MXPA96000735A - Procedure for the production and cultivation of hematopoyeti precursor decelulas - Google Patents

Abstract

The present invention relates to a method for the in vitro production of non-immortalized hematopoietic precursor cells of the erythrocyte development series, characterized in that cells containing a population of erythrocyte precursors are exposed, in a medium containing the usual components necessary for growth of erythrocytic cells, for at least a sufficient time with a combination based on growth factors containing at least one estrogen receptor ligand and at least one glucocorticoid receptor ligand and at least one, preferably at least two glucocorticoid receptor ligands. a tyrosine kinase receptor until the cells start to renew themselves

Description

"PROCEDURE FOR THE PRODUCTION AND CULTIVATION OF HEMATOPOYETIC PRECURSOR CELLS" Inventor (s): HARTMUT BEUG, German, domiciled in Kometengasse 11, A-1140 Vienna, Austria; OLIVER ESSELY, German, domiciled at Staudgasse 72, A-1180 Vienna, Austria; PETER STEINLEIN, German, domiciled in Schimmelgasse 10/2/7, A-1030 Vienna, Austria; EVA DEINER, German, domiciled in Goschlgasse 10/2, A-1030 Vienna, Austria and MAARTJE MARIE VON LINDERN, German, domiciled in Herndlgasse 26/5, A-1100 Vienna, Austria.

Causaire: BOEHRINGER INGELHEIM INTERNATIONAL GMBH, German company, domiciled at D-55216 Ingelheim am Rhein,, Germany Procedure for the production and culture of hematopoietic precursor cells The following invention relates to a process for the production and for the in vitro culture of hematopoietic precursor cells, in particular of the erit rocyta series of development. During normal hematopoiesis, pluripotent germ cells develop into precursor cells that are committed to a certain series of development (these precursors are called "imprisoned"); These cells are supposed to differ from the pluripotent germ cells in a double aspect: first, they are limited in their ability to differentiate into a single or a small number of specific developmental series. Secondly, according to the general opinion, such "imprisoned" precursor cells are unable to multiply continuously without simultaneous differentiation (this property is also designated as capacity for self-renewal) or they do so only transiently (Till and McCulloch, 1980). Therefore, it is assumed that the precursor cells, with their commitment to a certain series of development, begin a pre-established program of modifications in gene expression, at the end of which is the formation of a terminal differentiated cell. On the contrary, pluripotent germ cells are supposed to retain their ability to undergo numerous cell divisions without modifying their state of differentiation or gene expression. The program through which the precursor cells pass is, of course, compatible with the passage of numerous cell divisions, although it is assumed that the cells undergo, during each division, modifications, even when they are perhaps slight, of their state of differentiation or gene expression (Keller, 1992). This view that a fixed determination / differentiation program determines the development of "imprisoned" precursor cells was recently questioned in different ways: first, some observations suggest that normal "imprisoned" precursors can pass through long phases of the expansion, which points to a self-renewal or processes related to it. B lymphocyte precursors of muridic origin are continually renewed under a variety of culture conditions (stromal mast cell layers plus interleukin 7), but differ, under other conditions, in mature B cells (Rolink et al., 1991). Similarly, different murine granulocyte-murine macrophage colony forming cells (GM-CFC) can produce, depending on the concentration of GM-CFC, between 100 and more than 10,000 granulocytes and mature macrophages (Metcalf, 1980). Another phenomenon that is difficult to associate with a fixed program of the development of "imprisoned" precursors are leukemias. Although these stem in some cases from pluripotent germ cells, other leukemias are clearly derived from "imprisoned" precursors (Sawyers et al., 1991). In relation to the latter type, there is a concept repeatedly stated that the genetic modifications that take place in leukemia cells confer on them the abnormal capacity for self-renewal, a property that does not possess the corresponding normal precursor cell. While in the chronic phase of ielogenous leukopenia (CML) modified pluripotent stem cell clones outnumber the corresponding normal clones (perhaps by virtue of their enhanced capacity for self-renewal), other mutations that take place during the * blast crisis, lead to a massive growth of immature precursors and maturing cells of a series of special development, which is interpreted as a self-renewal of "imprisoned" abnormal precursors (Daley et al. , 1990; Elefanty et al., 1990; Kelliher et al., 1990). Recently, chicken cells have been shown that normal hematopoietic precursors, which are committed to the series of erroneous development, are capable, under specific conditions, for continuous self-renewal (Schroeder et al., 1993; Hayman et al. ., 1993). In this case, it was demonstrated that the combined action of TGFa ("transforming growth factor"), a ligand for the homolog in hens of the epidermal growth factor receptor / protooncogene-c-erbB (TGFaR / c-erbB; Lax et al. al., 1988) and estradiol induced the growth of normal precursors from the bone marrow of hens. These cells are termed, by virtue of their property to develop from cultures containing TGFa plus estradiol or SCF (germ cell factor), "SCF / TGFa precursor cells" (cells that develop in the presence of SCF are referred to as "cells"). SCF precursor cells "). The SCF / TGFß precursor cells express the proto-oncogene c-Kit, the estradiol receptor and TGFßR / c-erbB and are capable, in the presence of TGFa plus estradiol, of continuous self-renewal until the end of their normal life span in vitro . It was also shown that erroneous precursors, which are not different from normal CFU-E's ("erythrocyte colony forming units") in relation to all the properties investigated (called precursors of SCF), could be grown to from bone marrow using SCF from chickens (Hayman et al., 1993). In contrast to the SCF / TGFα precursors with the capacity for self-renewal, the SCF precursors lacked the expression TGFaR / c-erbB, and the cells showed in the presence of SCF only a transient self-renewal for a period of 7 to 10 days . When changed to differentiation factors (eri tropoyet ina plus insulin), both types differed in erythrocytes without differentiable kinetics. This pointed to the fact that the SCF / TGFα precursors are not the precursors of SCF precursors, as initially assumed by virtue of the fact that SCF / TGFα precursors are relatively rare (1 of 15,000 normal cells of the bone marrow), whereas the precursors of SCF are much more frequent (1 of 300-500; Hayman et al., 1993). However, these results left open the question whether the precursors of SCF / TGFa autorenovab 1 are derived from still immature precursors. One possible answer is that these cells represent a rare and separate type of cell that already appears in the bone marrow and develops from pluripotent precursors similar to a separate germline. An alternative answer would be that these cells come from normal CFU-E's that inherit the potential for self-renewal only under the effect of specific combinations of growth factors and hormones that are not normally active in er i t ropoyes is. Previous studies (Schroeder et al., 1993) showed that for the growth of SCF / TGFα precursors from the bone marrow there are two fundamental requirements: first, a certain duration - the growth never took place before 11 to 14 days; second, the dependence of both TGFa and estradiol, which was demonstrated by the fact that cell growth was completely inhibited by an estradiol antagonist, and was not manifested in the case of missing TGFα. In case the first response is correct and the precursors of SCF / TGFa are a type of cell that is always present in the normal bone marrow and only depends on TGFa and estradiol, other factors should not have an impact on the frequency of these cells; This simplified model however opposes two observations: first, it was found that the growth of SCF / TGFα precursors was strongly inhibited in the presence of chicken serum that had been treated with animal charcoal, whereas in serum treated with Freon or not treated it was not essentially affected. This allowed us to suppose that together with TGFa and estradiol, other factors that are eliminated by treatment with animal charcoal have an effect on SCF / TGFα precursors at any stage of their formation. In addition, it was observed that the bone marrow cells remained in SCF plus estradiol, they became stationary after 8 to 10 days, but at approximately 14fl day they started a slow growth again. These cells express TGFaR / c-erbB in a relatively high concentration (Hayman et al., 1993) and could be grown in TGFa plus estradiol, which suggests that these cells are precursors of SCF / TGFa that had developed from the original population of SCF precursors. The purpose of the present invention was to clarify the mechanisms involved in the formation of hematopoietic precursors of the eritectorial development series, which express c-Kit and TGFaR / c-erbB (in the context of the present invention referred to as " precursors of SCF / TGFa ") and, based on the recognitions obtained, to make available a method that allows in vitro culture of normal eritic precursor cells. In particular, a procedure should be made available that would enable mass preparation of human hematopoietic precursor cells not immortalized as well as, therefore, genetically unmodified. In the context of the present invention, it was first demonstrated for chicken cells that in the presence of SCF, TGFα, estradiol and certain unidentified hen serum factors, SCF / TGFα precursors are developed in cultures of purified SCF precursors. It was shown that in the culture of SCF precursors in the presence of a combination of SCF, TGFa, estradiol and undefined factors of normal or anemic chicken serum, a large number of these cells do not undergo a differentiation or apoptosis, rather, it begins, in a strictly time-dependent manner, to express increasing amounts of TGFaR / c-erbB, which, after 10 to 44 days, results in the obtainment of SCF / TGFa precursors. At this time, the expression of TGFaR / c-erbb in the cells is evidently high enough to allow proliferation in the presence of TGFa and estradiol in the absence of SCF. In the case of using sera from specially treated hens, it could be demonstrated that precursors of SCF / TGFα do not form when one of these three factors (SCF, TGFa or estradiol) was missing. On the other hand, the formation of the proliferating precursors in the presence of SCF, TGFa and estradiol was partially inhibited, when it was not eliminated, when the unidentified activity of the chicken serum was lacking. In further tests in the hen model it could be demonstrated that the activity in principle not identified can be replaced, at least in part, by two defined factors: 1. the glucocorticoid receptor ligand dexamethasone and 2. the receptor ligand of t-irosykinase, the insulin-like growth factor 1 (IGF-1). In addition, it could be assumed that eri t ropoyet ina is another factor that is responsible for the activity in serum 1 gal. When SCF precursors were cultured in SCF, TGFa and estradiol, SCF / TGFa precursors were accumulated in the culture until approximately two to two and a half weeks later they predominated in the culture. The expression of TGFaR / c-erbB increased with time when the SCF precursors were cultured in SCF, TGFa and estradiol. By virtue of the mass culture experiments carried out, it was not possible in principle to distinguish between two possibilities in the way in which the SCF / TGFα precursors were formed from the SCF precursor cultures. The first possibility (trivial) would be that from the beginning there is a small number of precursors of SCF / TGFa, which express c-erbB, in the normal bone marrow and, consequently, in the populations of SCF precursors, these cells gradually overcoming the SCF precursors (when the culture is carried out in the presence of SCF, TGFa and estradiol , which possibly helps these cells to an advantage in their growth.The nontrivial possibility and more interesting would be that in the bone marrow there are not present from the beginning erotranscriptive precursors that can proliferate only in TGFa and estradiol, but that the precursors of SCF / TGFa are induced for development from SCF precursors when all three factors are present, most components of chicken serum or dexamethasone and IGF-1 (see above).

The experiments carried out in the framework of the present invention demonstrated that the latter hypothesis is true, ie, that the precursors of SCF / TGFα can be developed from SCF precursors. The results obtained with hen cells showed that the normal erroneous precursors (precursors of SCF that in all the properties investigated resemble the precursors of "unit forming colony erite roe i tarias" (CFU-E )), are developed under the control of at least two growth factors (SCF, TGFa) plus a steroid hormone (estradiol) and an undefined activity in chicken serum, which was subsequently identified in part as dexamethasone plus IGF -1, to form another type of precursor er it roci tar ia (precursor SCF / TGFa). This other type of precursor is distinguished by its newly acquired expression of TFGaR / c-erbB (which corresponds to the mammalian EGF / TGFa receptor) and by its ability to undergo prolonged self-renewal as a reaction on TGFα and estradiol. The differentiation program of SCF / TGFα precursors after treatment with differentiation factors (EPO, insulin) greatly resembles the normal CFU-E precursor (Hayman et al., 1993). Since it has been supposed from erratic precursors that until now they are irreversibly committed to differentiation, experiencing a fixed program of 5 to 10 cell divisions, it was of great interest to discover the im- chickens that, under certain conditions, can acquire, through modification of their differentiation program ("change of development") a potential for self-renewal, with the premise that these discoveries would be valid for mammalian or even human cells. Within the framework of the present invention it is demonstrated that the results of the system with hens can be applied in surprising measure to human cells. There is a demand for human hematopoietic precursor cells that can be cultured in vitro, especially in relation to the transplantation of this type of cells in the treatment of cancer and AIDS patients. Another application of a transplant of this type is the treatment by gene therapy of chronic anemias, in which the maturation of erythrocytes is damaged, for example thalassemia and other genetically conditioned anemias. One of the few premises considered as definitive, which are necessary for the ability to successfully transplant blood cells, is the expression of CD34. However, it is unknown in which stage of development is the subpopulation of CD34 + cells that is actually responsible for successful transplantation, even though it is assumed that the series of development and the phase of cell differentiation obviously play a role. Autologous or allogeneic transplantation of haematopoietic precursor cells is linked with difficulties, one of the main problems in which a sufficient number of cells with the potential for proliferation necessary for the successful reconstitution of the hematopoietic system is to be transplanted and in which The criteria that determine this potential have not yet been sufficiently investigated. Until now, bone marrow cells from healthy donors were often used for allogeneic transplants; for autologous transplants, peripheral blood germ cells are used that are mobilized during the patient's recovery from chemotherapy and / or by treatment with recombinant growth factors. These methods are complex; in addition, they are associated with great inconveniences for the donor and, by virtue of hematological modifications of the patient, they provide few yields. Therefore, it was recently proposed, as an alternative, to use germ cells from healthy donors treated with cytokine.

An alternative considered to be very promising isalso, to use umbilical cord blood cells instead of bone marrow cells or CD34 * - positive peripheral blood cells, since most of the germ cells and hematopoietic precursors of the umbilical cord blood are found in an early development phase and has a greater potential for proliferation. However, in the case of a requirement of 5 x 10 -2 x 106 CD34 + cells per kg of body weight for transplantation of an adult, approximately 1.5 1 of umbilical cord blood would be necessary, this method encounters barriers in the treatment of adults. Therefore, there is a demand for a process that enables the mass culture of transplantable hematopoietic precursor cells by autologous or allogenic pathway. In the framework of the present invention, it was shown that the human erotification progenitor cells show, surprisingly, a behavior similar to that of the corresponding cells of hens, undergoing a modification in their program of differentiation, by virtue of which they inherit a potential of self-renewal. Like the cells of hens, the cells require SCF, estradiol and dexamethasone, in order to inherit the capacity for prolonged self-renewal. IGF-1 had a positive influence on the growth of hen and human hebrobots. Some of the cells in the culture of human precursors, which were obtained in the framework of the tests carried out, reacted on EGF receptor ligands, which is an additional sign that human cells resemble each other. in its behavior, under the influence of certain factors of growth and differentiation, to the precursors of SCF / TGFa of chicken. Accordingly, the present invention is also based on the decisive recognition that a modification in the differentiation program of human erctrocytic precursors must be manifested, by virtue of which they acquire the capacity for continuous growth. This modification of the differentiation program should be induced by the cooperation of factors that are ligands of representatives of the same groups of receptors, whose activation induces the development of self-renewing self-renewing precursors from the bone marrow of chickens. Accordingly, it relates to a process for the in vitro production of non-immortalized hematopoietic precursor cells of the eriteous development series, which is characterized in that cells containing a population of precursors er i troci ate , are exposed in a medium containing the usual components necessary for the growth of eritretary cells, at least for a time with a combination based on growth factors containing at least one ligand of the estrogen receptor and at minus one ligand of the lucocorticoid icoid receptor and at least one, preferably at least two ligands of a ti rosin receptor, until the cells begin to renew themselves, and because eventually the cells are continued to be cultured in a medium that contains the necessary factors for continuous self-renewal. By treating the cells with the combination of growth factors (hereinafter referred to as "combination of factors"), the cells undergo a modification of the differentiation program. This is accompanied by a modification of the expression pattern of the receptors that are again expressed or highly regulated by the action of the combination of factors, and / or by modification of the expression model of protein components of the cellular signal transmission pathways triggered by this or these epigenetic modifications. By "self-renewal" is meant the ability of cells to form daughter cells that during the following cell divisions no longer mature measurably, ie, in which no further possible accumulation of proteins of this type takes place, which are typical for mature cells, but which may also be expressed in small amounts in precursor cells. Another important criterion for self-renewal is that the ratio of mature cell proteins (terminally differentiated) (eg, hemoglobin) and proteins that are necessary for the function of each cell (so-called "house proteins", for example glycoprotein 1 ít icas) does not vary in a measurable way. Preferably, the method according to the invention is applied to human cells. A cell population enriched in CD34-positive cells of the bone marrow, peripheral blood or, in a particularly preferred embodiment, umbilical cord blood is preferably used as starting material. The enrichment can be carried out according to methods known from the literature; A perspective of this type of method is provided in the manual "Hemato-poietic Stem Cells, The Mulhouse Manual", 1994. Cells are cultured in vitro at least until their self-renewal is manifested. From a purely external point of view, cells with a self-renewing potential can be recognized because during a period of time corresponding to the in vitro lifespan of the cells (50 - 70 generations in the case of human cells) or a part of this duration of life, they are continuously divided into culture, that is to say they proliferate exponentially, as well as having a constant size and a relatively low content of erythrocyte proteins (for example hemoglobin). The person skilled in the art can establish in previous tests, with the help of these criteria, at what instant the cells have acquired a self-renewing potential and, correspondingly, define the duration of the crop. The self-renewal of the human cells of the eri trocyte development series obtainable within the framework of the present invention is distinguished by the fact that the cells dissolve, without recognizable differentiation, over a period of time substantially greater than that shown up to now. for BFU-E's (normal bursting colony forming units). The combination of factors is preferably a combination of at least three, preferably at least four factors, at least two of which are ligands of t-kinase receptors. On recipients of this type, their belonging to families and subfamilies, their ligands, as well as the signal transmission routes triggered by their activation, there is a great abundance of bibliography, and new representatives are continuously identified. The recipients of inquinase shots have in common that, after the binding of their ligand, they phosphorylate themselves to tyrosines. After this autophosphorylation, the phosphotyrosine radicals interact with specific topical molecules, thereby triggering the cellular response to the growth factors. The family of the t iros inquinase receptors is divided into different classes and subfamilies; they include the class to which the EGFR family belongs, HER2 / neu / c - erbB-2 and HER3 / c-erbB-3; the class to which the insulin receptor belongs, the "insulin-related receptor" and the IGF-1 receptor; the class that encompasses the PDGF receptor, the PDGFβ receptor, the MCSF-1 receptor and c-Kit; the class of fibroblast growth factor receptors (FGF receptor 1, FGF receptor 2, FGF receptor 3, FGF receptor 4) and the HGFR receptor (hepatocyte growth factor receptor). Some of these classes have the common feature that the domain of the kinase is interrupted by a sequence. In relation to the receptors of t iros inquinasa and their ligands, reference is made to the orientative article by Fantl et al. 1993, and Van der Geer, 1994, including the bibliography specifically cited therein to the different recipients. The combination of factors based on receptor ligands of t i ros inquinasa is composed of at least in each cas * or a ligand for receptors of different families within the receptors of t iros inquinasa. An example of a combination of this type is i) at least one ligand of a t-irosykinase receptor having a continuous domain of kinase and, ii) at least one ligand of a t-kinase receptor that possesses an interrupted kinase domain. with an insert.

Examples of representatives of the receptors defined in i) are the members of the family of EGF receptors (receptors 1-4 of the human epidermal growth factor); to this family belong the only partially identified recipients. The ligands of the receptors defined in i) are, among others, EGF, TGFa, NDF (neuronal differentiation factor; Peles and Yarden, 1993), including the variants resulting from differential splicing, heregulin, irregular anf, the glial growth factor, etc. (Fantl et al., 1993). Ligands of the receptors defined in ii) are, among others, the c-Kit ligand of SCF (germ cell factor), platelet-derived growth factor (PDGF) alpha and beta, all family members of the factor of growth of fibroblasts, CSF-1 (colony-stimulating factor 1) and vascular factors (for example VEGF, vascular endothelial growth factor) (Fantl et al., 1993). Beside them, there is a plurality of t-irosykinase receptors that can not be clearly associated with one of these two groups (whose ligands have only been known in part), whose activation by the corresponding ligands can determine the growth of precursor cells. human the corresponding ligands can also be used in the context of the present invention. These receptors include: the hepatocyte growth factor receptor (whose ligand is also called the "dissemination factor"; the findings obtained by Galimi et al., 1994, suggest that the hepatocyte growth factor receptor (HGFR) , which is supposed to activate the same signal transmission pathways as the EGF receptor, plays an important role in CD34 + cells and in human precursor erythrocyte cells resulting from the above), c-sea and c-ros ( whose ligands are not yet identified), several specific receptors of epithelial cells, whose ligands are unknown, a group of recently described receptors (among others by Tamagnone et al., 1993 and Kaipainen et al., 1993), cloned from cells er it roe i tarias, whose ligands are also still unknown, as well as the members of neurotrophic receptors (trk, trk-B, trk-C with ligands NGF, BNDF, etc.) and, in addition, receptor s of the insulin receptor family (insulin receptor, IGF-1 receptor, etc.). Without wishing to be limited to the theory, it should be essential for the triggering of the modification of the differentiation program that by means of the binding of the ligands and the activation of the receptors defined in i) and ii) determined with it different ways can be put into operation of signal transmission. Together with the two ligands of the tyrosine kinase receptors, the combination of factors contains iii) at least one estrogen receptor ligand and at least one ligand of the lucocort icoid receptor. In the context of the present invention, natural or synthetic steroid hormones which, like estradiol, activate the estrogen receptor or, like hydrocortase isone, activate the glucocorticoid receptor are suitable. In addition, the combination of factors may contain, possibly, progesterone receptor ligands, such as aldosterol and progesterone. It is common for these hormones that a) are of low molecular weight, that b) bind to receptors located in the nucleus, which represent transcription factors (proteins that modify the activity of the genes) regulated by the hormone in their activity and that c) in some of the systems that have now been investigated, they can modify the cell differentiation program In the framework of the present invention, it has been demonstrated that, together with an estrogen, a glucocorticoid, especially dexamethasone, is of decisive importance, for the growth of erythrocyte precursor cells of hens and human beings that are self-renewing., the combination of factors may contain iv) one or more additional factors. As additional factors iv) ligands which determine at least the acceleration of the modification of the differentiation program and, with it, a more efficient growth of the cells, are mainly considered. These factors are added to the environment, usually already at the beginning of the crop, taking into account that different factors may be necessary at different times during the modification of the differentiation program. In relation to the acceleration of the modification of the differentiation program, it may therefore be convenient to eliminate from the medium factors that are necessary for the unleashing of this process, but which can eventually be disregarded or even disadvantageous, in an instant. suitable that can be determined by serial tests. Additional factors are considered: 1. Ligands of receptors that act through phosphorylation with serine of target proteins (family of TGFβ receptors). Here, the ligands activin, inhibin, BMP, etc., are especially important. which also play a role in early embryonic development (Laufer, 1993; Hogan, 1993). 2. Ligands of other receptors for intracellular drugs, in particular IGF-1 or hepatocyte growth factor (HGF). 3. Representatives of the large group of cytokines or interleukins (growth factors and differentiation in the hematopoietic and immune system). Almost all of these cytokines bind to receptors that, by themselves, do not possess any known enzymatic activity, but some of them form complexes with intracellular, intracytic, and intracellular tumors. A perspective on this family of constant growth receptors and their ligands is given in Boulay and Paul, 1993. "It is essential for the action of a cytokine applicable within the framework of the present invention that it first stimulates the proliferation of immature precursors and, secondly, they do not have any activity that negatively influences the growth of the cell and / or triggers an apoptosis (programmed death of the cell). Preferred cytokines within the framework of the present invention are IL-1, IL-3, IL-11, IL-13-. EPO is particularly preferred. The population of cells obtained by the action of the combination of factors can be frozen after the start of self-renewal and, if required, thawed and then subsequently cultivated or transplanted directly, in order to take advantage of the self-renewal potential of the cells acquired in vitro for proliferation in vivo. However, the cells can be cultured beyond the period of time during which they acquire the potential of self-renewal, in order to obtain, within the population, a greater number of proliferating cells. The subsequent culture of the proliferating cells is carried out in the presence of the growth and differentiation factors that the cells require for continuous self-renewal. For chicken cells, TGFa is one of the factors that are necessary for the potential of continuous self-renewal and, consequently, for the cultivation of cells over a prolonged period of time in relation to obtaining a large number of cells. cells For human cells, preferred factors are used for subsequent culture of the cells, ligands of the type defined in section i), such as EGF and / or TGFa, and / or HGF, as well as SCF and in addition EPO and IGF-1. * The combination of factors suitable both for the induction of self-renewal and for the subsequent cultivation of proliferating cells is determined by testing the response of the cells and their growth behavior under the action of different mixtures of factors at different times; examples of tests of this type are represented, inter alia, in examples 4 b), 5, 7 b), 8. In this case, the mixture of factors is conveniently optimized so that different mixtures are first tested. of multiple components in order to identify the optimum effective mix. Then, from the optimum effective mixture, a factor is separated, step by step, and the behavior of the culture is compared with and without the factor. Overall, the combination of factors is optimized to achieve the fastest and most efficient growth of cells capable of self-renewing with as few factors as possible. The treatment carried out in the framework of the present invention with a combination based on SCF, TGFa, estradiol and other activity determined in hen and human cells an increase in the expression of biologically active TGFa / c-ErbB which also manifested itself in chicken cells by an autophosphorylate receptor increase after the addition of the ligand; the additional activity was, in the case of chicken cells, an activity not identified in greater detail in the chicken serum and, in the case of human cells, EPO.

