Tracing phenotypic reversibility of pancreatic β‐cells in vitro

Aims/Introduction: Studies have suggested that pancreatic β‐cells undergo dedifferentiation during proliferation in vitro. However, due to limitations of the methodologies used, the question remains whether such dedifferentiated cells can redifferentiate into β‐cells. Materials and Methods: We have established a method for cell tracing in combination with fluorescence‐activated cell sorter (FACS). Using this method, mouse pancreatic β‐cells labeled with green fluorescent protein (GFP) under the control of the insulin promoter are collected by FACS. These β‐cells can be traced and characterized throughout the culture process, even when insulin becomes undetectable, because the cells are also marked with monomeric red fluorescent protein (mRFP) driven by the CAG promoter. Results: When cultured with fetal mouse pancreatic cells, FACS sorted β‐cells lost GFP expression, but retained mRFP expression. The cells also lost expressions of genes characteristic of the β‐cell phenotype, such as Pdx1 and glucokinase, indicating dedifferentiation. More than 30% of such dedifferentiated pancreatic β‐cells were detected in S or G2/M phase. Furthermore, these dedifferentiated cells redifferentiated into insulin‐expressing cells on cultivation with a MEK1/2 inhibitor. Conclusions: Our data provide direct evidence that pre‐existing β‐cells can undergo dedifferentiation and redifferentiation in vitro, their phenotype is reversible and that dedifferentiation in β‐cells is associated with progression of the cell cycle. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00051.x, 2010)