WO2009126927A2 - Procédés de génération de cellules produisant de l'insuline - Google Patents

Procédés de génération de cellules produisant de l'insuline Download PDF

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WO2009126927A2
WO2009126927A2 PCT/US2009/040266 US2009040266W WO2009126927A2 WO 2009126927 A2 WO2009126927 A2 WO 2009126927A2 US 2009040266 W US2009040266 W US 2009040266W WO 2009126927 A2 WO2009126927 A2 WO 2009126927A2
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cells
insulin
cell
pdx
ihbecs
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WO2009126927A3 (fr
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Gordon Weir
Masaki Nagaya
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Joslin Diabetes Center, Inc.
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    • 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
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    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
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    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • This disclosure relates to methods of generating insulin-producing cells, and more particularly to methods of generating insulin-producing cells from intrahepatic bile epithelial cells (IHBECs).
  • IHBECs intrahepatic bile epithelial cells
  • the disclosure is based, inter alia, on the surprising discovery that IHBECs can be transdifferentiated into insulin-producing cells by various methods. Accordingly, this application provides methods of insulin-producing cells from IHBECs. These insulin- producing cells can introduced into subjects for the treatment of diabetic conditions. Accordingly, in one aspect the disclosure features methods of generating cell populations that include insulin-producing cells.
  • the methods include the steps of: (a) obtaining a population of intrahepatic biliary epithelial cells; (b) optionally, expanding the population of cells; (c) optionally, culturing the IHBECs or the expanded population of cells in a medium lacking insulin and serum; and (d) contacting the IHBECs or the expanded population of cells with one or more differentiation conditions for a time sufficient that at least a portion of the cells secrete insulin, thereby generating a cell population that includes insulin-producing cells.
  • the cell population that includes insulin-producing cells produces at least 0.1 ng/ml (e.g., at least
  • the cell population that includes insulin-producing cells secretes insulin in response to glucose or an insulin secretagogue.
  • expanding the population of cells includes culturing the cells in a three dimensional scaffold (e.g., a collagen gel).
  • the population of cells is expanded in a growth medium that includes insulin and/or serum.
  • a differentiation condition includes introducing into the population of cells a nucleic acid that expresses a transcription factor (e.g., Pdx-1, NeuroD, Pdx 1/VP16, Ngn3, Nkx ⁇ .l, or pancreas-specific transcription factor l ⁇ (Ptfl ⁇ )).
  • a transcription factor e.g., Pdx-1, NeuroD, Pdx 1/VP16, Ngn3, Nkx ⁇ .l, or pancreas-specific transcription factor l ⁇ (Ptfl ⁇ )
  • the nucleic acid is contained within a vector, e.g., a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, or a retroviral vector).
  • a differentiation condition includes inhibiting Notch signaling in the cells, e.g., by contacting the population of cells with a gamma-secretase inhibitor.
  • the differentiation condition includes inhibiting HNF6 expression in the cells, e.g., by introducing to the population of cells a nucleic acid that inhibits HNF6 expression.
  • a differentiation condition includes exposing the cells to one or more factors produced by pancreatic cells, e.g., fetal pancreatic cells.
  • a differentiation condition includes mixing the population of cells with a population of fetal pancreatic cells or contacting the population of cells with conditioned medium from fetal pancreatic cells.
  • the methods further include isolating the portion of the cells that secrete insulin, e.g., by cell sorting or immunological methods.
  • the cell population that includes insulin-producing cells or the isolated portion can be introduced into a subject, e.g., a human.
  • the initial population of intrahepatic biliary epithelial cells is obtained from the same subject to which the cell population that includes insulin-producing cells or the isolated portion is introduced.
  • the disclosure features a cell population comprising insulin- producing cells generated by any of the methods described herein.
  • the disclosure features methods of generating an insulin- producing cell.
  • the methods include providing an intrahepatic biliary epithelial cell or a cell derived from an intrahepatic biliary epithelial cell; and contacting the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell with one or more differentiation conditions for a time sufficient that the cell secretes insulin, thereby generating an insulin-producing cell.
  • the insulin- producing cell secretes insulin in response to glucose or an insulin secretagogue.
  • the differentiation condition includes introducing into the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell a nucleic acid that expresses a transcription factor (e.g., Pdx-1, NeuroD, Pdx 1/VP16, Ngn3, Nkx ⁇ .l, or pancreas-specific transcription factor l ⁇ (Ptfl ⁇ )).
  • a transcription factor e.g., Pdx-1, NeuroD, Pdx 1/VP16, Ngn3, Nkx ⁇ .l, or pancreas-specific transcription factor l ⁇ (Ptfl ⁇ )
  • the nucleic acid is contained within a vector, e.g., a viral vector (e.g., an adenovirus vector, an adeno-associated virus vector, or a retroviral vector).
  • a differentiation condition includes inhibiting Notch signaling in the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell, e.g., by contacting the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell with a gamma-secretase inhibitor.
  • a differentiation condition includes inhibiting HNF6 expression in the cells, e.g., by introducing to the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell a nucleic acid that inhibits HNF6 expression.
  • a differentiation condition includes exposing the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell to one or more factors produced by pancreatic cells, e.g., fetal pancreatic cells.
  • a differentiation condition includes mixing the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell with a population of fetal pancreatic cells or contacting the intrahepatic biliary epithelial cell or the cell derived from an intrahepatic biliary epithelial cell with conditioned medium from fetal pancreatic cells.
  • the disclosure features an insulin-producing cell generated by any of the methods described herein.
  • the disclosure features methods of treating a subject with an insulin deficiency (e.g., diabetes type I or type II) by generating a cell population that includes insulin-producing cells by a method described herein and introducing into the subject the cell population that includes insulin-producing cells or an isolated portion thereof.
  • an insulin deficiency e.g., diabetes type I or type II
  • the disclosure includes methods of generating a cell population comprising insulin-producing cells
  • the methods include obtaining a intrahepatic biliary epithelial cell and culturing (e.g., expanding) the cell in a first medium comprising insulin and serum for a first time period, e.g., to produce a cell population.
  • the resulting cell or cells can then be isolated from the first medium, or certain components of the first growth medium (e.g., insulin and/or serum) can be removed from the medium, before culturing (e.g., expanding) the cell or cells in a second medium lacking insulin and serum for a second time period to produce a population of intrahepatic biliary epithelial cells.
  • This population of intrahepatic biliary epithelial cells can then be contacted with at least one (e.g., one or more) of a nucleic acid encoding a transcription factor (e.g., Pancreatic and duodenal homeobox-1 (Pdx-1), NeuroD, Pdx 1/VP 16 (which is also known in the art or is marketed as Etoposide, Etopophos ® , Vepesid ® ), Neurogenin 3 (Ngn3), Nkx ⁇ .l, and pancreas-specific transcription factor l ⁇ (Ptfl ⁇ ), an inhibitor of Notch signaling, an inhibitor of HNF 6 expression, a fetal pancreatic cell, or a solution contacted by cultured fetal pancreatic cells for a time sufficient that at least a portion of the cells secrete insulin to generating a cell population comprising insulin-producing cells.
  • a transcription factor e.g., Pancreatic and duodenal homeobox-1 (Pd
  • At least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the cells in the cell population comprising insulin- producing cells are insulin producing cells.