In an embodiment of the invention, the combination of factors for the preparation of human hematopoietic precursor cells is composed of i) a ligand of a receptor of the family of EGF receptors and / or HGF receptor, ii) a ligand of c-Kit; iii) estradiol and dexamethasone, and iV) erythropoietin ina and IGF-1. In a particular embodiment of the invention, i) is EGF and / or TGFa and / or HGF, and ii) is SCF. If the factors in a sufficient concentration are co-constituents of the medium, for example in the form of serum components, they are not to be added separately. The usual components contained together with the combination of factors in the medium and necessary for the growth of the cells, such as vitamins, amino acids, etc., are usual for the person skilled in the art.; are contained in commercially available media or can be deduced from specialized manuals such as "Hematopoiet ic Stem Cells, The Mulhouse Manual", 1994, and scientific articles such as Sawada et al., 1990. Cells obtained according to the procedure according to the invention can be suspended, after separation of the culture medium, in a medium suitable for therapeutic application, for example in human serum albumin (HSA) or autologous plasma, and used for allogeneic or autologous transplantation. The method according to the invention can be used, inter alia, for the purpose of culturing from a stock of blood cells of an individual, whose production of CD34-positive cells was excited for example by treatment with cytokines, in the case of requirement for a hematopoietic cell transplant. These can be stored frozen, thawed if necessary, amplifi ed by in vitro culture and eventually used after an appropriate gene transfer, for therapeutic purposes in the patient. An example of a strategy in which human cells that are genetically modified and cultured in vitro can be used is the treatment of sickle-cell anemia by gene therapy. This hereditary disease appears mostly in the United States in a large number of patients of color. A possible way of proceeding consists in the cultivation of erroneous precursors of bone marrow, peripheral blood or (in the case of a prenatal diagnosis) umbilical cord blood, gene transfer from the locus of the human globin gene carrying the mutation. "hereditary persistence of fetal hemoglobin" (HPFH) and application of these genetically modified somatic cells (the band of germs is not affected) in the patient. The gene transfer in the cells obtainable according to the invention can be carried out with the aid of standard methods for the transfection of cells of this type. These include gene transfer by means of viral vectors (retroviruses, adenoviruses, adeno-associated viruses) or by non-viral systems based on receptor-induced endocytosis; compilations on usual methods are indicated for example by Mitani and Caskey, 1993; Jolly, 1994; Vile and Russel, 1994; Tepper and Mulé, 1994; Zatloukal et al., 1993, WO 93/0728.3.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1: Growth of SCF / TGFα precursors from SCF precursors Fig. 2: Expression and bioactivity of c-it and c-ErbB during proliferation of SCF precursors in SCF, TGFa and estradiol Fig. 3: Modification of the dependence of growth factors of SCF precursors that proliferate in SCF, TGFa and estradiol Fig. 4: Test strategy to clarify the formation of SCF / TGFα precursors Fig. 5: Evolution of SCF / TGFα precursors from precursors of SCF Figs. 6A, B: LD clones developed in SCF, TGFa and estradiol correspond to SCF / TGFα precursors: expression of bioactive c-ErbB and proliferation reaction on TGFα Figs. 7A, B: A factor in chicken serum facilitates the evolution of SCF precursors to give precursors of SCF / TGFa Fig. 8A: Requirement of estradiol and SCF during the evolution of SCF / TGFα precursors from precursors of SCF Fig.8B: Acceleration of the evolution of SCF precursors in SCF / TGFa precursors by anemic hen serum Fig. 9A: Acceleration of the transformation of SCF / TGFa precursors by dexamethasone Fig. 9B: Definition of similar growth factor to insulin (IGF-1) as one of the factors responsible for the activity in chicken serum Fig. 10: Growth of eritrocyte cells from human CD34 + peripheral blood cells Figs. 11A.B: Characterization of human erythrocyte precursors cultured in vitro Fig. 12A: Prolongation of the self-renewal potential of human CD34 + cells in umbilical cord blood by dexamethasone Fig. 12A: Increase in human cell growth peripheral blood by IGF-1 Fig. 13: Comparison of the properties of growing human erythrocyte precursors with gal 1a cells In the following examples, the materials and methods described by Hayman et al. were used, unless otherwise indicated. al., 1993. In the Examples 1-6 chicken cells were used, and in Examples 7 and 8 human cells.

Example 1 Enrichment of SCF / TGFa precursors by culture of SCF precursors in SCF, TGFa and estradiol In order to systematically establish whether SCF precursors contain SCF / TGFa precursors or whether they contain cells that can evolve towards this type of cells, they were seeded cells from a 6-day culture of purified SCF precursors (Hayman et al., 1993) in CFU-E medium containing 100 ng / ml of recombinant SCF, 5 ng / ml of TGFa and 5 x 10 M estradiol, and Cell proliferation was monitored by counting according to the system described by Hayman et al. *, 1993 (CASY-1, Schárfe system). (The use of the three factors was based on the consideration of keeping the SCF precursors alive as long as possible and at the same time stimulating the growth of possible SCF / TGFα precursors present from the start in culture or formed during the course of the study. culture). The result of this test is represented in figure 1. Surprisingly (and in strong "opposition to the results obtained in a comparative test from the culture of the same cells in TGFa plus estradiol), the cells showed only a weak transient decrease in their growth rate towards days 8 to 10, but continued to proliferate after them exponentially, rising from 25 to 30 doubling time to 18 to 22 h, after which an aging of the cells manifested ( Figure 1, unfilled circles.) In order to establish whether SCF precursors, which had been cultured in SCF, TGFa and estradiol, contained SCF / TGFα precursors or precursors of this type had been developed, and in order to To roughly evaluate its frequency, aliquots of the culture were taken at different instants, washed, transferred to CFU-E medium containing TGFa and estradiol but no SCF and the Cell number was compared to that of the culture that contained all three factors. (Cells were maintained by corresponding dilution with fresh medium at a density between 1 x 10 and 2 x 10 per ml and the cumulative cell number was calculated from the cell numbers obtained and from the corresponding cell factors. dilution (Schroeder et al., 1993; Hayman et al., 1993)) (Fig. 1, arrows). When the cells were removed on day 5 the combination of the three factors ceased immediately in their proliferation. The cell numbers remained almost constant until day 11. During this time, most of the cells underwent an apoptosis (these cells were not differentiated from the living cells by the cell counter), while some healthy lumps remained. "They started to form the culture around days 13 to 14. After that, the cells experienced a growth in the presence of TGFa plus estradiol that was not differentiable from the control culture in its kinetics (Figure 1, filled circles). A different behavior of the cells was observed when they were transferred, after culture in SCF, TGFa and estradiol, on day 12 to a medium with TGFa plus estradiol (Figure 1, filled squares). Up to this moment, only a part of the cells had suffered an apoptosis, while many others continued to grow, which translated as a transient decrease in the growth rate between days 13 and 16. Afterwards, the cells developed to a similar speed in TGFa plus estradiol to the control cells. After 18 days of culture, the transfer of the three factors to TGFa plus estradiol had no appreciable effect on cell proliferation (Figure 1, filled triangles), suggesting that at this time the culture consisted of all in precursors of SCF / TGFa.

Example 2 Increased expression of bioactive TGFaR / c-erbB in SCF precursors that are grown in SCF, TGFa and estradiol a) Expression and bioactivity of c-Kit and c-ErbB during the proliferation of SCF precursors in SCF, TGFa and estradiol In virtue of the results obtained in Example 1, it was assumed that the SCF precursors cultured in SCF, TGFa and estradiol were overcome in terms of growth by precursors of SCF / TGFα existing from the beginning or that had evolved in this type of precursors. The objective of the present assays was to demonstrate that the proliferating cells certainly express TGFaR / c-ErbB bioactive, which should be translated into the biochemical reactions expected from it (autophosphorylation lac ion) or biological reactions (stimulation of proliferation in corresponding assays) . For this purpose, on days 6, 12 and 20, aliquots of the culture as well as the control culture were taken (see Example 1), washed, incubated overnight in medium without growth factors, stimulated for 5 min with different combinations of factors and continued to be treated as described by Hayman et al., 1993, as well as for the transfer of phosphorus and the subsequent Western blot (using anti-TGFαR or c-ErbB antibodies) ). After a total of 6 days (after 3 initial days in SCF), the cells cultured in SCF, TGFa and estradiol showed the clear c-Kit phosphorylation expected by virtue of the reaction on SCF and expressed c-Kit in large amounts . In contrast, cells conforming to Western blot analysis contained only very small amounts of TGFaR / c-ErbB, and no autophosphorylation of c-ErbB was visible (these assays are depicted in Fig. 2A, indicating the arrows the TGFaR / c-ErbB 170 kD protein and the arrowheads the 140 kD SCF-R / c-Kit protein, the black circle in the lower fields indicates the position of a background band unrelated to TGFaR / c-ErbB). After 11 days, the expression of TGFaR / c-ErbB had clearly increased, as determined by Western blotting with c-ErbB. Additionally, a weak autophosphorylation reaction, even when clearly detectable, of the c-ErbB protein on the ligands could be detected (Figure 2B). As expected, the cells continued to express c-Kit autophosphorylable (Figure 2B). Similarly, cells tested after 20 days (at this time they were growing in both TGFa plus estradiol and also in SCF, TGFa and estradiol) expressed increased amounts of TGFaR / c-ErbB, which was clearly autophosphory and then reactive on TGFa (Figure 2C). Surprisingly, the cells continued to express lower amounts of TGFaR / c-ErbB than control cells that had been cultured from untreated bone marrow in TGFa plus estradiol. (Schroeder et al., 1993, Hayman et al., 1993). The results obtained show that precursors of SCF that are cultured in TGFa, SCF and estradiol, express already after 6 days, small amounts of TGFaR / c-ErbB and, after that, they continuously increase their expression during the following 8 to 14 days. b) Modification of the dependence of the growth factors of SCF precursors that proliferate in SCF, TGFa and estradiol In order to confirm that the TGFaR / c-ErbB detected biochemically represents the bioactive receptor, the cells further tested by the [H] -thymidine incorporation assay for their reaction on SCF, TGFa and estradiol, as described by Hayman et al. 1993. For this, aliquots of a culture of SCF precursors (5 days, graph A of Fig. 3) or of cells that had been activated for 11 or 20 days in SCF, TGFa and estradiol (graphs B and C of Fig. 3) were investigated as to their reaction on different factors (100 relative units correspond to the factor concentrations indicated below the symbols). It was shown that cells cultured for 6 days in SCF, TGFa and estradiol reacted, as expected, on SCF and estradiol, while no reaction was detected on TGFa (Figure 3). After 11 days in contact with these factors, the reaction of the cells on SCF and estradiol remained unchanged, but then a weak, even if clear, reaction on TGFa could be detected (Figure 3B). As expected, cells cultured for 20 days in SCF, TGFa and estradiol reacted strongly on all three factors, without any difference with respect to the control precursors of SCF / TGFα (Figure 3C). In summary, the results obtained show that SCF / TGFα precursors in self-renewal can be efficiently cultured from erroneous precursors that initially only react on SCF and that lack both detectable amounts of TGFaR / c-ErbB as well as capacity of continuous self-renewal for a longer time.

