JP2009017891A - Multipotent adult stem cell, source thereof, method of obtaining and maintaining the same, method of differentiation thereof, method of use thereof, and cells derived therefrom - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide mammalian multipotent adult stem cells (MASC), methods for obtaining, maintaining and differentiating MASC, and the use of MASC for therapy of diseases. <P>SOLUTION: The cells can differentiate into the mesodermic line, ectodermic line, and endodermic line individually, further, in at least one of these cell types. These cells and cell aggregates that can actually differentiate into one of these cell types are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

Related Patents This application is a US Provisional Patent Application No. 60 / 343,386 filed October 25, 2001, a US Provisional Patent Application No. 60 / 310,625 filed August 7, 2001, February 15, 2001. Claims the benefit of US Provisional Patent Application No. 60 / 269,062 filed on the same date and US Provisional Patent Application No. 60 / 268,786 filed February 14, 2001, which are incorporated herein by reference. included. Applicant also claims priority under WO 01/11011, 60 / 147,324 and 60 / 164,650, which are hereby incorporated by reference; any of which Can be used to practice the present invention. This application is a continuation-in-part of WO 01/11011 attached to Appendix 1 and part of this application. Materials included in the text by reference are not recognized as prior art.

The present invention relates generally to mammalian pluripotent adult stem cells (MASCs), and more particularly to methods for obtaining, maintaining and differentiating MASCs. Also provided is the use of MASC in the treatment of disease.

BACKGROUND OF THE INVENTION The creation of organs and tissues from stem cells, and their subsequent transplantation, offers promising treatments for several diseases, making stem cells the center of research in many areas. Stem cell technology offers promising new therapies for diabetes, Parkinson's disease, liver disease, heart disease, and autoimmune disease, to name a few. However, there are at least two major problems associated with organ and tissue transplantation.

  First, there is a lack of donor organs and tissues. Only 5 percent of organs needed for transplantation are available in the United States alone (Evans, et al. 1992). According to the American Heart Association, only 2,300 of the 40,000 Americans who needed a new heart in 1997 received the heart. The American Liver Society reports fewer than 3,000 donors for nearly 30,000 patients who die of liver failure each year.

  The second major problem is the possibility of incompatibility between the recipient's immune system and the transplanted tissue. Since the donor organ or tissue is recognized as a foreign body by the host immune system, immunosuppressive drugs must be given to the patient at a considerable cost both physically and financially.

  Xenotransplantation, i.e., transplantation of tissue or organs from another species, can provide another way to overcome the shortage of human organs and tissues. Xenotransplantation has the advantage of advance planning. The organ can be harvested while healthy and the patient can receive any beneficial pretreatment prior to the transplant operation. Unfortunately, xenotransplantation not only overcomes the problem of tissue incompatibility, but rather exacerbates it. In addition, according to the Centers for Disease Control, there is evidence that harmful viruses cross interspecies barriers. Pigs are potential candidates for organ and tissue donors, but interspecies infection of one or more viruses from pigs to humans has been documented. For example, more than 1 million pigs have recently been slaughtered in Malaysia (Butler, D. 1999) to contain the outbreak of Hendra virus, a disease that killed more than 70 people.

Stem cells: definition and uses The most promising source of organs and tissues for transplantation is therefore in the development of stem cell technology. Theoretically, stem cells can undergo self-renewing cell division to give daughter cells with the same phenotype and genotype indefinitely, and can finally differentiate into at least one final cell type. By creating a tissue or organ from the patient's own stem cells, or by genetically recombining a heterologous cell so that the recipient's immune system does not recognize it as a foreign body, a transplanted tissue is created, accompanied by infection or tissue rejection. The benefits associated with xenotransplantation can be provided without risk.

  Stem cells also provide the prospect of improving the outcome of gene therapy. The patient's own stem cells can be genetically modified in vitro and then reintroduced in vivo to produce the desired gene product. These genetically engineered stem cells have the potential to be induced to form a large number of cell types for implantation in specific parts of the body or for systemic administration. Alternatively, the heterologous stem cells can be genetically modified to express the recipient's major histocompatibility complex (MHC) antigen or no MHC antigen at all, from the donor without the risk of concomitant rejection It becomes possible to transplant the cells to the recipient.

  Stem cells are defined as cells that have a wide range of proliferative potential, differentiate into several cell lineages, and repopulate into tissues upon transplantation. A typical stem cell is an embryonic stem (ES) cell because it has unlimited self-renewal and pluripotent differentiation potential (Thomson, J. et al. 1995; Thomson, JA et al. 1998). Shamblott, M. et al. 1998; Williams, RL et al. 1988; Orkin, S. 1998; Reubinoff, BE, et al. 2000). These cells are derived from the inner cell mass of blastocysts (Thomson, J. et al. 1995; Thomson, JA et al. 1998; Martin, GR1981) or from primordial germ cells derived from post-transplant embryos (Embryonic germ cells or EG cells). ES and EG cells have been obtained from mice, and more recently from non-human primates and humans. When introduced into mouse blastocysts, ES cells can contribute to all tissues of the mouse (animal) (Orkin, S. 1998). Mouse ES cells are therefore totipotent. When transplanted into postnatal animals, ES and EG cells give rise to teratomas, which also show their pluripotency. ES (and EG) cells can be identified by positive staining using antibodies against stage-specific fetal antigen (SSEA) 1 and 4.

  At the molecular level, ES and EG cells express several transcription factors that are highly specific for these undifferentiated cells. These include oct-4 and Rex1, leukemia inhibitory factor receptor (LIF-R). The transcription factors sox-2 and Rox-1 are expressed in both ES and non-ES cells. Oct-4 is expressed in gastrulation embryos, early cleavage embryos, blastocyst inner cell mass cells, and embryonic carcinoma (EC) cells. In adult animals, oct-4 is found only in germ cells.

  Oct-4, together with Rox-1, causes transcriptional activation of the zinc finger protein Rex-1, and is also required to maintain ES in an undifferentiated state. The oct-4 gene is downregulated when cells are induced to differentiate in vitro. Several studies have shown that oct-4 is required to maintain the undifferentiated phenotype of ES cells and plays a major role in determining embryonic development and early stages of differentiation. Sox-2 is required with oct-4 to maintain the undifferentiated state of ES / EC and maintain mouse ES cells, but not to maintain human ES cells. Human or mouse primordial germ cells require the presence of LIF. Another prominent feature of ES cells is the presence of high concentrations of telomerase, which provides these cells with infinite self-renewal ability in vitro.

Stem cells are identified in most organs or tissues. The best characterized are hematopoietic stem cells (HSC). The mesoderm-derived cells are purified based on cell surface markers and functional characteristics. HSCs isolated from bone marrow (BM), blood, umbilical cord blood, fetal liver and yolk sac are progenitor cells that give rise to blood cells, i.e., subsequent translation reinitiates multiple hematopoietic lineages, Hematopoiesis can be started again for life. (Fei, R., et al., Patent Literature 1; McGlave, et al, Patent Literature 2; Simmons, P., et al, Patent Literature 3; Tsukamoto, etal, Patent Literature 4; Schwartz, et al, Patent Literature 5; see DiGuisto, et al, Patent Document 6; Tsukamoto, et al, Patent Document 7; Hill, B., et al, 1996) When transplanted into animals or humans that have received a lethal dose of radiation, HSC Newly established in erythroblasts, neutrophil-macrophages, megakaryocytes and lymphocyte hematopoietic cell pools. In vitro, hematopoietic stem cells can be induced to undergo cell division that regenerates at least several times, and can be induced to differentiate into the same lineage as seen in vivo. Thus, this cell meets the stem cell criteria. Stem cells that differentiate only to form cells of the hematopoietic lineage, however, provide the source of cells to repair other damaged tissue, such as heart or lung tissue damaged by high doses of chemotherapeutic drugs It is not possible.

  Another stem cell that has been extensively studied is the neural stem cell (NSC) (Gage F.H. 2000; Svendsen C.N. et al, 1999; Okabe S. et al, 1996). NSCs were first found in the subventricular region of the fetal brain and in the olfactory bulb. Until recently, it was believed that the adult brain did not contain cells with stem cell capabilities. However, several studies in rodents, more recently also non-human primates and humans, indicate that stem cells continue to exist in the adult brain. These stem cells can proliferate in vivo and continuously regenerate at least some neurons in vivo. When cultured ex vivo, NSCs can be induced to proliferate and can be induced to differentiate into various types of neurons and glial cells. When transplanted into the brain, NSCs can settle and give rise to neurons and glial cells. Therefore, both this cell and the hematopoietic stem cell satisfy the definition of stem cell.

  Clarkeetal. Reported that NSCs derived from Lac-Z transgenic mice injected into mouse blastocysts or chicken embryos contributed to some tissues of the chimeric mice or chicken embryos (Clarke, DL et al 2000). LacZ-expressing cells were found in varying degrees of mosaic not only in the central nervous system, but also in mesoderm derivatives and liver and intestinal epithelial cells, but not in other tissues including hematopoietic mechanisms. These studies therefore show that adult NSCs may have significantly greater differentiation potential than previously known, but ES or Furcht et al. (International Application No. PCT / USOO / 21387) and adults described herein, It was suggested that the pluripotent adult stem cells (MASCs) obtained from are not totipotent. The terms MASC, MAPC and MFC can be used interchangeably to describe pluripotent adult stem cells obtained from adults.

  The treatment of degenerative and traumatic brain disease can make great progress using cell replacement therapy. NSCs are found in the subventricular region (SVZ) and hippocampus of the adult mammalian brain (Ciccolini et al, 1998; Morrisonetal., 1999; Palmer et al., 1997; Reynolds and Weiss, 1992; Vescovi etal., 1999), can also be present in the upper garment and other non-neural areas of the brain (Doetschetal, 1999; Johansson et al, 1999; Palmer et al, 1999). Fetal or adult brain-derived NSCs can be induced to grow and differentiate into astrocytes, oligodendrocytes and functional neurons ex vivo (Ciccolini et al, 1998; Johansson et al, 1999; Palmer etal 1999; Reynoldset al, 1996; Ryder et al, 1990; Studer et al, 1996; Vescoviet al, 1993). In vivo, undifferentiated NSCs cultured at different times differentiate into glial cells, GABAergic and dopaminergic neurons (Flaxet al, 1998; Gage et al, 1995; Suhonen et al, 1996). The most widely used source of NSC is the allogeneic fetal brain, which raises both immunological and ethical issues. As an alternative, NSCs can be obtained from the own brain. It is not known whether prior neurological pathology affects the ability of NSCs to be induced to differentiate into cultured neurons and glial cells ex vivo, and additional surgery in the already diseased brain is the underlying disease This approach is less attractive because it can worsen

  The ideal source of neurons and glia for replacement strategies is cells that can be harvested from adult, readily available self-tissues other than the brain, and can be grown in vitro ex vivo or in vivo It can differentiate into a cell type that is lacking in the patient. Recent reports suggest that BM-derived cells acquire phenotypic characteristics of neuroectodermal cells when cultured in vitro under NSC conditions or enter the central nervous system (Sanchez- Ramoset al., 2000; Woodbury et al, 2000). The phenotype of BM cells with this ability is unknown. The ability for differentiation of cells that acquire neuroectodermal properties into other cell types is also unknown.

  Another tissue-specific cell with stem cell properties is a mesenchymal stem cell (MSC), first described by Fridenshtein (1982). MSCs are initially obtained from embryonic mesoderm and isolated from adult BM and can differentiate to form muscle, bone, cartilage, fat, bone marrow stroma, and tendons. During embryonic development, the mesoderm develops into tissues that produce limb bud mesoderm, bone, cartilage, fat, skeletal muscle and possibly endothelium. The mesoderm also differentiates into organ mesoderm, which can give rise to blood islands consisting of heart muscle, smooth muscle, or endothelium and hematopoietic progenitor cells. Early mesoderm or MSC may thus provide the origin of several cell and tissue types. Several MSCs have been isolated. (For example, Caplan, A., et al, Patent Document 8; Young, H., et al, Patent Document 9; Caplan, A., et al, Patent Document 10; Bruder, S., etal. Patent Document 11; Caplan, A., et al., Patent Document 12; Masinovsky, B., Patent Document 13; Pittenger, M., Patent Document 14; Jaiswal, N., etal., 1997 ,; Cassiede P., et al, 1996; Johnstone, B., et al, 1998; see Yoo, et al, 1998; Gronthos, S., 1994).

Of the many MSCs described, all show limited differentiation to form cells that are generally considered mesenchymal origin. To date, the most pluripotent MSCs reported were isolated by Pittenger, et al, SH2 ⁺ SH4 ⁺ CD29 ⁺ CD44 ⁺ CD71 ⁺ CD90 ⁺ CD106 ⁺ CD120a ⁺ CD124 ^- CD14 ^- CD34 ^- CD45 ^- phenotype It is a cell that expresses. This cell is capable of differentiating to form several cell types of mesenchymal origin, but obviously the differentiation potential is limited to cells of the mesenchymal lineage and the team that isolated the cell As noted, hematopoietic cells were never found during expansion culture (Pittenger, etal, 1999).

  Other tissue-specific stem cells have been identified, including gastrointestinal stem cells (Potten, C. 1998), epithelial stem cells (Watt, F. 1997), and hepatic stem cells, also called round cells (Alison, M. et al. 1998) Yes. Most of these are not well characterized by comparison.

  Compared to ES cells, tissue-specific stem cells have less self-renewal capacity and differentiate into multiple lineages but are not totipotent. There are no studies dealing with whether tissue-specific cells express the markers described above when found in ES cells. Furthermore, the degree of telomerase activity in tissue-specific or lineage-derived stem cells has not been fully investigated because it is difficult to obtain large numbers of highly amplified populations of these cells as part of the reason. Until recently, it was thought that tissue-specific stem cells could only differentiate into cells of the same tissue. Several recent publications suggest that adult organ-specific stem cells may be capable of differentiating into cells of various tissues. However, the true nature of these types of cells is not fully understood. Several studies have shown that cells transplanted during BM transplantation can differentiate into skeletal muscle (Ferrari1998; Gussoni1999). This can be considered within the range of possible differentiation of mesenchymal cells present in the bone marrow. Jackson announced that muscle satellite cells can differentiate into hematopoietic cells, which is also a phenotypic change within the visceral mesoderm of the embryo (Jackson 1999). Other studies have shown that stem cells from one germ layer (eg, visceral mesoderm) can differentiate into tissues that are thought to arise from another germ layer during embryogenesis. For example, endothelial cells or their progenitor cells detected in humans or animals that have undergone bone marrow transplantation are at least partially derived from bone marrow donors (Takahashi, 1999; Lin, 2000). Thus, MSC-like offspring derived from visceral mesoderm but not visceral nerve mesoderm are transplanted with the bone marrow to be injected. More surprisingly, there are reports showing that donor and hepatic epithelial cells and bile duct epithelial cells are found in donors in both rodents and humans (Petersen, 1999; Theise, 2000; Theise, 2000). Similarly, three groups have shown that NSCs can differentiate into hematopoietic cells. Finally, Clarke et al. Report that cells that can contribute to all tissues of tissue chimeric mice when called into blastocysts are called NSCs (Clarke et al, 2000).

It should be pointed out that most of these studies do not show definitively that single cells can differentiate into tissues of different organs. In addition, stem cells isolated from any organ may not necessarily be cells whose lineage has been determined. In fact, most researchers did not identify the phenotype of the starting cell. An exception was a study by Weissman and Grompe, who showed that cells that settled in the liver were present in Lin ^- Thy ₁ LowSca ₁ ⁺ bone marrow cells that were highly enriched in HSC. Similarly, the Mulligan group showed that bone marrow Sp cells highly enriched in HSC can differentiate into muscle and endothelium, and Jackson et al. Showed that muscle Sp cells are responsible for hematopoietic reconstitution (Gussoni et al. 1999).

  For transplantation of tissues and organs made from heterologous ES cells, the cells can be further genetically modified to inhibit the expression of certain cell surface markers, or chemotherapy immunosuppression to protect against transplant rejection. It is necessary to continue using the agent. Thus, ES cell research offers a promising new solution to the problem of limited supply of organs for transplantation, but with the need for immunosuppression to maintain transplantation of xenogeneic cells or tissues Problems and dangers remain. In order to provide immunologically compatible cells for therapies that control the majority of the population, it is necessary to establish an estimated 20 immunologically different strains of ES cells.

  Using cells from grown individuals rather than embryos as the origin of autologous or histologically matched allogeneic stem cells alleviates or overcomes the problem of tissue incompatibility associated with the use of transplanted ES cells, and Resolve the ethical tradeoffs associated with ES cell research. The greatest disadvantage associated with the use of autologous stem cells for tissue transplantation is its relatively limited differentiation potential so far.

Several stem cells have been isolated from mature organisms, especially humans, but these cells have limited potential to differentiate into multiple cell types despite being reported to be pluripotent. It is shown that.
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  Thus, fibroblasts, liver, osteoblasts, chondrocytes, adipocytes, although stem cells with multiple differentiation potentials have been previously isolated by other researchers and the present inventors No progenitor cells have been described that have the potential to differentiate into a wide variety of cell types, including skeletal muscle, endothelium, stroma, smooth muscle, cardiac muscle and hematopoietic cells. If cell and tissue transplantation and gene therapy provide the expected medical advances, stem or progenitor cells with the greatest or most extensive differentiation potential are sought. What is needed is an equivalent in ES cells and adults.

  BM, muscle, and brain are three tissues that have found greater apparent plasticity than previously thought. BM is found in several mesodermal (Ferrari G. et al, 1998; Gussoni E. et al, 1999; Rafii S. etal, 1994; Asahara T. et al., 1997; LinY. Et al, 2000; Orlic D et al, 2001; Jackson K. et al, 2001), endoderm (Petersen BEet al, 1999; Theise, ND etal, 2000; Lagasse E. et al, 2000; Krause D. et al, 2001), And neuroectodermal (Mezey D.S. et al, 2000; Brazelton TR, et al, 2000; SanchezRamos J. et al, 2000; Kopen G. et al, 1999) and skin (Krause, D. et al, 2001) including cells that can contribute to the structure. Muscle-derived cells can contribute to the hematopoietic mechanism (Jackson K. et al, 1999; Scale P. et al, 2000). NSCs can differentiate into hematopoietic cells (Bjornson C. et al, 1999; Shih C. etal., 2001), smooth muscle myoblasts (Tsai RY et al, 2000), and injected into mouse blastocysts There is also evidence that NSCs sometimes give rise to several cell types (Clarke, DL et al, 2000).

  This study shows that cells with the characteristics of pluripotent adult progenitor cells can be cultured and isolated from several different organs, namely BM, muscle and brain. The cells have the same morphology, phenotype, in vitro differentiation ability and very similar expressed gene profiles.

  The present invention is a pluripotent adult stem cell (MASC) isolated from a mammal, preferably a mouse, rat or human. The cells are derived from organs or tissues other than the embryo and have the ability to be induced to differentiate to form at least one differentiated cell type of mesoderm, ectoderm and endoderm origin. In a preferred embodiment, the organ or tissue from which MASC is isolated is bone marrow, muscle, brain, umbilical cord blood or placenta.

The present invention provides the following.
(Item 1) An isolated pluripotent adult stem cell (MASC) having the ability to be induced to differentiate to form at least one differentiated cell type of mesodermal, ectodermal and endodermal origin.
(Item 2) The cell according to item 1, which is derived from an organ or tissue other than a mammalian embryo.
(Item 3) Osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, myocardium, eyes, endothelium, epithelium, liver, pancreas, hematopoiesis, glia, neurons and oligodendrocytes 2. The cell of claim 1 having the ability to be induced to differentiate to form a cell selected from the group consisting of the cell type of the cell.
(Item 4) The cell according to item 1, wherein the organ or tissue is selected from the group consisting of bone marrow, muscle, brain, umbilical cord blood, and placenta.
(Item 5) The cell according to item 2, wherein the mammal is a mouse.
(Item 6) The cell according to item 2, wherein the mammal is a rat.

(Item 7) The cell according to item 2, wherein the mammal is a human.
(Item 8) The cell according to item 1, wherein differentiation is induced in vivo or ex vivo.
(Item 9) The cell according to item 3, wherein differentiation is induced in vivo or ex vivo.
(Item 10) The cell according to item 1, wherein the cell constitutively expresses oct4 and a high level of telomerase.
(Item 11) The cell according to item 10, wherein the cell is negative for CD44, MHC class I and MHC class II expression.
(Item 12) (a) The cell described in Item 1 is introduced into a blastocyst; (b) The blastocyst of (a) is transferred to a surrogate mother; and (c) The offspring is generated and born; A method for producing a normal non-human animal, comprising:
(Item 13) The animal according to item 12, wherein the animal is a chimera.
(Item 14) A composition comprising a population of MASCs and a medium for expanding the MASCs.
(Item 15) The composition according to item 14, wherein the culture medium contains epidermal growth factor (EOF) and platelet-derived growth factor (PDGF).
(Item 16) The composition according to item 15, wherein the medium further contains a leukemia inhibitory factor (LEF).
(Item 17) A composition comprising a population of fully or partially purified MASC progeny cells.
(Item 18) The composition according to item 17, wherein the progeny cells have the ability to further differentiate.
(Item 19) The composition according to item 17, wherein the progeny cells are terminally differentiated.
(Item 20) The progeny cells are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, myocardium, eyes, endothelium, liver, pancreas, hematopoiesis, glia, nerve 18. The composition according to item 17, which is a cell type of cells or oligodendrocytes.
(Item 21) (a) Obtaining tissue from a mammal; (b) Establishing a population of adherent cells; (c) Removing CD45 ⁺ cells from the population; (d) Recovering CD45 ⁻ cells; e) culturing CD45 ^- cells under expansion conditions to obtain an expanded cell population; a method for isolating and growing cells according to item 1, characterized in that each step is included.
(Item 22) An expanded cell population obtained by the method according to Item 21.
(Item 23) A method for differentiating MASC ex vivo, comprising the steps of the method according to Item 21, and further comprising culturing the proliferated cells in the presence of a target differentiation factor.
24. The differentiation factor is basic fibroblast growth factor (bFGF); vascular endothelial growth factor (VEGF); dimethyl sulfoxide (DMSO) and isoproterenol; and fibroblast growth factor 4 (FGF4) and 24. The method of claim 23, wherein the method is selected from the group consisting of hepatocyte growth factor (HGF).
(Item 25) Differentiated cells obtained by the method according to Item 23.

(Item 26) The differentiated cell according to item 25, wherein the cell is ectoderm, mesoderm or endoderm.
(Item 27) The cells are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, cardiac muscle, eyes, endothelium, epithelium, liver, pancreas, hematopoiesis, glia, nerve cells. The differentiated cell according to item 25, which is a cell type of oligodendrocyte.
28. The method of claim 21, further comprising the steps of administering the expanded cell population to a mammalian host, wherein the cell population is established and differentiated into tissue-specific cells in vivo. In vivo MASC, characterized in that the function of defective cells or organs is augmented, reconstituted or provided for the first time for injury, genetic disease, acquired disease or iatrogenic treatment How to differentiate.
(Item 29) The tissue-specific cells are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, myocardium, eyes, endothelium, epithelium, liver, pancreas, hematopoiesis, glia 29. The method according to item 28, which is a cell type of a nerve cell or oligodendrocyte.
(Item 30) The method according to item 28, wherein the MASC self-regenerates in vivo.
31. The method of claim 28, wherein the cells are administered with a pharmaceutically acceptable substrate.
(Item 32) The method according to item 31, wherein the substrate is biodegradable.
(Item 33) The method according to item 28, wherein the administration is performed by local injection, systemic injection, parenteral administration, oral administration, or intrauterine injection into an embryo.
34. The method of claim 33, wherein the local injection comprises catheter administration.
(Item 35) The disease is cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, degenerative or traumatic neurological disease, autoimmune disease, genetic defect, connective tissue disease, anemia, infection. 29. The method according to item 28, wherein the method is selected from the group consisting of symptoms and transplant rejection.
(Item 36) A differentiated cell obtained by the method according to item 28.
(Item 37) A therapeutic method comprising a step of administering a therapeutically effective amount of the cell according to item 11 to a patient in need thereof.
38. The method of claim 37, wherein no malformation is formed in the patient.
39. A method of treatment comprising administering to a patient in need thereof a therapeutically effective amount of MASC or its progeny cells.
40. The method of claim 39, wherein little or no pretreatment of the patient is required.
41. The method of claim 40, wherein the pretreatment comprises bone marrow function abolition by irradiation or chemotherapy.
(Item 42) The method according to item 39, wherein it is not necessary to induce resistance before or simultaneously with administration of MASC or progeny cells thereof.
43. The method of claim 39, wherein the patient's post-immunosuppressive treatment is reduced compared to conventional pharmacological doses.
44. The method of claim 39, wherein the progeny cells have the ability to further differentiate.
(Item 45) The method according to item 39, wherein the progeny cells are terminally differentiated.
46. The method of claim 39, wherein the MASC or progeny cells are administered by local injection, systemic injection, parenteral administration, oral administration, or intrauterine injection into the embryo.
47. The method of claim 46, wherein the local injection comprises catheter administration.
48. The method of claim 39, wherein the cells are administered with a pharmaceutically acceptable substrate.
(Item 49) The method according to item 48, wherein the substrate is biodegradable.
50. The method of claim 39, wherein the MASC or its progeny cells modify the immune system to resist viral, bacterial or fungal infection.
(Item 51) characterized in that MASC or its progeny reinforce, reconstitute, or provide for the first time the function of a defective cell or organ for injury, genetic disease, acquired disease or iatrogenic treatment 40. A method according to item 39.
(Item 52) The organ is bone marrow, blood, spleen, liver, lung, intestine, eye, brain, immune system, circulatory system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system, peripheral nervous system 52. The method of item 51, wherein the method is selected from the group consisting of: kidney, bladder, skin, epithelial appendages, breast-mammary gland, adipose tissue, and mucosa including mouth, esophagus, vagina and anus.
(Item 53) The method according to Item 51, wherein MASC self-regenerates in vivo.
(Item 54) The disease is cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, degenerative or traumatic neurological disease, autoimmune disease, genetic defect, connective tissue disease, anemia, infection. 52. The method according to item 51, wherein the method is selected from the group consisting of symptoms and transplant rejection.
(Item 55) The method according to item 44, wherein the progeny cells are differentiated ex vivo or in vivo.
(Item 56) The method according to item 45, wherein the progeny cells are differentiated ex vivo or in vivo.
(Item 57) The progeny cells are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscles, smooth muscles, myocardium, eyes, endothelium, liver, pancreas, hematopoiesis, glia, nerves 46. The method of item 45, wherein the method is selected from the group consisting of cells and oligodendrocytes.
58. MASC or its progeny cells home to and settle in one or more organs of the patient, thereby causing cells that are defective for injury, genetic disease, acquired disease or iatrogenic treatment or 40. A method according to item 39, wherein the function of the organ is augmented, reconfigured or provided for the first time.
(Item 59) The disease is cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, degenerative or traumatic neurological disease, autoimmune disease, genetic defect, connective tissue disease, anemia, infection. 59. The method of item 58, wherein the method is selected from the group consisting of symptom and transplant rejection.
60. The method of claim 58, wherein the injury is ischemia or inflammation.
(Item 61) The organ is bone marrow, blood, spleen, liver, lung, intestine, eye, brain, immune system, circulatory system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system, peripheral nervous system 59. The method of item 58, wherein the method is selected from the group consisting of: kidney, bladder, skin, epithelial appendages, breast-mammary gland, adipose tissue, and mucous membranes including mouth, esophagus, vagina and anus.
(Item 62) The method according to item 58, wherein the MASC self-regenerates in vivo.
(Item 63) The method according to Item 39, wherein the MASC or a progeny cell thereof promotes angiogenesis.
(Item 64) The method according to item 39, wherein the MASC or a progeny cell thereof is genetically modified to transport a therapeutic agent.
(Item 65) The method according to item 64, wherein the therapeutic agent is an anti-angiogenic agent.
66. The method of claim 39, wherein the administering is made via a three dimensional matrix comprising artificial blood vessels.
67. The method of claim 66, wherein the treatment is directed to abdominal aortic aneurysm, cardiac bypass surgery, peripheral vascular disease or coronary vascular disease.
(Item 68) Bone marrow, blood, spleen, liver, lung, intestinal tract, eye, brain, immune system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system, kidney, bladder, skin, epithelial appendage, MASC and pharmaceutically acceptable, characterized by the presence of MASC in an amount effective to create a tissue selected from the group consisting of breast-mammary gland, adipose tissue, and mucous membranes including mouth, esophagus, vagina and anus A therapeutic composition comprising a supported carrier.
(Item 69) (a) Remove MASC from the mammalian donor; (b) Expand MASC to form an expanded population of undifferentiated cells; and (c) Administer the expanded cells to the patient to obtain organs, tissues A therapeutic method for restoring the function of an organ, tissue or cell in a patient in need thereof, comprising restoring the function of a cell;
70. The method of claim 69, wherein the function is enzymatic.
(Item 71) A method according to item 69, wherein the function is genetic.
72. The method of claim 69, wherein the mammal donor is a patient.
(Item 73) The organ, tissue or cell is bone marrow, blood, spleen, liver, lung, intestine, eye, brain, immune system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system, peripheral nerve 70. The method of item 69, wherein the method is selected from the group consisting of: system, kidney, bladder, skin, epithelial appendage, breast-mammary gland, adipose tissue, and mucosa including mouth, esophagus, vagina and anus.
(Item 74) (a) A nucleic acid sequence encoding a donor's MHC antigen operably linked to a promoter is introduced into MASC ex vivo, and the MHC antigen is expressed by MASC; and (b) MHC antigen is transplanted Transplanting MASC into a patient so that the expression is at a level sufficient to inhibit rejection of the treated MASC; a method of inhibiting rejection of a heterogeneous MASC transplanted into the patient, comprising each step .
75. The method of claim 74, wherein the patient is the same species as the MASC provider or another mammalian species.
76. The method of claim 74, wherein the MASC is transplanted into the patient by local injection, systemic injection, parenteral administration, oral administration, or intrauterine injection into the embryo.
77. The method of claim 76, wherein the local injection comprises catheter administration.
78. The method of claim 74, wherein the cells are transplanted with a pharmaceutically acceptable substrate.
(Item 79) The method according to item 78, wherein the substrate is biodegradable.
(Item 80) (a) knocking out expression of MHC antigen in MASC by gene transfer; and (b) transplanting said gene transfer MASC into a patient; including each step, wherein said MHC antigen is not expressed by MASC; A method of inhibiting rejection of a heterologous MASC transplanted into a patient, characterized in that the rejection of the transplanted MASC is inhibited (Item 81) The patient is the same species as the MASC donor or another mammalian species Item 80. The method according to Item 80, wherein
82. The method of claim 80, wherein MASC is administered to the patient by local injection, systemic injection, parenteral administration, oral administration, or intrauterine injection into the embryo.
83. The method of item 80, wherein the local injection comprises catheter administration (item 84). The method of item 80, wherein the cells are implanted with a pharmaceutically acceptable substrate.
85. The method of claim 84, wherein the substrate is biodegradable.
(Item 86) (a) MASC is isolated; and (b) MASC is differentiated to form blood or blood components; blood or individual blood components are generated ex vivo, characterized in that each step is included. how to.
(Item 87) The method according to item 86, wherein the individual blood components are red blood cells, white blood cells, or platelets.
(Item 88) (a) Analyzing the genomic or proteomic organization of MASC or its progeny cells; (b) utilizing the analysis via computer analysis using bioinformatics and / or algorithms; and (c) new data And discovering a new drug by comparing the new data with a known database; and a method of drug discovery comprising each step.
(Item 89) (a) Analyzing MASC genomic or proteomic composition; (b) Inducing differentiation of MASC; (c) Analyzing genomic or proteomic composition of intermediate cells in the differentiation pathway; (d) Differentiation pathway Analyzing the genomic or proteomic organization of terminally differentiated cells; (e) characterizing the genomic or proteomic organization of MASC and its progeny using bioinformatics and / or algorithms; and (f) obtained in (e) Comparing data to identify pathway components; a method of identifying differentiation pathway components, comprising each step.
(Item 90) The method according to item 89, wherein the differentiation occurs in vitro.
(Item 91) The method according to item 89, wherein the differentiation occurs in vivo.
(Item 92) A method for comparing in vitro differentiation with in vivo differentiation, comprising a step of comparing the result of the method described in Item 90 with the result of the method described in Item 91.
(Item 93) (a) Analyzing the genomic or proteomic composition of MASC isolated from a healthy donor; (b) The genomic or proteomic composition of MASC isolated from a provider suffering from a disease or injury (C) characterize data from (a) using bioinformatics and / or algorithms; (d) characterize data from (b) using bioinformatics and / or algorithms; ) Comparing the data obtained in (c) with the data obtained in (d) to identify the molecular component of the disease or injury; a molecule of disease or injury characterized by comprising each step A method of identifying a component.
(Item 94) (a) identifying a product in MASC; and (b) isolating said product from MASC; having therapeutic, diagnostic or research utility, comprising each step A method to create a product with in vitro.
95. The method of claim 94, wherein the product is selected from the group consisting of proteins, lipids, glycoconjugates, DNA and RNA.
96. A method of inducing resistance to an antigen administered to a mammal in a mammal comprising the step of administering to the mammal an effective amount of MASC or its progeny cells after or simultaneously with administration of said antigen. Thereby, a mammalian humoral immune response to subsequent sensitization with said antigen is suppressed.
97. A method for removing toxins from a patient's blood, comprising the step of contacting the blood with MASC-derived cells ex vivo, said cells covering the inner surface of a hollow fibrous device.
(Item 98) The method according to item 97, wherein the cell is a kidney or a hepatocyte.
99. Contacting the patient's blood with MASC or its progeny cells ex vivo, wherein the MASC or its progeny cells are genetically modified to deliver a therapeutic agent, A method for transporting therapeutic products to a patient.
(Item 100) A method for testing the toxicity of a drug, comprising the step of contacting MASC or a progeny cell thereof with a drug ex vivo, and further observing the survival of said cell.
101. The method of claim 100, wherein the progeny cells are selected from the group consisting of liver, endothelium, epithelium and kidney cells. Examples of differentiated cells that can be obtained from MASC are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, heart muscle, eye, endothelium, epithelium, liver, pancreas, hematopoiesis, Glia, nerve cells or oligodendrocytes. Differentiation can be induced in vivo or ex vivo.

  The MASCs of the present invention are also summarized as cells that constitutively express oct4 and high concentrations of telomerase and are negative for CD44, MHC class I and MHC class II expression. The therapy is the cells administered to the patient in a therapeutically effective amount. The surprising advantage of this treatment is that teratomas do not form in vivo.

  An object of the present invention is to create a normal non-human animal containing MASC. Preferably, the animal is a chimera.

  Another embodiment of the invention is a composition comprising a population of MASCs and a medium that expands the population of MASCs. In some cases it may be beneficial for the medium to contain epidermal growth factor (EOF), platelet derived growth factor (PDGF) and leukemia inhibitory factor (LIF).

  The present invention also provides a composition comprising a population of fully or partially purified MASC progeny cells. The progeny cells may have the ability to further differentiate or may be terminally differentiated.

  In a preferred embodiment, the progeny cells are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, heart muscle, eye, endothelium, epithelium, liver, pancreas, hematopoietic, glia, It is a neuronal or oligodendrocyte cell type.

The present invention also obtains tissue from a mammal, establishes a population of adherent cells, removes CD45 ⁺ cells from the population, collects CD45 ⁻ cells and cultures under expanded conditions to obtain an expanded cell population This provides a method for isolating and growing MASCs. The object of the present invention is therefore to create an expanded cell population by this method.

  One embodiment of the present invention is a method of differentiating MASC ex vivo by isolating and expanding MASC, and then proliferating the expanded cells in the presence of a differentiation factor of interest. Preferred differentiation factors are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), dimethyl sulfoxide (DMSO) and isoproterenol; or fibroblast growth factor 4 (FGF4) and hepatocyte growth factor ( HGF). Another aspect of the invention is the differentiated cell itself.

  The present invention isolates and expands MASCs and then administers the expanded cell population to a mammalian host where the cell population settles and differentiates into tissue-specific cells in vivo, thereby causing injury, genetic disease, acquired Includes methods of differentiating MASCs in vivo such that the function of defective cells or organs is augmented, reconstituted, or provided for the first time for disease or iatrogenic treatment. Using this method, MASC can self-regenerate in vivo.

  Yet another embodiment of the present invention is a differentiated cell obtained by ex vivo or in vivo differentiation. In a preferred embodiment, the differentiated cell is ectoderm, mesoderm or endoderm. In another preferred embodiment, the differentiated cells are osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, skeletal muscle, smooth muscle, myocardium, eye, endothelium, epithelium, liver, pancreas, hematopoiesis , Glial, neuronal or oligodendrocyte cell types.

  An important application of this technology is a method of treating a patient by administering a therapeutically effective amount of MASC or its progeny. The progeny cells may have the ability to further differentiate or may be terminally differentiated. An unexpected advantage of this approach is that the need for pre- and / or post-treatment of patients with radiation, chemotherapy, immunosuppressive agents or other drugs or treatments is reduced or eliminated. Induction of tolerance before or during treatment is also not necessary.

  Such treatments include cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, degenerative or traumatic neurological disease, autoimmune disease, genetic defect, connective tissue disease, anemia, infectious disease A variety of diseases and conditions can be treated, including transplant rejection.

MASC or its progeny are administered via catheterization, systemic injection, parenteral administration, oral administration, or local injection including intrauterine injection into the embryo. Administration can take place with a pharmaceutically acceptable substrate, which can be biodegradable.

  MASC or its progeny administered to a patient modify the immune system to resist viral, bacterial or fungal infection.

  Surprisingly, no teratoma is formed when MASC or its progeny are administered to a patient.

  When administered to a patient, MASC or its progeny also reinforce, reconstitute, or provide for the first time the function of a defective cell or organ for injury, genetic disease, acquired disease or iatrogenic treatment You can also. Organs are bone marrow, blood, spleen, liver, lung, intestine, brain, immune system, circulatory system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system, peripheral nervous system, kidney, bladder, skin, epithelium Any of the appendages, breast-mammary gland, adipose tissue, and mucous membranes including mouth, esophagus, vagina and anus. Examples of diseases treatable by this method are cancer, cardiovascular disease, metabolic disease, liver disease, diabetes, hepatitis, hemophilia, degenerative or traumatic neurological disease, autoimmune disease, genetic defect, connective tissue disease, Anemia, infection and transplant rejection.

  MASC or its progeny home to one or more organs in the patient, thereby reinforcing the function of defective cells or organs for injury, genetic disease, acquired disease or iatrogenic treatment It has become established there as it is configured or offered for the first time, which is surprising and unexpected. In a preferred embodiment, the injury is ischemia or inflammation.

  In another preferred embodiment, MASC or its progeny promote angiogenesis.

  In another aspect of the invention, MASC or its progeny are genetically modified to deliver a therapeutic agent, preferably an anti-angiogenic agent.

  The present invention is MASC is bone marrow, blood, spleen, liver, lung, intestine, brain, immune system, bone, connective tissue, muscle, heart, blood vessel, pancreas, central nervous system, kidney, bladder, skin, epithelial appendages, A therapeutic composition comprising MASC present in an amount effective to produce tissue selected from the group consisting of breast-mammary gland, adipose tissue, and mucous membranes including mouth, esophagus, vagina and anus, and a pharmaceutically acceptable carrier. Offer things.

  The present invention further includes the step of removing MASCs from a mammalian donor to form an expanded population of undifferentiated cells and administering the expanded cells to a patient where the function of the organ, tissue or cells is restored, Therapies are provided to restore patient tissue or cell function. The function to be restored can be enzymatic or genetic. In a preferred embodiment, the mammalian donor is a patient.

  The present invention introduces a nucleic acid sequence encoding a recipient's MHC antigen linked to a promoter for manipulation ex vivo in MASC, where the MHC antigen is expressed by MASC and transplanted into the patient, where the MHC antigen is Provided is a method of inhibiting rejection of a heterologous MASC implanted in a patient, comprising a step expressed at a level sufficient to inhibit rejection of the transplanted MASC. The patient is the same species as the MASC donor or is another mammalian species.

  Another method of inhibiting the rejection of heterologous MASCs transplanted into a patient involves knocking out the expression of MHC antigens in the MASC by gene transfer and transplanting the transgenic MASC into the patient. MHC antigen is not expressed by MASC and transplant cell rejection is inhibited.

  One object of the present invention is a method of creating blood or individual blood components ex vivo by the process of isolating MASC and differentiating MASC to form blood or blood components. Preferably, the individual blood component is red blood cells, white blood cells or platelets.

  Another aspect of the invention is to analyze the genomic or proteomic organization of MASC or its progeny and use that analysis via computer analysis using bioinformatics and / or algorithms to collect new data and compare it to known databases. This is a method of drug discovery including a step of comparing and contrasting them.

  Further embodiments include analyzing the genome or proteomic organization of MASC or its progeny, inducing MASC differentiation in vitro or in vivo, analyzing the intermediate cell or proteome organization of the differentiation pathway, and the genome of the terminally differentiated cell of the differentiation pathway Or differentiation, including analyzing the proteomic composition, characterizing the genomic or proteomic composition of MASC and its progeny using bioinformatics and / or algorithms, and comparing the resulting data to identify pathway components This is a method for identifying components of a route. This method can be used to compare in vitro and in vivo differentiation, thereby characterizing the fundamental differences between the two systems.

  The present invention provides methods for in vitro production of products having therapeutic, diagnostic or research utility by identifying products in MASC and isolating the products from MASC. In a preferred embodiment, the product is a protein, lipid, glycoconjugate, DNA or RNA.

  The present invention includes a method for inducing resistance to an antigen administered to a mammal in a mammal, wherein the method suppresses the mammalian humoral immune response to subsequent sensitization with the antigen. As such, it comprises the step of administering to said mammal an effective amount of MASC or its progeny after or simultaneously with administration of said antigen.

  Also included is a method of removing toxins from a patient's blood comprising contacting the blood ex vivo with MASC-derived cells, wherein the cells cover the inner surface of a hollow fibrous device. In a preferred embodiment, the cell is a kidney or hepatocyte.

  One object of the present invention includes contacting the patient's blood ex vivo with MASC or progeny thereof, wherein the MASC or progeny thereof has been genetically modified to deliver a therapeutic agent. A method for transporting a product to a patient. A further object is a method for testing drug toxicity comprising contacting MASC or its progeny with said drug ex vivo and observing cell survival. In a preferred embodiment, the progeny cells are selected from the group consisting of liver, endothelium, epithelium and kidney cells.

  The following detailed description is presented for purposes of illustration, but is not intended to limit the invention to the particular embodiments described, and may be understood in conjunction with the accompanying drawings, which are incorporated herein by reference.

  FIG. 1 is a diagram showing the possibility of expansion of bone marrow (BM), muscle and brain-derived MASCs.

  FIG. 2 is a scatter plot showing gene expression in (A) muscle and brain MASC and (B) bone marrow and muscle MASC of gene expression.

FIG. 3 shows FACS analysis of MASC cultured with undifferentiated MASC and VEGF. The plot shows the specific antibody staining profile (thick line) versus the isotype control IgG staining profile (thin line). FIG. A shows the phenotype of undifferentiated MASC. MASC expresses low levels of p2 microglobulin, Flk1, Fit1 and AC133 but does not stain with any other anti-endothelial markers; Figure B shows the phenotype of MASC cultured with 10 ng / mL VEGF for 14 days Show. MASC expresses low levels of most markers associated with endothelial cells, but AC133 expression is lost; and Figure C shows the phenotype of MASC cultured with 10 ng / mL VEGF for 3-9 days . MASC lost AC133 expression by day 3 cultured with VEGF, acquired Tek and VE-cadherin expression by day 3, and acquired CD34 and HIP12 by day 9.

