IN VIRO GROWTH OF ISLOTES OF FUNCTIONAL LANGERHANS AND IN VIVO USE OF THE SAME BACKGROUND OF THE INVENTIONDiabetes 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 19Q4, diabetes was the direct cause of 35,000 deaths among Americans and was a contributory factor in another 95,000. Ocular complications of diabetes are the leading 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 infectionsREP: 23239 rapidly advance and not only cause the loss of teeth, but also cause a 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%. Clearly, the economic burden of diabetes is enormous. Every 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 $ 24,000 million dollars (statistics from the American Diabetes Association, 1988); however, the full 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 b insulin-independent diabetes. or IDD. Each type has a different prognosis, treatment and cause. Approximately 5 to 10% of patients with diabetes have IDD. IDD is characterized by a partial or complete inability to produce insulin, or normally due to the destruction of the insulin-producing β cells of the pancreatic islet of Langerhans. Patients with IDD would die without daily insulin injections to control their disease. 5 Little progress was made 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 0 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 β cells in IDD involves both humoral immune mechanisms (Baek eskov et al.1982; Baekkeskov et al .. 1990; Reddy et al., 1988; Pontesilli et al .. 1987) as immune mechanisms mediated by cells (Reddy et al .. 1988; Pontesilli et al .. 1987; Wang et al .. 1987). The humoral immunity is characterized by the appearance of antibodies against the membrane of the β cells (anti-69 kD and autoantibodies against the surface of the islet-cell), against the content of the β cells (autoantibodies anti-carboxypeptidase A .. anti ? -64 kD and / or anti-GAD), and / or against products secreted by β-cells (antiinsulin).
Although serum does not transfer IDD, autoantibodies against ß-cells occur at a very early age, raising the issue of an environmental trigger, 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 in 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., 1991). Adoptive transfer experiments using NOD mice (non-diabetic obese) as a model of human IDD, have firmly established a primary function of • 1 autoaggressive T cells in the pathogenesis of IDD (Bendelac et al., 1987; Mi 11er et al. 1988, Hanafusa et al., 1988, Bendelac et al., 1988). Unfortunately, the mechanisms underlying the destruction of pancreatic β cells are still unknown. Numerous strategies (eg, bone marrow replacement, immunosuppressive drugs, and immunizations with autoantigens) have been investigated as possible means of stopping the immune attack against pancreatic β-cells. However, for these approaches to be effective, individuals who eventually develop the 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 ß cells has already been destroyed. Because it is thought that the ß cells are a differentiated cell of the final stage, it is believed that the body has little capacity to regenerate new β cells. thus needing a regular therapy with insulin for life. Recently, one approach to solve this problem has been the transplantation of islet cells. The transplantation of islet cells has the advantage that the islets are allogeneous. which, in turn, can cause a loinmune 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. The new methods of the present invention have the advantage of the discovery that pluripotent stem cells still exist in the pancreas of adult individuals. The cells can be cultured in a nutrient 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). Later. this culture is left undisturbed for several weeks to allow stromal cells to be established. Once the stromal cell layer is mature, real cell differentiation can be initiated by impinging 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, islets can be recovered containing cells they produce; > insulin. glucagon, somatostatin and other hormones end crinas, using normal techniques. Previously, it was not known or suspected that pancreatic cells could be used to grow new islet cells, including Q cells, in culture. The fortuitous discovery of culture techniques to grow tissue similar to islet jn vi tro. it eliminates what had previously been a substantial and ancient barrier to diabetes research. The new methods and materials described herein.will make possible a better understanding of the mechanisms of diabetes. In addition, the ability to grow islet cells in culture will now make possible certain therapies for diabetes for the first time. For example, in accordance with the present invention, the new cellscultured 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 islet cell3 cultured are able to produce insulin invi vo. Thus, the present invention also relates to the use of enlarged islets in accordance with the present invention to be implanted in a mammalian species for the in vitro treatment of IDD. The present invention also greatly facilitates the b genetic engineering of islet cells to withstand subsequent immune destruction. For example. cultured islet cells can be transformed to express a protein or peptide that- - inhibit or prevent the destructive immune process.
Other proteins or peptides may also be expressed. Thus, the ability to grow islets functioned from the pancreatic cells of an individual, represents a great technical advance andfacilitates the use of new strategies for the treatment of IDD. The discovery that there are pluppotential stem cells in the adult pancreas avoids the need for fetal tissue as a source of cells. The present invention also relates toislet cells produced in vi tro 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 structures oftissue similar to islets.
