratory assays. Such assays include metabolic profiles; Differential CDC; Lipid profile; Fibrinogen levels; ABO rH blood type; Liver function tests; And determination of BUN / creatine levels. Patients were instructed to take the drugs (Diphenylhydramine (Benadryl ^™ ), 25 mg three times a day and prednisone, 10 mg) the day before transplantation.

Embryonic-like stem cells, either alone or in cord blood and spiked form, were taken from cryopreserved stock and thawed and stored at about 5 ° C. for about two days prior to transplantation.

Patients were moved to an outpatient diagnostic center equipped with all the equipment necessary for intravenous infusion, physiological monitoring and physical observation. Approximately 1 hour prior to transplantation, patients received oral administration of 25 mg xl of diphenylhydramine (Benadryl ^™ ) and 10 mg xl of prednisone. This is a preventive measure and reduces acute allergic reactions.

At the time of perfusion, 18 G in the peripheral vein was placed at one of the patient's tips and kept open by infusion of D5½ normal saline + 20mEq KC1 mEqKC1 at the TKO rate.

Patients were checked for heart rate, respiratory rate and body temperature, especially before transplantation.

Other monitoring such as electrocardiogram and blood pressure measurements may also be performed.

Embryonic-like stem cells were then injected at a rate of 1 unit per hour in 60 ml total delivered liquid volume. One unit is about 1-2 10 ⁹ total nucleated cells.

Alternatively the embryo-like stem cells in that unit can be delivered to cord blood with a total liquid volume of 60 ml. In this case the ratio of the number of embryonic-like stem cells to stem cells of cord blood is at least 2: 1.

The administered unit may also consist solely of cord blood.

Based on data from preclinical studies in mice, a total of 2.0 to 2.5 x 10 ⁸ cells per kilogram of body weight is administered. For example, a 70 kilogram patient will receive approximately 14-18 x 10 ⁹ total nucleated cells.

Patients should be immediately monitored for signs of allergic or hypersensitivity reactions that are discontinuation signals. After infusion, patients can be monitored in a lying position for at least 60 minutes and then resume normal activity.

Example 6

Treatment of Atherosclerosis Patients with Embryonic-like Stem Cells

The infusion protocol shown in Example 5 can also be used to administer embryonic-like stem cells alone or umbilical cord blood and spiked to atherosclerosis patients.

Embryonic-like stem cells or supplemented cell groups can be administered to asymptomatic patients, patients who are candidates for angioplasty, or patients who have recently undergone cardiac surgery.

The present invention is not to be limited in scope by the specific embodiments described herein.

Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such changes fall within the scope of the appended claims.

Just as each individual publication or patent or patent application is expressly individually incorporated completely by reference for all purposes, all references cited herein are fully incorporated for all purposes to the same extent by reference.                 

A citation of a publication is not to be construed as an admission that it was published prior to the filing date of this patent and that the present invention is not entitled to antedate such publication by virtue of prior invention.

Embryonic-like stem cells and mixed groups of embryo-like stem cells and stem cells of the present invention have a variety of uses and indications, such as, but not limited to, therapeutic, diagnostic and research applications for transplantation. Embryonic-like stem cells and mixed groups also include, for example, but are not limited to, vascular diseases, neurological diseases or disorders, autoimmune diseases or disorders, diseases or disorders including inflammation and diseases such as cancer or disorders associated therewith. Or for the treatment of abnormalities.

Claims (108)

Human hematopoietic stem or progenitor cells and A composition comprising isolated human placental stem cells having OCT-4 ⁺ , ABC-p ⁺ , SSEA3 ^−, and SSEA4 ⁻ properties, The placental stem cells are drained umbilical cord blood (cord blood), characterized in that obtained in the placenta removed by perfusion of residual blood. Human hematopoietic cord blood stem cells or progenitor cells and A composition comprising isolated human placental stem cells having OCT-4 ⁺ , ABC-p ⁺ , SSEA3 ^−, and SSEA4 ⁻ properties, The placental stem cells are drained umbilical cord blood (cord blood), characterized in that obtained in the placenta removed by perfusion of residual blood. The method according to claim 1 or 2, The composition is contained in a container. The method of claim 3, The container is sealed, air tight and sterilized. The method of claim 1, The stem cells or progenitor cells are characterized in that derived from umbilical cord blood or placental blood, fetal or neonatal hematopoietic stem cells or progenitor cells, adult cells or bone marrow stem cells or bone marrow progenitor cells. 6. The method of claim 5, The stem cells or progenitor cells are fetal or neonatal hematopoietic stem cells or precursor cells, characterized in that the composition. The method according to claim 1 or 2, The hematopoietic stem cells or progenitor cells with a plurality of cells, CD34 ⁺ and CD38 ^- composition, characterized in that. The method according to claim 1 or 2, The umbilical cord blood stem cells are characterized in that the plurality of cells are CD34 ⁺ and CD38 ⁺ . delete delete The first container contains human hematopoietic stem cells or progenitor cells, A second container is a kit containing isolated placental stem cells, drained from umbilical cord blood and obtained from the placenta removed by perfusion of residual blood, A kit comprising the composition of claim 1. The first container contains human hematopoietic umbilical cord blood cells, A second container is a kit containing isolated human placental stem cells, drained from umbilical cord blood and obtained from the placenta removed by perfusing residual blood, A kit comprising the composition of claim 2. The method according to claim 1 or 2, The number of placental stem cells and The number of hematopoietic stem cells or progenitor cells is At least a ratio of at least 1: 1. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete A plurality of cord blood stem cells and a plurality of ^{OCT-4 +, ABC-p} +, SSEA3 - and SSEA4 ^- the placenta, including, but stem cells, the kit characterized in that containing the cells in a separate container respectively. delete The method according to claim 1 or 2, The composition contains at least 5 × 10 ⁹ isolated nucleated cells, wherein the at least 5 × 10 ⁹ isolated nucleated cells are derived from placental stem cells that are OCT-4 ⁺ , ABC-p ⁺ , SSEA3 ⁻ and SSEA4 ⁻ . A composition comprising a. The method according to claim 1 or 2, At least 30% of the cells in said composition are placental stem cells. The method according to claim 1 or 2, At least 60% of the cells in said composition are placental stem cells. 43. The method of claim 42, At least 5 × 10 ⁹ The isolated nucleated cells contain cells received from a plurality of donors. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete