JPH06508762A - yolk sac stem cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

The present invention is directed to pre-yolk stem cells. In particular, the present invention provides hematopoietic reconstruction and treatment. Concerning the characterization, culture, and use of pre-vitelline stem cells for. Early stage before vehicle height formation Pre-vitelline stem cells isolated from the pre-embryonic yolk cells have mature leukocyte markers and major histocompatibility. Homogeneous morphology and primitive cell surface phenotype without expressing sex complex-encoded antigens shows. The cells can be cultured and expanded for long periods without changing their multipotency. Can be done. Therefore, pre-vitelline stem cells are used to regenerate the destroyed or defective human hematopoietic system. Construction and production of human blood cells and human antibodies, as well as human diseases, immune function, and vaccines. including the construction of large and small animal models for the testing of drugs and immunotherapies. It has a wide range of applications, not limited to these. 2. Background of the invention Multipotent stem cell populations can give rise to blood cells of diverse morphology and function ( Golde, 1991.5 cietific American. December: 86). Blood cell formation initially occurs in the embryonic yolk sac during early embryogenesis. It has been assumed that multipotent hematopoietic stem cells exist in the yolk sac because they can be detected or The characteristics of such cells remain poorly understood, and until now such cells have been (Moore and Metcalf) , 1970.18: 279). During fetal development, the stem cells are transferred to the fetal liver. It migrates and remains for a short time, eventually becoming a permanent site of blood cell formation in the adult. It moves to become the source of bone marrow. Research on the development of blood cells allows us to investigate hematopoiesis. Important growth and differentiation factors have been identified. Furthermore, histological typing techniques Using hematopoietic stem cells as a form of treatment for patients with existing or abnormal hematopoiesis Particularly dramatic progress has been made in 1, 2o1. Hematopoietic stem cells Multipotent stem cells can self-renew and also develop into lymphocytes, granulocytes, and macrophages. -) / divided into various lineages of blood cells including monocytes, red blood cells and selected cells (1'KukuC1kuta) et al., 1992 ．． Ann, Rev, Immunol, IQ: 759) 0 Stem cells are The mechanism of cell lineage transition is not fully understood. The mechanisms involved in stem cell replication are also unknown. The phenomenon is influenced in part by various growth and differentiation factors that specifically regulate hematopoiesis. It is clear that there is a lot going on. Other causes not yet confirmed There may be a combination of t + shi (Menocalf, 1989. Nature: 3 39:27),. Hematopoietic factors known to play a role in -1 include erythroboetin (EPO), granulocyte /7 clofer, ・=10 knee stimulation factor (G/M-C3F), m-granulocytes -11 pieces One Stimulus Factor (G-C3F), McFurage: 30 Stimulus Prisoner f (M -CS F), Interloy “Kinkawa-12 (II, -1-IL-, 12), and stem cell factor (SCF). Understanding hematopoiesis is important for the treatment of hematopoietic disorders. malignant transformation, immunodeficiency, genetic abnormality common and even viral infections are all of different lineages and at different stages of development. May have negative effects on blood cells. For example, basic knowledge of blood cell development is based on certain forms of hematopoiesis. Hematopoietie malignancy and anemia Helps make bone marrow transplantation successful in treatment. Traditional treatment uses whole bone marrow harvested from the iliac crest, but this method has certain limitations. There is a world. Bone marrow stem cells exist in extremely low concentrations, and they are in the final stages of differentiation. It may not even be at an early stage. Obstacles in bone marrow transplantation include The major histocompatibility complex (MHC) between donor and recipient is determined by HLA tissue typing. Must match. MHC class ■ and class ■ one at the main locus in the gene is important for preventing rejection of transplanted cells by the recipient, and What is important is that the donor cells produce an immune response called graft-versus-host disease against the host tissue. It is possible that it may also be a mediator. To promote graft acceptance by the host Immunosuppressants have often been used to treat patients, but this leaves patients susceptible to a wide range of opportunistic infections. make it easier to trollands tested the in vivo potential of embryonic cells. , day 7 embryonic mouse cells can colonize the hematopoietic system of normal unirradiated allogeneic mice. (Hollans, 1988. Br1tish, 1, Haem atol, 69: 437) L, (7) However, there is no Because we used embryonic cells, it is possible that the embryonic cell population actually contributed to this result. It was unknown. In a study on the effects of intrauterine cell transplantation, 9-day yolk sac cells were cells were injected into syngeneic fetuses that differed from the donor cells only at the β-globin locus (thoracic Toies et al., 1989. Proc, Natl, Acad, Sc i, U, S, A, 86: 7456). the donor cells induce hematopoiesis; I understand. Both of these inv:vo team studies isolated mice from mouse embryos. In addition, long-term culture and expanded embryonic yolk sac cells are used to increase their multipotency. There was no suggestion that he would be able to retain his abilities. Such a method is based on the species of origin of the yolk sac tissue. Diffusion chamber embedded inside (Schiff:/ (Symann) et al., Ex p. Hemat, 6: 749° 1978) or yolk sac cells in vitro ro methods leading to malignant transformation. For example, long-term culture cell lines with 10 to 13 are established from day-old mouse embryos, and they are frequently the source of tumor cells. (Globerson et al., 1987. Dif ferentiation 36:185). Therefore, the possibility of tumor formation This makes such long-term cultured cells undesirable for use in reconstructive therapy. 2.2. Major histocompatibility complex The MHC is a highly polymorphic gene complex (Bach and Sachs) (Sachs), 1987. New Eng, J, Med, 317: 489). It was first closely associated with the phenomenon of transplant rejection of tissue grafts. It was discovered by Subsequent research has shown that MHC class It has been conclusively demonstrated that this encoded antigen is a major target of transplant rejection. Ta. Such antigens are expressed by all body cells. NfHc class ■genes are found in a limited array of cells, most of which are related to the hematopoietic system. (array). They are counterattacks by allogeneic immune cells. It can also elicit responses. Studies on the expression of MHC antigens by embryonic yolk sac cells have yielded conflicting results. It was. Virinto studies yolk sac cells in mouse embryos aged 10 to 14 days. Billington and Jenkinson (T. transplantation 18:286.1974) indicates that these cells Expresses both H-2 and non-H-2 (mouse major and minor histocompatibility) antigens I discovered that. Bassey (Pat they) and Edidin's research (Transplantat). ion 15:211.1973), which can cause a strong immune response. It was reported that the antigen first appears in the 7th day of the embryo, but the latter suggests that these antibodies It was suggested that they do not appear in the uterus until day 9 or later. TI(EEARL Y DEVELOPMENT OF MAMMALS 219 (B alls) and Wild (eds.), Cambridge U, 21975 )checking. Heyner reported that the H-2 antigen was detected in 7-day mouse embryos. reported that it was detectable in the fetus (Hayner, 1973. splantation 16:675) o Furthermore, mice obtained on the 9th day of pregnancy It has been shown that yolk sac cells can initiate a graft-versus-host response in vitro. (Hofman and Groverson, 1973. Bur, J. , Immunol, 3:179) o However, Parr (Parr ) et al. showed that H-2 was present on the apical or basolateral membrane of the liver lobe within the mouse yolk sac even on day 200 of gestation. demonstrated the absence of antigen (Pearl et al., 1980. J, Exp. Med, 152: 945), thus, what is being said about the antigenicity of yolk sac cells? No consensus has been established. 