Methods of transdifferentiation and methods of use thereof
First Claim
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1. A method of manufacturing a population of human insulin producing cells, the method comprising the steps of:
- (a) obtaining adult human liver tissue;
(b) processing said liver tissue to recover primary adult human liver cells;
(c) propagating and expanding said primary adult human liver cells to a predetermined number of cells;
(d) transdifferentiating said expanded cells, wherein said transdifferentiating comprises;
(1) infecting said expanded cells with at least one expression vector, said infecting occurring at a first time period,(i) wherein said at least one expression vector comprises an adenoviral vector comprising a nucleic acid encoding a PDX-1 polypeptide and an expression vector comprising an adenoviral vector comprising a nucleic acid encoding a second pancreatic transcription factor polypeptide selected from NeuroD1 and Pax4, wherein said infecting with said adenoviral vectors occurs at the same time,or(ii) wherein said at least one expression vector comprises an adenoviral vector comprising a nucleic acid encoding a PDX-1 polypeptide and a second pancreatic transcription factor polypeptide selected from NeuroD1 and Pax4;
and(2) infecting said expanded infected cells of (1) with an adenoviral vector comprising a nucleic acid encoding a MafA polypeptide, said infecting occurring at a second time period, wherein said second time period is after said first time period;
and(e) harvesting said transdifferentiated expanded cells;
thereby manufacturing said population of human insulin producing cells having an increased insulin content, or increased glucose regulated insulin secretion, or increased glucose regulated C-peptide secretion, or any combination thereof, compared with control non-transdifferentiated liver cells.
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Abstract
Disclosed herein is a method for manufacturing a population of human insulin producing cells from non-pancreatic β-cells, wherein the resulting insulin producing cells have increased insulin content, or increased glucose regulated secretion of insulin, or a combination of both.
38 Citations
26 Claims
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1. A method of manufacturing a population of human insulin producing cells, the method comprising the steps of:
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(a) obtaining adult human liver tissue; (b) processing said liver tissue to recover primary adult human liver cells; (c) propagating and expanding said primary adult human liver cells to a predetermined number of cells; (d) transdifferentiating said expanded cells, wherein said transdifferentiating comprises; (1) infecting said expanded cells with at least one expression vector, said infecting occurring at a first time period, (i) wherein said at least one expression vector comprises an adenoviral vector comprising a nucleic acid encoding a PDX-1 polypeptide and an expression vector comprising an adenoviral vector comprising a nucleic acid encoding a second pancreatic transcription factor polypeptide selected from NeuroD1 and Pax4, wherein said infecting with said adenoviral vectors occurs at the same time, or (ii) wherein said at least one expression vector comprises an adenoviral vector comprising a nucleic acid encoding a PDX-1 polypeptide and a second pancreatic transcription factor polypeptide selected from NeuroD1 and Pax4; and (2) infecting said expanded infected cells of (1) with an adenoviral vector comprising a nucleic acid encoding a MafA polypeptide, said infecting occurring at a second time period, wherein said second time period is after said first time period; and (e) harvesting said transdifferentiated expanded cells; thereby manufacturing said population of human insulin producing cells having an increased insulin content, or increased glucose regulated insulin secretion, or increased glucose regulated C-peptide secretion, or any combination thereof, compared with control non-transdifferentiated liver cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A population of human insulin producing cells manufactured by a method comprising the steps of:
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(a) obtaining adult human liver tissue; (b) processing said liver tissue to recover primary adult human liver cells; (c) propagating and expanding said primary adult human liver cells to a predetermined number of cells; (d) transdifferentiating said expanded cells, wherein said transdifferentiating comprises; (1) infecting said expanded cells with at least one expression vector, said infecting occurring at a first time period, (i) wherein said at least one expression vector comprises an adenoviral vector comprising a nucleic acid encoding a PDX-1 polypeptide and an expression vector comprising an adenoviral vector comprising a nucleic acid encoding a second pancreatic transcription factor polypeptide selected from NeuroD1 and Pax4, wherein said infecting with said adenoviral vectors occurs at the same time, or (ii) wherein said at least one expression vector comprises an adenoviral vector comprising a nucleic acid encoding a PDX-1 polypeptide and a second pancreatic transcription factor polypeptide selected from NeuroD1 and Pax4; and (2) infecting said expanded infected cells of (1) with an adenoviral vector comprising a nucleic acid encoding a MafA polypeptide, said infecting occurring at a second time period, wherein said second time period is after said first time period; and (e) harvesting said transdifferentiated expanded culture; wherein said population of human insulin producing cells have an increased insulin content or increased glucose stimulated insulin secretion, or increased glucose regulated C-peptide secretion, or any combination thereof, compared with control non-transdifferentiated liver cells. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification