WO2008063934A2 - Procédés pour différencier les cellules bêta produisant de l'insuline à partir de cellules précurseurs pancréatiques - Google Patents

Procédés pour différencier les cellules bêta produisant de l'insuline à partir de cellules précurseurs pancréatiques Download PDF

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WO2008063934A2
WO2008063934A2 PCT/US2007/084327 US2007084327W WO2008063934A2 WO 2008063934 A2 WO2008063934 A2 WO 2008063934A2 US 2007084327 W US2007084327 W US 2007084327W WO 2008063934 A2 WO2008063934 A2 WO 2008063934A2
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cell
cells
insulin
pancreatic
anlagen
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WO2008063934A3 (fr
WO2008063934A9 (fr
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Kevan Herold
Salma Begum
Wei Chen
Virginia Papaiouannou
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The Trustees Of Columbia University In The City Of New York
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Type 1 diabetes TlDM.
  • Clinical studies have shown that anti-CD3 monoclonal antibody (rnAb) may be a useful agent to arrest diabetes at onset (2-4).
  • Other approaches for treatment and prevention of the disease are now in clinical trials sponsored by the NIH together with the Juvenile Diabetes Research Foundation (JDRF) and the American Diabetes Association (ADA). Despite this progress it is unclear whether these interventions alone will restore normal metabolic control or that the effects of these interventions will be long enduring.
  • JDRF Juvenile Diabetes Research Foundation
  • ADA American Diabetes Association
  • ⁇ cell mass is dynamic - it increases and decreases under normal and pathologic conditions.
  • Current understanding is that there is a continuous turnover of ⁇ cells during the life of a healthy individual.
  • insulin producing cells and perhaps their precursors are destroyed by the autoimmune process and regeneration fails because of the ongoing cell destruction. It follows, therefore, that if cell destruction were stopped and tolerance to autoimmunity were achieved, recovery of insulin producing cells could occur, in part by regeneration of islet tissue from endogenous stem cells.
  • the invention provides a method for identifying a factor capable of differentiating a non-insulin-producing precursor cell into a cell that produces insulin, the method comprising (a) separately preparing one or more cell membrane fractions from (i) an insulin- producing pancreatic beta cell and (ii) a non-insulin producing pancreatic beta cell; (b) identifying one or more factors contained in the membrane fraction of (i) which are not contained in the membrane fraction of (ii); (c) contacting a non-insulin-producing precursor cell with the factor identified in (b); and (d) determining whether production of insulin by the precursor cell in (c) is increased compared to a non-insulin-producing precursor cell in the absence of the factor, wherein determination of an increase in production indicates that the factor is capable of differentiating a non-insulin producing precursor cell into a cell that produces insulin.
  • the factor naturally occurs on an extracellular surface of the insulin-producing pancreatic beta cell.
  • the factor comprises a polypeptide.
  • the polypeptide comprises a hormone, a growth factor, a chemokine or a cytokine.
  • the factor is TGF-beta, interferon- gamma, hepatocyte growth factor, glucagon like peptide- 1, or any combination thereof.
  • the methods of the invention further comprise the step of isolating the factor identified in (b) from a fraction prepared in (a).
  • the method further comprises selecting insulin-producing cells which are responsive to glucose.
  • the selecting comprises conducting a glucose stimulation test.
  • the determining in (d) comprises flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), microscopy, or any combination thereof.
  • the determining in (d) comprises detecting BrdU, proinsulin, insulin, Pdx-1, or any combination thereof.
  • the precursor cell is a pancreatic anlagen precursor cell. In another embodiment, the precursor cell is an adult stem cell. In another embodiment, the precursor cell is an embryonic stem cell. In another embodiment, the precursor cell is a pancreatic stem cell.
  • the insulin-producing pancreatic beta cell of (a) is a beta TC3 cell cultured in the presence of serum, and wherein the non-insulin producing pancreatic beta cell of (a) is a beta TC3 cell cultured in the absence of serum.
  • the identifying in (b) comprises two-dimensional difference gel electrophoresis (DIGE), an iTRAQ gel, column chromatography, or any combination thereof.
  • the invention provides a method for differentiating a non-insulin-producing precursor cell into a cell that produces insulin, the method comprising contacting a non- insulin-producing precursor cell with a mature pancreatic islet cell, wherein the contacting comprises co-culture of the precursor cell and the islet cell in a cell culture.
  • the invention provides a method for differentiating a non-insulin-producing precursor cell into a cell that produces insulin, the method comprising contacting a non- insulin-producing precursor cell with a compound identified by a method of the invention.
  • the islet cell is contained within a whole pancreatic islet. In another embodiment, the islet cell is a beta cell. In one embodiment, the precursor cell is an adult stem cell. In another embodiment, the precursor cell is an embryonic stem cell. In another embodiment, the precursor cell is contained within pancreatic anlagen tissue. In another embodiment, the pancreatic anlagen tissue is MHC differentiated. In one embodiment, the cell that produces insulin is responsive to glucose. In one embodiment, the precursor cell comprises a detectable marker. In another embodiment, the detectable marker comprises a fluorescent protein. In another embodiment, the fluorescent protein is green fluorescent protein. In one embodiment, the precursor cell expresses CXCR4.
  • the invention provides a cell that produces insulin obtained by a method of the invention.
  • the cell is responsive to glucose.
  • the invention provides a method for treating diabetes in a subject, the method comprising administering an effective amount of a cell that produces insulin obtained by a method provided by the invention.
  • the administering comprises transplant of the insulin-producing cell into the subject.
  • Figures IA - IB C-peptide responses to the MMTT in an anti-CD3 mAb treated patient (A) and control subject (B) at study entry (diamonds), 6 mos (squares), and 12 months (triangles).
  • Figure 2 An increase in ⁇ cell proliferation precedes elevated glucose levels in NOD mice.
  • OGTT oral glucose tolerance test
  • Figure 3 ⁇ cell replication increases in NOD/scid recipients of diabetogenic spleen cells.
  • NOD/scid mice received 10 7 splenocytes from diabetic NOD mice.
  • ⁇ cell mass bars
  • proliferation closed symbols
  • the random glucose levels open symbols
  • Figure 4 Decreased ⁇ cell replication and increased ⁇ cell mass after treatment with anti-CD3 mAb.
  • Diabetic NOD mice were treated with anti-CD3 mAb (10 ⁇ g/d x 5) and ⁇ cell replication and mass were measured at diagnosis and 3 and 6 weeks after treatment with the mAb.
  • Islets from ICR mice were placed in culture for 6 days with media alone (A) or with Exendin-4 5 ⁇ g/ml (B). The islets were then dispersed into single cells and stained with SYTO- 13 (Molecular Probes)(X axis) and Ethidium Bromide (El 510, Sigma)(Y axis), that have previously been used for studies of apoptotic islet cells (1). There was a decrease in the percentage of apoptotic cells (R4)(SYTO-13- and EB+) from 6% to 3% of islet cells.
  • FIGS 7A - 7G Production of a novel fluorescent marker of chromatin that can be visualized in living or fixed tissue.
  • A The histone octomer core of nucleosome is marked with a fusion protein between human histone H2B and EGFP or EYFP.
  • B Transfected ES cells with stable integration of the transgene providing fluorescence in all nuclei.
  • C Mitotic and interphase nuclei at higher magnification.
  • D Mitotic (lower arrows) and pycnotic (upper arrow) nuclei can be distinguished.
  • E &
  • F Following germ line transmission of the transgene, the fusion protein marker can be visualized in either fixed or living embryos.
  • G Transgenic mice are viable and fertile, and the transgene is stable and ubiquitously expressed.
  • Figures 8A - 8 J Development of grafts of fetal pancreas transplanted under the kidney capsule.
  • A-E Grafted E12.5 pancreas and
  • F-J E15.5 pancreas recovered after 15 days, sectioned and stained for H&E, insulin, glucagon or amylase.
  • Figures 9A - 9D Mature islets induce proinsulin in pancreatic anlagen.
  • El 4.5 anlagen from GFP+ mice were cultured with (B) or without (A) or with 50 islets from adult mice or with insulin alone (C) for 6 days. After 4 days BrdU was added to the cultures and the cells were stained with anti-BrdU (Y axis) and anti-proinsulin (X axis) antibodies after 6 days. Staining of mature islets is shown in D. The numbers refer to the percentage of gated cells in the quadrant.
  • Figures 1OA - 1OB Islets enhance insulin expression when cultured with single anlagen cells.