3. Summary of the invention The present invention provides yolk sac stem cells, a method for isolating and culturing yolk sac stem cells, and a method for isolating and culturing yolk sac stem cells. A method for using yolk sac stem cells to reconstitute an allogeneic or xenogeneic hematopoietic system. related. The present invention shows that seven mouse yolk sacs isolated from mouse embryos before visible cartilage formation are C. D34'', ThY-1-1 MHC class I- and class H- homogeneous cell collection Based in part on the inventors' discovery that the group contains The long-term i n Vitro culture can increase the number with minimal differentiation, followed by appropriate cultivation. Treatment with hematopoietic growth and differentiation factors can give rise to mature blood cells of diverse lineages. can. Furthermore, the long-term cultured cells mediate antigen-specific immune responses and stimulate lymphohematopoietic functionally able to repopulate the blood cells and prolong the survival of animals whose hematopoietic systems have been destroyed. They can also mature into competent blood cells in vivo. Egg yolk diet of the present invention cells are successfully transplanted into allogeneic fetuses in utero and into non-immunosuppressed xenohosts. and these cells do not induce graft-versus-host and host-versus-graft reactions. Therefore, transplantation will result in tissue chimerism. Using the example of isolating mouse yolk sac cells and characterizing their cell surface phenotype The present invention will be explained. When a homogeneous population of egg yolk phagocytes is expanded in long-term culture, in It was found to retain pluripotency in vitro and in vivo. Specification The invention described in this article encompasses a wide variety of uses for egg yolk phagocytes. Ru. 4,

[Brief explanation of drawings]

Figure 1. FIG. 7 is a schematic diagram of the appearance of an O7 mouse embryo at about day 7 and day 845 of pregnancy. Figure 2. Mouse yolk sac cells from day 7 embryos were measured using flow cytometry. The cysts are apparently more homogeneous than cells from day 8.5 embryos. Figure 3. Mouse yolk sac cells from day 7 embryos express CD34 antigen but not Thy-LMHC class and class ■ antigens. Figure 4. Cultured egg yolk phagocytes can differentiate in vitro into mature blood cells, including (4A) monocytes, (4B) megakaryocytes, (4C) erythrocytes, and (4D) lymphocytes. Ru. Figure 5. Egg yolk meal recovered from recipient mouse pancreas after in vivo transplantation. It has been shown that the cells express mature leukocyte antigens by the donor cells. Figure 6. Antigen with serum from immunodeficient mice treated with yolk sac cells (Fig. 6A, lipopolymer Hemagglutination of red blood cells coated with glycosides and Figure 6B, human serum albumin. This study reveals the recovery of immune function by egg yolk phagocytes in vivo. Ru. Figure 7. Cultured egg yolk phagocytes repopulate chemically excised mouse pancreas and are the source of colony forming units in v:vo; (7A) Chemically excised mouse pancreas and fully repopulate. Comparison of pancreas; (7B) Repopulation on day 7 after yolk sac treatment Expanded pancreas; (7C) Repopulated pancreas 14 days after yolk sac treatment. Figure 8. Intrauterine injection of yolk phagocytes into allogeneic mice results in recombination in neonatal mice. This results in a woven chimera phenomenon. Figure 9. Long-term culture in sheep and goats given multiple high doses of yolk sac cells. Survival and differentiation of fed mouse yolk sac cells. 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to yolk sac stem cells, methods of isolating and culturing the yolk sac stem cells, and methods of using the yolk sac stem cells. Although the specific operations and methods described herein are illustrated using mouse yolk sac cells, they are merely illustrative for practicing the invention. Similar procedures and techniques are equally applicable to all mammalian species, including human subjects. Therefore, human yolk sac stem Cells may be isolated from the embryonic yolk sac prior to formation. CD34'', Th Y-1-1 and MI- (C class I- and class H-) cells may be cultured under the same conditions as described herein below. The development of the fertilized egg can be divided into three different stages: the fertilized egg, immediately after fertilization; Until the moment: the embryo, from the instant until the formation of all the progenitors, and the fetus, from the formation of the last progenitor until birth. The present invention, after confirming the course of development, but with immunodeficiency or rapidly increasing It takes advantage of the unique properties of embryonic yolk sac cells before they lose any of their ability to reproduce. When completely undifferentiated blastula or morula cells are transplanted into a developing animal, they cause a tumor. It is known to cause tumors. These totipotent, tumorigenic cells have no value for use in in vivo reconstructive therapy. However, according to the present invention, specific It is advantageous to transplant cells that have reached a certain stage of differentiation in which they have entered a biological order or lineage. Such cells can be used alone or to deliver genetic material or its expression products into specific tissues of the body, including blood cells. The cell is Transplant into a host before or after transformation with a foreign gene that is the target of taste, and create a target gene. It is necessary to use cells that have matured to the point where they lose totipotency, and fully mature cells will be rejected by the histoincompatible host. cormorant. After all, although it has just lost its totipotency, it still has specific tissue types. It is desirable to use cells that maintain pluripotency. Such cells also maintain the ability to colonize, thus facilitating their delivery to target tissues. Embryonic yolk sac stem cells confer special benefits in hematopoietic reconstitution. These cells are different from those in the embryo. The cells of the yolk sac develop within only a few different tissues. Among these tissues are the hematopoietic system, including red and white blood cells, and tissue of veins, arteries, and capillaries. Thus, by day 8 of mouse embryonic development, mesoembryonic cells within the yolk sac The vacuole begins to form a trap. The cells of the captured bird differentiate, and the surrounding cells become the inner part of the blood vessels in the future. The central cells first become mesenchymal cells, then red blood cells and white blood cells. Catchers establish communication to form a circulation network that extends within the embryo proper. Ru. Because the yolk phagocytes of the present invention do not express MMC antigens, they can mature in allogeneic and xenogeneic hosts. This proves their ability to escape immune rejection. It is something that In contrast, studies on bone marrow cells rely on using immunocompromised hosts. The culture method described herein is applicable to mass culture of lumbar yolk sac cells that maintain the yolk sac in its undifferentiated state, and is capable of providing a large number of receptors. Provide donor cells. 