title: Redifferentiation of expanded human pancreatic β-cell-derived cells by inhibition of the NOTCH pathway.
abstract: In vitro expansion of β-cells from adult human pancreatic islets would overcome donor β-cell shortage for cell replacement therapy for diabetes. Using a β-cell-specific labeling system we have shown that β-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key β-cell genes maintain open chromatin structure in expanded β-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced β-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace β-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.
authors.affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors.forename: Yael
authors.initials: Y
authors.lastname: Bar
authors.affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors.forename: Holger A
authors.initials: HA
authors.lastname: Russ
authors.affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors.forename: Elad
authors.initials: E
authors.lastname: Sintov
authors.affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors.forename: Leeat
authors.initials: L
authors.lastname: Anker-Kitai
authors.affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors.forename: Sarah
authors.initials: S
authors.lastname: Knoller
authors.affiliation: Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel. Electronic address: sefrat@post.tau.ac.il.
authors.forename: Shimon
authors.initials: S
authors.lastname: Efrat
mesh_terms: D000293:Adolescent; D000328:Adult; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000235:genetics; D054337:Cell Dedifferentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D044127:Epigenesis, Genetic / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000096:biosynthesis; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D051880:Receptors, Notch / Q000378:metabolism*; D015398:Signal Transduction; D000072056:Transcription Factor HES-1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D007328:Insulin; D051880:Receptors, Notch; D000072056:Transcription Factor HES-1; C078493:HES1 protein, human