  • Single anlagen cells (2 x 10 5 ) were dispersed from El 4.5 anlagen and cultured alone (A) or with 20 islets in microwells (B). After 4 days the cultures were pulsed with BrdU (Y axis) and stained with anti-proinsulin (X axis) after 6 days.
  • the numbers refer to the percentage of gated cells in each quadrant.
  • Figures HA - HH In situ hybridization for NeuroD and Pax4 expression in
  • Figure 12 Schematic depicting examples of experimental design which can be used in embodiments of the invention.
  • Figures 13A - 13E Adult islets enhance differentiation of E14.5 mouse pancreatic anlagen cells into proinsulin-producing beta cells.
  • Figure 14 Islets induce maturation of single pancreatic anlagen cells.
  • Figures 15A - 15D Fixed adult islets increase differentiation of E14.5 single pancreatic anlagen cells into proinsulin-producing beta cells.
  • Figures 16A - 16C Confocal analyses of insulin expression in pancreatic anlagen cell cultures. Cells were cultured in CC2 glass chamber slides for five days.
  • Figure 18 Confocal analyses of Pdx-1 expression in pancreatic anlagen cell cultures.
  • FIG 19 Insulin secretion of anlagen cell culture in response to glucose stimulations. 200 microliters of Krebs Ringer buffer containing the indicated concentrations of glucose was added to the cultures for 1 hour incubation. The supernatants were collected and insulin was measured by ELISA (Alpco).
  • Figures 2OA - 20B Islets without direct contact to pancreatic anlagen (A) have no effect on pancreatic anlagen cells in terms of beta cell differentiation and proliferation, compared to islets with direct contact to pancreatic anlagen (B). Cells were cultured for 7 days. BrdU pulse T: 40 h.
  • Figures 21A - 21E Effects of pancreatic anlagen on insulin production and proliferation of islet beta cells in vitro. Islets were co-cultivated with pancreatic anlagen with or without direct contact showed increased insulin production and higher beta cell proliferation as compared islet alone after 7 day in vitro culture. BudU pulse T: 40h.
  • Figures 22A - 22B Correction of diabetes in recipients of anlagen cells co- cultured with ⁇ TC3 cells.
  • Figures 23 A - 23B Induction of proinsulin in human pancreatic anlagen by co-culture with mature islets.
  • the invention provides a mechanism whereby differentiation of islet precursor cells can be directed towards mature insulin producing cells.
  • the invention also provides the use of pancreatic anlagen as a source of islet stem cells to correct diabetes. Together, these observations will allow the development of new approaches for clinical translation in which insulin-producing ⁇ cells may be differentiated from stem cells.
  • pancreatic ⁇ cells are formed by self duplication rather than differentiation from an initially insulin free stem cell(39). This study, however, evaluated ⁇ cells under physiologic conditions and not under circumstances such as islet inflammation or insulin resistance. Studies of pancreatectomized rats suggested that mature duct cells can dedifferentiate and serve as a multipotent progenitor cell. External stimuli can then direct the differentiation of these multipotent cells to endocrine, acinar, or mature duct phenotypes; these signals may be derived from blood vessels (40).
  • pancreatic anlagen The embryonic or fetal pancreas has a higher proportion of stem cells than the adult organ, and thus a greater potential for regeneration.
  • Cells that can differentiate into islet cells lie within the pancreatic anlagen during fetal development.
  • Intraperitoneal transplantation of pancreatic anlagen can reverse hyperglycemia induced in rats and mice with high doses of streptozotocin (49).
  • Selective differentiation of islet compared with acinar tissue over a 15-week period raised the possibility that the development of islet tissue may have been stimulated by the hyperglycemia in the rats.
  • Transplantation of xenogeneic pancreatic anlagen was also successful in reversing diabetes (rat into mouse) in the presence of costimulatory blockade.
  • the pancreatic anlagen represents a source of islet stem cells with the potential capability to restore normal ⁇ cell mass.
  • the invention provides methods to identify the factors that regulate differentiation of the precursor cells. There is very little known about these factors, but soluble factors released from islet cells have been shown to affect differentiation of embryonic stem cells — factors released by the developing pancreas stimulated endocrine pancreatic differentiation from embryonic stem cells (50). In the differentiated embryonic stem cells, the following have been observed: changes in the mRNA levels of insulin and PDX-I; co-expression of insulin, C-peptide and glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal.
  • Pancreatic anlagen as a source of islet precursor cells
  • Mature islets affect differentiation of developing pancreatic cells and stimulate differentiation of pancreatic anlagen into mature insulin producing cells.
  • the invention provides methods for identifying the signals that control differentiation of pancreatic anlagen cells into mature islets.
  • the methods of the invention also provide for the use of stem cells as a treatment for TlDM.
  • the factor(s) identified by the methods of the invention could be applied in order to differentiate a histocompatible stem cell.
  • pancreatic anlagen as a source of islet precursor cells (51 , 52).
  • the invention provides methods to resolve issues in order to bring this approach forward.
  • the response of transplanted tissue to the diabetic host environment is largely invisible at the cellular and gene expression level, being measured by the success of recovery of ⁇ cell mass and function.
  • the availability of a distinguishing marker of donor tissue that provides a means of monitoring and recovering transplanted tissue will be an invaluable source to gain a better understanding of the dynamics of ⁇ cell regeneration from different stem cell sources.
  • the methods of the invention provide for the use of tissues from mice with subcellularly localized GFP marker that will allow us to study pancreatic cell differentiation in living cells (see Example 6).
  • pancreatic anlagen itself could potentially be used as a source of cells for beta cell replacement therapies, other sources of adult or embryonic stem cells may also be used in the methods of the invention.
  • Pancreatic anlagen can be used in the methods of the invention to delineate factors that control differentiation.
  • the identified factors represent candidate molecules that may be applied to human stem cells in a clinical setting to enhance differentiation of precursor cells to insulin- producing islet cells.
  • the insulin-producing cells may then be tested in cell replacement therapies for their ability to prevent, reverse or treat diabetes in patients.
  • pancreatic buds are specified between the stomach and duodenum. These epithelial buds, which are marked by the expession of Pdxl, are the progenitors of all the endocrine and exocrine cells of the pancreas, which arise following branching and growth of the primordia into the surrounding mesenchyme (53-55).
  • the endocrine cells which make up less than 5% of the adult pancreas, are thought to arise by delamination from the epithelial primordia, aggregation into islets, followed by differentiation into ⁇ cells, as well as other endocrine cell types.
  • pancreatic structure involves an interaction between the epithelium and mesenchyme.
  • pancreatic anlagen epithelium is programmed to form ducts (52).
  • Mesenchyme is required for the formation of acinar structures and mesenchyme has proexocrine effects on differentiation of the epithelium.
  • NIH3 T3 cells have been shown to substitute for mesenchyme in the pro-exocrine effects (51).
  • the gene expression profile of cells at different stages of pancreatic development combined with transgenic studies have defined critical molecules in pancreas development and islet regeneration.
  • Pdxl is essential for early budding of the pancreatic primordial, FGF signaling as well as expression of transcription factors IsIl and Pbxl are required in the mesenchyme for growth promotion, and Neurogenin3 (Ngn3) appears to act as a genetic switch that specifies endocrine cell fate among pancreatic precursors, leading to the expression of NeuroD, Pax ⁇ and IsIl (56). Subsequently, in a fully differentiated and functioning pancreas, Pdxl again appears in the nucleus and is restricted to insulin producing cells, thus providing a useful marker for insulin producing cells (57, 58).
  • the invention provides a method for screening for factors capables of differentiating a non-insulin-producing precursor cell into a cell that produces insulin, the method comprising (a) separately preparing one or more cell membrane fractions from (i) an insulin-producing pancreatic beta cell and (ii) a non-insulin producing pancreatic beta cell; (b) identifying one or more factors contained in the membrane fraction of (i) which are not contained in the membrane fraction of (ii); (c) contacting a non-insulin-producing precursor cell with the factor identified in (b); and (d) determining whether production of insulin by the precursor cell in (c) is increased compared to a non-insulin-producing precursor cell in the absence of the factor, wherein determination of an increase in production indicates that the factor is capable of differentiating a non-insulin producing precursor cell into a cell that produces insulin.
  • the method may further comprise the step of isolating the factor identified in (b) from a fraction prepared in (a).
  • the method may also comprise selecting insulin- producing cells which are responsive to glucose, for example, by conducting a glucose stimulation test.