Example 3 Development of SCF / TGFα precursors from SCF precursors In order to clarify the issue of the origin of SCF / TGFα precursors from cultures of SCF precursors, the cloning method by limiting dilution (named in the following "LD cloning"). This method allows to analyze the proliferation behavior (and the differentiation behavior) of individual proliferative cells in a complex mixture of non-proliferative cells, since in the case of an adequate dilution it is possible to monitor the development of individual proliferating cells in individual cavities of Cell culture plates (96-well plates). The success of such a method naturally depends on a good cloning efficiency (10 to 50%) of the proliferating cells to be analyzed, a criterion that is met when the number of the prolific clones obtained is a linear function of the number of cells seeded, of until very few clones (1 to 10) for each plate of 96 wells. (The fact that this criterion is met for the explanation of the present question was demonstrated for the SCF and SCF / TGFa precursors of Hayman et al., 1993). Consideration of the mode in which LD cloning is performed between the two possible models (selective growth of rare SCF / TGFα precursors from SCF precursors or evolution of SCF precursors in SCF / TGFa precursors) is represented in figs. 4A and 4B, graphs on the right. In the left part of the figure the model is represented schematically in each case, and in the right part of the figure the expected result of the LD cloning, showing fig. 4A the model of the selective growth advantage of infrequent precursors in SCF, TGFa and estradiol and fig. 4B the alternative model that is based on a modification of the differentiation program of many or all of the precursors. In the case that, corresponding to the first model, the bone marrow contains both rare precursors of SCF / TGFα (one of 20,000) with the capacity for self-renewal and stable expression of c-ErbB, as well as precursors of SCF frequent (1 of 300) that proliferate transiently in the presence of SCF but are not capable of continuous self-renewal or express c-ErbB, then SCF should induce, after 4 to 6 days, many pro-active clones of precursors of SCF. Then, due to the differentiating or degenerating SCF precursors, the number of pro-active clones should decrease rapidly. In TGFa plus estradiol, a much smaller number of clones (1 in 20,000) should be obtained which, by virtue of the long-term self-renewal capacity of these clones, should remain essentially constant. In the presence of the three factors (SCF, TGFa and estradiol), the numbers of colonies should initially be as high as in SCF alone, but then they should decrease to the level obtained with TGFa plus estradiol (Figure 4A, graphic on the right). Corresponding to the second model, the bone marrow (and therefore the SCF precursors) contains from the beginning only a few precursors of SCF / TGFα, whereas the largest number of these cells develops from SCF precursors in a slow process that requires the presence of SCF, TFGA, estradiol (and chicken serum factors). Therefore, it would be expected that the frequency of the clones that develop in the presence of the three factors will not decrease or only weakly with time, which is in contrast with the expected behavior of this type of clones correspondingly to the first model (Figure 4B, graphic on the right). The frequency of clones that develop on the one hand in the presence of SCF and, on the other, in the presence of TGFa and estradiol, should correspond to the first model (Figure 4B, graphic on the right). a) LD Cloning of purified SCF precursors. 3-day, purified purified SCF precursors were prepared as described by Hayman et al., 1993.

The cells were then seeded in different concentrations (20 to 2500 cells per well of 96-well cell culture plate) in CFU-E medium containing only estrogen (control) or only SCF (plus estradiol antagonists ICI 164384 , in order to suppress the activity of estradiol contained in the serum), or TGFa plus estradiol or SCF, TGFa and estradiol. In order to guarantee a good cloning efficiency, 50 adherent myeloid cells were seeded in all the cavities as feeding layer. (Myeloid cells were obtained by preparing bone marrow cells and sowing them in the order of 5 x 10 cells / ml per 100 mm plate and treating them with 10 ng / ml of cMGF and SCF.) During the first two 2 to 3 days, the non-adherent or weakly adherent cells were suspended and then allowed to adhere on a larger plate). The immature healthy colonies were counted 4, 9 and 11 days (corresponding to a total cell age of 7, 12 and 14 days) after seeding of the cells. The result is represented in fig. 5A (except for the controls in Fig. 5, where very few colonies were obtained, the established frequencies are the result of counting more than 100 colonies from at least two different cell dilutions). As a control, the total efficiency of the cloning (differentiated undifferentiated colonies) was first determined, obtained 2 to 3 days after sowing with the purified SCF precursors in the different media (Figure 5A, left graph). It is observed that, in the presence of SCF, cloning rates of 10 to 20% were obtained, independently of the presence of esfradiol or TGFa. In media containing TGFa plus estradiol, or in controls that contained only estradiol, the few colonies visible at that time were too small for a count. More interesting were the results obtained with colonies containing more than 50% of healthy and immature cells. On day 7, the number of clones grown in SCF had only fallen to < 10", while clones that were cultured in SCF, TGFa and estradiol, were still present with a frequency of 10. The frequency of the clones developed in TGFa plus estradiol was even lower (2 x 10), whereas the clones The subsequent behavior of the clones developed in the different media supported the assumption that SCF / TGFα precursors were developed from SCF precursors.The immature clones, which only develop in SCF, descended at the time of 12 to 14 days at 3 x 10 or 1 x 10", approaching the background level (5 x 10) of the colonies developed on estradiol alone. As expected, the small number of colonies that had developed in TGF plus estrogen (2 x 10) did not vary with time. In agreement with the verification that SCF precursors can evolve in SCF / TGFα precursors (Figure 4B), a considerable part of the clones developed in SCF, TGFa and estradiol remained immature and with proliferation capacity, only decreasing the frequency (from 9 x 10 on day 7 to 5 x 10'2 on day 14, Fig. 5A). b) LD cloning of normal bone marrow cells In order to exclude that SCF precursors that have the capacity to acquire a self-renewal potential in the presence of SCF, TGFa and estradiol, before LD cloning they would have been previously selected by cloning in In vitro, tests were carried out with fresh and untreated bone marrow cells, in order to confirm the results obtained in a). In particular, it was to be determined whether erotification precursors with a potential for self-renewal could actually be formed with frequencies close to those of SCF precursors (1 of 3,000-5,000; Hayman et al., 1993) from individual cells, if they were grown in all three factors, while they remain infrequent (1 in 15,000) if they develop only in TGFa plus estradiol.

Normal bone marrow cells, prepared as described by Hayman et al., 1993, were seeded in an interval of 500 to 15,000 cells per well in CFU-E medium, which contained different combinations of factors at 4 different dilutions of the cells . { 500, 2,000, 6,000, 15,000) and immature colonies (containing more than 50% proliferative round cells) were counted at different instants after sowing. The result is represented in fig. 5B: after 4 days, the cells developed in SCF formed colonies with a frequency of 3 x 10 to 5 x 10. "Then, the frequency of the immature colonies progressively decreased, reaching after 3 days a frequency of 2 x 10" . In this case, an increasing proportion of cells in the differentiation manifested and then underwent an apoptosis. As expected, the clones developed in TGFa plus estradiol were from the beginning infrequent (6 x 10 to 8 x 10"), but the frequency remained essentially constant during the experiment, on the other hand, the clones developed in SCF, TGFa plus estradiol were found on days 4, 8 and 13 with a frequency of 3 x 10 -2 to 5 x 102. Therefore, the three factors SCF, TGFa and estradiol can actually induce the development of immature colonies of the bone marrow with a frequency that corresponds to that of SCF precursors after 4 days and that is similar to the frequency of cells in normal hen bone marrow that can form CFU-E colonies. Finally, it should be established if the three factors are really necessary to induce the growth of immature LD clones with a high frequency. In media containing individual factors (estradiol alone, TGFa, SCF plus ICI 164384, in order to suppress estradiol from the end-ogous serum), only very small numbers of immature clones were obtained (approximately 10). In TGFa plus SCF without estradiol, the clones behaved the same as in SCF alone, that is, they were frequent on day 4 and then decreased progressively (Figure 5B). Surprisingly, clones grown in SCF plus estradiol remained immature longer than those grown in SCF alone, but developed much more slowly compared to clones grown in TGFα plus estrogen or SCF or in TGFα plus estrogen. Since these clones had no similarity to the typical SCF / TGFα precursors (in relation to both the expression of c-ErbB and in vitro life span, see Example 4) they were not further investigated.

Example 4 Investigation of the in vitro lifespan as well as the expression of TGFaR / c-ErbB of SCF / TGFα precursors, which had been developed from SCF precursors. In order to investigate whether immature clones, obtained with a large Frequency by LD cloning of normal bone marrow cells or SCF precursors in SCF, TGFa plus estradiol, certainly represent typical precursors of SCF / TGFa, were investigated both in relation to their in vitro lifespan as well as their expression of TGFa / c -ErbB and its proliferation reaction on TGFa and other factors. The tests were carried out both in comparison with the clones grown only in TGFa plus estradiol, as well as with cells from mass cultures of SCF / TGFα precursors. a) Determination of the duration of life For the analysis of the in vitro life span, 10 to 12 healthy immature colonies were isolated, grown in SCF, TGFa and estradiol (obtained from 96-well plates with 500 seeded cells), or cultured in TGFa plus estradiol (from plates with 15,000 cells), were suspended and expanded in * their respective media until in a 100 mm plate 20 x 10 cells were obtained or the cells stopped growing due to reaching their lifespan in Clone-specific vitro (aging of the cells). Then, the developing clones were passed (diluted and transferred with fresh medium to new culture plates) until they were also aged, all the immature colonies that were obtained after growth for 13 days in SCF alone ( 6 colonies), as well as those from the control cultures (estradiol alone: 5 colonies; SCF only: 5 colonies; TGFa alone: 3 colonies; SCF plus TGFa: 8 colonies; SCF plus estradiol: > 15 colonies) were treated imi larly. Clones showing the predicted life span for that of the SCF / TGFa precursors were obtained only in SCF, TGFa plus estradiol and, as expected, in TGFa plus estradiol. 8 of the 12 clones developed with a high frequency in SCF, TGFa plus estradiol had a life span of 23 up to more than 28 generations (the remaining 4 had a lifespan of 12 to 15 generations). 7 of 10 clones that had developed in TGFa plus estradiol with a low frequency had a similar high life expectancy (23 to 31 generations, the lifespan of the remaining 3 were 15 to 17 duplications). This clearly demonstrates that the lifespan of SCF / TGFα precursors, which had evolved from SCF precursors in the presence of SCF, TGFa plus estradiol, was identical to that of authentic SCF / TGFα precursors. None of the colonies that had formed in the presence of individual factors or of SCF plus TGFα had a life span of more than 12 to 16 duplications. One clone, obtained in SCF plus estradiol, could be cultivated until generation 22, while another nine had a short life span (12 to 18 duplications). However, this clone was developed at a reduced rate, expressed very small amounts of TGFaR / c-ErbB and did not react on TGFa in a growth factor assay. Therefore, it can be assumed that these cells are rather an abnormal cell clone than authentic precursors of SCF / TGFa. b) Expression of TGFaR / c-ErbB and reaction on TGFa and other growth factors In order to determine if the LD clones obtained with a high frequency in SCF, TGFa plus estradiol express TGFaR / c-ErbB in similar amounts to those of the precursors of SCF / TGFa, grown in TGFa plus estradiol, were removed from the cells of 5 LD clones (2 clones were assembled by virtue of a small number of cells) cultured in the three factors, from 2 clones grown in TGFa plus estradiol , and from a mass culture of SCF / TGFα precursors, in each case all the factors overnight, the cells were lysed and investigated by Western blotting using anti-c-ErbB antibodies on the expression of TGFaR / c-ErbB. Fig. 6A, graph A (c-ErbB expression of LD clones from the bone marrow) clearly demonstrates that somewhat oscillating or similar amounts of TGFaR / c-ErbB were expressed in all three cell types, suggesting again that the clones of eri t rob shafts formed from SCF precursors in the presence of all three factors are authentic precursors of SCF / TGFa. In order to determine more quantitatively until the large number of LD clones obtained in the presence of the three factors resembles the SCF / TGFα precursors, another route was chosen: LD clones, induced by SCF precursors Purified by culture in all three factors (see Fig. 5B) were counted on day 13 and those plaques were chosen in which the majority of the cavities contained an immature culture. Then, the contents of all the cavities were suspended, washed in medium without factors and moved to new 96-well plates containing medium that had been completed by TGFa plus estradiol. The control LD clones, obtained from TGFa plus estradiol, SCF alone and estradiol alone, were treated in a similar manner. 3 days later (day 16) the clones were investigated for their proliferation capacity, measuring the incorporation of [H] thymidine (cavities with a number of counts of 5 times (in the case of individual colonies) or 10 times ( 2 or more colonies) above the basic value were counted as positive.From this analysis it was possible to calculate the frequency of clones incorporating thymidine (Figure 6B, Graph B, the filled rectangles show clones incorporating thymidine; striped boxes, all the clones.) The data obtained show that in essence all the healthy immature clones that had been cultured in TGFa plus estradiol and identified on day 13 incorporated thymidine on day 16, which confirmed that they were still proliferating. This was true for more than 50% of the clones (30 times more) that had been formed in the presence of the three factors. % of the few clones that had survived after 13 days in SCF only incorporated thymidine, while similarly rare clones that had developed in the presence of estradiol alone did not show any proliferation in TGFa plus estradiol. This made it possible to suppose that the clones formed in the controls did not represent typical precursors of SCF / TGFα, a finding that is confirmed by its short duration of life in vitro (see section a)). Finally, it was necessary to confirm that the LD clones, developed from SCF precursors with a high frequency in the presence of the three factors, showed a similar dependence of SCF, TGFa and estradiol as the precursors of SCF / TGFa. Figure 6B, graph C shows that a LD clone with the designation C6 (see Fig. 6A, graph A) showed a clear concentration-dependent reaction on the three factors, which corresponded almost to the behavior of a culture in mass of SCF precursors that had been cultured for 20 days in the presence of the three factors (see Fig. 3C), or precursors of SCF / TGFa, grown in TGFa plus estradioi alone.