  • liver parenchyma can provide for safe isolation of large numbers of intrahepatic biliary epithelial cells (IHBECs) for transdifferentiation.
  • IHBECs intrahepatic biliary epithelial cells
  • a considerable amount of liver tissue can be surgically removed with less potential for complications because of the capacity of the liver for regeneration (Michalopoulos et al., 1997, Science, 276:60-66).
  • the current methods provide for the generation of large numbers of IHBECs for differentiation and maintenance of long-term (>24 hour) viability of these cell preparations.
  • the capacity to generate large numbers of insulin-producing cells from IHBECs can allow for the production of sufficient numbers of cells for transplantation. If sufficient numbers of insulin-producing cells are generated, surplus cells can be frozen for later transplantation.
  • treating includes reducing or alleviating at least one adverse effect or symptom, e.g., absolute or relative insulin deficiency, fasting hyperglycemia, glycosuria, development of arteriosclerosis, microangiopathy, nephropathy, and neuropathy, of disorders characterized by insufficient insulin activity.
  • adverse effect or symptom e.g., absolute or relative insulin deficiency, fasting hyperglycemia, glycosuria, development of arteriosclerosis, microangiopathy, nephropathy, and neuropathy, of disorders characterized by insufficient insulin activity.
  • a cell that is "derived from” an animal is a cell that was taken from the animal, or a cell that is a progeny cell of a progenitor cell that was taken from the animal, e.g., removed from the animal surgically or by some other method.
  • FIG. 1 is an outline of an exemplary protocol for inducing the differentiation of IHBECs into insulin-producing cells (Group 3) and appropriate controls (Group 1 and Group 2).
  • FIG. 2 A is a representation of the macroscopic appearance of IHBECs with
  • Isolated IHBECs were suspended in rat tail collagen plus growth medium; the collagen gel containing the IHBECs gradually contracted. Scale bar, 10 mm.
  • FIG. 2B is a phase contrast light microscopy image of cultured IHBECs. Left, day 0 with CEFCM; right, after 5 days with CEFCM, IHBECs formed three-dimensional ductal cysts.
  • FIG. 2C is a phase contrast light microscopy image of cultured IHBECs at 14 day with CEFCM. Asterisk shows the same position as in panel A. The IHBECs had expanded to form ductal structures. Scale bar, 100 um.
  • FIG. 2D is a line graph depicting the diameter of the collagen gel over time.
  • FIG. 2E is a line graph depicting the number of IHBECs over time for culture with or without CEFCM. Bars show mean ⁇ SEM of five independent experiments. P ⁇ 0.05.
  • FIGs. 2F-2I are RT-PCR gels showing gene expression profiles of IHBECs at days 0 and 17 of culture, and at day 26 following transduction with AdV-Pdx- 1/VP 16.
  • Mouse liver (FIGs. 2F-2H) or islet (FIG. 21) was used as a control.
  • the oligonucleotide primers and cycle number used for semiquantitative PCR are shown in Table 1. All primers were designed to cross intron(s). Mouse liver and islets were used as controls. Gene expression studies in all groups were repeated at least 3 times with similar results.
  • FIG. 2F hepatocyte markers (tryptophan oxygenase [TO], tyrosine aminotransferase [TAT], cytochrome P450 1A2 [CYP1A2]).
  • FIG. 2G BEC markers (Cytokeratin [CK] 7 and 19, ⁇ -glutamyltranspeptidase [ ⁇ -GTP].
  • FIG. 2H endoderm progenitor markers (HNFsI ⁇ , 3 ⁇ , 4 ⁇ , and 6).
  • FIG. 21 islet markers (insulinl, glucagon, somatostatin, and pancreatic polypeptide).
  • FIGs. 2J-K are immunofluorescence micrographs depicting staining for CK 7 in IHBECs. IHBECs were positive for CK 7 (blue) at days 0 (FIG. 2J) and 17 (FIG. 2K). Propidium Iodide (PI, red) was used to show nuclei. Scale bar, 100 um.
  • FIG. 3 A is a pair of phase contrast light microscopy images of IHBECs cultured for 17 days with (right) or without (left) CEFCM. At 17 days in culture, the CEFCM- cells displayed signs of senescence (black arrows), including flattening and multinucleation, and numerous cytoplasmic vacuoles (white arrow heads).
  • FIG. 1A is a pair of phase contrast light microscopy images of IHBECs cultured for 17 days with (right) or without (left) CEFCM. At 17 days in culture, the CEFCM- cells displayed signs of senescence (black arrows), including flattening
  • 3B is a bar graph depicting the number of signs of senescence and cytoplasmic vacuoles per colony at day 17 for cells cultured with or without CEFCM. Bars show mean ⁇ SEM of 25 independent experiments. *, P ⁇ 0.095.
  • FIGs. 4A-4C are sets of micrographs of IHBECs transduced with AdV- Pdx-1 (FIG. 4A), NeuroD (FIG. 4B), or Pdx-l/VP16 (FIG. 4C) at the indicated MOI with each AdV expressing GFP as a reporter.
  • the left panel is a phase contrast light microscopy image
  • the middle panel depicts GFP fluorescence
  • the right panel depicts the two images merged together.
  • Scale bar 100 um.
  • FIGs. 5A-5D are RT-PCR gels showing gene expression profiles of IHBECs at day 14 of culture, Group 1 control cells (differentiation medium (DM only)), Group 2 control cells (AdV-GFP), and Group 3 cells transduced with AdV expressing Pdx-1,
  • FIG. 5A beta-cell related markers (insulin 1 and 2, glucagon, somatostatin, pancreatic polypeptide (PP), prohormone convertase (PC) 1 and 2, glucose transporter 2 (GLUT2)).
  • FIG. 5A beta-cell related markers (insulin 1 and 2, glucagon, somatostatin, pancreatic polypeptide (PP), prohormone convertase (PC) 1 and 2, glucose transporter 2 (GLUT2)).
  • FIG. 5B transcription factors (Neurogenin 3 (Ngn3), NeuroD, Nkx2.2, Paired box 4 (Pax4), Nkx ⁇ .l, Pax6, MafA, Pdx-1, and islet- 1 (IsIl)).
  • FIG. 5C exocrine cell related markers (pancreas-specific transcription factor l ⁇ (Ptfl ⁇ ), Amylase 2).
  • FIG. 5D early liver and pancreas markers (HNFl ⁇ and HNF6) and ⁇ -actin control.
  • FIG. 5E is a bar graph depicting quantitative Insulin 1 mRNA analysis for IHBECs (Group 1), AdV-GFP control cells (Group 2), and transduced cells (Group 3 (Pdx-1 :white column, NeuroD: dot column, Pdx-1 /VP16: black column)) assessed 7 days after transduction. There was no significant difference among the groups. Quantification of insulin mRNA levels was carried using the TaqManTM real-time PCR system. Mouse islets were used as a positive control and calculated for relative insulin 1 mRNA quantification. Data are presented as mean ⁇ SEM of three independent experiments.
  • FIGs. 6A-6D are sets of immunofluorescence micrographs of AdVs-P dx- 1/VP 16 transduced IHBECs at 7 days after transduction.
  • FIG. 6A triple staining for NeuroD (left, red), GFP (middle, green), and CK7 (right, blue).
