WO1997015310A1 - Croissance in vitro d'ilots de langerhans fonctionnels et utilisations in vivo de ces ilots - Google Patents

Croissance in vitro d'ilots de langerhans fonctionnels et utilisations in vivo de ces ilots Download PDF

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WO1997015310A1
WO1997015310A1 PCT/US1996/016952 US9616952W WO9715310A1 WO 1997015310 A1 WO1997015310 A1 WO 1997015310A1 US 9616952 W US9616952 W US 9616952W WO 9715310 A1 WO9715310 A1 WO 9715310A1
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cells
islet
cell
pancreatic
culture
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PCT/US1996/016952
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English (en)
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Ammon B. Peck
Janet G. Cornelius
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University Of Florida Research Foundation, Inc.
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Priority claimed from US08/547,746 external-priority patent/US6001647A/en
Application filed by University Of Florida Research Foundation, Inc. filed Critical University Of Florida Research Foundation, Inc.
Priority to AU74683/96A priority Critical patent/AU739771B2/en
Priority to JP9516734A priority patent/JPH11514877A/ja
Priority to EP96936866A priority patent/EP0871455A4/fr
Publication of WO1997015310A1 publication Critical patent/WO1997015310A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0678—Stem cells; Progenitor cells; Precursor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

Definitions

  • Ocular complications of diabetes are the leading cause of new cases of legal blindness in people ages 20 to 74 in the United States.
  • the risk for lower extremity amputation is 15 times greater in individuals with diabetes than in individuals without it.
  • Kidney disease is a frequent and serious complication of diabetes. Approximately 30 percent of all new patients in the United States being treated for end-stage renal disease have diabetes. Individuals with diabetes are also at increased risk for periodontal disease.
  • Humoral immunity is characterized by the appearance of autoantibodies to ⁇ cell membranes (anti-69 kD and islet-cell surface autoantibodies), ⁇ cell contents (anti- carboxypeptidase A,, anti-64 kD and/or antiGAD autoantibody), and/or ⁇ 531 PC17US96/16952
  • pancreatic cells Recent efforts to culture pancreatic cells, including efforts reported in the following publications, have focused on cultures of differentiated or partially differentiated cells which in culture have grown in monolayers or as aggregates.
  • the instant invention discloses a method and a structure wherein an islet- like structure is produced which has a morphology and a degree of cellular organization much more akin to a normal islet produced in vivo through neogenesis.
  • Coon et al. (WO 94/23572, 1994), disclosed a method for producing an expanded, non-transformed cell culture of pancreatic cells. Aggregated cultured cells are then embedded in a collagen matrix for implantation, with the attendant shortcomings notes for the Zayas et ai, supra structures and the distinctions noted with the structure produced according to the instant invention.
  • pancreas It could not be predicted that such a structure could be produced in vitro, particularly when one considers that during embryogenesis, islet development within the pancreas appears to be initiated from undifferentiated precursor cells associated primarily with the pancreatic ductal epithelium (Pictet et al., 1972) i.e. non-islet cells.
  • the ductal epithelium rapidly proliferates, then subsequently differentiates into the various islet-associated cell populations (Hellerstrom, 1984; Weir et al. , 1990; Teitelman et al., 1993; Beattie et al., 1994).
  • the cellular organization ofthe islet can be destroyed in diseases such at type I. insulin dependent diabetes (IDD), in which a progressive humoral and cell-mediated autoimmune response results in specific destruction of the insulin-producing ⁇ cells (Eisenbarth, 1986; Leiter et al., 1987).
  • ILD insulin dependent diabetes
  • the ⁇ cell is considered to be, for the most part, a differentiated end-stage cell, it is believed that the body has limited capacity to generate new ⁇ cells, thus necessitating regular life-long insulin therapy once the ⁇ -cell mass is destroyed.
  • the ⁇ -cell mass has been shown to increase and decrease in order to maintain euglycemia (Bonner-Weir et ai, 1994).
  • This neogenesis has been induced experimentally by dietary treatment with soybean trypsin inhibitors (Weaver et ai, 1985), high levels of interferon- ⁇ (Gu et ai, 1993), partial pancreatectomy (Bonner-Weir et al, 1993), wrapping ofthe head of the pancreas in cellophane (Rosenberg et al, 1992), specific growth factors (Otonkoski et ai, 1994) and the onset of clinical IDD.
  • the novel methods ofthe subject invention take advantage ofthe discovery that IPSCs exist even in the pancreas of adult individuals.
  • the cells can be cultured in a minimal, high amino acid nutrient medium that is supplemented with normal serum which is preferably derived from the same mammalian species which serves as the origin ofthe islet cells (homologous serum).
  • normal serum which is preferably derived from the same mammalian species which serves as the origin ofthe islet cells (homologous serum).
  • homologous serum normal serum
  • Several discrete phases of cell growth result in selection of IPSCs and subsequent progeny which are then induced to differentiate and form islet-like structures which are distinguishable from pseudo-islet or pseudo- pancreatic tissue ofthe prior art.
  • a first phase primary culture of cells from a pancreas are placed in a low serum, low glucose, high amino-acid basal medium.
  • the subject invention also greatly facilitates genetic engineering of islet cells to resist subsequent immunological destruction.
  • the cultured islet cells can be transformed to express a protein or peptide which will inhibit or prevent the destructive immune process.
  • Other useful proteins or peptides may be expressed.
  • expression of specific autoantigens, such as GAD, 64 kD islet cell surface antigens (see Payton et al. 1995), or any other markers identified on the differentiated pancreatic cells can be eliminated by standard gene knock-out or selection procedures to produce differentiated pancreatic cells which are not or are less susceptible to auto-immune attack. Methods for producing such mutant or knock out cell lines are well known in the art and include, for example, homologous recombination methods disclosed in U.S. Patent No.
  • the subject invention also concerns the islet cells produced in vitro according to the methods described herein. These cells are produced from a mammalian pancreatic cell suspension cultured in vitro and give rise to functional islet cells and islet-like tissue structures.
  • the subject invention further concerns the in vitro growth. propagation and differentiation of a pancreatic stem cell. i.e.. a progenitor cell or cells that give rise to the formation of all ofthe different types of cells and tissue that make up a normal pancreas.
  • Figure 8 shows an ecto pancreas.
  • Mature islet cells are differentiated cells which arise from IPSCs and which produce pancreatic hormones. Islet-like structures, or young-islets, are highly-organized structures of cells which we have discovered arise in culture from IPSCs (see Figure 3H, Figures 4A and 4B, and cross-section shown in Figure 5). The structures "bud” from foci formed by individual IPSCs after most ofthe cells, which are not IPSCs, which are placed into culture from dissociated pancreatic tissue, have died. Upon implantation ofthe islet-like structure, final differentiation occurs to produce fully mature islet cells.
  • mice is preferably about 1%.
  • a higher concentration is preferred, for example, about 5%.
  • the cell suspension prepared in the nutrient medium supplemented with normal serum and about 2.5-10 mM glucose is then incubated under conditions that facilitate cell growth, preferably at about 35-40 °C and, preferably, in an atmosphere of about 5% C0 2 . This incubation period is, thus, carried out utilizing standard procedures well known to those skilled in the art.
  • stromal or ductal epithelial cells proliferate and establish a monolayer which will ultimately give rise to islet-like structures.
  • the initiation of cellular differentiation can be brought about by re- feeding the cultures with Click's EHAA or like medium supplemented with normal serum as discussed above.
  • telomere growth factor a factor which up-regulate the Reg gene
  • factors which up-regulate the Reg gene such as hepatocyte growth/scatter factor, and other cellular growth factors, such as insulin-like-growth factor, epidermal growth factor, keratinocyte growth factor, fibroblast growth factor, nicotinamide.
  • IPSC cultures are optimized.
  • factors produced by the IPSC cultures in the course of differentiation which augment growth can be isolated, sequenced. cloned, produced in mass quantities, and added to IPSC cultures to facilitate growth and differentiation of those cultures.
  • the relevant factors are identified by concentrating IPSC culture supernates from early, intermediate and late stages of differentiation and testing for the ability of these concentrates to augment IPSC growth and differentiation.
  • Positive effects are correlated with molecular constituents in the concentrates by two dimensional gel electrophoresis of positive and negative supernates, purification and N- terminal sequencing of spots present only in the positive concentrates and subsequent cloning and expression ofthe genes encoding these factors.
  • islet-like structures and/or islet cells produced in vitro could reverse IDD
  • the islet-like structures were implanted into NOD mice.
  • Mice that received the isiet implants exhibited a reversal of insulin-dependent diabetes, whereas untreated NOD mice showed signs of clinical disease.
  • no autoimmune pathogenesis was observed during the duration of the implants.
  • islet implants of the subject invention can be used in vivo to treat diabetes in mammals, including humans.
  • pluripotent pancreatic stem cells which give rise to ISPCs. have been grown in vitro from pancreas cells isolated from a mammal.
  • a su ⁇ rising discovery using these in vitro grown cells in conjunction with the methods ofthe subject invention was the ability to grow and produce, in vivo, an organ that exhibited functional, mo ⁇ hological and histological features and characteristics similar to a normal pancreas, including cell differentiation, to form endocrine and exocrine tissues.
  • the ecto-pancreas (a pancreas-like organ situated at an abnormal site within the body cavity), produced in vivo according to the subject invention, represents a major scientific discovery and provides a novel means for studying, treating, reversing or curing a number of pancreas-associated pathogenic conditions including, but not limited to. pancreatitis, pancreatic cancer and IDD. This is accomplished by removal of the diseased tissue and implantation of the islet-like structures produced according to this invention. In addition, the islet-like structures can be implanted into the natural pancreatic site. Because this invention provides a method for culturing pancreatic stem cells and production of young islets in vitro, study ofthe growth and differentiation of this cell-type is now possible.
  • markers including cell-surface, intracellular, protein or mRNA
  • markers specific to every stage of this process, are also now readily identifiable through application of standard techniques including, but not limited to: production of antibodies, including monoclonal antibodies, to cells, cell surface markers, and cellular components which differ throughout the process of pancreatic stem cell maturation; production of T-lymphocytes which specifically respond to antigens expressed by the pancreatic cells at different stages in the maturation and differentiation process (see.
  • the in v/ ro-generated islet implants of this invention showed no signs of immunological attack over the time period studied here. It is possible that the autoantigen(s) are not expressed on cultured cells, or that the autoantigen(s) cannot be presented since culture dilutes out the macrophages, or such implants may induce peripheral tolerance.
  • the availability of long-term cultures of whole islets facilitates investigations into the pathogenesis of IDD. including the cellular recognition of ⁇ cells, the mode of islet infiltration, and the immune mechanisms of ⁇ cell destruction.
