Open Access Research article

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors

Guanghua Yang12, Karim Si-Tayeb12, Sébastien Corbineau12, Rémi Vernet1, Régis Gayon3, Noushin Dianat12, Clémence Martinet12, Denis Clay12, Sylvie Goulinet-Mainot12, Gérard Tachdjian5, Gérard Tachdjian5, Deborah Burks4, Ludovic Vallier6, Pascale Bouillé3, Anne Dubart-Kupperschmitt12* and Anne Weber12

Author affiliations

1 INSERM U 972, IFR 93, Bicêtre Hospital, and Paul Brousse Hospital, Villejuif F-94807, France

2 Université Paris-Sud, UMR-S972, IFR 93, Bicêtre Hospital and Paul Brousse Hospital, Villejuif F-94807, France

3 Vectalys SAS, Canal Biotech 2, 3 rue des satellites, Toulouse F-31400, France

4 CIBER de Diabetes y Obesidad, Centro de Investigación Principe Felipe, Eduardo Primo Yúfera 3, Valencia 46012, Spain

5 Department of Cytogenetics, Béclère Hospital, Clamart F-92141, France

6 Department of Surgery, The Anne McLaren Laboratory for Regenerative Medicine, Cambridge CB2 0SZ, UK

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Citation and License

BMC Biology 2013, 11:86  doi:10.1186/1741-7007-11-86

Published: 19 July 2013

Abstract

Background

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.

Results

We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.

Conclusions

We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Keywords:
Tissue-specific progenitor cells; Hepatoblasts; Purification; Non-integrating lentiviral vectors