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

Abstract

BackgroundHuman 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.ResultsWe 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.ConclusionsWe 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.

Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules

“…In midphase fetal liver, TGFβ promotes liver progenitor cells to undergo biliary differentiation [44]. Hence it is not surprising that the TGFβ inhibitor SB431542 has been used for the later stages of differentiation of progenitors to hepatocyte-like cells [16,45]. However, these studies used SB431542 in combination with other growth factors.…”

Generation of functional cholangiocyte‐like cells from human pluripotent stem cells and HepaRG cells

“…Our approach recapitulated the key stages of liver development and enabled us to stepwise differentiate hESCs and hiPSCs into definitive endoderm cells, hepatic progenitors, and hepatocyte-like cells in a fully defined medium without feeder cells or serum. 11 , 12 However, the question remains as to whether hepatic progenitors generated by way of different protocols, and in particular in our defined conditions, are able to generate cholangiocytes. In other respects, HepaRG is a well-characterized human hepatoma cell line which is permanently able to differentiate into mature hepatocytes from a stage of bipotent progenitors 13 and which is widely used for pharmacotoxicology assays.…”

Advanced cell‐based modeling of the royal disease: characterization of the mutated F9mRNA

“…Hepatic differentiation was induced using a protocol that mimics liver development in vivo , with a few modifications (Fig. A).…”

“…Hepatic differentiation was induced using a protocol that mimics liver development in vivo [5], with a few modifications ( Fig. 2A Activin A + 80 ng mL À1 FGF2 + 10 ng mL À1 bone morphogenetic protein (BMP) 4 (R&D Systems) + 10 lM Ly294002 (Calbiochem, Merck Millipore, Billerica, MA, USA) + 3 lM Chir99021 (Stemgent, Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 day without Chir99021.…”