5.1. Isolation of Yolk Phagocytes The embryonic yolk sac is the first identifiable site of hemocyte formation during ontogeny. Egg yolk phagocytes migrate to the liver of the fetus during embryogenesis and eventually migrate to the bones and fall off. They arrive as adults and differentiate into mature blood cells throughout their lifespan at -5. Embryonic development of the mammalian yolk sac is rapid and occurs within a narrow time frame. The mouse yolk sac is fully formed by the seventh stage of pregnancy, and blood is formed in the mesenchyme of the abdominal stem. It becomes detectable in the pre-yolk region and in the vicinity of the yolk. Shortly thereafter, a population of mesenchymal cells clumps together to form a trap. By day 8.5, extensive trap formation in the mouse yolk sac becomes easily visible microscopically. At this stage, the embryo Fetal development reaches a level where the fetal liver is formed, and previtelline cells begin to migrate into the fetal liver. As soon as the previtelline stem cells depart, the previtelline begins to atrophy. Similar phenomena occur in embryo development in other species, but the timing of the developmental phenomena is different. Varies between different species. In humans, the pre-yolk is formed on day IO of gestation, and the pre-yolk forms shortly thereafter. Growth occurs. Thus, human pre-yolk cells isolated at day 100 are comparable to those from day 7 mice. The previtelline is where blood cell formation is first established during development, and the previtelline cells are the most Eventually, they reach the bone marrow and become bone marrow hematopoietic cells, so the pre-yolk stage is where primitive hematopoietic cell precursors develop. It is inferred that it corresponds to the earliest part for life. The cells move into the hematopoietic differentiation pathway. As a result, they are no longer omnipotent. However, previtelline cells remain multipotent. These are the lymphocytic, myeloid, and erythroid lineages. This is because they have not yet transitioned into a specific blood cell lineage, as evidenced by their ability to become line cells. From now on, pre-yolk cells include bone marrow transplantation, but that It can be said that this is an ideal cell population for use in reconstructive therapy, including but not limited to. Canada Additionally, due to their primitive nature, these cells are not homologous, as evidenced by the absence of various maturation markers and cell surface expression of MHC transplant rejection antigens. It can be said that these cells can be uniquely used as a universal donor cell population even in heterogeneous and heterologous hosts. Pre-yolk isolation of the embryo can be performed using various surgical methods. Traditionally, the preyolk of mouse embryos has been dissected by the use of enzymatic digestion and mechanical separation with surgical excision. Let go. A gentler method of detaching the cells from the yolk sac membrane and separating them from each other is described in Section 6.2.1. There, the cells are dissociated by dipping the yolk pre-yolk into an EDTA solution to form a single-cell suspension. This method minimizes cell lysis due to physical forces and modification of surface orchids due to enzymatic treatment. Since the establishment of pre-yolk follicles indicates the initiation of cell differentiation and hemocyte formation, it is preferable to isolate pre-yolk cells prior to extensive fowl formation. using the methods described herein. It is possible to isolate a highly homogeneous large number of previtelline cells of day 7 mouse embryos (or human previtelline cells at a similar stage), and the cells obtained at this stage are, in principle, , the least migrated and least differentiated multipotent stem cell These stem cells should contain cells, and these stem cells should be directly cultured in long-term in vitro. It is suitable for use in in vivo therapy or as a carrier of a specific foreign gene for use in gene therapy. In order to maintain the pre-yolk cells for a long time, a relatively high concentration of blood, i.e. 15-20% The cells are grown in medium containing supernatant supplements. Various cytokines may be added to inhibit differentiation of the stem cells, such as leukemia inhibitory factor (LIF) or stem cell factor/c-kit ligand (SCF) or IL-3. These factors include, but are not limited to, SCF in combination with other cytokines. Such factors accelerate proliferation of cultured cells while inhibiting cell differentiation in VitrO. All were performed using pre-yolk cells grown in the presence of 10-100 U/- of LIF. However, a more potent (higher concentration of LIF may be used to suppress bidifferentiation). Similar results were obtained by growing cells using SCF (IL-3). Several other known hematopoietic factors such as , C3F and EPO are also present in individual cells. Combinations may be used depending on the needs to be selected for the cell type. example For example, when IL-3 and EPO are used in combination, cultured previtelline cells can be transformed into red blood cells. It helps propel the ball onto its path. Appropriate temperature, CO, humidity, humidity level and culture medium It is within the common knowledge in the art to maintain cells with a frequency of soil exchange. 5.2. Characterization of pre-vitelline cells is important because, as can be seen from the examples described herein, pre-vitelline cells obtained from mouse embryos are apparently more homogeneous than cells obtained at later stages. And Ru. Pre-yolk cells freshly isolated from day 7 and day 8.5 mouse embryos were Comparison was made by light scattering using the conventional method. 6.2.1 below. See section. Pre-yolk cells from a 7-day-old mouse embryo have a large cell size. It is evident that it is extremely homogeneous both in terms of size and shape. By day 8.5, distinct cell populations are clearly visible. This means that eggs at an earlier stage Previtelline cells can be obtained clonally, suggesting that diurnal differences in development are critical to the properties of previtelline cells. Another indicator of the primitive nature of early previtelline cells is the presence of various lineage-specific hemocyte markers. their cell surface phenotype regarding the expression of This form of analysis This can be most easily carried out by using a heterologous monoclonal antibody. When 7-day pre-yolk cells were reacted with antibodies, they were found to have all mature blood cell markers. The result was that the expression of Carr was lacking. In addition, such cells did not express MHC-encoded products, which are major targets of transplant rejection responses. Thus, pre-vitelline stem cells can be characterized as CD34'', Thy-1, MHC Krasny and Kras-2. Similarly, pre-vitelline stem cells obtained from day 100 embryos display the same cell surface phenotype. The CD34 and Thy-i markers have previously been shown to be associated with bone marrow hematopoietic stem cells (Spangrude et al., 1988.5 science 241:58). CD34 table when maturing Although the current state is attenuated, the existence of "rhy-t" is maintained and its density remains constant in mature blood. It increases in cells, especially T lymphocytes. The finding that previtelline stem cells are positive for CD34 expression is consistent with these cells being stem cells. But long The absence of ThY-1 expression suggests that pre-vitelline cells represent an earlier cell population than bone marrow stem cells, which express low levels of ThY- in the bone marrow microenvironment. suggest. In fact, when egg yolk phagocytes are cultured in vitro, a small proportion of the cells escape the action of LIF and begin to express 'rhy-i. This means that the expression Thy- This further suggests that this is a phenomenon occurring at a later stage of cell development. MHC-encoded classes and class molecules are responsible for the immune system between T, B, and antigen-presenting cells. Involved in epidemic regulation. These highly polymorphic molecules also serve as targets in major transplant rejection responses between genetically mismatched individuals. Therefore, HLA histology is now used to match donor and recipient at major MHC loci. routine clinical procedures to confirm graft