  • this technology facilitates islet transplantation. autologous islet replacement, development of artificial islets and reduces the need for insulin therapy. Accordingly, this invention provides a method for the in vitro growth of islet producing stem cells.
  • IPSCs. to produce an islet-like structure.
  • the method comprises culturing pancreatic cells from a mammalian species in a basal nutrient medium supplemented with normal serum at below about 0.5% and glucose at below about 1 mM. allowing the IPSCs to grow for at least about 3 weeks, and initiating cellular differentiation into mature islet cells by re-feeding the IPSCs in culture with a nutrient medium supplemented with normal serum at about 0.5-10% and glucose at about 2.5 mM-10 mM.
  • the pancreatic cells may be from any mammal, including humans and mice, and the serum is from the same species.
  • the medium preferably contains all ofthe amino acids essential to growth of cells from the species being cultured and in such quantity as to ensure that the culture does not become depleted.
  • the re-feed medium preferably contains glucose and serum in sufficient quantities to stimulate differentiation.
  • the cells are preferably re-fed frequently (about once per week).
  • This method produces islet cells and islet-like tissue structures.
  • This method also provides a source of endocrine hormones, including, but not limited to. insulin, glucagon and somatostatin, which can be recovered from the IPSC culture medium or which can be directly released into a mammal by implantation ofthe islet-like structures into the tissue of a mammal to produce a pancreas-like structure.
  • implantation provides a method for treating pancreatic disease in a mammal by implanting an islet-like structure to produce a pancreas-like organ in the mammal.
  • the islet cells or islet-like structure of this invention is genetically modified so as to not produce IDD autoantigens or
  • pancreatic stem cell may be encapsulated in an insulin, glucagon, somatostatin and other pancreas produced factor permeable capsule. Also provided is a method for analyzing the differentiation of pancreatic stem cells which comprises culturing at least one pancreatic stem cell in vivo, and inducing said at least one stem to begin differentiation into a pancreas-like structure.
  • This method also permits identification of mRNA or protein markers specific to a plurality of different stages in the differentiation process.
  • the protein markers may be expressed on the cell-surface, be secreted, or they may be intracellular.
  • a ligand binding molecule and a method for making a ligand-binding molecule which selectively binds to pancreatic stem cells or to more differentiated pancreatic cells is provided. The method comprises contacting a naive B- lymphocyte or T-lymphocyte with an identified protein marker, and culturing and expanding the B-lymphocyte or T-lymphocyte to obtain a population of cells which produce the ligand-binding molecule.
  • ligand-binding molecules thus provide a method of isolating a pancreatic stem cell or partially differentiated pancreatic cells at any stage between that of a pancreatic stem cell and a fully differentiated pancreatic cell.
  • This method comprises selecting the cell from a population of cells comprising the cell, which a specific ligand-binding molecule which binds to a protein marker expressed by the cell at a given state of differentiation.
  • the method comprises selecting and removing other cells from a population of cells comprising the cell with a specific ligand binding molecule which binds to a protein marker absent on the surface of the cell.
  • this invention provides a method for treating a mammal suffering from, or at risk of IDD, which comprises: a.
  • an IPSC modified so as not to express insulin dependent diabetes autoantigens in either the undifferentiated or in the differentiated state ofthe IPSC.
  • the autoantigen which is not expressed as a result ofthe modification is selected from GAD, 64 kD islet cell antigen, and HLA markers.
  • a method for in vitro neogenesis of islets from pluripotent stem or progenitor cells comprises: a. establishing a stromal, or "nurse", cell monolayer of ductal pancreatic epithelial cells which permits the generation of IPSCs; b. inducing stem/progenitor cell proliferation with culture conditions which promote cyclical regeneration of IPSCs and also prevent premature differentiation ofthe IPSC; and c. expanding and differentiating the IPSCs to produce an islet ⁇ like structure comprising ⁇ , ⁇ , and ⁇ cells.
  • the culture-generated islet-like structure is characterized by large, differentiated cells which stain with insulin-specific stain in the center ofthe islet-like structure; small differentiated cells which stain with glucagon-specific stain at the periphery; and proliferating and undifferentiated cells which do not stain with any of the endocrine hormone-specific stains in the inner cortex.
  • the structure is further characterized in that, upon breaking the structure into single cell suspensions by mechanical or other means in the presence of a proteolytic enzyme and subsequent staining of individual cells, individual cell populations which stain either with glucagon-specific stain ( ⁇ cells), insulin-specific stain ( ⁇ cells) or somatostatin-specific stain ( ⁇ cells) are observed.
  • the method of in vitro neogenesis of islets preferably comprises: a. dispersing and leaving undisturbed pancreatic cells in a minimal culture medium comprising little or no glucose, serum at a concentration below about 0.5%, essential amino acids for the cells ofthe species from which the pancreatic cells were obtained, and a lipid source, until about 99% ofthe cells in said culture have died (Phase I); b re- feeding the culture of step (a) with the minimal medium supplemented with about 1-10 mM glucose and about 0.5%-10% serum (but less than a toxic amount) and re-feeding about once a week until rapid proliferation occurs; c.
  • step (b) re-feeding the culture of step (b) with the minimal medium supplemented with 0.5%- 10% serum and about 10-25 mM glucose and, optionally, added growth or cellular factors (Phase III); d. allowing islet-like structures to bud into the medium; e. recovering the islet-like structure.
  • This process may be repeated several times by serially transferring epithelial cells plus early-stage, proliferating islet-like structures in culture in vitro.
  • Single cell suspensions of islet cells were prepared from whole islets isolated from the pancreas of 19-20 week old prediabetic male NOD UF mice, as detailed elsewhere (Shieh et aL, 1993). Typically, about 25% of the male mice in a NOD colony will have overt IDD at this age and will have severe insulitis.
  • the islet cells were re-suspended in glucose depleted or glucose- free Click's EHAA medium (Peck and Bach, 1973, supra; Peck and Click, 1973) supplemented with normal mouse serum (NMS) to 0.25%, plated in a 25 cm 2 tissue culture flask, and incubated at 37°C in a 5% C0 2 atmosphere.
  • the islet- infiltrating cells may dominate, thus permitting the establishment of immune cell lines, or second, ductal epithelial cells (often referred to a stromal cells in these cultures) may dominate, thus allowing the growth of a "nurse cell” monolayer. Growth of stromal-like cell monolayers appeared to result when islet-infiltrating cells were plated simultaneously but in limited numbers. Enrichment ofthe islet cells with decreased numbers of infiltrating cells can be achieved by gradient separation (Ja ⁇ e et al., 1991 , supra).
  • the rate of re-feeding can be, for example, at about one week intervals. Preferably, the rate of re-feeding is at about 5 to 6 day intervals. Small rounded cells appeared on top ofthe epithelial monolayers, almost as if by budding ( Figures IB and 3D, Stage II).
  • Figure IC Rapid re- feeding induced increasing numbers of cell clusters as well as increased numbers of cells within each cluster. Induction of islet ⁇ like structures (Stage III) was enhanced through re- feeding of cultures with EHAA medium supplemented with normal mouse serum (0.5%) and high levels of glucose ( 10 mM-25 mM and preferably about 16.7 mM glucose -
  • Mature islets (Stage IV) appeared as smooth spheroids composed of tightly clustered cells ( Figure 3F-3H). This differentiation appears to be enhanced when serum from NOD mice is used rather than serum from other strains of mice, and higher levels of insulin-like growth factor (IGF), epidermal growth factor (EGF) and/or hepatocyte growth factor (HGF) in the NOD mouse serum are believed to be responsible for this effect.
  • IGF insulin-like growth factor
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • the islet-like structures collected after natural detachment or removal from the stromal layers using a Pasteur pipette, were gently washed in medium, then broken into single cell suspensions by reflux pipetting.
  • FIG. 1 Single cell suspensions were prepared by cytocentrifugation, then stained for general mo ⁇ hology and insulin production.
  • the foci contained cells producing the endocrine hormones glucagon ( ⁇ cells), insulin ( ⁇ cells) and or somatostatin ( ⁇ cells).
  • ⁇ cells the endocrine hormones glucagon
  • ⁇ cells insulin
  • ⁇ cells the major population of cells stained positive with anti-insulin antibody, indicating the major cell type contained in the cultured islet is an insulin-producing ⁇ cell.
  • Figures IA through ID show the various cell types which develop during the culture process.
  • Figure 2 shows a well-developed islet obtained after the in vitro culture of cells according to the method of the subject invention.
  • the human cells can be suspended in Click's EHAA medium (or the equivalent thereof) supplemented with normal human serum.
  • the concentration of normal human serum used in the medium is about 0.25% - 1% in phases I and II, respectively, and 5% during subsequent phases.
  • the cultures should be left undisturbed with no re-feeding, preferably for several weeks (Phase
  • cell differentiation can be initiated by re-feeding the cultures with Click's EHAA medium supplemented with normal human serum and glucose as described in Example 1. Islet-like structures can subsequently be collected and single cell suspensions prepared for further propagation as described in Example 1.
  • mice were maintained on insulin injections for 4 days at the full daily dosage, and then for 2 days at the half daily dosage, after which the mice were completely weaned from further insulin treatment.
  • Control animals consisted of diabetic NOD mice that did not receive an implant.
  • control NOD mice showed a rapid onset of overt disease, including lethargy, dyspnea, weight loss, increased blood glucose levels (400-800 mg/dl), wasting syndrome, failure of wound healing and death within 18-28 days (Figure 7).
  • Implanted NOD mice maintained a blood glucose level of about 180-220 mg/dl (which is slightly about the normal range for mice), showed increased activity, rapid healing of surgical and blood-draw sites, did not develop dyspnea, and remained healthy until killed 55 days post-implant for histological studies (Figure 7). Similar observations have been seen with intra-splenic implants. These data are consistent with the concept that the implanted in vivo- generated islets provide the necessary insulin to maintain stable blood glucose levels over the time course of the experiment.
  • Example 4 In Vivo Production of Ecto-Pancreas Histological examinations ofthe implant sites in mice that were implanted with the islet cells as described in Example 3 revealed an additional characteristic of the in vitro generated islet- forming stem cells. Implanted cells which "leaked" from the implant site ofthe kidney underwent additional proliferation and differentiation and formed a highly structured ecto-pancreas. At first, the ecto-pancreatic tissue consisted entirely of proliferating exocrine cells which organized into an exocrine pancreas complete with innervating blood vessels. This exocrine pancreas progressed to form islet-like endocrine structures (see Figure 8).
  • the in vitro cell cultures produced according to the methods of the subject invention contain pluripotent 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 treatment of pancreatic diseases, including pancreatitis and pancreatic cancer.
