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In Vitro Proliferation of Cells Derived From Adult Human β-Cells Revealed By Cell-Lineage Tracing

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

Corresponding author: Shimon Efrat, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel. E-mail: sefrat{at}post.tau.ac.il

Abbreviations: CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein; EMT, epithelial-to-mesenchymal transition; MOI, multiplicity of infection; MSC, mesenchymal stem cell; NP40, Nonidet P-40; PARP, poly(ADP-ribose) polymerase; RIP-Cre, pTrip RIP405 nlsCRE DeltaU3

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.

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