FIG. 4 is a micrograph of mmASC colonization and in vivo differentiation. Slides were examined by fluorescence or confocal microscopy. Figures A, G, J, N, Q and S show identically stained tissues of control NOD-SCID animals not injected with mmASC. Figures AF show photomicrographs of bone marrow (BM) cytospins from control (A) and test (BF) animals stained with anti-β-gal-FITC antibody and PE-conjugated antibodies against various hematopoietic antigens. Show. AB: CD45, C: CD19, D: MAC1, E: GR1, F: TER119 and DAPI; FIGS. GI stained with anti-β-gal-FITC antibody and anti-CD45-PE antibody, control (G ) And micrographs of spleen sections from test animals (H, I). Donor-derived anti-β-gal ⁺ cells are found become a lump. H is 10x and I is 60x magnification; Figures J-M are micrographs of liver cross sections from control mice (J) and test animals (K-M) stained with anti-β-gal-FITC. Indicates. J-L is co-stained with mouse anti-CK-18 / anti-mouse-Cy5 plus CD45-PE and M is mouse anti-albumin / anti-mouse Cy3 antibody. J-K, L and M are 20-fold, 60-fold and 10-fold magnifications, respectively; FIGS. N-P show anti-β-gal-FITC plus mouse anti-pan-CK / anti-mouse-Cy5 antibody (N Figure 6 shows micrographs of intestinal sections from control mice and test animals (OP) stained with ~ P). N and P were co-stained with CD45-PE antibody. β-gal ⁺ Pan-CK ⁺ CD45 ⁻ epithelial cells cover 50% of the periphery of the villi (black arrow, figure P). CD45 was co-stained for (P) of the villi center of ^{Pan-CK - / β-gal} + cells (white arrows - Figure O); FIG Q~R mice in addition to the anti-beta-gal-FITC anti pan-CK / Shown are micrographs of lung cross sections from control mice (Q) and test animals (R) stained with anti-mouse-Cy5 and CD45-PE antibodies. Several β-gal ⁺ pan-CK ⁺ donor cells are seen lining the alveoli of the recipient animal (R). CD45 ⁺ / pan-CK ⁻ cells of hematopoietic origin are clearly seen from epithelial cells; and FIGS. ST are control mice (S) stained with anti-β-gal-FITC, anti-vWF-PE and TO-PRO3 and The micrograph of the blood vessel cross section of the thymic lymphoma (T) 16 weeks after transplantation which grew up in the test animal is shown.

β-gal ⁺ donor cells differentiated into vWF ⁺ endothelial cells within the recipient thymic lymphoma because tumor cells were not stained with anti-β-Gal antibody.

  FIG. 5 shows immunohistochemical evaluation using confocal fluorescence microscopy of MASC-derived endothelial cells. (A) MASC cultured in VEGF for 14 days. Typical membrane staining is seen for the adhesion receptor αvβ5 and for the adhesion binding proteins ZO-1, β- and γ-catenin. Scale bar = 50 μm. (B) MASC brightfield morphology on day 0 (top) and day 21 (bottom) after VEGF treatment. Bar = 25 μm.

  FIG. 6 is a photomicrograph of MASC-derived endothelial cells. FIG. A shows histamine-mediated release of vWF from MASC-derived endothelium. Staining with antibodies to myosin shows cytoskeletal changes with proliferation of myosin stress fibers and increased gap junctions (arrows) (representative example from 3 experiments). Scale bar = 60 μm; FIG. B shows that MASC-derived endothelium takes up a-LDL. After 7 days, the cells expressed Tie-1, but this time did not take up a-LDL. However, acquisition of vvWF expression on day 9 was accompanied by aLDL uptake (representative example of 10 experiments). Scale bar = 100 μm; and FIG. C shows angiogenesis by MASC-derived endothelium. After 6 hours, typical blood vessels could be seen (representative example of 6 experiments). Scale bar = 200 μm.

  FIG. 7 shows FACS analysis of MASC-derived endothelial cells. The plot shows the specific antibody staining profile (thick line) versus the isotype control IgG staining profile (thin line) (experimental> 3 representative examples). The numbers above the plot are the mean fluorescence intensity (MFI) for control IgG staining and specific antibody staining. Figure A shows flow cytometry analysis showing that hypoxia up-regulates Flk1 and Tek expression in MASC-derived endothelial cells; Figure B shows that hypoxia up-regulates VEGF production by MASC-derived endothelial cells Show. VEGF levels are measured by ELISA and results are shown as the mean ± SEM of 6 experiments; and FIG. C shows that IL-1a induces class II HLA antigen expression and increases adhesion receptor expression. The plot shows the specific antibody staining profile (thick line) against the isotype control IgG staining profile (thin line) (representative example of 3 experiments). The numbers above the plots show the MFI for control IgG staining and specific antibody staining.

  FIG. 8 is a photomicrograph of human MASC-derived endothelial cells. Figures C-F show either anti-human β2-microglobulin-FITC (Figure C) or anti-mouse-CD31-FITC (Figure D), and their fusion (Figure E), anti-vWF-Cy3 (Figure F) and A three-dimensional reconstruction of the fusion of three staining patterns (Figure G) is shown. Figures A and B show confocal images of one section stained with either anti-human β2-microglobulin-FITC and anti-vWF-Cy3, or anti-mouse CD31-Cy5 and anti-vWF-Cy3. Scale bar = 100 μm. Figure H shows advanced wound healing in perforated ears stained with anti-β2-microglobulin-FITC and anti-vWF in mice injected with human MASC-derived endothelial cells (top) or human foreskin fibroblasts s (bottom) Fig. 2 shows the formation of an angiogenic part. Scale bar = 20 μm. C = cartilage. D = dermis. Figure I shows that tumor angiogenesis is derived from endothelial cells generated from MASCs in vivo, resulting in highly vascularized portions in tumors stained with anti-β2-microglobulin-FITC, anti-vWF and TOPRO-3 Indicates. Scale bar = 20 μm.

  FIG. 9 is a diagram showing spike generation and expressed voltage-dependent sodium current in hMSC-derived neuronal cell-like cells. FIG. A shows a photomicrograph of cultured hMSC-derived neurons that showed spike generation and expressed voltage-dependent sodium current (the pipette shadow points to the cell). FIG. B is a diagram showing current fixation recordings obtained from hMSC-derived neurons. FIG. C is a diagram showing a current record obtained by subtracting leaks obtained from the same hMSC-derived neurons.

  FIG. 10 shows quantitative RT-PCR and Western blot analysis confirming the hepatocyte-like phenotype. Figures A and B show FGF4 and HGF, or FGF4 alone and mMASC (A) and hMASC (B) cultured in Matrigel ™ for 21 and 28 days, respectively. For αFP, Cyp2b9 and Cyp2bl3, transcripts were not detected in undifferentiated MASC, so the numbers below the blots are relative to the mRNA obtained from the liver. Li = mouse or human liver mRNA; NT = no template. As a representative example of the test of 5 mice and 1 human, FIG. C shows FGF4 and HGF or FGF4 alone and hMASC (B) cultured in Matrigel for 21 days. FH = FGF4 and HGF-induced hMASC on Matrigel, Huh = Huh7 cell line used as control.

  FIG. 11 shows a photomicrograph of hepatocyte-like cells. MASCs induced by FGF4 produce glycogen. Glycogen storage is seen as an accumulation of darkened areas (representative example from 3 trials). Scale bar = 25 μm.

Definitions Here, the following terms shall have the following meanings: “Expansion” shall mean the proliferation of cells without differentiation.

  An “intermediate cell” is a cell that has some characteristics but not all characteristics of MASC or its terminally differentiated progeny that occurs during MASC differentiation. The intermediate cell may be a progenitor cell determined by a specific pathway, but it must not be determined by a specific cell type.

  “Normal” shall mean animals that do not have illness, mutation, or malformation, ie, healthy animals.

  “Self-renewal” shall mean the ability of a cell to proliferate without being subjected to external stimuli. The presence of cytokines or other growth factors produced locally in the tissue or organ shall not be an external stimulus.

  “Homing” shall mean the ability of some MASCs or their progeny to migrate specifically to the site where additional cells are needed.

  “Expression knockout” shall mean the loss of function of a particular gene.

  As used herein, “genomic or proteomic organization” shall mean any cellular gene or protein component.

  “High levels of telomerase activity” can be associated with twice the level observed in the human immortal cell line MCF7. Soule et al. (1973) J. Cancer Inst. 51: 1409-1416.

Technical Applications MASC technology can be used to replace damaged, diseased, dysfunctional or dead cells in the mammalian body. In addition, these cells may be used with autologous or allogeneic cells, with or without natural or artificial carriers, substrates, or macromolecules, eg sickle cell disease, hemophilia or eg Gaucher disease, Neiman.・ Genetic, lost cells, abnormalities such as genetic mutations that affect protein functions such as “storage diseases” where products accumulate in the body due to processing defects such as Pick's disease, mucopolysaccharidosis, etc. The host can be injected to modify function or cells or organs. An example of dying or dead cell recovery can be the use of MASC or its differentiated progeny in the treatment of macular degeneration and other neurodegenerative diseases.

  Given the ability to “homing” these MASCs into host animal organs / tissues for integration, growth and differentiation, MASCs can probably be used to provide new endothelial cells also in the ischemic heart and myocardial cells themselves. There are possibilities and there are many other examples.

  There may be medical situations in which a temporary benefit to the function of a tissue or organ can have a desirable effect. For example, there is an example of a liver disorder patient who has connected enough bioartificial liver to allow normal liver function to be restored, preventing the need for liver transplantation. This is, for example, a serious and unmet medical need for a single liver disease called hepatitis C. There are 4-5 million Americans currently infected with hepatitis C, with an estimate that 50% of these people will have cirrhosis in the future and require liver transplantation. This is a major public health problem that requires relief. Hepatocytes derived from autologous or allogeneic MASCs can be transplanted in hepatitis C or other liver diseases. Such transplants provide temporary liver function so that the donor's own hepatocytes can recover, or permanently re-establish in the damaged liver and the donor cells restore normal liver function can do.

In addition to many cell therapies in which undifferentiated MASCs are administered to humans or other mammals and then differentiate into specific cells in the recipient, MASC progeny cells are differentiated ex vivo and subsequently purified cells or It can be administered as a mixture of cells to provide a therapeutic benefit. These undifferentiated MASCs can also be used as carriers or vehicles to transport drugs or molecules with therapeutic benefit. This can be used to treat any of a number of diseases including but not limited to cancer, cardiovascular disease, inflammatory disease, immune disease, infectious disease, and the like. Thus, by way of example, cells, possibly endothelial cells expressing new or high levels of angiogenic molecules, can be administered to a patient, which are incorporated into existing blood vessels to promote angiogenesis, for example in the heart; Similarly, it produces molecules that suppress angiogenesis and are incorporated into blood cells to suppress further formation in, for example, diabetic retinopathy or cancers where new angiogenesis is key to the etiology, spread and extent of the disease Endothelial cells can be obtained.

  The ability to settle in BM and form blood exvivo has important medical applications in the shadows. For example, with regard to blood exvivo production, blood transfusions and blood product injections are still in place around the world, and their safety is variable due to pathogen infection. Transfusions have led to an imminent risk of HIV, hepatitis B and C, and now mad cow disease or CJD, Creutzfeldt-Jakob disease. The ability to produce blood, particularly red blood cells in vitro, can provide a safe and reliable alternative to collecting blood from humans. There seems to be no complete replacement for donor blood collection. hMASC or its hematopoietic progeny can be injected into an animal, such as a sheep, in utero to form human hematopoietic cells and used as a source of human blood components or proteins for medical use. The same is true for hepatocytes, islets or many other cell types, but provides an alternative to producing human cells in vitro and animals are used as cell factories. This can assist in the blood shortage that is predicted to occur. hMASC can also be transplanted into human embryos to correct any one of several defects, if possible.

  Since these MASCs can generate clonal populations of specifically differentiated cells, they are a rich basis for drug discovery. This includes gene expression, analysis of gene expression, discovery of new gene activation patterns of activation, proteomics and protein expression and modification patterns around this. This is analyzed in bioinformatics using a database and an algorithm for analyzing these data compared to a public or self-owned database. Information about how a known drug or substance behaves can be compared to information obtained from MASC, its differentiated progeny, and available human populations. Pathways, targets, and receptors can be identified. New drugs, antibodies or other compounds can be found to produce biologically desirable reactions. Similarly, MASC and its differentiated offspring can be used as a monitor for unwanted reactions in combination with databases, bioinformatics and algorithms.

  These MASCs, derived from humans, mice, rats or other mammals, are the only bodies known to date that express very high levels of telomerase even in normal, non-malignant, slow-passage cells. It appears to be a cell (non-germ cell). Telomeres are elongated by MASC and are karyotypically normal. MASCs injected into mammals home to multiple organs, so newly arrived MASCs may be able to self-renew. Thus, MASCs are not only themselves, but are self-regenerated differentiated cell types that are damaged, dead, or otherwise have abnormal functions due to genetic or acquired disease But it has the potential to re-establish in the organ.

For example, in type I diabetes, islet insulin-producing beta cells are progressively lost. Various kidney diseases have progressive loss of function and in some cases, glomerular disappearance. In the case of diabetes, MASCs or differentiated progeny can home to the pancreas and induce islets to form themselves or by interaction with endogenous cells in the pancreas. This can have the effect of improving diabetes. Ultimately, MASC's ability to self-renew is controlled in vivo, ie promoted or suppressed, and differentiated progeny such as islet progenitor cells, hepatocyte progenitor cells, blood cell progenitor cells, nerve and / or cardiac progenitor cells Conditions, substances or drugs may be found that control, i.e. promote or inhibit, the transition to. MASCs can be used to find pathways, methods of activation and control that can induce endogenous progenitor cells to proliferate and differentiate in the organ.

  This same ability to re-establish, self-renew and also differentiate into cellular tissue or organ compartments can have numerous applications and unprecedented utility to meet the unmet medical need. For example, certain genetic diseases in which enzyme deficiency is present are treated by BM transplantation. This often helps the disease complications that the sequelae of the disease can remain in the brain or bone or elsewhere, but does not cure. MASCs and genetically engineered MASCs offer hope to improve a number of genetic and acquired diseases. They are also useful for diagnostic or research purposes and drug discovery.

  The invention also includes analyzing the genomic or proteomic organization of MASC and combining it with bioinformatics analysis, algorithmic computer analysis, collecting new databases, comparing them to known databases, and contrasting them; Methods are provided for discovery, genomics, proteomics, and pathway identification. This can lead to definition of targets for novel compounds, antibodies, proteins, small molecule organic compounds, or other biologically active molecules that may have therapeutic benefits, major components, pathways, novel genes and / or New patterns (proteomics) of gene and protein expression and protein modification can be identified.

  The following examples are provided to illustrate the invention, but do not limit the invention.

Selection, culture and characterization of mouse pluripotent adult stem cells (mMASC) Cell isolation and expansion All tissues were obtained according to guidelines from the University of Minnesota Institutional Animal Experimentation Committee. BM mononuclear cells (BMMNC) were obtained by Ficoll-Hypac separation of BM. BM was obtained from 5-6 week old ROSA26 mice or C57 / BL6 mice. Alternatively, muscle and brain tissue were obtained from 129 3 day old mice. Muscles from the proximal part of the forelimbs and hind limbs were removed and ground well. The tissue was treated with 0.2% collagenase (Sigma Chemical Co, St Louis, MO) for 1 hour at 37 ° C. and then with 0.1% trypsin (Invitrogen, Grand Island, NY) for 45 minutes. The cells were then well ground and passed through a 70 μm filter. The cell suspension was collected and centrifuged at 1600 rpm for 10 minutes. Brain tissue was incised and ground well. Cells were dissociated by incubation with 0.1% trypsin and 0.1% DNAse (Sigma) for 30 minutes at 37 ° C. The cells were then well ground and passed through a 70 μm filter. The cell suspension was collected and centrifuged at 1600 rpm for 10 minutes.

Expand BMMNC or muscle or brain suspension at 1xl0 ⁵ / cm ² [2% PCS supplemented low glucose Dulbecco minimal essential medium (LG-DMEM), platelet derived growth factor (PDGF) and epidermal growth factor (EGF) And leukemia inhibitory factor (LIF) were plated at 10 ng / mL each and maintained at 5xlO ³ / cm ² . After 3-4 weeks, CD45 ⁺ / glycophorin (Gly) -A ⁺ cells were removed from the cells collected with trypsin / EDTA using magnetic microbeads. The resulting CD45 ⁻ / Gly-A ⁻ cells were replated at 10 cells / well in FN-coated 96-well plates and expanded at cell densities between 0.5 and 1.5 × 10 ³ / cm ² . The possibility of MASC expansion was similar regardless of the tissue from which it was derived (Figure 1).

The characterization phenotypically of MASC, BM, from muscle and brain, MMASC cultured in FN is, CD13 ^+, CD44 ^-, CD45 ^-, class I and class II histocompatibility antigens - with low Flk1 and CKif, The characteristics of hMASC described in International Application No. PCT / US00 / 21387 were the same. Cells with type IV collagen, laminin or Mat
When cultured in rigel, the cell expansion during the first 2-3 months was greater, but the cells had MSC phenotypic characteristics, ie, expressed CD44 but not CD13. Similar to human cells, mMASC cultured in FN expressed oct-4 transcripts and LIF-R.

A continuously growing culture was obtained from approximately 1% of the wells seeded with 10 CD45 ⁻ / GlyA ⁻ cells. This suggests that the cells that can initiate cultured MASC are rare and probably less than 1 / 1,000 of CD45 ⁻ / GlyA ⁻ cells. MMA cultured in FN has a diameter of 8 to 10 μm, a large nucleus and a small cytoplasm. Some populations have been cultured for> 100 PD. Cell morphology and phenotype remained unchanged throughout the culture period.

  The mMASC after 40 and 102 PD were collected and the telomere length was evaluated. The telomere length was measured using a telomere length analysis kit of Pharmingen (New Jersey, USA) according to the instruction manual. The average telomere length (ATL) of mmASC cultured for 40 PD was 27 Kb. When retested after 102PD, ATL remained unchanged. For karyotype analysis of mMASC, cells were subcultured at a 1: 2 dilution for 12 hours before harvesting as described previously, harvested with trypsin-EDTA, treated with colcemid for 1.5 hours, followed by hypotonic KCl. Was lysed and fixed with acid / alcohol (Verfaillie et al., 1992). Cytogenetic analysis was performed monthly and showed normal karyotype except for one population that became hyperdiploid after 45 PD and was no longer used in the study.

Mouse MASCs obtained after 46 to> 80 PD were tested by quantitative (Q) -RT-PCR for the expression levels of oct4 and Rex1, two transcription factors important for maintaining the undifferentiated state of ES cells did. RNA was extracted from mouse MASC, offspring differentiated into neuroectodermal (1-7 days after addition of bFGF) and mouse ES cells. RNA was reverse transcribed and the resulting cDNA was amplified 40 times under the following reaction conditions (ABIPRISM7700, Perkin Elmer / Applied Biosystems): the first using 2 μL of DNA solution and 1XTaqMan SYBR Green Universal Mix PCR reaction buffer After denaturation (95 ° C for 10 minutes), 40 cycles of two-step PCR (95 ° C for 15 seconds, 60 ° C for 60 seconds). Primers are listed in Table 1.

  mRNA levels were normalized using GAPDH as a housekeeping gene and compared to levels in mouse ES cells. Oct4 and Rex1 mRNA were present at similar levels in BM, muscle and brain derived MASCs. Rex1 mRNA levels were similar in mmASC and mES cells, while oct4 mRNA levels were about 1,000 times lower in MASCs than ES cells.

The expression gene profiles of mouse BM, muscle and brain derived MASCs are very similar.To further evaluate whether MASCs from different tissues are similar, we expressed the expression gene profiles of BM, muscle and brain derived MASCs U74AAffimetrix It examined using the gene array. In summary, mRNA was extracted from 2 to 3xl0 ⁶ BM, muscle or brain-derived MASCs cultured during 45 cell divisions. CDNA preparation, hybridization with 6,000 mouse genes and U74A array containing 6,000 EST clusters, and data acquisition were performed according to the instructions (all from Affimetrix, SantaClara, CA). Data analysis was performed using GeneChip® software (Affimetrix). An increase or decrease in expression by a factor of 2.2 (IyerV.R. Etal., 1999; ScherfU. Et al., 2000; Alizadeh AA et al, 2000) was considered significant and the r ² value was analyzed using linear regression analysis Determined (Figure 2).

  Comparison of the expression gene profiles of MASCs derived from the three tissues showed that in BM-derived MASCs, less than 1% of the genes were expressed at levels that differed by more than 2.2 times compared to muscle-derived. Similarly, in BM-derived MASCs, only less than 1% of the genes were expressed at levels that differed by more than 2.2 times compared to brain-derived. Since the correlation coefficient between different MASC populations was> 0.975, MASCs from different tissues are very homologous and are consistent with the phenotypes described above and the differentiation features described in Example 5.

  Using mouse-specific culture conditions, maintain mmASC culture for over 100 cell divisions. mMASC culture was initiated using bone marrow from C57B1 / 6, ROSA26 and C57BL / 6 mice transfected with -HMG-LacZ.

Selection and culture of rat pluripotent adult stem cells (VMASC) BM and MNC from SpragueDawley or Wistar rats were obtained and seeded under the same conditions as for mmASC. After 21-28 days, CD45 ⁺ cells were removed from the cells and the resulting CD45 ⁻ cells were subcultured at 10 cells / well.

  Similar to mMASC, rMASC is expanded in culture for> 100 PD. Expansion conditions for rat MASC cultures required the addition of EGF, PDGF-BB and LIF, and culture in FN rather than type I collagen, laminin or Matrigel. rMASC was negative for CD44, CD45 and MHC class I and II and expressed high levels of telomerase. The ability of normal cells to grow beyond 100 cell divisions is unprecedented and unexpectedly contradicts the traditional theories over 20 years.

Rat MASCs after 42PD, 72PD, 80PD, and 100PD were collected to evaluate telomere length. Telomeres were not shortened during culture, as determined by Southern blot analysis after 42 PD, 72 PD, 80 PD, and 100 PD. Monthly cytogenetic analysis of rat MASC showed normal karyotype.

Human Pluripotent Adult Stem Cell (hMASC) Selection and Culture BM is an informed consent from healthy volunteer donors (2-50 years old) using guidelines from the University of Minnesota Committee on the use of human subjects in research Obtained later. BMMNC was obtained by Ficoll-pack density gradient centrifugation and depletedofCD45 ⁺ and glycophorin-A ⁺ cells were removed using magnetic microbeads (Miltenyii Biotec, Sunnyvale, Calif.).

Enlargement condition: 5x10 ³ pieces of CD45 ^- / GlyA ^- cells expansion medium 200 uL [1X insulin - transferrin - selenium (ITS), 1X linoleic acid bovine serum albumin ^{(LA-BSA), 10 -8} M dexamethasone, ^10-4 10% ng / ml in 58% DMEM-LG, 40% MCDB-201 (Sigma Chemical Co, St Louis, MO) supplemented with M ascorbic acid-2-phosphate (all Sigma), 100 U penicillin and 1,000 U streptomycin (Gibco) Dilute fetal calf serum (PCS) (Hyclone Laboratories, Logan, UT) containing EOF (Sigma) and 10 ng / ml PDGF-BB (R & D Systems, Minneapolis, Minn. The wells of 96-well plates coated with FN (Sigma) were seeded. The medium was changed every 4-6 days. Once grown to> 40-50% of the well, adherent cells were detached using 0.25% trypsin-EDTA (Sigma) and larger cultures coated with MASC expansion medium at 1: 4 dilution with 5 ng / ml FN The containers were replated and a cell density between 2-8 × 10 ³ cells / cm ² was maintained.

  Undifferentiated MASCs are CD31, CD34, CD36, CD44, CD45, CD62-E, CD62-L, CD62-P, HLA-class I and II, cKit, Tie, Tek, αvβ3, VE-cadherin, vascular endothelial cell adhesion The molecule (VCAM) and intracellular adhesion molecule (ICAM) -1 were not expressed. MASC expressed low / very low levels of β2-microglobulin, αvβ5, CDw90, AC133, Flk1 and Flt1, and high levels of CD13 and CD49b (FIG. 3).

Immunophenotyping analysis Immunofluorescence 1. Cultured cells were fixed with 4% paraformaldehyde and methanol-methanol at room temperature and incubated continuously for 30 minutes each with primary antibody and with or without secondary antibody . Between each step, the slides were washed with PBS / BSA. Cells were examined by fluorescence microscopy (ZeissAxiovert; CarlZeiss, Inc., Thornwood, NY) and confocal fluorescence microscopy (confocal 1024 microscope; Olympus AX70, Olympus Optical Co. LTD, Japan). To assess the frequency of different cell types in any culture, cells that stained positive for any antibody were counted in 4 fields (50-200 cells per field). 2. Collection tissue: Blood and BM cytospin specimens were fixed with acetone (Fisher Chemicals) for 10 minutes at room temperature. For solid organs, fresh frozen sections of tissue 5 μm thick were placed on glass slides and immediately fixed in acetone for 10 minutes at room temperature. After incubation with isotype serum for 20 minutes, cytospin preparations or tissue sections were stained sequentially for tissue specific antigen, β-gal and nuclear counterstain (DAPI or TO-PRO-3).

Cover slips were loaded using the Slowfade-antifade kit (MolecularProbes Inc., Eugene, OR, USA). Slides were examined by fluorescence microscopy and confocal fluorescence microscopy.

3. Antibody: Cells are fixed with 4% paraformaldehyde at room temperature or with methanol at −20 ° C. and continuously with primary antibody and FITC or Cy3-conjugated anti-mouse- or anti-rabbit-IgG antibody Each was incubated for 30 minutes. Between each step the slide was washed with PBS + 1% BSA. PE or FITC-conjugated anti-CD45, anti-CD31, anti-CD62E, anti-Mac1, anti-Gr1, anti-CD19, anti-CD3, and anti-Ter119 antibodies were obtained from BDPharmingen. GFAP (clone GA-5, 1: 400), galactocerebroside (GalC) (polyclonal, 1:50), MBP (polyclonal, 1:50), GABA (clone GB-69, 1: 100), parvalbumin (clone) PARV-19, 1: 2000), TuJl (clone SDL.3D10, 1: 400), NF-68 (clone NR4, 1: 400), NF-160 (cloneNN18, 1:40), and NF-200 (clone) N52, 1: 400), NSE (polyclonal, 1:50), MAP2-AB (clone AP20, 1: 400), Tau (polyclonal, 1: 400), TH (clone TH-2, 1: 1000), DDC (Clone DDC-109, 1: 100), TrH (clone WH-3, 1: 1000), serotonin (polyclonal, 1: 2000), glutamic acid (clone GLU-4, 1: 400), fast muscle myosin (clone MY -32; 1: 400 dilution) was obtained from Sigma. DAPI and TOPRO-3 were obtained from MolecularProbes. Antibodies against vWF (polyclonal; 1:50), Neuro-D (polyclonal, 1:50), c-ret (polyclonal, 1:50) and NurrI (polyclonal, 1:50) are from SantaCruzBiotechnology Inc., Santa Cruz, CA obtained. Antibodies against PSA-NCAM (polyclonal, 1: 500) are from Phanmingen, San Diego, CA, and serotonin transporter (clone MAB1564, 1: 400), DTP (polyclonal, 1: 200), Na gated potential channel (polyclonal, Antibodies to 1: 100), glutamate-receptors-5, -6 and -7 (clone 371, 1: 500) and NMDA (polyclonal 1: 400) receptors were obtained from Chemicon International, Temecula, CA. The anti-nestin (1: 400) antibody was donated by Dr. U. Lendahl, Lund University, Sweden. All antibodies against NSE (1:50) pan-cytokeratin (Cat. No. C-2562; 1: 100), CK-18 (C-8541; 1: 300), albumin (A-6684; 1: 100) are all Sigma Obtained from Polyclonal antibodies against Flk1, Flt1, Tek, HNF-1p were obtained from SantaCruzBiotechnology Inc., Santa Cruz, CA. Anti-nestin (1: 400) antibody was donated by Dr. U. Lendahl of Lund University, Sweden. Control-mouse, rabbit or rat IgG and FITC / PE / Cy3- and Cy5-labeled secondary antibodies were obtained from Sigma. Rabbit anti-β-gal-FITC antibody was obtained from Rockland Immunochemicals, USA. Obtained from TO-PRO-3 Molecular Probes Inc., DAP was obtained from Sigma.

B. X-GAL staining; Tissue sections were stained for β-galactosidase enzyme activity using Invitrogen's β-gal staining kit (pH 7.4). The instructions were followed except for the fixation step where the tissue sections were incubated for 5 minutes instead of 10 minutes.

C. FACS: For FACS, peel off undifferentiated MASC and use anti-CD44, CD45, CD13, cKit, MHC-class I and II, or b2-microglobulin (BDPharmingen) and secondary FITC or PE-conjugated antibodies Sequentially stained and fixed with 2% paraformaldehyde until analysis using a FACS-Calibur (Becton Dickinson).

Single-cell origin of differentiation lineage from MASCs Differentiation factors selected based on the differentiation ability of mMASC or rMASC based on what is described for differentiation into mesoderm, neuroectodermal, and endoderm of hMASC or ES cells Tested by adding (cytokine). Differentiation required reseeding of cells at 1 to ² × 10 ⁴ cells / cm ² in serum-free medium without EOF, PDGF-BB and LIF but with lineage specific cytokines. Differentiation is a tissue-specific marker [slow muscle myosin and MyoD (muscle), von-Willebrand factor (vWF) and Tek (endothelium), NF200 and MAP2 (neuroectoderm), and cytokeratin-18 and albumin (endoderm) )] Was determined by immunohistochemistry, RT-PCR, and functional tests.

MASC differentiation into neuroectodermal cells Palmer et al. Have shown that neural progenitor cells can be expanded in culture using PDGF-BB and that their differentiation can be induced by removing PDGF and adding bFGF as a differentiation factor. Based on these studies and studies conducted with hMASC, mmASC and rMASC were seeded in FN-coated wells without PDGF-BB and EGF but with 100 ng / mL bFGF. Progression of maturation of neuron-like cells was observed throughout the culture period. After 7 days, the majority of cells expressed nestin. After 14 days, 15-20% of MASCs are astrocytes (GFAP ⁺ ), 15-20% are oligodendrocytes (galactocerebroside (GalC) ⁺ ), and 50-60% are neurons (neural Filament-200 (NF-200) ⁺ ) acquired morphological and phenotypic characteristics. NF200, GFAP or GalC was not found in the same cell, suggesting that the neuron-like cells are unlikely to be hMASC or glial cells that inappropriately expressed neuronal markers. Neuronal cells also expressed Tau, MAP2 and NSE. Approximately 50% of neurons expressed gamma-aminobutyric acid (GABA) and parvalbumin, 30% expressed tyrosine hydroxylase and dopa-decarboxylase (DDC), and 20% expressed serotonin and tryptophan hydroxylase. Differentiation was similar when MASCs were expanded by more than 40PD or 90PD. Expression of neuroectodermal markers was confirmed by Q-RT-PCR performed as described in Example 1: MASC expresses otx1 and otx2 mRNA on day 2, and nestin mRNA is detected after 7 days It was done.

The effect of fibroblast growth factor (FGF) -8b as a differentiation factor was then tested. This is important for midbrain development in vivo and is used to induce dopaminergic and serotonergic neurons for hMASC from mouse ES cells in vitro. When densely grown hMASC (n = 8) was cultured with 10 ng / mL of FGF-8b + EGF, differentiation into cells that stained positive for neuronal markers but not positive for oligodendrocytes and astrocytes was observed . Neurons are GABA (GABA ⁺ ; 40 ± 4%), dopaminergic (DOPA, TH, DCC and DTP ⁺ , 26 ± 5%) and serotonergic (TrH, serotonin and serotonin-transporter ⁺ , 34 ± 6%) had neuronal characteristics. DOPA ⁺ neurons were stained with antibodies against Nurr1, suggesting differentiation into midbrain DA neurons. FGF-8b-induced neurons did not have the electrophysiological characteristics of mature neurons. Therefore, 3-week-old cells supported by FGF-8b were further cultured for 2-3 weeks with U-87, which is a glioblastoma cell line, and FGF-8b.

  Neurons acquire a more mature form with increased cell size and number, length, and neurite complexity, and reversibly blocked by electrophysiological features of mature neurons (1 μM tetrodotoxin (TTX)) The transient inward currents obtained were typical of voltage-activated sodium currents found only in mature neurons, along with transient time courses and voltage-dependent activation of inward currents).

  When hMASC (n = 13) was cultured with 10 ng / m brain-derived neural differentiation inducer (BDNF) + EOF, differentiation was limited to DOPA, TH, DCC, DTP and Nurr1-positive neurons. BDNF supports neuronal differentiation from ES cells and NSCs (Peault, 1996; Choi et al. 1998), but no studies have shown limited differentiation into DA-like neurons.

  Similar results were seen for bMA-induced mMASC and bFGF and BDNF-induced rMASC. Further studies on MASC-derived neurons are shown in Example 10.

MASC differentiation into endothelial cells As an example of mesoderm, differentiation into endothelium was induced. Undifferentiated mmASC or rMASC did not express the endothelial markers CD31, CD62E, Tek or vWF, but expressed low levels of Flk1. mMASC or rMASC were cultured with 10 ng / mL endothelial differentiation factor VEGF-B in FN-coated wells. After 14 days of VEGF treatment,> 90% of MASCs expressed Flt1, CD31, vWF or CD62 consistent with endothelial differentiation, regardless of the number of PDs passed. Like primary endothelial cells, MASC-derived endothelial cells formed blood vessels within 6 hours after reseeding on Matrigel.

Similarly, hMASC expresses Flk1 and Flt1, but not CD34, Muc18 (P1H12), PECAM, E- and P-selectin, CD36, or Tie / Tek. When hMASC is cultured in serum-free medium containing 2xl0 ⁴ cells / cm ² and 20 ng / mL vascular endothelial growth factor (VEGF), the cells express CD34, VE-cadherin, VCAM and Muc-18 after day 7. did. On day 14, the cells also expressed Tie, Tek, Flkl and Fltl, PECAM, P-selectin and E-selectin, CD36, vWF, and connexin-40. In addition, the cells were able to take up low density lipoprotein (LDL). Results from histochemical staining were confirmed by Western blot. In order to induce angiogenesis, MASC cultured with VEGF for 14 days was re-seeded on Matrigel containing 10 ng / mL VEGF-B, and 6 hours passed. Endothelial differentiation was not seen when hMASC cultured in> 2% PCS was used. Furthermore, endothelial cells did not arise when PCS was left in the medium during differentiation.

When subcultured with hMASC, at least a 1000-fold expansion was obtained, suggesting that endothelial progenitor cells generated from hMASC continue to have significant proliferative potential. Cell expansion was even greater when PCS was added to the culture after 7 days. When hMASC-derived endothelial cells were administered intravenously to NOD-SCI mice transplanted subcutaneously with human colon cancer, human endothelial cells were seen to contribute to angiogenesis in the tumor. Thus, incorporating recombinant endothelial cells to obtain therapeutic benefit, ie to inhibit angiogenesis, for example in cancer, or to promote angiogenesis in the limb or other organs such as the heart Is possible. Further studies on MASC-derived endothelial cells are shown in Example 9.

MASC differentiation into endoderm We tested whether mMASC or rMASC could differentiate into endoderm cells. Several different culture conditions were tested, including cultures in laminin, collagen, FN or Matrigel coated wells with the differentiation factors keratinocyte growth factor (KGF), hepatocyte growth factor (HGF) and FGF-4. . Approximately 70% of MASCs acquired morphological and phenotypic characteristics of hepatocyte-like cells when replated on Matrigel containing 10 ng / mL FGF4 + 10 ng / mL HGF. The cells became epithelioid cells, about 10% of the cells were binucleated, and about 70% of the cells were positive for albumin, cytokeratin (CK) -18, and HNF-1P.

  Endodermal-like cells generated in cultures containing FGF4 and HGF also measure urea levels in undifferentiated MASC and FGF4 and HGF-induced MASC supernatants using Sigma urea nitrogen kit 640 according to the instructions. The functional characteristics of hepatocytes, as determined by Urea was not detected in undifferentiated MASC cultures. Urea production was 10 μg / cell / hour 14 days after the addition of FGF4 and HGF, and was detectable at similar levels until day 25. This is equivalent to primary rat hepatocytes grown in a monolayer. The presence of albumin supports the concept of in vitro hepatocyte differentiation from MASC along with urea production. Further studies on MASC-derived hepatocytes are shown in Example 11.

  Given the likely presence of endoderm lineage progenitor cells, MASCs yield cells that form both exocrine and endocrine components of the liver and pancreas, and various cells of other endoderm-derived cell tissue lineages Probability is high.

  MASCs derived from muscle or brain can be used in mesoderm (endothelial cells), neuroectodermal (astrocytes and neurons) and endoderm (hepatocyte-like cells) using the methods described above for BM-derived MASCs. Induced to differentiate.

In order to demonstrate that the differentiated cells are derived from a single cell and that MASC is actually a “clone” pluripotent cell, a cultured cell with a retroviral vector introduced into MASC is created, and Its progeny were found to have a retroviral insert at the same site in the genome.

  Tested with two independently obtained ROSA26MASCs, two C57BL / 6MASCs and one rMASC population expanded between 40 and> 90 PD, and also for “clone” mice and “clone” rMASCs introduced with eGFP Went. There was no difference between eGFP-transfected and non-transfected cells. Of note, eGFP expression persisted in differentiated MASC.

Specifically, the eGFP tumor retrovirus vector was introduced into mouse and rat BMMNC cultured for 3 weeks in FN containing EGF, PDGF-BB and LIF for 2 consecutive days. CD45 ⁺ and GlyA ⁺ cells were then removed and the cells were subcultured at 10 cells / well. The eGFP-introduced rat BMMNC was expanded for 85 PD. Alternatively, mouse MASC expanded for 80 PDS was used. Subcultures of undifferentiated MASCs were made by seeding 100 MASCs from cultures maintained for 75 PD and re-expanding them to> 5xl0 ⁶ . Expanded MASCs were induced to differentiate into endothelium, neuroectodermal and endoderm in vitro. Lineage differentiation was demonstrated by staining with antibodies specific for these cell types as described in Example 4.

Single-cell origin of mesodermal and neuroectodermal progeny cells Retroviral marking was used to demonstrate that mesodermal and neuroectodermal differentiated progeny cells are of single-cell origin (Jordan et al. al, 1990; Nolta et al., 1996). The hMASC fraction obtained after 20 PD was transduced with MFG-eGFP retrovirus. eGFP ⁺ hMASC was diluted with non-introduced MASC from the same provider to give a final concentration of transfected cells of ~ 5%. The mixtures were seeded at 100 cells / well were expanded to culturing> 2xl0 ⁷ cells are obtained. Each of 5xl0 ⁶ MASCs was induced to differentiate into skeletal myoblasts, endothelial and neuroectodermal lineages. After 14 days under differentiation conditions, cells were harvested and used to identify retroviral integration sites and coexpression of eGFP with neuroectodermal, muscle and endothelial markers.

The myoblast differentiation, seeded hMASC to 2Xl0 ⁴ cells / cm ² in 2% PCS, EGF and PDGF-containing expansion medium, treated for 24 h in the same medium with 3μM 5-azacytidine. The cells were then maintained in expansion medium containing 2% PCS, EGF and PDGF-BB. For endothelial differentiation, HMASC at 2Xl0 ⁴ cells / cm ^2, not including EGF and PDGF has been cultured for 14 days in serum-free expansion medium containing VEGF-B of 10 ng / ml.

Immunofluorescence assessment showed that 5-10% of cells in culture were induced to differentiate positively for eGFP and skeletal muscle actin with 5-azacytidine, and 5-10% of cells co-stained for eGFP and vWF It was shown that 5-10% of the cells were induced to differentiate into eGFP and neuroectodermal co-stained for either NF-200, GFAP or MBP. To define the retroviral insertion site, the host genome flanking region of MASC and differentiated offspring was sequenced. The number of retroviral inserts in separate populations was between 1 and 7. As shown in Table 2, a single identical sequence adjacent to the retroviral insert was identified in muscle, endothelial and neuroectodermal cells mapped to chromosome 7 in population A16.

Primers specific to the 3 ′ LTR are designed, and primers specific to the adjacent genomic sequence are as shown in Table 3, and using real-time PCR, the retroviral insertion site is an undifferentiated cell and a differentiated cell. It was confirmed that they were the same. These results demonstrated that similar amounts of flanking and eGFP DNA sequences were present. Clone A12 contained two retroviral inserts located on chromosomes 1 and 7, respectively, and both flanking sequences could be detected not only in hMASC but also in muscle, endothelial and neuroectodermal strains. To determine whether this represents a single cell progeny or two cell progeny with two retroviral elements, real-time PCR was used to compare the relative amounts of eGFP flanking sequences on chromosomes 1 and 7. . Similar amounts of both flanking sequences were found in hMASC, muscle, endothelial and neuroectodermal cells, and a single cell with two retroviral inserts was the source of eGFP positive hMASC and differentiated progeny It was suggested that there was a high possibility of becoming. In other populations containing more than two retroviral inserts, we determine whether the insert is from multiple insertion sites in a single cell or from multiple cells that contribute to the eGFP positive fraction I couldn't. Nevertheless, our finding that progeny cells differentiated into muscle, endothelium and neuroectoderm in the two populations are derived from a single BM-derived progenitor cell is the result of three distinct mesodermal lineages and neuroectodermal We demonstrate for the first time that primordial cells can be cultured from BMs that differentiate at a single cell level in cells of different lineages.

Homing and colonization of mammalian MASC to many organs in the body. To investigate whether it has the ability to settle and differentiate into tissue-specific cells in vivo, it was tested. mMASC was grown from LacZ gene-introduced C57Black6 and ROSA26 mice as described in Example 1. 10 ⁶ mmASCs from LacZ mice were injected intravenously into the tail vein of NOD-SCID mice with or without 250Rad total body irradiation 4-6 hours prior to injection. Animals were sacrificed by cervical dislocation 4-24 weeks after injection.

Tissue collection blood and bone marrow: 0.5-1 ml of blood was obtained at the time of animal sacrifice. The femur and tibia were washed and BM was collected. For phenotypic analysis, red blood cells and erythrocytes in BM were removed using ice-cold ammonium hydrochloride (StemCell Technologies Inc., Vancouver, Canada) and 10 ⁵ cells were used for cytospin centrifugation. For serial transplantation, 5xl0 ⁷ cells from 2 femurs and 2 tibias were transplanted into individual secondary recipients by tail vein injection. Secondary recipients were sacrificed after 7-10 weeks.

Solid organs: Lungs were inflated with 1 ml of OCT compound (Sakura-Finetek Inc, USA) diluted 1: 4 in PBS. Samples of spleen, liver, lung, intestine, skeletal muscle, myocardium, kidney and brain of recipient animals were collected at -80 ° C in OCT and for quantitative PCR with RNALater (Ambion Inc., Austin, TX, USA) at -20 ° C.

mMASCs colonize and differentiate into tissue-specific cells in vivo Establishing β-gal / neomycin (NEO) transgene-containing cells (Zambrowiczetal., 1997) by immunohistochemistry for β-gal and by QPCR for NEO Tested. Immunohistochemistry and Q-PCR were performed as described in Examples 5 and 1, respectively. Primers are listed in Table 1.

Settlement, defined as the detection of more than 1% anti-β-gal cells, is shown in Table 4 and Figure 4 in the hematopoietic tissues (blood, BM and spleen) and lung, liver and intestinal epithelium of all recipient animals I was seen on the street.

BM (FIGS. 4B-F) and spleen (FIGS. 4H-I) β-gal ⁺ cells were co-stained with anti-CD45, anti-CD19, anti-Mad, anti-Gr1 and anti-TER119 antibodies. Similar results were seen for peripheral blood. Of note, although β-gal ⁺ CD3 ⁺ T cells were seen in chimeric mice, no β-gal ⁺ CD3 ⁺ T cells were found in either blood, BM or spleen. The reason for this is currently unknown.

  The hypothesis that colonization in the spleen occurs mainly as a mass of donor cells and when MASCs home to the spleen, like CFU-S, MASCs proliferate and differentiate locally to form donor cell colonies Matched. It is considered that the in vivo differentiation of mMASC into hematopoietic cells is not caused by contamination of HMA with mMASC. First, BMMNC has been depleted of CD45 cells by column selection prior to initiating mmASC culture. Second, early mesodermal or hematopoietic transcription factors, including brachyury (Robertsonetal, 2000), GATA-2 and GATA-1 (Weiss et al., 1995), are expressed in undifferentiated mMASC as shown by cDNA array analysis Not done. Third, the culture conditions used for mmASC are not supportive for HCS. Fourth, none of the attempts to induce hematopoietic differentiation in vitro from hMASC by culturing hMASC with hematopoietic support cells and cytokines (Reyeset al., 2001).

