OBJECTIVE-Expansion of insulin-producing β-Cells from adult human islets could alleviate donor shortage for cell- replacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of β-Cell markers in the cultured cells. Here, we report a genetic cell- lineage tracing approach for following the fate of cultured β-Cells. RESEARCH DESIGN AND METHODS-Cells dissociated from isolated human islets were infected with two lentiviruses, one expressing Cre recombinase under control of the insulin promoter and the other, a reporter cassette with the structure cytomegalovirus promoter-loxP-DsRed2-loxP-eGFP. RESULTS-β-Cells were efficiently and specifically labeled by the dual virus system. Label +, insulin- cells derived from β-Cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Isolated labeled cells could be expanded in the absence of other pancreas cell types if provided with medium conditioned by pancreatic non-β-Cells. Analysis of mouse islet cells by the same method revealed a much lower proliferation of labeled cells under similar culture conditions. CONCLUSIONS-Our findings provide direct evidence for survival and dedifferentiation of cultured adult human β-Cells and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human β-Cell proliferation under our culture conditions. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells using a genetic recombination approach that was previously restricted to transgenic animals.