acceptance in human transplant patients. In order. The absence of MHC antigens on the egg apparatus cell surface indicates hematopoietic reorganization. This strongly suggests the possibility of using such cells as universal donors in biomedical therapy, and this suggests the necessity of different tissue types and the necessity of restricting the use of only MHC-matched tissues as donor cells. Reduce. The development of adoptively transplanted yolk phagocytes into the environment of the host results in a mutually specific tolerance between the host and the donor cells, which leads to graft-versus-host and host-versus-graft reactions. Reduce the possibility of triggering. The yolk sac phenotype described above is observed in the majority of cells isolated from day 7 mouse embryos. Ru. Therefore, early isolation of yolk phagocytes results in highly homogeneous and purified stem cells. A population of cells is provided. This is in contrast to the purification procedures required for murine bone marrow hematopoietic stem cells of the CD34+ and 'rhy-i° phenotype. Such cells must be isolated and purified by successive selection steps, since they constitute less than 0.1% of the total cells in the bone marrow (Spangrud et al., 1991. Blood 78: 1395). On the other hand, egg apparatus stem cells require additional purification. They can be obtained in an essentially homogeneous state without the need for such cells, yet such cells retain their phenotypic and functional activity during long-term in vitro growth. 5.3. Functional Activity of Egg Yolk Phagocytes The pluripotency of egg apparatus stem cells to differentiate and mature into functionally competent blood cells of various hematopoietic lineages was tested by several in vitro and in vivo methods described herein. First, the existence of a multipotent population in long-term cultured yolk-competent cells was confirmed as follows. After 10 passages of 1 in vitro expansion, the vitelline competent cells were washed of LIF and exposed in culture to a cytokine combination containing IL-3, C3F and EPO at previously identified optimal concentrations for an additional 3 weeks. At the end of the period, the stimulated vitelline cells were prepared as blood smears and stained with hematoxylin. The results of this analysis indicate that red blood cells, granulocytes, megakaryocytes, and lymphocytes The appearance of blood cells that can be identified as In addition, donor cells were administered 4 weeks after in vivo injection into allogeneic 5CID mice. Similar studies were performed in vivo by harvesting cells. used in this study The egg yolk phagocytes used had been expanded for more than 40 passages in culture. Donor Cells Antibody specific for H-2' hablotibe and 3220 for B cells, for T cells Double staining of pancreatic, bone marrow, and thymocytes of 5CID mice was performed using antibodies against mature blood cell markers such as CD3 and Thy-1 for macrophages, and Mac-1 for macrophages. The results of this in vivo study This confirms in VitrO studies that yolk sac cells can give rise to mature T cells, B cells and macrophages/monocytes. In addition to morphological evidence of blood cell maturation from yolk phagocytes, adoptively transferred yolk phagocytes were tested for functional activity in the form of specific antibody production. One month after administering the egg yolk phagocyte injection, the mice were treated with lipopolysaccharide (LPS) or human serum albumin. immunization with either min (HSA). Mouse serum was serially diluted, reacted with the two antigens, and compared with normal mouse serum as a control. compared. LPS is a T cell independent antigen that directly activates polyclonal B cells. Ru. The high titers of LPS-specific antibodies in the serum of beige nude mice that retain egg apparatus cells after LPS immunization indicate the ability of the cells to produce B lymphocytes and plasma cells. This indicates the presence of a competent antibody. Furthermore, H3A, a T cell-dependent antigen, is weakly expressed in mice. elicited the production of specific, yet detectable, antibodies. Anti-H3A antibody response These T cells also require the help of antigen-presenting cells such as macrophages. This results in the development of mature and functional antibodies, as they are first activated. Provides evidence for the existence of T cells, B cells, and macrophages that cooperate and interact in life. As a corollary, this may lead to cytotoxicity, lymphinactivity and cytotoxicity. Other T cells and macrophages such as tocaine secretion, phagocytosis, antigen processing and presentation It has also been suggested that all enzyme-mediated functions can arise from the transplanted vitelline competent stem cells. In vivo transplantation of egg yolk phagocytes also allows the hematopoietic system to undergo chemical ablation or exposure to lethal doses. repopulated the pancreas of mice that had been previously destroyed by injection. This is due to a defect in the patient's lymphohematopoietic system due to a genetic disorder or acquired viral infection. or death with chemotherapy or radiation therapy to eradicate tumor cells in the bone marrow. This is similar to a situation where a patient's system is destroyed at will. Administration of egg yolk phagocytes resulted in the development of colony forming unit pancreata (CFU-8) in mice whose pancreas often took on a necrotic appearance after lethal dose irradiation or chemical resection. On the other hand, when egg yolk phagocytes were expanded over a period of time in vivo, this resulted in an apparently completely normal repopulation of the pancreas. assisted in the cultivation and restoration of the area. Additionally, mice treated with egg apparatus cells had longer survival times compared to untreated control groups. Therefore, long-term cultured yolk sac cells are effective for bone marrow reconstitution. It may be useful in a variety of settings where it may be applied as an effective therapeutic measure. Transfer of mature mouse egg cells to allogeneic fetuses and xenogeneic neonatal animals in utero The transplant did not induce graft rejection. The egg yolk phagocytes live in vivo. continued to exist, and established hematopoietic chimerism in the host's pancreas, liver, and peripheral blood. Thus, yolk phagocytes are universal donor cells in various mammalian species, including humans. It can be said that it is useful as a cell. (Margins below this page) 5.4. Use of Previtellular Stem Cells The absence of MHC antigen expression by previtelline stem cells provides a source of do6-cells for in vivo transplantation and reconstitution therapy. cells produce large numbers for more effective dosing. Use immediately after isolation from preyolk or long-term expansion in vitro to Can be used. Introduction of foreign genes into pre-yolk cells can be achieved by conventional methods during in vitro culture and/or in vivo gene therapy. Long-term culture cells may be used as a mixed population or progenitor cells may be cultured with CD34'', Thy-i, MHC class I- and class II-sources prior to in vivo use. Preselection may be based on initial phenotype or by limiting dilution cloning. 5.4.1. Human yolk sac cells in mice Human egg apparatus cells can be obtained, expanded in vitro, and transferred into immunodeficient or immunocompromised mice. Such mice contain the human hematopoietic system and are useful for studying human blood cell development in vivo, identifying novel hematopoietic growth and differentiation factors, and influencing various stages of blood cell formation. used in testing anti-cancer drugs as well as cytotoxic and/or inhibitory compounds. Can be used. Such chimeric mice, referred to herein as HumatoMouse(TM), are a normal 5CID/Hu mouse model in which the mice are reconstituted with human bone marrow stem cells. better than le. This is because FumatoMouse™ allows studies to outline the first phenomena of hematopoiesis. Furthermore, pre-vitelline cells can be implanted in utero into normal mouse fetuses for transplantation of human blood cells in a normal mouse environment. child Pre-vitelline cells such as the vitelline cells can be transferred with drug resistance genes to allow subsequent selective excision of only the host cells using the corresponding drug. It is important to note that SCI mice are not completely immunodeficient; It was observed that the degree of recovery was correlated with the age of the mouse. 