Significant levels of mmASC colonization were also seen in the liver, intestine and lung. Three-color staining immunohistochemistry was used to identify epithelial (CK ⁺ ) and hematopoietic (CD45 ⁺ ) cells in the same tissue section of the liver, intestine and lung. In the liver, β-gal ⁺ donor-derived cells formed a bundle of hepatocytes (CK18 ⁺ CD45 ⁺ or albumin ⁺ ), accounting for approximately 5-10% of any 5 μm section (FIGS. 4K-M). Some CK18 ⁺ CD45 ⁺ β-gal in a recipient origin ^- hematopoietic cells were clearly identified from epithelial cells. Albumin ⁺ β-gal ⁺ and CK18 ⁺ β-gal ⁺ cells established in hepatocyte bundles around the portal tract in a pattern seen in liver regeneration from hepatic stem cells and round cells (Alisonet al., 1998; Petersen et al., 1999). This and the fact that only 5 out of 20 slices contained donor cells were the concept that stem cells settled in some, not all, parts of the liver, where they proliferated and differentiated into hepatocytes. Match.

Intestinal colonization was also consistent with what is known about intestinal epithelial stem cells. In the intestine, each crypt contains 4-5 long-lived stem cells (Potten, 1998). The progeny of these stem cells undergo epithelial cells after several divisions in the middle and upper part of the crypt, which move upward, out of the crypt, and move to the adjacent villi. Donor-derived β-gal ⁺ panCK ⁺ CD45 ⁻ epithelial cells completely covered some villi (FIGS. 4O-P). In some villi, β-gal ⁺ panCK ⁺ CD45 ⁻ cells made up only 50% of the surroundings (black arrows, FIG. 4P), suggesting that colonization was in one but not both crypts. Some β-gal ⁺ panCK ⁻ cells were clearly seen in the center of the intestinal villi (white arrow, FIG. 4O). These cells were co-stained for CD45 (Figure 4P), indicating that they were donor-derived hematopoietic cells. In lung, the majority of donor cells β-gal ⁺ panCK ⁺ CD45 ^- While resulted alveolar epithelial cells, the majority of hematopoietic cells was a recipient origin ^{(panCK - CD45 + β-gal} -) (Figure 4R).

  The level of colonization detected by immunohistochemistry was consistent with the level measured by Q-PCR for NEO (Table 4). Establishment levels were similar in animals analyzed 4-24 weeks after intravenous injection of MASC (Table 4).

  There was no contribution to skeletal or myocardium. In contrast to epithelial tissue and hematopoietic mechanisms, little or no cell turnover was seen in skeletal or cardiac muscle in the absence of tissue injury. Therefore, a significant contribution of stem cells to these tissues is not expected. However, no colonization was seen in the two organs, skin and kidney, where epithelial cells undergo rapid turnover. In a blastocyst injection experiment (Example 8), it is shown that mmASC can differentiate into these cell types; a possible explanation for the absence of colonization in these organs in postnatal recipients is that This is a hypothesis that is currently being evaluated. Although mMASC differentiated ex vivo into neuroectodermal-like cells, no significant colonization of mMASC was seen in the brain, and rare donor cells found in the brain were not co-labeled with neuroectodermal markers. Two recent papers have shown that donor-derived cells with neuroectodermal characteristics can be detected in the brains of animals that have undergone BM transplantation. However, preparation methods for complete removal prior to transplantation or transplantation into new animals, which are conditions associated with the destruction of the blood brain barrier, were used. Cells were injected into unirradiated adult animals with complete or minimal damage to the blood brain barrier, or animals that received low doses of irradiation. This may explain that no mmASC colonization was seen in the CNS.

Confluent MASCs do not differentiate in vivo As a control, ROSA26-MASC was injected prior to injection and cultured until confluent. Confluent MASCs lose their ability to differentiate in ex vivo cells outside the mesoderm and behave like classical MSCs (Reyes, M. et al. 2001). Injection of 10 ⁶ confluent mMASCs did not cause significant levels of donor cell colonization. Only a few β-gal ⁺ cells were found in BM, but these cells were not co-labeled with anti-CD45 antibodies and MSCs could settle in tissues but differentiate into tissue-specific cells in response to local signals It shows that you can't do it anymore.

MASC-derived cells in mouse bone marrow were tested for BM derived from mice planted with ROSA26MASC, which can be transplanted continuously, to include cells that established in secondary recipients. 1.5 × 10 7 BM cells collected from the primary donor 11 weeks after intravenous injection of mMASC were transplanted into the irradiated secondary NOD-SCID recipient (FIG. 4: animals SR-1 and SR−
2). After 7 and 10 weeks, the secondary donor was sacrificed, and the donor animal's blood, BM, spleen, liver, lung and intestine were analyzed for NEO gene immunohistochemistry and Q- Analyzed by PCR. Similar to the primary recipient, a similar pattern of settlement was seen in the secondary recipient. 4-8% of BM, spleen and PB cells were β-gal ⁺ CD45 ⁺ ; 6 and 8% of intestinal epithelial cells were β-gal ⁺ pan-CK ⁺ and 4 and 5% of lung epithelial cells Was β-gal ⁺ pan-CK ⁺ . The level of colonization in the secondary donor's liver was lower than the primary recipient (5 and 8% β-gal ⁺ CK18 ⁺ versus 1 and 3%). This suggests that the mmASC can persist in the primary donor's BM and differentiate into hematopoietic cells as well as epithelial cells when transplanted to the secondary recipient.

  MASC-derived cells can produce insulin in vivo. NOD-SCID mice irradiated with ROSC26 mouse-derived MASCs were injected as described herein. The resulting MASC-derived cells co-stain for LacZ and insulin in a streptozotocin diabetes model.

Summary One of the crucial questions in “stem cell plasticity” is whether established and differentiated donor mMASCs function. The results described here show that one animal developed lymphoma in the thymus and spleen after 16 weeks, as is commonly seen in aged NOD-SCID mice (Prochazkaetal., 1992). Phenotypic analysis showed that this B-cell lymphoma was host-derived: CD19 ⁺ cells were β-gal ⁻ . Approximately 40% of CD45 ⁻ vWF ⁺ cells in the tumor vasculature were stained with anti-β-gal antibody, indicating that the angiogenesis in the tumor was partly derived from donor mMASC (FIG. 4T). This suggests that MASCs generate functional offspring in vivo. Similarly, higher levels of mmASC colonization and differentiation after low-dose irradiation (eg, hematopoietic mechanism and intestinal epithelium) (Table 4, p <0.001) indicate that mmASC is functional in host tissues. This suggests the possibility of contributing to

  These results indicated that mammalian MASCs were purified, expanded ex vivo, and injected intravenously, homed to various sites in the body, established in many organs, and cells in these various organs 1 Survived for months or longer. Such donor cells, undifferentiated progeny, and differentiated offspring are found by fluorescent markers in organs including but not limited to BM, spleen, liver and lung. These cells can be re-established in one or more compartments and used to enhance or restore cell or organ function.

Demonstration of in vitro hematopoiesis and erythropoiesis MASCs derived from human BM differentiate into a number of mesodermal lineages, including neuroectodermal, endodermal, and endothelial cells, at the single cell level. Since endothelium and blood are so closely related in ontogeny, it can be hypothesized that MASCs can differentiate into hematopoietic cells. GlyA, CD45 and CD34 negative eGFP-introduced human MASC (n = 20) was added to mouse yolk sac mesoderm cell line YSM5 and 10 ng / mL bFGF and VEGF as cell aggregate suspension for 6 days Co-cultured in serum-free medium. After 6 days, only eGFP ⁺ cells (ie MASC progeny) remained and YSM5 cells died.

The remaining cells are supplemented with 10ng / mL bone morphogenetic protein (BMP) 4, VEGF, bFGF, stem cell factor (SCF), Flt3L, hyperIL6, thrombopoietin (TPO), and erythropoietin (EPO) containing 10% fetal bovine serum The cells were transferred to the methylcellulose culture and cultured for 2 weeks. During these cultures, both adherent eGFP ⁺ cells and small round non-adherent cells forming numerous colonies attached to adherent cells were detected. Non-adhesive and adherent fractions were collected separately and cultured for 7 days in medium containing 10% FCS supplemented with 10 ng / mL VEGF and bFGF. Adherent cells were staining positive for vWF, formed blood vessels when seeded in ECM, were able to take up a-LDL, and showed endothelial properties. 5-50% of non-adherent cells were positive for staining by flow cytometry for human specific GlyA and HLA-class I. Gly-A ⁺ / HLA class I ⁺ cells were selected by FACS. With Wright-Giemsa staining, these cells showed the characteristic morphology and staining pattern of primitive erythroblasts. Cells were immunoperoxidase with benzidine ⁺ and human H ⁺ . By RT-PCR these cells expressed human-specific Hb-e but not Hb-a.

When reseeded for methylcellulose analysis containing 20% FCS and EPO, small erythroid colonies were seen after 10 days and 100% of these colonies were positive for staining for human-specific GlyA and Hb. The selection of MASC depends on the removal of CD45 ⁺ and GlyA ⁺ cells from the BM, and the cultured MASC is CD45 ⁻ and GlyA ⁻ and has always been examined using both FACS and cDNA array analysis, so to MASC Contamination of hematopoietic cells is very unlikely.

In vivo evidence of the pluripotent nature of MASC demonstrated by multi-organ chimeras after blastocyst injection of cells Important for therapeutic application of these cells is the proliferation and proper cell type of MASC Is the ability to differentiate in vivo. To date, the only cells capable of contributing to the entire group of tissues and organs in the body are ES cells. To analyze whether MASC can show the full potential of ES cells, determine the contribution to the formation of various tissues by introducing MASC into the early blastocyst and observing the fate of its progeny cells For analysis.

MASCs were generated from bone marrow of ROSA26 mice transfected with the β-galactosidase (β-gal) gene (Rafii, S., et al. 1994, Blood 84: 10-13) and expanded as described in Example 1. . One or 10-12 ROSA26MASC obtained after 55-65 PD were microinjected into 88 and 40 day 3.5 C57BL / 6 blastocysts, respectively. Blastocysts (8 / maternal) were transplanted into 16 foster mothers and mice were developed and born as shown in Table 5.

  Seven temporary mothers gave birth, consistent with the birth rates seen in other studies during this period. The number of individuals per litter varied between 1 and 8 for a total of 37 animals. Individuals born from microinjected blastocysts were similar in size to normal individuals and showed no obvious abnormalities.

Four weeks later, the animals were evaluated for chimerism by cutting the tail and assessing the contribution of β-gal / NEO transgene-containing cells to the tail by Q-PCR for NEO. The percent chimerism was determined by comparing the NEO copy number in the test specimen with the copy number in the tissue from the ROSA26 mouse according to the instructions (7700ABI PRISM Detector Software 1.6). Chimerism could be detected in 70% of blastocyst-derived mice injected with 10-12 MASCs and 50% of blastocyst-derived mice injected with one MASC micro-injected ( Figure 5). The degree of chimerism distributed between 0.1% and> 45%. After 6-20 weeks, the animals were sacrificed. Some mice were frozen in liquid nitrogen and thin sections were cut as described. Whole body mouse sections were stained with X-Gal. Thereafter, 1000 sets of digital images completely containing each section were collected to produce a composite image of individual whole body mouse sections. X-Gal staining was not seen in a representative non-chimeric individual (by Q-PCR for NEO) derived from a blastocyst injected with a single MASC. In contrast, the individual was 45% chimera according to R-PCR for NEO by tail cleavage analysis, and a single ROSA26-derived MASC contributed to all somatic tissues.

For other individuals, multiple organs were collected and analyzed for the presence of MASC-derived cells by X-GAL staining, staining with anti-β-gal-FITC antibody, and Q-PCR for NEO. Individuals with NEO ⁺ cells at the tail section, as shown by X-GAL staining and staining with anti-β-gal-FITC antibody, have ROSA26-derived MASCs in the brain, retina, lung, myocardium and skeletal muscle, Contributed to all tissues including liver, intestine, kidney, spleen, BM, blood, and skin.

In individuals arising from blastocysts microinjected with ROSA26MASC, chimerism was detected by X-GAL staining and anti-β-gal staining. β-gal ⁺ cells expressed markers typical of incorporated tissue. For NF200 and GFAP in the central nervous system and for dystrophin in skeletal muscle, β-gal ⁺ cells were co-stained with anti-β-gal ⁺ FITC and anti-NF200, or GFAP and TOPRO3 (observed at 20x magnification). Lung tissue was stained for anti-β-gal-FITC and pan-CK in alveoli and bronchi (also TOPRO3) (observed at 20x magnification). Skeletal muscle was stained with anti-β-gal-FITC, dystrophin-PE and TOPRO3 and observed at 20x magnification. The heart was stained with anti-β-gal-FITC, cardiac troponin-I-Cy3 and TOPRO3 and observed at 20x magnification. The liver was stained with anti-β-gal-FITC and pan-CK-PE and TOPRO3 (observed at 40x and 10x magnification). The intestine was stained with anti-β-gal-FITC, pan-CK-PE and TOPRO3 and observed at 20 times magnification. The kidney was stained with anti-β-gal-FITC (glomerus, tubule) and observed at 20x magnification. Bone marrow staining was observed for anti-β-gal-FITC and CD45-PE, GR1-PE and MAC1-PE. Spleen staining was observed for anti-β-gal-FITC and CD45-PE, CD3-PE and CD19-PE. The level of colonization estimated using Q-PCR for NEO was consistent with the level estimated by X-GAL and anti-β-gal-FITC staining.

Summary These data demonstrate for the first time that a single BM-derived MASC integrates into a developing mouse, yields cells of varying fate and contributes to the generation of tissues and organs in all three germ layers of the mouse. Since all living individuals had a thriving functional period regardless of the degree of chimerism, these studies also suggested that MASC could differentiate into three germ layer functional cells in vivo. To do. Whether MASCs contribute to germ cells when injected into blastocysts or after birth has not yet been tested.

Origin of Vascular Endothelial Progenitor Cells Angiogenesis, an in situ differentiation in which primitive endothelial progenitor cells called hemangioblasts differentiate and aggregate into endothelial cells to form the primary capillary network, is responsible for the development of the vascular system during embryogenesis (Hirashimaet al, 1999). In contrast, angiogenesis, defined as the formation of new blood vessels by the process of germinating existing blood vessels, occurs both during development and in postnatal life (Holashet al, 1999; Yang et al., 2001). Until recently, angiogenesis in postnatal life was thought to be mediated by the sprouting of endothelial cells from existing blood vessels. However, recent studies have suggested that endothelial “stem cells” can persist into adult life, where they may contribute to the formation of new blood vessels (Peichevet al, 2000; Linet al, 2000; Gehling et al. , 2000; Asahara et al., 1997; Shi etal, 1998), suggesting that angiogenesis in adults is at least partially dependent on the angiogenic process as it occurs. Endothelial progenitor cells have been isolated from BM and peripheral blood (Peichevet al., 2000; Watt et al., 1995). The ontogeny of these vascular endothelial progenitor cells is unknown.

  During development, endothelial cells are derived from the mesoderm. Flk1, a VEGF receptor 2, is a bipotential found in the aorta-genital ridge-medium kidney region (Medvinsky et al., 1996; Fong et al., 1999; Peault, 1996) and fetal liver (Fonget al., 1999) The sex stem cells are characterized by hemangioblasts and the determination of embryoid bodies to hemangioblasts is accompanied by the expression of Flkl (Ghoiet al., 1998; Choi, 1998). Whether hemangioblasts persist in adult life is unclear and only HSCs and vascular endothelial progenitor cells have been described. Like hemangioblasts, vascular endothelial progenitor cells express Flk1 (Peichevet al., 2000), and one paper suggested that HSCs express Flk1 in postnatal life (Ziegleret al., 1999). During development, the determination of hemangioblasts to the endothelial lineage is characterized by sequential expression of VE-cadherin, CD31, and a little later CD34 (Nishikawaetal, 1998; Yamashitaet al, 2000). In postnatal life, vascular endothelial progenitor cells have been selected from BM and blood using antibodies against AC133, Flk1, CD34, and H1P12 antibodies (Peichevetal, 2000; Lin et al, 2000; Gehling et al, 2000) . AC133 has also been found in other cells including NSCs (Uchida et al, 2000) and gastrointestinal epithelial cells (Corbeilet al, 2000). Upon differentiation into mature endothelium, the AC133 receptor is rapidly lost (Peichev et al, 2000; Gehling et al, 2000). Another receptor found on circulating endothelial cells is MUC18, a mucin, recognized by the H1P12 antibody (Linet al, 2000). MUC18 is lost during differentiation of vascular endothelial progenitor cells into the endothelium. CD34 is expressed on vascular endothelial progenitor cells and hematopoietic progenitor cells (Peichevetal., 2000; Baumhueter et al, 1994) and hepatic round cells (Crosby etal, 2001). This antigen is also lost upon differentiation of vascular endothelial progenitor cells to the endothelium. Most mature endothelial cells, except microvascular endothelial cells, no longer express CD34.

  In vitro generation of numerous endothelial cells that establish in vivo and contribute to angiogenesis from MASCs is described here for the first time. MASCs can be cultured and expanded for> 80 PD, and endothelial cells generated from MASC can expand at least a further 20 PD. MASC can therefore be an ideal source of endothelial cells for clinical treatment. Furthermore, since MASC are less mature than “angioblasts” ontogenously, this model appears to be useful for characterizing endothelium determination and differentiation.

hMASCs differentiate into cells with endothelial phenotypic characteristics MASCs were harvested and cultured as described in Example 3. To induce endothelial differentiation, MASCs were replated at 2 × 10 ⁴ cells / cm ² in FN-coated wells in serum-free expansion medium without EGF and PDGF-BB but with 10 ng / mL VEGF. Some PCS was added. The culture was maintained by changing the medium every 4 to 5 days. In some cases, cells were subcultured for 20 + PD under the same culture conditions at 1: 4 dilution after 9 days.

  To define the endothelial differentiation from MASC more extensively, FACS and immunohistochemical analysis of cells was performed after 3-18 days. Flkl and Fltl expression on undifferentiated MASC was low, maximal on day 9 and persisted until day 18. VE-cadherin (Peichevetal., 2000; Nishikawa et al., 1998) present in BM or vascular endothelial progenitor cells is not expressed in undifferentiated MASCs, but is expressed after 3 days of culture with addition of VEGF for 18 days Lasted until the eyes. MASC expressed low levels of AC133 found in endothelium as well as hematopoietic progenitor cells (Peichevetal., 2000; Gehling et al., 2000), but were no longer detectable after 3 days. CD34 present in endothelial and hematopoietic progenitor cells (Peichevetal., 2000; Asahara et al., 1997; Rafii et al., 30 1994) is not present in undifferentiated MASC (FIG. 4A), but from day 9 to 18 It was expressed until day one. MUC18, a mucin recognized by antibody H1P12, has been used to generate vascular endothelial progenitor cells from blood (Linetal., 2000). MASC was not stained with H1P12, but MASC treated with VEGF for 9 days was staining positive, but expression was lost by day 18.

  The endothelium-specific integrin αvβ3 (Eliceiri et al, 2000) was absent from undifferentiated MASC, while αvβ5 was expressed at very low levels. Integrin expression gradually increased during differentiation and was maximal by day 14 (Figure 5). The tyrosine kinase receptors Tie and Tek (Partanenet al, 1999) that are important for angiogenesis but not for endothelial differentiation were not expressed in MASC. Tek expression was observed after 3 days and Tie after 7 days (Figure 6). MASC also did not express vWF, but vWF was expressed after day 9 (Rosenbergetal, 1998; Wagner et al, 1982). Other mature endothelial markers including CD31, CD36, CD62-P (Tedder et al, 1995) (Figure 7) and adhesion-binding proteins ZO-1, p-catenin, and y-catenin (Figure 5) are detected after 14 days (Lietal, 1990; Van Rijen et al, 1997; Petzelbauer et al, 2000). VCAM or CD62-E was not expressed at high levels at any time during differentiation unless the endothelium was activated with IL-1α as described below. Endothelial differentiation was accompanied by the acquisition of β2-microglobulin and low levels of HLA-class I antigen but not HLA-class II.

Because higher concentrations of FCS support the growth of classical MSCs that differentiate only into osteoblasts, chondroblasts and adipocytes (Reyes et al, 2001; Pittenger et al, 1999), endothelial differentiation is 2% PCS It has been reported previously (Reyes et al, 2001) that it can only be obtained from MASCs expanded using or less, but not when expanded using 10% PCS. Endothelial differentiation cannot be induced when FCS is present during the first 7 days of differentiation. When non-confluent MASCs (<lx10 ⁴ cells / cm ² ) were induced to differentiate, no endothelium was seen. When MASCs were subcultured 9 days after exposure to VEGF using serum-free medium containing 10 ng / mL VEGF, the cells were able to undergo at least a further 12 PD. When 10% FCS and 10 ng / mL VEGF were added to the subculture medium, MASC-derived endothelial cells were able to undergo an additional 20 + PD regardless of the number of PD that had undergone MASC.

  Compared to undifferentiated MASC, the endothelial cells were larger and the nucleus / cytoplasm ratio was lower. The results were similar when MASC were used from cultures that had undergone 20 (n = 30) or 50+ (n = 25) PD.

Functional characteristics of MASC-derived endothelium. We tested whether hMASC-derived VEGF-induced differentiated progenitor cells have functional characteristics of endothelial cells. Endothelial cells respond to hypoxia by upregulating the expression of VEGF and the VEGF receptor Flkl and the angiogenic receptors Tie-1 and Tek (Kourembanasetal, 1998). hMASC and hMASC-derived endothelial cells were incubated at 37 ° C. in 20% or 10% O 2 for 24 hours. Cells were stained with antibodies to Flk1, Flt1, Tek and IgG controls, fixed with 2% paraformaldehyde and analyzed by flow cytometry. Furthermore, the VEGF concentration in the culture supernatant was measured using an ELISA kit (APbiotech, Piscataway, NJ). MASC-derived endothelial cells and undifferentiated MASCs were exposed to hypoxic conditions for 24 hours.

  Flk1 and Tek expression was significantly increased in MASC-derived endothelial cells exposed to hypoxia (FIG. 7), while the levels of these receptors were unchanged in undifferentiated MASCs. Furthermore, VEGF levels in culture supernatants of hypoxic endothelial cultures increased 4-fold (FIG. 7B), while VEGF levels in MASC cultures exposed to hypoxia did not change.

Next, it was tested whether MASC-derived endothelial cells up-regulate HLA-antigen and cell adhesion ligand expression in response to inflammatory cytokines such as IL-la (Meager, 1999; Steberet al, 2001) . 10 ⁶ MASCs and MASC-derived endothelial cells were incubated for 24 hours in serum-free medium with 75 ng / ml IL-1a (R & DSystems). Cells were fixed with 2% paraformaldehyde and stained with antibodies to HLA-class I, class II, β2-microglobulin, vWF, CD31, VCAM, CD62E and CD62P, or control antibodies, and FACScalibur (BectonDicki
nson).

  Significantly elevated levels of HLA-class I and II, β2-microglobulin, VCAM, ECAM, CD62E, CD62P were found in endothelial cells by FACS analysis (FIG. 7C).

In contrast, undifferentiated MASCs showed only Flk up-regulation.

  Another feature of endothelial cells is the uptake of LDL (Steinberg et al, 1985), in which MASCs induced to differentiate using VEGF are 2, 3, 5, 7, 9, 12, and 15 and 21 Incubated with LDL-dil-acil and tested. The dil-Ac-LDL staining kit was purchased from Biomedical Technologies (Stoughton, Mass.). Analysis was performed as per the instruction manual. Cells were co-labeled with either anti-Tek, -Tie-1 or -vWF antibodies. Three days later, Tek expression was detected, but a-LDL uptake was not detected. After 7 days, the cells expressed Tie-1, but did not take up a significant amount of a-LDL. However, acquisition of vWF expression on day 9 was accompanied by aLDL uptake (FIG. 6B).

Endothelial cells contain vWF that is stored in Weibel Pallade bodies and released in vivo when the endothelium is activated (Wagneret al, 1982). This can be induced in vitro by stimulating the cells with histamine (Rosenbergetal, 1998), which also results in activation of the intracellular skeleton of the cell (Vischer et al, 2000). MASC-derived endothelial cells were seeded at high density (10 ⁴ cells / cm ² ) on FN-coated chamber slides. After 24 hours, cells were treated with 10 μM histamine (Sigma) for 25 minutes in serum-free medium and stained with antibodies against vWF and myosin. Untreated and treated cells were fixed with methanol at −20 ° C. for 2 minutes, stained with antibodies against vWF and myosin, and analyzed using fluorescence and / or confocal microscopy. vWF was present throughout the cytoplasm of untreated endothelial cells. The cytoplasm of histamine-treated endothelial cells contained significantly lower amounts of vWF, and vWF was only detectable in the perinuclear region and was likely indicative of vWF present in the endoplasmic reticulum (FIG. 6A). Histamine treatment caused changes in gap junction expansion and an increase in the number of myosin stress fibers in the cytoskeleton (FIG. 6A).

Finally, endothelial cells were tested to determine if they can form “blood vessels” when seeded on Matrigel or extracellular matrix (ECM) (Haralabopouloset al, 1997). 0.5 ml extracellular matrix (Sigma) per well was added to the 24-well plate and incubated for 3 hours at 37 ° C. Added 10 ⁴ MASC and MASC derived endothelial cells per well in VEGF added serum-free medium of 0.5 ml, and incubated at 37 ° C.. As shown in FIG. 6C, culture of MASC-derived endothelial cells on ECM resulted in angiogenesis within 6 hours.

Breeding colonies of NOD-SCID mice in which hMASC-derived endothelial cells contribute to tumor-angiogenesis in vivo were established from mice obtained from Jackson Laboratories (BarHarbor, ME). Mice were kept free of specific pathogens and maintained in acid water and autoclaved feed. 60 mg of trimethoprim and 300 mg of sulfamethoxazole (Hoffmann-LaRoche Inc., Nutley, NJ) were given twice a week per 100 ml of water.

Three Lewis lung cancer spheroids were implanted subcutaneously in the shoulder. Three and five days after tumor implantation, mice were injected with 0.25 × 10 ⁶ human MASC-derived endothelial cells or human foreskin fibroblasts by tail vein injection. After 14 days, the animals were sacrificed, the tumors were removed and stored at −80 ° C. at low temperature using an OTC compound (Santura Finetek USA Inc, Torrance, Calif.). Furthermore, the cut ears for labeling the mice were also excised and stored at −80 ° C. at low temperature using an OTC compound. A 5 μm thick section of the tissue was placed on a glass slide and fixed and stained as follows.

  An anti-human-β2-microglobulin or HLA-class I antibody that recognizes both human and mouse endothelial cells in combination with an anti-mouse-anti-CD31 antibody and an anti-vWF, anti-Tek, or anti-Tie-1 antibody A computerized analysis of the branch length and number counted for each of the five sections showed that tumors that received human MASC-derived endothelial cells in mice had a vascular mass 1.45 ± 0.04 times greater than that of control mice. It showed that it had. These initial studies show that some human blood vessels in the tumor are co-labeled for either vWF, Tie, or Tek, but not for mouse-CD31, but for human-β2-microglobulin HLA-class I positive cells It was shown that human MASC-derived endothelial cells contributed to tumor angiogenesis in vivo.

  To better determine the degree of contribution, obtain a continuous 5 μm slide and stain with another method using either anti-human-β2-microglobulin-FITC or anti-mouse-CD31-Cy5 and anti-vWF-Cy3 did. All slides were examined by confocal microscopy. Different figures were then collected into 3 dimensions to determine the relative contribution of human and mouse endothelial cells to tumor vessels. When analyzing tumors obtained from animals injected with human MASC-derived endothelial cells, about 35% of tumor vessels are positive for anti-human-β2-microglobulin and vWF, while about 40% of endothelial cells are anti-human. Staining was positive for mouse CD31 antibody (FIGS. 8A-G). Tumors in animals that did not receive endothelial cells or received human fibroblasts did not contain endothelial cells that stained positive with anti-β2-microglobulin or anti-HLA-class-I antibodies.

  We also analyzed whether MASC-derived endothelial cells also contribute to wound healing angiogenesis. The area of the ear that was scored to label the mouse was then examined. Angiogenesis in ear wounds was partly derived from MASC-derived endothelial cells. Similar to blood vessels in the tumor, the percentage of human endothelial cells present in the wound's healed skin was 30-45% (FIG. 9H).

Undifferentiated hMASCs differentiate into endothelial cells in vivo 10 ⁶ undifferentiated MASCs were injected into the vein of 6-week-old NOD-SCID mice. Animals were raised for 12 weeks and then sacrificed. In one individual, a thymic tumor was detected, which is commonly seen in aged NOD-SCID mice (Prochazkaetal., 1992). The thymus was excised and stored in the OTC compound at -80 ° C. A 10-um thick section of tissue was placed on a slide glass and fixed and stained as follows.

  All hematopoietic cells were staining positive for mouse CD45 but not for human CD45, indicating that they were of mouse origin. Tumors were then stained with anti-human β2-microglobulin-FITC antibody and anti-vWF-Cy3 antibody that recognizes both human and mouse endothelial cells. About 12% of the vasculature was derived from hMASC (Figure 9I). These studies further confirmed that this hematopoietic element was not of human origin since no human β2-microglobulin was detected outside the vasculature.

Immunohistochemical and data analysis in vitro culture: to differentiate into MASC or endothelial undifferentiated 2 ^- 18 days induced MASC seeded in FN coated chamber slides, 4 minutes using 2% paraformaldehyde (Sigma), Fixed at room temperature. For cytoskeletal staining, chamber slides were fixed with methanol for 2 minutes at -20 ° C. For intracellular ligands, cells are permeabilized with 0.1 Triton-X (Sigma) for 10 minutes, followed by primary antibody (antibody) and FITC, PE or Cy5-conjugated anti-mouse-, goat- or rabbit-IgG antibody Each was incubated for 30 hours. Between each step, the slides were washed with PBS + 1% BSA. Primary antibodies against CD31, CD34, CD36, CD44, HLA-class I and II, β2-microglobulin were used at a 1:50 dilution. VCAM, ICAM, VE-cadherin, selectin, HIP12, ZO-1, connexin-40, connexin-43, MUC18, a _v b ₃ , a _v b ₅ , B-catenin and y-catenin (Chemicon) and Tek, Tie Primary antibody against vWF (SantaCruz) was used at 1:50 dilution. Stress fibers are antibodies against myosin (light chain 2
0 kD, clone no. MY-21; 1: 200). Secondary antibodies were purchased from Sigma and used at the following dilutions: anti-goat IgG-Cy-3 (1:40), anti-goat IgG-FITC (1: 160), anti-mouse IgG-Cy-3 (1 : 150) and anti-mouse IgG-FITC (1: 320), anti-rabbit-FITC (1: 160) and anti-rabbit-Cy-3 (1:30). TOPRO-3 was purchased from Sigma. Cells were examined by fluorescence microscopy using a Zeiss Axiovert microscope (CarlZeiss, Inc., Thomwood, NY) and confocal fluorescence microscopy using a confocal 1024 microscope (OlympusAX70, 5 Olympus Optical Co. LTD, Japan). .

Tumor or normal tissue: The tissue was cut into sections of 5-10 μm thickness using a cryostat. The sections were fixed with acetone for 10 minutes at room temperature and permeabilized with 0.1 TritonX for 5 minutes. Slides are incubated overnight for vWF, Tie or Tek, then secondary incubation with FITC, PE or Cy5-conjugated anti-mouse-, goat- or rabbit-IgG antibody, and mouse CD45-PE or human CD45-FITC, human β2 -Continuous incubation with antibodies against microglobulin-FITC, mouse CD31-FITC or TOPRO-3 for 60 minutes. Between each step, the slides were washed with PBS + 1% BSA. The slides were examined using a Zeiss Axiovert microscope by fluorescence microscopy and a confocal 1024 microscope by confocal fluorescence microscopy. The 3D reconstruction consisted of a collection of 350 photos in total, continuous 0.5 μm confocal photos from 5 μm thick slides. Slides were stained with vWF-Cy3 or double stained with human β2-microglobulin-FITC or mouse CD31-FITC. The photos from each slide were aligned with the next slide with Metamorph software (Universal Imaging Corp), and 3D reconstruction was done with 3D Doctor software (Ablesoftware Corp).

  The relative contribution of human (green) and mouse endothelial cells (colored in blue) to the three-dimensional blood vessels was calculated as cubic pixels of each color. The area of each color was also calculated as square pixels for the four vessels through the slide to obtain an accurate percentage of contribution. The area of each color was also calculated for four different major separate slides.

Summary The central finding of this study is that cells purified with BM from MSCs have the ability to differentiate into endothelial cells with functional characteristics indistinguishable from mature endothelial cells in vitro. Such endothelial cells contribute in vivo to angiogenesis in the context of wound healing and tumor formation, and undifferentiated MASCs differentiate into endothelial cells that contribute to tumor angiogenesis in response to local signals in vivo It has also been shown that it can.

Cells defined here precede hemangioblasts and hemangioblasts in ontogeny, so that the same cells that differentiate into endothelium also differentiate into other mesodermal cell types, as well as cells of non-mesoderm origin. Even cells precede.

  Although MASC has also been shown to differentiate into cells that express endothelial cell markers, it has been demonstrated that VEGF induces MASC function like endothelial cells. Endothelial cells modify lipoproteins during transport within the arterial wall (Adamset al, 2000). Endothelial cells maintain a permeation barrier through hemodynamic forces or cell-cell adhesion that expands when exposed to vasoactive agents such as histamine (Rosenberg et al, 1998; Li et al, 1990; Van Rijen et al, 1997; Vischer etal, 2000). Endothelial cells release procoagulant molecules to stop bleeding, such as vWF, tissue factor, and plasminogen activator inhibitor (Vischer et al, 2000) and alter the expression level of adhesion molecules in response to inflammation Regulates leukocyte exit (Meager, 1999; Steber etal, 2001). The endothelium also responds to hypoxia by producing VEGF and expressing VEGF receptors with the goal of increasing vascular density (Kourembanasetal, 1998). Thus, it has been demonstrated that endothelial cells generated from MASC can perform all these tasks when tested in vitro.

  Finally, it has been demonstrated that MASC-derived endothelial cells generated in vitro respond to angiogenic stimuli by migrating to tumor sites and contributing to tumor angiogenesis, similar to wound healing in vivo. This finding confirms that endothelial cells generated from MASC have all the functional characteristics of mature endothelium. The extent to which endothelial cells contribute to tumor angiogenesis and angiogenesis is 35-45%, a level similar to that described for other sources of endothelial cells (Conwayetal, 2001; Ribatti et al, 2001). Furthermore, angiogenic stimuli brought to the tumor microenvironment in vivo prove to be sufficient to collect MASC into the tumor bed and induce differentiation into endothelial cells that contribute to the tumor vasculature. It was. These studies therefore extend the studies reported by other groups that have shown that cells present in the bone marrow can contribute to new blood vessel formation in a process similar to angiogenesis (Peichevet al, 2000; Lin; et al, 2000; Gehling et al, 2000; Asahara et al, 1997), progenitor cells responsible for this process have been identified. This is the first report to the best of our knowledge that identifies cells present in postnatal BM as the very early progenitor cells of endothelial cells.

In addition to mesodermal cell types, a single adult BM-derived hMASC or to determine whether it can differentiate into functional neurons and astrocytes and oligodendrocytes ex vivo mMASC was tested. MMASC and hMASC were selected, cultured and expanded as previously described in Examples 1 and 3, respectively. Human neural progenitor cells (hNPC) were purchased from Clonetics (SanDiego, CA). hNPCs were cultured and differentiated as per the instruction manual.

Electrophysiology: The physiological properties of MASC-derived neurons were examined using standard whole cell patch clamp recordings. Voltage clamp and current clamp recordings were obtained using a Dagan 3900A patch clamp amplifier (Dagan Corporation, Minneapolis) retrofitted with a Dagan 3911 expansion unit. A patch pipette made of borosilicate glass was stretched with a Narishige pipette maker (model number PP-83) and polished using a Narishige microforge (model number MF-83). Patch pipettes (unit: mM) 142.0KF, 7.0Na ₂ S0 ₄ , 3.0MgS0 ₄ , 1.0CaCl ₂ , 5.0HEPES, ll.0EGTA, 1.0 glutathione, 2.0 glucose, 1.0 ATP (magnesium salt), 0.5 GTP (sodium salt) ). For most recordings, the fluorescent dye 5,6-carboxyfluorescein (0.5 mm; Eastman Kodak Chemicals) is also added to the pipette solution to visually confirm that the whole cell patch recording setup has been achieved using fluorescence microscopy. Added to. Pipette resistance ranged from 11-24 MΩ. Recording standard extracellular solution contained the following (in mM): 155NaCl, 5.0KC1, CaCl 2, 1.0MgCl 2, 10HEPES, glucose. For some experiments, 1 μM TTX was added to the standard control solution. The pH of the intracellular and extracellular fluids for recording was adjusted to 7.4 and 7.8 using NaOH, respectively. All reagents were obtained from Sigma. PClamp 8.0 (Axon Instruments, Foster City) was used for carrying out experiments and collecting and storing data. The data shown was corrected for a liquid phase interface potential of 8.4 mV, which was calculated using the JPCALC software package. Data offline analysis and image display were configured using Clampfit 8.0 (Axon Instruments, Foster City) and Prism (Graphpad, San Diego).

Transduction: Retroviral supernatants were prepared by incubating MFG-eGFP-containing PG13 cells provided by Dr. G. Wagemaker, University of Rotterdam, The Netherlands (Bierhuizenetal., 1997) with MASC expansion medium for 48 hours and filtered. And frozen at -80 ° C. MASCs were incubated with retroviral supernatant and 8 μg / ml protamine (Sigma) for 6 hours. This was repeated after 24 hours. Transduction efficiency was analyzed by FACS.

5 Gene microarray analysis: RNA was isolated from hMASC, bFGF or FGF-8b + EGF-derived cells using RNeasymini kit (Qiagene), digested with DNaseI (Promega) for 1 hour at 37 ° C, and repurified with RNeasy did. [32P] dATP-labeled cDNA probe prepared according to the instruction manual was hybridized with human Neurobiology AtlasArray (Clonetech # 7736-1, Clonetech Laboratories, Palo Alto, CA, USA) at 68 ° C. for 18-20 hours, and then 2 × SSC, Washing was performed 4 times for 30 minutes each at 68 ° C with 1% SDS, 0.1xSSC, 30 minutes at 68 ° C with 0.5% SDS, and once for 5 minutes at room temperature with 2xSSC. The array was read with a phosphoimager screen scanner (MolecularDynamics, Storm 860) and analyzed using AtlasImage 1.0 (Clontech). Greater than twice the difference between undifferentiated and differentiated cells was considered significant.

PCR analysis of retroviral inserts: PCR was used to amplify the flanking sequence 3 ′ from the 3 ′ LTR of the MFG vector in human genomic DNA. DNA from 10 ⁶ MASCs or progeny differentiated into endothelial or myoblasts or neuroectoderm was prepared from cells by standard methods. 300 ng of genomic DNA was digested with AscI and a splinkerette linker was ligated to the 5 ′ end (Devon R.S. et al., 1995). The two oligonucleotides used for the splinkerette linker were as follows: aattTAGCGGCCGCTTGAATTtttttttgcaaaaa (the hairpin loop forming sequence is lower case, upper case is the complementary part of the second splinkerette oligonucleotide), and agtgtgagtcacagtagtctcgcgttcgAATTAAGCGGCCGCTA, (underlined The sequence is also the sequence of the linker-specific primer (LS primer) used in the PCR and RT steps). 5'-biotin-T7 binding primer that recognizes sequences in the eGFP gene [biotin-ggc-cag-tga-att-gta-ata-cga-ctc-act-ata-ggc-tgg-CAC-ATG-GTC-CTG -CTG-GAG-TTC-GTG-AC; underlined indicates the minimal promoter sequence required for efficient in vitro transcription, capital letters are eGFP-specific sequences] and LS primer used to fold flanking regions 10 times Amplification using Advantage2 polymerase (Clontech). The biotin-labeled amplification product was captured using streptavidin-magnetic beads (streptavidin magnetic particles; Roche), and the resulting product was further amplified about 1,000 times using T7 RNA polymerase, and then treated with DNAase1. The resulting product was reverse transcribed using the agtgtgagtcacagtagtctcgcgttcsplinkerette primer according to the superscript II instructions (Gibco), followed by 30 nested PCRs using the 3'LTR primer [ggccaa gaa cag atg gaa cag ctg aat atg] . Adjacent sequences in human genomes derived from cells differentiated into endothelium, muscle, and neuroectodermal and undifferentiated MASCs were sequenced.

  In order to demonstrate the presence of identical insertion sites in multiple differentiated progeny, specific primers were created in the host flanking genome. Real-time PCR amplification (ABI PRISM 7700, Perkin Elmer / AppliedBiosystems) was used to quantitate flanking sequences compared to eGFP sequences. Reaction conditions for amplification were as follows: 2 μL of DNA solution, after first denaturation (95 ° C. for 10 minutes) using 1 × TaqMan SYBR Green Universal Mix (PerkinElmer / Applied Biosystems) PCR reaction buffer, 40 cycles of two-step PCR (95 ° C for 15 seconds, 60 ° C for 60 seconds). The primers used were as follows: clone A16: LTR primer = CCA-ATA-AAC-CCT-CTT-GCA-GTT-G; flanking sequence chromosome 7 = TCC-TGC-CAC-CAG-AAA-TAA- CC; clone A12 chromosome 7 sequence: LTR primer = GGA-GGG-TCT-CCT-CTG-AGT-GAT-T, flanking sequence = CAG-TGG-CCA-GAT-CTC-ATC-TCA-C; clone A12 chromosome 1 Sequence: LTR = GGA-GGG-TCT-CCT-CTG-AGT-GAT-T; flanking sequence = GCA-GAC-GAG-GTA-GGC-ACT-TG. The relative amount of flanking sequences was calculated using 7700ABIPRISM detection software 1.6 according to the instructions compared to the eGFP sequence.

Nerve transplantation: Newborn (day 1 to day 3) male Sprague Dawley rats (Charles River Laboratories) were used in this study. Rats were anesthetized by cold anesthesia. The skull was fixed using an improved stereotaxic head fixator, and the scalp was inverted to expose the skull. hMASC was collected using 0.25% trypsin / EDTA, washed twice and resuspended in PBS. The viability of hMASC was greater than 85%. The 2μl amount of hMASC suspended at a concentration of the phosphate buffer 0.7X10 ⁴ cells / [mu] l, with tapered glass micropipette attached to Hamilton syringe, it was stereotactically injected intracerebroventricularly with coordinates below (Mm from Bregma): AP-0.6, ML0.8, DV2.1, toothbar were set to -1. After the injection, the scalp was sutured to recover the pup rat.

  At 4 and 12 weeks after transplantation, the rats were anesthetized with chloral hydrate (350 mg / kg, i.p.), decapitated, the brain removed, frozen in powdered dry ice and stored at -80 ° C. Fresh frozen brains were cut using a cryostat and fixed with 4% paraformaldehyde for 20 minutes immediately before staining. Sections were incubated for 1 hour at room temperature with blocking / permeabilized solution containing 2% normal donkey serum (Jackson ImmunoLabs) and 0.3% triton X. For subsequent steps, primary and secondary antibodies were diluted with the same blocking / permeabilization solution. Primary antibodies (Chemicon mouse anti-human nucleus (1:25), anti-human cornea (1:25) and anti-NeuN (1: 200); DAKO rabbit anti-GFAP (1: 250), Sigma rabbit anti-NF200 (1 : 300) overnight at 4 ° C, washed 3 times for 10 minutes each with PBS, then incubated with secondary Cy3 (1: 200) and FITC (1: 100) antibodies (all JacksonImmunoLabs) for 2 hours at room temperature The slides were examined by fluorescence microscopy using a Zeiss Axiovert microscope and confocal fluorescence microscopy using a confocal 1024 microscope.

hMASC acquires neuronal, astrocyte and oligodendrocyte phenotypes when cultured with bFGF. Neuroectodermal differentiation was performed as described in Example 5. Briefly, cells with FN-coated chamber slides or culture plates, 60% DMEM-LG, 40 % MCDB-201 (SigmaChemical Co, St Louis, MO) 1XITS ^{include, 1X LA-BSA, 10 -8} M dexamethasone, 10 ^The cells were cultured in a serum-free medium supplemented with ⁻⁴ M ascorbic acid-2-phosphate (AA) (all Sigma), 100 U penicillin and 1,000 U streptomycin (Gibco). For some cultures we added lOOng / mL bFGF, while for other cultures we added 10ng / mL EOF + 10ng / mL FGF-8b (all R & DSystems). Cells were not subcultured, but the medium was changed every 3-5 days.