  • FIG. 6B triple staining for NeuroD (left, red), GFP (middle, green), and CK7 (right, blue).
  • FIG. 6B triple staining for
  • FIG. 6C triple staining for Pdx-1 (left, red), GFP (left, green), and CK7 (right, blue).
  • FIG. 6D triple staining for Pdx-1, (left, red), GFP (middle, green), and DAPI nuclear stain (right, blue).
  • FIGs. 6B and 6D there was complete overlap between transcription factors and DAPI in the nuclei.
  • FIGs. 6E-6F are sets of immunofluorescence micrographs of AdVs-P dx- 1/VP 16 transduced IHBECs at 7 days after transduction showing C-peptide staining for the confirmation of insulin synthesis.
  • the micrographs show triple staining for C-peptide (left, red), GFP (middle, green), and CK-7 (right, blue). Of 4000 cells counted, 2.8% in Pdx-l/VP16 transduced cultures stained strongly for C-peptide. Scale bars, 50 ⁇ m.
  • FIGs. 6G-6H are transmission electron micrographs of AdVs- Pdx-l/VP16 transduced IHBECs 7 days after transduction.
  • FIG. 6G shows secretory granules densely packed in the cytoplasm. Some granules show a clear halo surrounding a denser core, a morphology that is characteristic of insulin-containing granules. These cells remains apical microvilli which polarity of IHBECs (FIG. 6G, asterisks).
  • FIG. 6H shows a higher magnification view of the secretory granules, with the crystalline formation of the granular core. Scale bars, 1 ⁇ m.
  • FIG. 7B is a dot graph depicting insulin release in response to insulin secretagogues after 2 hour incubations by IHBECs (Group 1), control cells (Group 2), transduced cells (Group 3, Pdx-1 : white column, NeuroD: dotted column, Pdx-l/VP16: black column).
  • Insulin secretion was evaluated in serum-free DM or Krebs-Ringer bicarbonate buffer (KRBB) with 0.2% BSA supplemented with 5 mM glucose, 25 mM glucose, or 25 rnM glucose + 45 mM KCl, and quantitated by ELISA (detection limit of 0.04 ng/ml). Dotted arrows show undetectable insulin secretion.
  • FIGs. 8A-8B are scatter graphs depicting flow cytometry analysis of control and AdVs-Pdx-l/VP16 transduced IHBECs 7 days after transduction.
  • FIG. 8 A flow cytometry analysis of untransduced control groups (box 1, gated cells with low PI staining and GFP fluorescence).
  • FIG. 8B flow cytometry analysis of AdVs- Pdx-l/VP16 transduced IHBECs 7 days after transduction (box 2, gated live non-GFP-emitting cells; box 3, gated live, GFP-emitting cells effectively transduced with AdVs).
  • IHBECs and methods of introducing those insulin-producing cells into subjects for the treatment of disorders, e.g., diabetes.
  • the disclosure is based, inter alia, on the surprising discovery that IHBECs are capable of being transdifferentiated into insulin-producing cells by contacting the IHBECs or cells derived therefrom with various differentiation conditions.
  • IHBECs are the cells that comprise the epithelial cell lining of the intrahepatic biliary tree. IHBECs are distinct from epithelial cells of the extrahepatic ducts and the gall bladder. Methods of obtaining, isolating, and culturing IHBECs, including from human subjects, are known in the art. See, e.g., Joplin, 1994, Gut, 35: 875-878; Joplin et al., 1992, J. Clin. Invest., 90:1284-89; Auth et al., 2001, Hepatology, 33:519-529; and Ochiai et al., 2004, Pediatr. Surg. Int., 20:685-688.
  • IHBECs Exemplary methods of obtaining IHBECs include biopsy and perfusion of the liver with digestive enzymes, such as collagenase. IHBECs can be isolated from other liver cells by, e.g., differential density gradient centrifugation (Gall et al., 1985, Cell Biol. Int.
  • the cell or cells can be cultured in a liquid medium or on a support (e.g., a collagen gel support).
  • the medium can contain one or more of growth factors (e.g., EGF, HGF), insulin, and serum. See, e.g., Joplin et al., 1992, J. Clin. Invest., 90:1284-89; Ochiai et al., 2004, Pediatr. Surg. Int., 20:685-688.
  • IHBECs or cells derived from IHBECs are exposed to one or more differentiation conditions such that at least a portion of the differentiated cells secrete insulin.
  • the cells can be exposed to the one or more differentiation conditions simultaneously or consecutively.
  • the cells can be exposed to the differentiation conditions either in vitro or in vivo.
  • the differentiation conditions include increasing the expression or activity of one or more differentiation-related transcription factors in the cells, inhibiting the expression or activity of a component of the Notch signaling pathway in the cells, inhibiting the expression or activity of HNF 6 in the cells, exposing the cells to factors secreted by pancreatic cells (e.g., fetal pancreatic cells), or transplantation of the cells into a subject.
  • pancreatic cells e.g., fetal pancreatic cells
  • transplantation of the cells into a subject Typically, if the cells are exposed to the one or more differentiation conditions in vitro, the cells will be in a medium that does not contain insulin or serum.
  • a differentiation condition includes increasing the expression or activity of one or more transcription factors related to pancreatic lineage.
  • the one or more transcription factors can be selected from: pancreatic and duodenal homeobox 1 (Pdx-1; GenBank Accession No. NP 000200), neurogenic differentiation 1 (NeuroD; GenBank Accession No. NP 002491), Pdx-1 carrying the VP16 transactivator domain (Pdx-1 /VP16; see Kaneto et al, 2005, Diabetes, 54:1009- 22), neurogenin 3 (Ngn3; GenBank Accession No. NP_066279), NK6 transcription factor related, locus 1 (Nkx ⁇ .l; GenBank Accession No.
  • the one or more transcription factors can be expressed (e.g., exogenously expressed) within the cell by any means known in the art.
  • the cells may be transfected, transformed, or transduced using any of a variety of techniques known in the art. Any number of transfection, transformation, and transduction protocols known to those in the art may be used, for example those outlined in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y., or in numerous kits available commercially (e.g., Invitrogen Life Technologies, Carlsbad, Calif).
  • electroporation which may be performed on a variety of cell types, including mammalian cells, yeast cells and bacteria, using commercially available equipment.
  • Optimal conditions for electroporation are experimentally determined for the particular host cell type, and general guidelines for optimizing electroporation may be obtained from manufacturers.
  • Exemplary methods of expression include transduction with a virus that includes a nucleic acid that expresses the transcription factor, e.g., an adenovirus, an adeno- associated virus, or a retrovirus.
  • a virus that includes a nucleic acid that expresses the transcription factor, e.g., an adenovirus, an adeno- associated virus, or a retrovirus.
  • a differentiation condition includes inhibiting the expression or activity of a component of the Notch signaling pathway (Leach, 2005, J. Clin. Gastroenterol, 39:S78-82) in the cells.
  • Notch activity can be inhibited by contacting the cells with an antibody that binds specifically to Notch or a Notch ligand.
  • Notch signaling is inhibited by contacting the cells with an inhibitor of gamma-secretase.
  • the expression of a component of the Notch signaling pathway can be inhibited, e.g., by antisense or siRNA methods.