5CID mice have detectable natural killer cell and macrophern activity. Even a small proportion of mice reacquire T and B cell function as they mature. In this way, the normal 5CID mouse has the Fumatomouse (trademark) structure. It may not be the best host for growth. This is because their immune function can interfere with the analysis of donor pre-yolk cells. Steel mice have a mutation in the Steel locus encoding SCF, a ligand for the proto-oncogene C-Kit cell surface receptor. Mouse fetuses that are homozygous for this mutation are Only about 15 days of pregnancy survive before being miscarried due to the absence of a blood system and blood cell formation. Therefore, human previtelline cells can be injected into homozygous fetuses developing in utero, e.g., 8 months prior to miscarriage, to reconstitute their hematopoietic function. The resulting neonate should have a fully humanized system with no contributions from the host. This is because they normally do not survive to birth. The studies described herein demonstrate that cultured pre-yolk cells develop into mature blood cells in vivo. This suggests that these cells secrete growth and differentiation factors necessary to support their own development. Further improvements to the FumatoMouse™ model include the introduction of human growth and differentiation factor genes into the mouse. nothing. Several species of cytokines important for human blood cell formation, such as SCF, are species-specific and murine molecules can be effectively engineered to promote human cell growth and differentiation. If not used, transgenic 5CID or Steel mice can be configured to provide endogenous production of human cytokines of interest, such as IL-3, C8F, and SCF. Also, human previtelline cells can transfer the mouse receptor gene. Subsequent transfer of human previtelline cells into these mice A more complete and efficient human hematopoietic system should be developed in mice. 5, 4.2. Transplant xenografts using previtelline cells, i.e., repeated transfers of high-dose, long-term cultured mouse previtelline cells into sheep, allow these cells to persist in vivo, differentiate into mature lymphocytes, and develop into graft cells. It was shown that it does not transmit host diseases. Although mature donor mouse cells ultimately express MHC antigens in vivo, donor cells are abundant in the peripheral blood of the xenogeneic host. Graft rejection (host vs. graft) and The absence of a graft-versus-host reaction contributes to the primitive nature of pre-vitelline cells, particularly the lack of MHC antigen expression, causing these cells and the host immune system to “learn” from each other prior to MHC expression. xenotransplantation of solid organs has been performed in humans in situations where there is a paucity or presence of HLA-compatible organs. Pre-cells can be used, for example, to reconstitute the hematopoietic system of mammalian species in humans infected with HIV. Since non-human T cells cannot be infected by human old V, this method is Previtelline cells can also be used to transfer genes designed to disrupt old gene sequences involved in old replication prior to in vivo administration. can be entered. Such exogenously introduced genes may encode antisense RNA or ribozyme molecules that specifically interfere with HIV replication. Furthermore, the induction of tolerance by the transfer of xenogeneic pre-yolk cells can be applied to solid organs (including, but not limited to, heart, liver and kidney) from donor animals that share the same genetic make-up of the pre-yolk donor. may enable subsequent transplantation of. This opens up the possibility of using M) IC-incompatible yolk sac cells not only for reconstitution purposes but also as a first-step tolerogen for inducing special tolerance in recipients of subsequent organ transplants. enhance In addition, this form of pre-yolk cell transplantation may be applied in situations where a genetic defect has been detected in the fetus. Has a normal wild-type gene or an exogenously introduced gene Previtellular cells of humans or other mammals can be injected into the developing fetus in a routine procedure similar to that of intrauterine amniocentesis. Genetic diseases to which this method may be applicable include, but are not limited to, pyramidal cell anemia, thalassemia, and adenosine deaminase deficiency. Also, previtelline cells can be used in situations such as when a pregnant woman is diagnosed with old II, and reconstitution of the fetus with previtelline cells can prevent viral infection of the fetus. The ability of previtelline cells to grow in xenogeneic animals without irradiation or chemical treatment allows for large scale production of human hematopoietic cells and their secreted factors in vivo. Human previtelline cells are injected into large farm animals, blood is collected, and large amounts of human proteins or cells, such as red blood cells, lymphocytes, granulocytes, platelets, mole Noclonal antibodies and cytokines can be purified for clinical use. 5.5. Human Bone Marrow Replacement Therapy The protocol for the exchange of bone marrow cells in patients requiring bone marrow transplantation is It can be devised using cultured human yolk sac cells or xenogeneic yolk sac cells. Previtelline cells obtained from the 100th day of pregnancy human yolk sac were harvested using the procedure described herein. The cells can be isolated using cells, expanded in culture, and cryopreserved as donor cells for transplantation. Ablation of the recipient patient's bone marrow cells may not be required, but if it is used, it may be irradiated according to conventional methods (Kim et al., Radiology, 122:523.1977) or various Commonly used compounds (Busul) (Tutschka et al., Blood, 70:1382-1388°1987)). Pre-yolk cells can be introduced into a recipient using methods similar to bone marrow cells. Prior to in vivo transfer, pre-yolk cells may be transformed with a drug resistance gene, such as a methotrexate resistance gene. This allows subsequent use of high doses of the corresponding chemotherapeutic agent to completely eliminate less resistant host cells in the patient, without damaging the transferred previtelline cells. Postoperative patient management uses donor bone It would be the same as a transplant using bone marrow cells. High doses of previtelline cells obtained from allogeneic or xenogeneic sources may be or can be continuously infused into bone marrow recipients in the absence of radiation therapy. This represents a new method of bone marrow transplantation that does not involve immunosuppressing the recipient. 