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IN VITRO PROLIFERATION OF CELLS DERIVED FROM ADULT HUMAN BETA CELLS REVEALED BY CELL-LINEAGE TRACING

¹Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel
²UMR 7091, National Center for Scientific Research, Hôpital Pitié Salpêtrière, Pierre and Marie Curie University, 75013 Paris, France

Objective: Expansion of insulin-producing beta 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 beta-cell markers in the cultured cells. Here we report a genetic cell-lineage tracing approach for following the fate of cultured beta cells.

Research Design and Methods: Cells dissociated from isolated human islets were infected with 2 lentiviruses, one expressing Cre recombinase under control of the insulin promoter, and the other a reporter cassette with the structure CMV promoter-loxP-DsRed2-loxP-eGFP.

Results: Beta cells were efficiently and specifically labeled by the dual virus system. Label-positive, insulin-negative cells derived from beta 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 non-beta pancreatic 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 beta cells, and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human beta 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.

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