Example 5 Definition of the factors that are necessary for the modification of the SCF / TGFa precursor differentiation program from SCF / TGFa precursors The results obtained in the preceding Examples demonstrate that SCF precursors can evolve into SCF precursors. TGFa, that is, they acquire both the capacity for continuous self-renewal and for the expression of endogenous TGFaR / c-ErbB when they are cultured in the presence of the three factors. However, the fact that cultures of this type are decisively dependent on the presence of chicken serum that may contain small concentrations of TGFa, SCF and / or estradiol as well as additional factors not characterized, established barriers for the valuation of data and He threw several issues. It was unclear if SCF / TGFα precursors required small concentrations of SCF that, however, were present in chicken serum. Also, SCF precursors may require small amounts of a hen factor that functionally replaces TGFα and is also contained in chicken serum. Second, it was unclear at what point during the development of SCF / TGFα precursors from SCF precursors the various factors were used. And, finally, the question of what factor (s) in chicken serum was needed for growth induced by TGFα / estradiol of SCF / TGFα precursors of the bone marrow and whether this or these factors represent a new one remained unanswered. activity or a known factor, for example SCF. In order to answer these questions, it was necessary to create a load of chicken serum that was essentially free of endogenous hormonal and growth factor activities, but which still made it possible to complete the development of factor-dependent cells when the necessary growth factors were added from the outside. Initial trials had shown that the chicken hen treated with animal charcoal (Schroeder et al., 1992) strongly inhibited TGFa / estradiol-induced growth of SCF / TGFa precursors (even though it did not abolish it altogether), but did not impaired the growth rate of these precursors after their establishment. Therefore, for the present assays, chicken serum was used that had been released thoroughly from hormones and endogenous factors by treatment with Freon and subsequent treatment by three times with animal charcoal (Schroeder et al., 1992) (in what follows) , this spent serum is called "treated chicken serum"). Bone marrow cells were cultured in CFU-E medium containing Fetal calf serum treated with Freon and untreated chicken serum or treated chicken serum. The cells were cultured in SCF alone or in SCF, TGFa and estradiol and counted at the times indicated in Figure 7, the cumulative cell numbers determined as in Example 1 being plotted.

CFU-E, which was prepared with the treated hen serum (in figure 7, the unfilled squares mean purified chicken serum plus SCF, the filled squares mean purified chicken serum plus estradiol, the unfilled circles mean serum of normal chicken plus SCF and filled circles mean normal chicken serum with estradiol), allowed the growth of SCF precursors to the same extent as the control medium with untreated chicken serum, regardless of whether the cells had been grown in SCF alone or in SCF, TGFa more estradiol (Figure 7A, graph A). There was also no effect on the proliferation rate of cultures of SCF / TGFα precursors of 15 days of age, except for a weak effect when the cells began to age (Figure 7B, Figure C). Surprisingly, however, the serum of The treated hen slowed down the evolution of SCF precursors in SCF / TGFα precursors in the presence of SCF, TGFα and estradiol (Figure 7A, Figure B). After their late onset, the SCF / TGFα precursors formed in the treated chicken serum were, however, developed with the same30 vetocity than control cells in untreated chicken serum that had formed at least 5 days before (Figure 7A, Figure B). These observations made it possible to draw different opinions. ^ '-' '* "" clusions: first, the chicken serum contains a additional activity that favors the evolution of SCF precursors in SCF / TGFa precursors. And secondly, this activity is important for the change in development, but it does not harm the proliferation of SCF precursors before the modification nor is it important for the proliferation of established SCF / TGFα precursors. The availability of a treated chicken serum also allowed us to investigate at what point during the evolution of SCF / TGFα precursors the known factors were required. 3-day-old purified SCF precursors developed at a comparable rate in SCF plus TGFa, regardless of the presence or lack of estradiol (Figure 8A, Figure A), the estradiol present in the normal chicken serum used was suppressed again with ICI 164384). Accordingly, estradiol has no effect on the early proliferation of SCF precursors. The fact that these precursors developed at the same speed in media containing treated hen serum, SCF and estradiol with or without TGFa, demonstrates that TGFa is also superfluous and that the only factor required by early SCF precursors is SCF. During the modification of the differentiation program, a different model of growth factor requirements is formed. As shown in Figure 8A, graph A, the cells maintained in SCF plus TGFα without estradiol cease irreversibly to proliferate around days 8 to 10, which suggests that estradiol is necessary for the change. Previous results point to the fact that it is also necessary for the proliferation of established SCF / TGFα precursors (Schroeder et al., 1993). Another group of tests clearly shows that the SCF is necessary during the modification of the differentiation program. 6-day-old SCP precursors, established in media containing treated chicken serum and SCF, may evolve with poor efficacy in SCF / TGFα precursors when they are continued to be cultured in treated chicken serum containing all three exogenous factors. However, if they only contain TGFa and estradiol, under otherwise identical conditions, they lack this capacity completely (Figure 8A, Figure B). Therefore, the evolution of SCF / TGFα precursors depends on the presence of SCF during the modification of the differentiation program, while these same precursors, once established, are independent of SCF (see Fig. 7B, graphic C and bottom). Finally, the SCF precursors do not need any TGFa (Figure 7A, Figure A), but in their absence, no formation of SCF / TGFα precursors occurs even when untreated chicken serum is used (Schroeder et al. ., 1993). In summary, the tests carried out allow us to draw the following conclusion: the joint presence of SCF, TGFa and estradiol is necessary for the evolution of SCF / TGFα precursors from SCF precursors, while another unknown activity in the serum of chicken increases the effectiveness of its formation. Some data of experimental trials, which were carried out in a complementary way, allow us to suppose that this activity could be the er i t ropoyet ina de gallina, but they do not show it. It was found that anemic serum, in tests with growth factors, strongly stimulates the proliferation of SCF / TGFa precursors; an even more important finding was that the anemic serum increased the growth rate of SCF / TGFα precursors during and after their establishment, even when these cells were exposed to normal chicken serum plus SCF, TGFa and estradiol ("STE") (Fig. 8B, graph C). Finally, erbtoblasts that had been stimulated for self-renewal by a c-ErbB stably expressed in retroviruses, ie, an exogenous in-kinase t and which, after infection with another retrovirus, expressed the er-receptor. It could be stimulated three or more times in its rate of proliferation by recombinant human erythropoietin (EPO).

Example 6 Identification of two chicken serum factors that accelerate the t ansformation of SCF precursors in SCF / TGFa precursors or are necessary for their growth a) Ligands of the glucocorticoid receptor (eg dexamethasone) belong to hen serum factors that require SCF precursors for their evolution in SCF / TGFa precursors In the previous example it was shown that the evolution of SCF precursors in Precursors of SCF / TGFa requires, in addition to SCF, TGFa and estradiol, still other undefined factors of chicken serum that can be eliminated by treatment of serum with activated charcoal. In the presence of a chicken serum treated with active carbon, the evolution in SCF / TGFα precursors does not occur or it only occurs very inefficiently. Since steroid hormones are mostly eliminated by the treatment of serum with activated charcoal, other steroid hormones were investigated along with estradiol in terms of their activity during the transformation of the normal cell differentiation program. Firstly, glucocorticoid receptor ligands were tested, since a deficiency of glucocorticoids in humans leads, among others, to anemia and prevents the differentiation induced by DMSO in mouse cells. Previous trials have shown 1) that SCF cells do not need DMSO for their transient self-renewal, and 2) that established SCF / TGFa cells require small concentrations of glucocorticoids to grow. The cells do not develop if they are cultured in the presence of TGFa and estradiol in media in which both the fetal calf serum and the chicken serum were treated with active carbon. If 1 x 10"M dexamethasone is added to the same medium, the cells are stimulated to grow at a normal speed, and the cells may not grow even in untreated media when a glucocorticoid antagonist is added together with TGFa and estradiol. In order to directly test whether glucocorticoids (dexamethasone) accelerate the transformation of SCF precursors into SCF / TGFα precursors, an experiment was carried out in which SCF precursors were exposed for a short period (4). days, days 3-7 after isolation of the bone marrow, designated in the following period of induction) to different mixtures of factors (see Fig. 9) .The cells were then washed and seeded in a medium that contained only TGFa and estradiol (TE medium). Only SCF / TGFa precursors expressing c-ErbB, developed late, but not precursors of SCF or cells at an early stage of evolution can develop in this medium. tion in SCF / TGFa precursors (see Example 5). The results are represented in fig. 9. As a negative control, the cells (4-day old SCF precursors) were cultured during the induction period in the most average SCF with sera treated with activated charcoal (fetal calf serum and chicken serum). After the transfer to the TE medium, no growth of the cell could be observed for a long time. Only 9-10 days after transferring to the TE medium, SCF / TGFα precursors (Fig. 9, blank rhombuses) were developed, possibly derived from cells that were already present in the bone marrow as precursors of SCF / TGFa ( see Example 5). As a positive control, the SCF precursors were treated during the induction period of 4 days with SCF, TGFa and estradiol (Figure 9A, triangles in black). After transfer to the TE medium, the cells developed, as expected, much more rapidly and only a 5-day delay (latency phase) of growth was observed. This corresponds to the results represented in Example 1 (Fig. 1, arrows). A surprising result was obtained when the cells were treated during the induction period with SCF, TGFa, estradiol and dexamethasone. Not only did the cells develop during the induction period much more rapidly than in the controls, also after the transfer to the TE medium there was no appreciable delay and the cells continued to develop at a constant speed (Figure 9A, blank squares) ). This result shows that the addition of dexamethasone determined the transformation of virtually all of the SCF precursors into SCF / TGFα precursors. Additional investigations by the transfer of phospho-iros ina (Western blot with phosphothosin antibodies) resulted in these cells expressing the expected amounts of c-ErbB. The effect of dexamethasone could also be observed in cells that were only cultured in the presence of SCF. The addition of dexamethasone in the presence of SCF accelerated the growth of SCF / TGFα precursors more strongly than in the positive control (SCF, TGFa, estradiol, Fig. 9A, see triangles in black and white). As in Example 5, it was shown that the cells, in addition to SCF and dexamethasone, require the estradiol contained in the sera in small amounts, since the addition of the estradiol antagonist under the designation ICI 164384 (Schroeder et al., 1993) limited the growth of the cells to the extent observed in the negative control (Figure 9A, see blank diamonds and black circles). In addition, the cells required small concentrations of a c-ErbB ligand (unknown, contained in chicken serum). These results demonstrate i) that dexamethasone is necessary, in addition to TGFa and estradiol, for the growth of SCF / TGFα precursors with capacity for self-renewal, and ii) that this hormone greatly accelerates the transformation of SCF precursors into SCF precursors / TGFa. b) Growth of SCF / TGFa precursors: Insulin-like growth factor I (IGF-1) replaces, together with SCF, TGFa, estradiol and dexamethasone, the absolute necessary hen serum for the growth of all the tests carried out The former, with normal precursor cells of hens, capable of self-renewal, were linked to the presence of tested loads of chicken serum; It was not yet possible to define all the factors that can replace chicken serum. The results described in section a), in the sense that dexamethasone allows the growth of these cells in chicken sera treated in active carbon, led to a series of new tests to replace chicken serum by defined factors. The definition of these necessary factors for chicken cells constitutes the basis for the corresponding eventual requirements of human cells. Since a mixture of factors of SCF, TGFa, estradiol and dexamethasone optimally favored both the evolution and the growth of SCF / TGFa precursors, this mixture was used in mediums with and without chicken sera. As other possible factors for the substitution of chicken serum, insulin-like growth factor (IGF-1) and avian IL-6 (chicken growth factor, cMGF) were investigated. The tests were carried out in medium with (Fig. 9B, medium S13) and without chicken serum (Fig. 9B, Epo assay). Of the factors tested, only IGF-1 was effective. Fig. 9B shows that in the presence of SCF (S), TGFa (T), estradiol (E), dexamethasone (D) and IGF-1 (IG) both 16-day-old SCF / TGFα precursor cells (Figure 9B, circles in black, triangles in white) as well as bone marrow cells of 9 days of age, activated in SCF / TGFa and estradiol (Figure 9B, triangles in black and white) proliferated with the same speed in media with serum of chicken (symbols in black) and without chicken serum (symbols in blan-coT) This effect could be detected over a period of time of> 7 days.In the absence of IGF-1, the cells completely ceased in its growth after 2 days, the same result was obtained (no cell growth) when IGF-1 was replaced by cMGF.