Two weeks after reseeding with bFGF, as shown in Table 6, 26 ± 4% of the cells were astrocytes (GFAP ⁺ ), 28 ± 3% were oligodendrocytes (MBP ⁺ ), and 46 ± 5% Expressed a neuronal (NF200 ⁺ ) marker.

When hMASCs were replated at higher cell density (2x10 ⁴ cells / cm ² ) and differentiation was induced, cells with neuroectodermal phenotype were not detected, and cell-cell interaction was induced by bFGF-induced neurons. It was shown to inhibit ectoderm differentiation.

  When differentiation was induced using hMASC via 20 or 60 PD, there was no difference in the distribution of astrocyte-like, oligodendrocyte-like and neuronal-like cells. However, when hMASC expanded for 20 PD were cultured with bFGF,> 50% of the cells died, while> 70% of hMASC culture expanded for> 30 PD survived, neuron-like, astrocytes Acquired a oligodendrocyte-like or oligodendrocyte-like phenotype. This suggests that not all hMASCs can be induced to acquire neural features, but in vitro long-lived subpopulations of hMASC may be responsible for neuronal differentiation . The karyotype of hMASC has been shown to be normal regardless of the culture period (Reyesetal, 2001). The differentiation of hMASCs into neuroectodermal-like cells is therefore unlikely to be due to alteration of MASCs after prolonged culture.

Most astrocyte-like and oligodendrocyte-like cells died after 3 weeks. Progressive maturation of neuron-like cells was observed throughout the culture period. One week later, bFGF-treated hMASC was positive for staining for NeuroD, nestin, polysialic acid-added neuronal adhesion molecule (PSA-NCAM), and tubulin-β-III (TuJI) (Table 6). After 2 weeks, bFGF treated cells became staining positive for NF68, -160, and -200, NSE, MAP2-AB, and Tau. bFGF-induced neurons did not express GABA, serotonin or dopaminergic markers, but glutamate and glutamate receptors-5, -6 and -7 and N-methyl-D-aspartate (NMDA) receptors, and Na ⁺ gated potential channels were expressed.

Further confirmation of neuroectodermal differentiation was obtained from cDNA array analysis of two independent hMASC populations induced to differentiate using 100 ng / mL bFGF for 14 days. Nestin, otx1 and otx2 expression levels are consistent with immunohistochemical characterization, with> 2-fold increase in mRNA being nestin, GFAP, glutamate-receptors 4, 5, and 6, and glutamate, and some sodium Detected for the gate potential channel, but no increase in TH or TrH mRNA levels was seen. > 2 fold increase in mRNA levels is seen in mammalian achaete-scute homologue 1 (MASHI) mRNA (FrancoDel Arno et al., 1993), a transcription factor found only in the brain, and ephrin-A5 mRNA (O'Learyand Wilkinson , 1999). Astrocyte-specific markers GFAP and S100A5, and oligodendrocyte-specific markers myelin- oligodendrocyte glycoprotein precursor and myelin protein zero (PMZ), as well as huntingtin and the major prion protein precursor mRNA ,> 2-fold higher expression after exposure to bFGF. More than a 2-fold increase is also seen for several glycine receptors, GABA receptors, hydroxytryptophan receptor-A and neuronal acetylcholine receptors, glycine transporter proteins, synaptobrevin and synaptosome accessory protein (SNAP) 25 It was. Finally, bFGF induced the expression of BDNF and glial-derived neuronal differentiation inducing factor (GDNF).

Like hMASC, mmASC acquires neuronal, astrocyte and oligodendrocyte phenotypes when cultured with bFGF.

MASCs from other species were tested to determine if similar results were obtained. MMASC expanded between 40-90 PD were replated at the same conditions used for hMASC at 10 ⁴ cells / cm ² . After 14 days, mmASC acquired morphological and phenotypic characteristics of astrocytes (GFAP ⁺ ), oligodendrocytes (MBP ⁺ ) and neurons (NF-200 ⁺ , NSE ⁺ and Tau). NF200 and GFAP or MBP were never found in the same cell. In contrast to undifferentiated mMASC, bFGF-treated mMAS was significantly larger and neurites were extended> 40 μm.

A patch clamp was used to determine whether neuronal cell-like cells have neuronal functional characteristics and whether bFGF-inducible cells exhibit voltage-dependent Na ⁺ currents. Some cells expressed calcium current, and none of the mmASC-derived neuron-like cells (n = 59), despite the fact that 4 cells had evidence of spike generation mediated by calcium current, No sodium current or fast spike generation was seen. Thus, despite the expression of sodium gate potential channel mRNA and protein, bFGF-induced cells did not have a functioning voltage-dependent Na ⁺ current.

hMASC acquires a midbrain dopaminergic, serotonergic or GABA phenotype when cultured with EGF and FGF-8b. FGF-8b expressed in the midbrain hindbrain boundary and rostral forebrain induces differentiation of dopaminergic neurons in the midbrain and forebrain and serotonergic neurons in the hindbrain (Yeet al, 1998) . In Invitro, FGF-8b has been used to induce dopaminergic and serotonergic neurons from mouse ES cells (Leeet al., 2000).

HMASC (n = 8) expanded between 20 and 60 PD was replated at 2 × 10 ⁴ cells / cm ² in FN, ITS, AA, 10 ng / mL FGF-8b and 10 ng / mL EGF in serum-free medium . Over 80% of the cells survived for 3 weeks. FGF-8b and EGF induced differentiation into cells that stained positive for neuronal markers (FIG. 6) (Day 7: PSA-NCAM, Nestin and TuJI; Day 14: NF-68, NF-160, NF-200; Day 21: MAP2-AB, NSE, Tau, and Na ⁺ gated potential channels) did not induce oligodendrocytes and astrocytes. In contrast to our observations on differentiation induced by bFGF, cells seeded with EGF and FGF-8b at 10 ⁴ cells / cm ² did not differentiate. After 2-3 weeks, cells GABA resistance (GABA ^+, parvalbumin ⁺⁾ dopaminergic (TH ^+, DCC ^+, and DTP ⁺⁾ and serotonergic have the features of (TrH ⁺ and serotonin ⁺⁾ neurons (Table 6). The cells also expressed GABA-A receptor and glutamate receptor. Cells with a dopaminergic phenotype also include the nuclear transcription factor Nurr1 (Saucedo-Cardenas etal., 1998) expressed only in midbrain dopaminergic neurons and the glial cell line expressed in dopaminergic neurons Positively stained for an antibody against the proto-oncogene cRet (Truppet al., 1996), which is a membrane-bound receptor protein tyrosine kinase that is a component of the derived neuronal differentiation inducing factor (GDNF) receptor complex. This suggests that FGF-8b induces a phenotype consistent with midbrain dopaminergic neurons.

  Again, the results from immunohistochemical studies were confirmed by cDNA array analysis for hMASC induced to differentiate with FGF-8b + EGF for 14 days. Consistent with the immunohistochemical characterization, a> 2-fold increase in mRNA was detected for TH, TrH, glutamate, several glutamate receptors, and sodium gate potential channels. Since parvalbumin and GABA are not present on this array, their expression could not be confirmed by mRNA analysis. Consistent with the nearly limited neuronal differentiation seen by immunohistochemistry, there was no increase in expression of tGFAP, S100A5 mRNA, or PMZ mRNA, an oligodendrocyte-specific marker. Cells induced by FGF-8b + EGF expressed> 2 times more tyrosine kinase receptor (Trk) A, BDNF and GDNF, some glycine-, GABA- and hydroxytryptamine-receptors, and some synaptic proteins .

Co-culture with glioblastoma cell line U87 promotes neuronal maturation Regardless of the culture conditions used, hMASC-derived neurons do not survive more than 3-4 weeks in culture. Since none of the cultures contained glial cells after 3 weeks, neurons expressing both glutamate and glutamate receptors may have died due to glutamate toxicity (AndersonandSwanson, 2000). Alternatively, factors supplied by glial cells that are necessary for neuronal survival, differentiation, and maturation may not be present in culture (Blondelet al., 2000; Daadiand Weiss, 1999; Wagner et al., 1999) . To test this hypothesis, cells from a 3-week old FGF-8b + EGF culture were co-cultured with glioblastoma cell line U-87 in serum-free medium supplemented with FGF-8b + EGF for an additional 2 weeks.

  Glioma cell line U-87 [American Tissue Cell Collection (Rockville, MD)] was maintained in DMEM + 10% FCS (Hyclone Laboratories, Logan, UT). Cells from 3 week old FGF-8b + EGF containing cultures were labeled with the lipophilic dye PKH26 (Sigma) as per instructions. Labeled cells were replated with 1,000 U-87 cells in serum free medium containing FGF-8b + EGF on FN-coated chamber slides and maintained for an additional 2-3 weeks with the medium changed every 3-5 days. To ensure that PKH26 present in MASC-derived cells did not migrate to the U-87 cell line, U-87 cells were cultured for 7 days in BSA-containing medium and 20 μl PKH26 dye. No glioma cell label was detected.

Under these serum-free conditions, U-87 cells stopped growing but survived. In order to be able to confirm hMASC-derived cells by fluorescence microscopy, hMASC-derived neurons were labeled with the membrane dye PKH26 before co-culture with U-87 cells. FGF-8b + EGF-induced neurons cocultured with the same cytokine as U-87 cells for 3 weeks later survived for at least 2 more weeks. Neurons acquired a more mature morphology with increased cell size and increased neurite number, length and complexity. The electrophysiological characteristics of hMASC-derived PKH26-labeled neurons after co-culture with U-87 cells were evaluated by whole cell current fixation and potential fixation after current injection (FIG. 9B). By current fixation, 4 out of 8 KH26-labeled hMASC-derived cells present in the FGF-8b + EGF / U-87 culture showed spike generation against the injected current. The resting membrane potentials of spiked and non-spiked cells were -64.9 ± 5.5 mV and 29.7 ± 12.4 mV, respectively. For the individual cells tested, the input resistance of spiked and non-spiked cells was 194.3 (37.3) and 216.3 (52.5) MΩ, respectively. FIG. 9B shows an example of cells in which spike generation was observed. The upper figure shows a group of voltage recordings obtained from spike-producing cells under control conditions. DC current is first injected into the cells and kept in the range of -100 to -120 mV. A current injection procedure was then used to bring the membrane potential to different levels as shown in the middle figure. As shown in this example, a stage of depolarization current that is large enough to bring the cell to the action potential threshold causes a single spike. The figure below shows that cell spike generation can be blocked with 1 μm TTX, suggesting that the action potential is mediated by the Na gate potential channel. The current recording minus the leak (Figure 9C), obtained from the same cell voltage clamp mode, is an inward current that is transient in the time course and activated at a potential that is more positive than -58 mV. , And outward current. The transient inward current was reversibly blocked with 1 μm TTX. This pharmacology is typical of voltage-dependent Na ⁺ currents found only in mature and skeletal muscle cells, with a transient time course and voltage-dependent activation of inward currents (Sahetal., 1997 Whittemore et al., 1999). No skeletal muscle marker was detected in these neuron-like cells. These studies suggest that treatment with FGF-8b + EGF and co-culture with gliomas results in the basic characteristics of excitable neurons, TTX-sensitive voltage-dependent Na ⁺ currents, to the cells. .

HMASCs transplanted into the ventricle of neonatal rats differentiate into cells that express astrocytes and neuronal markers 1.4xl0 ^Four hMASCs were stereotactically placed on the side of Sprague Dawley rats from day 1 to day 3 after birth Injection into the ventricle. After 4 and 12 weeks, animals were sacrificed and analyzed for the presence of human cells and evidence that hMASC differentiated into neuroectodermal. Human cells identified by staining with antibodies against human nuclei or human nuclear membranes can be observed in SVZ up to 400 μm away from the lateral ventricle of the individual analyzed after 4 weeks, and after 12 weeks, Human cells could also be found deeper in the brain parenchyma, for example in the hippocampus and along the fornix. Some human cells have a typical astrocyte morphology and were positively stained with anti-GFAP antibody, while others were positively stained with anti-Neu-N antibody, NF-200 and anti-human nuclear membrane antibody. It was. Triple staining indicated that human nuclear antigen positive Neu-N positive cells did not co-express GFAP.

Summary The central finding of this study was the differentiation of MASCs derived from a single postnatal BM into cells with characteristics of mature neurons as well as characteristics of astrocytes and oligodendrocytes as well as mesodermal cells It can be guided to do. MASC showed time-dependent and culture-method-dependent maturation of cells with neuroectodermal properties. Double staining definitively demonstrated that neurons or glial cells were authentic and the results were not due to inappropriately expressed neuronal or glial markers. These results were confirmed at the mRNA level. Using retroviral marking tests, the host cell genomic region sequence adjacent to the retroviral vector was identical in all strains, so that neurons, astrocytes and oligodendrocytes also differentiate into muscle and endothelium. It was shown to be derived from one MASC. hMASC acquired not only the phenotype of mature neurons but also electrophysiological features, ie TTX-sensitive voltage-dependent Na ⁺ current. Finally, MASC was shown to be able to differentiate into cells that express markers of neurons and astrocytes in vivo.

Neurons differentiate into astrocytes and oligodendrocytes, as well as endothelium and muscle, using retroviral marking of hMASC in combination with PCR-based sequencing of genomic sequences flanking the retroviral insert 3'-LTR (Jordanet al, 1990), which clearly shows that MASCs can differentiate into cells of the mesodermal and neuroectodermal lineages at the single cell level. Show. The pluripotent adult stem cell or MASC, which has the ability to differentiate not only into mesodermal cell types but also into neuroectodermal cell types, has been newly named. Sanchez-Ramosetal. (Sanchez-Ramos et al, 2000) and Woodbury et al. (Woodbury et al., 2000) found that human or rodent MSC populations express astrocyte and neuronal markers in vitro. However, it was shown that the oligodendrocyte marker could not be expressed. However, neither test provided evidence that the same cells that acquired the neuroectodermal marker could differentiate into mesodermal cells. Furthermore, neither test showed that the cells expressing the neuronal marker also acquired functional neuronal features. Thus, although suggestive about neuronal differentiation, these reports did not decisively show neuronal and glial differentiation from MSCs.

  HMASCs transplanted into the rat ventricle of the rat neonate can migrate into the subneural region and into the hippocampus where they can differentiate into cells that express astrocytes and neuronal markers in response to local signals. Indicated. Migration and differentiation of NSCs to specific neuronal phenotypes occurs to a much greater extent in embryonic areas than in non-neural areas of the brain and in transplants in neonates compared to adult animals This model was therefore chosen (Bjorklund and Lindvall, 2000; Svendsenand Caldwell, 2000). Although hMASC is pluripotent and differentiates into cells outside the neuroectoderm, hMASC did not form teratomas. The number of cells that migrated outside the subventricular area was small after 4 weeks but increased after 12 weeks.

  MASCs can be isolated from postnatal BM and easily expanded and induced to differentiate into astrocyte, oligodendrocyte or neuronal cell types. One can avoid the availability of a suitable provider organization.

MASC differentiation into hepatocyte-like cells During embryonic development, the first sign of liver morphogenesis is thickening of the ventral endoderm epithelium, which occurs between embryonic day 7.5 and 8.5 in mice (ZaretK.S. , 2001). Little is known about the signals involved in initial endoderm formation and subsequent endoderm determination. In the early stages of gastrulation (e6-e7), the endoderm has not been determined even in the anteroposterior direction (Melton D., 1997). However, recent studies have shown that ex vivo exposure of the endoderm to FGF4 then lateralizes the early endoderm capable of expressing liver markers (Wells J. M. et al. t 1999). By embryonic day 8.5 in mice, the final endoderm forms the intestine and expresses HNF3P (Zaret K.S., 2000). The foregut endoderm is induced in the hepatic cell lineage by acidic (a) FGF and bFGF, both produced in the adjacent cardiac mesoderm (ZaretK.S., 2001), which is not the default fate of the pancreas but the fate of the liver Is necessary to induce (Zaret KS, 2001). The basic morphogenic proteins (BMPs) produced in transversum mesenchyme are also required because they increase the level of GATA4 transcription factors that enhance the ability of the endoderm to respond to FGF (ZaretK.S., 2001) And HNF3p are necessary for hepatic determination and, among other things, upregulate markers of hepatocyte-specific expression such as albumin, and thus are important effectors of downstream events leading to hepatocyte differentiation.

  In most cases, mature hepatocytes can undergo several cell divisions and are responsible for hepatocyte replacement. As a result, there is a great debate regarding the presence and function of liver stem cells. During extensive hepatic necrosis due to chemical injury, or when hepatocytes are treated with chemicals that block their growth, a population of smaller circular cells called round cells appear and grow (Petersen, BE, 2001) . These round cells may constitute the “stem cell” part of the liver. Circular cells reside within the smallest unit of the biliary tract called the herring canal and travel from there to the liver parenchyma (TheiseN.D., Et al., 1999). Round cells are amphoteric and produce both hepatocytes and biliary epithelium in vitro and in vivo. Round cells express several hematopoietic markers such as Thy1.1, CD34, Flt3-receptor, and cKit, and also express αFP, CK19, γ-glutamyl-transferase, and OV-6. The origin of round cells is unknown (Petersen, B.E., 2001; KimT.H. el al, 1997; Petersen, B.E., 2001).

Until recently, hepatocytes were believed to be obtained only from cells of endodermal origin and their progenitor cells. However, recent studies suggest that nonendodermal cells also form hepatocytes in vivo and in vitro (Petersen, BE, 2001; Pittenger MFet al., 1999). After bone marrow (BM) transplantation, round cells are derived from donor BM (TheiseN.D., Et al., 1999). Transplantation of enriched hematopoietic stem cells (HSC) into FAH ^-/- mice, an animal model of type I tyrosinemia, results in the proliferation of a large number of donor LacZ ⁺ hepatocytes, and the animals have liver biochemical functions (Lagasse E. et al., 2000). Furthermore, single HSCs may not only reestablish hematopoietic mechanisms but also contribute to the lung, skin, liver and intestinal epithelium (Krause DS etal., 2001). Exocrine pancreatic tumor cells treated with dexamethasone (Dex) in the presence or absence of oncostatin M (OSM) in vitro can acquire a hepatocyte phenotype (ShenC.N. et al., 2000). Finally, mouse embryonic stem (ES) cells spontaneously acquire a hepatocyte phenotype, and this process is facilitated by treatment with aFGF, HGF, OSM, and Dex (Hamazaki T. et al., 2001 ).

  It has been individually demonstrated that a single HSC can differentiate not only into mesodermal and neuroectodermal cells in vitro, but also into cells with morphological, phenotypic and functional characteristics of hepatocytes.

mMASC, rMASC, and hMASC acquire a hepatocyte-like phenotype when cultured with FGF4 and / or HGF. mMASC, rMASC and hMASC were selected and cultured as described. Various extracellular matrix (ECM) components and cytokines known to induce hepatocyte differentiation from in vivo or ES cells (Zaret) to determine the optimal conditions for differentiation of MASCs into hepatocyte-like cells KS, 2001) was tested for the effect of mMASC or rMASC on hepatocyte differentiation. Since differentiation requires cell cycle arrest, the effect of cell density was also tested. To demonstrate differentiation into hepatocyte-like cells, the cells were stained with antibodies against albumin, CK18, and HNF3p after 14 days.

Optimal differentiation of mMASC or rMASC into albumin, CK18, and HNF3p positive epithelioid cells is shown in Table 7A with MASC 21.5 × 10 ³ cells / cm ² in the presence of 10 ng / ml FGF4 and 20 ng / ml HGF. It was seen when sowed on Matrigel. After 14 days, the percentage of albumin, CK18, and HNF3p positive epithelioid cells was 61.4 ± 4.7%, and 17.3% of the cells were binuclear. When seeded on FN, only 53.1 ± 6.3% of the cells were stained and fewer binucleated cells (10.9%), but differentiated into CK18 and HNF3p positive epithelioid cells.

  Incubation with either FGF4 or HGF resulted in albumin, CK18, and HNF3p positive epithelioid cells, but albumin, CK18 when treated with both FGF4 and HGF as shown in Table 7A The proportion of HNF3p positive cells was higher. Addition of aFGF, bFGF, FGF7, BMP, or OSM did not increase the percentage of cells that were positive for hepatocyte markers, whereas 1% DMSO and 0.1 mM to 10 mM sodium butyrate were positive for hepatocyte markers Did not support differentiation of mmASC or rMASC.

When testing cell densities from 2.5 to 25 × 10 ³ cells / cm ² , the highest percentage of cells with hepatocyte markers was found for cells seeded at 21.5 × 10 ³ cells / cm ² . No hepatocyte differentiation was seen when cells were seeded at <12.5 × 10 ³ cells / cm ² .

hMASC was seeded at 3-30 × 10 ³ cells / cm ² in 10 ng / mLFN or 1% Matrigel containing aFGF, bFGF, FGF7, 1% DMSO, HGF, and / or FGF4. Only cells treated with 10 ng / ml FGF4 alone, 20 ng / ml HGF alone, or a combination of both differentiated into epithelioid cells expressing albumin, CK18, and HNF3p. The hMASC seeded with 15-30X10 ³ cells / cm ² while differentiated into epithelioid cells, hMASC seeded at 3x10 ³ cells / cm ² died. Like mMASC or rMASC, the percentage of albumin, CK18, and HNF3p positive epithelioid cells is higher when hMASC is cultured in Matrigel (91.3% ± 4.4) than in FN (89.5% ± 5.4) And as shown in Table 7B, the proportion of binuclear cells was higher in Matrigel (31.3%) than in FN (28.7%).

Characterization of the phenotype of MASC differentiation into hepatocyte-like cells. Hepatocyte differentiation is marked by early (HNF3P, GATA4, CK19, αFP) and late (CK18, albumin, HNF1α) markers by immunofluorescence and confocal microscopy. Were further evaluated over time. The mmASC or rMASC seeded on Matrigel containing FGF4 and HGF increased in diameter from 8 μm to 15 μm as shown in Table 8A. On days 21-28, approximately 60% of the cells were epithelioid, surrounded by smaller round or spindle cells. Undifferentiated mmASC or rMASC did not express any liver-specific transcription factor or cytoplasmic marker. After 4 days, the cells expressed HNF3J3, GATA4 and αFP, low levels of CK19, and very few cells were staining positive for HNF1α, albumin or CK18. On day 7, the large epithelioid cells were positive for staining for HNF30, GATA4, HNF1α and increased staining for albumin and CK18. Only a few cells expressed αFP. After 14, 21, and 28 days, the large epithelioid cells were positive for staining for GATA4, HNF30, HNF1α, CK18 and albumin, but not for αFP or CK19. Smaller cells surrounding the epithelioid nodules were positive for staining for CK19 and αFP.

HMASCs seeded on Matrigel containing FGF4 and HGF, or FGF4 alone, increased from 10-12 μm in diameter to 20-30 μm by day 21. After 7 days, cells expressed HNF3p, GATA4 and low levels of CK19, while only a few cells were positive for albumin or CK18. After 14 and 21 days,> 90% of epithelioid cells stained positive for GATA4, HNF3p, HNF1α, HNF4, CK18 and albumin, whereas only a small number of cells stained positive for αFP or CK19, as shown in FIG. 10B. there were.

Hepatocyte-like cells are derived from the same single hMASC that differentiated into neuroectodermal and endoderm. A single mMASC or rMASC is shown to differentiate into endothelium, neuroectodermal, and CK18 and albumin positive endoderm cells. Has been. It has also been shown that a single hMASC differentiates into mesoderm and neuroectoderm. The same single hMASC was tested to be able to differentiate into hepatocyte-like cells. MASCs were obtained as described and cultured and expanded. For differentiation, 0.015 μg / ml fibronectin (FN), 0.01-8 μg / ml collagen (SigmaChemicalCo, St. Louis, MO) or 1% Matrigel (Becton) at 25 × 10 ³ cells / cm ² at 25 × 10 ³ cells / cm ^2. -Dickinson), serum-free medium [60% low glucose DMEM (DMEM-LG; Gibco-BRL, Grand Island, NY), 40% MCDB-201 (Sigma), 1X insulin / transferrin / selenium, 4.7 μg / ml linoleic acid, 1 mg / ml bovine serum albumin (BSA), 10 ^-8 M dexamethasone, lO ^-4 M ascorbic acid-2-phosphoric acid (all Sigma), 100 U / ml penicillin, addition of 100 U / ml streptomycin (Gibco)] 2% PCS (Hyclone, Logan Utah) and 10 ng / mL each of epidermal growth factor (EGF) (Sigma), leukemia inhibitory factor (LIF; Chemicon, Temecula, CA) and platelet-derived growth factor (PDGF-BB; R & D Systems, Minneapolis, MN). hMASCs were seeded at 15-30 × 10 ³ cells / cm ² at 0.1 μg / ml FN or 1% Matrigel in the same medium without LIF (Reyes M., 2002). After 8-12 hours, media is removed, cells are washed twice with phosphate buffer (PBS) (Fischer), and 5-50 ng / ml HGF, aFGF, bFGF, FGF4, FGF7, or OSM; or 10 mg / The cells were cultured in a serum-free medium supplemented with ml dimethyl sulfoxide (DMSO) or 0.1-1 mM sodium butyrate.

Transduction of eGFP into hMASC was performed using retrovirus-containing eGFP-CDNA and expanded to> 5 × 10 ⁶ cells. 20% were induced to differentiate into muscle, endothelium, neuroectoderm and endoderm. For clone A16, a single retroviral insertion site was present in undifferentiated MASCs and mesodermal and neuroectodermal differentiated cells, and EGFP ⁺ clone A16MASC differentiated into CK18 and albumin positive cells. The same insertion site is present in FGF4-treated MASCs made from the same cell population (5'-TAGCGGCCGCTTGAATTCGAACGCGAGACTACTGTGACTCACACT-3 ', chromosome 7), and a single hMASC differentiates into endoderm other than mesoderm and neuroectodermal Prove that.

Quantitative RT-PCR shows that FGF4 and HGF induce hepatocyte determination and differentiation. Quantitative RT-PCR confirmed hepatocyte differentiation for early (HNFSp, GATA4, CK19, αFP) and (CK18, albumin, HNF1α, cytochrome P450) markers of hepatocyte differentiation. RNA was extracted from 3xl05 MASCs or MASCs induced to differentiate into hepatocytes. mRNA was reverse transcribed and cDNA was amplified as follows: ABIPRISM7700 (Perkin Elmer / Applied Biosystems) was used to perform initial denaturation (10 ° C. at 95 ° C. with 2 μL of DNA solution, 1XTaqMan SYBR Green Universal Mix PCR reaction buffer). Min) followed by 40 cycles of two-step PCR (95 ° C for 15 seconds, 60 ° C for 60 seconds). The primers used for amplification are listed in Table 9.

mRNA levels are normalized using β-actin (mouse and human) or 18S (rat) as housekeeping genes and newly isolated rat or mouse hepatocytes, 7
MRNA levels in rat hepatocytes cultured for one day or mRNA derived from human adult liver RNA purchased from Clontech, Palo Alto, California.

  On day 0, mmASC and rMASC expressed low levels of albumin, αFP, CK18, CK19, TTR, HNF3P, HNF1α and GATA4 mRNA, but not CYP2B9 and CYP2B13 (mouse) or CYP2B1 (rat) mRNA (Figure 10). After treatment with mMASC or rMASC with FGF4 and HGF, HNF3J5 and GATA4 mRNA expression increased on day 2, peaked by day 4, slightly decreased and became constant by day 14. αFP and CK19 mRNA increased from the second day, reached a maximum by the fourth day, and decreased thereafter. TTR mRNA increased after the 4th day, and reached the maximum level by the 7th day. CK18, albumin, HNF1α and P450 enzyme mRNA increased from day 7 and peaked on day 14. Between days 14 and 21, MASCs induced by FGF4 and HGF expressed albumin, TTR, CK18, CYP2B9 and CYP2B13 (mouse) and CYP2B1 (rat) mRNA.

  Undifferentiated hMASC expressed low levels of albumin, CK18, and CK19, CYP1B1, but not αFP (FIG. 10) and CYP2B6 mRNA. Albumin, CK18, CK19, and CYP1B1 mRNA levels were significantly increased in FGF4 alone or hMASC cultured with FGF4 and HGF for 14 days compared to day 0 (MASC) culture. Albumin, CK18 and CYP1B1 mRNA levels continued to increase and were higher on day 28. Although CYP1B1 is not a specific hepatocyte marker, its up-regulation suggests hepatocyte determination and maturation. Low levels of CYP2B6, 0.5% to 1.0% of fresh liver mRNA, could be seen on days 14 and 21, but not on day 0. MRNA levels of immature hepatocyte markers (CK19 and αFP) decreased as differentiation progressed and were higher in cultures induced with FGF4 alone, whereas mRNA levels of mature hepatocytes (CK18 and albumin) were induced by FGF4 and HGF. It was higher in sex hMASC.

Western blot demonstrates that FGF4 + HGF induces hepatocyte determination and differentiation Expression of hepatocyte-specific gene expression was also confirmed by Western blot and performed as described in Reyes et al. (2000). Antibodies against αFP, human albumin and CK18 were diluted 1: 1000 with blocking buffer. Goat anti-β-actin (1: 1000) was obtained from SantaCruz. Secondary antibodies were HRP-conjugated goat anti-mouse and HRP-conjugated donkey anti-goat (Amersham, Arlington Heights). ECL was performed according to the instruction manual (Amersham). Undifferentiated hMASC did not express CK18, albumin, or αFP protein (FIG. 1OB). After treatment with FGF4 alone or with FGF4 and HGF for 35 days, hMASC expressed albumin and CK18 but not αFP, consistent with histochemical analysis.

mMASC, rMASC, and hMASC acquire functional activity of hepatocytes Using five different assays, determine whether cells with morphological and phenotypic characteristics of hepatocytes also have functional characteristics of hepatocytes did.

  Urea production and secretion by hepatocyte-like cells was measured at various times throughout the differentiation. The urea concentration was measured by colorimetric analysis (Sigma model 640-1) as per the instruction manual. Rat hepatocytes grown in monolayers and fetal mouse liver primordium were used as positive controls and media as negative controls. This assay can detect a minimum urea concentration of 10 mg / ml. Since this assay also measures ammonia, the samples were evaluated before and after the addition of urease.

  Urea or ammonia was not detected in the medium alone. Undifferentiated MASC did not produce urea. After treatment with FGF4 and HGF, urea production by MASC increased in a time-dependent manner. The time course of urea production was similar in mouse and rat cultures. For hMASCs treated with FGF4 and HGF, urea was not detected on day 4 and was similar to mouse and rat culture up to day 12, exceeding urea in mouse and rat culture on day 21. The level of urea produced in MASC-derived hepatocytes was similar to that in monolayer cultures of rat primary hepatocytes. For all three species, cells differentiated on Matrigel produced significantly more urea compared to FN.

Albumin production was measured at various time points throughout the differentiation. Rat albumin concentration was determined by a competitive enzyme-linked immunoassay (ELISA) described previously (Tzanakakis ES, et al., 2001; Wells JM et al. Y 2000). Human and mouse albumin concentrations were measured using a similar ELISA method, replacing human or mouse albumin and anti-human or anti-mouse albumin antibodies with rat components where appropriate. Peroxidase-conjugated anti-human albumin and control human albumin were obtained from Cappel. Peroxidase-conjugated affinity purified anti-mouse albumin and control mouse albumin were obtained from BethylLaboratories (Montgomery, Texas). To ensure the specificity of the ELISA, human, mouse and rat antibodies were incubated for 2 hours at 37 ° C. in 3% BSA in distilled water (dH ₂ O). The ELISA had a sensitivity of at least 1 ng / ml.

  Undifferentiated MASCs did not secrete albumin. After treatment with FGF4 and HGF, mmASC, rMASC and hMASC produced albumin in a time-dependent manner. As seen for urea production, cells differentiated on Matrigel produced a greater amount of albumin than when cultured on FN. Although albumin was not detected on day 3 in human culture, mouse, rat, and human cells secreted similar levels of albumin. The level of albumin produced in mouse, rat and human MASC-derived hepatocytes was similar to that seen in monolayer cultures of rat primary hepatocytes.

Next, cytochrome P450 activity was assessed using PROD analysis on aggregates of MASC-derived hepatocytes and spherical colonies of rat primary hepatocytes. mMASC-hepatocyte aggregates were prepared by seeding FGF4 and HGF-treated mMASC from day 14 on a non-tissue culture plate at 5 × 10 ⁴ cells / cm ² and using a shaker at 10 rpm for 5 hours. did. Cell aggregates were transferred to a Primaria petri dish and densified for 4 days in the presence or absence of 1 mM phenobarbital. hMASC-hepatocyte aggregates were prepared by the hanging drop method. In summary, 10 ³ hMASCs treated with FGF4 and HGF for 24 days were placed in 100 μL droplets in the presence or absence of 1 mM phenobarbital. After 4 days, aggregates were collected and cytochrome P450 activity was assessed by PROD analysis. Pentoxyresorufin (PROD) (MolecularProbes, Eugene, Oregon) is O-dealkylated by cytochrome P450 and changes from a non-fluorescent compound to a fluorescent compound resorufin (Tzanakakis E.S. et al., 2001). As a result, cytochrome P450 (CYP) activity is estimated from the fluorescence intensity generated by PROD metabolism. Measurement and detection of resorufin in situ was performed using confocal microscopy as described (Tzanakakis E.S. et al., 2001).

  PROD activity was not found in undifferentiated mMASC or hMASC aggregates. However, mMASCs (18 days with FGF4 and HGF) and hMASCs (28 days, FGF4 alone) induced to form aggregates had significant PROD activity. The PROD activity of MASC-derived hepatocyte aggregates was similar to that of rat primary hepatocyte aggregates. Several different cells have P450 activity, but upregulation of P450 activity by phenobarbital is only seen in hepatocytes. Therefore, P450 was also tested in the presence or absence of phenobarbital. Without phenobarbital, some P450 enzymes partly participate in PROD metabolism, giving these samples elevated fluorescence values. In contrast, in phenobarbital-induced aggregates, PROD activity was almost completely inhibited by mouse cytochrome Cyp2b9, Cyp2blO, and Cyp2bl3, rat cytochrome Cyp2bl / 2 (TzanakakisE.S. Etal, 2001), and CYP2B6 in humans. Metabolized. Thus, the increased fluorescent activity is less than the actual increase in protein expression of a defined cytochrome P450 enzyme. When aggregates were incubated with phenobarbital for 96 hours, there was a 32% to 39% increase in PROD activity, with functional hepatocyte-specific Cyp2b9, Cyp2blO, and Cyp2bl3 in mMASC and CYP2B6 in hMASC-derived hepatocytes. Suggested to exist.

  MASC-derived hepatocytes were evaluated for their ability to take up LDL by incubating FGF4-treated hMASC with LDL-dil-acil. Cells were co-labeled with anti-CKl8 or anti-Pan-CK and HNF-3p or GATA4 antibody. After 7 days, low levels of a-LDL uptake were detected and increased to a maximum on day 21.

Another metabolic function of hepatocytes is glycogen production or gluconeogenesis. The level of glycogen storage was analyzed by periodate Schiff (PAS) staining on days 3, 7, 14, and 21 for FGF4- and HGF-induced mouse MASC and FGF-induced hMASC. For PAS, slides were oxidized with 1% periodic acid for 5 minutes and washed 3 times with dH2O. Thereafter, the slides were treated for 15 minutes with Schiff's reagent, in dH2O 5 ^- washed 10 minutes and then washed in dH2O and stained for 1 minute with Mayer'shematoxylin. Glycogen storage was first seen by day 4 and the maximum level was seen after day 21 (Figure ll).

Hepatocytes Isolation and culture hepatocytes 4 ^- 6 week old male was as taken according the Sprague-Dawley rats (. SeglenP.O, 1976). The survival number of hepatocytes after collection ranged from 90 to 95%. Hepatocytes were cultured as described (Tzanakakis ES et al., 2001; Tzanakakis ES et al, 2001). To form monolayers, hepatocytes were cultured on 35 mM Fischer plate medium (Fischer Scientific, Pittsburgh, PA) coated with 8 μg / cm ² collagen (Cohesion Technologies, Palo Alto, Calif.). To form spheroids, hepatocytes were cultured in a 35-mmPrimaria ™ petri dish. (BectonDickinson). Under both conditions, the seeding density was 5 × 10 ⁴ cells / cm ² . After 12 hours of initial plating, the medium was changed to remove dead and non-adherent cells. The medium was changed every 48 hours thereafter.

Summary It has been shown that single MASCs derived from postnatal mouse, rat and human BM can differentiate into endoderm cell types with hepatocyte phenotype and function in vitro. MASCs cultured under cell differentiation conditions expressed primordial and mature hepatocyte markers in a time-dependent manner, and the markers were shown by immunofluorescence microscopy of double and triple labeled cells. Since non-hepatocyte markers were not co-expressed with hepatocyte antigens, the protein expression profile was hepatocyte specific and not pseudo. Results from immunohistochemistry were confirmed by Western blot. In addition, RT-PCR is a transcription factor HNF3β and GATA4 known to be important for endoderm determination, and transcription factors such as HNF3β required for subsequent hepatocyte differentiation, and for example CK19, CK18, αFP and albumin Up-regulation of cytoplasmic proteins such as

  FGF4 alone, or both FGF4 and HGF, have been shown to differentiate MASCs into cells with morphological and phenotypic characteristics of hepatocytes, but may show acquisition of hepatocyte functional characteristics. Unless possible, this alone does not prove that the cells have differentiated into hepatocytes. Therefore, several functional tests were performed in combination to identify functioning hepatocytes. mMASC, rMASC or hMASC produced urea and albumin, contained phenobarbital-induced cytochrome P450 activity, could incorporate Dil-acil-LDL, and contained glycogen granules. Urea production is specific for hepatocyte activity, but renal tubular epithelium also produces urea (Hedlund E. et al., 2001). In contrast, albumin production is a specific test for the presence and metabolic activity of hepatocytes (Hedlund E. et al., 2001). Cytochrome P450 is found in hepatocytes but is also present in many other cell types (Jarukamjorn K. et al., 1999).

However, Cyp2bl activity in rats (Tzanakakis ES et al., 2001), Cyp2b9 and Cyp2bl3 in mice (Li-Masters T. et al., 2001; Zelko I. Et al., 2000), and CYP2B6 in humans are relatively It is considered hepatocyte specific. The presence of these forms of P450 was shown by RT-PCR. When P450 activity is inducible by phenobarbital, the specificity for hepatocytes is further improved, as shown (RaderD.J. Et al., 2000).

LDL uptake is seen in hepatocytes (OhS.H. et al., 2000), but other cells such as endothelium have similar abilities (Avital I. et al., 2001). Finally, only hepatocytes can produce and store glycogen. Taken together, these functional studies show that mouse, rat or human-derived MASCs treated with FGF4 and HGF in vitro not only express hepatocyte markers but also have functional characteristics consistent with hepatocyte metabolic activity. Show.

Several studies have shown that BM-derived cells can differentiate into hepatocyte-like cells in vivo and in vitro (Petersen B.E. et al., 1999; Theise ND et al., 2000; Krause DS et al., 2001; Pittenger M.F. et al., 1999; Wang S. et al., 2001; Lagasse E. et al., 2000). However, most studies do not address the phenotype of BM cells that differentiate into cells with a hepatocyte phenotype. It is unknown whether cells positive for staining for hepatocyte markers have the functional characteristics of hepatocytes, and whether cells that differentiate into hepatocytes can also differentiate into mesodermal cells such as hematopoietic cells. . Lagasseet al. Showed that cKit ⁺ Thy ₁ ^low Sca1 ⁺ Lin ⁺ -cells present in mouse BM differentiate into cells with hepatocyte function as well as hepatocyte phenotype (Lagasse E. et al. ., 2000). Despite the fact that only 50 cells can be transplanted, this study does not prove that the same cells that differentiate into hematopoietic cells also differentiate into hepatocytes. It was. Krauseet al. Showed that single cells can re-establish in the hematopoietic mechanism and give rise to rare hepatocytes. However, functional evaluation of hepatocytes was not performed (Krause DSet al., 2001). Avita et al. Is a Thy-1 ⁺ cell in mouse BM β2m ⁻ is albumin, HNF4, C / EBPct, and cytochrome P4503A2 mRNA and protein (Wilmut I., et al., 1997), usually in the liver We recently reported that the phenotype of hepatocyte progenitor cells is observed. Thus, the presence of such hepatocyte progenitors in BM may explain the in vivo differentiation of bone marrow into hepatocytes noted in recent studies (Krause DS et al, 2001; Lagasse E. et al., 2000).

  Retroviral marking was used (Reyes M. et al., 2001; Jiang Y., 2002) to address the question of whether cells producing functional hepatocyte-like cells also produce other cell types. It has recently been shown that cells expressing albumin, GK18 and HNF1α can arise from the same mmASC and rMASC that differentiate into cells with endothelial and neuroectodermal phenotypes (JiangY., 2002) . Similar results have been confirmed for hMASC. Extending a recently published study that showed obtaining cells with mesodermal and neuroectodermal phenotypes and functions from singlehMASC (Reyes M., 2002), the same single hMASC is also It is shown here that they differentiate into cells having a morphology and phenotype. Thus, MASCs that do not express hepatocyte markers and do not have functional hepatocyte activity are present in the BM, and depending on the culture conditions, this may indicate hepatocyte phenotype and hepatocyte functional characteristics, or mesodermal and It is shown for the first time to acquire phenotypic and functional characteristics of neuroectodermal cells.

Transplantation of LacZ gene-introduced MASC to treat hemophilia mice MASC was obtained from ROSA26 mice containing β-gal / NEO transgene (10 ⁶ cells / mouse) and hemophilia mice without prior irradiation (N = 5) was injected intravenously. Animals were sacrificed 1 month (N = 2) and 2 months (N = 3) after MASC transplantation. Bone marrow cytospin and frozen sections of liver, spleen, skeletal muscle, heart, lung and intestine were stained for the presence of β-gal antigen using FITC-conjugated anti-β-gal antibody and pan-cytokeratin or CD45. Tissues were also analyzed for β-gal gene by Q-PCR as described in Example 6.

Preliminary analysis showed by immunohistochemistry and Q-PCR that 1 out of 3 individuals (M3) analyzed 2 months after injection had 0.1% lung epithelial cells derived from donor cells. Immunohistochemistry showed that <1% CD45 ⁺ donor cells were established in spleen, bone marrow and intestine in individual M5. The tissue of individual M4 had some donor-derived cells immunohistochemically; PCR data for this individual is awaiting results.

  All publications, patents and patent applications are hereby incorporated by reference into the present disclosure as if individually incorporated by reference. In the preceding specification, the invention has been described with reference to specific preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, while the invention allows and is described herein. It will be apparent to those skilled in the art that certain details may be varied considerably without departing from the basic principles of the invention. (References)

  (Pluripotent adult stem cells and methods for isolating them) Part of the present invention is through a grant from the National Institutes of Health / National Allergic Infectious Diseases for MorayamaReyes (Grant No. IF31-AI-Gn10291) Received support from the US government.

  The present invention relates generally to methods for isolating stem cells, stem cells isolated by the methods, and therapeutic uses of such stem cells. More particularly, the present invention relates to isolated bone marrow-derived progenitor cells that have the ability to differentiate to form cells of various cell lineages, as well as methods of isolating such cells and isolated by such methods. And methods for inducing specific differentiation of cells, as well as specific markers present in those cells, such as proteins and transcription factors.