  • a differentiation condition includes inhibiting the expression or activity of hepatocyte nuclear factor 6 (HNF6; human mRNA sequence GenBank Accession No. NM 004498).
  • HNF6 hepatocyte nuclear factor 6
  • the expression of HNF 6 can be inhibited, e.g., by antisense or siRNA methods.
  • insulin-secreting cells can be isolated following differentiation.
  • the insulin-secreting cells can be isolated by cell sorting or immunological selection methods to obtain a population of cells in which at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the cells in the population express and/or secrete insulin.
  • cells can be administered to a subject by injection or implantation of the cells into target sites in the subject.
  • the cells can be inserted into a delivery device which facilitates introduction by injection or implantation of the cells in the subjects.
  • delivery devices include tubes, e.g., catheters for injecting cells and fluids in to the body of a recipient subject.
  • the tubes additionally have a needle, e.g., a syringe, through which the cells of the disclosure can be introduced into the subject at a desired location.
  • the pancreatic cells can be inserted into such a delivery device, e.g., a syringe, in different forms.
  • the cells can be suspended in a solution or embedded in a support matrix when contained in such a delivery device.
  • the term "solution” includes a pharmaceutically acceptable carrier or diluent in which the cells of the disclosure remain viable.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid to the extent that easy syringability exists.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • Solutions of the disclosure can be prepared by incorporating the pancreatic cells described herein in a pharmaceutically acceptable carrier or diluent and as require other ingredients enumerated above, followed by filtered sterilization.
  • Support matrices in which cells, e.g., insulin-secreting cells, can be incorporated or embedded include matrices that are recipient-compatible and that degrade into products that are not harmful to the recipient. Natural and/or synthetic biodegradable matrices are examples of such matrices. Support matrices include plasma clots; collagen matrices; basement membrane matrices (e.g., MatrigelTM basement membrane matrix) (Bonner-Weir et al, 2000, Proc. Natl. Acad. Sci.
  • thermoreversible gelation polymer (TGP) matrices thermoreversible gelation polymer (TGP) matrices; alginate (Duvivier-Kali et al., 2001, Diabetes, 50:1698- 1705); and synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.
  • TGP thermoreversible gelation polymer
  • alginate Duvivier-Kali et al., 2001, Diabetes, 50:1698- 1705
  • synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.
  • Other example of synthetic polymers and methods of incorporating or embedding cells into the matrices are known in the art. See, e.g., U.S. Pat. No. 4,298,002 and U.S. Pat. No. 5,308,701.
  • the matrices provide support and/or protection (e.g., protection from immunological responses) for the cells, e.g., insulin-secreting cells,
  • Common methods of administering cells include implantation of cells in a pouch of omentun (Yonda et al., 1989, Diabetes, 38: 213-216); intraperitoneal injection of the cells (Wahoff et al., 1994, Transplant. Proc, 26:804); implantation of the cells under the kidney capsule of the subject (see, e.g., Liu et al., 1991, Diabetes, 40:858-866; Korgren et al., 1998, Transplantation, 43: 509-514; Simeonovic et al., 1982, Aust. J. Exp. Biol. Med.
  • the cells can be embedded in a support matrix.
  • Cells can be administered in a pharmaceutically acceptable carrier or diluent as described herein.
  • diabetes is a general term to describe diabetic disorders as they are recognized in the art, e.g., Diabetes Mellitus. Diabetes Mellitus is characterized by an inability to regulate blood glucose levels. The two most prevalent types of diabetes are known as Type I and Type II diabetes. The term also encompasses the myriad secondary disorders caused by diabetes, both acute and chronic, e.g., diabetic complications, e.g., hypoglycemia and hyperglycemia, retinopathy, angiopathy, neuropathy, and nephropathy. Examples of diabetes include insulin dependent diabetes mellitus and non-insulin dependent diabetes.
  • Type 1 diabetes Insulin dependent diabetes mellitus
  • Type 2 diabetes mellitus is a metabolic disease of impaired glucose homeostasis characterized by hyperglycemia, or high blood sugar, as a result of defective insulin action which manifests as insulin resistance, defective insulin secretion, or both.
  • a patient with Type 2 diabetes mellitus has abnormal carbohydrate, lipid, and protein metabolism associated with insulin resistance and/or impaired insulin secretion.
  • the disease leads to pancreatic beta cell destruction and eventually absolute insulin deficiency. Without insulin, high glucose levels remain in the blood.
  • the long term effects of high blood glucose include blindness, renal failure, and poor blood circulation to these areas, which can lead to foot and ankle amputations. Early detection is critical in preventing patients from reaching this severity.
  • the majority of patients with diabetes have the non-insulin dependent form of diabetes, currently referred to as Type 2 diabetes mellitus.
  • Exemplary models of Type I diabetes include: Komeda diabetes-prone rat (Yokoi, 2005, Exp. Anim., 54: 111-115); streptozocin (STZ) treated animals; BBDP rat (Hillebrands et al, 2006, J. Immunol, 177:7820-32); NOD mouse (Aoki et al, 2005, Autoimmun. Rev., 4:373-379); the G ⁇ ttingen minipig (Larsen et al., 2004, ILAR J., 45:303-313); and several non-human primate models (Gaur, 2004, ILAR J., 45:324-333).
  • Exemplary models of Type II diabetes include: a transgenic mouse having defective Nkx-2.2 or Nkx-6.1; (U.S. Pat. No. 6,127,598); Zucker Diabetic Fatty fa/fa (ZDF) rat. (U.S. Pat. No. 6,569,832); Rhesus monkeys, which spontaneously develop obesity and subsequently frequently progress to overt type 2 diabetes (Hotta et al., 2001, Diabetes, 50: 1126-33); and a transgenic mouse with a dominant-negative IGF-I receptor (KR-IGF-IR) having Type 2 diabetes-like insulin resistance.
  • KR-IGF-IR dominant-negative IGF-I receptor
  • Step 1 the IHBEC-rich fraction is isolated from adult mouse liver with 1 x 10 5 viable cells/mouse being recovered.
  • Adult 8 week male C57/BL6 mice were obtained from Taconic Farms (Germantown, NY). All animals were bred and maintained under pathogen- free conditions in accordance with housing and husbandry guidelines.
  • To isolate IHBECs a two-step liver perfusion was performed (Nagaya et al., 2006, Hepatology, 43:1053-62).
  • the remnant tissues were collected and minced, transferred into a flask, and then treated with 0.02% soybean trypsin inhibitor (GIBCO, Invitrogen Corporation, California, CA) and 0.04% collagenase solution (collagenase D, Roche, Indianapolis, IN) for 7 minutes.
  • the digested tissues were suspended in Dulbecco's modified Eagle's medium (DMEM, GIBCO) with 10% FBS and 100 mg/L penicillin and 100 mg/L streptomycin, and centrifuged at 150 x g for 1.5 minutes. The pellet was resuspended in medium, filtered sequentially through 250, 100, and 40 ⁇ m nylon mesh. Small cell aggregates on the 40 ⁇ m mesh were retrieved. 1 x 10 5 viable cells were typically recovered.