  • Example 6 Analysis of Islet-Like Structures Photomicrographies of serial sections of immature, culture-generated islet-like structures and sections thereof (shown in Figures 4 and 5, respectively) again demonstrate the uniformity of growth. Large, somewhat differentiated cells which stain weakly with insulin are observed in the islet center. Small differentiated cells which stained with glucagon were apparent at the periphery, while a significant number of immature, proliferating, and undifferentiated cells which did not stain with any ofthe endocrine hormone antibodies were present in the inner cortex.
  • the islet-like structures were collected following detachment from the epithelial monolayers, gently washed in medium, then broken into single cell suspensions by mechanical means, such as reflux pipetting in the presence of a proteolytic enzyme such as 0.25% trypsin. Slides of single cell suspensions were prepared by cytocentrifugation and stained for general mo ⁇ hology or cellular content. Several mo ⁇ hologically distinct mature and immature cell types are observed following H/E staining.
  • pancreatic tissue is dispersed in a culture medium.
  • the dispersed pancreatic cells are subjected to limit dilution according to methods well known in the art.
  • serial ten-fold dilutions are conducted after an initial evaluation ofthe number of cells/mL in the dispersed sample, such that the final dilution yields, at the most, an average of 0.3 cells per microtitier well or other container suitable for this type of dilution experiment.
  • the cells are allowed to remain undisturbed until foci begin to develop.
  • These foci are stem cells which have arisen from a single pluripotent stem cell or IPSC and which can each be cultured to yield an islet-like structure for implantation to form a pancreas-like structure.
  • Example 8 Identification of Markers Associated with Different Stages of Pancreatic Stem Cell Differentiation, and Production of Ligand Binding Molecules Specific to Each Stage of Differentiation
  • Foci of isolated stem cells produced according to Example 7 or by an analogous method are analyzed both prior to and after induction of differentiation according to Example 1 or by a similar method.
  • the cells at each stage, from stem cell to fully committed differentiated pancreatic cells. are analyzed as follows:
  • Nucleic Acid At each stage of differentiation, including the undifferentiated progenitor cells and the fully differentiated pancreatic cells. mRNA is isolated. This RNA is used to make 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 message expressed at each stage of pancreatic stem cell development. Accordingly, message not present in stem cells but present in fully differentiated pancreatic cells is identified by hybridizing the cDNA from each stage and isolating message that remains unhybridized.
  • differentiated display PCR arbitrarily primed PCR or RDA-PCR (see above) may be used.
  • message unique to each stage is identified by subtraction of message present at other stages of differentiation.
  • gene products at each stage ofthe differentiation process are identified by expressing the product encoded by the subtracted message.
  • Antibodies including monoclonal antibodies, are then produced by using these gene products as antigens according to methods well known in the art (see, Goding, J.W., 1986). These antibodies are subsequently used to isolate cells from any given stage of differentiation based on affinity for markers expressed on the cell surface ofthe pancreatic cell.
  • identification of specific markers which are expressed on the surface ofthe differentiated pancreatic cells allows production of knock ⁇ out lines of pancreatic cells by site-directed mutagenesis using the identified sequences to direct mutations in stem cells according to methods such as those disclosed in U.S. Patent No. 5,286,632, supra; U.S. Patent No. 5.320,962; U.S. Patent No. 5.342,761 ; and in WO 90/1 1354; WO 92/03917; WO 93/04169: and WO 95/17911. Selection of mutant cells which do not produce the knocked-out gene product is accomplished using the antibodies to the specific gene product selected against to provide clones of cells in which that product is absent.
  • B. Protein Markers At each stage of differentiation, including the undifferentiated progenitor cells and the fully differentiated pancreatic cells, antibodies are generated to whole cells and subcellular fractions, according to standard methods known in the art. As specific examples of this process: a) Production of rat anti-mouse IPSC mAbs: To enhance selection of B lymphocytes activated against IPSC-specific antigens, rats are immunized with normal mouse tissue followed by treatment with cyclophosphamide on day 7 post-immunization. Cyclophosphamide selectively kills the reactive B cells, leaving the rats unresponsive to normal mouse antigens. On day 14 post-immunization, the rats are re-challenged with cells collected from various stages of mouse IPSC cultures. Three to four weeks after this secondary challenge, the rats are re-immunized with
  • IPSC culture cells for three days, then fused with the SPO/2 myeloma partner. Positively reacting antibodies are selected and cloned.
  • b) Production of Mouse Anti-Human IPSC mAbs Mouse anti- human IPSC mAbs are prepared using the same procedure as described above for the production of rat anti-mouse mAbs, except that mice are immunized with normal human tissue and then re-challenged after cyclophosphamide treatment with cells from various stages of human IPSC cultures.
  • c) Use of Anti-IPSC mAbs in the Identification of Various Differentiation Stages of Islet Cell Growth The mAbs raised against
  • IPSC cultured cells are used to sort by FACS or any other means known in the art. such as in magnetically stabilized fluidized beds (see below), the various cell populations defined by these reagents. Sorted cell populations are examined for their stages of differentiation (e.g., co-expression of insulin, glucagon, somatostatin, ⁇ -galactosidase. tyrosine hydroxylase, the Reg-gene to name a few) and their growth capacity (e.g., their ability to initiate IPSC cultures).
  • stages of differentiation e.g., co-expression of insulin, glucagon, somatostatin, ⁇ -galactosidase. tyrosine hydroxylase, the Reg-gene to name a few
  • growth capacity e.g., their ability to initiate IPSC cultures.
  • Reagents which define cell surface and differentiation marks of cells involved in the neogenesis of islets are useful for the scientific community in this area of research.
