HES-1 is involved in adaptation of adult human beta-cells to proliferation in vitro.
OBJECTIVE
In vitro expansion of beta-cells from adult human islets could solve the tissue shortage for cell replacement therapy of diabetes. Culture of human islet cells typically results in <16 cell doublings and loss of insulin expression. Using cell lineage tracing, we demonstrated that the expanded cell population included cells derived from beta-cells. Understanding the molecular mechanisms involved in beta-cell fate in vitro is crucial for optimizing expansion and redifferentiation of these cells. In the developing pancreas, important cell-fate decisions are regulated by NOTCH receptors, which signal through the hairy and enhancer of split (HES)-1 transcriptional regulator. Here, we investigated the role of the NOTCH signaling pathway in beta-cell dedifferentiation and proliferation in vitro.


METHODS
Isolated human islets were dissociated into single cells. beta-Cells were genetically labeled using a Cre-lox system delivered by lentiviruses. Cells were analyzed for changes in expression of components of the NOTCH pathway during the initial weeks in culture. HES-1 expression was inhibited by a small hairpin RNA (shRNA), and the effects on beta-cell phenotype were analyzed.


RESULTS
Human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the NOTCH pathway and downregulation of the cell cycle inhibitor p57. Inhibition of HES-1 expression using shRNA resulted in significantly reduced beta-cell replication and dedifferentiation.


CONCLUSIONS
These findings demonstrate that the NOTCH pathway is involved in determining beta-cell fate in vitro and suggest possible molecular targets for induction of beta-cell redifferentiation following in vitro expansion.
Bar
Yael
Y
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
Russ
Holger A
HA
Knoller
Sarah
S
Ouziel-Yahalom
Limor
L
Efrat
Shimon
S
D000328:Adult; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D019070:Cell Lineage / Q000502:physiology; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000502:physiology*; D034741:RNA, Small Interfering; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D000072056:Transcription Factor HES-1; D015854:Up-Regulation / Q000502:physiology
D051792:Basic Helix-Loop-Helix Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018398:Homeodomain Proteins; D034741:RNA, Small Interfering; D051880:Receptors, Notch; D000072056:Transcription Factor HES-1; C078493:HES1 protein, human