  • DMEM Dulbecco's modified Eagle's medium
  • Isolated IHBECs were cultured with collagen embedded floating culture method (CEFCM). Viable isolated cells were suspended in DMEM/F12 (GIBCO) on ice containing 0.3 mg/mL collagen (Collagen type I rat tail, Becton Dickenson (BD), Franklin Lakes, NJ). Then 1.0 x 10 4 cells/ml/well were plated in a 12-well dishes. The collagen containing the aggregates was allowed to solidify and was then incubated at 37 0 C for 2 hours.
  • CEC collagen embedded floating culture method
  • GM growth medium
  • DMEM/F12 supplemented with 5% FBS, 5% NuSerumTM IV Serum (BD), 0.5 ⁇ g/mL insulin-transferrin-sodium selenite (ITS) (GIBCO), 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 "7 M dexamethasone (Sigma-
  • GM was changed every 2 days.
  • Fig. 2A left and 2B left show macroscopic and phase contrast microscopic views at days 0 and 1 , respectively. After about 5 days with
  • IHBECs formed three-dimensional ductal cysts (Fig. 2B, right) and rapidly expanded their number about 15-fold within 2 weeks (Fig. 2E).
  • the collagen gel containing the expanding cells gradually shrank over 14 days (Fig. 2D).
  • IHBECs initially expanded but then started to die at around 8 days (Fig. 2E).
  • RT-PCR and immunofluorescent staining were carried out with hepatocyte, biliary epithelial cell (BEC), and islet markers.
  • the cDNA products were then diluted to a concentration corresponding to 20 ng/ ⁇ l.
  • RT-PCR was performed using PCR Master Mix (ABgene, New York, NY) to monitor the transcription of genes related to the genesis of insulin-producing cells.
  • the primers were complementary to the mRNA sequences of the genes of interest and are listed in Table 1.
  • the primers were designed to cross exon-exon boundaries to exclude the possibility that genomic DNA was amplified. The exception was MafA, which lacks an intron. All primers had a calculated Tm of 60 0 C.
  • ABI 7300 real-time PCR system (Applied Biosystems) was used according to the manufacturer's instructions for 36 cycles.
  • the probe and primer sets of mouse insulin 1 (assay identification no. Mm01259683_gl) and ⁇ -Actin (part no. 4352341E) were purchased from Applied Biosystems; they are specific for mouse and do not bind to cDNA of mouse kidney, or liver.
  • cDNA Twenty ng cDNA was applied to each well, and levels of mRNA were determined as the average of triplet aliquots. Levels of insulin mRNA expression were normalized to those of the internal control ⁇ -Actin. Data were compared with results from mouse liver run in parallel.
  • Cells at day 0 expressed BEC markers (Fig. 2G), but neither hepatocyte (Fig. 2F) nor islet (Fig. 21) markers.
  • IHBECs expressed a number of hepatocyte nuclear factor (HNF) family genes (HNF l ⁇ , HNF3 ⁇ , HNF4 ⁇ , HNF6; Fig. 2H) considered to be endoderm progenitor markers (Wilson et al, 2003, Mech. Dev., 120:65-80).
  • HNF hepatocyte nuclear factor
  • Immunostaining for CK7 and CKl 9 was performed essentially as previously described (Nagaya et al, 2006, Hepatology, 43:1053-62). Briefly, cells on dishes were fixed in cold absolute ethanol after three rinses with phosphate-buffered saline (PBS). Primary antibodies included mouse anti-cytokeratin 7 (CK7; Dako Cytomation) and rabbit anti-cytokeratin 19 (CKl 9), rabbit anti-NeuroD (1 :200; Cell Signaling Technology, Inc., Danvers, MA). AlexaTM488-conjugated and AlexaTM594-conjugated antibodies (Molecular Probes, Eugene, OR) were used as secondary antibodies.
  • PI VECTASHIELDTM Mounting Medium with PI; Vector Laboratories, Ltd., Peterborough, England. Cultures processed with secondary antibodies only were used as negative controls. Liver and pancreas sections were stained as positive controls. Day 0 and day 17 cells stained positive for both for CK7 (Figs. 2J- 2K) and CKl 9. Dispersed IHBECs at day 14 were seeded onto collagen-coated dishes and cultured for 3 days (Step 3). The cells were washed with Ca 2+ -free HEPES-buffered saline, and a 0.1% collagenase solution was added for 20 minutes to dissolve the collagen gel and release the aggregated IHBECs fragments.
  • the cells were then counted with trypan blue on a hemocytometer. After counting, the dispersed IHBECs were allowed to settle, then resuspended in GM and plated onto collagen-coated, 35-mm plastic dishes (BD) or 12 well dishes (BD) and maintained at 37 0 C in a humidified 5% CO 2 incubator. The IHBECs formed colonies and expanded about 1.4-fold during these 3 days.
  • Contrast light microscopy of cultured IHBECs at day 17 without CEFCM displayed signs of senescence, including enlargement, flattening (Fig. 3 A, black arrows), and increased number of cytoplasmic vacuoles (Fig. 3 A, white arrows).
  • the IHBECs subjected to CEFCM manipulation had fewer senescent cells or cytoplasmic vacuoles (Fig. 3B).
  • DM serum- and insulin-free differentiation medium
  • DM serum- and insulin-free differentiation medium
  • GlutaMAXTM-I Supplement serum- and insulin-free differentiation medium
  • the IHBECs were randomly divided into 3 groups.
  • the cells were maintained in untreated condition (Group 1), transduced with adenovirus (AdV) expressing green fluorescent protein (GFP) (Group 2), or transduced with AdV expressing Pdx-1, NeuroD, and Pdx-l/VP16 (Group 3).
  • AdV adenovirus
  • GFP green fluorescent protein
  • AdV AdV expressing Pdx-1, NeuroD, and Pdx-l/VP16
  • the media were changed every 2 days.
  • DM Control Group 1
  • IHBECs started to express some endocrine progenitor genes (Ngn 3, NeuroD, Nkx ⁇ .l and Pdx-1) but lacked insulin mRNA (Fig. 5A). Therefore, the IHBECs were then transduced with AdVs expressing Pdx-1, Neuro D, and Pdx-l/VP16.
  • AdVs expressing Pdx-1, NeuroD, and Pdx-1 /VP 16 driven by a cytomegalovirus (CMV) promoter were prepared with the AdEasy TM Adenoviral Vector System (Stratagene, La Jolla, CA) as previously described (Kaneto et al, 2005, Diabetes, 54:1009-22; Yatoh et al., 2007, Diabetes Metab. Res. Rev, 23:239-249). These AdVs all carried the reporter GFP.
  • the control adenovirus (AdVs-GFP) was prepared in the same manner. The integration of each gene into the adenovirus was done by transfection into the adenovirus packing 293 cell line according to the manufacturer's instructions.
  • Adenovirus titers were further increased up to 1 x 10 10 plaque forming units (PFU)/ml with Vivapure AdenoPACKTM 100 purification kits (Vivascience, Edgewood, NY).
  • the medium was changed to serum- free DMEM medium containing purified recombinant AdV-Pdx-1, -Neuro D, or Pdx-l/VP16 and incubated for 2 hours at 37 0 C.
  • MOI multiplicity of infection
  • IHBECs were infected at MOIs of 1, 10, 25, 50, 100, or 200 for 2 hours at 37 0 C.
