In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing.
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.


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
Beta-cells were efficiently and specifically labeled by the dual virus system. Label(+), insulin(-) 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 pancreatic non-beta-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.
Russ
Holger A
HA
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
Bar
Yael
Y
Ravassard
Philippe
P
Efrat
Shimon
S
D000328:Adult; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000637:transplantation / Q000821:virology; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016086:Lentivirus / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D016133:Polymerase Chain Reaction
C045073:Cre recombinase; D019426:Integrases