EXAMPLE 7 Culture of Human Eritrocyte Cells Which Have Similarity to the Precursors of SCF of Hen and SCF / TGFa of Gal 1 ina a) Provisional definition of the conditions that make possible the growth of human erythrocyte precursors from bone marrow or peripheral blood. Tests were carried out with human hematopoietic cells. The assumption on which these trials were based was that the human precursors have a lifespan in vitro similar to that of human fibroblasts (50 to 70 generations), which represents the basis for the determination of erythrocyte precursors. - human beings capable of self-renewal. The bone marrow or peripheral blood of healthy donors served as a source for these trials. From these sources, immature blood cells expressing the surface antigen of CD34 cells were enriched by immunoaffinity chromatography according to the method described by Shpal 1 et al., 1994. Enriched cells were seeded, as in the preceding examples , in a modified CFU-E medium (Hayman et al., 1993) containing human serum (Sigma) instead of chicken serum and human transferrin saturated with iron (Sigma) instead of conalbum. The medium was supplemented with 20 ng of TGFa (Promega), 20 ng of recombinant EGF (Promega, EGF was used for the case that the hypothetical member of the EGF receptor family present in eri trocytes cells does not have TGFa as a functional ligand), 100 pg of purified human SCF (Promega), 5 x 10 M estradiol (in some assays that were useful for the characterization of the cells, other factors were added to the medium, such as IL-3, IL-1). and LIF). Cell growth was monitored by cell counting and the cell types present in the cultures were analyzed by centrifugation on slide and histochemical staining on hemoglobin and histological dyes (Beug et al., 1982). (i) Bone marrow assays Initial trials of cultivating erythrocyte precursors from human bone marrow in modified CFU-E medium, containing human serum, human transferrin saturated with iron, 20 ng TGFa (Promega), 20 ng of Recombinant EGF (Promega), 100 ng of purified human SCF (Promega), 5 x 10 M estradiol and other different factors (in each case 10 ng of IL-3, IL-6, IL-1 and LIF) per milliliter (ml ), did not show any success in principle. However, when recombinant EPO was added to the medium (3 international units / ml), it was possible to culture erroneous precursors that remained immature for 13 days but that in essence all differed on day 16. During this time , cell numbers increased by 25 to 50 times; a more accurate determination was not possible by virtue of low cell numbers (only 2 x 10 cells initially seeded, thus less than 10 cells after 3-5 days). The proliferating cells obtained resembled human pro-erythroblasts and were surprisingly similar to the normal precursor cells of chickens (Fig. HA, graphs A and B, see below). During the first couple of days of the culture, as well as after day 15, many nuclear reenucleotides, enucleating cells and erythrocytes were visible, indicating that the differentiating reticulocytes differed in normal cultures in erythrocytes. and they could also carry out the enucleation process in a normal way (core ejection). Without EPO, the cultures did not develop and contained very few immature eri trocytes. Mainly, they contained mature monoblasts, as well as different types of immature granulocytes (neutrophils, eosinophils, mast cells). ii) Tests with peripheral blood cells The one described in i) was repeated with 40 x 10 6 CD34 + cells, enriched from human peripheral blood. 2 x 10 cells / ml in modified CFU-E medium plus SCF, TGFα and EGF, estradiol and human recombinant EPO were seeded in tissue culture plates and the cells were counted at the indicated times, the mean cell volumes being determined in a electronic cell counter of the CASY-1 type, Schárfe system. Given that the initial number of cells was greater than in the assay carried out with the bone marrow, the kinetics of proliferation of the culture could be accurately monitored. Fig. 10A shows that the numbers of cells decreased during the first 2 to 3 days, which has to be attributed to the maturation and / or death of partially differentiated precursor cells. Then, the cells proliferated exponentially with doubling times between 20 and 30 h until day 15, after which no further growth was observed. The total increase in the number of cells during this growth phase was > 300 times Fig. 10A also shows that the cells retained their size during the exponential growth phase (cell diameter between 9 and 10 μm, cell volume between 500 and 600 cells), which is a first indication that they remained immature . Given that antigen markers were not available that differentiate human proer it from other myeloid or pluripotent precursors, for the realization of these tests, and the detection is not really definitive by histological staining, indirect methods were used in order to determine the percentage of errictor precursors in the cultures: first, aliquots were stained at regular intervals by benzene-acid, which represents a very sensitive detection of hemoglobin (Graf and Beug, 1978). At day 6, the cultures already contained 14% of benzidine-positive cells and on days 10 and 11, these values had increased to 51 or 63%. Since a pure culture of chicken SCF / TGFa precursors contains between 30 and 60% of benzidine-positive cells, it can be deduced from these results that approximately on day 10 the crop consisted predominantly of erotic precursors. This assumption could be confirmed in an assay in which the cells were induced to differentiate: an aliquot of the 10-day-old culture was washed and resuspended in modified CFU-E medium containing 10 U / ml of human recombinant EPO plus 10 ng / ml insulin or IGF-1 (insulin-like growth factor 1). A parallel aliquot additionally contained IL-3 (10 ng / ml). The data in fig. 10B show that the cell numbers increased approximately 3 times, decreasing their cell volume at the same time considerably, as is to be expected for erigerative cells in differentiation. A staining with acid benzidine after 2 days provided >; 95% of benzidine-positive cells in the culture that had only received EPO / insu 1 ina. This indicates that most of the cells present before the induction of differentiation should have been eritrified, particularly since in the differentiating cultures only very few apoptotic cells were visible after cytocentrifuging and histological staining. (see below). The addition of IL-3 possibly delayed differentiation; after 2 days only 66% of benzidine-positive cells were detected, and the cells grew somewhat more rapidly, while their cell volume decreased more slowly (Fig. 10B). b) Characterization of proliferating cells in SCF, TGFa, estradiol and EPO In order to establish whether the erithrocyte precursors obtained in a) by culture in SCF, TGFα plus EGF, estradiol and recombinant EPO correspond to the precursors of * SCF / Hen TGFa cultured in the preceding examples, the following two batches of assay were chosen: First, the cell types present in the cultures were characterized by centrifugation on slides and histological staining and combined histochemistry on hemoglobin (Beug et al. , 1992, see the phases defined there). With this, the time that hemoglobin-negative or weakly positive, immature proletariants would have to be established in the crops should be established in order to obtain an indication of whether errant progenitors were indeed self-renewing, such as would be expected from the growth kinetics and the size distribution (Figure IOA). The proer i t robus differed in the staining used for other cells by means of a large central cell nucleus, a strongly basophilic cytoplasm, a characteristic overlap of the cytoplasm border and a weak but differentiable staining of myeloid cells with neutral benzidine. Fig. 11A, graph A (proliferative cells, bone marrow after 7 days, CD34 + cells after 10 days) and B (cells differentiated after 10 days of proliferation and 4 days of differentiation), shows that a large percentage of the cells that are In the culture, they are similar to prole and idina-negative benzodiazepines and some myeloid cells were present. These results were obtained until day 14 and then clearly increased the percentage of maturing cells. In contrast to this, cells obtained after induction of 4-day differentiation (see above) were represented by reticulocytes as well as mature and enucleating erythrocytes (Figure 11A, Figures A and B), which is a further confirmation that the cells maintained in SCF, TGFa, estradiol and EPO were really unable to be incorporated into the differentiation induced by the above factors. Fig. HA shows by slide on slide and histological and histochemical staining combined on hemoglobin p-feparac ions characterized by human bone marrow (BM) and CD34 + cells (CD34) (graph A), each photographed under green light ( top.) and. blue light (lower part) in order to establish histological features and hemoglobin staining. Er = enucleated erythrocytes and erythrocytes; R = reticulum; Pe = probri troblasts; M = myeloid cells. Fig. HA, graph B shows CD34 + cells cultured for 10 days that were induced for 4 days for differentiation and photographed in a typical manner.

A clear demonstration that cell-like SCF / TGFα precursors can actually be obtained from human erroneous precursors was obtained by investigating whether the cells express both c-Kit and also a member of the c-receptor family. ErbB / EGF and proliferate as a reaction on the respective ligands. Since in principle no culture could be obtained that underwent a self-renewal during the 50 to 70 divisions expected, it was considered that most of the cells in the cultures, particularly in the early stages, correspond to precursors of SCF and that the precursors of SCF / TGFa had only been formed with lower efficacy, which probably has to be attributed to suboptimal conditions of the culture. Therefore, the reactivity of human bone marrow cells on different growth factors was tested by different tests with growth factors (Leutz et al., 1984; Hayman et al., 1993). The results of these tests are represented in fig. 11B, graph C (factors of self-renewal TGFa / EGF, SCF) and in fig. 11B, graph D (differentiation factors (EPO, IL-3)). For these tests, CD34 + cells were cultured for 8 days, washed, and as described in Example 4, they were tested for their dependence on growth factors, with the difference that serum-free CFU-E was used. human. A concentration of relative growth factors of 100 corresponded to 400 ng / ml of recombinant SCF, in each case 40 ng / ml of TGFa or EGF; 10 ng / ml of recombinant human IL-1, 20 U / ml of recombinant human EPO, 40 ng / ml of recombinant human IL-3 and 10 ng / ml of recombinant mouse LIF. Lo-r values represented are the average values of triple determinations. The cells showed a strong reaction on SCF and, what was still more significant, a weak but clear reaction on a mixture of TGFα and SCF. On the contrary, no reaction was observed on the two cytokines IL-1 and LIF that act on very early pluripotent hematopoietic cells. This allows to conclude that the cells that react on SCF represent erroneous "imprisoned" precursors. As expected, the cells reacted with the same intensity on the differentiation factors EPI and IL-3, which also confirms that the culture contains predominantly erigenetic cells.