  BACKGROUND OF THE INVENTION The development of organs and tissues from stem cells and subsequent transplantation has provided a promising treatment for many disease states, thus making stem cells the center of research in many areas. Using stem cells to form organs and tissues for transplantation offers promising alternative treatments for diabetes, Parkinson's disease, liver disease, heart disease, and autoimmune disease, to name a few . However, there are at least two important problems associated with organ and tissue transplantation. First, there is a shortage of donor organs and tissues. Only 5% of the organs required for transplantation in the US are available to recipients (Evans, et al., J. Am. Med. Assoc. (1992) 267: 239-146). According to the American Heart Disease Association, only 2,300 of the 40,000 Americans who need a new heart in 1997 can receive the heart, and the US Liver Disease Fund It has been reported that there are fewer than 3,000 donors for nearly 30,000 patients who die. A second important issue is a potential incompatibility between the transplanted tissue and the recipient's immune system. The donor organ or tissue is recognized by the host immune system as a foreign body and needs to be provided with a rejection inhibitor to the patient, knowing that it is both economically and physically burdensome.

  Xenotransplantation, ie, transplantation of tissue or organs from another species, can provide an alternative means to overcome human organ and tissue deficiencies. Xenotransplantation will provide an advanced plan for transplantation that allows organs to be harvested while still healthy and further allows the patient to undergo beneficial pretreatment prior to transplantation surgery. Unfortunately, xenotransplantation cannot overcome the problem of tissue incompatibility and rather exacerbates it. Furthermore, according to the Centers for Disease Control, there is evidence that harmful viruses cross species barriers. Although pigs have become promising candidates for organ and tissue donors, cross-species infection of multiple viruses from pigs to humans has been demonstrated. For example, more than 1 million pigs have recently been slaughtered in Malaysia to prevent the development of Hendra virus, a disease with fatal consequences infecting more than 70 humans (Butler, D. , Nature (1999) 398: 549).

Stem cells: definition and use Thus, the most promising source of organs and tissues for transplantation is in the development of stem cell technology. Theoretically, stem cells can undergo self-renewal cell division and give rise to daughter cells that are identical in phenotype and genotype for an indefinite period of time, eventually becoming at least one definitive cell type. Can differentiate. Xenotransplantation by generating tissues or organs from the patient's own stem cells or by genetically recombining xenogeneic cells so that the recipient's immune system does not recognize them as foreign, without the risk of concomitant infection or tissue rejection The transplant can be created to provide the benefits associated with the.

  Stem cells also offer the potential to improve the outcome of gene therapy. The patient's own stem cells can be genetically modified in vitro and reintroduced in vivo to produce the desired gene product. These genetically engineered stem cells will have the ability to be induced to differentiate to form multiple cell types for transplantation at a specific site of the body or for systemic application. Alternatively, the heterologous stem cells are genetically engineered to express the recipient's major histocompatibility complex (MHC) antigen, or not express any MHC, to the recipient without risk of concomitant rejection. Transplantation of those cells from can be made possible.

  A stem cell is defined as a cell that has a mitotic ability that can be referred to as infinite in some humans, differentiates into different cell lines, and regenerates the tissue at the time of transplantation. A typical stem cell is an embryonic stem cell having unlimited self-renewal ability and pluripotent differentiation ability. The cells can be obtained from the inner cell mass of the blastocyst or from primordial germ cells (embryonic germ cells or EG cells) derived from the post-transplant embryo. Until now, ES and EG cells have been obtained from mice, but recently they have also been obtained from non-human primates and humans. When ES cells are introduced into mouse blastocysts or blastocysts of other animals, they can differentiate into all tissues of the mouse (animal). ES and EG cells form teratomas when transplanted into postnatal animals, again indicating its pluripotency. ES (and EG) cells can be identified by positive staining with SSEA1 and SSEA4 antibodies.

  At the molecular level, ES and EG cells express many transcription factors that are very specific for these undifferentiated cells. Examples are oct-4 and Rex-1. In addition, LIF-R, sox-2 and Rox-1 transcription factors are also found, but the latter two are also expressed in cells other than ES cells. Oct-4 is a transcription factor expressed in embryos before gastrulation, in early-stage embryos, cells in the inner cell mass of blastocysts, and embryonic tumor (EC) cells. Oct-4 is downregulated upon inducing cell differentiation in vitro, and in adult animals oct-4 is found only in germ cells. Several studies have shown that oct-4 is required for maintenance of the undifferentiated phenotype of ES cells and plays an important role in determining the early stages of embryogenesis and differentiation . Oct-4, together with Rox-1, activates transcription of the zinc finger protein Rex-1, and is also required to maintain ES cells in an undifferentiated state. Similarly, sox-2, together with oct-4, maintains the undifferentiated state of ES / EC cells and is required to maintain mouse ES cells (different in humans). The presence of LIF is required in human and mouse primordial germ cells. Another feature of ES cells is the presence of telomerase, so that these cells have unlimited self-renewal ability in vitro.

  Stem cells have been identified in many organ tissues. The most frequently examined are hematopoietic stem cells. The cells are mesoderm-derived cells that have been isolated based on cell surface markers and functional characteristics. Hematopoietic cells are isolated from bone marrow, blood, umbilical cord blood, fetal liver, and yolk sac and provide the recipient's life-long haematopoietic action to form multiple hematopoietic cell lines (see Fei, R., et al. McGlave, et al., US Pat. No. 5,460,964; Simmons, P., et al., US Pat. No. 5,677,136; Tsukamoto, et al ., US Pat. No. 5,750,397; Schwartz, et al., US Pat. No. 5,759,793; DiGuisto, et al., US Pat. No. 5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,716,827; Hill, B., et al., Exp. Hematol. (1996) 24 (8): 936-943). Hematopoietic stem cells regenerate red blood cells, neutrophil macrophages, bone marrow megakaryocytes, and lymphoid hematopoietic cell pools when transplanted into animals or humans irradiated with lethal radiation levels. Hematopoietic cells can be allowed to undergo at least some self-renewal cell division in vitro and differentiate into the same cell line as seen in vivo. That is, this cell satisfies the standard as a stem cell. However, since these stem cells differentiate only into hematopoietic cells, they cannot serve as a source of cells for repairing other damaged tissues such as the heart and lungs damaged by administration of a large amount of chemotherapeutic agents.

  The second well-studied stem cell is the neural stem cell (Gage FH: Science 287: Science 287: 1433-1438, 2000; Svendsen CN etal., Brain Path 9: 499-513, 1999; Okabe S et al., Mech Dev 59: 89-102, 1996). Neural stem cells were first identified from the subventricular region of the fetal brain and the olfactory bulb. Until recently, it was thought that adult brains did not already contain stem cell-capable cells, but some studies in rodents and more recently non-human primates and humans Was still present in the adult brain. These stem cells can proliferate in vivo, and can continuously regenerate at least some of the neurons in vivo. Neural stem cells can be grown in culture ex vivo and differentiated into different types of neurons and glial cells. Neural stem cells can engraft and form neurons and glial cells when transplanted into the brain. Therefore, this cell also meets the stem cell criteria.

Mesenchymal stem cells (MSCs) are first derived from embryonic mesoderm and isolated from adult bone marrow, but differentiate into muscle, bone, cartilage, fat, bone marrow stroma, and tendons. Is possible. In embryogenesis, mesoderm develops into limb bud mesoderm, bone-forming tissue, cartilage, fat, skeletal muscle, and possibly endothelium. The mesoderm also differentiates into visceral mesoderm, which produces blood islands consisting of heart muscle, smooth muscle, or endothelium and hematopoietic progenitor cells. Thus, primitive mesoderm or mesenchymal stem cells can be the source of many different cells and tissues. Mesenchymal stem cells are the third tissue specific cells listed as stem cells and were first described by Fridenshtein (Fridenshtein, Arkh. Patol., 44: 3-11, 1982). Many mesenchymal stem cells have been isolated so far (cf. Caplan, A., et al., US Pat. No. 5,486,359; Young, H., et al., US Pat. No. 5). Caplan, A., etal., US Pat. No. 5,811,094; Bruder, S., et al., US Pat. No. 5,736,396; Caplan, A., etal. US Pat. No. 5,837,539; Masinovsky, B., US Pat. No. 5,873,670; Pittenger, M., US Pat. No. 5,827,740; Jaiswal. N., et al., J. CellBiochem. (1997) 64 (2): 295-312; Cassiede P., et al., J. BoneMiner. Res. (1996) 11 (9): 1264-1273; Johnstone, B., et al. , (1998) 238 (1): 265-272; Yoo, et al., J. Bone Joint Surg. Am. (1998) 80 (12): 1745-1757; Gronthos, S., Blood (1994) 84 ( 12): 4164-4173; Makino, S., et al., J. Clin. Invest. (1999) 103 (5): 696-705). Many of the mesenchymal stem cells described so far all show limited differentiation potential and differentiate only into differentiated cells generally considered to be derived from mesenchyme. The most pluripotent mesenchymal stem cell reported so far is a cell isolated by Pittenger et al., And the phenotype of this cell is SH2 ⁺ SH4 ⁺ CD29 ⁺ CD44 ⁺ CD71 ⁺ CD90 ^+. CD106 ⁺ CD120a ⁺ CD124 ⁺ CD14 ⁻ CD34 ⁻ CD45 ⁻ . This cell can differentiate into many types of cells derived from mesenchyme, but as the research team that isolated the cell said it did not identify any hematopoietic cells during growth culture, It is clear that the differentiation potential is limited to mesenchymal cells (Pittenger, et al., Science (1999) 284: 143-147).

  To date, other stem cells such as digestive stem cells, epidermal stem cells, and hepatic stem cells, also called oval cells, have been identified (Potten C, Philos Trans R Soc Lond B Biol Sci353: 821-30, 1998; WattF, Philos. Trans R Soc Lond B Biol Sci 353: 831, 1997; AlisonM et al, Hepatol 29: 678-83 1998). Many of these cells have not been well examined.

  Compared to ES cells, tissue-specific stem cells have lower self-renewal capacity and differentiate into multiple cell lines but do not have pluripotency. It is not clear whether tissue-specific cells express markers as described above for ES cells. In addition, telomerase activity in tissue-specific stem cells has not been fully studied, partly due to the difficulty in obtaining large numbers of dense populations of these cells. is there.

  Until recently, it was thought that tissue-specific stem cells could only differentiate into cells of the same tissue, but many recent papers could allow adult tissue-specific stem cells to differentiate into cells of different tissues Has been suggested. Many studies have shown that cells transplanted during bone marrow transplantation can differentiate into skeletal muscle (Ferrari Science 279: 528-30, 1998; Gussoni Nature 401: 390-4, 1999). This is believed to be within the potential differentiation potential of mesenchymal cells present in the bone marrow. Jackson mentions in his paper that muscle satellite cells can differentiate into hematopoietic cells, which is also an example of phenotypic conversion in the visceral mesoderm (JacksonPNASUSA 96: 14482- 6, 1999). Some studies have shown that stem cells from one layer of the embryo (eg, visceral mesoderm) can differentiate into tissues thought to be derived from different layers of the embryo during embryogenesis. For example, endothelial cells or progenitor cells detected in humans and animals that have undergone bone marrow transplantation are at least partially derived from bone marrow donors (Takahashi, Nat Med 5: 434-8, 1999; Lin, Clin Invest 105: 71-7, 2000). That is, cells derived from visceral mesoderm that are not derived from visceral mesoderm such as MSC are introduced together with the transplanted bone marrow. A further surprising report is that hepatic and biliary epithelial cells are derived from donor bone marrow in rodents and humans (Petersen, Science 284: 1168-1170, 1999; Theise, Hepatology 31: 235-40, 2000; Theise, Hepatology 32: 11-6, 2000). Similarly, three research groups have shown that neural stem cells can differentiate into hematopoietic cells. Finally, Clarke et al. Reported that neural stem cells injected into blastocysts can differentiate into all tissues of chimeric mice (Clarke, Science 288: 1660-3, 2000).

It should be pointed out here that many of these studies do not concludely prove that a single cell can differentiate into tissues of different organs. In fact, most researchers have not identified the phenotype of progenitor cells. An exception is the work by Weissman and Grompe. This study shows that cells engrafted in the liver are present in Lin ^- Thy ₁ LowSca ₁ ⁺ bone marrow cells with highly enriched hematopoietic stem cells. Similarly, Mulligan's research group has shown that bone marrow SP cells highly enriched in hematopoietic stem cells can differentiate into muscle and endothelium, and Jackson et al. It has been shown to be due to cells (Gussoniet al., Nature 401: 390-4, 1999).

  To transplant tissues and organs generated from different types of embryonic stem cells, the cells can be further genetically modified to inhibit the expression of certain cell surface markers, or chemotherapeutic immunosuppressants can be used to prevent transplant rejection. It is necessary to use it continuously. Thus, while embryonic stem cell research offers a promising alternative to the limited supply of transplanted organs, it is problematic because of the need for immunosuppression to establish transplanted xenogeneic cells and tissues. With danger. Estimating 20 immunologically distinct embryonic stem cell lines to establish immunocompatible cells that can be treated to cover the majority of the population (Wadman, M., Nature ( 1999) 398: 551).

Using adult-derived cells rather than embryo-derived as a source of autologous or heterologous genotyped stem cells not only solves the problem of tissue incompatibility associated with the use of transplanted embryonic stem cells, but also embryonic stem cells The ethical dilemma associated with this research will also be resolved. To date, the greatest difficulty associated with the use of autologous stem cells for tissue transplantation is limited cell differentiation. Many stem cells have been isolated so far from fully developed organisms, especially humans. However, although these cells have been reported to have pluripotency, their ability to differentiate into different types of cells was limited.

  That is, stem cells having pluripotency have been isolated so far by the present inventors and other researchers, but fibroblasts, osteoblasts, chondrocytes, adipocytes, skeletal muscle, endothelium, stroma, No progenitor cells have been described so far that have the ability to differentiate into a wide variety of cells of different cell lines such as smooth muscle, cardiac muscle, and hematopoietic cells. Stem cells or progenitor cells with the highest differentiation potential are required for future advances in therapy by cell and tissue transplantation and gene therapy. Therefore, there is a need for cells that are equivalent to embryonic stem cells in adults.

  SUMMARY OF THE INVENTION The present invention provides isolated pluripotent mammalian stem cells that are surface antigens CD44, CD45, and HLA class I and II negative. In addition, the cells may be surface antigen CD34, Muc18, Stro-1, HLA class I negative, oct3 / 4 mRNA positive, and hTRT mRNA positive. In particular, the cells of the present invention comprise the surface antigens CD31, CD34, CD36, CD38, CD45, CD50, CD62E and CD62P, HLA-DR, Muc18, Stro-1, cKit, Tie / Tec, CD44, HLA class I, and 2 -May be negative for microglobulin and positive for CD10, CD13, CD49b, CD49e, CDw90, Flk1, EDF-R, TGF-R1 and TGF-R2, BMP-R1A, PDGF-R1a and PDGF-R1b. The present invention provides isolated, pluripotent, non-embryonic, non-germline cells that express the transcription factors oct3 / 4, REX-1, and ROX-1. Also provided is an isolated pluripotent cell derived from a postnatal mammal that is responsive to growth factor LIF and has a receptor for LIF.

  The cells of the present invention described above can be induced to differentiate into at least one type of differentiated cell derived from mesoderm, ectoderm, and endoderm. For example, the cells of the present invention include osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stromal cells, skeletal muscle cells, smooth muscle cells, cardiomyocytes, endothelial cells, epithelial cells, hematopoietic cells, glial cells, It is possible to induce differentiation into at least cells of neuronal cells and oligodendrocyte cell types. As such cells, human cells or mouse cells can be used. The cells can be obtained from a fetus, newborn, child, or adult. The cells can be obtained from organs such as bone marrow, liver and brain.

  The present invention further provides a differentiated cell obtained from the above pluripotent adult stem cell, in which case the progeny cells are bone cells, chondrocytes, adipocytes, fibroblasts, bone marrow stromal cells, skeletal cells It can be a muscle cell, smooth muscle cell, cardiomyocyte, endothelial cell, epithelial cell, endocrine cell, exocrine cell, hematopoietic cell, glial cell, neuronal cell, or oligodendrocyte. Differentiated progeny cells can be skin epithelial cells, liver epithelial cells, pancreatic epithelial cells, pancreatic endocrine cells or islet cells, pancreatic exocrine cells, gastrointestinal epithelial cells, kidney epithelial cells, or epidermis related structures (such as hair follicles) . Differentiated progeny cells may form soft tissue or teeth around the teeth.

The present invention provides an isolated transformed pluripotent mammalian stem cell as described above, in which case the insertion of a preselected isolated DNA into the genome of the cell, preselected isolation It can be modified by replacing a segment of the cell genome with DNA or deleting or inactivating at least a portion of the cell genome. This modification can be carried out by inserting DNA by incorporating a viral vector, or by transduction with a virus using a DNA virus, RNA virus or retroviral vector. It is also possible to inactivate a part of the cell genome of the isolated transformed cell using an antisense nucleic acid molecule having a sequence complementary to the sequence of the part to be inactivated in the cell genome. Furthermore, it is possible to inactivate a part of the cell genome using a ribozyme sequence that targets the sequence of the part of the cell genome to be inactivated. The modified genome has a gene sequence of a selectable or screenable marker gene that is expressed to allow differentiation between progeny cells having the modified genome or progeny thereof and progeny cells having the unmodified genome. You may do it. For example, as a marker, green, red, yellow fluorescent protein, β-gal, Neo, DHFR ^m , or hygromycin can be used. The cells of the invention are capable of expressing genes that can be regulated by inducible promoters and other regulatory mechanisms that regulate the expression of proteins, enzymes or other cell products.

  The present invention provides cells that express telomerase at high levels and are capable of maintaining long telomeres after growth in vitro compared to lymphocyte telomeres obtained from the same donor. . Telomeres may have a length of about 11-16 KB after growth in in vitro culture.

  The present invention provides a cell differentiation solution for promoting continuous proliferation or differentiation of undifferentiated pluripotent stem cells, which comprises a factor that regulates the expression level of oct3 / 4.

The present invention provides a method for isolating pluripotent adult stem cells (MASC). In this method, bone marrow mononuclear cells were depleted of CD45 ⁺ glycophorin A ⁺ cells, CD45 ⁻ glycophorin A ⁻ cells were recovered, and the recovered CD45 ⁻ glycophorin A ⁻ cells were seeded on a matrix coating and seeded. Cells are cultured in a medium supplemented with growth factors. In the depletion step, negative selection using a monoclonal or polyclonal antibody may be performed. The growth factor can be selected from PDGF-BB, EGF, IGF, and LIF. In the last step, it is also possible to culture in a medium supplemented with dexamethasone, linolenic acid and / or ascorbic acid.

  The present invention relates to a method for culturing isolated pluripotent adult stem cells, which comprises adding cells to a serum-free or low serum medium containing insulin, selenium, bovine serum albumin, linolenic acid, dexamethasone, and platelet-derived growth factor. Is provided. As a serum-free or low serum medium, it is possible to use low glucose DMEM mixed with MCDB. Insulin may be present at a concentration of about 10 to about 50 μg / ml. The serum-free or low-serum medium can contain an effective amount of transferrin at a concentration greater than 0 and less than about 10 μg / ml, with selenium present at a concentration of about 0.1 to about 5 μg / ml. Bovine serum albumin may be present at a concentration of about 0.1 to about 5 μg / ml, linolenic acid may be present at a concentration of about 2 to about 10 μg / ml, and dexamethasone is about 0.005 to about It may be present at a concentration of 0.15 μM. The serum-free medium or low serum medium can contain about 0.05 to 0.2 mM L-ascorbic acid. Serum free or low serum medium is about 5 to about 15 ng / ml platelet-derived growth factor, about 5 to about 15 ng / ml epidermal growth factor, about 5 to about 15 ng / ml insulin-like growth factor, 10-10, 000 IU of leukemia inhibitory factor can be included. The present invention further provides a cultured clonal population of mammalian pluripotent adult stem cells isolated according to the above method.

  The present invention provides a method for permanently and / or conditionally immortalizing MASC-derived cells and differentiated progeny, comprising the step of introducing telomerase into the MASC or differentiated progeny. .

  The present invention provides for administration of fully allogeneic pluripotent stem cells, induced hematopoietic stem cells, or progeny cells to a mammal for subsequent tissue transplantation derived from pluripotent stem cells and other organ transplants. It provides a method for reconstituting a mammal's hematopoiesis and immune system by inducing immune tolerance in an animal.

  The present invention provides a method for proliferating undifferentiated pluripotent stem cells into differentiated hair follicles by administering an appropriate growth factor and proliferating the cells.

  The present invention provides many uses for the cells described above. For example, the present invention is a method for using an isolated cell, wherein the population of cells is transplanted in utero to chimerize the cell or tissue, so that a human or animal after transplantation before or after birth The present invention provides a method for correcting a genetic abnormality by producing a human cell in a human body and producing an enzyme, protein or other product having a therapeutic effect in the human body or animal body. The present invention is further a method for using the above cells in gene therapy for a patient in need of therapeutic treatment, by introducing into the cells a preselected isolated DNA encoding the desired gene product. The present invention provides a method for genetically modifying cells, proliferating the cells in culture, and introducing the cells into the patient to produce a desired gene product.

  The present invention provides a method for growing a patient's tissue by growing said isolated pluripotent adult stem cell in culture and contacting an effective amount of said expanded cell with the damaged tissue of the patient in need of repairing the damaged tissue. It provides a method of repairing. The cells can be introduced into the patient's body by local or systemic injection. The cells can be introduced into the patient's body with a suitable matrix implant. Such matrix implants may provide additional genetic material, cytokines, growth factors and other factors that promote the proliferation and differentiation of the cells. The cells can be encapsulated in polymer capsules or the like prior to introduction into the patient's body.

  The present invention is a method for inducing an immune response against an infectious agent in a human patient, wherein a clonal population expanded with pluripotent adult stem cells is genetically modified in culture to produce a protective immune response against the infectious agent. There is provided a method comprising expressing one or more preselected antigen molecules to cause and introducing an effective amount of genetically modified cells to induce an immune response in a patient. This method may further include a step of differentiating pluripotent adult stem cells into dendritic cells before the second step.

The present invention is a method for using MASCs to identify genetic polymorphisms associated with physiological abnormalities, wherein MASCs are simply derived from a statistically significant population of individuals from whom phenotypic data can be obtained. Separating the MASC from the statistically significant population of individuals, establishing a MASC culture, identifying at least one gene polymorphism in the cultured MASC, and the cultured MASC And the differentiation pattern exhibited by the MASC having the normal genotype and the differentiation pattern exhibited by the MASC having the identified gene polymorphism are related to the at least one gene polymorphism. And a method of characterizing an abnormal metabolic process.

  The present invention further provides a method for treating cancer in a mammal, wherein a pluripotent adult stem cell is genetically modified to produce a tumor toxic protein, an anti-angiogenic protein, or a protein expressed on the surface of a tumor cell. The present invention provides a method comprising a step of expressing together with a protein related to immune response stimulation to an antigen, and a step of introducing an effective anticancer amount of the genetically modified pluripotent adult stem cell into the mammal.

  The present invention provides a method of using MASC to characterize a cellular response to a biological or physiological factor, comprising isolating MASC from a statistically significant population of individuals, said statistically significant Establishing a plurality of MASC cultures by culturing and proliferating MASCs obtained from an individual population; contacting the MASC cultures with one or more biological or physiological factors; and Providing a method comprising identifying one or more cellular responses to a physiological factor and comparing the one or more cellular responses of a plurality of MASC cultures obtained from the statistically significant population of individuals. Is.

  The present invention further provides a method for using specifically differentiated cells for therapy, comprising administering the specifically differentiated cells to a patient in need thereof. The present invention further provides a method of utilizing genetically engineered pluripotent stem cells to selectively express endogenous genes or transgenes, as well as MASCs grown in vivo to treat disease to animals. The utilization method utilized for the transplantation / administration of is provided. For example, it is possible to treat blindness caused by neuroretinal diseases caused by macular degeneration, diabetic retinal disease, glaucoma, retinitis pigmentosa, etc. by using pluripotent stem cells, ie neural retinal cells derived from MASC. is there. The cells of the present invention can be used to establish cells in the body of mammals, and can administer autologous, allogeneic, and heterologous cells to produce tissue-specific metabolic and enzymatic properties of the mammal. It is possible to restore or modify coagulation, structural function, etc. The cells of the present invention can be used for fixing cells in the body of a mammal, and can induce cell differentiation in vivo. The cells of the present invention can also be used to administer differentiated stem cells into the body of a mammal. It is possible to treat a disease state such as a genetic disease, a degenerative disease, a cardiovascular disease, a metabolic accumulation disease, a neurological disease, or cancer by using the cells of the present invention and progeny thereof in vitro or in vivo. It is possible to create a gingival-like material for treating periodontal disease using the cells of the present invention. The cells of the present invention can be used to generate pluripotent stem cell-derived skin epithelial tissues that can be used for skin transplantation and plastic surgery. It is possible to strengthen muscles such as penis and heart using the cells of the present invention. The cells of the present invention can be used to produce blood for therapeutic use ex vivo, or to produce human hematopoietic cells and / or blood for use in humans in the body of an animal before or after birth. The cells of the present invention can be used as a therapeutic tool to help patients recover from chemotherapy or radiation therapy in cancer treatment or autoimmune disease treatment or to induce immune tolerance in the recipient. AIDS and other infectious diseases can be treated using the cells of the present invention.

In particular, myocarditis, cardiomyopathy, heart failure,
It is possible to treat heart diseases such as heart attack disorders, high blood pressure, atherosclerosis, heart valve dysfunction. It is possible to treat diseases associated with central nervous system deficiency or damage by utilizing genetically engineered pluripotent mammalian stem cells or differentiated progeny thereof. Furthermore, using the cells differentiated into pluripotent mammal-derived stem cells or nerve-related cells, it affects stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia associated with AIDS, spinal cord injury, brain and other nervous systems It is possible to treat diseases associated with neurological deficits or degeneration such as metabolic diseases.

  Differentiating and proliferating hematopoietic cells, pancreatic islets or β cells, hepatocytes and other cells in vitro or in vivo using progeny differentiated into stem cells or stromal cells derived from pluripotent mammals It is possible to support. The stem cells of the present invention or their progeny differentiated into cartilage can be used to treat indirect or cartilage diseases such as cartilage rupture, cartilage thinning, and osteoarthritis. In addition, stem cells of the present invention or progeny differentiated into osteoblasts are used to produce bone fractures, non-healing fractures, osteoarthritis, and tumors that have metastasized to bone, such as prostate cancer, breast cancer, multiple myeloma, etc. It is possible to improve pathological conditions that adversely affect bone, such as bone “holes”.

  The present invention further provides kits that provide immunization to induce a protective immune response in human patients. This kit contains a medium and antibody for isolating pluripotent adult stem cells from bone marrow aspirate, a medium and cellular factors for culturing isolated pluripotent adult stem cells, and a multiplicity of antigen molecules. A genetic element for genetically modifying a capable adult stem cell is included. The kit may further contain a medium and cellular factors effective in differentiating pluripotent adult stem cells into tissue-specific cells. A viral vector can be used as a genetic element, and this viral vector may have a nucleotide sequence encoding one or more antigens derived from bacteria or viruses. As this genetic element, it is also possible to use a plasmid having a nucleotide sequence encoding a bacterial, viral or parasitic antigen. This plasmid may be packaged with calcium phosphate transfection elements. As a genetic element, it is possible to use a vector having a nucleotide sequence encoding an antigen common to cancer cells. It is also possible to use a vector having a nucleotide sequence encoding a parasitic antigen as a genetic element.

  The present invention further provides a method for gene profiling of pluripotent stem cells as described above, and a method of using this gene profiling in a data bank. The present invention further provides methods of use using genetically profiled pluripotent stem cells as described above in a database to assist in drug discovery.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1a and 1b are photographs of undifferentiated MASCs of the present invention. Cells lacking CD45 expression and glycophorin A expression were selected by immunomagnetic bead depletion and FACS. Cells recovered after sorting were small blasts (FIG. 1a). DMEM, 10 ng / ml IGF, 10 ng / ml EGF, 10 ng / ml PDGF-BB, transferrin, selenium, bovine serum albumin, dexamethasone, linolenic acid, insulin, and ascorbic acid in wells of a 96-well plate coated with fibronectin 5000 cells were seeded in a synthetic medium consisting of Small colonies of adherent cells formed after 7-21 days (FIG. 1b).

FIG. 2 is a graph showing the growth rate of MASCs in culture. DMEM, 10 ng / ml IGF, 10 ng / ml EGF, 10 ng / ml PDGF-BB, transferrin, selenium, bovine serum albumin, dexamethasone, linolenic acid, insulin, and ascorbic acid in wells of a 96-well plate coated with fibronectin CD45 ⁻ / GlyA ⁻ cells were seeded on a synthetic medium comprising 2% FCS and without. When the cells reached confluence, the cells were collected by trypsinization and subcultured twice weekly at a dilution ratio of 1: 4 under the same culture conditions.

FIG. 3 shows the telomere length of MASCs from a 35 year old donor cultured at a replated density of ² × 10 ³ cells / cm ² during 23 and 35 cell divisions. The telomere length was determined by a conventional method. The telomere length was 9 kB. This was 3 kB longer than the telomere length of blood lymphocytes obtained from the same donor. When telomere length was evaluated after 10 and 25 cell divisions, respectively, and telomere length was evaluated after 35 cell divisions, no change was observed. As controls, HL60 cells (short telomeres) and 293 cells (long telomeres) were measured.

  FIG. 4 shows the general protocol for culture, transduction, differentiation, and confirmation of differentiation used by the inventors for the MASCs of the present invention. As shown in the figure, transduction with a retroviral vector having eGFP was performed after culturing. The MASC that reached almost confluence was prepared in the presence of 10 μg / mL protamine in MASC medium (DMEM, 2% FCS, EGF, PDGF-BB, transferrin, selenium, bovine serum albumin, dexamethasone, linolenic acid, insulin, and ascorbic acid. ) Was exposed to PA317 supernatant containing MFG-eGFP prepared in (6) for 2 consecutive days. Cells were trypsinized 24 hours after the last transduction and selected by FACS. 30-70% of MASCs were eGFP positive. Using an ACDU apparatus on the FACS, 1 to 100 eGFP positive cells were selected per well of a 96-well plate containing FN-coated and the same medium. Of these wells, MASC progeny occurred in approximately 2 wells per plate with 10 cells per well. From here, clones were grown in culture. 8-10 passage populations of expanded cells were induced to differentiate along different differentiation pathways. Differentiation was confirmed by the method shown in the figure.

  FIG. 5 shows the differentiation protocol used by the inventor to induce differentiation of the MASCs of the present invention to form osteoblasts, chondroblasts, and adipocytes as indicated. In the figure, cytokines necessary for inducing terminal differentiation and appropriate tests for demonstrating the induction of terminal differentiation are shown.

FIG. 6 shows immunohistochemical staining of bone shaloprotein on day 15 in culture and bone shalo on days 7, 11, and 14 after induction with 10 ⁻⁷ M dexamethasone, β-glycerophosphate, and 10 mM ascorbic acid. Shows Western blot analysis of proteins. The middle panel shows the results of toluidine blue staining for cartilage and the results of Western blot analysis for type II collagen on days 7, 11 and 14, serum-free with 100 ng / mL TGF-β1 added. It shows that MASCs were differentiated into chondrocytes after micromass culture in the medium. In the bottom panel, the results of oil red staining on day 14 and Western blot analysis for PPARg show differentiation after treatment of MASC with 10% horse serum.

  FIG. 7 shows a Western blot of muscle protein. Panel A shows the results of culturing MASC reaching confluence with 3 μM 5-azacytidine for 24 hours. Following this, cultures were maintained in MASC growth media (DMEM, 2% FCS, EGF, PDGF-BB, transferrin, selenium, bovine serum albumin, dexamethasone, linolenic acid, insulin, and ascorbic acid). Differentiation was assessed by Western blot. Five days after induction with 5-azacytidine, Myf5, Myo-D and Myf6 transcription factors were detected in approximately 50% of the cells. After 14-18 days, the expression level of Myo-D was significantly reduced, but Myf5 and Myf6 remained high. On the 4th day after induction, desmin and skeletal muscle actin were detected, and skeletal muscle myosin was detected on the 14th day. Immunohistochemistry showed that 70-80% of cells expressed mature muscle protein after 14 days (not shown). Treatment with 5-azacytidine or retinoic acid expressed Gata4 and Gata6 in the first week of culture. In addition, low levels of troponin T were detected after day 2, indicating that cardiac muscle troponin T was found in embryonic skeletal muscle, thus producing fetal muscle. Smooth muscle actin was detected 2 days after induction and remained high until 14 days later. Panel B was maintained in serum-free medium for 14 days, and 100 ng / mL PDGF was added as the only cytokine to MASC that reached confluence. The presence of smooth muscle markers was assessed by Western blot. Smooth muscle actin was detected after the second day and smooth muscle myosin was detected after the sixth day. On day 15, approximately 70% of the cells were positively stained with anti-smooth muscle actin / myosin antibody by immunohistochemistry. The presence of myogenin was confirmed after the 4th day and desmin was observed after the 6th day. After 2 to 4 days, Myf5 and Myf6 proteins were detected and remained high until day 15. Myo-D was not detected.

  In Panel C, confluent MASCs were exposed to retinoic acid and then cultured in serum-free medium supplemented with 100 ng / mL bFGF. Cells were then analyzed by Western blot. Two days later, Gata4 and Gata6 were expressed, and remained high until the 15th day. Myocardial troponin T was expressed after 4 days, cardiac troponin I was expressed after 6 days, and ANP was detected after 11 days (not shown). On day 15, these myocardial proteins were detected in over 70% of cells by immunohistochemistry (not shown). From the second day onward, the transcription factor Myf6 was found. Desmin expression began on day 6 and myogenin expression on day 2. Skeletal muscle actin was also confirmed. The inventors have further confirmed that natural contraction rarely occurs in the culture and propagates a distance of several mm.

  FIG. 8 is a photomicrograph showing a state in which myoblasts and myotubes form a multinucleated myotube by fusion. The MASCs transduced with eGFP for 24 hours with 5-azacytidine and maintained in MASC growth media were obtained from MASCs that were not transduced with eGFP obtained from the same donor. Co-cultured with myoblasts. In order to induce myotubes, MASC-derived myoblasts (obtained by inducing untransformed MASCs with 5-azacytidine for 24 hours and then maintaining in MASC growth medium for 14 days) with 10% horse serum and DMEM Incubated in. When multinucleated cells are formed, myotubes are incubated with PKH26 (red membrane dye), washed, and myotubes transduced with eGFP prepared as described above in the presence of 10% horse serum. And co-cultured. Two days later, the cells were examined under a fluorescence microscope. The micrograph shows a state where eGFP positive myoblasts are fused with myotubes labeled with PKH26.

  FIG. 9 shows the method used in the present invention to induce endothelial cell differentiation from the MASCs of the present invention, and the markers used to detect endothelial cell differentiation.

  FIG. 10 shows a series of photographs of immunofluorescent staining for von Willebrand factor and the CD34 marker and Western blot analysis for the endothelial cell surface receptor Tie / Tek to confirm endothelial cell differentiation. Pluripotent adult stem cells (MASCs) expressed Flk1, but did not express CD34, PECAM, E- and P-selectin, CD36, Tie / Tek and Flt1. When MASC was cultured in serum-free MASC medium supplemented with 20 ng / ml VEGF, it was confirmed that by the 14th day, CD34 was expressed on the cell surface and the cells expressed vWF (immunofluorescence method). In addition, the cells expressed Tie / Tek on days 7, 11 and 14 as shown by Western blot analysis. Angiogenesis was observed when cells induced by VEGF were cultured on Matrigel or type IV collagen.

  FIG. 11 shows that MASCs were cultured with SCF, Flt3-L, Tpo, and Epo for 14 days, and then cultured in SCF and EGF-containing MASC medium using hematopoietic support feeder AFT024, whereby MASCs were astrocytes, It is a series of photomicrographs showing differentiation into oligodendrocytes and neural cells. Cells were labeled with antibodies against glial fibrillary acidic protein (GFAP) (astrocytes), galactocerebroside (GalC) (oligodendrocytes), and neurofilaments-68 and 200 (neurons).

  FIG. 12 is a series of photomicrographs showing that neurons develop when culturing low density MASCs in fibronectin-coated wells with 100 ng / mL bFGF. 20 ± 2% of cells are positively stained for β-tubulin III, 22 ± 3% are positively stained for neurofilament-68, 50 ± 3% are positively stained for neurofilament-160, and 20 ± 2% Positive staining for neurofilament-200, 82 ± 5% positive staining for neuron specific enolase (NSE) and 80 ± 2% positive staining for microtubule associated protein 2 (MAP2). The number of axons per neuron increased from 3 ± 1 to 5 ± 1 and 7 ± 2 after 2, 3-4 weeks after differentiation. Although not shown in the figure, after 2 weeks of culture, 26 ± 4% of the cells were GFAP positive and 28 ± 3% were GalC positive, whereas after 4 weeks GFAP or GalC positive cells. Decreased.

  FIG. 13 shows RT-PCR results and Western blot analysis for GFAP, myelin basic protein (MBP), and neurofilament-200, x, x, and days after induction of MASC by bFGF.

  FIG. 14 shows the effect of 100 ng / mL bFGF or 10 ng / mL FGF-9, FGF-8, FGF-10, FGF-4, BDNF, GDNF, or CNTF on neurogenesis from MASCs. The properties of the differentiated cells were determined by immunohistochemistry using antibodies against GFAP, GalC, neurofilament 200, tyrosine hydroxylase (TH), GABA and parvalbumin, and acetylcholine (CAT). When cultured with bFGF for 3 weeks, MASC differentiated into neurons, astrocytes, and oligodendrocytes. GABA, parvalbumin, tyrosine hydroxylase, DOPA-decarboxylase, or tryptophan hydroxylase was not detected. When cultured for 3 weeks with 10 ng / mL FGF-9 and EGF, MASCs yielded astrocytes, oligodendrocytes, and GABAergic and dopaminergic neurons. Incubating MASC with 10 ng / mL FGF-8 and EGF for 3 weeks gives rise to both GABAergic and dopaminergic neurons. When MASC was cultured with 10 ng / mL FGF-10 and EGF for 3 weeks, astrocytes and oligodendrocytes were produced, but no neurons were generated. Treatment of MASC with 10 ng / mL FGF-4 and EGF for 3 weeks differentiated into astrocytes and oligodendrocytes, but not into neurons. When MASC was treated with 10 ng / mL BDNF and EGF, differentiation into only tyrosine hydroxylase positive cells was observed. When cultured with GDNF, MASC differentiated into GABAergic and dopaminergic neurons. When cultured, differentiation into GABAergic neurons only was observed after 3 weeks.

  FIG. 15 shows the effect of transplantation of undifferentiated MASCs around the parietal infarction caused by ligation of the middle cerebral artery in the brain of Wistar rats. Rats were continuously administered cyclosporine and the function of the paralyzed limbs was examined 6 weeks after MASC injection. As a control, animals were injected with a medium adjusted with physiological saline or MASC. The results of testing for the stereotaxic function of the extremities 6 weeks after transplantation of MASC or control solution are shown. Only in the rat transplanted with MASC, a functional improvement comparable to the level of animals subjected to sham transplantation was seen.

FIG. 16 shows the effect of transplantation of undifferentiated MASCs around the parietal infarction caused by ligation of the middle cerebral artery in the brain of Wistar rats. Rats were continuously administered cyclosporine and the function of the paralyzed limbs was examined 6 weeks after MASC injection. The animals were killed after 2 and 6 weeks to determine the neuronal phenotype. Since the brain after transplantation of eGFP ⁺ cells emits autofluorescence, it was necessary to perform immunohistochemical analysis of the graft. At 2 weeks, the majority of eGFP ⁺ cells were detected in the transplant area itself. After 6 weeks, eGFP ⁺ cells migrated outside the graft. At the second week, cells stained with anti-eGFP antibody maintained a spherical shape, and their diameters ranged from 10 to 30 μm. After 6 weeks, eGFP ⁺ cells had greatly shrunk, and neurites extending to normal brain tissue were seen in the transplanted area. The presence of human cells was confirmed by staining with NuMa, a human specific nuclear antibody (not shown). This antibody allows double and triple staining with immunofluorescent antibodies and will be used in the future to identify human cells in the graft.

The present inventors detected a small population of nestin positive cells in the same place as NuMa positive cells and GFP ⁺ cells using human-specific anti-nestin antibodies. This indicates neuronal ectoderm differentiation. Furthermore, the inventors confirmed positive staining for β-tubulin III, neurofilaments -68 and -160, oligo markers, and GFAP. This indicates differentiation into nerve cells and glial cells.

  FIG. 17 shows immunohistochemical and western blot analysis for cytokeratins 18 and 19 after treating MASCs with HGF and KGF. After 14 days, large epithelial-like cells expressing psycherokeratin 18 and 19 were seen.

DETAILED DESCRIPTION OF THE INVENTION It is not known whether genetic reprogramming occurs in stem cells committed to a lineage similar to what occurs in a “cloning process” or “trans-differentiation”. The present invention enables pluripotent stem cells that remain after purification from various organs (eg, bone marrow, liver, brain) and further cultured in vitro to proliferate without apparent senescence. And it can be differentiated into a variety of cell types different from the tissue from which the stem cells are derived. The phenotypes of stem cells from various organs with “plasticity” are similar (CD45 ⁻ CD44 ⁻ HLA ⁻ DR ⁻ HLA class I ⁻ oct3 / 4 mRNA ⁺ and hTRT ⁺ ). Furthermore, the characteristics of such stem cells are similar to those of, for example, primordial germ cells (the stem cells can be their immediate progeny).

The present invention has evidence that a small fraction of bone marrow cells, as well as cells in the brain and liver, express genes (oct-4, Rex-1) that are usually found only in ES and EG cells. Furthermore, the present invention detects eGFP ⁺ cells in the bone marrow and brain of newborn mice transformed with the oct-4 / eGFP construct, and the oct-4 expressing cells are in tissues other than germ cells in the late embryonic stage. Is present. Therefore, a small number of stem cells remain even after adulthood and survive in various organs having pluripotency. This explains the plasticity seen in stem cells derived from different organs.

Selection and phenotype of pluripotent stem cells The present invention relates to bone cells, cartilage, adipocytes, fibroblasts, bone marrow stromal cells, skeletal muscle, smooth muscle, cardiac muscle, endothelium, epithelial cells (keratinocytes), hematopoietic cells, glia Pluripotent adult stem cells (MASCs) isolated from adult human, mouse (or other species), newborn, or fetus, capable of differentiating into progenitor cells of cells, neurons, and oligodendrocytes ). These cells exhibit a differentiated phenotype closer to embryonic stem cells than any adult-derived stem cells described so far.

  The pluripotent adult stem cells described here have been isolated by a method developed by the present inventors. The inventors have identified a number of specific cell surface markers that characterize MASC. By using the method of the present invention, it is possible to isolate pluripotent adult stem cells from adults, juveniles or fetuses of humans, mice and other species. Furthermore, in mice these cells are isolated from the brain and liver. Therefore, bone marrow aspirate, brain or liver tissue and possibly other organs are collected and positive well known to those skilled in the art based on surface markers expressed on the cells as specified by the inventors Alternatively, by using the negative selection method, it has become possible for those skilled in the art to isolate the cells without carrying out cumbersome experiments.