5.6. Identification of Novel Markers for Previtellular Cells Mouse previtellular cells express CD34 but do not express any of the other known leukocyte markers. Preyolk cells may express other early markers that have not yet been identified. If so, the previous failure to identify these unique molecules may be due to their reduced expression in more mature cells or even stem cells after migration from the yolk sac to other sites. It can be. Therefore, previtelline cells can be used to generate antibodies to these cell surface antigens in order to identify and characterize such unknown markers. In addition, polyclonal clones that recognize novel antigenic markers expressed by pre-yolk cells The production of monoclonal antibodies and monoclonal antibodies is within the scope of the invention. A variety of procedures known in the art can be used to produce pre-yolk cell antibodies. For antibody production For this purpose, various host animals can be prepared such as viable pre-vitellular cells, fixed cells or membrane preparations (including, but not limited to, rabbit, hamster, mouse, rat, etc.). can be immunized by injection with Different adjuvants can improve immunity depending on the host species. These adjuvants are Freund's (complete and complete). mineral gels such as aluminum hydroxide, surface-active substances, e.g. Zolecithin, pluronic polyol, polyanion, peptide, oil emulsion tion, keyhole limbet hemocyanin, dinitrophenol, and potentially beneficial human adjuvants such as BCG (tuberculosis Calmette-Guerin) and Corynebacterium parvum. Monoclonal antibodies to novel antigens related to pre-yolk cells have been developed for continuous passage of cells in culture. may be prepared using any technique that provides for the production of antibody molecules by cell systems. Ru. These include the hybridoma technology first described by Kohler and Milstein (1975, Nature 256.495-497), and the more recent human B cell hybridoma technology CKosbor et al. 1983. [mmunology Today 4 Near 2; Cote et al., 1983. Proc. Natl, Acad, Sci, 80:2026-2030) and EBV-Ha including, but not limited to, hybridoma technology (COIe et al., +985. Monoclonal Antibodies and Cancer Therapy. Alan R, Li5s, Inc., pp. 77-96). Genes from mouse antibody molecules of appropriate antigen specificity are generated together with genes from human antibody molecules. Techniques developed for the production of "chimeric antibodies" by splicing genes can be used (eg, Morrison et al., 1984. Proc, Natl. Acad, Sci. 81:6851-6855; Neuberger et al., 1984. Nature, 312: 604-608HTakeda et al., 1 985. Nature 314:452-454). Alternatively, the techniques described for the production of waterway antibodies (US Pat. No. 4,946,778) can be employed to produce waterway antibodies. Syngeneic, allogeneic and xenogeneic hosts can be immunized with previtelline cells, which can be prepared in viable or fixed form or with extracted membrane fragments. Noclonal antibodies are effective in mature macrophages, granule cells, T cells, and B cells. They can be differentially screened by selective binding to pre-yolk cells rather than to pre-yolk cells. Antibody fragments containing binding sites for that molecule can be produced by known techniques. For example, this fragments such as F(ab')2 fragments that can be produced by pepsin digestion of antibody molecules. and F(ab”)2 fragments by reducing the disulfide bridges. Fab fragments including, but not limited to: BALB/C7 mice, C57BL/67 mice, beige nude X-linked immunodeficient (BNX) mice, and C3H/5CID mice were purchased from Jaclyn Laboratories (Bar Harbor, ME) and purchased from Edison Animal Biotech. The animals were housed in the animal facility of the Edison Animal Biotechnology Center. 6゜1.2. Yolk Sac Isolation On day 7 of pregnancy (the day of plugging was counted as day 08), female mice were sacrificed by cervical dislocation and the uteri containing the fetuses were placed in Delbetzko's saline phosphate buffered solution (PBS). ) + penicillin and streptomycin antibiotics (final concentration: 1.000 units of potassium penicillin G and 1000 μg of streptomycin sulfate) cells/ml) in a Petri dish. Under a laminar air flow bench, each uterine fragment containing the fetus was removed aseptically by dissection with the aid of a dissecting microscope. Each fetus, surrounded by the encapsulated decidua, was transferred to a separate Vetri dish containing PBS+peninosilicate streptomycin. Encapsulation The decidua was opened with watchmaker's forceps and each fetus was transferred to an individual Petri dish where the yolk sac tissue was incubated in 002% EDTA in PBS for 15-30 min at 4 °C. The amnion, placenta, fetus, and Reichard's membrane were dissected across the membrane. The pre-yolk cells in single cell suspension were then washed in PIJS before culturing. 6.1.3. Culture conditions Isolated pre-yolk cells were incubated with 18% heat-inactivated fetal calf serum and 0.2 Mm β-methane. captoethanol, 50 μg/ml gentamicin and 10% LIF. The cells were grown in alpha medium (Sigma) supplemented with mustard medium (100-1000 u/ml of the LIF producing cell line, Ch. LIFD). Collage cells The cells were grown without a feeder layer in cells or gelatin-coated dishes and incubated at 37°C in 5% CO2 in air. Medium was changed every other day. 6, 1.4. Flow Cytometry Analysis 10 6 pre-yolk cells were washed twice in cold BPS containing O, IBSA and sodium azide. Cell pellets were suspended in the same buffer containing test antibodies for 30 minutes at 4°C. Cells were then washed twice in cold BPS and analyzed by flow cytometry. Thy-t, Ly-1, LY-2, Mac-1, MHC class I and class II-specific antibodies The body was purchased from Boehringer Mannheim. Anti-Ml/70, anti-H2', and anti-H2'' antibodies were purchased from Pharmingen (San Jego, CA). Anti-CD34 was used as the hybridoma supernatant. 6.1.5. Induction BALB/c yolk sac cells were grown to approximately 50% confluency in medium containing vitelline IF. I set it. Cells were harvested, washed, and medium containing growth factors was added. Growth factor used The offspring received various combinations of LIF (100-1000 U/ml), 5CF (50 tJ/ml), EPO (1-25 LI/ml), IL-2 (10-200 [J/ml), and IL-3 (10-200 U/ml). reach confluence The medium was changed every 2 days until the pre-vitellular cells were plated in a 1=4 ratio into fresh gelatinized 35 mm culture dishes. On day 5 and month 211, cells were stained with blood. Prepared for color. Day 0, Day 5, and Day 211 cells were analyzed by flow cytometry for the appearance of differentiated blood cells. 6, 1.6. Blood aggregation assay Lipopolysaccharide (LPS) conjugated to trinitrophenol (TNP) and human serum albumin (ISA) conjugated to TNP were administered to BNX and 5CID mice, respectively (both of which were pre-injected intraperitoneally with 108 Mouse yolk sac thin (receiving cysts) were injected intraperitoneally at 20 μg/mouse. A second injection of antigen was given one week later, the animals were bled 7 days later, and the serum was assayed for the presence of specific antibodies. Two-fold serial dilutions of mouse serum were performed in microtiter plates. Dinitrophenol (DNP)-coated sheep red blood cells (SRBC) were added to each added to the well. Plates were incubated for 1 hour at room temperature. Check the test results It was evaluated visually. The diffusion pattern of 5RBCs showed a positive TNP-specific antibody response. Negative wells had a small compact pellet of 5 RBCs. In mice, the yolk sac is fully formed by day 7 of gestation and continues until day 8.5. A trap formation is usually seen. Therefore, in order to isolate homogeneous undifferentiated previtelline cells, mouse fetuses were surgically removed prior to the formation of visual traps, preferably on day 7 of pregnancy. The vitelline true region of the fetus was separated by excision and the outer surface of the yolk sac was immersed in cold EDTA, which resulted in the separation of the previtelline cells from the membrane into a single cell suspension (Figure 1). Physical extravasation of pre-yolk cells obtained from day 7 and day 8.5 fetuses Freshly isolated 7-day-old cells revealed a much more uniform cell shape and cell size than 8.5-day-old cells when compared by flow cytometry analysis. This clearly shows that pre-yolk cells were a homogeneous population at day 7, but by day 8.5, differentiation activity had already occurred resulting in a mixed population of cells in the yolk sac. (Figure 2). Therefore, day 7 pre-yolk cells were used for all in vitro and in vivo studies described herein below. 