Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors
Open accessAbstract
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.
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99% of APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin-19, and were further cultured into more mature cells that resembled hepatocytes.
“Surprisingly, 99% of these APOA-II-GFP-positive cells expressed the hepatoblast markers, such as α-fetoprotein and cytokeratin-19, and were further cultured into more mature cells that resembled the hepatocytes [93].”
From Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through (2022) · cites this paper as [93]
In Results > Purification of hepatic progenitors by FACS (page not provided), more than 99% of the sorted cells were GFP-positive, and the sorted cells expressed AFP and CK19. That 99% figure describes GFP positivity, not the proportion expressing both markers. The Discussion (page not provided) says the sorted cells could differentiate further into more mature hepatocytes. (LLM, conf=0.84)
Experimental — accuracy not yet measuredhESCs encoding GFP driven by the liver-specific APOA-II promoter can be differentiated into hepatic progenitors at day 16.
“For example, human embryonic stem cells (hESCs) encoding for green fluorescent protein (GFP) driven by the liver-specific apolipoprotein A-II (APOA-II) promoter can be differentiated into hepatic progenitors at day 16.”
From Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through (2022) · cites this paper as [93]
Purified integrase-deficient LV facilitated the generation of a population of purified hESC-derived hepatic progenitors that were devoid of integrated viral DNA.
“In a previous study, purified integrase-deficient LV facilitated the generation of a population of purified hESC-derived hepatic progenitors that were devoid of integrated viral DNA.”
From Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through (2022) · cites this paper as [93]
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- Authors
- Yang, G., Si-Tayeb, K., Corbineau, S., Vernet, R., Gayon, R., Dianat, N., Martinet, C., Clay, D., Goulinet-Mainot, S., Tachdjian, G., Tachdjian, G., Burks, D., Vallier, L., Bouillé, P., Dubart-Kupperschmitt, A., & Weber, A.
- Affiliations
- Inserm (FR)
- Université Paris-Sud (FR)
- Centro de Investigacion Principe Felipe (ES)
- University of Cambridge (GB)
- Venue
- BMC Biology
- Published
- 2013-07-19
- Volume
- 11
- Issue
- 1
- Pages
- 86-86
- ISSN
- 1741-7007 (ISSN-L)
- PMC
- PMC3751548
- OpenAlex
- W1980952042
- Cited by
- 26
- References
- 33
- Citations by year
- 2026: 1 · 2025: 1 · 2022: 3 · 2021: 1 · 2020: 2 · 2019: 1 · 2018: 3 · 2017: 5 · 2016: 2 · 2015: 4 · 2014: 3
- FWCI
- 1.34
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- BMC Biology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- HAL (submittedVersion, other-oa)
- Funders
- Wellcome Trust (079249)
- European Commission (223317, 278152)
- Agence Nationale de la Recherche (2010-RFCS-004)
- Institut National de la Santé et de la Recherche Médicale
- Fondation pour la Recherche Médicale
- Agence de la Biomédecine
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Genetics › Mesenchymal stem cell research
- Keywords
- human embryonic stem cells, hepatocyte differentiation, HIV integrase inhibitors, GFP reporter, cell purification, alpha-fetoprotein, cytokeratin 19, albumin secretion, cell therapy safety, drug screening
- MeSH terms
- Cell Differentiation, Cell Line, Cell Separation, DNA, Viral, Flow Cytometry, Genetic Vectors, Humans, Liver, Organ Specificity, Promoter Regions, Genetic, Transduction, Genetic, Biomarkers, Lentivirus, Apolipoprotein A-II, Virus Integration, Genes, Reporter, Hepatocytes, Green Fluorescent Proteins, Cytochrome P-450 CYP3A, Embryonic Stem Cells
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