Example 8 Growth factors and additional steroid hormones induce in cultures of human pro-biloblasts, capable of terminal differentiation, self-renewal for a long time (> 20 generations). In Example 6 results were obtained in the system with hens that were of potential importance for the growth of human proletards, capable of self-renewal: 1. Of the factors in principle not defined in the system with hens that, in addition to SCF, TGFa and estradiol, are necessary for the evolution of SCF precursors in SCF precursors / TGFa, two factors could be identified: the steroid hormone dexamethasone and the general growth factor, insulin-like growth factor (IGF-1). 2. Therefore, the effect of dexamethasone on the self-renewal behavior of human proerbism was investigated more precisely. Likewise, it was investigated whether IGF-1, which in the system with hens makes hen serum cells independent, shows in human cells at least growth-promoting properties. The results obtained were surprising: it was possible to increase the multiplication of human progerms during their lifetime in vitro from 200-1000 times to more than 100,000 times. This enabled a more accurate characterization of cell populations obtained through colony assays and FACS analysis. It was also possible to investigate with much greater precision than in Example 7 the differentiation behavior of proletards, since sufficient cells were available. ~ *) Effect of dexamethasone Human CD34 + cells from umbilical cord blood, purified as described in Example 7, part (ii), were seeded in medium plus Epo, huSCF, TGFa and estradiol, as described in Example 7. To a second culture were added additionally to the above factors, so-called "factor mix", dexamethasone 1 x 10 M. The growth of the cells was monitored until a recognizable multiplication was completed. The results are represented in fig. 12A. As expected, the cells grew exponentially in the "factor mix" until 13/14 and showed a cell multiplication of 1000-2000 times (Figure 12A, black circles). Unexpectedly, the parallel cultivation that had been cultivated in the "factor mix" plus dexamethasone proliferated exponentially until at least the 18th day and then ceased only gradually in its growth (Figure 12A, blank squares), so that a multiplication of the cells was obtained 150,000 times. If one starts from the fact that always a part of the cells appears in spontaneous differentiation and, therefore, only a part of the population of immature cells is available for the conservation of the self-renewal potential of the culture, then these data show that The proer it robles of human umbilical cord blood are able, in the presence of Epo, SCF, TGFa, estrogen and dexamethasone, to retain their self-renewal potential for at least 20 generations of cells. This is essentially more than even the 7-10 cell divisions that experience human BFU-E's within their normal developmental potential (Sawada et al., 1990). This demonstrates, i) that human eritrocyte precursor cells, similar to the corresponding cells of the hen, can be induced, by combination of in-kinase ligands and steroid receptor ligands, to a modification authentic of its development potential, that is, a long-term self-renewal. b) Effect of IGF-1 CD34 + cells from peripheral blood of an adult person (obtained as in Example 7, part ii)) were cultured in the "factor mix" plus dexamethasone until the cells began to grow exponentially . After40 ng / ml of recombinant human IGF-1 (Promega) was added to an aliquot of the cells. As shown in fig. 12B, the cells with IGF-1 (circles in black) grew significantly faster than without this growth factor (blank squares). Given that the medium used contained 15% fetal calf serum as well as 4% serum of human umbilical cord blood (of which it is expected that they contain a basal IGF-1 concentration), an increase in the rate was obtained. certainly relatively sparse growth which, however, shows that the cells react on IGF-1. This is also apparent from assays in which the cells were grown overnight without a factor, then stimulated for 5 and 10 minutes with IGF-1, lysed and investigated in the transfer of phosphotosin (see Example 2) as for the phosphorylation of the receptors. The cells, thus treated, showed in the transfer of phosphothoses to an autophosphorylation of the intracellular chain of 90 kD IGF-1 receptors as well as the 130 kD protein IRS-1 ("insulin receptor substrate"). . c) More accurate characterization of human erythroblasts capable of self-renewal with the help of colonies and surface markers * The surprising ability of human proeri troblast? jSa cultured in the "mixture of factors" with and without dexamethasone, allowed to advise It is convenient to investigate these cells with greater precision in terms of their development potential and their position within the series of erroneous development. For this, two types of methods were used: First, the cells were planted in semi-liquid media with appropriate combinations of cytokines and 10 days later the type of colonies developed was counted. The following types of colonies were differentiated: i) colony forming units that explode (BFU-E), colonies consisting of 1000 a > 20,000 cells that exclusively contain erythrocytes and, therefore, indicate that the starting cell is an immature precursor that is committed ("imprisoned") to the series of eritrocyte development; ii) a mixture of BFU, large colonies with > 20,000 cells which, in addition to erythrocytes, contain cells from at least one other series of development and, therefore, target pluripotent stem cells; and iii) granulocyte / macrophage colony forming units (CFU-GM), colonies based on 100 a > 1,000 cells that do not contain erythrocytes but only myeloid cells (macrophages and / or granulocytes) and, therefore, come from non-eritrite precursor cells. Secondly, the cells were tested, with the aid of suitable antibodies and FACS analysis, for the expression of surface markers that are specific for cells of certain development series and degrees of maturation. Although there are no surface markers that, when used individually, exclusively recognize proer it rob humans, can be determined with great certainty, by combination of several markers are expressed on eri t rob immature and / or mature, with specific markers for myeloid cells (CD 33) and lymphoid cells (CD-3, CD-19) the cells belong to the series of erythrocyte development. The CD71 antibody (a-t transferase receptor) is suitable for this purpose, which certainly weakly stains all the proliferating cells, but strongly marks the erythrocyte cells. Together with CD 117 (c-Kit, it is only expressed on very immature eri trocytes cells, similar to BFU-E, as well as pluripotential precursors and certain myeloid cells (mast cells)) and GPA (a-glycophorin, specific for partially mature eritrocyte cells), the CD 71 antibody allows the safe determination of proverbodies (bright CD 71, CD107 from positive to weakly positive, GPA negative or weakly positive, CD33, CD3, CD19 negative ). In order to test cells of the corresponding cultures for their belonging to series of development and to their degree of maturation within the series of erite roe i taria development, aliquots of the cells of the cultures shown in fig. 12A on day 13 and day 16 and were subjected to the tests represented in Tables I to III. The cells taken from the culture on day 16 without dexamethasone were furthermore separated according to the density: in the system with hens only cells with a density </ p> are immature. 1070 g / cm, cells with a density > 1,072 g / cm are in all cases partially mature and only require 1-2 days for maturation in erythrocytes, experiencing only a few cell divisions. Corresponding fractions were prepared from the human cells and tested separately for the formation of colonies and surface markers. The results are represented in Tables I to III. They show that the predominant type of colony is formed, both after 13 and after 16 days of BFU-E 's, that is, of immature erythrocyte precursors. The dexamethasone effect of maintaining the cells for a longer time in an immature state, characterized by self-renewal, is also clear from the BFU-E figures: cultures with dexamethasone contain, both after 13 and after 16 days, a 2-2.5 times greater number of immature colony forming pre-cores as well as dexamethasone. The data also show that the cell population consists, in a very predominant part, of "imprisoned" erythrocyte precursors. Both after 13 and 16 days, more than 90% of the colony-forming cells are purely erythrocyte precursors, with the proportion of pluripotent precursors (mixture of BFU's) and "imprisoned" myeloid precursors, in each case only 3-6%. Interestingly, dexamethasone also stimulates the content of 3-6 fold pluripotencial precursors, while the effect on myeloid precursors is much weaker. As expected, the densest fraction (>; 1.072 g / cm) of 16-day-old umbilical cord blood cells were unable to form colonies in semi-liquid Methocel medium. His more mature character was also confirmed by the analysis of the markers. Tables II and III further show that the results of the analysis of the markers in FACS completely confirm the conclusions drawn from the colonies trials. All cultures contained only a small proportion of myeloid cells (10-20% after 13 days, 5-7% after 16 days, no lymphoid cells as well as only CD34-positive cells, around 5%, data not shown.About 85% of the cells are strongly positive CD 71, but only a small% of the cells are positive GPAs.The effect of dexamethasone of maintaining the precursor cells in an immature state is also clear from the expression of CD 107 (c-Kit): on day 16 (when the culture in dexamethasone was already clearly decreasing in growth rate, see Fig. 12A), only 21% of the cells were c-Kit positive, whereas in the parallel culture maintained in the presence of dexamethasone, still growing exponentially, still more than 50% of the cells were t-positive c-Ki.Also the partially mature state of the densest fraction of the 16-day-old culture it could be confirmed by an lysis markers: only 53% of cells were CD 71-positive (maturing cells lose receptor transferr ina), while 66% of the cells were positive GPA. In summary, the characterization of umbilical cord blood cultures yielded the following summaries: i) Cultures are composed very predominantly in immature precursors similar to proerblasts that are committed to the series of erotification development. , but that can still form large eri trocal colonies (BFU-E). The doping with pluripotent cells and cells from other series of development is less than 10%. ii) The effect of dexamethasone, together with other factors inducing the ability of human proletarotroblasts for long-term self-renewal (more than 16 cell divisions) is clearly reflected in the analyzes of colonies and markers: both the ability to form BFU-E (and mixture of BFU-E's) as the ability to express c-Kit is decisively reinforced by dexame asona. d) Regulation of the differentiation of probes and troblasts from umbilical cord blood cultured in vitro: application of the results obtained in the system with chickens. An essential advantage of normal erythrocyte precursor cells, capable of self-renewal in the hen system was that the cells, after the removal of "self-renewal factors" (SCF, TGFa and estradiol) and replacement of these factors by differentiation factors (Epo, insulin) are differentiated, with a normal kinetics and experiencing the expected number of cell divisions, in mature erythrocytes (Hayman et al., 1993). In Example 7 it was already demonstrated that this observation was transferable, in principle, to human robbery: the human-grown prover it was grown in vitro matured in recombinant human Epo and insulin in enucleating erythrocytes (core expellers) ( Example 7, Fig. 10A, graphs A and B). The presence of larger quantities of human proetroblasts clearly capable of self-renewal allows us to invest quantitatively and induce differentiation. In addition, it was now possible to analyze the influence of other factors on the differentiation program of the cells. In the system with chickens it was possible to demonstrate that SCF, in the presence of Epo and insulin, can strongly delay the eri-rociation differentiation or prevent it practically during the first 4-5 days (Hayman et al., 1993). In addition, the thyroid gland hormone T3 (tr iiodo-t-ronin), especially together with ligands of the RXR co-receptor, was able to accelerate erotification differentiation and to completely cancel out the determined retardation. by SCF of the erotification differential (Schroeder et al., 1992, Beug et al, 1994). Therefore, it was interesting to know if also these observations obtained in the system with hens were valid for the human system. While a clear SCF effect, which pointed to a delay in differentiation, was detectable in purified human BFU-E's (Dai et al., 1991, Sawada et al., 1991), direct investigations of the effect of T3 on the development of purified erythrocyte precursors. The tests were carried out with 16-day-old cells of the culture maintained with the "factor mixture" and dexamethasone. The cells were separated by centrifugation, washed in medium without factors and cultured at a density of 1-2 x 10 cells / ml in different differentiation media. The differentiation medium contained 2% human serum (umbilical cord blood) and no other additive (Figure 13, blank squares, no factor) or 10 units / ml human recombinant Epo plus 10 ng / ml insulin (Figure 13). Fig. 13, Epo, Ins, squares in black), Epo, Ins plus 100 ng of human SCF (Fig. 13, SCF, Epo, Ins, circles in white) and the above factors plus tr iyodo-t ironina 200 nM and 10 M cis-ret acid (Fig. 13, SCF, Epo, Ins, T3, lig.RXR, circles in black). During differentiation, the cells were cultured at a density of 2-4 x 10 cells / ml and fresh factors were added daily. At the indicated times the cell volume was determined in an electronic cell counter of the CASY-1 type, Schárfe system, (see Example 7). At the same time, the hemoglobin content of aliquots of cells of a known number of cells was determined by photometric measurement (Kowenz et al., 1987). The results are represented in fig. 13. While, as expected, in the absence of Epo / Ins, the hemoglobin content per cell volume was increasing (Figure 13, blank squares), a strong increase was observed in the presence of Epo / insulin (approx. 8 times) of the hemoglobin content / volume of cells (Figure 13, squares in black). Surprisingly, SCF delayed Epo / insulin-induced erythrocyte differentiation in a manner very similar to the chick system (Fig. 13)., circles in white), while the addition of thyroid gland hormone (T3) plus RXR ligand completely eliminated this delay of differentiation by SCF (Fig. 13, black circles), again exactly analogously to the data obtained in the system with chickens. In summary, these data show that prover i trob humans capable of self-renewal in culture mature in the culture depending on eri t ropoyet ina, accumulating hemoglobin erythrocytes. This process is delayed by SCF and accelerated by T3 (as in purified human BFU-E's, Sawada et al., 1991).

