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

  1. Holger A. Russ1,
  2. Yael Bar1,
  3. Philippe Ravassard2 and
  4. Shimon Efrat1
  1. 1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel
  2. 2Unité Mixte de Recherche 7091, National Center for Scientific Research, Hôpital Pitié Salpêtrière, Pierre and Marie Curie University, Paris, France
  1. 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

Abstract

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.

Footnotes

  • Published ahead of print at http://diabetes.diabetesjournals.org on 3 March 2008. DOI: 10.2337/db07-1283.

    Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-1283.

    The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    See accompanying commentary, p. 1457.

    • Accepted February 26, 2008.
    • Received September 10, 2007.
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  1. Diabetes vol. 57 no. 6 1575-1583
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