A. Isolation of Pluripotent Adult Stem Cells (MASC) from Human Bone Marrow To select pluripotent adult stem cells (MASC), standard techniques well known to those skilled in the art (see Muschler, GF, et al., J Bone Joint Surg. Am. (1997) 79 (11): 1699-709, Batinic, D., et al., Bone Marrow Transplant. (1990) 6 (2): 103-7) Bone marrow mononuclear cells are obtained from bone marrow aspirate. Pluripotent adult stem cells are present in bone marrow (or other organs such as liver and brain), but do not express normal leukocyte antigen CD45 or erythroblast-specific glycophorin-A (Gly-A). This mixed population of cells is subjected to Ficoll-Hypaque separation. The cells are then subjected to negative selection using anti-CD45 and anti-Gly-A antibodies to remove the CD45 ⁺ and Gly-A ⁺ cell population and recover the remaining approximately 0.1% of bone marrow mononuclear cells. It is also possible to inoculate cells into fibronectin-coated wells, and after culturing for 2 to 4 weeks as described below, CD45 ⁺ and Gly-A ⁺ cells can be removed. Alternatively, cells are isolated by performing a positive selection using a combination of the cell-specific markers described herein identified by the inventors, such as leukemia inhibitory factor (LIF) receptor. Both positive and negative selection are methods well known to those skilled in the art, and many monoclonal and polyclonal antibodies suitable for performing negative selection are also well known in the art (see Leukocyte Typing V, Schlossman, et al. al., Eds. (1995) Oxford University Press), commercially available from many sources. Furthermore, methods for separating mammalian cells from a mixture of cell populations are described in US Pat. No. 5,759,793 (magnetic separation) by Schwartz et al. And Basch, et al., J. Immunol. Methods ( 1983) 56: 269 (Immunoaffinity Chromatography) and Wysockiand Sato, Proc. Natl. Acad. Sci (USA) (1978) 75: 2844) (Fluorescence Chromatographic Cell Analysis Separation Method) (Figure 1A). . The recovered CD45 ⁻ / GlyA ⁻ cells are seeded in culture dishes coated with 5-115 ng / ml (preferably about 7-10 ng / ml) of serum fibronectin or other suitable matrix coating. The cells are 1-50 ng / ml (preferably about 5-15 ng / ml) platelet-derived growth factor-BB (PDGF-BB), 1-50 ng / ml (preferably 5-15 ng / ml) epidermal growth factor ( EGF), 1-50 ng / ml (preferably 5-15 ng / ml) of insulin-like growth factor (IGF) or 100-10,000 IU (preferably about 1000 IU) LIF of 10 ⁻¹⁰ to 10 ⁻⁸ M Maintained in Dulbecco's Minimum Essential Medium (DMEM) or other suitable cell culture medium supplemented with dexamethasone or other suitable steroid, 2-10 μg / ml linolenic acid, and 0.05-0.15 μM ascorbic acid. The Other suitable media include MCDB, MEM, IMDM, RPMI and the like. Cells can be maintained in serum-free, in the presence of 1-2% fetal calf serum, or in 1-2% human AB serum or autologous serum (FIG. 1B).

The present inventors have found that pluripotent adult stem cells (MASC) cultured at low density express leukemia inhibitory factor receptor (LIF-R) and express small amounts of CD44 or do not express pluripotent stem cells It was shown that when cells having the characteristics of (MSC) were cultured at high density, LIF-R was not expressed but CD44 was expressed. 1-2% of CD45 ⁻ GlyA ⁻ cells are CD44 ⁻ , and less than 0.5% of CD45 ⁻ GlyA ⁻ cells are LIF-R ⁺ . The cells selected by the fluorescence chromogenic cell analysis separation method (FACS) were subjected to quantitative RT-PCR (real-time PCR) to quantify oct-4 mRNA. Concentration of mRNA oct-4 is, CD45 not sorted ^- compared to ^cells, CD45 ^{- -} GlyA GlyA - CD44 ^- cells at 5 fold ^higher, CD45 ^- GlyA - was 20-fold higher in ^{LIF-R +} cells. Sorted cells were seeded in MASC medium supplemented with 10 ng / ml EGF, PDGF-BB, and LIF. Frequency of MASC starts to grow, not sorted CD45 ^- GlyA ^- compared to cells CD45 ^- GlyA ^- 30 times higher in ^{LIF-R + cells,} CD45 ^- GlyA ^- CD44 ^- was three times higher in the cell.

When the human cells were replated at a cell density of less than 0.5 × 10 ³ cells / cm ² , the culture died without much growth. When replated at a cell density of more than 10 × 10 ³ cells / cm ² every 3 days, the cells stopped dividing before 30 divisions as described below. This also lost the ability to differentiate. When replated at a cell density of ² × 10 ³ cells / cm ² every 3 days, more than 40 divisions were normally seen, and cell populations with more than 70 divisions were also seen. Cell doubling time was 36-48 hours for the first 20-30 divisions. After this, the cell doubling time extended to 60-72 hours (FIG. 2).

When pluripotent adult stem cells (MASC) obtained from 5 donors (2 to 55 years old) were cultured at a seeding density of ² × 10 ³ cells / cm ² until 23 to 26 divisions, the length of telomeres was It was 11-13 kB. This was 3-5 kB longer than the telomere length of blood lymphocytes obtained from the same donor. The telomere length of the cells obtained from two donors was measured after 23 and 25 cell divisions, respectively, and re-measured after the 35th cell division, no change was observed. These MASC karyotypes were normal. (FIG. 3) B. FIG. Isolation of Pluripotent Adult Stem Cells (MASC) from Mouse Tissue Bone marrow was obtained from C57 / BL6 mice and mononuclear cells or cells from which CD45 and GlyA positive cells were removed were used under the same culture conditions as those used in human MASC (10 ng / mL human PDGF-BB and EGF). When bone marrow mononuclear cells were seeded, hematopoietic cells were removed by removing CD45 ⁺ cells 14 days after the start of culture. Similar to human MASC, the culture was replated at a seeding density of 2000 cells / cm ² every two cell divisions. In contrast to that seen in human cells, when fresh mouse bone marrow mononuclear cells from which CD45 ⁺ cells were removed on day 0 were seeded in MASC medium, the cells did not grow. When mouse bone marrow mononuclear cells were seeded and CD45 ⁺ cells were removed from cultured cells after 14 days, cells similar in morphology and phenotype to human MASCs appeared.

  When cultured with PDGF-BB and EFG alone, cell doubling was slow (greater than 6 days) and the culture could not be maintained for more than 10 cell divisions. By adding 100-10000 ng / mL (preferably 1000 IU) of LIF, cell proliferation was greatly improved and the number of cell divisions exceeded 70.

Bone marrow, brain, or liver mononuclear cells obtained from 5-day-old FVB / N mice were seeded in MASC medium with EGF, PDGF-BB, and LIF on fibronectin. After 14 days, CD45 ⁺ cells were removed and the cells were maintained under MASC culture conditions as described above. Cells that were similar in morphology and phenotype to human MASC and mouse bone marrow C57 / B16 MASC grew during culture initiated with bone marrow, brain, or hepatocytes from FVB / N mice.

  C. Pluripotent adult stem cell (MASC) phenotype Human pluripotent adult stem cells (MASC) obtained after human MASC 22-25th cell division were subjected to immunophenotypic analysis by fluorescence chromogenic cell analysis separation (FACS). As a result, the cells were CD31, CD34, CD36, CD38. , CD45, CD50, CD62E, and -P, HLA-DR, Muc18, STRO-1, cKit, Tie / Tek are not expressed, CD44, HLA-class I, and β2 microglobulin are expressed at low levels, CD10 , CD13, CD49b, CD49e, CDw90, Flk1 (N> 10).

In cultures replated at a cell density of ² × 10 ³ cells / cm ² , when the number of cell divisions exceeds 40, the phenotype becomes more uniform and cells expressing HLA-class I or CD44 are not seen ( N = 6). When cells grow to high confluence, they will express high levels of Muc18, CD44, HLA-class I and β2-microglobulin, similar to the phenotype reported for pluripotent stem cells (MSCs). (N = 8) (Pittenger, Science (1999) 284: 143-147).

By immunohistochemical analysis, human MASCs cultured at a seeding density of ² × 10 ³ cells / cm ² expressed EGF-R, TGF-R1 and -2, BMP-R1A, PDGF-R1a and -B, and MASC A portion (1-10%) of the cell population was stained with anti-SSEA4 antibody (Kannagi R, EMBO J 2: 2355-61, 1983).

The present inventors evaluated the expression gene profile of human MASC cultured over 22 to 26 cell divisions at a seeding density of ² × 10 ³ cells / cm ² using a cDNA array manufactured by Clonetech, and the following: Got profile.

  A. Pluripotent adult stem cells (MASC) are CD31, CD36, CD62E, CD62P, CD44-H, cKit, Tie; ILI, IL3, IL6, IL11, G-CSF, GM-CSF, Epo, Flt3-L, or CNTF HLA-class I, CD44-E and Muc18 mRNA are expressed at low levels.

  B. MASC is a cytokine BMP1, BMP5, VEGF, HGF, KGF, MCP1; cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130, LIF-R, activin R1 and -R2, TGFR-2, BMP -R1A; expresses mRNA of adhesion receptors CD49c, CD49d, CD29 and CD10.

  C. MASC are required with hTRT and TRF1; POU-domain transcription factor oct-4 c sox-2 (oct-4 to maintain the undifferentiated state of ES / EC. UwanoghoD, Mech Dev 49: 23-36 , 1995), sox-11 (neurogenesis), sox-9 (chondrogenesis, LefebvreV, Matrix Biol 16: 529-40, 1998); homeodomain transcription factors Hoxa4 and -a5 (designation of cervical and thoracic skeletons; Airway organ formation, PackerAI, Dev Dyn 17: 62-74, 2000), Hox-a9 (bone marrow hematopoiesis, LawrenceH, Blood 89: 1922, 1997), Dlx4 (designation of forebrain and peri-head structure, Akimenko MA, J Neurosci 14: 3475-86, 1994), MSX1 (embryonic mesoderm, adult heart and muscle, cartilage and bone formation, Forst-Potts L, DevDyn 209: 70-84, 1997), PDX1 (pancreas, Offield MF, Development 122 : 983-95, 1996).

  D. The presence of oct-4, LIF-R, and hTRT mRNA was confirmed by RT-PCR.

  E. Furthermore, by RT-PCR, Rex-1 mRNA (required with oct-4 to maintain the undifferentiated state of ES. Rosfjord E, Biochem Biophys Res Common 203: 1795-802, 1997) and Rox-1 Of mRNA (required with oct-4 for transcription of Rex-1 Ben-ShushanE, Cell Biol 18: 1866-78, 1998) was shown to be expressed in MASC.

  Oct-4 is a transcription factor expressed in embryos before gastrulation, early-stage embryos, cells in the inner cell mass of blastocysts, and fetal (EC) cancer cells (Nichols J, et alCell 95: 379-91, 1998) and down-regulated when cells are induced to differentiate. Oct-4 expression plays an important role in determining the early stages of embryogenesis and differentiation. Oct-4, together with Rox-1, activates transcription of Rex-1, a zinc finger protein that is also required to maintain ES undifferentiated (RosfjordE, Rizzino A. Biochem Biophys Res Commun 203: 1795). -802, 1997; Ben-Shushan E, etal., Mol Cell Biol 18: 1866-78, 1998). Furthermore, sox-2 expressed in ES / EC and other more differentiated cells is required along with oct-4 to maintain the undifferentiated state of ES / EC (UwanoghoD, Rex M, Cartwright). EJ, Pearl G, Healy C, Scotting PJ, Sharpe PT: Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests aninteractiverole in neuronal development. Mech Dev 49: 23-36, 1995). Although the presence of LIF is required for the maintenance of mouse ES cells and primordial germ cells, this condition is less clear for human and non-human primate ES cells.

We observe that oct-4, Rex-1, and Rox-1 are expressed in human and mouse bone marrow derived pluripotent adult stem cells (MASC) and mouse liver and brain derived MASCs did. Human MASC expresses leukemia inhibitory factor receptor (LIF-R) and is positively stained with SSEA-4. Although it is still unclear whether the growth of human MASC is affected by LIF, early experiments have shown that human MASC are enriched by sorting LIF-R ⁺ cells. In contrast, growth of mouse MASC is facilitated by LIF. Finally, oct-4, LIF-R, Rex-1 and Rox-1 mRNA levels increase after the 30th cell division in human MASC culture, resulting in cells with a more uniform phenotype. In contrast, these markers are not expressed in MASCs cultured at high density. This is related to aging before 40 cell divisions and the loss of the ability of cells to differentiate into other than chondroblasts, osteoblasts and adipocytes. Thus, the presence of oct-4 is a marker that indicates the presence of the most primitive cells in MASC culture along with Rex-1, Rox-1, sox-2, and LIF-R.

The present inventors examined mouse transformants for the eGFP gene which is the promoter of oct-4. In these animals, eGFP expression is found in primordial germ cells as well as in postnatal germ cells. We tested whether eGFP positive cells were found in the bone marrow, brain, and liver of these postnatal animals when MASC expressed oct-4. EGFP ⁺ cells (1% of the brightest cells) were selected from the bone marrow, brain, and liver of 5 days old mice. When evaluated by fluorescence microscopy, less than 1% of cells sorted from the brain and bone marrow were eGFP ⁺ . Oct-4 mRNA was detected by Q-RT-PCR in the sorted cell population. Sorted cells were seeded under conditions capable of supporting mouse MASC (wells supplemented with EGF, PDGF, LIF and coated with fibronectin). Cells survived but did not grow. When transferred to mouse embryonic fibroblasts, cell proliferation was observed. When seeded again under MASC support conditions, cells with MASC morphology and phenotype were detected.

  2. Mouse MASC As in human cells, when C57 / BL6 MASC is cultured with EGF, PDGF-BB and LIF added, it becomes CD44 and HLA-class I negative, is positively stained with SSEA-4, and oct-4 , LIF-R, ROX-1 and sox-2 transcripts were expressed. Similarly, MASCs obtained from FVB / N bone marrow, brain, and liver expressed oct3 / 4 mRNA.

Culture of pluripotent adult stem cells Isolated pluripotent adult stem cells (MASC) as described herein can be cultured using the methods of the present invention. Briefly, culture in a low serum or serum-free medium is preferred for maintaining the cells in an undifferentiated state. The serum-free medium used for culturing the cells as described herein is supplemented as shown in Table I.

MASCs expressed LIF-R, and some cells expressed oct-4, which was tested to see if culture was improved by adding LIF. When 10 ng / mL LIF was added to human MASC, there was no effect on short-term cell growth (cell doubling time up to 25 cell divisions, oct-4 expression levels were the same). Compared to that in human cells, when fresh mouse bone marrow mononuclear cells from which CD45 ⁺ cells were removed on day 0 were seeded in MASC culture, no cell proliferation was seen. When mouse bone marrow mononuclear cells were seeded and CD45 ⁺ cells were removed after 14 days from cultured cells, cells similar in form and phenotype to human MASCs appeared. This indicates that early growth of mouse MASC requires factors secreted by hematopoietic cells. When cultured with only PDGF-BB and FEG, the doubling time of the cells was slow (longer than 6 days) and the culture could not be maintained for more than 10 cell divisions. Addition of 10 ng / mL LIF significantly improved cell proliferation.

  Cells established in culture can be frozen and stored as frozen stock using DMEM supplemented with 40% FCS and 10% DMSO. Other methods for preparing frozen stocks of cultured cells are well known to those skilled in the art.

  Differentiation induction of MASC into unipotent progenitor cells and tissue-specific cell types The pluripotent adult stem cell (MASC) of the present invention uses appropriate growth factors, chemokines, and cytokines, for example, various cells derived from mesoderm. It is possible to induce differentiation so as to form many cell lines such as cells derived from neuroectodermal (glia cells, oligodendrocytes and neurons) and cells derived from endoderm.

A. Visceral mesoderm 1. Osteoblasts: Confluent pluripotent adult stem cells (MASC) from 10 ⁻⁶ to 10 ⁻⁸ M (preferably about 10 ⁻⁷ M) dexamethasone, β-glycerophosphate and 5 to 20 mM (preferably 10 mM) ) And ascorbic acid. In order to prove the presence of osteoblasts, Von Kossa staining method (CaPO4 silver reduction reaction), or antibodies to osteoshaloprotein, osteonectin and osteocalcin (immunohistochemistry / Western) were used. After 14 to 21 days of culture, more than 80% of the cells were positively stained by these antibodies (FIGS. 5 and 6).

  2. Chondroblasts: Pluripotent adult stem cells (MASC) were trypsinized and cultured as micromass suspension cultures in serum-free DMEM supplemented with 50-100 ng / mL (preferably 100 ng / mL) TGF-β1. A minute cartilage aggregate was formed at the bottom of the test tube and stained positively with toluidine blue. Initially, type I collagen was detected throughout the micromass (day 5), but after 14 days it was detected only in the outer layer of fibrous cartilage. After 5 days, type II collagen was detected, and after 14 days, the micromass was strongly stained. Staining for bone shaloprotein was negative or weakly positive in the outer layer of fibrous cartilage. Different staining intensities were obtained for osteonectin, osteocalcin, and osteopontin. The presence of type II collagen was confirmed by Western blot and RT-PCR. Furthermore, when RT-PCR was performed on the cells collected after 5 days, the presence of CART1 and CD-RAP1 as transcription factors specific to cartilage was shown (FIGS. 5 and 6).

3. Adipocytes: To induce adipocyte differentiation, about 10 ⁻⁷ to about 10 ⁻⁶ M (preferably about 10 ⁻⁷ M) dexamethasone, about 50 to about 200 μg / ml (preferably about 100 μg / ml) Medium supplemented with about 20% insulin or about 20% horse serum. Adipocyte differentiation is detected by light microscopy, oil red staining, or detection of lipoprotein lipase (LPL), adipocyte lipid binding protein (aP2), or peroxisome proliferator-activated receptor (PPAR) Is done. Methods for detecting cell markers and products are known to those skilled in the art and include detection methods using specific ligands such as troglitazone (TRO) and rosiglitazone (RSG) that bind to PPARγ.

  4). Expression gene profile of cartilage and bone The present inventors examined genes expressed upon differentiation into osteoblasts and chondroblasts. Specifically, the expression gene profile of MASC (n = 3) and MASC switched to osteoblast and chondroblast culture conditions over 2 days was examined to see a uniform relative conversion to these two specific cell lines. It was determined using a cDNA array of Clonetech and Invitrogen. Some of the detected changes are listed in Table 2. This is by no means a definitive assessment of the expressed gene profiles of MASCs, osteoblasts, and chondroblasts. However, this result shows that differentiation of MASC into bone and cartilage brings about marked and diverse changes in the expressed gene profile, and many cells in culture can induce differentiation along a predetermined pathway. It is consistent with the finding that there is.

5). Bone-expressed gene profile by subtractive hybridization: We used the subtraction method to investigate the genetic differences between undifferentiated pluripotent adult stem cells (MASCs) and unipotent progeny cells . Polyadenylated mRNA was extracted from undifferentiated MASC and cells were induced to differentiate into an osteoblast cell line for 2 days. Subtraction and amplification of cDNAs with different expression levels was performed using PCR-selection kits sold by Clonetech without modification, as recommended by the manufacturer. Sequence analysis began with genes expressed in day 2 osteoblast cultures, not undifferentiated MASCs.

  1) The present inventors determined the sequences of 86 cDNA sequences having different expression levels. The inventors have confirmed by Northern blotting that mRNA is actually specifically expressed in progeny osteoblasts on the second day, not in MASC. The resulting sequences were compared (using the BLAST algorithm) with the following database. That is, SwissProt, GenBank protein and nucleotide collections, ESTs, murine and human EST contigs.

  2) Sequences were classified by homology. 8 are transcription factors, 20 are involved in cell metabolism, 5 are involved in chromosome repair, 4 are involved in the apoptotic pathway, 8 are involved in mitochondrial function, 14 are adhesion receptor / ECM elements 19 were known EST sequences with unknown function, and 8 were novel sequences.

  3) The present inventors used pluripotent adult stem cells (MASCs) obtained from 3 individual donors, which were induced to differentiate into bone over 12h, 24h, 2d, 4d, 7d, and 14d. Q-RT-PCR was performed on two of the sequences. The two genes were expressed in the first 2 and 4 days of induction of differentiation, respectively, and then down-regulated.

  4) The present inventor further analyzed genes that are present in undifferentiated MASC but not in osteoblasts on the second day. As a result, 30 genes with different expression levels were sequenced, 5 of which were EST sequences or unknown sequences.

  B. Muscle Differentiation of pluripotent adult stem cells (MASCs) into cells with any muscular phenotype requires that the MASCs reach confluence prior to differentiation induction.

  1. Skeletal muscle: To induce skeletal muscle differentiation, pluripotent adult stem cells (MASCs) that have reached confluence are treated with about 1 to about 3 μM (preferably about 3 μM) of 5-azacytidine in MASC growth medium for 24 hours. To process. The culture is then maintained in MASC medium. Differentiation is assessed by Western blot and immunohistochemistry. Differentiation into skeletal muscle in vitro can be performed by standard methods well known to those skilled in the art and by Western blotting or immunohistochemistry using commercially available antibodies, Myf-5, Myo-D, Myf-6, It can be demonstrated by detecting a series of activations of myogenin, desmin, skeletal muscle actin and skeletal muscle myosin. Five days after induction with 5-azacytidine, Myf5, Myo-D, and Myf6 transcription factors are detected in about 50% of the cells. After 14 to 18 days, the expression level of Myo-D decreased significantly, while the expression levels of Myf5 and Myf6 remained high. Desmin and skeletal muscle actin were detected early on day 4 after induction, and skeletal muscle myosin was detected on day 14. By immunohistochemistry, it was confirmed on day 14 that 70-80% of the cells expressed mature muscle protein. Gata4 and Gata6 were expressed in the first week of culture by treatment with 5-cytidine. Furthermore, low levels of troponin-T were detected after 2 days. Two days after induction, smooth muscle actin was detected and remained high for 14 days. When 20% horse serum was added, fusion of myogenic cells to multinucleated myotubes was observed (FIG. 7). The inventors have shown that it is possible to fuse lineage-converted myogenic cells with myocytes of different differentiation pathways using a double fluorescent label (FIG. 8).

  2. Smooth muscle: A pluripotent adult stem cell (in a serum-free medium supplemented with a high concentration (about 50 to about 200 ng / ml, preferably about 100 ng / ml) of platelet-derived growth factor (PDGF) without adding a growth factor. It is further possible to induce smooth muscle cells by culturing MASC). The cells preferably have reached confluence at the start of differentiation. Terminally differentiated smooth muscle cells can be identified by detecting the expression of desmin, smooth muscle specific actin, and smooth muscle specific myosin by standard methods well known to those skilled in the art. Smooth muscle actin was detected after 2 days and smooth muscle myosin was detected after 14 days. About 70% of the cells were positively stained with anti-smooth muscle actin antibody and anti-smooth muscle myosin antibody. The presence of myogenin was confirmed after 4 days, and desmin was confirmed after 6 days. Two to four days later, more Myf5 and Myf6 proteins were detected and remained high until day 15. Myo-D was not detected (FIG. 7).

3. Myocardium: Myocardial differentiation is performed as described above using about 5 to about 200 ng / ml (preferably about 100 ng / ml) of basic fibroblast growth factor (bFGF) in a standard serum-free medium without the addition of growth factors. It is possible by adding. After MASC reaching confluence was exposed to μM (preferably about 3 μM) of 5-azacytidine and then 10 ⁻⁵ to 10 ⁻⁷ M (preferably 10 ⁻⁶ M) retinoic acid, then in MASC growth medium Cultured. Alternatively, after culturing MASC with either one of these inducers or a combination of both, 50-200 ng / ml (preferably 100 ng / ml) FGF2, or 5-20 ng / ml (preferably 10 ng / ml) May be cultured in a serum-free medium supplemented with a combination of BMP-4 and 100 ng / ml FGF2. The inventors confirmed the expression of a protein consistent with cardiomyocytes. Gata4 and Gata6 were expressed as early as day 2 and remained high until day 15. Myocardial troponin T was expressed after day 4, and cardiac troponin I was expressed after day 6. ANP was detected after the 11th day. These myocardial proteins were detected in more than 70% of the cells on day 15 by immunohistochemistry. The transcription factor Myf6 was confirmed after the second day. Desmin expression began on day 6 and myogenin expression on day 2. Skeletal muscle actin was also confirmed. When the culture was maintained for more than 3 weeks, the cells formed syncytium. The inventors further confirmed that natural contraction rarely occurred in the culture and propagated a distance of several mm (FIG. 7).

  C. Endothelial cells Pluripotent adult stem cells (MASC) expressed Flk1, but did not express CD34, PECAM, E- and P-selectin, CD36, Tie / Tek and Flt1. When MASC was cultured in serum-free MASC medium supplemented with 20 ng / ml VEGF, it was confirmed that CD34 was expressed on the cell surface by day 14 and the cells expressed vWF (immunofluorescence method) (FIGS. 9, 10). ). In addition, the cells expressed Tie, Tek, Flk1 and Flt1, PECAM, P-selectin and E-selectin, and CD36. The results of histochemical staining were confirmed by Western blot. Angiogenesis was observed when cells induced by VEGF were cultured on Matrigel or type IV collagen (FIGS. 9, 10).

D. It hematopoietic cells multipotent adult stem cells (MASC) differentiate into CD34 ⁺ endothelial cells are also CD34 recent studies ^- that the FLK ⁺ cells can be induced to differentiate into endothelial cells and hematopoietic cells shows Therefore, it was examined whether MASC could be induced to differentiate into hematopoietic progenitor cells. In MASC medium containing PDGF-BB and EGF prepared by AFT024 feeder, a mesenchymal cell line derived from fetal liver capable of supporting mouse and human regenerative stem cells ex vivo and supplemented with 5% FCS and 100 ng / ml SCF. MASCs were replated on type IV collagen. Cells recovered from these cultures expressed cKit, cMyb, Gata2, and G-CSF-R, but not CD34 (RT-PCR). Since hematopoiesis is induced by factors released by embryonic visceral endoderm, we co-cultured βGal ⁺ mouse EB and human MASC in the presence of human SCF, Flt3-L, Tpo, and Epo did. In two separate experiments, a small population of βGal ⁻ cells expressing human CD45 were detected.

E. Stromal cells: The inventors induced "stromal cell" differentiation by culturing pluripotent adult stem cells (MASC) with IL-1α, FCS, and horse serum. In order to prove that these cells have hematopoietic support ability, the feeder was irradiated with 2,000 cGy, and CD34 ⁺ umbilical cord blood cells were seeded in contact with the feeder. After 1, 2, and 5 weeks, progeny cells were replated in the methylcellulose assay to determine the number of colony forming cells (CFC). The CFC increased 3 to 5 fold after 2 weeks and was maintained after 5 weeks. This was similar to the case where CD34 ⁺ cells were cultured in contact with AFT024, a feeder cell derived from mouse fetal liver.

  F. Neuronal cells Surprisingly, pluripotent adult stem cells (MASC) induced by VEGF, hematopoietic cytokines SCF, Flt-L, Tpo and Epo in EGF-containing MASC medium prepared by AFT024, a hematopoietic support feeder. ) Differentiated into glial fibrillary acidic protein (GFAP) positive astrocytes, galactocerebroside (GalC) positive oligodendrocytes, and neurofilament positive neurons (FIG. 11). Therefore, the inventors hypothesized that the production of FGF2 by the AFT024 feeder and the addition of EGF to the culture induced differentiation into neural cells in vitro.

  Thus, the inventors investigated the influence of FGF2, which is known to play an important role in neurogenesis and ex vivo culture of neural progenitor cells, on MASC-derived neurogenesis. The culture of human bone marrow derived MASC (n = 7) cultured with EGF and PDGF-BB added is switched to a medium containing 50 to 500 ng / mL (preferably 100 ng / mL) of FGF2 in a culture with less than 50% confluence. 2 to 4 weeks later, differentiation into astrocytes, oligodendrocytes, and cells having a neuronal phenotype was confirmed (FIG. 11). After 2 weeks of culture, 26 ± 4% of the cells were GFAP positive, 28 ± 3% were GalC positive, and 46 ± 5% were neurofilament-200 positive. When examined again after 3 weeks, the number of GFAP or GalC positive cells decreased, but 20 ± 2% of cells stained positive for β-tubulin III and 22 ± 3% positive for neurofilament-68. 50 ± 3% stained positive for neurofilament-160, 20 ± 2% positively stained for neurofilament-200, 82 ± 5% positively stained for neuron specific enolase (NSE), 80 ± 2 % Positively stained for microtubule associated protein 2 (MAP2) (FIG. 12). GABA, parvalbumin, tyrosine hydroxylase, DOPA-decarboxylase, and tryptophan hydroxylase were not detected. The number of axons per neuron increased from 3 ± 1 to 5 ± 1 and 7 ± 2 after 2, 3-4 weeks after differentiation. Differentiation into astrocytes, oligodendrocytes, and cells with neuronal characteristics is confirmed by Western blot and RT-PCR analysis in that GFAP, myelin basic protein (MBP) and neurofilament-200 are present in FGF2-treated cells. However, it was confirmed by proving that it does not exist in MASC.

  FGF-9 is first isolated from the glioblastoma cell line and induces glial cell division in culture. FGF-9 is found in vivo in neurons in the cerebral cortex, hippocampus, substantia nigra, brainstem motor nucleus, and Purkinje cell layer. When MASC was added with 5-50 ng / mL (preferably 10 ng / mL) of FGF-9 and EGF and cultured for 3 weeks, astrocytes, oligodendrocytes, GABAergic neurons, and dopaminergic neurons were generated. . During the development of the central nervous system, FGF-8 expressed by the forebrain at the mid / hindbrain boundary induces differentiation of midbrain and forebrain dopaminergic neurons along with sonic hedgehog. It has been found that adding 5-50 ng / mL (preferably 10 ng / mL) of FGF-8 and EGF to MASC and culturing for 3 weeks produces both GABAergic and dopaminergic neurons. FGF-10 is found in trace amounts in the brain, and its expression is limited to the hippocampus, thalamus, midbrain, and brainstem, and is selectively expressed in neurons and not in glial cells. Incubation of MASC in 5-50 ng / mL (preferably 10 ng / mL) FGF-10 and EGF for 3 weeks resulted in astrocytes and oligodendrocytes, but no neurons. FGF-4 is expressed in the notochord and is required for midbrain regionalization. Treatment of MASC with 5-50 ng / mL (preferably 10 ng / mL) FGF-4 and EGF for 3 weeks differentiated into astrocytes and oligodendrocytes, but did not differentiate into neurons.

  Other growth factors that are specifically expressed in the brain and affect neurogenesis in vivo and in vitro include brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and ciliary body There are neurotrophic factors (CNTF). BDNF belongs to the nerve growth factor family that promotes in vitro differentiation of NSCs, human epithelial cells, and neural progenitor cells into neurons and promotes in vivo axon generation of hippocampal neural stem cells. Consistent with the known function of BDNF to support survival of nigral dopaminergic neurons, treatment of MASC with 5-50 ng / mL (preferably 10 ng / mL) BDNF and EGF results in tyrosine Differentiation into only hydroxylase positive neurons was observed. GDNF belongs to the TGF superfamily. GDNF is expressed in the preneural ectoderm early in neurogenesis, indicating an important role of GDNF in neurogenesis. GDNF promotes the survival of peripheral neurons and muscle motor neurons, and has neurotrophic factor activity and ability to promote differentiation. It has been found that 5-50 ng / mL (preferably 10 ng / mL) GDNF induces differentiation of MASCs into GABAergic and dopaminergic neurons. CNTF was first isolated from the ciliary ganglion and belongs to the gp130 family of cytokines. CNTF promotes nerve survival early in development. CNTF increases the number of GABAergic and cholinergic neurons in the culture of rat fetal hippocampal neurons. Furthermore, CNTF prevents GABAergic neuron cell death and promotes GABA uptake. CNTF of 5-50 ng / mL (preferably 10 ng / mL) showed a similar GABAergic induction effect on MASC, which differentiated into GABAergic neurons only after 3 weeks of exposure to CNTF.

The fate of MASCs transplanted into rat brains was also investigated. 50,000 eGFP ⁺ MASCs were stereotaxically implanted around the parietal infarct induced in Wistar rats that were continuously administered cyclosporine. The stereotaxic function of the limbs was tested 6 weeks after transplantation with saline, MASC conditioned medium, or MASC. Only in the rat transplanted with MASC, a functional improvement comparable to the level of the animal subjected to sham transplantation was seen (FIG. 15).

The animals were killed after 2 and 6 weeks to determine the neuronal phenotype. Since the brain after transplantation of eGFP ⁺ cells emits autofluorescence, immunohistochemical analysis of the graft was performed. At 2 weeks, the majority of eGFP ⁺ cells were detected in the transplant area itself (FIG. 16). After 5 weeks, eGFP ⁺ cells migrated outside the graft. At the second week, cells stained with anti-eGFP antibody maintained a spherical shape, and their diameters ranged from 10 to 30 μm. After 6 weeks, the cells stained with the anti-eGFP antibody were greatly reduced, and dendrites extending to normal brain tissue were seen in the transplanted area. The presence of human cells was confirmed by staining with NuMa, a human specific nuclear antibody. If this antibody is used, double and triple staining with an immunofluorescent antibody is possible, and human cells in the graft can be identified.

The present inventors detected a small population of nestin positive cells in the same place as NuMa positive cells and GFP ⁺ cells using human-specific anti-nestin antibodies. This indicates neuronal ectoderm differentiation. Furthermore, the inventors confirmed positive staining for tubulin III, neurofilaments -68 and -160, oligo markers, and GFAP. This indicates differentiation into neurons and glial cells (not shown in the figure).

  G. Epithelial cells The inventors used confluent pluripotent adult stem cells (MASC) (n = 4) in combination with 10 ng / mL hepatocyte growth factor (HGF) alone or in combination with keratinocyte growth factor (KGF). Processed. After 14 days, large epithelioid cells expressing the HGF receptor, cytokeratins 8, 18, and 19 were confirmed. The presence of cytokeratin 19 indicates the possibility of differentiation into biliary epithelial cells. By changing the substrate from fibronectin to collagen gel or matrigel, the incidence of cytokeratin 18-expressing cells having the shape of epithelial cells was improved (FIG. 17).

Single Cell Origin of Differentiated Multiple Cell Lines In order to confirm whether pluripotent adult stem cells (MASCs) are clones with each other, the inventors performed selection using FACS1, eGFP ⁺ transduced with MFG-eGFP Ten cells were seeded per well and cultured to 10 ⁸ cells. Transduction was performed as follows. MASCs replated 24 hours ago were exposed to MFG-eGFP or MSCV-eGFP and 10 μg / mL protamine packaged in PG13 cell line for 6 consecutive hours over 6 hours. Transduced 40-70% of MASC. The expression of eGFP was maintained ex vivo for at least 3 months and maintained in many cells after differentiation. When one cell was sorted, no growth was seen in more than 1,000 wells, but when seeded with 10 cells per well, cell growth was seen in 3% of the wells. . Only 0.3% of the wells showed further growth to more than 10 ⁷ cells. These cells were then induced to differentiate into all types of mesoderm-derived cells (osteoblasts, chondroblasts, adipocytes, skeletal and smooth muscle cells, and endothelium). Again, differentiation was confirmed by immunohistochemistry and Western blotting. The cells were further subjected to inverse PCR to prove that the sequences of human DNA sandwiching the inserted viral DNA were similar. The inventors have confirmed that the retroviral gene has been inserted into the same site in the human genome of three separate clones of MASC and differentiated offspring.

MASC engraftment The inventors conducted a study to investigate whether pluripotent adult stem cells (MASCs) engraft and are maintained in vivo.

1. The inventors injected eGFP ⁺ MASC intramuscularly into NOD-SCID mice. Four weeks later, the animals were killed and the muscles were examined to determine if teratomas had developed as described above for human ES cells. No teratoma occurred in 5 out of 5 animals. eGFP positive cells were detected.

2. The inventors injected eGFP ⁺ MASCs into SCID mouse embryos intrauterine IV. The animals were examined immediately after birth. PCR analysis confirmed the presence of eGFP ⁺ cells in the heart, lung, liver, spleen, and bone marrow.

  3. The inventors stereotactically transplanted MASCs into the brains of rats that were intact or developed infarctions. MASCs acquired a neuronal phenotype and were maintained for at least 6 weeks.

Application example of MASC Osteoblasts The pluripotent adult stem cells (MASCs) of the present invention induced to differentiate into bone cells are used as cell therapy or tissue regeneration in osteoporosis, Paget's disease, fractures, osteomyelitis, osteonecrosis, chondrogenic dysfunction, Osteogenesis imperfecta, hereditary multiple exostoma, multiple epiphyseal dysplasia, Marfan syndrome, mucopolysaccharidosis, neurofibromatosis, or scoliosis, focal malformation, spina bifida, hemivertebra, It can be used for reconstruction of fused vertebrae, limb malformations, reconstruction of tissue damaged by tumors, and reconstruction after infections such as otitis media.

  2. Chondrocytes Chondrocytes formed by inducing differentiation of the pluripotent adult stem cells (MASCs) of the present invention are cell therapy, age-related diseases and injuries, sports-related injuries, or rheumatoid arthritis, arthritic psoriasis, Can be used for tissue regeneration, outer ear reconstruction, nasal reconstruction, and cricoid cartilage reconstruction in certain diseases such as Reiter arthritis, ulcerative colitis, Crohn's disease, ankylosing spondylitis, osteoarthritis .

  3. Adipocytes Adipocytes obtained from pluripotent adult stem cells (MASC) can be used for remodeling in reconstruction and cosmetic surgery and for the treatment of type II diabetes. In reconstructive surgery, adipocytes differentiated by the method of the present invention are used for reconstruction of defective tissue by, for example, breast reconstruction after mastectomy or other surgical operations such as removal of tumor from the face or hands. Is possible. In cosmetic surgery, the fat cells obtained from the cells of the present invention by the method of the present invention can be used in various methods such as breast enlargement and removal of aging skin. Furthermore, the adipocytes obtained in this way provide an in vitro model system that is effective for the study of fat restriction.

  4). Fibroblasts Fibroblasts derived from MASC can be used for cell therapy and tissue repair to promote healing of trauma and provide a supporting element for connective tissue such as a foundation for cosmetic surgery. .

  5). Skeletal muscle The skeletal muscle cells obtained by inducing differentiation of MASC are cell therapy and tissue repair in the treatment of Dushanne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, skeletal muscle myopathy, and reconstruction to repair skeletal muscle damage It can be used for surgery.

  6). Smooth muscle cells obtained by inducing differentiation of MASC are used for cell therapy, tissue repair in the treatment of abnormal development of the digestive system such as esophageal obstruction, intestinal obstruction, intussusception, intestinal infarction surgery and colon colon It can be used for tissue replacement after anastomosis.

The smooth muscle cells formed from the MASCs of the present invention can further be used for the reconstruction of blood vessels damaged by bladder or uterine reconstruction, neovascularization, such as atherosclerosis or aneurysms. Smooth muscle progenitor cells (mesangial cells) can be used as an in vitro model of glomerular disease and cell therapy, or for tissue regeneration in diabetic neuropathy. Smooth muscle progenitor cells can also be used to repair distal convoluted tubules and paraglomerular tissue dense plaques that are important in blood pressure regulation.

  7. Cardiomyocytes Cardiomyocytes derived from pluripotent adult stem cells (MASC) treat valve replacement, congenital heart failure or congestive heart failure due to cardiomyopathy or endocarditis, or heart tissue damaged by myocardial infarction Useful for cell therapy and tissue repair. Cells can be highly effective, particularly by local administration by injection. Microglial cells differentiated from MASC are useful for the treatment of spinal cord injury and neurodegenerative diseases such as Huntington's disease, Parkinson's disease, multiple sclerosis and Alzheimer's disease, and the repair of tissues damaged by infections affecting the central nervous system. It is possible to use. Microglia genetically modified to produce cytokines can also be used for transplantation to treat central nervous system infections that are difficult to access due to the blood-brain barrier. Glial cells produce growth factors or growth factor inhibitors to regenerate neural tissue after attacks due to multiple sclerosis, amyotrophic lateral sclerosis and brain tumors, and to regenerate neural tissue after spinal cord injury It can also be used for this purpose.

  8). Stromal cells Stromal cells derived from the pluripotent adult stem cells (MASC) of the present invention can be used for bone marrow replacement after chemotherapy and as transplanted cells for bone marrow transplantation. For breast cancer, for example, bone marrow aspirate is taken from the patient before a powerful chemotherapy regimen is performed. Such chemotherapy damages tissues, especially the bone marrow. MASCs isolated from the patient's bone marrow can be cultured and expanded to obtain autologous cells sufficient for re-establishment of bone marrow cells. Because these cells can differentiate into different tissues, locally or systemically introduced cells migrate to other damaged tissues and the differentiation of these cells is induced by cellular factors present in the tissue environment The advantage of being proliferated is obtained.

  9. Endothelial cells Multipotent adult stem cells (MASC) are differentiated into endothelial cells by the method described above, and the endothelial cells are used for the treatment of factor VIII deficiency and the production of angiogenic factors for neovascularization. It is possible. This endothelial cell further provides an in vitro model for tumor suppression using an angiogenesis inhibitor and an in vitro model of vasculitis, hypersensitivity, and coagulation disease. By using such cultured endothelial cells and high-speed screening methods well known to those skilled in the art, it is possible to screen thousands of useful compounds having a therapeutic effect more quickly and cost-effectively.

  10. Hematopoietic cells Pluripotent adult stem cells (MASCs) can differentiate into hematopoietic cells. It is therefore possible to regenerate the bone marrow after high dose chemotherapy using the cells of the present invention. Collect bone marrow aspirate from the patient before chemotherapy. Stem cells are isolated, cultured and induced for differentiation by the method of the present invention. This is followed by reintroduction of the mixture of differentiated and undifferentiated cells into the patient's bone marrow cavity. Currently, clinical trials using hematopoietic stem cells are underway for this purpose, but the stem cells of the present invention can be further differentiated into cells that can replace not only bone marrow but also cells damaged by chemotherapy of other tissues. It has the further advantage of being. By further differentiating hematopoietic cells derived from MASC of the present invention into blood cells and storing them in a blood bank, it is possible to solve the problem of insufficient blood for transfusion.

  11. Neuroectodermal cells Microglia differentiated from MASC were damaged by spinal cord injury and treatment of neurodegenerative diseases such as Huntington's disease, Parkinson's disease, multiple sclerosis and Alzheimer's disease, and infections affecting the central nervous system It can be used for tissue repair. Microglia genetically modified to produce cytokines can also be used for transplantation to treat central nervous system infections that are difficult to access due to the blood-brain barrier. Glial cells produce growth factors or growth factor inhibitors to regenerate neural tissue after attacks due to multiple sclerosis, amyotrophic lateral sclerosis and brain tumors, and to regenerate neural tissue after spinal cord injury It can also be used for this purpose. MASCs differentiated into oligodendrocytes and astrocytes can be transplanted, for example, into demyelinating tissue, particularly the spinal cord, where MASCs form myelin sheaths in the surrounding neural tissue. This method has proved effective in mice when embryonic stem cells are used as a source of oligodendrocytes and astrocyte progenitors (Brustle, O., et al., Science (1999)). 285: 754-756). The MASCs of the present invention have the additional advantage of not only having the broad differentiation characteristics of embryonic stem cells, but also providing autologous cells for transplantation.

  The cells of the present invention are used for cell replacement therapy and / or gene therapy, for example, mucopolysaccharidosis, leukodystrophy (globoid cell leukodystrophy, Canavan disease), fucosidosis, GM2 gangliosidosis, Niemann-Pick disease, San Filip syndrome It is possible to treat congenital neurodegenerative diseases and storage diseases such as Wolman disease and Tay-Sachs disease. Also, the MASC of the present invention can be used for traumatic diseases such as stroke, central nervous system hemorrhage, central nervous system trauma, peripheral nervous system diseases such as spinal cord injury and syringomyelia, retinal detachment, macular degeneration and other degenerative retinal diseases and diabetes. It can be used for the treatment of retinal diseases such as retinal diseases.

  12 Ectodermal epithelial cells Furthermore, the epithelial cells of the present invention are used for cell replacement therapy and / or gene therapy to treat or alleviate the symptoms of skin diseases such as alopecia, burn wounds and albinism It is possible.

  13. Endodermal epithelial cells Epithelial cells derived from MASCs of the present invention can be used for cell replacement therapy and / or gene therapy to treat multiple organ diseases or alleviate their symptoms. Using this cell, for example, accumulative diseases such as mucopolysaccharidosis, leukodystrophy, GM2 gangliosidosis, hyperbilirubin diseases such as Krigler-Najjar syndrome, eg ornithine decarboxylase deficiency, citrullinemia, and argininosuccinic acid Ammonia diseases such as congenital abnormalities of the urea cycle such as infectious diseases, amino acid and organic acid abnormalities such as phenylketonuria, congenital hypertyrosinemia, and α1-antitrypsin deficiency, and coagulation such as factor VIII and factor IX deficiency It is possible to treat or alleviate congenital liver diseases such as diseases. The cells can also be used to treat acquired liver disease due to viral infection. The cells of the present invention can also be used for ex vivo applications such as the production of artificial liver (similar to kidney dialysis), the generation of coagulation factors, and the production of proteins and enzymes produced by liver epithelial cells. It is.