  • the determining in step (d) can be carried out using one or more techniques known to one of ordinary skill in the art, for example, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), or microscopy. Determining in (d) may also be carried out by detecting BrdU, proinsulin, insulin, or Pdx-1. Examples of techniques that can be used to carry out the identifying in step (b) include two- dimensional difference gel electrophoresis (DIGE), an iTRAQ gel, or column chromatography. In the iTRAQ gel approach, the factor can be identified by its ability to stimulate differentiation of the precursor cells into insulin-producing cells in culture.
  • DIGE two- dimensional difference gel electrophoresis
  • iTRAQ gel the factor can be identified
  • a factor identified using the methods of the invention may naturally occur on the extracellular surface of the insulin-producing pancreatic beta cell.
  • the factor may comprise a polypeptide, a nucleic acid, or a small molecule.
  • factors include hormones, growth factors, chemokines or cytokines. Additional examples of factors include, but are not limited to, TGF-beta, interferon-gamma, hepatocyte growth factor, and glucagon like peptide- 1.
  • Non-limiting examples of precursor cells that can be used in the methods of the invention include pancreatic anlagen precursor cells, pancreatic stem cells, adult stem cells, or embryonic stem cells.
  • An example of an insulin-producing pancreatic beta cell is a beta TC3 cell ( ⁇ TC3) cultured in the presence of serum.
  • An example of a non-insulin producing pancreatic beta cell is a beta TC3 cell cultured in the absence of serum.
  • the invention provides a method for differentiating a non-insulin-producing precursor cell into a cell that produces insulin.
  • the method comprises contacting a non- insulin-producing precursor cell with a mature pancreatic islet cell, wherein the contacting comprises co-culture of the precursor cell and the islet cell in a cell culture for a minimum of about 6 days.
  • the invention also provides a method for differentiating a non-insulin-producing precursor cell into a cell that produces insulin.
  • the method comprises contacting a non- insulin-producing precursor cell with a factor identified by a factor screening method of the invention.
  • the method can be carried out, for example, by adding about 50 islets to about 100,000 - 200,000 precursor cells and co-culturing the cells for a minimum of about 6 days.
  • Islet cells used in the methods of the invention may be contained within a whole pancreatic islet; the islet cell can be a beta cell.
  • Precursor cells that can be used in the invention include adult stem cells and embryonic stem cells.
  • the precursor cell may be contained within pancreatic anlagen tissue; the pancreatic anlagen tissue may be MHC differentiated.
  • the precursor cell may also comprise a detectable marker, for example a fluorescent protein such as green fluorescent protein.
  • the precursor cell may express CXCR4.
  • the invention provides a cell that produces insulin obtained by a method of the invention.
  • the cell may be responsive to glucose.
  • the invention provides a method for treating diabetes in a subject, the method comprising administering an effective amount of a cell that produces insulin obtained by a method provided by the invention.
  • the administering comprises transplant of the insulin-producing cell into the subject.
  • the methods of the invention provide a means for understanding the differentiation of islet stem cells from pancreatic anlagen.
  • the anlagen contains all of the stem cells involved in development of mature pancreatic cells.
  • the invention provides methods to study the development of mature islet cells in vivo following immune modulation. The methods can also be used to obtain new information about the acquisition of immune stimulatory properties during development of ⁇ cells.
  • the invention provides that differentiation of these precursor cells can be directed by factors in mature islet cells. These signals may be important for differentiation and identifying them is an important translational goal for implementing the use of embryonic stem cells for ⁇ cell replacement.
  • EXAMPLE 1 BETA CELL RESPONSE IMPROVES WHEN AUTOIMMUNE DIABETES IS HALED BY ANTI-CD3 mAb THERAPY
  • Residual ⁇ cell mass is a key determinant of metabolic control in patients with
  • TlDM TlDM.
  • This notion is supported by data from the Diabetes Control and Complications Trial and other observational and prospective studies in which metabolic control was found to be easier with residual insulin production, a result of increased ⁇ cell mass (5-7).
  • the clear differences in success at normal metabolic control in patients with Type 1 and Type 2 diabetes may be due to the residual insulin production in the latter group.
  • residual ⁇ cell function may be important in other metabolic responses such as counterregulatory responses to hypoglycemia ⁇ ).
  • a clinical goal, therefore, of interventional studies in TlDM is to maintain or even improve ⁇ cell mass in patients. Because TlDM is an autoimmune disease, a means to induce immunologic tolerance is necessary in order to prevent the continuous destruction of ⁇ cells.
  • EGF can increase ⁇ cell proliferation and mass (16-20).
  • ⁇ cell proliferation occurs most commonly in response to insulin resistance and hyperinsulinemia. This may be due to the interdependence between insulin signaling and ⁇ cell proliferation (21).
  • streptozotocin treatment of mice a small population of insulin containing cells reappeared in the islets that could be greatly enhanced by administration of insulin and restoration of normoglycemia (22).
  • Mice lacking insulin receptor substrate-2 (irs-2) develop ⁇ cell failure due to decreased proliferation/increased apoptosis of ⁇ cells (23-25).
  • PI3K phosphatidylinositol 3-kinase
  • Forkhead transcription factors of the FoxO family have been shown to play a critical role in cell proliferation and cell survival.
  • Haploinsufficiency of FoxO 1 reverses ⁇ cell failure in Irs2-/- mice through partial restoration of ⁇ cell proliferation by increased expression of the transcription factor Pdxl, which has suggested that FoxOl is a key distal mediator of Insr signaling (26).
  • ⁇ cell proliferation requires translocation of FoxOl from the nucleus to the cytoplasm.
  • Transgenic expression of a constitutively nuclear FoxOl in liver and ⁇ cells causes diabetes because of increased expression of glucogenic enzymes, and this same transgene prevents ⁇ cell proliferation in response to insulin resistance (27).
  • GLP-I receptor agonists may also stimulate ⁇ cell replication and/or differentiation.
  • One study reports the development of hypoglycemia with hyperinsulinemia and nesidioblastosis in a patient after gastric bypass procedure (28). Similar findings in other patients have been reported (29).
  • the mechanism of increased ⁇ cell mass by GLP-I receptor agonists has been studied by several investigators. Decreased apoptosis was found in the islets of mice administered streptozotocin and treated with GLP-I receptor agonist (31).
  • the GLP-I receptor agonist Exendin-4 enhanced pancreas regeneration following partial pancreatectomy in rats and treatment with Exendin-4 increased the rates of diabetes reversal following treatment of diabetic NOD mice with anti-lymphocyte serum (32, 33).
  • Inflammation is also a stimulus of ⁇ cell proliferation
  • mice with mutations of receptors for these cytokines that will receive adoptive transfer of wild type diabetogenic spleen cells may be studied using mice with mutations of receptors for these cytokines that will receive adoptive transfer of wild type diabetogenic spleen cells.
  • immune modulatory agents there is not a continuous increase in ⁇ cell mass, and permanent remission of TlDM is not seen.
  • ⁇ cell proliferation occurs as part of the inflammatory process, and there are mechanisms of ⁇ cell replication that are activated under physiologic and pathologic conditions. Studies can be designed to identify the basis for the induction of ⁇ cell proliferation that occurs during insulitis so that new approaches can be developed to induce proliferation without destruction of ⁇ cells.
  • Beta cell proliferation increases during development of diabetes in NOD mice
  • Beta cell mass and ⁇ cell replication rates were determined by analysis of histologic sections of pancreata from non-obese diabetic (NOD) mice beginning at 6 weeks of age, and compared to age matched NOD/scid mice, ⁇ cell mass and replication were determined by modifications of techniques involving area of insulin+ cells in 3 - 5 sections of pancreas, and staining for insulin and Ki67 (27, 64) The changes in these measurements were correlated with random and stimulated (1 mg/gm body weight) glucose levels.
  • NOD non-obese diabetic
  • Inflammatory infiltrate in the islets enhances beta cell proliferation
  • CD4+CD25+ regulatory T cells that were isolated from NOD mice successfully treated with anti-CD3 mAb were mixed with diabetogenic splenocytes at the time of adoptive transfer into NOD/scid recipients and compared ⁇ cell proliferation in these recipients to those receiving CD4+CD25- cells from the same donors.
  • CD4+CD25+ Tregs were mixed with diabetogenic splenocytes, transfer of diabetes was prevented whereas when CD4+CD25- cells were mixed, all recipients developed diabetes.
  • ⁇ cell mass was preserved in the non-diabetic mice, ⁇ cell proliferation was significantly reduced.
  • CD4+CD25- or CD4+CD25+ cells were harvested from NOD mice successfully treated with anti-CD3 mAb and mixed (2 x 10 6 cells) with diabetogenic splenocytes and transferred to NOD/scid recipients. The rates of diabetes were determined after 8 weeks or when the incidence of diabetes stabilized in the control mice, ⁇ cell mass and replication rates were determined by immunohistochemistry as noted above. *p ⁇ 0.05, ** ⁇ 0.02 CD4+CD25- vs CD4+CD25+ recipients
  • EXAMPLE 4 IDENTIFICATION OF CELLS AND MEDIATORS RESPONSIBLE FOR PROLIFERATION OF BETA CELLS DURING THE INFLAMMATORY RESPONSE
  • the proportion of ⁇ cells that are newly formed during insulitis can be determined and the ability of subsets and clonal populations of lymphocytes as well as their products to induce islet proliferation can be tested.
  • Using a molecular approach one can specifically address the role of IFN- ⁇ and TGF- ⁇ , two cytokines previously shown to induce ⁇ cell proliferation.
  • NOD mice The objective of these studies is to identify the cells and mediators of ⁇ cell proliferation in order to develop new approaches to stimulate the expansion of ⁇ cells in the absence of ⁇ cell death. To understand the basis for this effect, the contribution of the proliferating ⁇ cells to the total ⁇ cell mass can be assessed. One can then determine whether T cells, and which T cells are responsible for ⁇ cell proliferation by adoptive transfer of purified T cells, T cell subsets, and even T cell clones. One can then take a molecular approach to address the likely ligands and molecules that may be involved in this replicative process. The following pathways can be studied: First, studies have shown that constitutive nuclear expression of FoxOl impairs ⁇ cell proliferation in response to insulin.
  • Translocation of FoxOl from the nucleus involves activation of the PD kinase pathway and is the mechanism thought to account for the expansion of ⁇ cell mass in settings of insulin resistance.
  • IFN- ⁇ The role of IFN- ⁇ can be tested by studying the proliferation of ⁇ cells in NOD/scid mice with a mutated form of the IFN- ⁇ receptor, following adoptive transfer of diabetogenic spleen cells. Third, recent studies have suggested that TGF- ⁇ may stimulate ⁇ cell replication (36). To study the role of TGF- ⁇ , NOD/scid mice with a conditional ⁇ cell knock-out of the TGF- ⁇ RIl can be used as recipients of adoptively transferred diabetogenic splenocytes (78).
  • a limitation of the studies of ⁇ cell proliferation in all models of insulitis is that there are a number of simultaneous changes ongoing that may affect calculations of ⁇ cell turnover and make it difficult to know whether or not the ⁇ cell proliferation that is observed actually accounts for changes in ⁇ cell mass. For example, the percent of proliferating insulin+ ⁇ cells is increased during development of diabetes in the NOD mouse but the overall mass is decreasing with time. Because the mass is affected by both proliferation and death, it is difficult to understand the significance of the proliferation measurements since the total number of ⁇ cells is changing. Some studies have suggested that sustained ⁇ cell apoptosis in TlDM implies that there is ongoing regeneration (79).
  • the extended use of BrdU to label ⁇ cells has been used for labeling of cells for as long as 16 weeks (80).
  • Example 3 The data shown in Example 3 indicates that the ⁇ cell proliferation rate is high in prediabetic mice of this age. After 2 weeks, the mice can be sacrificed and the proportion of total ⁇ cells that are BrdU labeled can be determined by immunohistochemical staining. This can be compared with the same proportion measured in NOD/scid mice that receive BrdU over the same period of time. If the ⁇ cell proliferation accounts for the recovery of ⁇ cell mass in mice treated with anti-CD3 mAb and in prediabetic NOD mice, the proportion of labeled cells may be higher in the anti-CD3 mAb treated or the prediabetic mice than in NOD/scid controls.
  • T cells and/or T cell products are responsible for ⁇ cell proliferation.
  • Islet cells can be isolated from GFP+ mice (see Example 7) and cultured in absence or presence of T cells that have been activated with PHA. Proliferation of ⁇ cells can be detected by flow cytometry after 6 day culture by pulsing with BrdU and measuring incorporation by intracellular staining of the GFP+ cells on the dispersed (by trypsin) cells as shown in Example 7. In order to have sufficient cells for analysis in each assay, approximately 200 islets can be used. One can also test whether supernatants from activated T cells contain molecules that stimulate ⁇ cell replication.
  • Splenocytes from diabetic NOD mice can be activated with anti-CD3 mAb 145-2C11 (1 ⁇ g/ml for 48 hrs), and the supernatant from the cultures can be added to cultures of islets from NOD/scid mice.
  • One can detect proliferation of ⁇ cells by BrdU/proinsulin co-staining after 6 days in culture.
  • Identifying cells responsible for ⁇ cell replication One can determine whether
  • T cells, CD4 and/or CD8 T cells, or their products are responsible for ⁇ cell proliferation by adoptively transferring purified populations of T cells into NOD/scid recipients.
  • splenocytes or subpopulations of splenocytes purified with magnetic beads can be infused into NOD/scid recipients.
  • the recipient mice can be followed for development of diabetes, and ⁇ cell proliferation and cell death can be studied. Briefly, pancreases is resected, cleared of fat and lymph nodes, weighed and oriented in a cassette so that the entire width and length can be viewed in a saggital section and fixed.
  • pancreatic sections are immunostained for insulin using a mouse anti-insulin primary antibody (Sigma, St. Louis, MO) followed by anti-GP antibody conjugated to Vector Red (Vectastain ABC kit, Vector, Burlingame, CA).
  • Vector Red Vector Red
  • Nuclear staining of Ki67 is done with biotinylated antibody (Novocastra, Newcastle upon Tyne, UK) and developed with 3,3 " diamino benzidine (DAB) (Sigma, St. Louis, MO).
  • Ki67+ T cells can be separated from proliferating ⁇ cells by co-staining with insulin; to further exclude that the Ki67+ cells are T cells sections will be stained with anti-CD3 mAb.
  • Sections are counterstained with methylene green.
  • the number of Ki67+/insulin+ cells is determined for each section.
  • the area of ⁇ cells and total pancreatic area are calculated using image analysis software (Spot software v.3.3, Diagnostic Instruments, Inc., Sterling Hts, MI).
  • Total ⁇ cell mass per pancreas is calculated by multiplying the volumetric ratio occupied by ⁇ cells and total pancreatic cells by the total pancreatic weight in grams.
  • a neogenesis index is determined using a modification of a previously described protocol (81).
  • the number of Ki67+/insulin+ cells/ ⁇ cell and non- ⁇ cell area is calculated. For each pancreatic sample, an average of -100 hpfs are sampled or 8 - 10 sections from each pancreas.
  • Group sizes of 8-10 animals can be analyzed in this manner.
  • epithelial markers such as mouse specific cytokeratin isoforms 7, 19 and 20 and carbonic anhydrase II together with Ki67.
  • epithelial markers such as mouse specific cytokeratin isoforms 7, 19 and 20 and carbonic anhydrase II together with Ki67.
  • cells can be stained with a cocktail of non- ⁇ cell antibodies (anti-glucagon, anti- somatostatin, and anti-pancreatic polypeptide, Sigma, St. Louis MO) (81)
  • TUNEL assays can be performed using the ApopTag in situ apoptosis detection kit (Intergen, NY). Apoptotic ⁇ cells can be identified with TUNEL+ brown nuclei and apoptosis-specific morphological criteria (e.g. nuclear condensation, apoptosis bodies)(82). The apoptotic index (AI) will be calculated as % apoptosis-positive ⁇ cells/ ⁇ cell area, which can be identified by co-staining with insulin.
  • ApopTag in situ apoptosis detection kit Intergen, NY.
  • Apoptotic ⁇ cells can be identified with TUNEL+ brown nuclei and apoptosis-specific morphological criteria (e.g. nuclear condensation, apoptosis bodies)(82).
  • the apoptotic index (AI) will be calculated as % apoptosis-positive ⁇ cells/ ⁇ cell area, which can be identified by co-staining with insulin.
  • CD8+ T cells to induce ⁇ cell proliferation and transfer diabetes into NOD/scid recipients.
  • These cells can be purified from splenocytes from diabetic NOD by magnetic bead separation. The purity of the cells can be confirmed by flow cytometry prior to transfer. Similarly, one can determine whether clonal populations of diabetogenic T cells induce proliferation by transferring either BDC2.5 or A14 transgenic T cells into NOD/scid recipients. (NOD transgenic mice expressing these transgenes are available and have been used them in studies of NOD mice (83).) In previous studies with these cells, diabetes develops about 2 weeks after transfer of these cells into NOD/scid mice.
  • the adoptive transfer model can be used in which diabetogenic splenocytes are transferred into genetically modified NOD/scid mice in order to determine the mechanisms involved in ⁇ cell proliferation. Specifically, one can determine whether the pathway involved is the P 13 kinase pathway or a previously unrecognized pathway, and whether candidate mediators IFN- ⁇ and/or TGF- ⁇ are involved. In each case, the genetically modified mice can be backcrossed to NOD/scid mice for 3 - 5 generations in order to fix the MHC.
  • mice, heterozygous for the scid mutation can then be intercrossed and the homozygous animals can be used for breeding and as recipients in adoptive transfer studies with diabetogenic splenocytes.
  • the homozygous scid mice can easily be identified by the absence of circulating lymphocytes. The following mice can be used for these studies:
  • mice heterozygous for the IFN yRBnull allele and fixed to homozygosity for linkage markers delineating all known Idd loci of NOD origin were intercrossed.
  • these mice develop autoimmune diabetes.
  • Carriers of the disrupted IFN yRBnull allele are identified by polymerase chain reaction (PCR) using the primer set 5- GCTATTCGGCTATGACTGGG-3 (SEQ ID NO:1) and 5-
  • GAAGGCGATAGAAGGCGATG-3 (SEQ ID NO:2), which generates a 706-bp product from within the neor insert.
  • Presence of the intact wild-type IFNyRB allele is typed by PCR with the primer set 5-TCGTTTTCCCAGCTTGCGGCC-3 (SEQ ID NO:3) and 5- CAAGCTATATTCCACCTGGTA-3 (SEQ ID NO:4), which generates a 132-bp product.
  • This latter primer pair does not amplify aPCR product from the IFN yRBnull allele.
  • the IFN yRBnull mice are available from Jackson Laboratory (Strain No. 004352).
  • TGF- ⁇ RH Transgenic loss of TGF- ⁇ RH in islets: One can prepare recipients that lack expression of TGF- ⁇ RII selectively in islet cells.
  • the mice (86) contain a floxed TGF- ⁇ RII. They will be backcrossed to NOD for at least 3 generations and crossed to NOD RIP-cre mice (available from the Jackson Laboratory). Expression of TGF ⁇ RII in various tissues can be identified by PCR and confirm that the lack of expression is limited to islets.
  • the transgenic and non-transgenic backcrossed mice can be used as recipients of splenocytes from diabetic NOD mice, and the mice can be followed for development of diabetes and ⁇ cell proliferation studied as described above.
  • TGF ⁇ RII mice Use of the TGF ⁇ RII mice depends on exclusive expression of the ere transgene in the islets. However, some studies have reported leakiness of expression of RIP- cre in the hypothalamus. If there is evidence of leakiness of the RIP-cre transgene or difficulties in breeding the mice, one can use other RIP-cre mice which have previously been successfully used for knock out of tissue specific expression and in which leakiness of the transgene is not a problem (87) (88).
  • mice in each of these treatment groups were compared and the rates of ⁇ cell replication were similar in mice treated with anti-CD3 mAb (0.82 ⁇ 0.3%) or anti-CD3 mAb+ exendin-4 (0.87 ⁇ 0.47%), but the variability between individual mice was great.
  • a dose related inhibition of ⁇ cell apoptosis is observed when Exendin-4 is added to cultures ( Figure 6).
  • GFP+ pancreatic anlagen can be transplanted into NOD mice rendered tolerant to autoimmune diabetes by treatment with anti-CD3 mAb to study the development of ⁇ cells in this clinically relevant setting and to identify the fate of islet stem cells.
  • An important feature of the proposed transplantation protocol is the ability to positively identify all cellular derivatives of the donor tissue by virtue of a ubiquitous fluorescent transgene (65, 66).
  • the introduction of jelly fish GFP genes into different species has provided genetically stable fluorescent markers that can be detected in living tissues (65, 67-69).
  • a superior, subcellularly localized GFP marker with high sensitivity and high resolution at the cellular level has recently been developed using a histone-GFP fusion ( Figure 7) (66).
  • Histone-GFP fusions have been successfully used in tissue culture cells and in transient transgenics of other species, such as C. elegans and zebrafish, for the sensitive analysis of chromosome dynamics in living cells (65).
  • the fusion protein provides superior resolution compared with a standard nuclear-localization sequence, and all chromatin, including interphase nuclei, mitotic figures, and pyknotic nuclei can be visualized in living cells (Figure 7).
  • mice have great potential as donors in transplanation studies for visualizing dynamic subcellular events in an organismal context while allowing the unequivocal identification and tracking of donor cells (70, 71), and also the possibility of recovering donor cells by fluorescence activated cell sorting (FACS) (72).
  • FACS fluorescence activated cell sorting
  • the H2B-EGFP transgene has been backcrossed onto NOR (non-obese resistant) mice (73, 74).
  • NOR non-obese resistant mice
  • the fluorescent transgene can be put onto the NOD background as a source of tissue for transplantation, but in light of the anormalities associated with NOD pancreas development (see Example 9), a closely related strain can be used that does not have the predisposition to develop diabetes.
  • NOR is a recombinant inbred strain that inherits 88% of the genome from NOD and 12% from the C57BLKs/J strain.
  • the NOR and NOD strains share the diabetogenic major histocompatibility (MHC) haplotype H- 2g7 (Iddl) but differ at 5 of the remaining 18 Idd loci (74).
  • MHC diabetogenic major histocompatibility
  • the H2B-EGFP transgene is transferred by a cross to NOR mice.
  • Fl mice carrying the transgene were identified by fluorescence of an ear punch under UV light and were backcrossed to NOR mice.
  • the offspring were genotyped for the transgene and screened with serotyping of peripheral blood monocytes (PBMC) by flow cytometry.
  • PBMC peripheral blood monocytes
  • the PBMC of each progeny were stained with anti- mouse MHC I (H-2K d , PE-conjugated, BDbioscences) and MHC II (I-A k , APC-conjugated, BDbioscences) antibodies.
  • MHC I H-2K d
  • MHC II I-A k
  • ICR mice to develop an efficient transplantation protocol for embryonic pancreatic anlagen.
  • anatomical differences in mouse compared with rat may create difficulties stabilizing transplanted tissue on the peritoneum as mice lack peritoneal folds that are present in the rat.
  • transplantation under the kidney capsule a standard transplantation site that has been used in many other studies (reviewed in (75)).
  • Using the renal transplantation site one can examine the growth and differentiation of pancreatic anlagen from embryos between embryonic day 12.5 (E12.5) and E15.5 in non-diabetic ICR mice.
  • pancreas were dissected from random-bred ICR embryos at E 12.5 to El 5.5 and transplanted between 3 and 7 whole fetal pancreas under the renal capsule of adult ICR mice. After 15-21 days, the grafts were recovered and analyzed by histology and immunohistochemistry. In the grafts of E 15.5 pancreas, both exocrine and endocrine tissue were found ( Figures 8H and 8J). Clusters of insulin producing ⁇ cells were observed ( Figure 8H), but there was a lack of glucagon- expressing peripheral ⁇ cells ( Figure 81), indicating that these islets are not mature.
  • the pancreatic anlagen can differentiate into islet cells and correct chemically- induced diabetes in a surprisingly short time, possibly through selective differentiation of the islet compared with the acinar compartment (see review in (49, 75).
  • donor tissue carrying a novel, nuclear-localized fluorescent transgene that marks all chromatin By using donor tissue carrying a novel, nuclear-localized fluorescent transgene that marks all chromatin, one can study proliferation dynamics in the transplanted tissue and recover transplanted tissue to determine gene expression profiles. Two previous studies have shown that there may be redevelopment of endogenous islets following induction of tolerance and reversal of diabetes with islet transplants, although the source of the new islet cells is not clear (46, 89). If evidence of ⁇ cell development is observed in the host, one can determine their donor or host origin using the fluorescent marker. If the cellular replacement therapy fails, one can determine the stage of developmental arrest, as well as the effects of hyperglycemia on this process by following the fluorescently-tagged donor cells.
  • H2B-EGFP histone 2B-enhanced green fluorescent protein
  • Example 4 beginning approximately 1 week after the hyperglycemia is discovered. At this time, hyperglycemia is persistent and there is little chance of a return to normal glucose level following treatment with anti-CD3 mAb. After 5 consecutive days of treatment with anti- CD3 mAb, the mice will receive a transplant of either adult islets or pancreatic anlagen, isolated from transgenic mouse embryos at two different embryonic stages. Pancreatic anlagen can be manually dissected from E12.5 and E15.5 embryos and maintained briefly on ice in 50:50 Dulbecco' Modified Eagles Medium:Ham's Fl 2 containing 25 nM prostaglandin El and 5 :g/ml iron saturated transferrin.
  • mice that receive transplants blood glucose levels can be measured 3x weekly and compared to pre- transplant levels and to mice that are treated with exogenous insulin to maintain glucose control in near normal range. The glucose levels should be corrected between 10-30 days after transplantation (49). To determine the effects of the transplant on insulin production, the mice can be given a glucose tolerance test, prior to, 3 weeks after, and 5 weeks after transplantation, and the insulin response will be measured. In addition, to determine whether there has also been expansion of alpha cell mass, the glucagon response to insulin-induced hypoglycemia can be measured. After obtaining a blood sample for glucose and glucagon, 2 U/kg of insulin is administered and another sample is obtained after 15 min (90).
  • glucose tolerance tests can be performed to stimulate insulin production and measure serum insulin levels. After drawing baseline serum samples, the mice will be given a glucose load ip (1.0 mg/gm) and a serum sample will be collected after 15, 30, 60, and 120 minutes for measurement of glucose and insulin. Insulin can be measured by ELISA for mouse insulin (APCO, NH). The lower limit of detection of this assay is approximately 0.1 mg/ml, but the sensitivity can be improved by reducing the dilution of serum in the assay buffer (1 :5).
  • Total ⁇ cell mass can be determined using techniques outlined in Example 4.
  • the entire transplanted tissue can be harvested and adhering tissue removed.
  • the tissue can be weighed and stained for insulin.
  • the glucose and insulin levels can be determined in mice after the removal of the transplanted tissue.
  • This calculation of total ⁇ cell mass does not take into account the possibility that cells may have migrated from the pancreatic anlagen to other tissues or that there may have been regeneration of pancreatic islets. This will be indicated by the failure of hyperglycemia to return following removal of the transplanted tissue.
  • the cells from the anlagen are genetically tagged with a fluorescent marker, they can be easily identified in dissected organs and in sections. If migration of transplanted cells to other organs is involved, ⁇ cell mass will be estimated by a combination of fluorescence microscopy and the methods described in these Examples.
  • transplanted tissue can be isolated at different intervals after transplantation and subjected to fluorescence activated cell sorting (FACS) for subsequent gene expression analysis using the PancChip5.0 to determine whether the patterns of gene expression correspond to the early, newborn, or adult patterns of expression (91).
  • FACS fluorescence activated cell sorting
  • the recipients' pancreas can be harvested and subjected to similar histologic and immunohistochemical analysis. If ⁇ cell tissue is found in the host pancreas, the source of the tissue can be determined using fluorescence microscopy to detect donor cells harboring the H2B-EGFP transgene.
  • mice treated with anti-CD3 mAb can be compared to mice that do not receive the mAb, and the efficacy of different sources of stem cell for ⁇ cell recovery can be assessed. It may be that the earlier anlagen are less immunogenic and will therefore provide a better source of replacement cells, even in the absence of anti-CD3 mAb. On the other hand, the high proportion of ⁇ cell progenitors present at the later stage, especially in comparison with the low proportion of stem cells thought to persist in adult islets, may result in this source of stem cells leading to faster or more complete recovery. In control animals, receiving transplants but no mAb treatment, there will either be no islet development or a developmental arrest in islet development.
  • pancreatic anlagen tissue transplanted The volume of pancreatic anlagen tissue transplanted or the transplant site may be limiting factors in recovery from the diabetic state, although in another study, 10 similarly staged rat pancreatic anlagen transplanted to a peritoneal site successfully rescued chemically-induced diabetes (49). If no recovery is observed in mAb treated mice, one can increase the number of anlagen or experiment with different developmental stages.
  • EXAMPLE 7 INDUCTION OF BETA-CELL DIFFERENTIATION OF PANCREATIC
  • pancreatic anlagen can provide important information about factors that control islet development and differentiation, expression of islet antigens, and sources of islet stem cells that are needed for translation of the new technology of stem cells and cellular replacement for clinical treatment of TlDM.
  • An understanding of the factors that control differentiation of islet cells from stem cells from anlagen may suggest approaches that might be used to stimulate differentiation of islet precursor cells from other sources.
  • co-culture with mature islets of Langerhans can stimulate induction of insulin producing cells from pancreatic anlagen. E14.5 GFP+ pancreatic anlagen was obtained and each anlagen was co-cultured with 50 islets from adult 129 mice.
  • EXAMPLE 8 MATURE ISLETS CELLS MODIFY THE DEVELOPMENT OF PANCREATIC PRECURSOR CELLS PRESENT IN THE PANCREATIC ANLAGEN
  • islets are co-cultured with 2 x 10 5 anlagen cells in each microwell (U bottom).
  • the cultures are maintained at 37 0 C, 5% CO2 for 6 days.
  • BrdU is added to the cultures.
  • the anlagen is cleared of the mature islets and dispersed into single cells with trypsin.
  • the cells are analyzed by flow cytometry after permeabilization and staining with anti-proinsulin (PE) and anti-BrdU (APC)(R&D Systems).
  • the cells are analyzed by flow cytometry.
  • An electronic gate is placed around GFP+ cells to identify cells that arise from the anlagen and exclude mature islet cells.
  • the GFP+ anlagen can be co-cultured with vascular endothelial cells from trypsinized aorta, hepatocytes, and with thymocytes.
  • Other sources of islet cells can be tested including ⁇ TC3 and ⁇ TCl cells, which produce glucagon rather than insulin(94) (95).
  • the anlagen cells (2 x 10 s ) can be cultured with 2 x 10 4 cells.
  • Islets will be isolated from mice that are treated with streptozotocin (STZ) (200 mg/kg) and are hyperglycemic (> 250 mg/dl) prior to isolation. They can then be added to the co-culture experiments as described. Identification of a soluble factor vs. cell fraction
  • Islets can be cultured in media for 6 days and this media can then be added to anlagen for 6 additional days followed by flow analysis. If there is no effect of the media on anlagen differentiation, one can then test whether cellular components of the islet are responsible.
  • An islet homogenate can be produced by homogenizing islets in media and this homogenate will be added to the islets directly (96). If the homogenate induces differentiation, further studies can involve fractionation of the homogenate to identify the component responsible for the effect.
  • studies can involve cell fractionation and further investigations to identify the molecule(s) responsible.
  • studies can address whether the effect of the islets is to promote differentiation or to redirect precursor cells towards a pathway of ⁇ cell differentiation.
  • the techniques for harvesting anlagen have been established as have the use of the proposed molecular markers (see Example 7).
  • a potential problem in the interpretation of studies of lineage commitment is the limited number of markers for other endocrine cell lineages such as ⁇ cells, particularly since the majority of islet cells are ⁇ cells, and increased differentiation into this lineage may be difficult to identify.
  • GLP-I agonists may increase ⁇ cell mass (28, 30). This may occur by decreasing the rate of ⁇ cell death or by the involvement of enhancement of ⁇ cell proliferation (31 , 32).
  • the effects of the receptor agonists on pancreatic stem cells have not been studied. To study this, one can compare differentiation of anlagen cells in the presence and absence of Exenin-4 in cultures. For these studies, Exendin-4 (California Peptide Research, Napa, CA) can be added at a concentration of 100 nM to the anlagen cultures described above (31). At the end of the 6 day culture period, the cells can be dispersed and stained for insulin, proliferation (by BrdU uptake) and analyzed as described above. RNA can also be prepared from the anlagen and the expression of markers of cell lineage will be determined by real time PCR and with the PancChip as described below.
  • the media used in the studies contained 17mM glucose.
  • the effects of the islets may be altered by their response to glucose - thus elevated glucose levels might induce factor(s) that would enhance ⁇ cell differentiation.
  • To determine whether glucose induces islets to mediate the effects on differentiation one can compare the effects of islets on ⁇ cell differentiation in media containing a low (1.6 mM) to high (16 mM) glucose concentration.
  • MFI mean fluorescence index
  • GFP+ cells allows one to sort anlagen from mature islets that are present in the same culture.
  • Anlagen from wild type mice can be cultured in the presence or absence of islets from adult GFP mice at a ratio of 50 islets/anlagen.
  • the mature islets can be separated from the anlagen.
  • GFP labeled islets rather than anlagen to ensure that the mature islet cells are completely removed from the co-cultures before the glucose stimulated insulin release is determined.
  • the absence of cells from the islet donor can be verified by visualizing the anlagen tissue under UV light.
  • the anlagen can then be placed back into culture for 30 minutes with media containing glucose at a concentration of 1.6 mM.