The present invention also relates to 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 vivo use of pancreatic stem cells grown in vi tro. 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 vivo from pancreatic cells grown in vi tro. it 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 1A to ID show growing cells in accordance with the methods of the present invention. Figure 2 shows an islet grown in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, functional Langerhans islets can be grown for the first time in in vitro cultures. The techniques of the present invention result in cell cultures that can produce insulin, glucagon, somatostatin or other hormones. end crinas. Other useful proteins can also be produced. for example, transforming the islet cells with DNA that codes for the proteins of interest. The ability to grow these functional cell cultures makes it possible for those skilled in the art to carry out procedures that previously were not possible. The method of the present invention includes making suspensions of stem cells from the pancreas of a mammal. Preferably, the stem cells will be from the pancreas of a prediabetic mammal. Nevertheless. it is also contemplated in the present invention that cells from mammals already showing clinical signs of diabetes can be used. Cell suspensions are prepared using standard techniques. After. The cell suspension is cultured in a nutrient medium that facilitates cell growth. 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 available to those skilled in the art. Technicians in the field could prepare and use other equivalent nutritious media. The medium used to suspend the islet cells is advantageously supplemented with normal serum from the same mammalian species from which the islet cells originate. Thus, in the case of mouse islets, the medium is supplemented with normal mouse serum, whereas in the case of human and medium islet cells it is supplemented with normal human serum. The preparation of normal serum is well known to those skilled in the art. The concentration of the normal serum used in the cell culture of the present invention may vary from about 0.5 to about 10%. but for mice, about 1% is preferable. For human serum, a higher concentration, for example about 5%, is preferred. The cell suspension prepared in the nutrient medium supplemented with normal serum is subsequently incubated under conditions that facilitate cell growth, preferably at about 35 to 40 * C and, preferably, with an atmosphere of about 5% C0_. This incubation period, then, is carried out using the normal procedures known to those skilled in the art. After. the cell culture is preferably left undisturbed and without feeding for several weeks. Preferably, the cultures are not disturbed for at least 3 weeks. During this time, the stromal cells proliferate and become a monolayer. which will eventually make the islet cells arise. The initiation of cell differentiation can be carried out by realimating the cultures with EHAA Click medium supplemented with normal serum, as described above. Rapid feedback was found to induce extensive islet foci formation with considerable cellular differentiation. Upon histological examination of the cells in the islet-like structures, at least 3 distinct types of cells are identifiable and appear similar to islet cells prepared from islets of control mice. The time necessary for cell differentiation to occur within these foci diminished as the frequency of real imentation increased. It was possible to propagate and expand the islet producing crops. through serial transfers of stromal cells derived from islets, more 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 metabolic problems of IDD.
In order to determine whether the islet-like structures and / or the islet cells produced in accordance with the present invention can reverse the IDD. Skele-like structures were implanted in NOD mice. The mice that received the islet implants exhibited a regression of the insulin-independent diabetes. whereas 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 the cytotoxic interferon derived from T cells. The availability of long-term complete islet cultures can also be used in research on the pathogenesis of IDD, including the cellular recognition of ß cells, the mode of infiltration to the islets and the immune mechanisms of the destruction of the ß cells. 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 in accordance with the methods of the present invention. It is very useful to teach students important aspects related to cell differentiation and function. In a further embodiment of the present invention, pancreatic pluripotent stem cells have been grown in vi tro from pancreatic cells isolated from a mammal. A surprising discovery using these cells grown in vi f.ro in conjunction with the methods of the present invention was the ability to grow and produce, in vivo, an organ exhibiting endocrine and exocrine tissues morphologically and histologically functional. The ectopáncreas (an organ similar to the pancreas located in an abnormal site within the corporal cavity) produced in vivo in accordance with the present invention, represents a great scientific discovery and provides a new means to study, to treat. reverse or cure a number of pathogenic disorders associated with the pancreas.