Table I Type of cell Type of colony (for 105 cells) BFU-E mixture of BFU's CFU-GM 1 2250 60 70 2 5750 400 200 3 1200 45 115 4 < 1 < 1 < 1 5 2500 105 100 6 ND ND ND ND = not detectable 1: umbilical cord blood (13 days, factor mix) 2: umbilical cord blood (13 days, factor mix + dexamethasone) 3: umbilical cord blood (16 days, factor mix, immature fraction <1,070 g / cm) 4: umbilical cord blood (16 days, factor mix, mature fraction> 1.072 g / cm) 5: umbilical cord blood (16 days, factor combination + dexamethasone) 6: peripheral blood (CD34 *, 9 days, factor mix + dexamethasone) Table II Cell type Surface marker of immature cell er i t roci tar io CD71 CD117 GPA (receptor of (c-Kit, re (g 1 icofor ina) t ransferr ina) ceptor of SCF) 1 ND ND 66% 2 ND ND 20% 3 88% 21% 9% 4 5 533 %% 2 211 %% 66% 84% 51% 7% 6 80% 65% 30% ND = not detectable 1: umbilical cord blood (13 days, factor mix) 2: umbilical cord blood (13 days, factor mix + dexamethasone) 3: umbilical cord blood (16 days, factor mix, immature fraction <1,070 g / cm) 4: umbilical cord blood (16 days, factor mix, mature fraction> 1.072 g / cm I 5: umbilical cord blood (16 days, factor mix + dexamethasone) 6: peripheral blood (CD34 +, 9 days, factor mix + dexamethasone) Table III Cell type Surface marker of non-erigerative cell cell CD33 CD3, CD19 (large.) (B cells, T cells) 1 10% < 0.1% 2 20% < 0.1% 3 5% < 0, 1% 4 < 0, 1% < 0.1% 7% < 0, 1% 6 5% ND ND = large non-detectable. = granulocyte cells 1: umbilical cord blood (13 days, factor mix) 2: umbilical cord blood (13 days, factor mix + dexamethasone) 3: umbilical cord blood (16 days, factor mix, immature fraction <1,070 g / cm) 4: umbilical cord blood (16 days, factor mix, mature fraction> 1.072 g / cm) 5: umbilical cord blood (16 days, factor mix + dexamethasone) 6: peripheral blood (CD34, 9 days, factor mix + dexamethasone) Bibliography Beug, H., Palmieri, S., Freudenste in, C, Zentgraf, H and Graf, T., 1982, Cell 28, 907. Beug, H., Dóderlein, G. and Zenke, M., 1992, in Nuclear Processes and Oncogenes, (PA Sharp, ed.) P. 53, Academic Press Inc., Hartcourt Brace Jovanovich, San Diego Publishers. Beug, H., Müllner, E.W. and Hayman, M.J., 1994. Curr. Op. Cell Biol. 6, 816-824. Boulay, J.L. and Paul, W.E., 1993, Current Biology 3, 573-581. Dai, C.H., Krantz, S.B. and Zsebo, K.M., 1991. Blood 78, 2493- -2497. Daley, G.Q., Van Elten, R.A. and Baltimore, D., 1990, Science 247, 824-830. Elefanty, A.G., Hariharan, I.K. and Cory, S., 1990, EMBO J. 9, 1069-1078. Fantl, W.J. et al., 1993, Annual Reviews of Biochemistry 62, 453-481. Galimi, F., Bagnara, G.P., Bonsi, L., Cottone, E., Follenzi, A., Simeone, A. and Comoglio, P.M., 1994, J. Cell. Biol. 127, 1743-1754. Graf, T. and Beug, H., 1978, Biochi. Biophys. Minutes 516, 269-299. Hayman, M.J., Meyer, S., Martin, F., Steinlein, P. and Beug, H., 1993, Cell 74, 157-169. Hogan, B., 1993, Current Biology 170-172. Jolly, D., 1994, Cancer Gene Therapy 1, 51. Kaipainen, A., Korhonen, J., Pajusola, K., Aprelikova, O., Persian, M.G. , Terman, B.I. and Alitalo, K., 1993. J. Exp. Med. 178, 2077-2088. Keller, G., 1992, Curr. Opinion in I munology 4, 133-139. Kelliher, M.A., McLaughlin, J., Witte, O.N. and Rosenberg, N., 1990, Proc. Natl. Acad. Sci. USA 87, 6649-6653. Kowenz, E., Leutz, A., Doderlein, G., Graf, T. and Beug, H., 1987. In Modern. Trends in Human-Leukemia VII, R. Neth, R.C. Gallo, M.F. Greaves and H. Kabisch, eds. (Heidelberg: editorial Springer), pgs. 199-209. Laufer, E., 1993, Current Biology 3, 306-308. Lax, I., Johnson, A .. Howk, R .. Sap, J., Bellot, F., Winkler, M., Ullrich, A., Vennstrom, B., Schless inger, J. and Givol, D., 1988, Mol. Cell. Biol. 8, 1970-1978. Leutz, A., Beug, H. and Graf, T., 1984, EMBO J. 3, 3191-3197. Metcalf, D., 1980, Proc. Natl. Acad. Sci. USA 77, 5327-5330. Mitani, K. and Caskey, C.T., 1993, Trends in Biotechnology 11, 162-166. Peles, E. and Yarden, Y., 1993, Bioessays 15, 815-824. Rolink, A., Kudo, A., Karasyama, H. and Kikuchi, Y., 1991, EMBO J. 10, 327-336. Sawada, K., Krantz, S.B., Dai, C.H., Koury, S.T., Horn, S.T., Glick, A.D. and Civin, C.I., 1990. J. Cell. Physiol. 142, 219-230. Sawada, K., Krantz, S.B., Dai, C.H., Sato, N., Ieko, M., Sakurama, S., Yasukouchi, T. and Nakagawa, S., 1991, J.

Cell. Physiol. 149, 1-8. Sawada, K., et al., J. Cell. Physiol. 142, 219-230. Sawyers, C.L., Denny, C.T. and Witte, O.N., 1991, Cell 64, 337-350. Schroeder, C., Gibson, L., Zenke, M. and Beug, H, 1992, Oncogene 7, 217-227. Schroeder, C., Gibson, L., Nordstrom, Ch. And Beug, H., 1993, EMBO J. 12, 951-960. Shpall, E.J., Jones, R.B., Bearman, S.I., Franklin, W.A., Archer, P.G. , Curiel, T., Bitter, M., Claman, HN, Stemmer, SM, Purdy, M., Myers, SE, Ha, L., Taffs, S., Heimfeld, S., Hallagan, J., Berenson , RJ, 1994, J. Clin. Oncol. 12, 28-36. Tamagnone, L., Partanen, J., Armstrong, E., Lasota, J., Ohga i, K., Tazunoki, T., LaForgia, S., Huebner, K. and Alitalo, K., 1993, Oncogene 8, 2009-2014. Tepper, R.I. and Mulé, J.J., 1994, Human Gene Therapy 5, 153. Ti 11, J.E. and McCulloch, M., 1980, Biochim. Biophys. Act 605, 431-459.

Van der Geer, P., Hunter, T. and Lindberg, R.A., 1994, Annual Review Cell Biol. 10, 251-337. Vile. R. and Russel, S., 1994, Gene Therapy 1, 88. Zatloukal, K., Schmidt, W., Cotten, M., Wagner, E., Stingl, G. and Birnstiel, ML, 1993, Gene 135, 199. Hematopoiet ic Stem Cells, The Mulhouse Manual, 1994, eds. Wunder, E., Sovalat, H., Henon, P.R. and Serke, S. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property. 'tAf- * r - "-

Claims (18)

1. - Procedure for the in vitro production of non-immortalized hematopoietic precursor cells of the eritherapeutic development series, characterized in that cells containing a population of erroneous precursors are exposed, in a medium containing the usual components necessary for the growth of erigeous cells, for at least sufficient time with a combination based on growth factors containing at least one estrogen receptor ligand and at least one ligand of the glucocorticoid receptor and at less one, preferably at least two ligands of a t-receptor in-kinase receptor until the cells themselves begin to renew themselves, and because eventually the cells continue to be cultured in a medium containing the factors necessary for continuous self-renewal.

2. Method according to claim 1, characterized in that the cells are human cells.

3. Method according to claim 2, characterized in that a population of cells enriched with CD34-positive cells is used as starting cells.

4. Method according to claim 3, characterized in that a population of "bone marrow, peripheral blood or umbilical cord blood cells" is used

5. Method according to one of the preceding claims, characterized in that the combination of factors

6. - Method according to claim 5, characterized in that the combination of factors contains at least two ligands that bind to receptors of different classes of t-receptor in-kinase receptors.

7. Method according to claim 6, characterized in that the ligands are chosen from groups that bind to receptors with a differently structured kinase domain

8. Process according to claim 7, characterized in that the ligands are chosen from the groups i) ligands that bind to in-kinase receptor receptors that have a continuous kinase domain, and ii) ligands that bind to receptors that are e t inos kinase possessing a kinase domain interrupted by an insertion.

9. Method according to claim 8, characterized in that the combination of factors contains i) a ligand of a receptor of the family of EGF receptors and / or of the HGF receptors and ii) a ligand of c-Kit.

10. Method according to claim 9, characterized in that the combination of factors contains i) TGFa and / or EGF and / or HGF, ii) SCF, iii) dexamethasone and / or hydrocort isone, and estradiol.

11. Method according to one of the preceding claims, characterized in that the combination of factors also contains one or several other factors that accelerate the acquisition of the potential of self-renewal.

12. Procedure according to claim 11, characterized in that the one or more other factors are chosen from the cytokine and / or ligand groups of t-receptor in-kinase and / or serine kinase receptors.

13. Method according to claim 12, characterized in that the cytokine is in tropoyet ina.

14. Process according to claim 12, characterized in that the t-irosin-kinase receptor ligand is IGF-1.

15. Method according to one of claims 10 to 14, characterized in that the combination of factors comprises i) TGFa and / or EGF and / or HGF, ii) SCF, iii) dexamethasone and estradiol, iv) er it ropoyet ina e IGF-1.

16. Method according to one of the preceding claims, characterized in that the cells are further cultivated after acquiring the potential for self-renewal in the presence of factors that are necessary for the continuous self-renewal of the cells.

17. Method according to claim 16, characterized in that human cells are further cultured in the presence of at least one ligand of the family of EGF receptors or of the HGF receptor, SCF, eri t ropoyet ina and IGF-1.

18. Process according to claim 17, characterized in that the ligand is EGF and / or TGFa and / or HGF. In testimony of which I sign the present in this City of Mexico, D.F., on February 23, 1996. By: BOEHRINGER INGELHEIM INTERNATIONAL GMBH