  • such reagents greatly facilitate the isolation (or enrichment) of IPSCs per se. Isolation of IPSCs permits testing ofthe efficacy of re-implanting IPSCs instead of whole islets into IDD patients, or even implantation directly into the pancreas, circumventing the need for extra-pancreatic implants.
  • these antibodies are used to isolate cells from any given stage of differentiation based on affinity for markers expressed on the cell surface ofthe pancreatic cell. Identification of specific markers which are expressed on the surface of the differentiated pancreatic cells allows production of knock-out lines of pancreatic cells. Cells which do not produce the undesirable gene product are selected by using the antibodies to select for clones of cells in which that product is absent.
  • markers significant to T-cell recognition and destruction of differentiated pancreatic cells are identified by activating naive T-cells with whole pancreatic cells or subcellular fractions thereof, across the differentiation process. Identification of markers significant to T-cell activation allows subsequent modification of the stem cells to eliminate these marks and thereby produce cells which, in the differentiated state, are resistant to autoimmune destruction.
  • pancreatic stem cells or partially or completely differentiated pancreatic cells can be isolated according to methods well known in the art. Accordingly, the methods for hematopoietic stem cell isolation disclosed in U.S. Patent No. 5,061.620; 5,437,994; 5,399,493: in which populations of pure stem cells are isolated using antibodies to stem cell markers, are hereby inco ⁇ orated by reference as if fully set forth herein. Likewise, the methods for mammalian cell separation from mixtures of cells using magnetically stabilized fluidized beds (MSFB), disclosed in U.S. Patent No.
  • MSFB magnetically stabilized fluidized beds
  • Cells isolated according to the methods of Example 9 or like methods are cultured according to the method of Example 1 or like culturing method.
  • Factors significant in inducing differentiation are assayed by adding different factors to the growth medium and observing the differentiation inducing effect on the cells.
  • conditioned culture media from various cells can be tested, and factors which cause pancreatic stem cell differentiation are isolated using induction of differentiation as a purification assay.
  • Other factors such as glucose, other chemicals, hormones and serum fractions are similarly tested to isolate the significant differentiation inducing factors.
  • Factors produced at different stages of differentiation are isolated and analyzed from the conditioned culture medium of cells at each stage of the differentiation process. These factors are likewise tested for their autocrine effect on stem cells and further differentiation of partially differentiated stem cells.
  • Example 11 Genetic Modification of Pancreatic Stem Cells to Produce Autoantibody. CTL Resistant, and HLA Modified Differentiated Pancreatic Cells
  • Pancreatic stem cells cultured according to Example 1 or 2 or isolated according to Example 8 are subjected to genetic modification according to any method known in the art to produce autoantibody and CTL resistant stem and differentiated pancreatic cells, according to methods such as those disclosed in U.S. Patent No. 5.286,632, supra; U.S. Patent No. 5,320,962, supra; U.S. Patent No. 5,342,761. supra; and in WO 90/1 1354, supra; WO 92/03917, supra; WO 93/04169, supra; and WO 95/17911, supra.
  • selection of resistant stem cells is accomplished by culturing these cells in the presence of autoantibody or IDD associated CTLs or CTLs activated with IDD specific autoantigens.
  • pancreatic stem cell and differentiated cell are modified, optionally by an iterative process, in which the stem cell is exposed to normal, allogeneic lymphocytes, and surviving cells selected.
  • a site directed mutagenesis approach is used to eliminate the HLA markers from the surface ofthe stem or differentiated cells, and new stem cells thereby generated or isolated from pancreas-like structures are used to implant into a recipient mammal in need of such implantation.
  • the adeno-associated virus (AAV) vector system carrying the neomycin-resistance gen, neo is used.
  • AAV can be used to transfect eukaryotic cells (Laface, 1988).
  • the pBABE- bleo shuttle vector system carrying the phleomycin-resistance gene is used (Morgenstein, 1990). This shuttle vector can be used to transform human cells with bacteria-derived genes.
  • a) Transfection of IPSCs Cultured IPSCs are transfected with either the retroviral segment ofthe pBABE-2-bleo vector by electroporation or the AAV-neo vector by direct infection.
  • Adherent cells from established IPSC cultures are removed gently from the tissue culture flasks using C-PEG buffer (phosphate buffered saline supplemented with EDTA and high glucose). These cells are suspended in DMEM and 10% fetal rat serum containing the retroviral stock, and in the case of pBABE- bleo, subjected to electroporation. (Since electroporation can be a fairly harsh procedure compared to direct viral infection the cells subject to electroporation are examine for viability. Viability ofthe IPSC culture cells is determined by their ability to exclude vital dye and analysis of injury- associated cell products such as glycosaminoglycans and hydroperoxides.)
  • Neomycin or phleomycin resistant cultured cells are tested for the presence ofthe appropriate transfecting viral DNA.
  • Cells are removed from the culture flasks using C-PEG buffer and digested in lysis buffer containing proteinase K.
  • DNA is phenol/chloroform extracted, then precipitated in ethanol/sodium acetate.
  • Proviral DNA is identified using nested PCR.
  • PCR primers are used which amplify the entire open reading frame ofthe appropriate resistance gene.
  • the PCR product is used as template.
  • Selected intemal 5' and 3' primers are used which amplify an intemal sequence of known base pair size.
  • c) Stability of Transformation The long-term stability of the transformations is determined by maintaining long-term growing cultures ofthe transfected cells and periodically testing them for the presence of pro-viral DNA, as described above.