  Further, the epithelial cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate symptoms of biliary diseases such as biliary cirrhosis and biliary atresia.

  Furthermore, the epithelial cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate symptoms of pancreatic diseases such as pancreatic occlusion, pancreatitis, and α1-antitrypsin deficiency. is there. Furthermore, pancreatic epithelial cells can be obtained from the cells of the present invention, and nerve cells can be obtained, whereby β cells can be generated. These cells can also be used for the treatment of diabetes (subcutaneous transplantation, intrapancreas or intrahepatic transplantation). Furthermore, the epithelial cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate symptoms of intestinal epithelial tissues such as intestinal obstruction, inflammatory bowel disease, intestinal infarction, and intestinal resection. Is possible.

  14 Modification of MASCs to confer senility under non-optimal culture conditions Pluripotent adult stem cells (MASCs) have long telomeres (12 kb) that do not differ in cells obtained from donors of different ages . When ex vivo culture of MASCs, telomeres are not shortened in ex vivo culture for longer than 4 months (longer than 35 cell doublings). This can be longer. Telomerase is present in MASCs obtained from people of all ages. When MASC is cultured in a confluence state, aging begins and telomeres begin to shorten. MASC is transduced / transfected with a telomerase-containing construct so that cell senescence is achieved because it is preferable to perform long-term growth in relatively dense cultures for production, commercial reasons, etc. It is possible to prevent. Although these cells can be used for in vivo transplantation, it is preferred to remove telomerase from the cells prior to transplantation. This can be done by constructing the construct such that telomerase is located between the two LoxP sites. This makes it possible to excise telomerase with Cre recombinase. Cre can be transduced / transfected into target cells using a second vector / plasmid or as part of a telomerase-containing construct. Cre is a constitutively active form or binding of one or more mutant ligands of human estrogen receptor (ER) that is not induced by, for example, natural estrogen receptor (ER) ligand but can be induced by 4-hydroxy tamoxifen (OHT) It can be introduced as an inducible enzyme by fusing the domain to the protein or by using a drug induction system such as tetracycline or rapamachine induction.

  15. Transplantation approach to prevent immune rejection a. Universal donor cells Pluripotent adult stem cells (MASCs) can be genetically engineered into cells and universal donor cells for gene therapy to treat diseases such as genetic diseases and replace enzymes. Undifferentiated MASCs do not express HLA-I type, HLA-II type antigen or β2 microglobulin, but some of the differentiated progeny express at least type I HLA antigen. MASC can be transformed into a universal donor cell by depleting HLA-I and HLA-II antigens and optionally introducing HLA antigens from the recipient patient, thereby facilitating the cells Therefore, it is possible to prevent NK cell-mediated cytotoxicity and to prevent unlimited virus growth in cells and malignant transformation of cells. HLA antigens are deleted by introducing stop codons as in the case of chimeric cells, by homologous recombination, or by introducing point mutations into the promoter region, or by introducing point mutations into the first exon of the antigen. It is possible. Host HLA antigens can be transferred by introducing HLA antigen cDNA into target cells by transduction or transfection with viruses such as retroviruses, lentiviruses, adeno-associated viruses and the like. Using MASC, it is possible to adjust a specific protein to a predetermined amount or concentration in the body or blood.

  b. Intrauterine transplantation to avoid recognition by the immune system Multipotent adult stem cells (MASCs) are used for engraftment in the uterus to correct genetic abnormalities or to develop the host immune system before the development of the host immune system It is possible to introduce cells so that they are not recognized by the cell. This provides a method for mass production of human cells such as blood in the body of an animal, and a method for correcting genetic defects in human fetuses by transplanting cells that produce normal proteins or enzymes It is also possible to use as.

16. Gene therapy Until now, human cells used for gene therapy have been basically limited to bone marrow and skin cells. This is because other cell types are difficult to extract from the body, grown in culture, genetically modified, and successfully reimplanted to the patient from whom the tissue originated (Anderson, WF, Nature ( 1998) 392: 30; Anderson, WF, Scientific American (1995) 273: 1-5; Anderson, WF, Science (1992) 256: 808-813). The pluripotent adult stem cells (MASCs) of the present invention are extracted and isolated from the body, proliferated in culture in an undifferentiated state or induced to differentiate, and can be transformed using various methods, particularly viral transduction. It is possible to modify. The uptake and expression of genetic material can be demonstrated and the expression of foreign DNA is stable throughout the developmental process. Vectors such as retroviruses for inserting foreign DNA into stem cells are well known to those skilled in the art (Mochizuki, H., et al., J. Virol (1998) 72 (11): 8873-8883; Robbins , P., et al., J. Virol. (1997) 71 (12): 9466-9474; Bierhuizen, M., Et al., Blood (1997) 90 (9): 3304-3315; Douglas, J. , etal., Hum. Gene Ther. (1999) 10 (6): 935-945; Zhang, G., et al., Biochem. Biophys. Res. Commun. (1996) 227 (3): 707-711) . When transduced with retroviruses, green fluorescent protein (eGFP) expression persists in terminally differentiated muscle cells, endothelial cells, and c-Kit positive cells derived from isolated MASCs. This indicates that the expression of the retroviral vector introduced into MASC persists throughout differentiation. Final differentiation was induced from cultures that had been pre-transduced with retroviral vectors and started with 10 eGFP ⁺ cells that were selected several weeks during the initial MASC culture period.

  Although hematopoietic stem cells have limited differentiation potential, they have been shown to be useful in gene therapy (see Kohn, DB, Curr. Opin. Pediatr. (1995) 7: 56-63). . The cells of the present invention were proved by the sustained expression of green fluorescent protein in terminally differentiated myocytes, endothelial cells and c-Kit positive cells despite the introduction of retroviral vectors into undifferentiated stem cells. Thus, it provides a wide variety of differentiated cells capable of maintaining transduced or transfected DNA when terminally differentiated.

  The MASC of the present invention also has other advantages compared to hematopoietic stem cells for gene therapy. The stem cells of the present invention can be isolated from bone marrow aspirate obtained under local anesthesia, grown in culture and transfected with foreign genes relatively easily. A reasonable number of hematopoietic stem cells used for similar purposes must be isolated from at least 1 L of bone marrow, and it is difficult to grow the cells in culture (see, Prockop, DJ, Science (1997). 276: 71-74).

  Examples of candidate genes for gene therapy include apolipoprotein E (which shows a correlation with the risk of Alzheimer's disease and cardiovascular disease), MTHFR (a high homocysteine level and a correlation between stroke and mutant). ), Factor V (correlated with the risk of blood clot), ACE (correlation between risk of heart disease and variant), CKR-5 (correlation with resistance to HIV) HPRT (hypoxanthine-guanine phosphoribosyltransferase, developing Leish-Nyhan disease due to deficiency), PNP (purine nucleoside phosphorylase, deficiency leading to severe immunodeficiency disease), ADA (adenosine deaminase, deficient causing severe Leading to complex immunodeficiency diseases), p21 (candidate gene for treatment of telangiectasia in capillary dilatation P47 (defect has been correlated with a lack of neutrophil oxidase activity in patients with chronic granulomatous disease), Genbank accession numbers M55067 and M38755) Rb (for tumorigenesis) Related retinoblastoma susceptibility gene, Genbank accession number M15400), KVLQT1 (potassium channel protein, correlation between abnormal and cardiac arrhythmia. Genbank accession number U40990), dystrophin gene (related to Duchenne muscular dystrophy) Genbank accession numbers M18533, M17154 and M18026), CFTR (transmembrane transmembrane conductance regulator associated with cystic fibrosis, Genbank accession number M28668), phosphatidylinositol 3-kinase (related to capillary dilatation ataxia, Accession number U26455), and genes encoding VHL (deletions or mutations of this protein have been shown to be associated with von Hippel-Lindau disease) (Latif, F., et al., Science (1993) ) 260: 1317-1320). Other diseases that can be effectively treated by the use of these genetically modified cells include Factor IV deficiency, adenosine deaminase deficiency (related to severe combined immunodeficiency disease or SCID), diabetes, and glucocerebrosidase , Α-yluronidase blood luck.

  These novel genes can be regulated by inducible promoters so that enzyme levels can be adjusted. These inducible promoter systems may have a human estrogen receptor (ER) mutant ligand binding domain attached to the protein to be produced. This requires patients to take tamoxifen to express the protein. Alternatively, a tetracycline-on / off system, RU486 or a rapamycin induction system can be used. A further way to perform relative selective expression is to use a tissue specific promoter. For example, in the brain, it is possible to introduce a transgene whose expression is regulated by a neuron-specific enolase promoter (Ad-NSE) or a glial fibrillary acidic protein (GFAP) promoter. Expression is realized. Similarly, it is also possible to obtain expression only in endothelial cells by using the Tec promoter or VE-cadherin promoter.

  Genetically modified MASCs can be introduced locally or injected systemically. These human stem cells with more limited differentiation potential secrete protein for at least 8 weeks after systemic injection into SCID mice when transfected with the gene for factor IX (Keating, A., et al., Blood (1996) 88: 3921). The MASC of the present invention has a broader differentiation ability than any non-embryonic stem cells reported so far, and migrates to different tissues, where cell differentiation is induced by factors such as cytokines and growth factors. , Providing further advantages in systemic or local administration. Differentiated cells become part of the surrounding tissue, but retain the ability to produce the protein product of the induced gene.

For example, in Parkinson's disease, clinical studies have shown that mesencephalic dopamine neurons obtained from human fetal carcasses can survive and function in the brain of Parkinson's disease patients. PET scans show that [ ¹⁸ F] fluorodopa uptake in the area surrounding the cell graft increases after transplantation and remains in that state for some patients for at least 6 weeks (see Dunnett). , S and A. Bjorklund, Nature (1999) 399 (Suppl.) A32-A39; Lindvall. O., Nature Biotech. (1999) 17: 635-636; Wagner, J., et al., NatureBiotech. (1999 ) 17: 653-659). Unlike embryonic cells, the isolated MASCs described by the present invention allow rapid delivery of cells for transplantation, while differentiation making embryonic cell transplantation a promising alternative for disease treatment. Maintaining the performance.

  In the treatment of AIDS, the MASC of the present invention can be genetically engineered to produce Rev10, a trans-dominant negative mutant of Rev that inhibits the function of wild-type Rev produced in HIV-infected cells. It is possible (Bevec, D. et al., Proc. Natl. Acad. Sci. USA (1992) 89: 9870-9874; Ranga, U., et al., Proc. Natl. Acad. Sci. USA (1998 ) 95 (3): 1201-1206). When MASC is induced to differentiate into hematopoietic lineage cells and is introduced into the patient's body, MASC restores the supply of T cells in deficient HIV-infected patients. Because these genetically modified cells have the mutant RevM10, they acquire resistance to the lethal effects of infection by many HIV strains.

  Genetically modified MASCs can be encapsulated in an inactive carrier to produce secreted proteins while protecting cells from the host immune system. Techniques for microencapsulation of cells are well known to those skilled in the art (see Chang, P., et al., Trendsin Biotech. (1999) 17 (2): 78-83). Examples of materials for cell microencapsulation include polymer capsules, alginate-poly-L-lysine-alginate microcapsules, poly-L-lysine-barium alginate capsules, barium alginate capsules, polyacrylonitrile / polyvinyl chloride (PAN / PVC). ) Hollow fibers and polyethersulfone (PES) hollow fibers. For example, US Pat. No. 5,639,275 (Biege, E., etc.) (Baetge, E.) describes long-term and stable biologically active molecules using biocompatible capsules in which genetically engineered cells are encapsulated. An apparatus and method for efficient expression is disclosed. Such biocompatible immunoisolating capsules, together with the MASCs of the present invention, provide a method for treating many physiological diseases such as diabetes and Parkinson's disease.

  For example, in diabetic patients, heterologous stem cells that have been genetically modified to produce insulin at a physiologically therapeutic level can be encapsulated for delivery into the patient's tissue. Alternatively, autologous stem cells obtained from the patient's own bone marrow aspirate can be transduced with retroviruses as described above. These cells are genetically modified to produce physiologically therapeutic levels of insulin and then encapsulated and introduced into the patient's tissue as described by Chang and Baetge. This allows the cells to stay in the tissue and produce insulin over time.

  Another advantage of the microencapsulation of cells of the present invention is that various cells producing molecules that exert a biological therapeutic effect can be encapsulated in the microcapsules. The MASCs of the present invention can be induced to differentiate into multiple different cell lines, each of which can be genetically modified to produce a therapeutically effective level of biologically active molecule. MASCs with different genetic elements can be encapsulated together to produce different biologically active molecules.

  The MASC of the present invention can overcome the most difficult barrier in gene therapy by genetic modification ex vivo. For example, a patient's bone marrow aspirate is collected and MASC is isolated from this aspirate. The MASC is genetically modified to express one or more desired gene products. This MASC can be screened or selected ex vivo to identify successfully modified cells and these cells can be reintroduced locally or systemically into the patient. Alternatively, it is also possible to genetically modify MASC and induce differentiation by culturing to obtain a specific cell line for transplantation. In either case, a source of stably transfected cells that can express the desired gene product by the transplanted MASC is provided. The method provides an immunologically safe method for producing transplanted cells, particularly when the patient's own bone marrow aspirate is the source of MASC.

  This method can be used to treat diabetes, cardiomyopathy, neurodegenerative diseases, and adenosine deaminase deficiency by just counting a few of them. For example, in diabetes, MASCs can be isolated, genetically modified to produce insulin, and transplanted into a diseased patient. If the disease is related to autoimmunity, MASCs can be genetically modified to express modified MHC to avoid immune surveillance or not to express MHC. By using an adenoviral vector that expresses the E3 region of the viral genome, MHC expression was suppressed in transplanted pancreatic islet cells. As the inventors have demonstrated, the cells of the present invention can be stably transfected or transduced and thus provide a more permanent source of insulin for transplantation in diabetic patients. .

  In particular, donor MASCs that have been genetically modified to alter the expression of MHC, and in particular autologous MASCs that have been genetically modified to express the desired hemoglobin gene product, are particularly useful for cell therapy for the treatment of sickle cell anemia and thalassemia. It is valid.

Methods for Genetically Modifying MASCs Cells isolated by the methods described herein can be genetically modified by introducing DNA or RNA into the cells by various methods well known to those skilled in the art. These methods roughly fall into four large categories. (1) for example, a retrovirus (such as lentivirus), simian virus 40 (SV40), adenovirus, Sindbis virus, and viral introduction method including the use of DNA or RNA viruses such as bovine papilloma virus, (2) Chemical introduction methods such as calcium phosphate transfection and DEAE dextran transfection method, (3) Membrane fusion introduction method using membrane vesicles filled with DNA such as liposome, erythrocyte ghost, and protoplast, (4) Micro Physical introduction methods such as injection, electroporation, or direct “naked” DNA introduction. Pluripotent adult stem cells (MASCs) are genes that are inserted by inserting a preselected isolated DNA, replacing a segment of the cell genome with a preselected isolated DNA, or deleting or inactivating at least a portion of the cell's genome. It is possible to modify. Deletion and inactivation of at least a part of the cell genome can be performed by various methods such as genetic recombination, antisense method (including the use of peptide nucleic acid or PNA), ribozyme method and the like. Insertion of one or more preselected DNA sequences can be performed by homologous recombination or viral insertion into the genome of the host cell. It is also possible to introduce a desired gene sequence into a cell, particularly into a cell nucleus, using a plasmid expression vector or a nuclear localization sequence. Methods for inducing polynucleotides into the nucleus are well known in the art. Genetic material induces the gene of interest positively or negatively through the use of a specific chemical / drug, disappears after administration of a specific drug / chemical, or a chemical (such as a tamoxifen-responsive mutant estrogen receptor) It can be introduced using a promoter that can be labeled to allow induction by or expression in specific cell compartments (such as cell membranes).

Homologous recombination Calcium phosphate transfection utilizes a plasmid DNA / calcium ion precipitate and can be used to isolate or introduce plasmid DNA incorporating a target gene or polynucleotide into cultured MASC. Briefly, plasmid DNA is mixed with a solution of calcium chloride and then added to the phosphate buffered solution. When a precipitate has formed, this solution is added directly to the cultured cells. Treatment with DMSO or glycerol can be used to improve transfection efficiency, and bis-hydroxyethylaminoethanesulfonate (BES) can be used to increase the level of stable transfectants. . Calcium phosphate transfection systems are generally commercially available (eg, ProFection®, Promega Corp, Madison, Wis.).

  The DEME dextran transfection method is also well known to those skilled in the art and is preferred over the calcium phosphate transfection method when transient transfection is desired because it is often more efficient.

  Since the cells of the present invention are isolated cells, microinjection is particularly effective for introducing genetic material into the cells.

  Briefly, cells are first placed on the stage of an optical microscope. While observing a magnified image with a microscope, a glass micropipette is inserted into the nucleus to inject DNA or RNA. This method is advantageous because it allows the desired genetic material to be introduced directly into the nucleus and avoids degradation of the injected polynucleotide in the cytoplasm and lysosomes. This method has been used effectively to modify the germ cells of transformed animals.

  The cells of the present invention can be genetically modified by electroporation. First, target DNA or RNA is added to the suspension of cultured cells. When this DNA / RNA-cell suspension is placed between two electrodes and an electric pulse is applied, the membrane is temporarily permeable by opening small holes in the outer membrane of the cell. When the target polynucleotide enters the cell from the small hole opened in the membrane and the electric field is interrupted, the small hole is closed in about 1 to 30 minutes. Introduction of DNA or RNA for genetic modification of cells by a liposome can be performed using a cationic liposome that forms a stable complex with a polynucleotide. Dioleyl phosphatidylethanolamine (DOPE) or dioleyl phosphatidylcholine (DOPC) can be added to stabilize the liposome complex. A recommended reagent for the liposome introduction method is Lipofectin® (Life Technologies, Inc.), which is generally commercially available. For example, Lipofectin® is a mixture of N- [1- (2,3-dioleyloxy) propyl] -NNN-trimethylammonium chloride, which is a cationic lipid, and DOPE. Introduction of linear DNA, plasmid DNA or RNA can be performed in vitro or in vivo by a liposome introduction method. Liposomes are a preferred method because liposomes can contain large pieces of DNA and can be administered to specific cells or tissues while protecting the polynucleotide from degradation. Numerous other introduction systems relying on the liposome method are commercially available, such as Effectene ™ (Qiagen), DOTAP (Roche Molecular Biochemicals), FuGene6 ™ (Roche. Molecular Biochemicals) and Transfectam (registered trademark) (Promega). The gene transfer efficiency by cationic lipid is determined by the purified G glycoprotein of the vesicular stomatitis virus envelope as in the method of Abe et al. (Abe, A., etal. (J. Virol. (1998) 72: 6159-6163)). It can be enhanced by using purified virus or cell envelope components such as (VSV-G).

  It is possible to introduce target DNA into MASC using gene transfer methods that have been shown to be effective in introducing DNA into primitive and established mammalian cell lines using lipopolyamine-coated DNA. . This method is described by Loeffler, J. et al. And Behr, J .; (Loeffler, J. and Behr, J. Methods in Enzymology (1993) 217: 599-618).

  Naked plasmid DNA can be directly injected into tissue mass consisting of cells differentiated from isolated MASC. This method has been shown to be effective in introducing plasmid DNA into skeletal muscle tissue, and expression in mouse skeletal muscle has been observed over 19 months after a single intramuscular injection. Actively dividing cells take up naked plasmid DNA more efficiently. For this reason, it is advantageous to stimulate cell division prior to treatment with plasmid DNA.

  It is also possible to introduce a gene into MASC in vivo or in vitro using gene introduction by microprojectile. For the basic procedure of microprojectile gene transfer method, see J.A. Wolff, Gene Therapeutics (1994), page 195. Briefly, the microbeads, usually gold or tungsten particles, having a particle size of 1-3 microns are coated with plasmid DNA encoding the target gene. The coated particles are placed on a carrier sheet inserted above the firing chamber. The carrier sheet is accelerated towards the holding screen after launch. The retention screen acts as a barrier to prevent further movement of the carrier sheet, where the polynucleotide-coated particles are typically driven by a flow of helium gas toward a target surface such as a tissue mass consisting of differentiated MASCs. . Microparticle injection methods have been described and are well known to those skilled in the art (see, Johnston, SA, et al., Genet. Eng. (NY) (1993) 15: 225-236). ; Williams, RS, et al., Proc. Natl. Acad. Sci. USA (1991) 88: 2726-2730; Yang, NS, et al., Proc. Natl. Acad. Sci. USA (1990) 87: 9568 -9572).

  As described by Sevetyen et al. (Nature Biotech. (1998) 16: 80-85), more efficient expression can be achieved by binding a signal peptide to plasmid DNA and incorporating this DNA into the cell nucleus. Is possible.

  It is possible to genetically modify the MASC of the present invention and its progeny using a viral vector. Similar to the physical methods described above, for example, one or more target genes, polynucleotides, antisense molecules and ribozyme sequences are introduced into cells using a viral vector. Viral vectors and methods for introducing DNA into cells using them are well known to those skilled in the art. Examples of viral vectors that can be used to genetically modify the cells of the present invention include adenovirus vectors, adeno-associated virus vectors, retrovirus vectors (such as lentivirus vectors), alphavirus vectors (eg, Sindbis vectors), And herpes virus vectors.

  Many retroviral vectors have been developed to efficiently introduce DNA into non-dividing cells, but retroviral vectors are effective in transducing actively dividing cells (Mochizuki, H , et al., J. Virol. (1998) 72: 8873-8883). Packaging cell lines for retroviruses are well known to those skilled in the art. The packaging cell line provides the viral proteins necessary for the generation of the viral vector capsid and the maturation of the viral particles. In general, such viral proteins include proteins encoded by retroviral genes such as gag, pol, and env. Select a suitable packaging cell line from known cell lines to generate ecotropic, xenotropic, or amphotropic retroviral vectors, thereby providing a specific specificity to the retroviral vector system. .

  In general, retroviral DNA vectors are used with packaging cell lines to produce the desired target sequence / vector combination within the cell. Briefly, a retroviral DNA vector is a plasmid DNA having two retroviral LTR sequences near the multiple cloning site and the SV40 promoter. The first LTR is located 5 ′ of the SV40 promoter functionally linked to the sequence of the target gene cloned in the multicloning site, and the 3 ′ second LTR is located after the multicloning site. To do. After its formation, the retroviral DNA vector is introduced into the packaging cell line by calcium phosphate transfection as described above. After about 48 hours of virus production, the viral vector containing the target gene sequence is harvested.

  A method for gene transfer into specific types of cells by retroviral vectors is shown by Martin et al. (J. Virol. (1999) 73: 6923-6929). This method uses a single-chain variable fragment antibody against a high-molecular-weight melanoma-associated antigen, a surface glycoprotein fused to the envelope of an amphotropic murine leukemia virus, to target the target gene with a vector using a vector. Has been introduced. For example, when it is desirable to introduce a targeted gene into a target cell, such as when genetically modifying a differentiated cell, it is fused to an antibody fragment against a specific marker expressed by each cell line differentiated from the MASC of the present invention. It is possible to introduce genes into these cells with the aim of using the retroviral vector thus prepared.

It is also possible to genetically modify the cells of the present invention using a lentiviral vector. Many such vectors are described in the literature and are well known to those skilled in the art (Salmons, B. and Gunzburg, WH, "Targeting of Retroviral Vectors for Gene Therapy," Hum. Gene. Therapy (1993) 4: 129-141). . These vectors have been shown to be effective in genetically modifying human hematopoietic stem cells (Sutton
, R., et al., J. Virol. (1998) 72: 9683-9697). Lentiviral packaging cell lines have already been described (see Kafri, T., et al., J. Virol. (1999) 73: 576-584; Dull, T., et al., J. Virol. (1998) 72: 9683-9697).

  Attempts to introduce DNA into cells expressing erythropoietin receptors using recombinant herpesviruses such as type I herpes simplex virus (HSV-1) have been successful (Laquerre, S. et al. , et al., J. Virol. (1998) 72: 9683-9697). It is possible to genetically modify the cells of the present invention using these vectors, and the inventors have proved that the cells of the present invention were stably transformed with viral vectors.

  Adenovirus vectors have high transformation efficiency, can introduce DNA fragments of up to 8 Kb, and can infect both dividing and differentiated cells. Many adenoviral vectors have been described in the literature and are well known to those of skill in the art (see Davidson, BL, et al., Nature Genetics (1993) 3: 219-223; Wagner, E., et al., Proc. Natl. Acad. Sci. USA (1992) 89: 6099-6103). Methods for inserting target DNA into adenoviral vectors are well known to those skilled in the art of gene therapy, as are methods for introducing target DNA into specific types of cells using recombinant adenoviral vectors. (See, Wold, W., Adenovirus Methods and Protocols, Humana Methods in Molecular Medicine (1998), Blackwell Science, Ltd.). Binding affinity for certain types of cells has been demonstrated by modification of the viral vector fiber sequence. An adenoviral vector system for regulating protein expression in gene transfer has been described previously (Molin, M., et al., J. Virol. (1998) 72: 8358-8361). Previously, systems have been described for propagating adenoviral vectors with receptor specificity that have been genetically modified to allow targeted transformation of specific cell types (Douglas, J., et. al., Nature Biotech. (1999) 17: 470-475). A sheep adenovirus vector found in a recent report eliminates the possibility of pre-existing humoral immunity disruption (Hofmann, C., et al., J. Virol. (1999). 73: 6930-6936).

  Furthermore, adenovirus vectors that enable gene transfer and stable gene expression targeting specific cells using molecular conjugate vectors are available. This vector is constructed by concentrating plasmid DNA having a target gene together with polylysine. This polylysine is bound to adenovirus that has lost its ability to divide (Schwarzenberger, P., et al., J. Virol. (1997) 71) 8563-8571).

  Alphavirus vectors, particularly Sindbis virus vectors, for transforming the cells of the invention are also available. These vectors are commercially available (Invitrogen, Carlsbad, Calif.), For example, US Pat. No. 5,843,723, Xiang, C .; (Science (1989) 243: 1188-1191), Bredenbeek, P. et al. J. et al. (J. Virol. (1993) 67: 6439-6446) and Frolov, I. et al. (Proc. Natl. Acad. Sci. USA (1996) 93: 11371-11377).

  The inventors have shown that MASC has good transformation ability when using the eGFP-MND lentiviral vector and eGFP-MGF vector described by Robbins et al. (J. Virol. (1997) 71 (12): 9466-9474). It was shown to have. Using this method, PA3-17 packaging cells (described by Miller, AD, and C. Buttimore (Mol. Cell. Biol. (1986) 6: 2895-2902) are derived from NIH3T3 fibroblasts. 30 to 50 hours after exposure to a green fluorescent protein (eGFP) vector containing a supernatant prepared in a combination of an amphotropic packaging cell line) and protamine (8 mg / ml) for 4.6 hours and a short exposure. % MASC can be transformed. Expression of eGFP is seen throughout the culture period of undifferentiated MASC. In addition, transgenes have been successfully introduced into MASC by transfection with Lipofectamine.

  Transfection or transduction of target cells can be demonstrated using genetic markers in a manner well known to those skilled in the art. For example, Aequorea victoria green fluorescent protein has been shown to be an effective marker in the identification and tracking of genetically modified hematopoietic cells (Persons, D., et al., Nature Medicine (1998) 4: 1201-1205). . Other selectable markers include β-Gal gene, truncated nerve growth factor receptor, drug selection markers (NEO, MTX, hygromycin, etc.) and the like.

  17. MASC is useful for tissue repair The stem cells of the present invention can also be used for tissue repair. The inventors have determined that the pluripotent adult stem cells (MASC) of the present invention are fibroblasts, osteoblasts, chondrocytes, adipocytes, skeletal muscles, endothelium, stromal cells, smooth muscles, cardiac muscles, hematopoietic cells, etc. Proved to differentiate into many types of cells. For example, transplanting MASCs that have been induced to differentiate into osteoblasts by the methods described earlier herein promotes the repair process, strengthens weakened bones, and remodels joint surfaces It is possible. It is possible to reshape the surface of articular cartilage by injecting MASC differentiated into chondrocytes by the method described above into the joint. Caplan et al. (US Pat. No. 5,855,619) describe a biomatrix implant consisting of a contracted gel matrix containing mesenchymal stem cells. This implant is specifically designed to repair tissue defects such as tendons, ligaments, meniscuses and muscles. For example, cartilage can be formed by adding chondrocytes so as to be in contact with a porous three-dimensional scaffold formed of collagen, synthetic polyglycolic acid fiber, synthetic polylactic acid fiber or the like. The inventors can obtain an implant that promotes tissue repair by differentiating the MASC of the present invention into, for example, chondrocytes, and accumulating it in or around a scaffold material such as collagen, synthetic polyglycolic acid or synthetic polylactic acid. Showed that.

  In addition, the cells of the present invention are introduced into a specific anatomical site by means of a matrix, where they are initially introduced by a specific growth factor encoded in a plasmid that is contained in the matrix or contained in the matrix to be taken up by the cell. It is possible to direct the growth of cell populations. For example, it is possible to incorporate DNA into the pores of the matrix in the foaming process when forming a specific polymer matrix. When the polymer used in the foaming process expands, the DNA is confined in the pores, thereby controlling the release of the plasmid DNA and allowing the sustained release of the DNA. A method for preparing such matrices is described by Shea et al. (Nature Biotechnology (1999) 17: 551-554).

  Bonadio, J .; (Nature Medicine (1999) 5: 753-759), plasmid DNA encoding cytokines, growth factors and hormones can be confined in a gene-activated polymer matrix carrier. When this biodegradable polymer is transplanted, for example, in the vicinity of a broken bone, and MASC is transplanted there, MASC takes up DNA and produces cytokines, growth factors and hormones to increase the local concentration. Healing is accelerated.

  It is possible to produce a tissue or organ for transplantation using the cells provided by the present invention, that is, MASC isolated by the method of the present invention. According to Oberpenning et al. (NatureBiotechnology (1999) 17: 149-155), muscle cells obtained from the outer surface of the canine bladder and lining cells obtained from the inner surface of the canine bladder are cultured, and a tissue sheet is prepared from the culture. It has been reported that a functional bladder can be formed by coating the outside of a small polymer sphere with muscle cells and the inside with lining cells. When the sphere was inserted into the urinary system of a dog, the sphere began to function as a bladder. According to Nicklason et al. (Science (1999) 284: 489-493), it was reported that a vascular graft material having a predetermined length could be produced from cultured smooth muscle and endothelial cells. Other methods for forming tissue layers from cultured cells are well known to those skilled in the art (see, Vacanti, et al., US Pat. No. 5,855,610). These methods are particularly effective when used in combination with the cells of the present invention having a broader differentiation ability than any of the non-embryonic stem cells described so far.

  The MASC of the present invention can restore cardiomyocytes by injecting directly into the tissue damage area or systemically, thereby engrafting the cells in the heart tissue. This method is particularly effective when combined with angiogenesis. Both injection methods and methods for promoting angiogenesis are well known to those skilled in the art. The MASCs of the present invention have broad differentiation potential that provides a more diverse source of cells for heart or other tissue repair utilizing these methods.

  The MASCs of the present invention are useful, for example, for the purpose of regenerating bone marrow after high dose chemotherapy. Collect bone marrow aspirate from the patient before chemotherapy. Stem cells are isolated, cultured and induced for differentiation by the method of the present invention. This is followed by reintroduction of the mixture of differentiated and undifferentiated cells into the patient's bone marrow cavity. Currently, clinical trials using hematopoietic stem cells are being carried out for this purpose. The MASC of the present invention can be further differentiated into cells that can replace not only bone marrow but also cells damaged by chemotherapy of other tissues. It has the further advantage of being.

  It is also possible to change the histocompatibility antigen of allogeneic donor-derived stem cells using the method described by Lawman et al. (International Patent Application Publication No. WO 98/42838). By using this method, a frozen stock is prepared and stored to form a panel of transplanted bone marrow that can be used for administration to patients who cannot remodel their bone marrow, such as in leukemia patients. Is possible.

  The patient's immune system cells and blood cells can be recovered by, for example, isolating autologous stem cells from the patient, culturing the cells to expand the cell population, and then reintroducing the cells into the patient Is possible. This method reduces the immune system and bone marrow cells by radiation and / or chemotherapy for therapeutic purposes, such as patients diagnosed with multiple myeloma, non-Hodgkin's lymphoma, autoimmune disease or solid tumor This is especially effective when necessary.

The patient's blood and immunity with allogeneic cells of the present invention or isolated by the method of the present invention for the treatment of leukemia, autoimmune diseases, genetic diseases such as sickle cell anemia and thalassemia Restoration of cell lines can be performed especially when the histocompatibility antigen is changed by the method described by Lawman et al. (International Patent Application Publication No. WO 98/42838).

  To achieve the objectives described herein, autologous or allogeneic MASCs of the present invention can be injected directly into a tissue site in a differentiated or undifferentiated state, with or without genetic modification. , Administered systemically, on or near the surface of an acceptable matrix, or in combination with a pharmaceutically acceptable carrier.

  19. MASC provides a model system for studying differentiation pathways The cells of the present invention are also useful for further study of developmental processes. For example, Ruley et al. (International Patent Application Publication No. WO 98/40468) describe vectors and methods for inhibiting the expression of specific genes and obtaining the DNA sequence of the inhibited genes. Cells of the present invention can be treated with a vector as described by Ruley, thereby inhibiting the expression of genes that can be identified by DNA sequence analysis. It is possible to induce differentiation of these cells and examine the effect of the modified genotype / phenotype.

  For example, Hahn et al. (Nature (1999) 400: 464-468) show that when a specific combination of genes previously shown to be correlated with cancer is introduced into normal human epithelial fibroblasts, the cells become cancerous. It was proved that differentiation can be induced.

  Control of gene expression using vectors having inducible expression elements provides a method for examining the effect of specific gene products on cell differentiation. Inducible expression systems are well known to those skilled in the art. One such system is No, D.D. (Proc. Natl. Acad. Sci. USA (1996) 93: 3346-3351).

  Using MASC, it is possible to examine the effects of specific genetic changes, toxicants, chemotherapeutic drugs and other drugs on the developmental pathway. Tissue culture methods well known to those skilled in the art allow large-scale culture of hundreds of thousands of cell samples from different individuals, for example, rapid screening of suspected teratogenic or mutagenic compounds Can be performed.

  For the purpose of studying developmental pathways, it is possible to treat MASCs with certain growth factors, cytokines and other drugs, including compounds suspected of teratogenicity. MASC can also be genetically modified by the methods and vectors previously described. Furthermore, it is possible to modify the expression of the natural gene sequence by modifying MASC by antisense technology or treatment with proteins introduced into cells. For example, a desired peptide or polypeptide can be introduced into a cell using a signal peptide sequence. One particularly effective method for introducing polypeptides and proteins into cells is described by Rojas et al. (Nature Biotechnology (1998) 16: 370-375). According to this method, a polypeptide or protein product that moves through the cell membrane and into the cell by being introduced into the medium can be obtained. Any number of proteins can be used in this way to investigate the effect of the target protein on cell differentiation. It is also possible to bind VP22, which is a herpes virus protein, to a functional protein and transport it into cells using the method described by Phelan et al. (Nature Biotech. (1998) 16: 440-443).

  The cells of the present invention have a defective phenotype for testing their potential effectiveness as chemotherapeutic agents and gene therapy vectors by genetic manipulation by introducing foreign genes, suppressing expression of genomic DNA, or excising DNA. It is possible to create differentiated cells with.

  20. MASC provides various types of differentiated and undifferentiated cultured cells for high-throughput screening. The pluripotent adult stem cell (MASC) of the present invention can be obtained, for example, by culturing in a multi-well culture plate such as 96-well. It is possible to obtain a system for performing high-throughput screening for cytokines, chemokines, growth factors, or pharmacological compositions in pharmacogenomics or pharmacogenetics. The MASC of the present invention provides the only system that can differentiate cells into specific cell lines from the same individual. Unlike many primary cultures, these cells can be maintained in culture and observed over time. Treatment of multiple cultures of cells from the same individual and different individuals with the target factor, and the influence of the cellular factor on different types of differentiated cells with the same gene composition, or from individuals with different gene compositions It is possible to determine whether there is a difference in the effect on similar types of cells. Therefore, for example, it is possible to screen cytokines, chemokines, pharmacological compositions, and growth factors quickly and cost-effectively, and to clarify the effects more clearly. Cells isolated from large populations and characterized for the presence or absence of genetic polymorphisms, particularly single nucleotide polymorphisms, can be stored in cell culture banks for various screening methods. For example, a pluripotent adult stem cell obtained from a statistically significant population that can be determined by methods well known to those skilled in the art allows polymorphisms associated with enhanced positive or negative responses to a wide range of substances. An ideal system for high throughput screening to identify is given. Examples of such substances include pharmacological compositions, vaccine preparations, cytotoxic substances, mutagens, cytokines, chemokines, growth factors, hormones, inhibitors, chemotherapeutic agents, and other compounds or factors. Host. The information gained from these studies can be widely applied in the treatment of infectious diseases, cancer, and many metabolic diseases.

  In methods that utilize MASCs to characterize cellular responses to biological or pharmaceutical agents, or combinatorial libraries of such agents, MASCs are isolated from a statistically significant population, the culture is grown, and one or more Contact with biological or pharmaceutical substances. MASCs can be induced to differentiate before or after growth in culture when differentiated cells are the desired target of a particular biological or pharmaceutical agent. By comparing one or more cellular responses between MASC cultures obtained from multiple individuals of a statistically significant population, it is possible to determine the effect of the biological or pharmaceutical substance. It is also possible to screen different compounds such as compounds in a combinatorial library using MASCs having the same gene composition or cells differentiated from these cells. Gene expression systems used in combination with cell-based high-throughput screening have been described previously (see, Jayawickreme, C. and Kost, T., Curr. Opin. Biotechnol. (1997) 8: 629- 634). A high-capacity screening method used to identify an inhibitor of endothelial cell activation is described by Rice et al., Which utilizes a cell culture system for primitive human umbilical vein endothelial cells (Rice, etal Anal. Biochem. (1996) 241: 254-259). The cells of the present invention provide a variety of terminally differentiated and undifferentiated cells for high-throughput screening methods used in identifying a large number of targeted biological and pharmaceutical agents. Most importantly, the cells of the present invention provide a source of cultured cells from a population of diverse genes that have different responses to biological and pharmaceutical substances.

  MASCs are provided as frozen stocks, alone or with pre-packaged media and culture supplements, and separately packaged, appropriate factors at an effective concentration to induce differentiation of specific cell types Provided with. MASC can also be provided as a frozen stock containing cells prepared by methods well known to those skilled in the art and induced to differentiate by the methods described above.

  21. MASC and genetic profile Gene mutations have a direct and indirect effect on disease susceptibility. In the direct case, a single nucleotide change that results in a single nucleotide polymorphism (SNP) also changes the amino acid sequence of the protein, directly affecting the disease and susceptibility to the disease. The resulting functional change of the protein can often be detected in vitro. For example, certain APO lipoprotein E genotypes have been shown to be associated with the onset and progression of Alzheimer's disease in certain patients.

  DNA sequence abnormalities can be detected by dynamic allele-specific hybridization, DNA chip technology, and other methods well known to those skilled in the art. It is estimated that the coding region of the protein in the human genome is only about 3%, and it is estimated that there are probably 200,000 to 400,000 common SNPs in the coding region.

  In previous research methods using SNP-related gene analysis, samples for gene analysis were obtained from a large number of individuals capable of phenotypic characterization. Unfortunately, the genetic correlations obtained with these methods were limited to the identification of specific polymorphisms associated with easily identifiable phenotypes and did not give further information on the cause of the disease .

The MASC of the present invention provides the elements necessary to bridge between the identification of genetic elements associated with a particular disease and the final phenotype found in patients with that disease. Briefly, MASCs are first isolated from a statistically significant population of individuals from which phenotypic data can be obtained (see Collins, et al., Genome Research (1998) 8: 1229-1231). The resulting MASC sample is then cultured and expanded, and the secondary culture of cells is stored as a frozen stock. This frozen stock can be used to obtain a culture for use in later developmental experiments. Multiple gene analyzes can be performed on the expanded cell population to identify gene polymorphisms. For example, single nucleotide polymorphisms can be identified in a large sample population in a relatively short time using currently available methods such as DNA chip technology well known to those skilled in the art (Wang, D., et al. ., Science (1998) 280: 1077-1082; Chee, M., et al., Science (1996) 274: 610-614; Cargill, M., et al., Nature Genetics (1999) 22: 231-238 Gilles, P., et al., Nature Biotechnology (1999) 17: 365-370; Zhao, LP, et al., Am. J. HumanGenet. (1998) 63: 225-240). Methods for SNP analysis have also been described so far by Syvanen, Xiong, Gu, Collins, Howell, Buteow and Hoogendoorn (Syvanen, A., Hum. Mut. (1999) 13: 1-10), (Xiong, M. and L. Jin, Am. J. Hum. Genet. (1999) 64: 629-640), (Gu, Z., et al., Human Mutation (1998) 12: 221-225), (Collins, F., et al., Science (1997) 278: 1580-1581), (Howell, W., etal., Nature Biotechnolo
gy (1999) 17: 87-88), (Buetow, K., et al., NatureGenetics (1999) 21: 323-325), (Hoogendoorn, B., et al., Hum. Genet. (1999) 104 : 89-93).

  When a specific polymorphism is associated with a specific disease phenotype, cells obtained from an individual identified as the carrier of that polymorphism are used as a control, cells obtained from non-carrier individuals, It is possible to investigate occurrence abnormalities. The MASC of the present invention can be induced to differentiate into specific types of cells using the predetermined methods described herein and other predetermined methods well known to those skilled in the art. It provides an experimental system for studying developmental abnormalities associated with genetic diseases. For example, when a specific SNP is associated with a specific neurodegenerative disease, the polymorphism using both undifferentiated MASC and MASC differentiated into nerve-derived cells such as neuronal progenitor cells and glial cells Can be investigated. By tracking cells that exhibit specific polymorphisms during differentiation, they are sensitive to drug sensitivity, chemokine and cytokine responsiveness, responsiveness to growth factors, hormones, and inhibitors, and to changes in receptor expression and / or function It is possible to identify genetic elements that affect responsiveness. This information is extremely useful in exploring treatment methods for diseases caused by genes or those with a genetic predisposition.

  In the present method for identifying genetic polymorphisms associated with physiological abnormalities using MASCs, MASCs are isolated from statistically significant populations that can obtain phenotypic data (statistically). A significant population is defined by those skilled in the art as a sufficiently sized population containing elements having at least one genetic polymorphism), and the culture is grown to establish a MASC culture. Next, using the DNA obtained from the cultured cells, the gene polymorphism in the cultured MASC obtained from the population is specified, and the cells are induced to differentiate. Abnormalities associated with a particular genetic polymorphism by comparing the differentiation pattern exhibited by MASC with normal genotype with the differentiation pattern exhibited by MASC having the identified genetic polymorphism or showing a response to a candidate drug It is possible to identify and characterize metabolic processes.

  22. MASC enables safe vaccine administration MASCs of the present invention can be used as antigen-presenting cells by genetic modification so as to produce antigenic proteins. For example, using an autologous or allogeneic progenitor cell that has undergone multiple genetic modifications, the plasmid embedded in a biodegradable matrix and the progenitor cell of the present invention so as to be released slowly to transfect the target cell In combination, it is possible to stimulate an immune response to one or more antigens, and the maximum effect of the immune response may be enhanced by slow release of antigen-presenting cells. It is known to those skilled in the art that multiple administrations of certain antigens over time can enhance the immune response in the final antigen challenge. In addition, in the method of Zhang et al. (Nature Biology (1998) 1: 1045-1049), MASC can be used as an antigen-presenting cell to induce T cell tolerance to a specific antigen.

Many vaccine formulations currently in use use additive chemicals and other substances. Examples include antibiotics (suppressing bacterial growth in vaccine culture), aluminum (adjuvant), formaldehyde (inactivate bacterial products against toxoid vaccine), monosodium glutamate (stabilizer), egg protein ( Ingredients of vaccines produced using growing chicken eggs), sulfites (stabilizers), and thimerosal (preservatives). Due to some of these additional ingredients, there is currently widespread social concern regarding the safety of vaccine formulations. For example, thimerosal contains mercury and consists of a combination of ethyl mercury chloride, thiosalicylic acid, sodium hydroxide, and ethanol. In addition, although not critical, some studies suggest a relationship between certain vaccine components and potential complications such as diseases commonly associated with autoimmunity. Therefore, more effective vaccine therapies are sought, and social cooperation for vaccine research will be easily obtained if the comfort of vaccination methods increases.

  The MASC of the present invention can be differentiated into dendritic cells, and when these dendritic cells present antigens to the T cells, the T cells are activated to produce a response to foreign substances. Such dendritic cells can be genetically modified to express foreign antigens using the methods described above. An advantage of such a vaccine administration method is that a plurality of antigens can be presented by one genetically modified cell.

  Heterogeneous differentiated or undifferentiated MASC vaccine vectors provide the additional advantage that the immune system is stimulated by foreign cell surface markers. In vaccine design experiments, it has been shown that stimulation of an immune response by multiple antigens enhances the immune response to a particular antigen in a vaccine formulation.

  For example, hepatitis A, hepatitis B, chickenpox, polio, diphtheria, pertussis, tetanus, Lyme disease, measles, mumps, rubella, influenza B (Hib), BCG, Japanese encephalitis, yellow fever, and Immunologically effective antigens have been identified for rotavirus.

  A method for inducing an immune response against an infectious agent in a human subject using the MASC of the present invention is to expand a clonal population of pluripotent adult stem cells in culture so as to express one or more predetermined antigenic molecules. It is possible to genetically modify the proliferated cells to stimulate a protective immune response against the infectious agent and introduce an effective amount of genetically modified cells into the subject to induce the immune response. Methods for administering genetically modified cells are well known to those skilled in the art. An effective amount of genetically modified cells in inducing an immune response is the amount of cells that express a sufficient amount of the desired antigen to produce an antibody response that can be measured by methods well known to those skilled in the art. Preferably, an antibody response is a protective antibody response that can be detected by resistance to a disease when challenged with an appropriate infectious agent.

  23. MASC and Cancer Treatment The pluripotent adult stem cell (MASC) of the present invention provides a novel vehicle for cancer treatment. For example, MASC can be induced to differentiate into endothelial cells or progenitor cells that engraft endothelial tissue when administered locally or systemically. These cells are used for angiogenesis (angiogenesis) to supply blood to the neoplastic tumor, and thus divide and proliferate in the endothelial tissue. It is possible to inhibit angiogenesis and block blood flow to the tumor by genetically manipulating the cells so that the cells die by stimulation of an externally introduced factor. An example of a factor introduced from outside is tetracycline, which is an antibiotic. In that case, cells are transfected or transduced with a gene such as Caspase or BAD that induces cell death under the control of a tetracycline-responsive factor. Tetracycline responsive factors have been described previously in the literature (Gossen, M. & Bujard, H., Proc. Natl. Acad. Sci. USA (1992) 89: 5547-5551) and in vivo in endothelial cells. (Sarao, R. & Dumont, D., Transgenic Res. (1998) 7: 421-427) and commercially available (CLONETECH Laboratories, Palo Alto, CA).

  Further, undifferentiated MASC or MASC differentiated into a tissue-specific cell line may be transformed into a predetermined product that inhibits tumor cells or inhibits angiogenesis (pigment epithelium inducing factor (PEDF) described by Dawson et al. Dawson, et al., Science (1999) 285: 245-248)) can be genetically modified and transported to the extracellular environment. For example, Koivunen et al. Describe cyclic peptides having predetermined amino acid sequences that selectively inhibit MMP-2 and MMP-9 (a matrix metalloprotease that has been shown to be associated with tumorigenesis). This cyclic peptide has been shown to block tumor growth and invasion in animal models and specifically target new blood vessels in vivo (Koivunen, E., Nat. Biotech. (1999) 17: 768-774). If it is desired to deliver the cell to the tumor site, produce a tumor inhibitory product, and then destroy it, the cell is further genetically modified to have a cell death promoting protein that promotes cell death under the control of an inducible promoter Is possible.

  MASCs are isolated from patients, cultured ex vivo, and genetically modified ex vivo to express the antigen, particularly in combination with a receptor that has been shown to be associated with a high immune response to the appropriate antigen. Since it can be reintroduced into a subject to cause an immune response against a protein expressed by tumor cells, it further provides a vector for administration of a cancer vaccine.

  24. MASC, or kit containing elements for MASC isolation and culture The pluripotent adult stem cells (MASC) of the present invention are provided as a kit with appropriate packaging materials. For example, MASCs are provided in the form of frozen stocks with appropriate factors and media for culturing in an undifferentiated state as packaged herein. Furthermore, it is also possible to provide a separately included differentiation-inducing factor as described above.

  According to the present invention, a kit containing an effective amount of an appropriate factor for isolating and culturing patient stem cells is also provided. If the clinician collects the bone marrow aspirate from the patient, the anti-CD45 and anti-glycophorin A are in the kit, and then the stem cells are selected by the method described herein, It is only necessary to culture the cells according to the method of the present invention using the existing medium. The composition of the basic medium is described above.

  One aspect of the invention is the preparation of a kit for isolating MASCs from human subjects under clinical conditions. It is possible to isolate MASCs from simple bone marrow aspirates using the kit components included. It is clinically possible to obtain a population of antigen presenting cells (APCs) from a patient's own bone marrow sample by using a differentiation factor, medium, and additional kit elements containing instructions for inducing MASC differentiation in culture. This is possible for engineers. As a further material to be included in the kit, a polynucleotide administration vector encoding an appropriate antigen expressed and presented by differentiated APC can be provided. For example, it is possible to provide, for example, a plasmid having the gene sequence of the protective antigen of hepatitis B surface antigen, hepatitis A, adenovirus, Plasmodium falciparum and other infectious agents. These plasmids can be introduced into cultured APCs using, for example, calcium phosphate transfection materials and instructions included in the kit. It is also possible to provide additional material for reinjecting the genetically modified APC into the patient's body, thereby providing an auto-vaccine administration system.

  The invention is further illustrated by the following detailed examples.

  Examples Example 1 Isolation of MASCs from bone marrow mononuclear cells Bone marrow mononuclear cells were obtained from bone marrow aspirated from the posterior iliac crest of more than 80 volunteers.

As shown in Table 3, 10-100 cm ³ of bone marrow was obtained from each subject. Table 3 shows the approximate number of mononuclear cells isolated from each subject. Mononuclear cells (MNC) were obtained by centrifuging bone marrow on a Ficoll-Paque density gradient (Sigma Chemical Co., St. Louis, MO) (Sigma Chemical Co, St Louis, MO). Bone marrow MNCs are incubated with CD45 and glycophorin A microbeads (MiltenyiBiotec, Sunnyvale, Calif.) For 15 minutes, and this sample is placed in front of the SuperMACS magnet to obtain CD45 ⁺ / Gly. -A ⁺ cells were removed. The extracted cells were 99.5% CD45 ⁻ / Gly-A ⁻ .

As shown in Table 3, the removal of CD45 ⁺ / GlyA ⁺ cells recovered CD45 ⁻ / GlyA ⁻ cells that comprised about 0.05-0.10% of total bone marrow mononuclear cells.

Cells that did not express CD45, a common leukocyte antigen, or glycophorin-A (GlyA), a marker for erythroid progenitor cells, were selected. CD45 ⁻ / GlyA ⁻ cells constitute 1/10 ³ of bone marrow mononuclear cells. CD45 ⁻ / GlyA ⁻ cells were coated with fibronectin and seeded in wells supplemented with 2% FCS, EGF, PDGF-BB, dexamethasone, insulin, linolenic acid and ascorbic acid. A small population of adherent cells formed after 7-21 days. We used a limiting dilution assay to determine the frequency of cells producing adherent cell populations, one per 5 × 10 ³ CD45 ⁻ / GlyA ⁻ cells.

When colonies (about 10 ³ cells) appeared, the cells were recovered by trypsin treatment and replated at a dilution ratio of 1: 4 every 3 to 5 days under the same culture conditions. The cell density was kept at 2-8 × 10 ³ cells / cm ² . Cell doubling time was 48-60 hours. As a result of performing immunophenotypic analysis by fluorescent chromogenic cell analysis separation method (FACS) after 10 to 12 cell divisions, the cells were CD31, CD34, CD36, CD38, CD45, CD50, CD62E and CD62-P, Muc18, cKit. , Tie / Tek, and CD44 were not expressed. The cells did not express HLA-DR or HLA-class I at all and expressed β2 microglobulin at low levels. Cells were strongly positively stained with antibodies against CD10, CD13, CD49b, CD49e, CDw90, Flk1. This MAS
The C phenotype did not change until cell division exceeded 30 (n = 15). Among 2-50 year old donors, MASC cultures were established that contained cells that could divide more than 30 times from more than 85% of donors and could differentiate into all mesodermal cells (see below). MASCs were grown for 10 donors until cell division exceeded 50 times. When cells were cultured in serum-free medium supplemented with 10 ng / mL IGF, cell doubling time increased (> 60 hours) but cell division exceeded 40 times. The cells cultured in serum-free medium were negative for HLA-class I and CD44, as seen in cells cultured in medium without IGF and supplemented with 2% FCS, as described below. Differentiated into germinal phenotype.

When cells were seeded on type I collagen or laminin instead of fibronectin, the cells expressed CD44 and HLA-DR but were unable to grow until cell division exceeded 30 times. When EGF or PDGF was omitted, the cells died without dividing, whereas when these cytokines were added at high concentrations, MASC proliferated initially, but division stopped when cell division exceeded 20-30 times. Even when high concentrations of dexamethasone were added, cell division did not exceed 30 times. Cells were cultured in medium supplemented with more than 2% FCS and expressed CD44, HLA-DR, and HLA-class I. Similarly, CD44, HLA-DR and HLA-class I, and Muc-18 were obtained in high density cultures (8 × 10 ³ cells / cm ² ), which is similar to the phenotype described for MASC. ing. Proliferation ability was lost in high-density culture or culture with high concentration of FCS, and cell division was stopped after 25 to 30 times.

We attempted to clone the MASC by reseeding the MASC at 1 cell / well when the culture was established. More than 2000 cells obtained from 3 donors were seeded one by one in a 96-well plate coated with FN and containing the same medium. No cell growth was seen in any well. It is noteworthy that when cells were seeded at 10 cells / well, cell growth was seen in about 4% of the wells. Of these wells, 5% of the progeny were able to grow until the number of cells exceeded 10 ⁷ .

  When MASCs obtained from 5 donors (2 to 50 years old) were cultured over 15 cell divisions, the telomere length was 11 to 16 kB. In 3 donors this was 3 kB longer than the telomere length of blood lymphocytes from the same donor. Regarding telomere length of cells obtained from one donor, no change was found when telomere length was measured after 15, 30, and 45 cell divisions. Cytogenetic analysis was performed on MASCs recovered after 30 cell divisions and showed a normal karyotype.

Example 2 Differentiation of MASCs To induce osteoblast differentiation, serum-free medium was supplemented with 10 ⁻⁷ M dexamethasone, 10 mM ascorbic acid, and 10 mM glycerophosphate. Osteoblast differentiation was confirmed by detection of calcium mineralization, alkaline phosphatase expression, and production of bone sialylproteins, osteopontin, osteocalcin and osteonectin, which are relatively specific for bone development (see FIG. 7).

To induce cartilage differentiation, serum-free medium was supplemented with 100 ng / ml TGF-1 (P & D Systems, Minneapolis, Minn.) As previously described. The cells were induced to differentiate in the state of adhering to fibronectin or in suspension culture, and differentiated chondrocytes were formed by either method. Differentiation forming chondrocytes was confirmed by detection of type II collagen and glycosaminoglycan aggrecan (see FIG. 7).

To induce adipocyte differentiation, 10 ⁻⁷ M dexamethasone and 100 μg / ml insulin were added to the medium. In addition, adipocyte differentiation was induced by replacing the serum-free medium with a medium containing 20% horse serum. Adipocyte differentiation was detected by detection of LPL and aP2.

  In order to induce skeletal muscle cell differentiation, MASCs with a confluence of over 80% were treated with 3 μM 5-azacytidine for 24 hours and then maintained in MASC medium supplemented with EGF and PDGF-BB. On day 5 after the treatment, expression of muscle-specific protein was observed. On the second day of induction, Myf5, Myo-D, and Myf6 transcription factors were detected. After 14 to 18 days, the expression level of Myo-D was significantly reduced, but Myf5 and Myf6 were kept at high levels. On the 4th day after induction, desmin and sarcomeric actin were detected, and on the 14th day, fast contracting myosin and slow contracting myosin were detected (FIG. 7). By immunohistochemistry, it was confirmed that 70-80% of cells expressed mature muscle protein after 14 days. We have shown that 20% horse serum is added to fuse myogenic cells into multinucleated myotubes (FIG. 7). Of note, 5-azacytidine treatment further induced Gata4 and Gata6 expression in the first week of culture and myocardial troponin T expression after 14 days. Furthermore, smooth muscle actin was detected 2 days after induction, and remained high until the 14th day.

  When 100 ng / mL PDGF was added as the only cytokine to MASC that had reached confluence by maintaining in serum-free MASC medium for 14 days, differentiation into smooth muscle cells was observed. These cells expressed smooth muscle markers. The presence of myogenin was confirmed on the 4th day and the presence of desmin after 6 days. Smooth muscle actin was detected after the second day, and smooth muscle myosin was detected after 14 days. After 14 days, about 70% of the cells were positively stained with anti-smooth muscle actin and myosin antibody. Furthermore, after 2 to 4 days, Myf5 and Myf6 proteins were detected and these remained high until day 15, but Myo-D was not detected (FIG. 7).

  Myocardial differentiation was induced by adding 100 ng / ml basic fibroblast growth factor (bFGF) to the standard serum-free medium previously described herein. The cells had reached confluence at the start of bFGF treatment. Add 100 ng / ml 5-azacytidine, 100 ng / ml bFGF, and 25 ng / ml bone morphogenetic proteins 2 and 4 (BMP-2 and BMP-4) to the medium to induce further development of heart tissue did. At the beginning of the treatment for inducing cardiac tissue differentiation, the cells had reached over 80% confluence. Gata4 and Gata6 expression was seen on day 2 and remained high until day 15. Expression of Myf6 and desmin was observed after the second day, and expression of myogenin was observed after the sixth day. Expression of cardiac troponin T was observed after day 4, and expression of cardiac troponin I and ANP was observed after day 11. On day 15, these mature myocardial proteins were detected in more than 70% of cells by immunohistochemistry. When the culture was continued for 3 weeks or more, the cells formed syncytium. We further confirmed that spontaneous contractions occur rarely in culture and propagate a distance of several millimeters (FIG. 7). Here again, Myf5, Myf6 and smooth muscle actin were detected after the sixth day.

To induce endothelial cell differentiation ex vivo by day 15-20, vascular endothelial growth factor (VEGF) was added to serum-free medium without other growth factors at a concentration of 20 ng / ml. Endothelial cell differentiation was confirmed by immunofluorescent staining to detect cellular proteins and receptors associated with endothelial cell differentiation. The results are shown in FIG.

Hematopoietic cell differentiation was adjusted with an AFT024 feeder, a mesenchymal cell line derived from fetal liver supporting mouse and human regenerative stem cells ex vivo, and PDFF-BB supplemented with 5% FCS and 100 ng / mL SCF and It was induced by culturing MASCs in wells coated with type IV collagen while containing EGF-containing MASC medium. Cells recovered from these cultures expressed cKit, cMyb, Gata2, and G-CSF-R, but not CD34 (RT-PCR). Since hematopoiesis is induced by factors released by embryonic visceral endoderm, we co-cultured βGal ⁺ mouse EB and human MASC in the presence of human SCF, Flt3-L, Tpo, and Epo did. In two separate experiments, a small population of βGal ⁻ cells expressing human CD45 were detected.

Differentiation of “stromal cells” was induced by culturing MASC with IL-1α, FCS, and horse serum. In order to prove that these cells have hematopoietic support ability, the feeder was irradiated with 2,000 cGy, and CD34 ⁺ umbilical cord blood cells were seeded in contact with the feeder. Two weeks later, progeny cells were replated in the methylcellulose assay to determine the number of colony forming cells (CFC). CFCs grew 3-5 times.

  MASC cultures that reached confluence were treated with hepatocyte growth factor (HFG) and KGF. After 14 days, the cells expressed MET (HGF receptor), cytokeratins 18 and 19, which have been shown to be associated with the development of hepatic epithelial cells.

Example 4 Transduction into adult bone marrow-derived MASC After MASC culture was established after about 3 to 10 generations of subculture, MASC was transduced with MASC by retrovirus for 2 consecutive days. The retroviruses used were MFG-eGFP or MND-eGFP-SN constructs, courtesy of Donald Kohn (Donald Kohn, MD, LA Childrens Hospital, Los Angeles, CA). Both vectors were packaged in PA317, an amphotropic cell line, or PG13, a gibbon leukemia packaging cell line. The producer feeder cells were cultured in MASC growth medium for 48 hours to prepare retroviral supernatant. The supernatant was filtered and frozen at −80 ° C. until use. MASCs that reached near confluence were subcultured in MASC growth medium. After 24 hours, the medium was replaced with a retrovirus-containing supernatant supplemented with 8 g / mL protamine (Sigma), and cultured for 5 hours. This was repeated after 24 hours. Two to three days after the last transduction, using a Consort computer on a FACS Star Plus flow cytometer (both Becton Dickinson Inc.) (Becton Dickinson Inc.) on a 96-well plate coated with 5 ng / mL FN EGFP ⁺ cells were selected at 10 cells per well. Expression of eGFP was seen in 40-85% of adherent cells. Ten eGFP ⁺ cells per well were sorted into fibronectin-coated 96-well plates using an automated cell placement unit (ACDU) on a fluorescence activated cell sorter. Cells were cultured for 1-7 months in MASC growth medium. After 3-4 weeks, adherent cells reached confluence in 3-4% wells. The cells were cultured again and expanded. Fewer than 1 well per plate was produced that produced progeny that grew to more than 10 ⁷ cells (an additional 48 hours of cell division). Therefore, it has 1/10 ⁷ to 1/10 ⁸ of bone marrow cells having further proliferation ability.

  The expanded clonal cell population was divided into 5-10 populations. Some of the cells were cryopreserved in an undifferentiated state, and some were induced to differentiate into osteoblasts, chondrocytes, stromal cells, skeletal and smooth muscle cells, and endothelial cells. It is known that cells are present in differentiated cells by immunohistochemistry and / or Western blot to prove that differentiation has occurred along a given pathway and to identify the type of tissue The protein was examined.

  In order to show that the cell population originated from one cell, the single cell sorting method or the ring cloning method was used. However, since MASC is an adherent cell, two FACS and ring cloning were used instead of one by two. Cells may have been selected. The origin of all differentiated cell clones was investigated using the random integration of retroviruses. Because of random viral integration, the host cell DNA located on either side of the retroviral LTR is cell specific. All daughter cells resulting from cell division can be identified based on the presence of a retrovirus at the same location in the host cell's genome.

  The flanking host DNA on both sides of the inserted retrovirus 3'-side and 5'-side LTRs was amplified by Inverse polymerase chain reaction (PCR). Inverse PCR was performed according to a protocol provided courtesy of Jan Norta (Jan Nolta, Ph.D., LA Children Hospital, Los Angeles, CA). Briefly, DNA is extracted from undifferentiated MASC and differentiated progeny, cleaved with Taq1 (Invitrogen) (Invitrogen), fragments are ligated and Inverse PCR is performed to perform 5 ′ flanking host cells. The DNA sequence was obtained. It was demonstrated that all differentiated cell lines were derived from a single cell by using such inverse PCR or Southern blot analysis primarily for hematopoietic stem cells. After amplifying the flanking DNA, the sequence of 200 to 300 bases was determined, and a primer specifically recognizing the flanking DNA was designed. Subsequently, PCR was performed on undifferentiated cells and differentiated cells using a primer specific for flanking DNA and a primer recognizing 5 'long terminal repeat (LTR) to amplify DNA from differentiated progeny. In each of the three samples examined, a sequence specific to one cell was identified as the flanking sequence of the 5 'LTR. This sequence was the same in undifferentiated cells and differentiated cells. This proves that all cells with “mesoderm” origin originate from a single cell.

  Using this method, the present study confirmed that osteoprogenitor cells are present in the bone marrow and can differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts, and bone marrow stromal cells. did. We further demonstrated that a single bone marrow-derived cell can give rise to cells from the viscera and visceral mesoderm. Furthermore, it was proved that the karyotype of cells cultured for 9 months or more was normal, and the vigorous proliferation ability was due to the nature of the cells as stem cells and not due to tumor formation or immortalization.

Embodiment 5 FIG. Generation of glial cells and nerve cells from living bone marrow mesenchymal stem cells Differentiated neurons have finished dividing and little or no neuronal regeneration has been observed in vivo. If the function of the defective tissue can be restored by introducing a new neural stem cell (NSC) having the ability to divide into the defective region of the brain, great progress in the treatment of neurodegenerative and traumatic diseases of the brain Become. It has been previously discovered that MASCs selected from postnatal bone marrow that differentiate into all mesodermal cells can also differentiate into neurons, oligodendrocytes, and astrocytes.

The culture of MASC was established as described in Example 1. Neurogenesis was induced as follows. Neurons, astrocytes, and oligodendrocytes were generated in a medium consisting of a medium for neural differentiation. This medium contains: 10-95% DMEM-LG (preferably about 60%), 5-90% MCDB-201 (preferably about 40%), 1 × ITS, 1 × LA-BSA, 10 ⁻⁷ to 10 ⁻⁹ M dexamethasone (preferably 10 ⁻⁸ M), 10 ⁻³ to 10 ⁻⁵ M ascorbic acid 2-phosphate (preferably about 10 ⁻⁴ M), and 0.5 to 100 ng / mL EGF (preferably about 10 ng / mL). This medium may further contain one or more of the following cytokines in order to induce differentiation into specific types of cells.

  5-50 ng / mL bFGF (preferably about 100 ng / mL)-astrocytes, oligodendrocytes, neurons (type unknown).

  5-50 ng / mL FGF-9 (preferably about 10 ng / mL)-astrocytes, oligodendrocytes, GABAergic and dopaminergic neurons.

  5-50 ng / mL FGF-8 (preferably about 10 ng / mL)-no induction on dopaminergic, serotonergic, and GABAergic neurons, glial cells.

  5-50 ng / mL FGF-10 (preferably about 10 ng / mL)-no induction into astrocytes, oligodendrocytes, neurons.

  5-50 ng / mL FGF-4 (preferably about 10 ng / mL)-no induction into astrocytes, oligodendrocytes, neurons.

  5-50 ng / mL BDNF (preferably about 10 ng / mL)-dopaminergic neurons only.

  5-50 ng / mL GDNF (preferably about 10 ng / mL)-GABAergic and dopaminergic neurons.

  5-50 ng / mL GDNF (preferably about 10 ng / mL) —GABAergic neurons only.

Growth factors for inducing differentiation of MASC into neural cells were selected based on knowledge in embryonic development of the nervous system or knowledge obtained from studies evaluating NSC differentiation in vitro. All media were serum free and supplemented with EGF, a potent ectoderm inducer. FGF plays an important role in the development of the nervous system. When human postnatal bone marrow-derived MASCs were cultured in the presence of 100 ng / mL bFGF and 10 ng / mL EGF, differentiation into astrocytes, oligodendrocytes, and neurons was observed. Astrocytes are identified as GFAP (glial-fibrillar-acidic-protein) positive cells, oligodendrocytes are identified as glucocerebrosid positive (GalC), neurons are NeuroD, tubulin IIIB (Tuji), synaptophysin, and neuronal The filaments 68, 160, and 200 are identified as cells that in turn express. These cells do not express markers for GAGA-, dopaminergic, and serotonergic neurons.

  FGF-9 is first isolated from the glioblastoma cell line and induces glial cell division in culture. FGF-9 is found in vivo in neurons of the cerebral cortex, hippocampus, substantia nigra, brainstem motor nucleus, and Purkinje cell layer. When 10 ng / mL FGF-9 and EGF were added to MASC and cultured for 3 weeks, astrocytes, oligodendrocytes, GABAergic neurons, and dopaminergic neurons were generated. During the development of the central nervous system, FGF-8 expressed by the forebrain at the mid / hindbrain boundary induces differentiation of midbrain and forebrain dopaminergic neurons along with sonic hedgehog. It has been found that adding 10 ng / mL FGF-8 and EGF to MASC and culturing for 3 weeks produces both GABAergic and dopaminergic neurons. FGF-10 is found in trace amounts in the brain, and its expression is limited to the hippocampus, thalamus, midbrain, and brainstem, and is selectively expressed in neurons and not in glial cells. Incubation of MASC in 10 ng / mL FGF-10 and EGF for 3 weeks resulted in astrocytes and oligodendrocytes, but no neurons. FGF-4 is expressed in the notochord and is required for midbrain regionalization. Treatment of MASC with 10 ng / mL FGF-4 and EGF for 3 weeks differentiated into astrocytes and oligodendrocytes, but not into neurons.

  Other growth factors that are specifically expressed in the brain and affect neurogenesis in vivo and in vitro include brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and ciliary body There are neurotrophic factors (CNTF). BDNF belongs to the nerve growth factor family that promotes in vitro differentiation of NSCs, human epithelial cells, and neural progenitor cells into neurons and promotes in vivo axon generation of hippocampal neural stem cells. Consistent with the known function of BDNF to support the survival of substantia nigra dopaminergic neurons, treatment of MASC with 10 ng / mL BDNF and EGF results in differentiation into only tyrosine hydroxylase positive neurons. It was seen. GDNF belongs to the TGF superfamily. GDNF is expressed in the preneural ectoderm early in neurogenesis, indicating an important role of GDNF in neurogenesis. GDNF promotes the survival of peripheral neurons and muscle motor neurons, and has neurotrophic factor activity and ability to promote differentiation. GDNF has been found to induce the differentiation of MASCs into GABAergic and dopaminergic neurons. CNTF was first isolated from the ciliary ganglion and belongs to the gp130 family of cytokines. CNTF promotes nerve survival early in development. CNTF increases the number of GABAergic and cholinergic neurons in the culture of rat fetal hippocampal neurons. Furthermore, CNTF prevents GABAergic neuron cell death and promotes GABA uptake. CNTF showed a similar GABAergic induction effect on MASC, and MASC differentiated only into GABAergic neurons after 3 weeks of exposure to CNTF.

As stated above, some hematopoietic cytokines such as IL-11 and LIF have been shown to be NSC trophic factors. Furthermore, in vitro studies of neural progenitor cells have shown that SCF, Flt3L, EPO, TPO, G-SCF, and SCF-1 act at an early stage of neural cell differentiation, whereas IL5, IL7, IL9 and IL11 have been shown to act in later neural maturation stages. Early-acting cytokine (10 ng / mL thrombopoietin (Amgen courtesy) (Amgen Inc., Thousand Oaks, CA)), 10 ng / mL granule cell colony stimulating factor (Amgen), 3 U erythropoietin (Amgen), and 10 ng / Ng interleukin 3 (R & D systems) followed by 14 ng / ml fetal liver tyrosine kinase 3 ligand (Immunex Inc.) (Immunex Inc, Seattle, WA) and 15 ng / ml SCF (Amgen) MASCs were induced by culturing for 1 month in medium conditioned by mouse fetal liver feeder layer AFT024 supplemented (by courtesy of Dr. Ihor Lemichka, Princeton University, NJ). Neurons generated under such conditions express neurofilament 68, but do not express neurofilament 200 and are immature.

  In some cultures, retrovirus was used to transduce MASC with an eGFP carrying vector (as described in Example 4 above). Differentiated glia and neuronal cells continued to show eGFP expression. This indicates that these cells can be genetically modified without interfering with their differentiation. Thus, undifferentiated MASCs can generate neural stem cells that later give rise to astrocytes, oligodendrocytes, and neurons.

  MASC is one of the major problems in NSC transplantation, because it is easy to isolate from post-natal bone marrow, grow ex vivo, and induce differentiation into glial cells and specific neural cells in vitro The problem that difficult donor tissue is difficult to obtain is solved.

  The cells of the present invention can be used for cell replacement therapy and / or gene therapy, for example, mucopolysaccharidosis, leukodystrophy (globoid cell leukodystrophy, Canavan disease), fucosidosis, GM2 gangliosidosis, Niemann-Pick disease, San Philip syndrome It is possible to treat or alleviate the symptoms of congenital neurodegenerative diseases and storage diseases such as Wolman disease and Tay-Sachs disease. The MASC of the present invention can treat or alleviate the symptoms of acquired neurodegenerative diseases such as Huntington's disease, multiple sclerosis and Alzheimer's disease. Further, the MASC of the present invention can be used for traumatic diseases such as stroke, central nervous system hemorrhage, central nervous system trauma, peripheral nervous system diseases such as spinal cord injury and syringomyelia, retinal detachment, macular degeneration and other degenerative retinal diseases and diabetes. It can be used for the treatment of retinal diseases such as retinal diseases.

Example 6 Generation of Hematopoietic Cells Hematopoietic stem cells (HSC) are mesoderm-derived cells. HSC has long been thought to be derived from yolk sac mesoderm. There is ample evidence that primordial erythroid cells are derived from yolk sac. It is not clear whether the finished hematopoietic cells are derived from yolk sac cells. A recent series of studies in chicken embryos, mice and human fetuses have shown that the finished hematopoietic cells are derived from native embryos, ie, mesodermal cells present in the AGM region. In humans, a small population of Flk1 ⁺ cells develops in the dorsal aorta on days 22-35 and differentiates into CD34 ⁺ endothelial or hematopoietic cells. These are thought to be cells that colonize the fetal liver. Cells with hematopoietic potential are derived from the dorsal aorta, but for these cells to differentiate and commit to mature hematopoietic cells, the endothelial environment needs to migrate to the liver, which is advantageous for the generation of hematopoietic cells. In contrast, cells remaining in the AGM region do not occur in hematopoietic cells.

  Some clones in the MASC culture of the present invention have hematopoietic potential. This MASC differentiates into endothelial cells to form what appears to be an embryoid body. This cell aggregate differentiates into hematopoietic cells. This microscopic suspension aggregate is trypsinized and replated on FN, type IV collagen or ECM. The medium is 5 to 1000 ng / mL in MASC medium containing 0.5 to 1000 ng / mL PDGF-BB (preferably about 10 ng / mL) and 0.5 to 1000 ng / mL EGF (preferably about 10 ng / mL). Complemented with mL SCF (preferably about 20 ng / mL) or composed of a combination of IL3, G-CSF, Flt3-L and SCF (2-1000 ng / mL, preferably about 10-20 ng / mL) It was used. Alternatively, 5% FCS and 1 in MASC medium containing 0.5-1000 ng / mL PDGF-BB (preferably about 10 ng / mL) and 0.5-1000 ng / mL EGF (preferably about 10 ng / mL). A solution prepared by supplementing ˜1000 ng / mL SCF (preferably about 100 ng / mL) with AFT024 cells was used. Expression of cKit, cMyb, Gata2 and G-CSF-R is observed in cells collected from any of these cultures (RT-PCR / immunohistochemical method) and may be capable of differentiation into hematopoietic cells. Indicated.

Example 7 Generation of Epithelial Tissue Applicants further demonstrated the occurrence of epithelial tissue. Briefly, blood vessels were first coated with 1-100 ng / mL fibronectin and 1-100 ng / mL laminin, other ECM products such as type IV collagen and Matrigel. The medium used consists of the following. 10-95% DMEM-LG, 5-90% MCDB-201, 1 × ITS, 1 × LA-BSA, 10 ⁻⁷ to 10 ⁻⁹ M dexamethasone (preferably 10 ⁻⁸ M), 10 ⁻³ to 10 ⁻⁵ M ascorbic acid diphosphate (preferably 10 ⁻⁴ M). One or more of the following cytokines may be added to this medium.

  0.5-100 ng / mL EGF (preferably about 10 ng / mL).

  0.5-1000 ng / mL PDGF-BB (preferably about 10 ng / mL).

  0.5-1000 ng / mL HGF (hepatocyte growth factor) (preferably about 10 ng / mL).

  0.5-1000 ng / mL KGF (Keratinocyte Growth Factor) (preferably about 10 ng / mL).

  Some of the cells were positive for pancytokeratin and positive for cytokeratin 18 and 19 and these cells were shown to be derived from the endoderm (hepatic epithelial cells, bile duct epithelial cells, pancreatic acini). Cells and gastrointestinal epithelial cells). Some cells have shown the presence of H-Met, a hepatocyte growth factor receptor, specific for liver and kidney epithelial cells. Some cells showed the presence of keratin, a characteristic of skin epithelial cells.

The cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate symptoms of multiple organ diseases. Using this cell, for example, accumulative diseases such as mucopolysaccharidosis, leukodystrophy, GM2 gangliosidosis, hyperbilirubin diseases such as Krigler-Nager syndrome, such as ornithine decarboxylase deficiency, citrullinemia, and argininosuccinic aciduria Ammonia diseases such as congenital abnormalities of the urea cycle such as infectious diseases, amino acid and organic acid abnormalities such as phenylketonuria, congenital hypertyrosinemia, and α1-antitrypsin deficiency, and coagulation such as factor VIII and factor IX deficiency It is possible to treat or alleviate congenital liver diseases such as diseases. The cells can also be used to treat acquired liver disease due to viral infection. The cells of the present invention can also be used for ex vivo applications such as the production of artificial liver (similar to kidney dialysis), the generation of coagulation factors, and the production of proteins and enzymes produced by liver epithelial cells. It is.

  Further, the cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate symptoms of biliary diseases such as biliary cirrhosis and biliary atresia.

  Furthermore, the cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate the symptoms of pancreatic diseases such as pancreatic occlusion, pancreatitis, and α1-antitrypsin deficiency. . Furthermore, pancreatic epithelial cells can be obtained from the cells of the present invention, and nerve cells can be obtained, so that β cells can be generated. These cells can also be used for the treatment of diabetes (subcutaneous transplantation, intrapancreas or intrahepatic transplantation).

  Furthermore, the epithelial cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate symptoms of intestinal epithelial tissues such as intestinal obstruction, inflammatory bowel disease, intestinal infarction, and intestinal resection. Is possible.

  Further, the epithelial cells of the present invention can be used for cell replacement therapy and / or gene therapy to treat or alleviate skin diseases such as alopecia, skin wounds such as burn wounds and albinositis. .

Example 8 MASC, Cartilage and Bone Expressed Gene Profiles We have determined the expression gene profile of human MASCs cultured for 22-26 cell divisions at a seeding density of ² × 10 ³ cells / cm ^{2 from} Clonetech. ) And Invitrogen cDNA arrays, and the following profiles were obtained. Furthermore, the inventors evaluated the change in gene expression when MASC was induced to differentiate into cartilage and bone for 2 days.

  -Pluripotent adult stem cells (MASC) are CD31, CD36, CD62E, CD62P, CD44-H, cKit, Tie, ILI, IL3, IL6, IL11, G-CSF, GM-CSF, Epo, Flt3-L, or It does not express CNTF receptor mRNA and expresses low levels of HLA-class I, CD44-E and Muc18 mRNA.

  MASCs are cytokines BMP1, BMP5, VEGF, HGF, KGF, MCP1; cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130, LIF-R, activin R1 and -R2, TGFR-2, BMP-R1A; expresses mRNA of adhesion receptors CD49c, CD49d, CD29 and CD10.

  -MASC expresses mRNA of hTRT, oct-4, sox-2, sox-11, sox-9, hoxa4, -5, -9, Dlx4, MSX1, PDX1.

  -Oct-4, sox-2, Hoxa4, 5, 9; Dlx4, PDX1, hTRT, TRF1, cyclin, cdk, syndecan-4, dystroglycan, integrin α2, α3, β1, FLK1 in both cartilage and bone , LIF-R, RAR-α, RARγ, EGF-R, PDGF-R1a and -B, TGF-R1 and -2, BMP-R1A, BMP1 and 4, HGF, KGF, MCP1 expression is lost or reduced did.

  -Osteoblast differentiation is HOx7, hox11, sox22, cdki, syndecan-4, decorin, lumican, fibronectin, osteoshaloprotein, TIMP-1, CD44, β8, β5 integrin, PTHr-P, leptin-R, VitD3 -Associated with increased acquisition / expression of FGF-R3, FGF-R2, estrogen-R, wnt-7a, VEGF-C, BMP2.

  -Cartilage differentiation is Sox-9, FREAC, hox-11, hox7, CART1, Notch3, cdki, type II collagen, fibronectin, decorin, cartilage glycoprotein, cartilage oligomeric matrix protein, MMP and TIMP, N-cadherin, CD44, It was associated with the acquisition of α1 and α6 integrins, VitD3-R, BMP2, BMP7.

Example 9 Characterization of genes with different expression levels in MASCs and osteoblasts by subtractive hybridization We have subtracted to identify genetic differences between undifferentiated MASCs and committed progeny. The method was used. Polyadenylated mRNA was extracted from undifferentiated MASCs and cells were induced to differentiate into osteoblast lines for 2 days. A PCR selection kit obtained from Clonetech was used to subtract and amplify cDNAs with different expression levels based on the manufacturer's recommendations without any changes. The gene sequences expressed in day 2 osteoblast cultures were analyzed, but the gene sequences expressed in undifferentiated MASCs were not analyzed.

  The sequences of 86 cDNA sequences with different expression levels were determined. No expression of mRNA was observed in MASC, and it was confirmed by Northern blotting that mRNA was actually specifically expressed in the progeny of osteoblasts on the second day. These sequences were compared to the following database (by BLAST algorithm). SwissProt, GenBank protein and nucleotide collection, ESTs, mouse and human ESTconfigs.

  Sequences were classified by homology. 8 are transcription factors, 20 are involved in cell metabolism, 5 are involved in chromatin repair, 4 are involved in the apoptotic pathway, and 8 are involved in mitochondrial function. 14 were adhesion receptor / ECM components, 19 were known EST sequences with unknown function, and 8 were novel sequences.

  Two of the new sequences were subjected to Q-RT-PCR over 12 hours, 24 hours, 2 days, 4 days, 7 days, and 14 days on MASCs obtained from 3 donors and induced to differentiate into bone. . The gene was expressed during the first 2 and 4 days of differentiation, respectively, and then down-regulated.

Genes present in undifferentiated MASC but not in day 2 osteoblasts were analyzed. The sequences of 30 genes with different expression levels were determined, 5 of which were EST sequences or known sequences. It was confirmed by Northern blotting that these genes are present in MASC but not in day 2 osteoblasts.

Example 10 Establishment of MASC A study was conducted to determine if MASCs established and survived in vivo.

eGFP ⁺ MASC was injected intramuscularly into NOD-SCID mice. Four weeks later, the mice were killed and examined for teratoma in the muscle as reported for human ES cells. No teratoma was found in 5 out of 5 animals. eGFP positive cells were detected. In addition, eGFP ⁺ MASCIV was injected intrauterine into SCID mouse fetuses. Animals were evaluated immediately after birth. PCR analysis showed the presence of eGFP ⁺ cells in heart, lung, liver, spleen, and bone marrow.

  Stereotaxic transplantation of MASCs into normal or infarcted brains of rats revealed that the cells acquired a phenotype found in neurons and this phenotype was maintained for at least 6 weeks. These studies have shown that human MASCs are established in vivo and differentiate organ-specifically without developing into teratomas.

  Furthermore, these studies have shown that MASCs are significantly different from embryonic stem cells and germ cells. MASCs are a new class of pluripotent stem cells that can be obtained from different organs of adults and children.

Example 11 Example of Selection, Proliferation, and Characterization of Mouse-Derived MASC Pluripotent adult stem cells (MASC) can be obtained from mouse bone marrow and can also be present in organs other than bone marrow.

1. Identification of MASCs from mouse bone marrow The researchers selected MASCs from mouse bone marrow. Bone marrow was obtained from C57 / BL6 mice and mononuclear cells or cells depleted of CD45 and GlyA positive cells (n = 6) were used under the same conditions as used in human MASC (10 ng / mL human PDGF-BB and EGF). When bone marrow mononuclear cells were seeded, CD45 ⁺ cells were depleted 14 days after the start of culture to remove hematopoietic cells. As in human MASC, the cultures were replated at a cell density of 2000 cells / cm ² after every two cell divisions.

In contrast to observations in human cells, no growth was seen when fresh mouse mononuclear cells depleted of CD45 ⁺ cells on day 0 were seeded in MASC medium. When mouse bone marrow mononuclear cells were seeded and after 14 days the cultured cells were depleted of CD45 ⁺ cells, cells with morphology and phenotype similar to human MASCs appeared. This indicates that factors secreted by hematopoietic stem cells may be required for the initial growth of mouse MASC. When cultured with PDGF-BB and EFG alone, the cell doubling time was long (greater than 6 days) and the culture could not be maintained over 10 cell divisions. By adding 10 ng / mL LIF, the proliferation rate of the cells was increased, and more than 70 cell divisions were possible. When cultured on laminin, type IV collagen, or Matrigel, cell proliferation was seen but the cells were CD44 ⁺ and HLA-class I positive. As in human cells, C57 / BL6 MASC cultured with LIF on fibronectin-coated dishes are CD44 and HLA-class I negative, positively stained by SSEA-4, oct-4, LIF-R, and The sox-2 transcription factor was expressed.

  MASCs obtained from mouse bone marrow can be induced to differentiate into cardiomyocytes, endothelium and neuroectodermal cells by the method used for induction of human MASC differentiation. Therefore, it can be said that C57B16 mouse-derived MASC corresponds to human bone marrow-derived MASC.

2. MASC is also present in tissues other than bone marrow The inventors investigated whether MASCs are present in other tissues such as the liver and brain. Bone marrow, brain and liver mononuclear cells obtained from 5 day-old FVB / N mice treated with collagenase and trypsin were seeded onto MASC medium supplemented with EGF, PDGF-BB and LIF on fibronectin. After 14 days, CD45 ⁺ cells were removed and the cells were maintained under MASC culture conditions as described above. In cultures initiated with bone marrow, brain, or hepatocytes, cells with morphology similar to mouse MASCs obtained from human MASC and bone marrow of C57 / B16 mice proliferated. These cells expressed oct-4 mRNA.

The inventors further examined mice transformed for the eGFP gene which is the promoter of oct-4. In these animals, eGFP expression was seen in primordial germ cells as well as in postnatal germ cells. Since MASC expresses oct-4, we tested whether eGFP positive cells are found in the bone marrow, brain, and liver of these animals after birth. EGFP ⁺ cells were sorted from the bone marrow, brain and liver of 5 day old mice (brightest 1% cells). When examined by fluorescence microscopy, less than 1% of cells sorted from the brain and bone marrow were eGFP ⁺ . Oct-4 mRNA was detected in the selected population by Q-RT-PCR. Sorted cells were seeded under MASC support conditions (wells coated with fibronectin and added with EGF, PDGF, LIF). Cells survived but did not grow. When transferred to mouse embryonic fibroblasts, cell proliferation was observed. Further transfer to MASC medium resulted in cells with morphology and phenotype similar to MASC obtained from human bone marrow or bone marrow of C57 / B16 or FVB / N mice by classical MASC selection and culture methods.

  While the invention has been described with reference to different specific and preferred embodiments and methods, it will be appreciated that many changes and modifications may be made without departing from the scope of the invention. All references, patents and patent documents are incorporated as if individually incorporated.

FIG. 1 is a diagram showing the possibility of expansion of bone marrow (BM), muscle and brain-derived MASCs. FIG. 2 is a scatter plot showing gene expression in (A) muscle and brain MASC and (B) bone marrow and muscle MASC of gene expression. FIG. 3 shows FACS analysis of MASC cultured with undifferentiated MASC and VEGF. FIG. 4 is a photomicrograph of mMASC colonization and in vivo differentiation. FIG. 5 shows immunohistochemical evaluation using confocal fluorescence microscopy of MASC-derived endothelial cells. FIG. 6 is a photomicrograph of MASC-derived endothelial cells. FIG. 7 shows FACS analysis of MASC-derived endothelial cells. FIG. 8 is a photomicrograph of human MASC-derived endothelial cells. FIG. 9 is a diagram showing spike generation and expressed voltage-dependent sodium current in hMSC-derived neuronal cell-like cells. FIG. 10 shows quantitative RT-PCR and Western blot analysis confirming the hepatocyte-like phenotype. FIG. 11 shows a photomicrograph of hepatocyte-like cells.

Claims (1)

Invention described in the specification .