Cell populations, methods of transdifferentiation and methods of use thereof

• US 9,982,236 B2
• Filed: 06/13/2014
• Issued: 05/29/2018
• Est. Priority Date: 06/13/2013
• Status: Active Grant

• Current Assignee: Orgenesis Ltd. (Orgenesis Inc.), Tel Hashomer Medical Research Infrastructure And Services Ltd.
• Original Assignee: Orgenesis Ltd. (Orgenesis Inc.), Tel Hashomer Medical Research Infrastructure And Services Ltd.
• Inventors: Ferber, Sarah
• Primary Examiner(s): Johnson, Kara
• Application Number:
  US14/897,262
• Publication Number:
  US 20160130559A1
• Time in Patent Office: 1,446 Days
• CPC Class Codes: A61K 35/38 Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2506/14 from hepatocytes
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2830/30 being an enhancer not forming part of the promoter region
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

The present invention provides methods for the sequential and temporally-regulated administration of pancreatic transcription factors to induce non-pancreatic cells to transdifferentiate and mature along the pancreatic β-cell lineage. The present invention also provides methods for identifying, isolating and enriching transdifferentiation predisposed cells and methods for treating a degenerative pancreatic disorder such as diabetes.

Claims

1. A method of producing a transdifferentiated population of cells having a mature pancreatic beta cell phenotype and function comprising:

a. obtaining an adult mammalian liver cell population enriched for cells predisposed to transdifferentiation, wherein the enriched population is obtained by providing a population of adult mammalian liver cells and isolating a sub-population of cells comprising;

(i) an active wingless-type MMTV integration site family (Wnt) signaling pathway;

or

(ii) increased expression from a glutamine synthase response element (GSRE);

or

(iii) an increased expression of homer scaffolding protein 1 (HOMER1), lysosomal associated membrane protein 3 (LAMP3), or bone morphogenetic protein receptor type 2 (BMPR2), or any combination thereof;

or

(iv) a decreased expression of ATP binding cassette subfamily B member 1 (ABCB1), integrin subunit alpha 4 (ITGA4), ATP binding cassette subfamily B member 4 (ABCB4), or prion protein (PRNP), or any combination thereof;

or any combination thereof;

b. contacting said enriched adult mammalian liver cell population with a pancreatic and duodenal homeobox (PDX-1) polypeptide or a nucleic acid encoding a PDX-1 polypeptide under conditions to allow uptake of the said polypeptides or nucleic acids at a first time period;

c. contacting the population of cells of step (b) with a paired box gene 4 (Pax-4) polypeptide, or a neurogenic differentiation 1 (NeuroD1) polypeptide, or a nucleic acid encoding a Pax-4 polypeptide, or a nucleic acid encoding a NeuroD1 polypeptide under conditions to allow uptake of the said polypeptides or nucleic acids at a second time period; and

d. contacting the population of cells of step (c) with a MAF bZIP transcription factor A (MafA) polypeptide or a nucleic acid encoding a MafA polypeptide under conditions to allow uptake of the polypeptide or the nucleic acid at a third time period;

thereby producing a transdifferentiated population of cells having a mature pancreatic beta cell phenotype and function.

2. The method of claim 1, wherein cells comprising an active Wnt signaling pathway comprise increased expression of Wnt Family Member 3A (Wnt3a), or beta-catenin, or signal transducer and activator of transcription 3 (STAT3) binding elements, or any combination thereof, or decreased expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK1) or Dickkopf WNT signaling pathway inhibitor 3 (DKK3), or activated protein C (APC), or any combination thereof.

3. The method of claim 1, wherein said obtaining comprises incubating an adult mammalian population of liver cells with Wnt3a.

4. The method of claim 1, wherein the second time period is at least 24 hours after the first time period or is at the same time as the first time period, and wherein the third time period is at least 24 hours after the second time period.

5. The method of claim 4, wherein when the second time period is at the same time as the first time period, the third time period is at least 48 hours after the second time period.

6. The method of claim 4, wherein when the second time period is at the same time as the first time period, the nucleic acid encoding PDX-1 polypeptide further encodes NeuroD1 polypeptide or Pax-4 polypeptide.

7. The method of claim 1, wherein said enriched population of liver cells comprises pericentral liver cells.

8. The method of claim 1, wherein said transdifferentiated predisposed cell population comprises an increased percent (%) of transdifferentiated cells having said mature pancreatic beta cell phenotype and function compared with the % of mature transdifferentiated cells produced from a non-enriched population.

9. The method of claim 1, wherein said mature pancreatic beta cell phenotype and function comprises increased insulin production, or increased glucose regulated insulin secretion, or increased glucose regulated C-peptide secretion, or any combination thereof, compared to a control population of non-transdifferentiated adult mammalian liver cells.

10. The method of claim 9, wherein said increased insulin production, increased glucose regulated secretion of insulin, or increased glucose regulated secretion of C-peptide is in combination with decreased gene expression of Isl1 or reduced production of somatostatin, or any combination thereof.

11. The method of claim 1, wherein said mature pancreatic beta cell phenotype comprises an increased expression of the glucagon gene compared to a control population non transdifferentiated of adult mammalian liver cells.