  • GFP + cells were counted by microscope (Olympus, Tokyo, Japan) to determine the transduction efficiency. Transduction efficiency was describes as percentage of GFP + cells in all cells. Seven days after transduction, the cells were harvested and evaluated.
  • the 50 MOI recombinant adenovirus transductions for Pdx-1, NeuroD, or 100 MOI for Pdx-l/VP16 resulted in efficient expression of the transgenes under the control of CMV promoter having 30-50% transduction efficiency with high cell survival (Figs. 4A-4C).
  • Example 3 Gene Expression Profiles of IHBEC-Derived Insulin-Producing Cells To analyze the molecular events occurring in IHBECs during the series of culture steps, gene expression profiles of transcription factors and pancreas-related genes at day 26 of culture were determined by RT-PCR as described above (Figs. 5A-5D). Whereas IHBECs at Step 1 only expressed biliary epithelial cell markers (Fig. 2G) and early liver and pancreas markers (Fig. 2H), At the end of Step 4, Group 1 (medium control) cells expressed the pancreatic progenitor markers Ngn3, NeuroD, Nkx ⁇ .l, Pdx-1 (Fig.
  • pancreas-specific transcription factor l ⁇ pancreas-specific transcription factor l ⁇ (Ptfl ⁇ ) (Fig. 5C).
  • Pdx-1, NeuroD or Pdx-l/VP16 Group 3
  • Pdx-1 or Pdx-1 /VP 16 transduced cells faintly expressed amylase 2 (Fig. 5C).
  • Expression of Ptfl ⁇ was found in all groups of the cells (Fig. 5C).
  • Insulin 1 mRNA expression in all groups was assessed on day 26 by quantitative RT-PCR (Figs. 5 A, 5E).
  • Pdx-1 /VP 16 was the most effective inducer of insulin expression in IHBECs, however, compared with primary mouse islets, the expression of insulin mRNA was low (Adv-Pdx-1, 6.18 ⁇ 0.45 x 1(T 5 ; Adv-NeuroD, 5.81 ⁇ 0.54 x 10 ⁇ 5 , Adv-Pdx-l/VP16, 3.86 ⁇ 1.71 x 10 5 compared to adult mouse islets).
  • Insulin 1 mRNA was not detected in IHBECs cultured in DM medium control (Group 1), or Adv-GFP control cells (Group 2) (Fig. 5E).
  • Insulin release into the culture medium was measured 7 days after transduction (Fig. 7A). Insulin levels in culture supernatants was measured using the Ultra Sensitive Insulin ELISA (Crystal Chem, Inc, IL) (detection limit of 0.04 ng/ml). The fold stimulation was calculated for each culture by dividing the insulin concentration in the stimulation supernatant by the insulin concentration in the basal supernatant. After 48 hours in culture, AdVs-Pdx-1, NeuroD, and Pdx-l/VP16 transduced IHBECs all released insulin.
  • Pdx-1 /VP 16 transduced cells had significantly higher insulin secretion than other transduced cells (Pdx-1, 1.1 ⁇ 0.1; NeuroD, 8.0 ⁇ 1.4; Pdx-l/VP16, 17.3 ⁇ 3.1 ng / 1.2 x 10 5 cells) (Fig. 7A).
  • the large amount of insulin released over 48 hours is surprising considering that only a small percentage of cells (about 3%) were more highly differentiated.
  • Cells of control groups 1 and 2 did not detectably release insulin (Fig. 7A). The release of insulin into the culture medium in response to various stimuli was monitored for group 3 cells (Fig. 7B).
  • the cells were subjected to static incubation in Krebs-Ringer bicarbonate buffer (KRBB; 133 mM NaCl, 4.69 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 25 mM HEPES, 2.52 mM CaCl 2 ⁇ H 2 O, 5 mM NaHCO 3 ) supplemented with 5 mM glucose and 0.2% BSA.
  • KRBB Krebs-Ringer bicarbonate buffer
  • the concentration of the insulin secreted into the buffer solution was measured as described above.
  • PI propidium iodide
  • nested PCR was performed. Multiplex single-cell RT-PCR analysis was performed according to a previously described method with some modifications (Miyamoto et al., 2002, Dev. Cell, 3:137-147). Briefly, reverse-transcription was performed followed by a first round of PCR. Sorted single cells were deposited into 96-well U-bottom plates (BD) with 7.5 ⁇ l lysis and RT buffer containing gene-specific reverse primers for CK 19, Neuro D, Pdx-1, Insulin 1, Insulin 2, Glucagon, Somatostatin, PP, and HPRT.
  • BD 96-well U-bottom plates
  • the lysis buffer contained the following: [Ix First Strand Buffer (Invitrogen), 10 rnM DTT (Invitrogen), 1 mM dNTPs (New England BioLabs, Ipswich, MA), 0.5% TritonTM X-IOO (Sigma), 0.1% bovine serum albumin, 10 U/ ⁇ l M-MLV Reverse Transcriptase (Invitrogen), 0.1 U/ ⁇ l RNase inhibitor (Invitrogen), 0.4 pM reverse primers]. Retrieved cells were lysed by pipetting several times in the plate, and then cell lysates were transferred to a 96-well optical Reaction Plate with Barcode (Applied Biosystem, Foster City, CA).
  • the samples were incubated for generation of cDNA by reverse transcription and at 94 0 C for 30 seconds to inactivate the enzyme.
  • the first- round PCR was carried out in the same plate by addition of premixed PCR buffer containing the gene-specific forward primers [Ix GeneAmp® PCR Gold Buffer (Applied Biosystem), 2.5 mM MgCl 2 , AmpliTaqTM Gold 0.1 U/ ⁇ l, 0.1 pM forward primers (Table I)] .
  • the total volume of each of the 1st PCR reactions was 20 ⁇ l, and PCR was performed with the following parameters: 36 cycles of 30 seconds at 94 0 C, 90 seconds at 60 0 C, 90 seconds at 72 0 C.
  • the 2nd-round PCR was performed as follows: 1 ⁇ l of the first-round PCR reactions was plated into new PCR plates, which contained mixed buffer [Ix GeneAmp® PCR Gold Buffer (Applied Biosystem), 2.5mM MgCl 2 , AmpliTaqTM Gold (Applied Biosystem) 0.1 U/ ⁇ l, and 0.25 pM forward primers (Table I)] .
  • each gene was analyzed separately using nested gene-specific primers (Table 1) with the following parameters: 35 cycles of 36 seconds at 94 0 C, 90 seconds at 60 0 C, 90 seconds at 72 0 C. Aliquots of 2nd-round PCR products were subjected to gel electrophoresis. Two hundred pg of total RNA isolated from mouse islets was used as a positive control.
  • GFP + cells Twenty three percent Of GFP + cells expressed both the insulin 1 and 2 genes. Importantly, these insulin 1 and 2 expressing cells expressed other marker genes including islet genes (glucagon, somatostatin, and PP) and CKl 9 (Table 2). In Groups 1 and 2, no cells exhibited insulin gene expression (Tables 3-4). Greater than 94% of Pdx-l/VP16 transduced cells expressed CK19, and all cells that expressed insulin genes also expressed CKl 9.