As used in the present. the term "growth" refers to the maintenance of cells in a living state and may include, but is not limited to. the propagation and / or the differentiation of the 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 of procedures that illustrate, including the best OR embodiment, the practice of the present invention. These examples should not be considered as limiting. All percentages are given by weight and all solvent mixing ratios are given in volume, unless indicated otherwise. Example 1 - Culture of functional Langßrhaps islets Single cell suspensions of islet cells were prepared from whole islets isolated from the pancreas of a pre-diabetic male NOD / UF mouse of 19-0 20 weeks of age, as detailed in another document (Shieh et al., 1993). Typically, approximately 25% of the male mice in a NOD colony will have an open IDD at this age and will suffer from severe insulitis. The islet cells were resuspended in EHAA 5-Click medium supplemented with 1% murine normal serum (NMS) (Peck and Bach 1973, Peck and Click, 1973), inoculated in a 25 cm cell culture flask and incubated at 37"C with 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 lines; or second, stromal-like cells can dominate, thus allowing the growth of a "mother cell" monolayer. The growth of stromal-like cell monolayers appeared to result when the cells that were infiled to the islet were seeded simultaneously but in limited numbers. Enrichment of islet cells with decreased numbers of infiltrating cells can be achieved by gradient separation (Jarpe et al., 1991). 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. From this monolayer of cells, small rounded cells appeared, almost as if from the layer of stromal cells. Differentiation of the cultures was initiated by realimating the cultures with EHAA Click medium supplemented with SNM and a sugar solution comprising glucose or sucrose or other equivalent sugars. Typically sugar is glucose. The glucose concentration can be between about 10 mM to 25 mM. but typically it is between 15 and 17 mM. Preferably, the concentration of glucose in the medium is about 16 mM. Techniques for realizing cell cultures in v t ro are known in the art and typically include removing from about 50 to about 90% of the old nutrient medium and adding fresh medium to the culture flask. Fast i d feedback induced the formation of increasing numbers of islet growth centers (referred to herein as foci) exhibiting cell differentiation. The frequency of feedback can be, for example, at intervals of one week. Preferably, the frequencyof feedback is at intervals of approximately 5 to 6 days. In the peak production, as many as 150 to 200 bulbs appeared simultaneously in a single flask of 2 25 cm cell culture.cell proliferation and differentiation, the organization of the islet was carried out and still the islet appeared surrounded itself by a capsular material. The islets generally grew to a constant size (although several grew to a size approximatelydouble the overall size), then detached from the stromal layers to float in the medium. These free-floating islands tended to break in a period of 46 to 2 hours. similarly to isolated pancreatic islets grown under similar conditions. The islet-like structures, collected after natural detachment or removal of the stromal layers using a Pasteur pipette. they were carefully washed with the medium and then broke to form cell suspensions alone by reflux pipetting. Single cell suspensions were prepared by cytocentrifugation. then they were stained to observe the general morphology and the production of insulin. The foci contained cells that produced the endocrine hormones glucagon (cells a), insulin (ß cells) and / or somatostatin (cells 6). In addition, a large part of the cell population gave positive staining with anti-insulin antibodies, indicating that the major cell type contained in the cultured islet was an insulin-producing β-cell. Figures IA to 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.
Example 2 - Culture of human islet cells To culture human islet cells, a procedure similar to that described in Example 1 was used. The method of the present invention is particularly advantageous, since it is not necessary to use fetal cells to start the culture cell phone. In a preferred embodiment, human cells can be suspended in Click's EHAA medium (or a medium equivalent thereto) supplemented with normal human serum. Preferably, the concentration of human normal serum used in the medium is about 5%. Crops should be left unaltered without feedback, preferably for several weeks. After about 4-5 weeks in culture, actual cell differentiation can be initiated by impinging the cultures with Click EHAA medium supplemented with normal human serum and glucose, as described in Example 1. Subsequently, the islet-like structures are can collect and cell suspensions alone can be prepared for further propagation, in the manner described in Example 1. Example 3 - Implantation of growing islet cells n vi tro To test the efficacy of these islet-like structures generated n vi tro to reverse the complications of IDD, approximately 150 to 200 foci plus some stromal cells grown in accordance with the method of the present invention from pancreatic tissue of NOD mice. were taken from the cell culture flask by reflux pipetting. Next, the cells were implanted underneath the renal capsule of syngenic diabetic NOD mice. maintained by daily injections of insulin. The implant was performed by puncturing the renal capsule with a hypodermic needle, twisting a small 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 clamped 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 at daily doses of half, after which the mice were completely separated from any additional treatment with insulin. The control animals consisted of diabetic NOD mice that did not receive an implant. NOD control mice, when withdrawn from daily insulin injections, showed a rapid onset of open disease, including lethargy, dyspnea, weight loss, increased blood glucose concentration. Exhaustion syndrome, failures to heal wounds and death, within a period of 3 weeks. The implanted NOD mice maintained a blood glucose level of approximately 180 mg / dl (which is slightly above the normal range for mice), showed increased activity, quickly healed the surgical wounds and the sites from which blood was taken, they did not develop dyspnea and remained healthy until they were sacrificed for histological studies. Similar observations had been made with intrasplenic implants. 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 additional characteristic of islet-forming stem cells generated in 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 first, 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 endocrine structures similar to islets. Thus, in vitro cell cultures produced in accordance with the methods of the present invention contain pluppotent pancreatic stem cells capable of regenerating a completely new pancreas. The growth of a pancreas containing both exocrine and endocrine tissue provides new methods for the treatment of pancreatic diseases, including pancreatitis and pancreatic cancer. 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, which should be within the spirit and scope of the appended claims. References Eisenbarth. G.S. (1986) N. Engl. J. Med. 314: 1360. Cahil, G.F. and H.O McDevitt (1981) N. Engl. J.
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Peck (1993) Autoimmunity 15: 123. Peck A.B. and F.H. Back (1973) J. Immunol. Methods 3: 147 Peck A.B. and it. Click (1973) European J. Immunology 3: 382. 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 an antecedent, what is contained in the following is claimed as property.