6, 2.2. Cell surface phenotype of pre-yolk cells freshly isolated from day 7 mouse fetuses. Several known leukocyte malignancies were detected by reactivity of pre-yolk cells with monoclonal antibodies. The markers were immediately examined for their cell surface expression. This kind of uncultured Yolk sac vesicles express CD34 but not Thy-1, MHC class antigens and class II antigens (Figure 3). Expression of CD34 by pre-yolk cells is This is consistent with the fact that these are primitive stem cells. This is because CD34 is currently the first detectable marker for bone marrow hematopoietic stem cells. Absence of MHC antigen expression at this stage may be due to genetically mismatched hosts during in vivo transfer. Importantly, the possibility of rejection of these cells is greatly reduced. Furthermore, the lack of Thy-i expression indicates that the pre-vitelline cells of the present invention represent an earlier cell population in ontogeny than the Thy-i "hematopoietic stem cells found in the bone marrow, and thus may not be associated with either trait. This indicates that it should contain a pluripotent population that is less committed to different cell lineages. 6, 2.3. Long-term maintenance of pre-vitelline cells Pre-vitelline cells isolated from a 7-day-old fetus were established in culture in the presence of 10-100 U/ml of leukemia inhibitory factor (1, IF) without the use of a feeder layer. did. Cells increased in number and had a doubling time of approximately 18 hours. Such cultured cells can be grown in continuous culture. The cells were propagated in vitro for over 41 passages covering a period of over 9 months in culture. Also, previtelline cells were able to proliferate in stem cell factor with similar results. LIF can inhibit previtelline cell differentiation over extended periods of in vitro proliferation. However, the effect of LIF is incomplete. After all, a small fraction of the cultured cells began to express species differentiation markers comprising Thy-molecules and MMC-encoded molecules. It is et al. However, the majority of long-term cultured cells retained their initial cell surface phenotype. Furthermore, such cells remained pluripotent, as demonstrated by their ability to give rise to mature blood cells in vrtro and nvlvo, as described below. Cells with the initial phenotype in long-term culture may be obtained by cell sorting or by repeated limiting dilution cloning. 6, 2.4. DIFFERENTIATION OF PRE-VIETRO CELLS IN VITRO After one month of in vitro culture in the presence of LIF, pre-vitelline cells were differentiated in response to various known hematopoietic growth factors including IL-3, IL-2, and EPO. Differentiation into mature blood cells of all lineages These abilities were tested. When cultured in IL-3 and EPO, the appearance of red blood cells could be easily detected in the yolk incubation medium. In response to C3F and IL-3, yolk phagocytes induce megakaryocytes and condylar cells. matured into granule cells. Figure 4 is a blood stain of egg yolk culture grown in the presence of a combination of cytokines, in which the appearance of various blood cell lineages can be identified. In addition, this The expression of various leukocyte surface markers (including CD34, CD45, LFA, MAC-1, Ly-1 and Ly-2) by these cells becomes detectable. 6, 2.5. IN VIVO differentiation of egg yolk phagocytes Egg yolk phagocytes originating from BALB/C Long-term cultures of cells were injected into allogeneic C3H/5CID mice after 22 passages in vitro. After 4 weeks, the pancreas and liver of treated animals were treated with monoclonal anti- The body was analyzed for the presence of donor cells. Donor cells were identified by antibodies specific for donor H-2' habrotibs. Double staining experiments using two antibodies further demonstrated that the aliquots consisting of donor cells expressed CD3, Thy-1, B220 and Ml/70 (Figure 5). Therefore, these results indicated that long-term cultured mouse yolk sac cells can spontaneously differentiate into T cells, B cells and macrophages in vivo. 6, 2.6. Production of immunocompetent cells by IN VIVO egg yolk phagocytes Egg yolk preparation To examine whether the follicles are capable of giving rise to functionally mature blood cells, long-term cultured yolk cells were transferred in vivo into allogeneic 5CID or BNX mice and tested for specific antibody production. BNX mice receive egg yolk phagocytes When immunized with LPS and subsequently immunized with LPS one month later, specific antibody titers were detected in the serum. (Figure 6). Since LPS is a polyclonal B cell activator, this result indicates the presence of functionally active antibody producing B cells. Furthermore, when 5CID mice were injected with egg yolk phagocytes and subsequently immunized with ISA (which is a T cell-dependent antigen), antibody responses were again detected, indicating that long-term cultured yolk phagocytes had differentiated. In v: Suggests that cells can become immunocompetent T cells and B cells in vo. 6, 2.7. Chemotherapy drug that repopulates chemically ablated mouse pancreas with yolk phagocytes A class of agents have been effective and used as bone marrow ablative agents in bone marrow transplant operations (Floersheim and Rus-zkiewicz, 1969. Nature 222:854). One of the most effective drugs used to replace it is the drug busulfan (Tutschka et al., 1987. Bfood 70:1382). Careful titration of the busulfan dose and the use of an inbred strain (C57BL/6) of mice of a specific age and weight (3-4 weeks old) allowed for complete bone marrow ablation in these mice. However, a dose of busulfan was determined that would not directly kill them. These blocks The dose of sulfan is 11-14 days after treatment if they do not receive a bone marrow transplant. This results in the ultimate death of the mouse. This dosage is 1. P, single dose by injection 65 mg of busulfan (per gram of body weight) administered per day. C57BL/6 mice were treated with this dose of busulfan and then busulfan 1. of 106 syngeneic cultured vitelline cells 24 hours after treatment. P. When receiving injections, recipient animals received pancreatic cancer on days 7 and 144 of busulfan treatment. Visceral repopulation was revealed (Figure 7). On day 7, pancreatic colonization in the recipient animals was observed, indicating the early stages of pancreatic repopulation due to transplantation. Additionally, 122 days after treatment, the pancreas of control busulfan-treated mice that did not receive vitelline implants and Comparison of the pancreases of these animals receiving explants showed significant differences in pancreatic viability. The pancreas of the control animals was dark, almost black, and clearly necrotic, whereas the pancreas of the recipient animals was red/pink, clearly normal and healthy. Furthermore, the survival time of mice treated with egg yolk phagocytes was extended by 18-20 days. 6, 2.8. A long-term cultured yolk sac cell line in which intrauterine administration of yolk phagocytes produces tissue chimerism was tested for its ability to survive in an allogeneic host. 10.000-50.000 BALB/c yolk sac cells after 13-20 passages in vitro were injected intrauterinely into 8-day-old fetuses of 7BL/6 mice. At birth, the neonatal pancreas and liver were collected and analyzed for the presence of donor cells. Since the donor cells were of the H-21 hablotype, they were identified by flow cytometry analysis using a monoclonal antibody specific for the H-2d antigen. Figure 8 represents the results from the two neonates tested, which revealed that donor cells were present in significant numbers in both the liver and pl,i of the recipient mice. Crab is shown. Therefore, intrauterine administration of egg yolk phagocytes to MHC-incompatible mice may lead to tissue chimerism, and cell survival and homing of cells to lymphohematopoietic organs. Tissue chimerism was preserved even when mouse tissues were harvested at one month of age. 6, 2.9. Xenotransplantation of egg yolk phagocytes results in long-term survival of cells in vivo. To test the feasibility of using yolk phagocytes in seed transplantation and reconstitution, long-term cultured