  • Example 7 Transdifferentiation by Inhibition of Notch Signaling To enhance transdifferentiation, the Notch signaling pathway is inhibited in cells with and without transduction using adenoviral (AdV) vectors expressing key transcription factors.
  • AdV adenoviral
  • notch ligands (jagged 1 and 2, delta-like 1-3) of cells interact with their receptors Notch 1-3 on adjacent cells. This interaction leads to the cleavage of Notch by a ⁇ -secretase activity, such that the notch intracellular domain (NICD) is released and translocates to the nucleus where it regulates transcription.
  • ⁇ -secretase inhibitors have been used to block Notch signaling: ⁇ -secretase inhibitor II (Calbiochem, 50 ⁇ M) in DMSO was effective in epithelial cells when added to cells for 6 hours (Chojnacki et al, 2003, J.
  • DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S- phenylglycine t-butyl ester; 10 or 20 ⁇ M, Calbiochem
  • DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S- phenylglycine t-butyl ester; 10 or 20 ⁇ M, Calbiochem
  • Notch signaling may be quiescent (Jensen et al., 2000, Nat. Genet., 24:36-44; Miyamoto et al., 2003, Cancer Cell, 3:565-576).
  • Environmentally- induced alterations in Notch signaling in IHBECs in vitro can lead to accelerated pancreatic endocrine differentiation.
  • Either ⁇ -secretase inhibitor II or DAPT are added to the cell culture at Step 4 of transdifferentiation (Fig. 1).
  • the efficacy of the inhibition is determined in cells at 9 days in DM by real-time PCR for mRNA of the Notch family member hesl (a key downstream mediator of Notch signaling as a measure of effectiveness) and for insulin 1 mRNA.
  • Western blotting is carried out for Hesl and an ELISA assay is used to measure insulin content in transdifferentiated cells. Further, SOX9 expression is determined by antibody staining.
  • Example 8 Transdifferentiation by Inhibition of HNF 6 Expression
  • AdV adenoviral vectors expressing key transcription factors
  • HNF6 is an early transcription factor controlling the initiation of BEC differentiation (Clotman et al, 2002, Development, 129:1819-28). Knockdown of HNF6 with siRNA enhances the transdifferentiation of IHBECs. That transdifferentiation of
  • IHBECs is enhanced by short-term down-regulation of HNF6 expression, because the knockdown enhances dedifferentiation.
  • HNF6 siRNAs are used to dedifferentiate HNF6 siRNAs.
  • FITC and Cy TM -3 fluorophore-labeled Luciferase (Luc) siRNA GL2 duplexes are used for the assessment of siRNA delivery into IHBECs.
  • the sense and antisense strands of the 2'-ACE RNAs are: 5'-[FLUOROPHORE]-CGUACGCGGAAUACUUCGAdTdT-3'
  • the mouse HNF6-siRNA is obtained (one cut domain, family member 1; NM_008262A; Abgene, Rochester, NY) as an annealed, predesigned siRNA. Samples are divided into 3 groups: (1) untreated controls, (2) HNF6-siRNA transfected, and
  • Luciferase-siRNA (Luc-siRNA) transfected IHBECs as a nonspecific siRNA controls.
  • the siRNA is packaged using LipofectamineTM 2000 (Invitrogen) at a DNA-to-liposome ( ⁇ g/ ⁇ L) ratio of 1 :3, following the manufacturer's recommendations in a delivery volume of 100 ⁇ L. Dose titrations are optimized. Samples are transfected and incubated at 37 0 C, 5% CO 2 for 48 hours. The Cy3-labeled Luc-siRNA is used for fluorescent imaging of siRNA- transfected IHBECs in vitro, luciferase assay, and as a nonspecific siRNA control for quantitative RT-PCR. The FITC-labeled Luc-siRNA is used for FACS analysis of single- cell IHBECs suspensions to determine transfection efficiency and duration of the efficacy.
  • LipofectamineTM 2000 Invitrogen
  • a combination of inhibition of Notch signaling pathway by ⁇ -secretase inhibitor or DAPT and knockdown of HNF 6 by siRNA manipulations is used for transdifferentiation with and without using adenoviral (AdV) vectors expressing key transcription factors.
  • AdV adenoviral vectors expressing key transcription factors.
  • the efficacy of the combination is determined in cells at 9 days in DM by real-time PCR for insulin 1 mRNA and an ELISA assay to measure insulin content.
  • AAV a non-pathogenic parvovirus
  • AAV serotype 8 vectors which have low immunogenicity and high transduction efficiency, long-term and stable expression of the delivered transgene, and persistence in the liver, with transduction efficiencies comparable to that of AdVs
  • AdVs AdVs
  • AAV-NeuroD and AAV- Pdxl/VP16 are produced, purified using cesium chloride density centrifugation, and titered by quantitative dot blot analysis.
  • AAV-I, 2, 5, 6, and others are also produced and tested.
  • Example 11 Enhanced Differentiation to Insulin-Producing Cells in vivo
  • IHBECs Enhanced differentiation to insulin-producing cells is fostered by the in vivo environment.
  • the transplanted cells are characterized for function, structure, mass and their capacity to reverse diabetes.
  • Differentiated or undifferentiated insulin-secreting IHBECs are transplanted into STZ-diabetic ICR-scid mouse recipients to determine their capacity for further maturation.
  • ICR/scid mice are given STZ (180-220 mg/Kg BW - freshly dissolved in citrate buffer, pH 4.5) with a single intra-peritoneal injection. Once non-transplanted animals reach blood glucose levels greater than 350 mg/dL for at least 10 days, the IHBECs are transplanted.
  • IHBECs are aspirated into a 200 ⁇ l pipette tip connected to a 1 ml Hamilton syringe and allowed to sediment. The tip is then connected to PE-50 polyethylene tubing, into which the IHBECs are injected. The tubing is then folded and centrifuged for 1.5 minutes at 1200 rpm, and the tip again connected to the Hamilton syringe. With the mouse under light anesthesia, the left kidney is exposed via a flank incision. A capsulotomy is performed in the lower kidney pole, the tubing inserted beneath the capsule and gently advanced to the upper pole. IHBECs are gently injected and the capsulotomy cauterized with a disposable low-temperature cautery device.
  • the cells are transplanted to the renal subcapsular space, and cells are also transplanted with a basement membrane matrix (e.g., MatrigelTM basement membrane matrix) (Bonner-Weir et al, 2000, Proc. Natl. Acad. Sci. USA, 9:7999-8004) or a thermoreversible gelation polymer (TGP) matrix subcutaneously or into the epididymal fat pad.
  • a basement membrane matrix e.g., MatrigelTM basement membrane matrix
  • TGP thermoreversible gelation polymer
  • the number of the cells used for transplants is 5,000,000 for non-transduced cells and 50,000 for transduced cells, respectively.
  • body weight and glucose levels are determined once a week.
  • Blood is obtained from the snipped tail and glucose is measured with a portable glucose meter (Precision QID). Grafts are initially evaluated at 3 weeks, and also evaluated at later time points.
  • IHBECs are manipulated to inhibit the Notch signaling pathway (as described in Example 7) and knockdown HNF6 (as described in Example 8) in vitro. 50,000 to 5,000,000 cells are then transplanted into mice as described in Example 11. Blood glucose levels and weights of the mice are monitored.
  • Insulin-producing cells for transplantation are generated using a combination of AAVs-NeuroD or Pdx- 1 /VP 16 with inhibition of the Notch signaling pathway and/or knockdown of HNF 6 gene.
  • the cells are transplanted following transdifferentiation with or without purification of insulin-producing cells by FACS using the anti- ⁇ -cell antibody IC2 (Brogren et al., 1986, Diabetologia, 29:330-333; Aaen et al., 1990, Diabetes, 39:697- 701; Moore et al., 2001, Diabetes, 50:2231-36).
  • Transduced cells, which produce insulin are retrieved on an ARIA cell sorter with the Summit software (BD).
  • the cells are dispersed, and Propidium Iodide (PI) is used for exclusion of dead cells.
  • PI Propidium Iodide
  • IHBECs and IHBEC-derived cells are also placed into alginate capsules and transplanted into the peritoneal cavity.
  • simple alginate microcapsules 900-1,100 mm in diameter
  • poly lysine coating are used, which have been used previously for successful transplants in mice (Duvivier-Kali et al., 2001, Diabetes, 50: 1698-1705).
  • IHBEC derived cells are resuspended in 1.2 ml of 1.5% (w/v) ultrapurified sodium alginate, and the droplets are made with an electrostatic droplet generator.
  • the gel beads are formed by crosslinking with BaCl 2 (10 mM).
  • Encapsulated IHBECs are suspended in 5 ml of sterile phosphate-buffered saline and transplanted into the peritoneal cavity of diabetic mice through a small ventral incision using a 50 ml pipette. Mice are monitored with twice weekly measurement of non-fasting blood glucose and body weight.
  • IHBEC-derived cells obtained from mouse insulin promoter GFP mice (MIP-GFP) are mixed with pieces of mouse pancreas from stage El 5. The GFP allows for identification of insulin-producing cells derived from the IHBECs. Transdifferentiation is monitored in vitro and following transplantation into STZ-diabetic ICR-scid mice.
  • MIP-GFP mouse insulin promoter GFP mice

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  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de génération d'une population de cellules ayant des cellules produisant de l'insuline, comprenant l'obtention d'une population de cellules épithéliales biliaires intrahépatiques; l'agrandissement de la population de cellules; la mise en culture de la population de cellules agrandie dans un milieu manquant d'insuline et du sérum; et la mise en contact de la population de cellules avec un état de différenciation pendant un temps suffisant pour qu'au moins une partie des cellules sécrètent de l'insuline.
PCT/US2009/040266 2008-04-11 2009-04-10 Procédés de génération de cellules produisant de l'insuline WO2009126927A2 (fr)

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WO2012168434A1 (fr) * 2011-06-08 2012-12-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Reprogrammation partielle de cellules somatiques en cellules souches tissulaires induites (its)
US8835400B2 (en) 2010-10-08 2014-09-16 Mina Therapeutics Limited RNA molecules that upregulate insulin production
EP2975116A1 (fr) * 2014-07-16 2016-01-20 Max-Delbrück-Centrum für Molekulare Medizin Reprogrammation induite par Tgif2 des cellules hépatiques pour cellules progénitrices pancréatiques et leurs utilisations médicales
WO2016108237A1 (fr) * 2014-12-30 2016-07-07 Orgenesis Ltd. Procédés de transdifférenciation et procédés d'utilisation de ceux-ci
US9982236B2 (en) 2013-06-13 2018-05-29 Orgenesis Ltd. Cell populations, methods of transdifferentiation and methods of use thereof
US10202601B2 (en) 2013-11-22 2019-02-12 Mina Therapeutics Limited C/EBPα short activating RNA compositions and methods of use
US10494608B2 (en) 2015-04-24 2019-12-03 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
US10975354B2 (en) 2017-05-08 2021-04-13 Orgenesis Ltd. Transdifferentiated cell populations and methods of use thereof
US11060062B2 (en) 2017-10-26 2021-07-13 University Of Copenhagen Generation of glucose-responsive beta cells

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US8835400B2 (en) 2010-10-08 2014-09-16 Mina Therapeutics Limited RNA molecules that upregulate insulin production
US8916534B2 (en) 2010-10-08 2014-12-23 Mina Therapeutics Limited Methods of inducing insulin production
US11365414B2 (en) 2010-10-08 2022-06-21 Mina Therapeutics Limited Methods of inducing insulin production
US9885046B2 (en) 2010-10-08 2018-02-06 Mina Therapeutics Limited Methods of inducing insulin production
US10774331B2 (en) 2010-10-08 2020-09-15 Mina Therapeutics Limited Methods of inducing insulin production
WO2012168434A1 (fr) * 2011-06-08 2012-12-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Reprogrammation partielle de cellules somatiques en cellules souches tissulaires induites (its)
US9982236B2 (en) 2013-06-13 2018-05-29 Orgenesis Ltd. Cell populations, methods of transdifferentiation and methods of use thereof
US10947509B2 (en) 2013-06-13 2021-03-16 Orgenesis Ltd. Cell populations, methods of transdifferentiation and methods of use thereof
US10633659B2 (en) 2013-11-22 2020-04-28 Mina Therapeutics Limited C/EBPα short activating RNA compositions and methods of use
US10202601B2 (en) 2013-11-22 2019-02-12 Mina Therapeutics Limited C/EBPα short activating RNA compositions and methods of use
EP2975116A1 (fr) * 2014-07-16 2016-01-20 Max-Delbrück-Centrum für Molekulare Medizin Reprogrammation induite par Tgif2 des cellules hépatiques pour cellules progénitrices pancréatiques et leurs utilisations médicales
US10526577B2 (en) 2014-07-16 2020-01-07 Max-Delbrueck-Centrum Fuer Molekulare Medizin TGIF2-induced reprogramming of hepatic cells to pancreatic progenitor cells and medical uses thereof
KR20170102308A (ko) * 2014-12-30 2017-09-08 오르제네시스 엘티디. 전환분화 방법 및 이의 사용 방법
EA035360B1 (ru) * 2014-12-30 2020-06-02 Ордженисис Лтд. Способы трансдифференцировки и способы их использования
EP3698803A1 (fr) * 2014-12-30 2020-08-26 Orgenesis Ltd. Procédés de transdifférenciation et leurs procédés d'utilisation
US10179151B2 (en) 2014-12-30 2019-01-15 Orgenesis Ltd. Methods of transdifferentiation and methods of use thereof
WO2016108237A1 (fr) * 2014-12-30 2016-07-07 Orgenesis Ltd. Procédés de transdifférenciation et procédés d'utilisation de ceux-ci
KR102487883B1 (ko) 2014-12-30 2023-01-11 오르제네시스 엘티디. 전환분화 방법 및 이의 사용 방법
US11617769B2 (en) 2014-12-30 2023-04-04 Orgenesis Ltd. Methods of transdifferentiation and methods of use thereof
US10494608B2 (en) 2015-04-24 2019-12-03 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
US11613736B2 (en) 2015-04-24 2023-03-28 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
US10975354B2 (en) 2017-05-08 2021-04-13 Orgenesis Ltd. Transdifferentiated cell populations and methods of use thereof
US11060062B2 (en) 2017-10-26 2021-07-13 University Of Copenhagen Generation of glucose-responsive beta cells

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