  • An aliquot of the media can be taken and the concentration of glucose can then be increased to 16 mM.
  • Another aliquot of media can be taken after 30 minutes and the concentration of insulin the aliquots can be compared to each other and to insulin release by islets under the same conditions.
  • the islet cultures can involve 100 islets which contain about 10 5 cells - the approximate number of cells that are present in each anlagen.
  • the embryonic stage at which the co-cultures are initiated are an important determinant of the commitment to ⁇ cell development.
  • commitment to the ⁇ cell lineage may be necessary to promote differentiation of insulin+ cells, and therefore, islets may not affect differentiation of anlagen from time points earlier than El 5.5.
  • islets may not affect differentiation of anlagen from time points earlier than El 5.5.
  • Anlagen can be isolated from embryos as young as El 2.5. Cultures with anlagen from El 2.5 — birth can be compared to identify the stage at which islet differentiation can be affected. At each stage, the anlagen tissue can be characterized by expression of the lineage markers described below.
  • the anlagen derived (GFP+) cells can be stained for glucagon.
  • the GFP+ cells can be sorted by FACS from the dispersed anlagen after 6 days in co-culture with mature islets. Because there is a risk of losing cells during the sorting procedure, one can start with a pool of about 5 anlagen, which will yield about 5 x 10 5 cells.
  • RNA can be prepared from the sorted cells and the expression of the following genes can be determined by real time PCR: a) Neurogenin 3 (ngn3) is a member of a family of basic-helix-loop-helix (bHLH) transcription factors, required for endocrine cell fate determination in the developing mouse pancreas (53). Ngn3 is expressed in islet cell progenitors and functions as a proendocrine gene driving islet cell differentiation (97). b) Nkx2.2, a member of the mammalian NK2 homeobox transcription factor family, is required for the final differentiation of pancreatic ⁇ cell, and in its absence, ⁇ cells are trapped in an incompletely differentiated state (98).
  • ngn3 is a member of a family of basic-helix-loop-helix (bHLH) transcription factors, required for endocrine cell fate determination in the developing mouse pancreas (53). Ngn3 is expressed in islet cell progenitors and functions as a proendocrine gene
  • Isletl a LIM homeodomain protein, is expressed in all classes of islet cells in the adult and its expression in the embryo is initiated soon after the islet cells have left the cell cycle (99).
  • Pax ⁇ a transcription factor, is required for islet cell number, morphology and hormone gene expression. It has an essential role in maintaining normal ⁇ cell function after birth, but not for ⁇ cell neogenesis during late embryonic development and early postnatal stages (100).
  • NeuroD a basic helix-loop-helix transcription factor, functions as a regulatory switch for endocrine pancreatic development.
  • pancreatic islet morphogenesis is abnormal and overt diabetes develops due in part to inadequate expression of the insulin gene (101).
  • Pax4 a transcription factor, operates in the ⁇ / ⁇ cell lineage and mice lacking Pax4 completely lack these cell lineages at birth (76).
  • Pdxl is a homeodomain transcription factor originally identified as an activator of the insulin and somatostatin genes (102-105). Early in development, Pdxl is widely expressed in pancreas and is required for cell proliferation but not for generation of the earliest insulin-synthesizing or glucagons-synthesizing cells (106, 107) (108).
  • Pdxl At approximately E14.5, high levels of Pdxl become restricted to ⁇ -cells (104). High Pdxl expression remains restricted to ⁇ -cells in adult pancreas, and its activity is essential for maintaining the functionality of these cells (99, 109).
  • Ptfla and Hesl are markers of exocrine cell lineage (110, 1 11)
  • Brn4 is a marker of alpha cell lineage (112).
  • the PancChip 6.0 contains 13,059 mouse cDNAs chosen for their expression in various stages of pancreatic development, many of which are not found on commercially available arrays. This new version of the PancChip includes 1000 full-length clones of particular importance to the field of pancreatic development and pathways relating to glucose homeostasis.
  • the chip includes cDNAs from every assembly found to be expressed in the pancreas. Therefore, by comparing the results of the Chip analysis of anlagen in the absence of presence of islets, one can obtain information pertaining to all aspects of pancreatic differentiation. To avoid the obstacle of amplification of the cDNA before preparation of cRNA for chip analysis, one can pool anlagen cells sorted from replicate cultures. Approximately 10 anlagen would be predicted to give a sufficient cell yield for each chip. This will require pooling anlagen from 2 pregnant donors.
  • pancreas of NOD neonates has a number of abnormalities (60, 61) suggesting that the genetic predisposition leading to autoimmune disease later in life may have its origins in fetal developmental anomalies.
  • Studies have shown increases in fibronectin in early post-natal pancreas from NOD compared to B6 of BALB/c mice, with migration of macrophages to the sites of increased fibronectin (62).
  • Enlarged and irregularly shaped islets and an increase in the endocrine/exocrine area were also reported. These abnormalities may have their origins in fetal development.
  • a wave of ⁇ cell apoptosis that occurs early in the neonatal period is believed to be the time of initiation of insulitis (63).
  • developmental abnormalities in the NOD pancreas would represent a likely initiation event involved in diabetes pathogenesis.
  • pancreas of NOD neonates has a number of abnormalities (60, 61) indicating that the genetic predisposition leading to autoimmune disease later in life may have its origins in fetal developmental anomalies. Studies can be designed to investigate the development of the pancreas in NOD mice using molecular markers of different developmental stages.
  • abnormalities are present in the pancreas in prediabetic NOD mice from birth onwards (60, 61). Abnormalities include increased apoptosis of ⁇ cells, elevated levels of some types of antigen presenting cells and FasL positive cells, abnormalities of extracellular matrix protein expression, and high percentages of immature islets reflecting islet neogenesis related to neonatal ⁇ cell hyperactivity. These differences indicate that from birth onwards there is an intricate relationship between endocrine and immune events in the NOD mice suggesting that the tissue specific- autoimmune reaction could arise from developmental phenomena taking place during fetal life.
  • Changes in the developmental sequence or timing of molecular events may lead directly to the anomalies seen at birth in the NOD pancreas, and these anomalies may be related to the eventual onset of autoimmune disease. Differences in the development of NOD pancreas could be related to possible outcomes of transplantation therapy if, for example, transplantation of fetal pancreas recruits or stimulates development of residual endogenous NOD islets.
  • pancreas starts to develop at E9.5 as separate dorsal and ventral evaginations from foregut endoderm, known as the pancreatic buds (54). These grow, branch, differentiate and eventually merge to form the definitive pancreas.
  • the first endocrine cells appear as early as El 0.5 and produce glucagon and/or insulin (112, 1 14, 115).
  • the pattern and timing of expression of genes involved in the differentiation of different pancreatic cell types, endocrine, exocrine and ductal, of the pancreas can be examined using in situ hybridization and immunohistochemistry and islet structure can be studied using anti-insulin, anti-glucagon, and anti-amylase antibodies.
  • Staged-matched embryos from NOD, NOR, and ICR mice can be collected in cold PBS from stage E 8.5 to E 17.5.
  • Embryos from E8.5 to El 0.5 can be processed as whole mounts following opening of the abdomen and removal of non-gut tissues and organs to expose the gut for proper visualization of the pancreatic bud.
  • pancreas can be dissected out along with the attached stomach and duodenum.
  • Embryos and isolated pancreas can be fixed in 4% paraformaldehyde overnight followed by PBT wash and dehydration in methanol. Samples can be stored in methanol at -20°C for in situ hybridization according to protocols standard in the laboratory (118).
  • pancreas can be fixed in 4% PFA for three hours at 4 0 C, cryoprotected overnight in 30% sucrose, embedded in Tissue Tek OCT compound, and frozen on dry ice for storage at -8O 0 C until used. Cryostat sections can be cut for immunohisotchemistry according to protocols standard in the laboratory.
  • the initiating event in TlDM remains unclear. Studies in the NOD mouse indicate that it occurs very early in life and may involve ⁇ cell death. This Example shows that the initiating event in diabetes may be linked to abnormalities in pancreatic development.
  • EXAMPLE 10 ADULT ISLET BETA CELLS ENHANCE DIFFERENTIATION OF MOUSE EMBRYONIC PANCREATIC PRECURSOR CELLS INTO BETA CELLS
  • Pancreas anlagen was isolated from GFP+ mouse embryos at age of E14.5-15.5 and cultured with or without mature islets on the organotypic inserts for 7 days in serum free DMEM/F-12 medium containing B-27 supplement. The BrdU was added for labeling proliferating cells. After culturing, the embryonic pancreatic anlagen and adult islets were dispersed into single cells and stained with anti-BrdU and anti-proinsulin mAbs. Beta cell proliferation and differentiation was evaluated by FACS analysis.
