Original Research ArticleGenetically modified C3A cells with restored urea cycle for improved bioartificial liver
Introduction
Chronic liver diseases, which have a mortality rate of 2 million deaths per year, represent a major global public health problem [1]. The recent report on the growing burden of liver disease in Europe points at the increasing number of cirrhosis and liver cancer as the major reason for the problem [2]. Viral infection, excessive drug and alcohol consumption can also cause acute and acute-on-chronic liver failure (ALF and ACLF, respectively). Although ALF is a relatively rare event, its mortality rate of over 80% makes treatment of those suffering from it extremely challenging [3]. Both acute and chronic liver disease require costly liver transplantation (LT) — currently the only definitive treatment for liver failure. However, the major limiting factor for the LT is donor organ shortage. Emerging alternatives to LT include such experimental procedures as hepatocyte transplantation and use of bioartificial liver (BAL) devices. These techniques could represent feasible therapies bridging the time for whole organ transplantation or supporting the regeneration of the liver. Although promising, all these cell-based therapeutic strategies require an adequate source of liver parenchymal cells. Isolated human hepatocytes are ideal for such applications and their capabilities are still being examined [4]. However, as for LT, the main problem is the limited availability of human liver tissue. Additionally, severe limitations for the use of human primary hepatocytes for liver failure therapies are donor variability and the fact that these cells do not proliferate and quickly dedifferentiate in the culture which leads to the loss of their specific functions. Therefore, seeking alternative liver cells sources has gained a lot of interest in recent years [5,6]. Beside well-studied porcine primary hepatocytes [7] and cell lines derived from human hepatocellular carcinoma (HCC): HepaRG [8], HepG2 [9] and its subclone — C3A [[10], [11], [12], [13]], hepatic progenitor cells [14], hepatocyte-like cells obtained from stem cells [15], and iHEPs differentiated from induced pluripotent stem cells (iPSCs) [16] have become a new promising approach. Although hepatocyte-like cells can bring hope, no alternative cell source can yet replace the functionality and efficacy of primary human hepatocytes.
The two most advanced BALs, HepatAssist [7] and ELAD [10], utilize porcine primary hepatocytes and C3A cells, respectively. The use of the porcine hepatocytes is controversial due to potential zoonotic disease transmission, protein–protein incompatibility, and possible immune response [6]. Thus, the biocomponent of choice for current BAL application are highly differentiated human liver tumor-derived cell lines. On the other hand, although HepG2/C3A cells perform a number of physiological functions similar to human hepatocytes, they also have some metabolic deficiencies. First of all, they do not metabolize ammonia efficiently due to their urea cycle being non-functional. This is because the expression levels of arginase I (ARG1) and ornithine transcarbamylase (OTC) genes are too low [17,18]. Very few studies addressed these issues.
Here we report, for the first time, the successful genetic modification of the C3A cells and establishment of the C3A_AO cell line stably overexpressing hARG1 and hOTC genes.
Section snippets
Cell lines and cell culture
Human Hepatocellular Carcinoma — C3A (CRL-10741), Human Osteosarcoma — HOS (CRL-1543), and Human Embryonic Kidney 293T — HEK293T (CRL-11268) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured under standard conditions in a high glucose DMEM (Dulbecco’s Modified Eagle Medium, Sigma Aldrich, Poznan, Poland) supplemented with a 10% fetal bovine serum — FBS (Biological Industries Inc., Beit-Haemek, Israel) and 1% non-essential amino
The efficiency of C3A transduction with lentiviral vectors (LVs) carrying sequences of human ARG1 and OTC genes
To evaluate LV-ARG1- and LV-OTC-mediated transductions, modified C3A cell lines were concurrently labelled with mCherry fluorescent marker protein and analyzed by fluorescence microscopy and flow cytometry (Supplementary Fig. S1). The percentage of mCherry-positive C3A cells in the whole studied population was 31% and increased after second and third rounds of transductions to achieve 43% and 53%, respectively. For further analysis, triple-transduced C3A cells have been chosen.
Subsequently, we
Discussion
This study reports the first establishment of genetically modified C3A cells stably overexpressing two urea cycle genes coding for ARG1 and OTC. The hepatoma C3A cells used for ELAD construction, the most successful BAL hybrid device that had already been tested in patients with hepatic failure [12,13,21,22], are characterized by strong contact inhibition, unlimited expansion in vitro, high albumin production, the ability to grow in glucose-deprived medium, and activity of some of the
Conclusion
Although the physiological capabilities of the new cell line need to be further examined, at this stage of our study we may conclude that the engineered cells are able to convert ammonia to urea more effectively than C3A cells. Moreover, the use of LVs for genetic modifications opens the way for further improvement of the engineered cells by introducing greater number of copies of transgenes at any required ratios.
Authors’ contributions
Concept and design — KDP, AS; experiments and procedures — AS, KEZ, AW, MG, BB; data analysis and presentation — KDP, AS, AW, KEZ, MG, BB, MC, JM; writing of article — KDP, MC, JM, DGP. All authors read and approved the final manuscript.
Funding
Nalecz Institute of Biocybernetics and Biomedical Engineering statutory funding. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
CRediT authorship contribution statement
Krzysztof Dariusz Pluta: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing - original draft, Writing - review & editing. Anna Samluk: Conceptualization, Investigation, Methodology, Formal analysis. Agnieszka Wencel: Investigation, Methodology. Karolina Ewa Zakrzewska: Investigation, Methodology, Formal analysis. Monika Gora: Investigation, Methodology, Formal analysis. Beata Burzynska: Investigation, Methodology, Formal analysis. Malgorzata
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Karolina Zakrzewska’s present address: Institute of Fundamental Technological Research, Polish Academy of Sciences, Warsaw, Poland.