BALB/c yolk sac cells and yolk-viable cells in pusher sheep and Injected into a goat. Sheep received 40xlO' mouse yolk sac cells intravenously on day 3 of life, and goats received the same cell dose on day 7 of life. After 4 days, both movements The subjects received a second dose of 200xlO' cells. After another 4 days, a final injection of 60xlO6 cells was given, followed by antibody staining and flow cytometry approximately 1.5 months later. Peripheral blood mononuclear cells were collected for avian analysis. Figure 9 demonstrates that a significant number of blood cells obtained from the sheep were reactive with anti-H-26 antibodies. There were fewer donors in the goat's peripheral blood, but donor cells could nevertheless be detected. Furthermore, cells expressing the mouse T cell markers t,y-i were present from both animals. However, none of the animals had cells positive for the mouse macrophage marker Mac-1, consistent with the fact that macrophages are not normally present in peripheral blood. The results of this experiment are revealed in several ways. It illustrates the possibility of xenogeneic reconstitution using mouse yolk sac cells. None of the animals were pretreated with irradiation or cytotoxic agents. High cell doses and repeated injections do not induce graft rejection. won. Also, both animals are normal and healthy as they do not exhibit graft-versus-host reactions. It was clear that The consistent finding that a larger number of donor cells can be recovered from sheep than from goats may be a result of the goats being older before receiving their first cell injection. Younger sheep when given the first cell dose produced a more effective induction of tolerance. However, there was still acceptance of goat donor cells in the absence of prior immunosuppressive treatment. If induction of tolerance is the mechanism underlying this observation, this could mean that the tolerized host also has the same hub of the original donor. Other solid organs, including hearts, livers and kidneys, from xenogeneic donors that share rotibunds It further suggests that government can be accepted. Finally, the expression of T cell markers indicates normal differentiation and maturation in vitro and the absence of macrophages in peripheral blood. presence suggests proper homing of the correct cell lineage in the host after intravenous administration of previtelline cells. 7. Example: IN VIVO Transformation of Previtellular Cells In this example, both untransformed vitelline cells and a retroviral vector containing the foreign gene of interest were transformed. inject into the target animal. The foreign gene used was the growth hormone gene (bGH). Previtellular cells recovered from day 8 C57/S mouse fetuses are cultured in an STO feeder layer system until approximately 20 x 1.06 cells are produced per culture flask (150 cm). Cells were placed in new flasks when cell density was greater than 80%. All experiments were performed using cells of 10 or more passages. Pre-yolk cells (2-4XIO') were injected into newborn mice on day 3 to 5 after birth. P. Injected. Two months after injection of pre-yolk sac cells (positive results were observed with infection as early as two weeks after yolk sac injection), animals received 1. V, for injection After transformation with the plasmid pLJPCKbGH, the cells received 1 x 10 viral particles of a replication-defective retroviral vector produced from the Moloney murine leukemia strain Mulligan ψ2 packaging cell line. Of the 145 animals treated by this procedure, 1.12 were tested in the ELISA assay. The test result was positive for bGH in the serum. Below is a breakdown of the positive bGH levels in these test animals. 20-59 ng/ml = 56 mice 60-100 ng/ml = 20 mice 100-200 ng/ml = 14 mice 200-500 ng/ml = 13 mice >500 ng/ml = Equal pairs of 9 mice Animals were injected with retroviral vectors rather than pre-vitelline cells. these good None were positive for bGH. Retroviral vectors can specifically transfect injected pre-yolk cells. It is thought that the pre-vitelline cells secrete factors that have an effect on nearby cells, making them more susceptible to transfection. 7th day FIG, IA FJG, 78 FjG, 4A FIG, 4B FIG, 4C FfG, 4D Gu-FL2\Fluorescence 2 <----FL2\Fluorescence 2 <----FL2\Fluorescence 2 -F, L2X Fluorescence 2 Gu-FL2X Fluorescence 2 ・(?)人-','7 (!!,j□□(,su°toshi(su・纂〉(J)・1C)・i, tube, j)...9go-, 10'-'', (7,. 81) Designated countries EP (AT, BE, CH, DE. DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), AU, CA, JP, KR (72) Inventor Antoczak, Michael Earl. 26 75 Sam's Road, Albany, Ohio 45710 United States

Claims (1)

[Claims] 1. Table of CD34+, Thy-1-, MHC class I- and MHC class II- Fruits of yolk sac stem cells that are present and capable of differentiating into mature blood cells in vivo A cellular composition comprising a qualitatively homogeneous population. 2. 2. The composition of claim 1, wherein the yolk sac stem cells are isolated from the yolk sac prior to blood island formation. 3. 3. The set of claim 2, wherein said yolk sac stem cells are isolated from a mouse yolk sac on day 7 of pregnancy. A product. 4. 3. The set of claim 2, wherein said yolk sac stem cells are isolated from a human yolk sac on day 10 of pregnancy. A product. 5. Table of CD34+, Thy-l-, MHC class I- and MHC class II- yolk sac stem cells comprising isolating a substantially homogeneous population of yolk sac stem cells exhibiting a phenotype. A method of producing a cellular composition of cells. 6. In the presence of substances that suppress cell differentiation, CD34+, Thy-l-, MHC Substantial homogeneity of yolk sac stem cells exhibiting class I- and MHC class II-phenotypes A method of expanding the cellular composition of yolk sac stem cells comprising culturing a population of yolk sac stem cells. 7. The method of claim 6, wherein said substance is a leukemia inhibitory factor. 7. The method of claim 6, wherein said substance is a stem cell factor. A method for hematopoietic reconstruction comprising administering the yolk sac stem cells of claim 1 to an animal. 10. 10. The method of claim 9, wherein said cellular composition is administered intravenously. 11. 10. The method of claim 9, wherein said cellular composition is administered intrauterinely. 12. 10. The method of claim 9, wherein the animal is a mouse. 13. 10. The method of claim 9, wherein the animal is a sheep. 14. 10. The method of claim 9, wherein the animal is a goat. 15. 10. The method of claim 9, wherein said animal is a human. 16. 16. The method of claim 15, wherein the human is infected with human immunodeficiency virus. 17. Animals other than humans that have a hematopoietic system reconstituted with yolk sac stem cells. 18. 18. The animal of claim 17, wherein said animal is a mouse. 19. 18. The animal of claim 17, wherein the animal is a sheep. 20. 18. The animal of claim 17, wherein the animal is a goat. 21. In the presence of growth factors, CD34+, Thy-l-, MHC class I- and exhibits an MHC class II-phenotype and is unable to differentiate into mature blood cells in vivo. in vitro comprising culturing a substantially homogeneous population of yolk sac stem cells capable of A method for producing blood cells in o. 22. Growth factors include EPO, IL-2, IL-3, G-CSF, M-CSF, G 22. The method of claim 21, wherein the method is M-CSF, or a combination thereof. 23. A method for producing blood cells in an animal, the method comprising: (a) CD34+, Thy-l- , exhibits MHC class I- and MHC class II-phenotypes, and has been developed in vivo. Injecting animals with a substantially homogeneous population of yolk sac stem cells that can differentiate into mature blood cells And (b) A method comprising collecting blood cells from the animal. 24. CD34+, Thy-l-, MHC class I- and MHC class II- Administering a substantially homogeneous population of phenotypically allogeneic or xenogeneic yolk sac stem cells A method of immunologically tolerizing an animal, comprising: