MXPA98003263A - In vitro growth of functional langerhans yuso in vivo de los mis - Google Patents

Abstract

The present invention relates to new methods that make it possible, for the first time, to grow functional islets in in vitro cultures. The present invention also relates to the use of islet-like structures grown in vitro to be implanted in a mammal for the in vivo treatment of diabetes. The present invention also relates to a process for using islets implants grown in vitro to grow an organ in vivo that has the same functional, morphological and histological characteristics as those observed in normal pancreatic tissue. The ability to grow these cells in vitro and organs in vivo opens up important new paths for research and treatment related to diabetes.

Description

IN VITRO GROWTH OF ISLOTES OF FUNCTIONAL LANGERHANS AND IN VIVO USE OF THEMSELVES DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Diabetes is a major public health problem. As presented in 1987, the Report on the National Large-Scale Plan to Combat Diabetes, commissioned by the National Diabetes Counseling Office, is known to have six million people in the United States with diabetes and an additional five million have the disease which has not yet been diagnosed. Each year, more than 500,000 new cases of diabetes are identified. In 1984, diabetes was the direct cause of 35,000 deaths among Americans and was a contributing factor in another 95,000. Ocular complications of diabetes are the main cause of new cases of legal blindness in people aged 20 to 74 years in the United States. The risk of amputation of lower limbs is 15 times higher in individuals with diabetes than in individuals without it. Kidney disease is a frequent and serious complication of diabetes. Approximately 30% of all new patients, in the United States, who are treated for end-stage renal disease, have diabetes. Individuals with diabetes are also at increased risk of periodontal disease. Periodontal infections progress rapidly and not only cause the loss of teeth, but also cause a compromised metabolic function. Women with diabetes are at risk for serious complications of pregnancy. Current statistics suggest that the death rate of children of mothers with diabetes is approximately 7 percent. Clearly, the economic burden of diabetes is enormous. Each year, patients with diabetes or its complications spend 24 million patient days in hospitals. A conservative estimate of total annual costs attributable to diabetes is at least $ 24 trillion (statistics from the American Diabetes Association est., 1988); however, the overall economic impact of this disease is even greater, because additional medical expenses are often attributed to the specific complications of diabetes rather than to diabetes itself. Diabetes is a chronic complex metabolic disease, which results in the body's inability to properly maintain and utilize carbohydrates, fats and proteins. It is the result of the interaction of several environmental and hereditary factors and is characterized by high concentrations of glucose in the blood caused by a deficiency in the production of insulin or an inability to use it. Most cases of diabetes fall into two clinical types: Type I or juvenile diabetes and Type II or adult diabetes. Type I diabetes is often referred to as Insulin-Dependent Diabetes, or IDD. Each type has a different prognosis, treatment and cause. Approximately 5 to 10 percent of patients with diabetes have IDD. IDD is characterized by a partial or complete inability to produce insulin, normally due to the destruction of insulin-producing β cells of the pancreatic islet of Langerhans. Patients with IDD may die without daily insulin injections to control their disease. There was little progress in resolving the pathogenesis of diabetes until the mid-1970s, when evidence began to accumulate suggesting that Type I IDD had an autoimmune etiopathogenesis. It is now generally accepted that IDD is the result of a progressive autoimmune response, which selectively destroys the insulin-producing β cells of the pancreatic islet of Langerhans in genetically predisposed individuals. Autoimmunity against ß cells in IDD involves both humoral immune mechanisms (Baekkeskov et al., 1982, Baekkeskov et al., 1990; Reddy et al. , 1988; Pontesiiii et ai. , 1987) as immune mechanisms mediated by cells (Reddy et al., 1988, supra, Pontesilli et al., 1987, supra, Wang et al., 1987). The humoral immunity is characterized by the appearance of antibodies against the membrane of the β cells (anti-69 kD antibodies and of the cell surface of the islet), against the content of the β cells (autoantibodies anti-carboxypeptidase Ai, anti-64 kD and / or anti-GAD), and / or against products secreted by β-cells (anti-insulin). Although the serum does not transfer the IDD, autoantibodies against β cells occur at a very early age, raising the issue of environmental activation, possibly involving an antigenic mimicry. The presence of immunological reactivity mediated by cells in the natural course of IDD is evidenced by an inflammatory lesion of the pancreatic islets, called insulitis. Insulin, in which infiltrates of inflammatory / immune cells are clearly observed by histology, has been shown to be comprised of numerous cell types, including T and B lymphocytes, monocytes and natural killer cells (Signore et al., 1989; Jarpe et al. al., 1991). Adoptive transfer experiments using diabetic non-obese NOD mice; As a result of human IDD, they have firmly established a primary role of autoaggressive T lymphocytes in the pathogenesis of IDD 'Benaelac, et al. , 1987; Miller et al. , 1988; Hanafusa et al. , 1988; Bendelac et al. , 1988). Unfortunately, the mechanisms underlying the destruction of pancreatic β cells are still unknown. Recent efforts to cultivate pancreatic cells, including the efforts reported in the following publications, have focused on cultures of differentiated or partially differentiated cells, which have been grown in culture in monolayers or as aggregates. In contrast to these reports, the present invention describes a method and structure in which a structure similar to the islet is produced, which has a morphology and a degree of organization much more similar to a normal islet produced in vi vo through the neogenesis. Gazdar et al. , (1980) described a continuous, clonal cell line that secretes insulin and somatostama established from a transplantable rat islet cell tumor. However, the cells described were tumopgen cas and not pluppotential. Brothers, A. J. (WO 93/00441, 1993), described cells that secrete the hormone including pancreatic cells, maintained in long-term culture. However, the cultivated cells differed, as opposed to the stem cells, which were selected in a primary stage for their hormone-secreting phenotype, as opposed to their ability to regenerate a structure similar to that of the pancreas. Korsgren et ai. , described a selection of compounds in vi tro for their potential to induce the differentiation of fetal porcine pancreatic cells. The present invention does not depend on the use of fetal tissue. Nielsen, J. H., (WO 86/01530, 1986) described a method for the proliferation of beta cells completely or partially differentiated. However, this description depends on the fetal tissue as a source of islet cells for growth in culture. McEvoy et al. (1982), described a method for culturing fetal rat islet tissue and compared the effect of serum on the maintenance, growth and differentiation of cells A, B, and D in a defined medium. Again, the source of islet cells is fetal tissue. Zayas et al. (EP 0 363 125, 1990), described a process for the proliferation of pancreatic endocrine cells. The process depends on the use of fetal pancreatic tissue, and a synthetic structure, including collagen, must be prepared to embed these cells for implantation. Aggregates thus produced from cultured cells after implantation require 60-90 days before they have any effect on blood glucose levels, and require 110-120 days before approaching euglycemia. In contrast, the present invention provides structures similar to islets that grow in vi tro, which do not require collagen or other synthetic means for retention of their organization, which, after implantation, produce much faster effects on the glycemic state of the receiver. Coon et al. (WO 94/23572, 1994), described a method to produce an expanded cell culture, not transformed from pancreatic cells. The aggregated cultured cells were then embedded in a collagen matrix for implantation, with the concomitant disadvantages highlighted by the structures of Zayas et al. , supra, and the distinctions noted with the structure produced in accordance with the present invention. In spite of previous reports, the present invention, where functional islet-like structures containing cells expressing insulin, glucagon and / or somatostatin can be implanted in clinically diabetic mammals, which subsequently remain healthy (after the elimination of the treatment with insulin), it's surprising. Because the conventional and immunofluorescent histology of the pancreatic islets of Langerhans (Lacey et al., 1957; Baum et al. , 1962; Dubois, 1975; Pelietier et ai. , 1975; Larsson et al. , 1975), along with the formation of recent three-dimensional images (Brelje et al., 1989), have revealed a remarkable cellular architecture and organization of the pancreatic islets ideal for rapid responses, in addition to finally controlled, to changes in glucose levels in blood. It could not be predicted that such a structure could be produced in vi tro, particularly when it is considered that during embryogenesis, the development of the islet within the pancreas seems to be initiated from undifferentiated precursor cells associated mainly with the epithelium of the pancreatic duct (Pictec et al. al., 1972), that is, cells not belonging to the islet. The duct epithelium proliferates rapidly, then differentiates subsequently into the different cell populations associated with the islet (Hellerstro, 1984, Weir et al., 1990, Teitelman et al., 19093, Beattie et al., 1994). The islets are organized into spheroidal structures in which the ß cells that produce insulin form a nucleus surrounded by a mantle of non-ß cells. For the most part, glucagon-producing cells (if the islet was derived from the dorsal lobe) or alternatively, cells? peptide producers, pancreatic (if the islet was derived from the ventral lobe), they reside within the outer cortex (Brelje et al., supra, 1989; Weir et al., supra 1990). The atostatin producing d cells, which are dendritic in nature, reside within the inner cortex and extend pseudopodia to innervate the cells to (or PP) and the β cells. These spherical islet structures tend to sprout from the epithelium of the duct and move short distances to the surrounding exocrine tissue. The vascularization induced by angiogenesis results in a direct arterial blood flow to the mature islets (Bonner-Weir et al., 1982, Teitelman et al., 1988, Menger et al., 1994). Since blood glucose can stimulate the proliferation of β cells, vascularization can act to further increase the number of β cells. Similarly, neurogenesis leads to the innervation of the islets with sympathetic, parasympathetic neurons and peptidemagines (Weit et al., Supra, 1990). That ability to produce structures similar to functional islets in vi tro that can be implanted to produce structures similar to the pancreas, is therefore very remarkable. Unfortunately, the cellular organization of the islets can be destroyed in diseases such as insulin-dependent diabetes (IDD), type I, in which a humoral and cell-mediated autoimmune response, progressive, results in the specific destruction of β-cells. insulin producers (Eisenbarth, 1986; Leiter et al., 1987). Because β-cells are considered to be mostly different end-stage cells, it is believed that the body has a limited capacity to generate new β-cells, hence the need to regulate insulin therapy throughout of life, once the mass of β cells has been destroyed. However, in experimental animals, the mass of β cells has been shown to increase and decrease in order to maintain euglycemia (Bonner-Weir et al., 1994). Plasticity can occur through two routes of islet growth: first, by neogenesis, or growth of new islets by differentiation of the pancreatic duct epithelium, and second, by hypertrophy, or expansion through the duplication of pre-existing β cells . During embryogenesis, the ß cell mass initially expands from the differentiation of new cells, but in the late fetal stages the ß differentiated cells are duplicated. Duplication is then probably the main means of expansion after birth, but the capacity for duplication seems to decrease with age. Adult islet cells have been shown to replicate in response to stimuli known to initiate the growth of neonatal islet cells, eg, glucose, growth hormone, and various peptide growth factors (Swenne, 1992; Hallerstrom et al. , 1988; Bonner-Weir et al. , 1989, Marynissen et al. , 1983; Neilsen et al. , 1992; Brelje et al. , 1993). These observations suggest that the low level of growth of ß cells in adults can accommodate functional demands. For example, during pregnancy or chronic obesity, the ß cell mass increases significantly and is reversible because, after finishing pregnancy or after weight loss, an increase in ß cell mortality via apoptosis rapidly reduces the ß cell mass. It is generally accepted that all types of pancreatic endocrine cells differ from the same epithelium of the duct (Pictet et al., 1972, supra, Hellerstrom, 1984, supra, Weir et al., 1990, supra, Teitelman et al., 1993, supra), but it is uncertain if they are derived from a common mother / precursor cell. In the normal adult pancreas, approximately 0.01% of the cells within the duct epithelium will express islet cell hormones and may be stimulated to undergo morphogenic changes to form new islets, reminiscent of neogenesis. This neogenesis has been experimentally induced by dietary treatments with soybean trypsin inhibitors (Weaver et al., 1985), high levels of interferon-? (Gu et al., 1993), partial pancreatectomy (Bonner-Weir et al., 1993), cellophane pancreatic head envelope (Rosenberg et al., 1992), specific growth factors (Otonkoski et al., 1994). ) and the appearance of clinical IDD. Recently, attention has been focused on the Reg gene (Watanabe et al., 1994, Otonkoski et al., 1994), identified in a cDNA library subtracted from rat islets in regeneration, as a control element in the neogenesis of islet ß cells. Regeneration of the Reg gene (for example, by the growth factor of hepatocytes / scattering factor) induces the proliferation of the ß cells resulting in an increase in mass, while deregulation of the Reg gene (for example, by nicotinamide) induces the differentiation of "pre-ß" cells to mature cells. Thus, a population of precursor / stem cells remains in the adult pancreatic ducts and the differentiation of this population can be educated in response to specific stimuli. This action may actually occur continuously at low levels.

OR Although intensive efforts have been made to reproduce the neogenesis of the smallest islets, minimal success has been achieved. Now, for the first time, the conditions that allow the growth and expansion of mammalian derived islet producing stem cells (IPSC) in culture, as well as their differentiation into islet-like structures, are described. Numerous strategies have been investigated (for example, bone marrow replacement, immunosuppressive drugs and immunizations with autoantigens) as possible means of counteracting the immune attack against pancreatic β cells. However, for these methods to be effective, individuals who eventually develop clinical disease must be identified. Often, patients are identified too late for effective intervention therapy, since the immune attack has progressed to a point where a large percentage of the β cells have already been destroyed. Because ß cells are thought to be a differentiated cell from the final stage, it is thought that the body has little capacity to regenerate new β cells, thus necessitating regular insulin therapy for life. A recent method to solve this problem has been the transplantation of islet cells. The transplantation of islet cells has the disadvantage that the islets are allogeneic, which, in turn, can cause an alloimmune response. Thus, there would be great advantages in growing islets of Langerhans containing functional β cells directly from patients with IDD.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the discovery that functional islets containing insulin-producing β-cells, as well as other types of islet cells, can be grown in long-term cultures from pluripotent stem cells alone, which result in islet producing stem cells, IPSC. The new methods of the present invention take advantage of the discovery that IPSCs still exist in the pancreas of adult individuals. The cells can be cultured in a minimal nutritional medium containing a high concentration of amino acids, which is supplemented with normal serum, which is preferably derived from the same mammalian species that serves as the origin of the islet cells (homologous serum). . Several discrete phases of cell growth result in the selection of IPSC and subsequent progeny, which are then induced to differentiate and form islet-like structures, which are distinguishable from the pseudoislotic and pseudopancreatic tissue of the prior art. In a first phase, the primary culture of cells of a pancreas is placed in a basic medium, low in serum, low in glucose, high in amino acids. Subsequently, this culture is left undisturbed during several plantings to allow stromal cells to be established and to allow the vast majority of differentiated cells to die. Once the stromal cell layer is mature, cellular differentiation can be initiated by feeding the cell culture with a medium with a high content of amino acids supplemented with normal homologous serum plus glucose. After an additional period of growth, functional islets containing cells that produce insulin, glucagon, somatostatin, and other endocrine hormones can be recovered using standard techniques. Previously it was not known or suspected that non-islet cells derived from the pancreas (cells derived from the duct) could be used to grow new islet cells, including β cells, in culture. The fortuitous discovery of culture techniques to grow islet-like tissue in vitro eliminates what had previously been a substantial and ancient barrier to diabetes research. The new methods and novel materials described in the present, will make possible a better understanding of the mechanisms of diabetes. In addition, the ability to produce islet-like structures from IPSC in culture will now enable certain therapies for diabetes for the first time. For example, in accordance with the present invention, new cultured cells from diabetic individuals can be implanted in a patient as a way to control or eliminate the need for insulin therapy for the patient, because the islets and / or the cultured islet cells are capable of producing insulin in vivo. Thus, the object of the invention also relates to the use of islets grown in accordance with the present invention to be implanted in a mammalian species for the in vitro treatment of the IDD. The object of the invention also greatly facilitates the genetic engineering of islet cells to withstand subsequent immunological destruction. For example, cultured islet cells can be transformed to express a protein or peptide that will inhibit or prevent the destructive immune process. Other useful proteins or peptides can be expre. In addition, the expression of specific autoantigens, such as the cell surface antigens of the 64 kD islet cell, GAD, (see Payton et al., 1995), or any other markers identified on differentiated pancreatic cells, can be eliminated by the procedures of extermination or standard genetic selection to produce differentiated pancreatic cells which are not or are less susceptible to an autoimmune attack. Methods for producing such mutant or killed cell lineages are well known in the art and include, for example, the homologous recombination methods described in U.S. Patent No. 5,286,632; U.S. Patent No. 5,320,962; U.S. Patent No. 5,342,761; and WO 90/11353; WO 92/03917; WO 93/04169; WO 95/17911, all of which are incorporated herein by reference. In addition, a universal donor cell was produced by preparing a modified stem cell that does not express human leukocyte antigen (HLA) markers when the cells differentiate into a structure similar to the pancreas (see especially WO 95/17911 supra). Thus, the ability to grow functional islets in vi tro from pancreatic cells of an individual represents a great technical advance and facilitates the use of new strategies for the treatment of IDD. The discovery that there are pluripotent stem cells in the adult pancreas (without exclusion), avoids the need to use fetal tissue as a source of cells.

The present invention also relates to islet cells produced in vitro in accordance with the methods described herein. These cells can be produced from a suspension of pancreatic cells of mammals cultured in vi tro and can give rise to functional islet cells and islet-like tissue structures. The present invention also relates to the growth, propagation and differentiation in vi tro, of a pancreatic stem cell; that is, a cell or progenitor cells that can give rise to the formation of all the different types of cells and tissue that make up a normal pancreas. In addition, the present invention relates to the in vi ve use of pancreatic stem cells grown in the vi to produce an "ectopáncreas" that exhibits functional, morphological and histological characteristics similar to those observed in the normal pancreas. Thus, the ability to produce a functional "ectopáncreas" in vi vo from pancreatic cells grown in vitro, can be used to treat, reverse or cure a wide variety of pancreatic diseases that are known to result in damage or destruction of the pancreas.

BRIEF DESCRIPTION OF THE FIGURES Figures LA to ID show growing cells in accordance with the methods of the present invention. Figure 2 shows a structure similar to an islet grown in accordance with the present invention. Figures 3A through 3H show sequential stages in the development of a structure similar to the islet in vi vo of 3A, which shows a few cells after a few weeks in culture, which have survived and begin to "sprout" (Figure 3B, dark structure in the upper right part of the field), and divide (Figure 3C several places in the field), and to form highly organized structures (Figures 3D-3H) under the culture conditions described here. Figure 4 shows photomicrographs of the structures shown in Figures 3G-3H, showing the highly organized morphology thereof. Figure 5 shows the H / E staining of cross sections with structure similar to the islet showing the highly organized morphology of the structure with β cells in the center and glucagon producing cells in the periphery.

Figures 6A through 6F show a series of iographs in which an islet-like structure, such as that shown in Figure 3H, was harvested from a primary culture. In Figure 6B, the structure has disintegrated, and most of the cells have died, but in Figure 6C a new structure is developed. In the Figure 6D, several new structures have been formed. This series of passage steps can be repeated a number of times until the IPSC decreases. In this case, when the structure disintegrates, as in Figure 6E, instead of new structures being formed, the differentiated cells multiply, as shown in Figure 6F. It is this type of proliferated differentiated cells that are thought to have been produced by researchers such as Coon et al. (see WO 94/23572, supra). Figure 7 shows the data of control and NOD implant mice after cessation of insulin therapy. Figure 8 shows an ectopáncreas.

ABBREVIATIONS AND DEFINITIONS IPSC are the Islet Production Stem Cells. IPSCs are a small population of cells derived from epithelial cells of the duct (ie, these cells are cells derived from the pancreas, but not from islet 1 differential) discovered in fetal or adult pancreas, which, according to this invention, have the capacity to give rise to structures similar to islands in vi tro. When the epithelial cells of the duct are implanted in vi ve, a structure similar to the pancreas is formed. When the structure similar to the pancreas and the cells of the epithelium of the duct are implanted in another place different from the natural pancreatic iocalization in vi, the structure similar to the pancreas is known as ectopáncreas. The pluripotent pancreatic stem cells are cells discovered in the pancreas which give rise to IPSC. Mature islet cells are differentiated cells which come from IPSC and produce pancreatic hormones. The islet-like structures, or young islets, are highly organized cell structures which have been discovered to arise in IPSC cultures (see Figure 3H, Figures 4A and 4B, and cross section shown in Figure 5). The "outbreaks" of foci structures formed by individual IPSCs after most of the cells, which are not IPSC, which are placed in culture from the dissociated pancreatic tissue that have died. After the implantation of the islet-like structure, the final differentiation occurs to produce completely mature islet cells.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, the islets of functional Langerhans may for the first time be grown in in vitro cultures. The techniques of the present invention result in cell cultures which can produce insulin, glucagon, somatostatin or other endocrine hormones. Other useful proteins can also be produced, for example, by transforming the islet cells with the DNA encoding the proteins of interest. The ability to grow those functional cell cultures allows those skilled in the art to carry out other procedures. which previously were not possible. In the following description, the term islet-like structure should be read as an interchangeable term with the term "young islets", because those structures produced in vi tro have the majority of islet attributes produced in vivo during normal neogenesis. . The immature nature of these structures allows for in vivo implantation with rapid differentiation and vascularization adequate to provide a functional replacement to damaged or otherwise compromised islets in receptors such as diabetic or prediabetic mammals, in need of such treatment. The method of the present invention involves making cell suspensions, including stem cells from the pancreas of a mammal. Preferably, the stem cells could be from the pancreas of a prediabetic mammal. However, it is contemplated in the present invention that islet producing, IPSC stem cells from mammals already showing clinical signs of diabetes can be used. Cell suspensions are prepared using standard techniques. Then, the cell suspension is cultured in a nutrient medium that facilitates the growth of the IPSC, and which at the same time severely compromises the sustained growth of other differentiated or mature cells different from the IPSC. In a preferred embodiment, the nutrient medium is one that has a high concentration of amino acids. One such means is known as Click EHAA media and is well known and readily available to those skilled in the art (Pec and Bach, 1973, incorporated herein by reference for this purpose). Those skilled in the art could prepare and use other equivalent nutrient media. What is required for such means is that they have little or no glucose (less than about 1 mM) and less serum (less than about 0.05%). The high concentrations of amino acids are preferably amino acids which are known to be essential for the cells of the species being cultivated, and to provide a carbon source for the cultured cells. In addition, at least one rudimentary lipid precursor, preferably pyruvate, is provided. Those conditions are so stressful that most types of * differentiated cells do not survive. Surprisingly, however, after prolonged culture of the cells from pancreatic tissue without refeeding (approximately 3 weeks) the IPSC survive and after prolonged culture, they begin to proliferate. "Subsequent culture phases employ media supplemented with normal serum from the same mammalian species from which the islet cells originate.So, in the case of mouse islets, the medium is supplemented with normal mouse serum, while in the case of human islet cells, the medium is supplemented with normal human serum.The preparation of normal serum is well known to those skilled in the art.The concentration of normal serum used in the cell culture method of the present invention, it can vary from about 0.5% to about 10%, but for mice it is preferable about about 1%. For human serum, a higher concentration, for example about 5%, is preferred.

The cell suspension prepared in the nutrient medium supplemented with normal serum and with approximately 2.5-10 mM glucose, is subsequently incubated under conditions that facilitate cell growth, preferably at about 35-40 ° C and, preferably, with an atmosphere of approximately 5% C02. This incubation period, then, is carried out using normal procedures well known to those skilled in the art. During this time, the stromal or epithelial cells of the duct proliferate and establish a monolayer, which eventually results in islet-like structures. The initiation of cell differentiation can be carried out by realizing the cultures with EHAA Click medium or the like, supplemented with normal serum, as described above. It was found that rapid feedback induces an extensive formation of islet foci and islet-like structures with considerable cell differentiation. It has been found that cell differentiation is increased by the inclusion of relatively high concentrations of glucose (approximately 10-25 mM and preferably 16.7 mM) in the fed medium. In addition, it was contemplated that any number of other biological factors, including, but not limited to, factors which excessively regulate the Reg gene, such as the epatocyte growth / diffusion factor, and other factors of cell growth, such as the insulin-like growth factor, epidermal growth factor, keratinocyte growth factor, fibroblast growth factor, nicotinamide, and other factors that modulate cell growth and differentiation can be added to crops to optimize and control the growth and differentiation of IPSCs. Using any of these different factors, or combinations of them, in different stages, at different planting densities and in different sowing times in the course of IPSC differentiation, IPSC crops are optimized. In addition, the factors produced by IPSC cultures in the course of differentiation which increase growth can be isolated, sequenced, cloned, produced in bulk quantities, and added to IPSC crops to facilitate the growth and differentiation of those crops. . The relevant factors are identified by the concentration of the IPSC culture supernatants from the primary, intermediate, and final stages of the differentiation, and by testing the capacity of those concentrates to increase the growth and differentiation of the IPSC. The positive effects are correlated with the molecular constituents in the concentrates by gel electrophoresis, two-dimensional, positive and negative supernatants, purification and N-terminal sequencing of spots present only in the positive concentrates and the subsequent cloning and expression of the genes they encode for those factors. After histological examination of the cells in the islet-like structures, at least 3 cell types were identifiable and appear to be similar to islet cells prepared from islets of control mice. The time necessary for cell differentiation to occur within these foci decreased as the frequency of feedback increased. It was possible to propagate and expand islet-producing crops, through serial transfers of stromal cells derived from islets, plus foci of islets, to new culture flasks. This facilitates the generation of sufficient numbers of islets, as required for use in the methods described herein, for example, to reverse the etiological problems of IDD. In order to determine if the islet-like structures and / or the islet cells produced in accordance with the present invention could reverse the IDD, islet-like structures were implanted in NOD mice. The mice that received the islet implants exhibited withdrawal of insulin-dependent diabetes, whereas the untreated NOD mice showed signs of clinical disease. In addition, autoimmune pathogenesis was not observed during the duration of the implants. Thus, the islet implants of the present invention can be used in vivo, for the treatment of diabetes in mammals, including humans. In a preferred embodiment of the present invention, the progress of diabetes can be slowed down or stopped, by reimplantation of autologous islets engineered to be resistant to specific factors involved in the immune attack. For example, the islets can be manipulated in such a way that they are resistant to cytotoxic T cells (see for example, Durinovic et al., 1994, identification of islet-specific T cells and T cell receptor sequences, which are similar to insulin-inducing T cells of diabetic mice; and Ellias and Cohen, 1994, identification of peptide sequences useful in the therapy of diabetes in NOD mice by suppressing the production of specific diabetogenic T cell clones; Conrad et al. , 1994, description of an islet cell superantigen, attached to the membrane, which activates the pronferation of T cells that infiltrate the islet; Santamaría et al. , 1994, description of the requirement of coexpression of B7-1 and TNFa for diabetes and destruction of islet cells; any of these antigens can be eliminated according to known methods to improve the resistance of the implanted islets against the immunological attack). The availability of long-term cultures of whole islets can also be used in research on the pathogenesis of IDD, including cellular recognition of β-cells, the mode of islet infiltration and the immune mechanisms of cell destruction. H.H. In addition, this technology will facilitate the transplantation of the islets, the replacement of autologous islets and even the development of artificial islets. The growth of these cells and islet-like structures in accordance with the methods of the present invention is very useful for teaching students important aspects related to cell differentiation and function. In a further embodiment of the present invention, pancreatic pancreatic stem cells have been grown, which give rise to IPSC from pancreatic cells isolated from a mammal. A surprising discovery using these cells grown in vi tro in conjunction with the methods of the present invention was the ability to grow and produce, in vivo, an organ that exhibited functional, morphological and histological characteristics and characteristics similar to those of a pancreas. normal, including cell differentiation to form endocrine and exogenous tissues.

E? Ectopancreas (an organ similar to the pancreas located at an abnormal site within the body cavity) produced mv vo in accordance with the present invention, represents a great scientific discovery and provides a new means to study, treat, reverse or cure a number of pathogenic disorders associated with the pancreas, including, but not limited to, pancreatitis, pancreatic cancer and IDD. This is achieved by removing the diseased tissue and implanting islet-like structures produced according to this invention. In addition, islet-like structures can be implanted in the natural pancreatic site. Because this invention provides a method for culturing pancreatic stem cells and the production of young islets, it is now possible to study the growth and differentiation of this type of cells. Accordingly, all known methods of cell culture, purification, isolation and analysis can be carried out to support the significant issues with respect to how many cell types are involved in the differentiation of pancreatic cells. These methods include, but are not limited to, the classification of fluorescence activated cells (FACS), the use of magnetic beads (as in, for example, the use of commercially-available DYNA BEADS which are specifically adapted for this purpose), the use of of magnetically stabilized fluidized beds (MSFB, see U.S. Patent No. 5,409,813), and any of a number of other methods known in the art. The way to this process is now sensitive to dissection. The identification of markers (including cell surface, intracellular, protein or mRNA), specific for each stage of this process, are also now easily identifiable through the application of standard techniques including, but not limited to: antibody production, including monoclonal antibodies, to cells, cell surface markers and cellular components which differ through the maturation process of pancreatic stem cells; production of T lymphocytes, which respond specifically to antigens expressed by pancreatic cells at different stages in the maturation and differentiation process (see, for example, Wegmann et al., 1993); identification and elimination of cell surface markers recognized by T cells and which, therefore, result in the destruction of differentiated β cells if present (see the references above); identification of significant factors that affect the different stages of maturation and the different factors produced by the cells that are differentiating; Subtractive hybridization of nucleic acids isolated from cells at different stages in the maturation process, it is allowed to highlight the significant gene products for each aspect of cell differentiation; Differential deployment PCR (see, Liang et al., 1992); PCR arbitrarily primed (see, Welsh et al., 1992); PCR for the analysis of representative differences (RDA-PCR) (see, Lisitsyn, 1993); encapsulation of unique pancreatic progenitor cells or populations thereof for implantation in appropriate host organisms, thereby providing the advantages that such methods have been demonstrated in the implantation of other types of progenitor or engineered cells (see, Altman et al. , 1994); genetic design of pancreatic progenitor cells to produce cells less susceptible to autoimmune attack, such as deactivating autoantigen genes, or insertion of genes that increase resistance; other genes which can be inserted into IPSCs include those that provide altered cell surface antigens or which provide different biochemical properties to the cells' internal environment; these include genes which express enzymes which can increase or decrease the sensitivity of cells to glucose or genes which increase or decrease the response of cells to growth factors; in addition, genes can also be introduced which increase or decrease the production of insulin, glucagon or somatostatin; examples of how such types of modifications can be introduced in IPSCs include electroporation, viral vectors, transfection or any of a number of other methods well known in the art (see, for example, WO 95/17911, WO 93/04169; WO 92/03917, WO 90/11354, U.S. Patent No. 5,286,632, WO 93/22443, WO 94/12650, or WO 93/09222, all of which are incorporated by reference for this purpose); production of universal donor (deactivated) cells which, for example, have suppressed or otherwise modified antigens in human leukocytes (see, WO 95/17911, supra). Because this process does not depend on the use of fetal tissue, it is possible to remove pancreatic tissue from a mammal that has IDD or is at risk of developing IDD, grow a structure similar to the islet in vivo and reimplant that structure in the individual to produce physiologically relevant amounts of insulin in response to fluctuations in blood glucose. In view of the foregoing description and the exemplary support that follows, it should be recognized that the scope of the appended claims extends to various embodiments and aspects of this invention, which those skilled in the art will recognize are achieved by this significant invention. It should also be recognized that the data presented here reveals that neogenesis within isolated pluripotent stem / progenitor stem islets is possible, but involves several distinct stages of growth, including: 1) Establishment of a cell monolayer estrus at, or "nurse" ", of cells of the ductal epithelium, which allows the generation of IPSC; 2) Induction of proliferation of stem / progenitor cells with specific culture conditions which promote the cyclic regeneration of the IPSC and also prevent the preferential differentiation of the IPSC; 3) Expansion and differentiation of cells a, ß and d. This step is dictated by the culture environment, since differences in crop nutrients and growth factors result in islets containing different percentages of various islet cell types. Identification of the in vi tro conditions that induce ß cells to their final maturation stage, i.e., formation of insulin-containing granules and glucose response can now be achieved. The present factor m vi which achieves this final differentiation is identified by the addition of JO cell extracts or growth factors to the IPSC cultures. Primary IPSC cultures have been maintained for up to 10 months and secondary cultures for an additional 14-16 months each capable of expanding and differentiating to form islet-like structures. Although the ability to grow functional slides from pre-diabetic adults represents a major technical advance and focuses attention on possible new strategies to achieve a cure for IDD, perhaps the most important aspect of this work is the demonstration that stem cells / pluripotential progenitors, which give rise to the IPSC, and structures similar to the pancreas when implanted, exist in the islets of both normal and (pre) diabetic adults. This discovery will eliminate the need to use either fetal, allogeneic or xenogeneic tissue for the transplantation of β cells in patients with IDD; develop novel strategies to reverse hypoglycemia in vi; study immological responses to newly implanted islets; and / or create islets resistant to immune attack. We intend to speculate, based on the data presented here, that the well-documented period of removal in patients with type I IDD after the initial attack of the disease may actually represent the time when the growth of stem cells is induced, only for to be later overwhelmed by the autoimmune reaction in progress. Since it is thought that the re-implantation of autologous islets requires cells designed to be immune to the immune attack, the identification and culture of islet stem cells as described herein is essential for the genetic engineering efforts described herein. Surprisingly, the islet implants generated within this invention showed no signs of immune binding during the period of time studied here. It is possible that autoantigens are not expressed on cultured cells, or that autoantigens can not be presented because the culture dilutes the macrophages, or such implants can induce peripheral tolerance. The availability of long-term complete islet cultures facilitates investigations of the pathogenesis of IDD, including cellular recognition of β-cells, mode of islet infiltration, and immune mechanisms of β-cell destruction. In addition, this technology facilitates islet transplantation, replacement of autologous islet, development of artificial islets and reduces the need for insulin therapy. Accordingly, this invention provides a method for the growth of intracellular islet producing stem cells. IPSC, to produce a structure similar to the islet. The method comprises culturing pancreatic cells of a mammalian species in a basic nutrient medium supplemented with normal serum below about 0.5% and glucose below about 1 mM, allowing the IPSC to grow for at least 3 weeks, and initiating the Cell differentiation in mature islet cells by feeding the IPSCs in culture with a nutrient medium supplemented with normal serum at approximately 0.5-10% and glucose at approximately 2.5 mM-10 mM. Pancreatic cells can be from any animal, including humans and rats, and the serum is from the same species. The medium preferably contains all the amino acids essential for the growth of the cells of the species being cultivated and in such quantity to ensure that the culture does not decay. After the reaction, the fed medium preferably contains glucose and serum in amounts sufficient to stimulate differentiation. Furthermore, according to this method, once differentiation has begun, the cells are preferably fed back frequently (approximately once a week). This method produces islet cells and structures: tissue similar to islets. This method also provides a source of endocrine hormones, including, but not limited to, O insulin, glucagon and somatostatin, which can be recovered from the IPSC culture medium or can be released directly into a mammal by implantation of islet-like structures in the tissue of a mammal to produce a structure similar to the pancreas. Tai implantation provides a method for treating pancreatic diseases in a mammal by implanting an islet-like structure to produce an organ similar to the pancreas in the mammal. In one embodiment, the islet cells or islet-like structure of this invention are genetically modified so as not to produce autoantigens for IDD or HLA markers so that self-antigens associated with insulin-dependent diabetes, other than insulin, are not expressed, or that have been modified so that they do not express human leukocyte antigens, when said stem cells differentiate into such pancreatic-like organ. In addition, the pancreatic stem cell can be encapsulated in insulin, glucagon, somatostatin and another permeable capsule of factor produced by the pancreas. A method is also provided for analyzing the differentiation of pancreatic stem cells, which comprises culturing at least one pancreatic stem cell, and inducing at least the mother to begin differentiation into a structure similar to the pancreas. This method also allows the identification of specific protein mRNAs or markers for a plurality of different stages in the differentiation process. Protein markers can be expressed on the cell surface, to be secreted, or they can be intracellular. In another aspect of this invention there is provided a ligand binding molecule and a method for making a ligand binding molecule which selectively binds pancreatic stem cells or more differentiated pancreatic cells. The method comprises contacting an infected T lymphocyte or T lymphocyte with an identified protic marker, and culturing and expanding the B lymphocyte or T lymphocyte to obtain a population of cells that produce the ligand binding molecule. These ligand-binding molecules thus provide a method for isolating a pancreatic stem cell or partially differentiated pancreatic cells at any stage between a pancreatic stem cell and a fully differentiated pancreatic cell. This method comprises selecting the cell from a population of cells comprising the cell, a specific ligand binding molecule which binds to a protein marker expressed by the cells at a given stage of differentiation. Alternatively, the method comprises selecting and removing another cell from a population of cells comprising the cell with a specific ligand-binding molecule which binds to an absent protein tag on the surface of the cell. In still another aspect, this invention provides a method for treating a mammal that suffers from, or is at risk of, IDD, which comprises: a. remove the pancreatic tissue of the mammal; b. cultivate pluripotent pancreatic cells present in the pancreatic tissue in vi tro to generate structures similar to the islets; and c. implant such structures similar to the islets in the mammal. In a further aspect of this invention, modified IPSCs are provided so that they do not express self-antigens of insulin-dependent diabetes in any undifferentiated or differentiated state of the IPSC. Preferably, the autoantigen that is not expressed as a result of the modification is selected from GAD, 64 kD islet cell antigen, and HLA markers. As part of the method of this invention, a method is provided for the islet neogenesis from pluripotent stem or progenitor cells, which comprises: a. establish a monolayer of stromal cells, or "wet nurses", of ductal pancreatic epithelial cells which allow the generation of IPSC; b. induce the proliferation of the adre / progenitor cell with culture conditions that promote the cyclic regeneration of IPSC and also prevent the mature differentiation of the IPSC; and c. expand and differentiate the IPSC to produce a structure similar to the islet comprising a, ß and d cells. Preferably, the islet-like structure generated in cultures is characterized by large, differentiated cells, which are stained with insulin-specific staining in the center of the islet-like structure; small differentiated cells, which stain with specific staining for glucagon in the periphery; and proliferate undifferentiated cells which do not stain with any of the stains specific for endocrine hormone in the inner cortex. The structure is further characterized because, after disruption in structure in single cell suspensions by mechanical or other means in the presence of a proteolytic enzyme and subsequent staining of individual cells, populations of individual cells are stained either with specific staining. for giucagon (cells a), insulin specific staining (ß cells) or specific staining for somatostatin (d cells) are observed. The method of islet neogenesis according to this invention preferably comprises: a. disperse and lyse undisturbed pancreatic cells in a minimal culture medium comprising little or no glucose, serum at a concentration less than about 0.5%, essential amino acids for the cells of the species from which the pancreatic cells were obtained, and a lipid source, up to about 99% of the cells in the culture have died (Phase I); b. feed the culture of step (a) with the minimal medium supplemented with approximately 1-10 mM glucose and approximately 0.5% -10% serum (but less than a toxic amount) and perform approximately once a week until it occurs a rapid proliferation; c. feed the culture of step (b) with the minimum medium supplemented with 0.5% -10% of serum and approximately 10-25 mM of glucose and, optionally, add growth or cellular factors (Phase III): d. allow the islet-like structures to sprout in the middle; and. recover the structure similar to the islet. This process can be repeated several times by serially transferring epithelial cells, more structures similar to proliferating islets, in the primary stage, in culture m vi tro. As used herein, the term "growth" refers to the maintenance of cells in a living stage, and 4J may include, but is not limited to, the propagation and / or differentiation of cells. The term "propagation" refers to an increase in the number of cells present in a culture as a result of cell division. The following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be taken as limiting. All percentages are by weight and all proportions of solvent mixture are by volume unless otherwise indicated.

Example 1 - Culture of functional Langerhans islets Single cell suspensions of islet cells were prepared from whole islets isolated from the pancreas of prediabetic male NOD / UF mice aged 19-20 weeks, as detailed in another document (Shieh et al., 1993). Typically, approximately 25% of the male mice in the NOD colony will have an open IDD at this age and will suffer from severe insulitis. The islet cells were resuspended in glucose-free or glucose-free Click EHAA medium (Peck and Bach, 1973, supra, Peck and Click, 1973) supplemented with 0.25% normal mouse serum (SRN), in a 25 cm cell culture flask, and incubated at 37 ° C in an atmosphere of 5% CO, -. At this stage two results are possible: first, the cells that infiltrate the islet can dominate, thus allowing the establishment of immune cell lineages; or second, duct epithelial cells (often referred to as stromal cells in those cultures) can dominate, thus allowing the growth of a "mother cell" monolayer. The growth of stromal-like cell monolayers appeared to result when the cells infiltrating the islet were seeded simultaneously but in limited numbers. Enrichment of islet cells with a smaller number of infiltrating cells can be achieved by gradient separation (Jarpe et al., 1991). The vast majority (> 99%) of the original cells died during this incubation period, leaving a small number of epithelial-like cells attached to the culture dish (Figures IA and 3A, stage I). The stromal cell cultures, when left undisturbed for 4-5 weeks (ie, without refeeding), proliferated to cover the entire lower surface of the culture vessel (Figures 3C and 3D). The differentiation and expression of endocrine hormone from the cultures was initiated by feeding the cultures with EHAA Click medium supplemented with SRN and a solution of • • sugar that included glucose or sucrose or other equivalent sugars. Typically sugar is glucose. The glucose concentration can be between approximately 4 0. 25 mM to 10 mM, but typically it is around 2.5 mM. Also, normal NOD or SRN serum was preferably included at about 0. 5%. Techniques for reacting cell cultures in vi tro are well known in the art and typically include removing from about 50% to about 90% of the old nutrient medium and adding new media to the culture flask. Rapid reactivity induced the formation of increasing numbers of islet growth centers (referred to herein as foci) exhibiting cellular differentiation. The frequency of feedback can be, for example, at intervals of one week. Preferably, the frequency of reactive is at intervals of approximately 5 to 6 days. Small rounded cells appeared on the epithelial monolayers, almost as if they sprouted (Figures IB and 3D, Stage II). In peak production, as many as 50-100 foci were presented simultaneously in a single 25 cm2 (4 in.) Cell culture flask. Each individual rounded cell underwent rapid proliferation, with daughter cells forming cell agglomerates (Figure IC). The rapid reactivity induced increasing numbers of cell agglomerates as well as a greater number of cells within each agglomerate. The induction of islet-like structures (Stage III) was increased through the refeeding of the cultures with supplemental EHAA medium with normal mouse serum (0.5%) and high glucose levels (10 mM - 25 mM and preferably approximately 16.7 mM glucose - see Figures ID and 3E-3F). As cell proliferation and differentiation proceeded, the organization of the islet was carried out and still the islet appeared surrounded itself by a capsular material. The mature islets (Stage IV) appeared as smooth spheroids composed of highly agglomerated cells (Figure 3F-3H). This differentiation seems to increase when serum from NOD mice is used instead of serum from other strains of mice, and high levels of insulin-like growth factor (IGF), epidermal growth factor (EGF) and / or growth factor of hepatocytes (HGF) in mouse NOD serum are thought to be responsible for this effect. The islets generally grew to a constant size (approximately 100-150 μ, Figures 3, although fusion of two agglomerates resulted in an islet approximately twice the overall size), then detached from the stromal layers to float in the medium . These free-floating islands tended to rupture over a period of 48 to 72 hours, similar to what was observed when the pancreatic islets were isolated from uninvited sources and then cultured under similar conditions. Next 4 / serial rounds of this process could be conducted (see Figures 6A-6D and example 5 below). The islet-like structures, collected after natural detachment or removal of stromal layers using a Pasteur pipette, were carefully washed with the medium, then broken to form cell suspensions alone by reflux pipetting. Cell suspensions alone were prepared by cytocentrifugation, then stained to observe the general morphology and insulin production. The foci contained cells that produced the endocrine hormones glucagon (cells a), insulin (ß cells) and / or somatostatin (cells d). In addition, a large part of the cell population gave positive staining with anti-insulin antibody, indicating that the major cell type contained in the cultured islet was an insulin-producing β cell. Figures IA and ID show the various cell types that develop during the cultivation process. Figure 2 shows a well-developed islet obtained after cell culture in accordance with the method of the present invention. 40 Example 2 - Culture of Human Islet Cells To culture human islet cells, a similar procedure as described in Example i was used. The process of the present invention is particularly advantageous, since it is not necessary to use fetal cells to initiate cell culture. In a preferred embodiment, human cells can be suspended in Click EHAA medium (or a medium equivalent thereto) supplemented with normal human serum. Preferably, the concentration of human normal serum used in the medium is approximately 0.25% - 1% in phases I and II, respectively, and 5% during subsequent phases. Crops should be left unaltered without feedback, preferably for several weeks (Phase I). After about 4-5 weeks in culture, cell differentiation can be initiated by replenishing the cultures with Click EHAA medium supplemented with normal human serum and glucose, as described in Example 1. Subsequently, the islet-like structures can be Collect and cell suspensions may be prepared alone for further propagation, in the manner described in Example 1.

ETemplo 3 - Implementation of Islet Cells in In Vitro Growth To test the efficacy of these similar islet structures generated m vitro to coat the complications of IDD, approximately 150 to 200 foci were grown, plus some stromal cells m alive according to the method of the present invention from pancreatic tissue. of NOD mice were taken from the cell culture flask by reflux pipetting. Afterwards, the cells were implanted below the renal capsule of diabetic NOD singenie mice, maintained by daily injections of insulin. The implant was performed by puncturing the renal capsule with a hypodermic needle, screwing a thin capillary tube through the puncture site in the kidney, and injecting the islet foci directly into the region of the cortex. The capillary tube was carefully removed and the site of the needle was cauterized. The surgical incision of each implanted mouse was held until the skin showed signs of having healed. The implanted mice were maintained with insulin injections for 4 days at full daily doses, and then for 2 days with half of the daily doses, after which the mice were completely separated from any additional insulin treatment. The control animals consisted of diabetic NOD b mice that did not receive an implant. or? At 8-14 days after insulin withdrawal, control NOD mice showed a rapid onset of open disease, including lethargy, dyspnea, weight loss, increased blood glucose levels (400-800 mg / dL) ), wasting syndrome, deficiency of wound healing and death within 18-28 days (Figure 7). The implanted NOD mice were maintained at a blood glucose level of approximately 180-220 mg / dL (which is slightly above the normal range for mice), showed increased activity, quickly healed the surgical wounds and sites from where Blood was taken, they did not develop dyspnea and remained healthy until they were sacrificed 55 days after implantation to perform histological studies (Figure 7). Similar observations had been made with intrasplenic implants. These data are consistent with the concept that the islets generated implanted implanted provide the insulin needed to maintain stable blood glucose levels during the time course of the experiment.

Example 4 - In Vivo Production of Ectopáncreas Histological examinations of implant sites in mice that were implanted with islet cells in the manner described in Example 3 revealed an Ol characteristic. of the islet-forming stem cells generated m vi tro. The implanted cells, which "flooded" the implant site of the kidney, presented an additional proliferation and differentiation and formed a highly structured ectopáncreas. At the beginning, the ectopáncreas tissue consisted entirely of proliferating exocrine cells, which were organized in a complete exocrine pancreas, with inervant blood vessels. This exocrine pancreas progressed to form islet-like endocrine structures (see Figure 8). Thus, the cell cultures produced in accordance with the methods of the present invention contain pluripotent pancreatic stem cells capable of regenerating a completely new pancreas. The growth of a pancreas that contains both exocrine and endocrine tissue provides new methods for the treatment of pancreatic diseases, including pancreatitis and pancreatic cancer.

Example 5 - Long Term Propagation of IPSC The long-term (> 1 year) spread of IP5C was achieved through the serial transfer of small numbers of epithelial cells plus a few proliferating islet-like structures, in the primary stage to o ¿ new culture containers Cells from a single 25 cm 'tissue culture vessel had been successfully expanded to 5-10 150 cm tissue culture vessels. Interestingly, the serial transfer uniformly resulted in "fusion" islet structures, similar to detached islet-like structures, while new stromal monolayers were formed (Figures 6A-6B). However, serially transferred crops produced new islets faster than primary crops and in larger numbers (up to 200-250 structures per square inch of culture-figures 6C-6D). However, eventually, after many rounds of growth and series production of islet-like structures, a point was generally reached where after the islet-like structure "melts", only differentiated cells proliferate (see Figures 6E). -6F). The same may occur in the absence of the formation of observable islet-like structures, if primary pancreatic tissue grows in primary culture under conditions that do not kill most differentiated cells.

Example 6 - Analysis of Islet-like Structures Photomicrographies of serial cuts of islet-like structures generated in culture, immature and cuttings thereof (shown in Figures 4 and 5, respectively) again demonstrate the uniformity of growth. Large, somewhat differentiated cells were observed which were faintly stained with insulin in the center of the islet. Small differentiated cells which were stained with glucagon were evident in the periphery, although a significant number of immature, proliferating and undifferentiated cells were not stained with any of the endocrine hormone antibodies that were present in the inner cortex. To more accurately determine the cellular phenotypes present within the islets that grew in vitro, structures similar to the islets were collected after they were detached from the epithelial monolayers, gently taken to a medium, then broken into suspensions of a single cell by mechanical means, such as reflux pipetting in the presence of a proteolytic enzyme such as 0.25% trypsin. Smears of single-cell suspensions were prepared by cytocentrifugation and stained to determine the general morphology or ceiiar content. Several types of ae were stored Mature and immature morphologically distinct cells after staining with H / E. In addition, populations of macular cells stained with anti-glucagon (cells a), anti-msulin (ß cells), or anti-somatostatin (cells or), indicate the prolific nature of stem / progenitor cells that give rise to similar structures to islets. These observations emphasize: first, the aerial staining of endocrine hormones suggests that the cells of islets generated m vi tro remain relatively immune and, therefore, capable of greater differentiation after m vi ve implantation, and second, the fact of > 100% of the cells could contribute to staining with endocrine hormone that indicates that some cells must express both glucagon and insulin simultaneously, according to what was recently reported by Teitel et al. (Teitelman et al., 1993 supra).

Example 7 - Pancreatic Cell Limiting Dilution-Single Pancreatic Stem Cell Cloning According to the methods described above, the pancreatic tissue was dispersed in a culture medium. To isolate unique stem cells for clone production! of differentiated pancreatic cells, pancreatic cells or were subject to limiting ancestor and outside the methods Dien conocíaos in the art. Thus, for example, serial additions were made after an initial evaluation of the number of cells / mL in the sample alone, so that the final dilution gave, at least, a mean of 0.3 cells per well. microtituador or other container aaecuaao to this type of dilution experiment. Subsequently, the cells were allowed to remain without perturation until the beginning of the Aesarro io the foci. These foci are stem cells which emerged from a single mare piuppotenciai or IPSC cell and which can be cultured to form a structure similar to the islet for implantation to form a structure similar to the pancreas. Example 8 - Identification of Markers Associated with Different Stages of Differentiation of Pancreatic Stem Cell and Production of Specific Ligand Binding Molecules for Each Differentiation Stage 0 Foci of isolated maar cells produced according to Example 7 or by an analogous method was analyzed both before and after induction of differentiation according to Example 1 or by a similar method. The cells at each stage, on the stem cells to compromised differentiated pancreatic cells were analyzed as siyue: A. Nucleic Acid: At each stage of differentiation, including undifferentiated progenitor cells and fully differentiated pancreatic cells, the mRNA was isolated. This RNA was used to produce a cDNA according to standard methods known in the art (Maniatis et al., 1982) including, but not limited to, PCR-dependent amplification methods using universal primers, such as poly A Each amplification represents a library of messages expressed at each stage of pancreatic stem cell development. Consequently, the message not present in the stem cells but present in the fully differentiated pancreatic cells has been identified by hybridizing the cDNA of each step and isolating the message that remains unhybridized. Likewise, methods such as differential flash PCR, arbitrarily primed PCR or RDA-PCR (see above) can be used. In this way, a unique message was identified for each stage by subtracting the message present in the other stages of the differentiation. Also, by this method, the genetic products were identified at each stage of the differentiation process, expressing the product encoded by a subtracted message. Antibodies were then produced, including monoclonal antibodies using their gene products as antigens according to methods well known in the art (see Goding, J. W., 1986). These antibodies were subsequently used to isolate s from a given stage of differentiation based on affinity for markers expressed on the surface of pancreatic s. In addition, the identification of specific markers which are expressed on the surface of differentiated pancreatic s allow the production of out-of-combat pancreatic lineages by site-directed mutagenesis using the sequences identified to direct mutations in stem s according to methods such as those described in U.S. Patent No. 5,386,632, supra; U.S. Patent No. 5,320,962; U.S. Patent No. 5,342,761; and in WO 90/11354; WO 92/03917; WO 93/04169; and WO 95/17911. The selection of mutant cells that do not produce the knockout gene product is achieved by using the antibodies for the specific genetic product selected to provide clones of cells in which that product is absent.

B. Protein Markers: At each stage of differentiation, including undifferentiated progenitor cells and fully differentiated pancreatic cells, antibodies were generated with whole cells and subcellular fractions, according to standard methods known in the art. As specific examples of this process: a) Production of rat anti-rat anti-IPSC mAb: To improve the selection of activated B lymphocytes against specific antigens for IPSC, rats were immunized with normal mouse tissue followed by treatment with cyclophosphamide 7 days after immunization. Cyclophosphamide selectively killed reactive B cells, leaving the rats unresponsive to normal mouse antigens. On day 14 after immunization, the rats were challenged with cells harvested from various stages of mouse IPSC cultures. Three to four weeks after this second challenge, the rats were reinmunized with IPSC culture cells for three days, then fused with SPO / 2 associated myeloma. The antibodies that reacted positively were selected and cloned.

B. Production of Mouse Human Anti-IPSC mAbs: Mouse human anti-IPSC mAbs were prepared using the same procedure described above for the production of rat anti-mouse mAb, except that mice used were immune to a normal human antigen and then challenged again after treatment with cyclophosphamide with cells from various stages of human IPSC cultures.

C. Use of Anti-IPSC mAbs in the Identification of Various Stages of Differentiation in Islet Cell Growth: The mAbs directed against the cultured IPSC cells were used to classify by FACS or by any other means in the art, such as fluidized beds magnetically stabilized (see above), the different cell populations defined by these reagents. The classified cell populations were examined by their stages of differentiation (eg, co-expression of insulin, glucagon, somatostatin, β-galactosidase, tyrosine hydroxylase, the Reg gene namely a few) and their growth capacity (eg, their ability to start IPSC crops). The reagents that define the cell surface of the differentiation markers of the cells involved in islet neogenesis are useful for the scientific community in this area of research. In addition, such reagents greatly facilitate the isolation (or enrichment) of IPSC per se. The isolation of the IPSC allows to test the efficacy in the reimplantation of the IPSC instead of complete islets in patients with IDD, or even implantation directly in the pancreas, increasing the need for extrapancreatic implants. In addition, these antibodies were used to isolate cells from any given stage of differentiation based on the affinity of markers expressed on the cell surface of pancreatic cells. The identification of specific markers that are expressed on the surface of differentiated pancreatic cells allows the production of non-functional pancreatic cell lineages. Cells that do not produce the undesirable kennic product were selected using the antibodies to select clones of cells in which that product was absent. Analogously, significant markers were identified for the recognition of T cells and destruction of differentiated pancreatic cells by the activation of candid T cells with complete pancreatic cells or subcellular fractions thereof, through the differentiation process. The identification of significant markers for the activation of the T cells allows the subsequent modification of the stem cells to eliminate those markers and therefore produce the cells that, in the differentiated state, are resistant to autoimmune destruction.

Example 9 - Isolation of Pancreatic Cells in Different Stages of Differentiation Using the labels and ligand binding molecules identified according to Example 8, pancreatic stem cells or partially or completely differentiated pancreatic cells could be isolated according to methods well known in the art. Accordingly, methods for the isolation of hematopoietic stem cells described in U.S. Patent Nos. 5,061,620; 5,437,994; 5,399,493; populations in which the pure stem cells were isolated using antibodies to markers of the stem cells, are incorporated herein by reference in their entirety as set forth. Similarly, methods for the separation of mammalian cells from cell mixtures using magnetically stabilized fluidized beds (MSFB), described in U.S. Patent No. 5,409,813, are incorporated herein by reference in their entirety as set forth. Antibodies for the markers identified at each stage of the differentiation of the pancreatic stem cells were attached to magnetized beds, and the cells were passed through the magnetized bed stabilized magnetically. The cells that adhered to the antibody bound to the magnetizable beds, or the cells that flowed through the bed were isolated. Any of a number of other methods known in the art can be used to isolate specific cells for this purpose. Those methods include, but are not limited to, destruction of the complement of undesirable cells; cellular selection; immunoaffinity chromatography, elutritiado; and soft agar isolation techniques (see, Freshrey, R.I. 1988), Example 10 - Analysis of Factors that Activate the Differentiation of Pancreatic Stem Cells and Factors Produced in Different Stages of Stem Cell Differentiation Cells isolated according to the methods of Example 9 or similar methods were cultured according to the method of Example 1 or similar culture methods. The significant factors for the induction of differentiation were tested by adding different factors to the growth medium and observing the inductive effect of the differentiation on the cells. In this way, conditioned culture media of several cells could be tested, and the factors that produce the differentiation of the pancreatic stem cells were isolated using the induction of differentiation as a purification assay. Other factors such as glucose, other chemicals, hormones and serum fractions are tested in a similar way to isolate the significant differentiation inducing factors. The factors produced in the different stages of the differentiation were isolated and analyzed from the conditioned culture medium of the cells at each stage of the differentiation process. These factors were also tested to determine their autonomous effect on stem cells and the additional differentiation of partially differentiated stem cells.

Example 11 - Genetic Modification of Pancreatic Stem Cells to Produce CTL-Resistant Antibodies and Differentiated Pancreatic Cells Modified with HLA Pancreatic stem cells cultured according to Example 1 or 2 or isolated according to Example 8 were subjected to genetic modification according to any method known in the art to produce differentiated pancreatic and antibody resistant cells and CTL, according to methods such as those described in U.S. Patent No. B, 6,632, supra; U.S. Patent No. 5,320,962, s pra; U.S. Patent No. 5,342,761, supra; and in WO 90/11354, supra; WO 92/03917, supra; WO 93/04169, supra; and WO 95/17911, supra. Alternatively, selection of resistant stem cells was achieved by culturing those cells in the presence of autoantibody or CTL associated with IDD or CTL activated with IDD-specific autoantigens. As a result of these techniques, cells were generated that have greater resistance to destruction by antibody-dependent mechanisms or T lymphocytes. Such cells were implanted into an appropriate host in an appropriate tissue as described above in Examples 3 and 4 to provide a structure similar to the pancreas , which had greater resistance to destruction by autoimmune processes. Likewise, the antigen profile of human leukocytes of pancreatic stem cells and differentiated cells was modified, optionally by an iterative process, in which the stem cells were exposed to normal cells, allogeneic lymphocytes and selected survivors. Alternatively, a site-directed mutagenesis method was used to remove HLA markers from the surface of the stem or differentiated cells, and thus new stem cells were generated or isolated from the pancreas-like structures used for the treatment. implant in a recipient mammal in need of such an implant.

In a specific example, the associated adenovirus vector system (AAV) containing the neomycin resistance gene, neo, was used. AAV can be used to transfect eukaryotic cells (Laface, 1988). In addition, the pBABE-bleo launch vector system containing the phleomycin resistance gene (Morgenstein, 1990) was used. This launch vector can be used to transform human cells with genes derived from bacteria. a) IPSC transfection: Cultured IPSCs were transfected with either the retroviral segment of the pBABE-2-bile vector by electroporation or by the AAV-neo vector by direct infection. The adherent cells of established IPSC cultures were gently removed from the culture vessels using C-PEG buffer (phosphate buffered saline supplemented with EDTA and a high glucose content). These cells were suspended in DMEM and 10% of fetal rat serum containing the retroviral standard, and in the case of pBABE-bleo, it was electroporated. (Since electroporation can be a very discordant procedure compared to direct viral infection, cells subjected to electroporation were examined for viability.) The viability of IPSC culture cells was determined by their ability to exclude vital dye and analysis of cellular products associated with damage such as glycosaminoglycans and hydroperoxides). Secondary cultures of transfected cells were established. The recultivated cells were selected for their resistance to phleomycin or neomycin, respectively. b) Identification of Proviral DNA in Transformed Cells: Cultured cells resistant to neomycin or phleomycin were tested for the presence of the appropriate transfectant viral DNA. The cells were removed from the culture vessels using C-PEG buffer and digested in lysis buffer containing proteinase K. The DNA was extracted with phenol / chloroform, then precipitated in ethanol / sodium acetate. The proviral DNA was identified using nested PCR. For the first reaction, PCR primers were used which amplify the entire open reading frame of the appropriate resistance gene. For the second PCR reaction, the PCR product was used as a standard. Internal selected 5 'and 3' primers were used which amplify an internal sequence of known base pair size. The final PCR product was detected by ethidium bromide staining of agarose gels after electrophoresis and / or Southern blotting. o / c) Transformation Stability: The long-term stability of the transformations was determined by maintaining the long-term growth of the cultures of the transfected cells and periodically testing them for the presence of proviral DNA, as described above. These studies provide information on the efficacy and reproducibility of transfection procedures using IPSC as target cells. In addition, they establish a second basis for the use of IPSC transformed in the treatment of patients with IDD.

Example 12 - Encapsulation of In Vitro Generated Islets and Implantation in a Mammal Methods of cell encapsulation are well known in the art (see, for example, Altman et al., 1984, Trans Am. Soc. Art. Organs., 30: 382-386, incorporated herein by reference, in which human insulinomas were enclosed in selectively permeable macrocapsules). Accordingly, the isolated isolated generated islets, optionally genetically modified according to Example 11, or structures similar to the pancreas produced according to Examples 3 and 4, were encapsulated in an encapsulant permeable to insulin, glucagon and somatostatin. . Preferably such an encapsulant is hypoallergenic, is easily and suitably stable in a target tissue, and provides greater protection to the implanted structure, so as to ensure a differentiation into a functional entity without destruction of the differentiated cells. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that those skilled in the art will be able to suggest various modifications or changes in light thereof including the spirit and scope of the application and the scope of the appended claims.

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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, property is claimed as contained in the following:

Claims (48)

1. A method for the growth of islet producing stem cells, IPSC to produce islet cells or an islet-like structure, characterized in that it comprises culturing pancreatic cells of a mammalian species, in a mass nutrient medium supplemented with normal serum at less than 0.5% and glucose below approximately 1 mM, which allows the IPSC to grow for at least about 3 weeks, and initiate cell differentiation in mature islet cells, realizing the IPSC in culture with a nutrient medium supplemented with normal serum at approximately 0.5-10% and glucose at approximately 2.5 mM-10 mM.

2. The method according to claim 1, characterized in that the pancreatic cells are human islet cells and the serum is normal human serum.

3. The method in accordance with the claim 1, characterized in that the pancreatic cells are mouse islet cells and the serum is normal mouse serum.

4. A method according to claim 1, characterized in that the nutrient medium comprises a nutrient medium with a high content of amino acids.

5. The method in accordance with the claim 1, characterized in that the culture medium used to feed the cell culture, further comprises glucose.

6. The method according to claim 1, characterized by the differentiation of the cultured stem cells, is initiated at approximately 4 to 5 weeks of culture growth, by re-feeding the pancreatic cell culture with a nutrient medium supplemented with normal homologous serum.

7. The method according to claim 1, characterized in that after the cell differentiation initiated by the refeeding of the culture, the culture is fed back at intervals of approximately one week.

8. The method according to claim 1, characterized in that the normal serum is obtained from the same mammalian species from which the islet cells were obtained.

9. The method according to claim 1, characterized in that the islet-like tissue structure is produced after differentiation of the IPSC.

10. An islet cell produced by the method according to claim 1.

11. An islet-like tissue structure produced by the method of claim 9.

12. A method for producing an endocrine hormone which comprises culturing pancreatic cells according to claim 1, and recovering endocrine hormones from pancreatic cell culture.

13. The method according to claim 12, characterized in that the hormone is a human hormone.

14. The method in accordance with the claim 12, characterized in that the hormone is a mouse hormone.

15. The method in accordance with the claim 12, characterized in that the differentiation starts at approximately 4 to 5 weeks of the growth of the culture, by means of the IPSC cell culture feedback with a nutritive medium supplemented with normal serum.

16. The method according to claim 12, characterized in that the endocrine hormone is selected from the group consisting of insulin, glucagon and somatostatin.

17. A method for producing a pancreatic-like organ in a mammal, characterized in that it comprises implanting an islet cell or an islet-like structure produced by the method according to claim 1, into the tissue of the mammal.

18. A method for treating a pancreatic disease in a mammal, characterized in that it comprises producing a similar organ? 1 pancreas in the mammalian m vi, in accordance with the method of claim 17.

19. An organ similar to the pancreas, produced by implanting a structure similar to the islet in a mammal, characterized in that a structure similar to the islet is produced by growing the islet producing stem cells, IPSC, present in pancreatic cells from a mammalian species in a basic nutrient medium supplemented with normal serum at less than about 0.5% and glucose below about 1 irJM, allow the IPSC to grow for at least about 3 weeks , and initiate cellular differentiation in mature islet cells by feeding the IPSCs in culture with a nutrient medium supplemented with normal serum at approximately 0.5-10% and glucose at approximately 2.5 mM-10 mM.

20. The method according to claim 1, characterized in that the islet-like structure comprises cells that are selected from the group consisting of α cells, β cells and d cells.

^ 21. The method in accordance with the claim 17, characterized in that the islet-like structure or islet cells implanted in the mammal are autologous to the recipient mammal of the implant. 0

22. The method according to the claim 17, characterized in that the mammal is a human.

23. The organ similar to the pancreas according to claim 19, characterized in that the organ is produced in a human.

24. A mammal, other than a human, having an organ similar to the pancreas produced in accordance with the method of claim 17.

25. A mammal, according to claim 24, characterized in that the mammal is a mouse.

26. A method for producing an organ similar to the pancreas in a mammal, which was characterized in that it comprises implanting at least one pancreatic stem cell in the tissue of a mammal.

27. An organ similar to the pancreas, characterized in that it is produced in a mammal, according to the method of claim 26.

28. The method according to claim 26, characterized in that at least one pancreatic stem cell has been modified so that it does not express the autoantigens associated with insulin-dependent diabetes, other than insulin, or that have been modified so that they do not express leukocyte antigens. humans, so that the stem cells differentiate in the organ similar to the pancreas. 36

29. The method according to claim 26, characterized in that at least one pancreatic stem cell is encapsulated in an insulin permeable capsule, glucagon, so atostatma or other factor produced by the pancreas.

30. A method for analyzing the differentiation of pancreatic stem cells, characterized in that it comprises culturing at least one pancreatic stem cell m vi tro.

31. The method in accordance with the claim 30, characterized in that it further comprises inducing at least one stem cell to begin to differentiate into a structure similar to the pancreas.

32. The method in accordance with the claim 31, characterized in that it further comprises identifying specific mRNA or protein markers by a plurality of the different stages in the differentiation process.

33. The method in accordance with the claim 32, characterized in that the protein markers are expressed on the cexuiar surface, are secreted, or are intracellular.

34. A method for labeling a ligand binding molecule which selectively binds to pancreatic stem cells or to more differentiated pancreatic cells, characterized in that it comprises contacting a cannulated nnfocito 3 or nnfocito with an identified protein marker in accordance with the method of Claim 33, and culturing and expanding such a B lymphocyte or T lymphocyte to obtain a population of cells that produce such a ligand binding molecule.

35. A ligand binding molecule, characterized in that it is prepared according to the method according to the method of claim 34.

36. The ligand binding molecule, according to claim 35, characterized in that it is an antibody, a monoclonal antibody, or a T cell receptor.

37. A method for isolating a pancreatic stem cell or partially differentiated pancreatic cell at any stage between a pancreatic pancreatic pancreatic cell and a fully differentiated pancreatic cell, characterized in that it comprises selecting the cell from a population of cells comprising such cells, with the molecule of ligand binding according to claim 35, which binds to a cell surface protein marker expressed by such cell in the differentiation step, or select and remove others from a population of cells comprising tai cell with the ligand binding molecule according to claim 35, which binds to an absent cell surface protein marker on the surface of such a cell.

38. An isolated cell, characterized in that it was isolated in accordance with the method according to claim 37.

39. A pancreatic stem cell or a population of pancreatic stem cells.

40. A method for treating a mammal that suffers from, or is at risk of IDD, characterized in that it comprises: a. remove pancreatic tissue from a mammal; b. cultivate pancreatic cells pluripotences present in the pancreatic tissue, m alive, to generate structures similar to islets; and c. implant structures similar to islets in the mammal.

41. A modified IPSC, characterized because it does not express antigens of insulin-dependent diabetes either in the undifferentiated or differentiated state of the IPSC.

42. The modified IPSC according to claim 41, characterized in that the autoantigen, was not expressed as a result of the modification is selected from GAD, antigen from an islet cell surface of 64 kD, and HLA markers.

43. The process according to claim 5, characterized in that the differentiation is improved by the inclusion of approximately 10-25 mM of glucose, growth factor / fibrosa hepatocytes, growth of keratinocytes, fibroblast growth factor, epidermal growth factor , insulin-like growth factor, nicotinamide, or autologous growth factors produced by the IPSC.

44. The method in accordance with the claim 43, characterized in that the glucose concentration in the fed medium is approximately 16.7 mM.

45. A method for the neogenesis in islet of islets from pluripotent stem or progenitor cells, characterized in that it comprises: a. establish a monolayer of stromal cells or "nurse" cells of the pancreatic epithelium of the duct, which allow the generation of IPSC; b. induce the proliferation of the mother / progenitor cell with culture conditions that promote the cyclic regeneration of the IPSC, and also prevent the mature differentiation of the IPSC; and c. expand and differentiate the IPSC to produce a structure similar to the islet comprising cells a, β, and d.

46. A method for the long-term propagation of IPSC, characterized in that it comprises the serious transfer of epithelial cells from the closest stages, which proliferate in islet-like structures in in vitro cultures.

47. A structure similar to an islet generated by culture, characterized by differentiated, large cells, which are stained with the specific staining for insulin in the center of the structure similar to the islet; yi small differentiated cells, which are stained with specific staining for glucagon in the periphery; and proliferating and undifferentiated cells were observed which do not stain with any of the stains specific for the endocrine hormones in the inner cortex; the structure is further characterized because, after the disruption of the structure in a single cell suspension by mechanical means in the presence of a proteolytic enzyme and the subsequent staining of the individual cells, individual cell populations which are stained either with specific staining for glucagon (cells a), specific staining for insulin (ß cells) or specific staining for somatostatin (cells d).

48. The method of islet neogenesis in vi tro, according to claim 45, characterized in that it comprises: a. Dispersing and releasing undisturbed pancreatic cells in a minimal culture medium comprising little or no glucose, serum at a concentration less than about 0.5%, essential amino acids for the cells of the species from which the pancreatic cells were obtained, and a rudimentary lipid source, up to approximately 99% of the cells in such a culture died (Phase I); b. Feed the culture of step (a) with the minimal medium supplemented with approximately 1-10 mM glucose and approximately 0.5-10% serum (but less than a toxic amount of) and feed back approximately one week until rapid proliferation occurs; c. feed the culture of step (b) with the minimum medium supplemented with 0.5-10% serum and glucose at approximately 10-25 mM and, optimally, add cell or growth factors (Phase III); d. allow structures similar to the islets to sprout in the middle; and. recover the structure similar to the islet.