  • Example 12 Encapsulation of In Vitro Generated Islets and Implantation Into a Mammal
  • isolated in vitro generated islets optionally genetically modified according to Example 1 1. or pancreas-like structures produced according to examples 3 and 4, are encapsulated in an insulin, glucagon and somatostatin permeable encapsulant.
  • encapsulant is hypoallergenic.

Abstract

La présente invention se rapporte à de nouveaux procédés qui, pour la première fois, permettent de développer des îlots fonctionnels dans des cultures in vitro. La présente invention se rapporte également à l'utilisation de ces structures de type îlots cultivées in vitro en vue de l'implantation chez un mammifère dans le cadre d'une thérapie in vivo du diabète. La présente invention se rapporte en outre à un procédé utilisant ces implants d'îlots cultivés in vitro pour développer un organe in vivo qui possède les mêmes caractéristiques fonctionnelles, morphologiques et histologiques que celles observées dans le tissu pancréatique normal. La possibilité de faire se développer ces cellules in vitro ainsi que des organes in vivo ouvre d'importantes perspectives de recherche et de thérapie liées aux diabètes.
PCT/US1996/016952 1995-10-25 1996-10-23 Croissance in vitro d'ilots de langerhans fonctionnels et utilisations in vivo de ces ilots WO1997015310A1 (fr)

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WO2001001130A1 (fr) * 1999-06-25 2001-01-04 Proteome Sciences Plc PROCEDES ET COMPOSITIONS SE RAPPORTANT AU DYSFONCTIONS DES ILOTS PANCREATIQUES ET DES CELLULES $g(b)
WO2001032839A2 (fr) * 1999-10-29 2001-05-10 Mcgill University Milieu servant a la preparation de cellules dedifferenciees
EP1224259A1 (fr) * 1999-09-27 2002-07-24 University Of Florida Inversion de diabetes dependant de l'insuline par des cellules souches insulaires, des cellules insulaires progenitrices et des structures de type insulaire
US6436704B1 (en) 2000-04-10 2002-08-20 Raven Biotechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
EP1240518A1 (fr) * 1999-12-13 2002-09-18 The Scripps Research Institute Marqueurs pour identifier et isoler les progeniteurs des cellules alpha et beta d'ilots pancreatiques
WO2002088335A1 (fr) * 2001-04-24 2002-11-07 Ajinomoto Co., Inc. Cellules souches et procede d'extraction de ces cellules
EP1257282A1 (fr) * 1999-12-06 2002-11-20 The General Hospital Corporation Cellules souches pancreatiques et leur utilisation en transplantation
EP1301589A1 (fr) * 2000-06-30 2003-04-16 Amcyte, Inc. Culture de cellules embryonnaires pancreatiques presentant un stade de developpement intermediaire specifie
EP1309673A2 (fr) * 2000-08-09 2003-05-14 ES Cell International Pte Ltd. Cellules souches du pancrease
WO2004108910A1 (fr) * 2003-06-03 2004-12-16 Organ Recovery Systems, Inc. Selection et propagation de cellules progenitrices
WO2006060709A2 (fr) * 2004-12-02 2006-06-08 Organ Recovery Systems, Inc. Voies biologiques dans des cellules progenitrices
US7101546B2 (en) 2001-12-21 2006-09-05 Amcyte, Inc. In situ maturation of cultured pancreatic stem cells having a specified, intermediate stage of development
US7202080B2 (en) 2001-03-29 2007-04-10 Ixion Biotechnology, Inc. Method for transdifferentiation of non-pancreatic stem cells to the pancreatic differentiation pathway
US7371576B2 (en) 2002-09-06 2008-05-13 Reneuron, Inc. CD56 positive human adult pancreatic endocrine progenitor cells
US8110399B2 (en) 2000-10-06 2012-02-07 The General Hospital Corporation Stem cells of the islets of langerhans and their use in treating diabetes mellitus
KR101452877B1 (ko) * 2013-08-05 2014-10-22 사회복지법인 삼성생명공익재단 조기-내피전구세포 및 췌도세포를 포함하는 세포이식물의 제조방법
US9428733B2 (en) 2005-10-05 2016-08-30 Akifumi Matsuyama Method for obtaining pancreatic endocrine cells from adipose tissue-origin cells

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US6638765B1 (en) 1999-02-04 2003-10-28 Mcgill University Platform for the differentiation of cells
WO2000046351A3 (fr) * 1999-02-04 2001-07-19 Univ Mcgill Plate-forme de differenciation cellulaire
AU781750B2 (en) * 1999-02-04 2005-06-09 Mcgill University Platform for the differentiation of cells
WO2000046351A2 (fr) * 1999-02-04 2000-08-10 Mcgill University Plate-forme de differenciation cellulaire
WO2001001130A1 (fr) * 1999-06-25 2001-01-04 Proteome Sciences Plc PROCEDES ET COMPOSITIONS SE RAPPORTANT AU DYSFONCTIONS DES ILOTS PANCREATIQUES ET DES CELLULES $g(b)
AU781477C (en) * 1999-06-25 2005-11-10 Proteome Sciences Plc Methods and compositions relating to pancreatic islet and beta-cell dysfunction
AU781477B2 (en) * 1999-06-25 2005-05-26 Proteome Sciences Plc Methods and compositions relating to pancreatic islet and beta-cell dysfunction
EP1224259A1 (fr) * 1999-09-27 2002-07-24 University Of Florida Inversion de diabetes dependant de l'insuline par des cellules souches insulaires, des cellules insulaires progenitrices et des structures de type insulaire
EP1224259A4 (fr) * 1999-09-27 2005-04-27 Univ Florida Inversion de diabetes dependant de l'insuline par des cellules souches insulaires, des cellules insulaires progenitrices et des structures de type insulaire
WO2001032839A2 (fr) * 1999-10-29 2001-05-10 Mcgill University Milieu servant a la preparation de cellules dedifferenciees
WO2001032839A3 (fr) * 1999-10-29 2001-11-01 Univ Mcgill Milieu servant a la preparation de cellules dedifferenciees
US6866843B2 (en) 1999-12-06 2005-03-15 Viacell, Inc. Method of transplanting in a mammal and treating diabetes mellitus by administering a pseudo-islet like aggregate differentiated from a nestin-positive pancreatic stem cell
JP2003523323A (ja) * 1999-12-06 2003-08-05 ザ ジェネラル ホスピタル コーポレーション 膵臓幹細胞および移植におけるその使用
EP1257282A4 (fr) * 1999-12-06 2003-05-02 Gen Hospital Corp Cellules souches pancreatiques et leur utilisation en transplantation
US7544510B2 (en) 1999-12-06 2009-06-09 The General Hospital Corporation Stem cells of the islets of Langerhans and their use in treating diabetes mellitus
US7537756B2 (en) 1999-12-06 2009-05-26 The General Hospital Corporation Stem cells and their use in transplantation
US7438902B2 (en) 1999-12-06 2008-10-21 The General Hospital Corporation Stem cells and their use in transplantation
EP1257282A1 (fr) * 1999-12-06 2002-11-20 The General Hospital Corporation Cellules souches pancreatiques et leur utilisation en transplantation
US6923959B2 (en) 1999-12-06 2005-08-02 The General Hospital Corporation Method of pre-inducing a state of immune tolerance before organ transplantation
EP1240518A4 (fr) * 1999-12-13 2006-05-17 Scripps Research Inst Marqueurs pour identifier et isoler les progeniteurs des cellules alpha et beta d'ilots pancreatiques
EP1240518A1 (fr) * 1999-12-13 2002-09-18 The Scripps Research Institute Marqueurs pour identifier et isoler les progeniteurs des cellules alpha et beta d'ilots pancreatiques
JP2003530102A (ja) * 2000-04-10 2003-10-14 レイヴェン バイオテクノロジーズ， インコーポレイテッド ヒト膵臓上皮前駆細胞血栓ならびにその単離および使用の方法
US7078231B2 (en) 2000-04-10 2006-07-18 Raven Biothechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
US6436704B1 (en) 2000-04-10 2002-08-20 Raven Biotechnologies, Inc. Human pancreatic epithelial progenitor cells and methods of isolation and use thereof
EP1301589A1 (fr) * 2000-06-30 2003-04-16 Amcyte, Inc. Culture de cellules embryonnaires pancreatiques presentant un stade de developpement intermediaire specifie
EP1301589A4 (fr) * 2000-06-30 2004-06-16 Amcyte Inc Culture de cellules embryonnaires pancreatiques presentant un stade de developpement intermediaire specifie
EP1309673A2 (fr) * 2000-08-09 2003-05-14 ES Cell International Pte Ltd. Cellules souches du pancrease
US8110399B2 (en) 2000-10-06 2012-02-07 The General Hospital Corporation Stem cells of the islets of langerhans and their use in treating diabetes mellitus
US7202080B2 (en) 2001-03-29 2007-04-10 Ixion Biotechnology, Inc. Method for transdifferentiation of non-pancreatic stem cells to the pancreatic differentiation pathway
WO2002088335A1 (fr) * 2001-04-24 2002-11-07 Ajinomoto Co., Inc. Cellules souches et procede d'extraction de ces cellules
US7101546B2 (en) 2001-12-21 2006-09-05 Amcyte, Inc. In situ maturation of cultured pancreatic stem cells having a specified, intermediate stage of development
US7371576B2 (en) 2002-09-06 2008-05-13 Reneuron, Inc. CD56 positive human adult pancreatic endocrine progenitor cells
US9068169B2 (en) 2002-09-06 2015-06-30 Reneuron, Inc. CD56 positive human adult pancreatic endocrine progenitor cells
WO2004108910A1 (fr) * 2003-06-03 2004-12-16 Organ Recovery Systems, Inc. Selection et propagation de cellules progenitrices
WO2006060709A3 (fr) * 2004-12-02 2006-07-06 Organ Recovery Systems Inc Voies biologiques dans des cellules progenitrices
WO2006060709A2 (fr) * 2004-12-02 2006-06-08 Organ Recovery Systems, Inc. Voies biologiques dans des cellules progenitrices
US9428733B2 (en) 2005-10-05 2016-08-30 Akifumi Matsuyama Method for obtaining pancreatic endocrine cells from adipose tissue-origin cells
KR101452877B1 (ko) * 2013-08-05 2014-10-22 사회복지법인 삼성생명공익재단 조기-내피전구세포 및 췌도세포를 포함하는 세포이식물의 제조방법
WO2015020267A1 (fr) * 2013-08-05 2015-02-12 사회복지법인 삼성생명공익재단 Procédé pour préparer un implant de cellule comprenant des cellules progénitrices endothéliales précoces et des cellules des îlots pancréatiques

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CA2235509A1 (fr) 1997-05-01
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