  • ⁇ TC3 cells functioned as well as islets in term of enhancing embryonic B-cell differentiation (1.7 fold increase in insulin+ anlagen cells compared to culture without ⁇ TC3). Induction of anlagen differentiation required cellxell contact.
  • Our studies suggest that mature islet cells deliver a signal that accelerates differentiation of pancreatic stem cells into mature insulin+ cells.
  • Beta cell replacement therapy represents a promising approach for treatment of diabetes. Advances in stem cell research provide a good opportunity to generate beta cells with various sources of stem cells.
  • pancreatic anlagen which consists of many islet stem cells or precursor beta cells, can be used as a source of islet stem cells.
  • Rodent pancreatic anlagen can differentiate into islets and correct STZ-induced diabetes in mice (Roger et al. Am J Physiol Endocrinol Metab 286:E502-E509 (2004)).
  • mature islet beta cells may regulate maturation of pancreatic islet stem cells or progenitor beta cells.
  • Islets are isolated from adult mice, and pancreatic anlagen (PA) is collected from mouse embryos. Then, a certain number of islets together with one or two PA onto a cell insert and cultured them with serum free medium supplemented with B27, which favors endocrine precursor cell growth. To maximize cell contact between the anlagen cells and the islets, dispersed single anlagen cells were co-cultured with or without islets in a 96-well plate. To monitor cell proliferation, cells were labeled with BrdU. At the end of culture, FACS, ELISA and immunohistochemistry are examples of techniques that may be used to characterize anlagen cell growth and differentiation.
  • Results also show that insulin is not responsible for cell differentiation.
  • the differential effect of islets was mediated by direct contact among anlagen and islets. This finding suggested that signals originate from the surface of islets.
  • islets were fixed with BD cytofix/cytoperm buffer, then, dispersed single anlagen cells were cultured with certain number of fixed islets in 96 well plate. As shown in Figures 14 and 15, on day 7 of culture, around 60% ⁇ 76% anlagen cells developed into proinsulin-producing beta cells when co-cultured with fixed islets. There was a trend of dose-dependency on differentiation. The peak time point of maturation in anlagen cells was around day 7.
  • beta TC3 cells fixed adult thymocytes, mouse lung endothelial cells, which are derived from endode ⁇ n. Only beta TC3 cells enhanced differentiation of anlagen cell into beta cells. The non-beta cells had no effect on differentiation of anlagen cells.
  • Beta cell differentiation in anlagen cells was observed with confocal microscopy ( Figures 16A — 16C). Few insulin+ cells are observed in the anlagen cell monolayer culture ( Figure 16A). However, many insulin+ cells are visible in anlagen cells cultured with fixed islets ( Figures 16A and 16B). These mature beta-cells had small nuclei and condensed chromatin, showing typical morphology of differentiated cells.
  • Pdx-1 expression which is normally only seen in mature beta-cells, was also analyzed. As shown in Figure 18, very few Pdx-1 positive cells could be found in anlagen cell culture alone. A significantly increased number of Pdx-1 positive anlagen cells seen in the presence of fixed islets. Studies can also be designed to co-localize both Pdx-1 and insulin in individual cells. [00140] To characterize functions of newly developed beta cells, the glucose stimulation test was conducted with these cells. As shown in Figure 19, even at low glucose level, a significant amount of insulin was detected in anlagen cells cultured with fixed islets as compared to anlagen cells along. At high glucose concentrations, the difference in insulin release was more evident between the two groups.
  • the insulin secretion pattern of new beta cells was not typical to the pattern generally seen for adult beta cells. This could result from lack of 3D culture condition, pre-exposure to high concentration of glucose in culture medium, or contamination of insulin in the medium. Thus, the newly developed beta cells responded to glucose stimulation with respect to insulin secretion.
  • mature islet beta-cells are able to induce differentiation of embryonic islet stem cells or precursor cells into insulin-producing beta cells.
  • the differentiation effect of mature beta cells is dependent on cell surface membrane bound signals.
  • New developed beta cells exhibit similar characteristics of mature beta cells.
  • the invention provides an in vitro model for future investigation of beta-cell differentiation and methods for generating mature beta-cells form human fetal pancreatic anlagen which may provide a cellular therapy for treatment of diabetes.
  • EXAMPLE 11 REMISSION OF HYPERGLYCEMIA IN MICE WITH ANLAGEN CELLS THAT HAVE BEEN CO-CULTURED WITH FIXED BETA CELLS ( ⁇ TC3)
  • mice were induced in immune deficient, NOD/scid mice with a single injection of streptozotocin (200 mg/kg). After hyperglycemia was established, the mice received a transplant of an equal number of anlagen cells that had been cultured with ( Figure 22A) or without ( Figure 22B) fixed ⁇ TC3 cells for 6 days, mixed with human vascular endothelial cells. The recipient mice also were initially treated with an insulin pellet that was removed on day 21 in all of the mice.
  • EXAMPLE 12 INDUCTION OF PROINSULIN+ CELLS IN HUMAN PANCREATIC
  • Topp B Promislow K, deVries G, Miura RM, Finegood DT. A model of beta-cell mass, insulin, and glucose kinetics: pathways to diabetes. J Theor Biol 2000;206(4):605-19.
  • Tsai E Sherry N, Palmer J, Group tD-S, Herold KC.
  • Kitamura T Nakae J
  • Kitamura Y et al.
  • the forkhead transcription factor Foxol links insulin signaling to Pdxl regulation of pancreatic beta cell growth. J Clin Invest 2002; 110(12): 1839-47.
  • Lammert E Cleaver O
  • Melton D Induction of pancreatic differentiation by signals from blood vessels. Science 2001 ;294(5542):564-7.
  • Wilson ME Kalamaras JA, German MS. Expression pattern of IAPP and prohormone convertase 1/3 reveals a distinctive set of endocrine cells in the embryonic pancreas. Mech Dev 2002;115(l-2):171-6.
  • Wilson ME Scheel D, German MS. Gene expression cascades in pancreatic development. Mech Dev 2003; 120(1 ):65-80.
  • Harrelson Z, Kelly RG, Goldin SN, et al. Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 2004;131(20):5041-52.

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Abstract

La présente invention concerne des procédés pour identifier un facteur capable de différencier une cellule précurseur ne produisant pas d'insuline en une cellule qui produit de l'insuline. La cellule précurseur peut être une cellule précurseur pancréatique dérivée du tissu pancréatique. La présente invention concerne également des procédés pour différencier une cellule précurseur ne produisant pas d'insuline en une cellule qui produit de l'insuline. Ce procédé comprend la mise en contact d'une cellule précurseur ne produisant pas d'insuline avec une cellule des îlots pancréatiques matures, la mise en contact comprenant la co-culture de la cellule précurseur et de la cellule des îlots dans une culture cellulaire. L'invention concerne en outre des cellules produisant de l'insuline produites par un procédé de l'invention et des procédés d'utilisation de telles cellules pour traiter les diabètes.
PCT/US2007/084327 2006-11-09 2007-11-09 Procédés pour différencier les cellules bêta produisant de l'insuline à partir de cellules précurseurs pancréatiques WO2008063934A2 (fr)

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CN115029299A (zh) * 2018-11-07 2022-09-09 杭州瑞普晨创科技有限公司 JAK2抑制剂在胰岛β细胞诱导分化中的应用

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* Cited by examiner, † Cited by third party
Title
DOR Y. ET AL.: 'Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell diferentiation' NATURE vol. 429, May 2004, pages 41 - 46, XP002388399 *
OUZIEL-YAHALOM L. ET AL.: 'Expansion and redifferentiation of adult human pancreatic islet cells' BIOCHEM. BIOPHYS. RES. COMM. vol. 341, no. 2, January 2006, pages 291 - 298, XP005265708 *
WEI C. ET AL.: 'Adult Islet Beta-Cells Enhance Differentiation of Mouse Embryonic Pancreatic Precursor Cells into Beta-Cells' DIABETES vol. 55, no. SUPPL. 1, June 2006, page A32 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029299A (zh) * 2018-11-07 2022-09-09 杭州瑞普晨创科技有限公司 JAK2抑制剂在胰岛β细胞诱导分化中的应用
CN115029299B (zh) * 2018-11-07 2023-11-14 杭州瑞普晨创科技有限公司 JAK2抑制剂在胰岛β细胞诱导分化中的应用

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