Development of a Bmi1+ Cardiac Mouse Progenitor Immortalized Model to Unravel the Relationship with Its Protective Vascular Endothelial Niche.

The adult mammalian heart has been demonstrated to be endowed with low but real turnover capacity, especially for cardiomyocytes, the key functional cell type. The source, however, of that turnover capacity remains controversial. In this regard, we have defined and characterized a resident multipotent cardiac mouse progenitor population, Bmi1+DR (for Bmi1+ Damage-Responsive cells). Bmi1+DR is one of the cell types with the lowest ROS (Reactive Oxygen Species) levels in the adult heart, being particularly characterized by their close relationship with cardiac vessels, most probably involved in the regulation of proliferation/maintenance of Bmi1+DR. This was proposed to work as their endothelial niche. Due to the scarcity of Bmi1+DR cells in the adult mouse heart, we have generated an immortalization/dis-immortalization model using Simian Vacuolating Virus 40-Large Antigen T (SV40-T) to facilitate their in vitro characterization. We have obtained a heterogeneous population of immortalized Bmi1+DR cells (Bmi1+DRIMM) that was validated attending to different criteria, also showing a comparable sensitivity to strong oxidative damage. Then, we concluded that the Bmi1-DRIMM population is an appropriate model for primary Bmi1+DR in vitro studies. The co-culture of Bmi1+DRIMM cells with endothelial cells protects them against oxidative damage, showing a moderate depletion in non-canonical autophagy and also contributing with a modest metabolic regulation.

Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-ß-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.

Viral integration promotes SV40T-induced immortalization by disturbing the expression of DNA/chromosome- and ECM-associated functional genes.

Lentivirus containing simian virus 40 large T antigen (SV40T) is routinely used to induce cell immortalization. However, the roles of viral integration itself in this progress is still controversial. Here, we transformed primary mouse embryonic fibroblasts (MEFs) with SV40T lentivirus and studied the roles of viral integration in the immortalization using RNA sequencing (RNA-seq) and whole genome sequencing (WGS). During the immortalization, differentially expressed genes (DGEs) are enriched in viral infection and several diverse activities. However, DEGs between immortalized and aging cells are significantly enriched in DNA/chromosome- and extracellular matrix (ECM)-associated activities. Gene regulatory network (GRN) analysis shows that although p53 is a key regulatory factor, many other transcription factors also play critical roles in the process, like STAT1. Of these DEGs, 32 genes have viral integration in their coding and/or regulatory regions. Our findings suggest that viral integration may promote SV40T-mediated immortalization by disturbing the expression of DNA/chromosome- and ECM-associated genes.

Establishment and functional characterization of immortalized rabbit dermal papilla cell lines.

Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. In this study, we introduced the SV40 large T antigen (SV40T) into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DPC lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). These cell lines displayed early passage morphology and high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA, IGF1, ALPL, FGF2, BMP2 and TGFß2), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Lagging Strand Initiation Processes in DNA Replication of Eukaryotes-Strings of Highly Coordinated Reactions Governed by Multiprotein Complexes.

In their influential reviews, Hanahan and Weinberg coined the term 'Hallmarks of Cancer' and described genome instability as a property of cells enabling cancer development. Accurate DNA replication of genomes is central to diminishing genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase (Pol-prim), during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair are discussed.

Immortalization Reversibility in the Context of Cell Therapy Biosafety.

Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines.

Immortalization of primary cells derived from the endangered Ryukyu long-furred rat.

The Ryukyu long-furred rat is an endangered species confined to the southernmost three small islands of Japan (Amami-Oshima, Tokunoshima, and Okinawa). Its population is rapidly decreasing because of roadkill, deforestation, and feral animals. To date, its genomic and biological information are poorly understood. In this study, we successfully immortalized Ryukyu long-furred rat cells by expressing a combination of cell cycle regulators, mutant cyclin-dependent kinase 4 (CDK4R24C) and cyclin D1, together with telomerase reverse transcriptase or an oncogenic protein, the Simian Virus large T antigen. The cell cycle distribution, telomerase enzymatic activity, and karyotype of these two immortalized cell lines were analyzed. The karyotype of the former cell line immortalized with cell cycle regulators and telomerase reverse transcriptase retained the nature of the primary cells, while that of the latter cell line immortalized with the Simian Virus large T antigen had many aberrant chromosomes. These immortalized cells would be valuable for studying the genomics and biology of Ryukyu long-furred rats.

Establishment and characterization of immortalized human vocal fold fibroblast cell lines.

PURPOSE: Vocal fold scarring is abnormal scar tissue in the lamina propria layer of the vocal fold. To facilitate investigation of vocal fold scarring, we established and characterized immortalized human vocal fold fibroblast (iHVFF) cell lines. METHODS: Human vocal fold fibroblasts were immortalized by introducing Simian virus 40 large T antigen (SV40TAg) by transfection. Successfully transfected fibroblasts were sorted using flow cytometry. Immunofluorescence cytochemistry and western blot were applied to analyze the expression of fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Cell proliferation rate was measured by CCK-8 assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA expression level. RESULTS: The iHVFFs continued to proliferate for more than 30 generations and appeared spindle-shaped. The expression of Vimentin and α-SMA were detected in both iHVFFs and primary fibroblasts, and enhanced expression of FAP was observed in iHVFFs. Furthermore, iHVFFs exhibited an increased proliferative capability compared with the primary fibroblasts. RT-qPCR results suggested that collagen type III alpha 1 chain (COL3A1), interleukin-6, cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), hepatocyte growth factor (HGF) in the iHVFFs significantly increased, whereas transforming growth factor-ß1 (TGF-ß1), elastin and matrix metallopeptidase-1 (MMP-1) expression significantly downregulated. No differences in mRNA expression of α-SMA, fibronectin and collagen type I alpha 2 chain (COL1A2) were noted between iHVFFs and primary fibroblasts. CONCLUSION: iHVFFs can be used as a novel tool cell for future researches on the mechanisms of pathogenesis and treatment of vocal fold scarring.

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding.

Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring-shaped hexameric helicases that initially encircle dsDNA but transition to ssDNA for function as replicative helicases. How the duplex is initially unwound, and the role of the two helicases in this process, is poorly understood. We recently described an initiation mechanism for eukaryotes in which the two helicases are directed inward toward one another and shear the duplex open by pulling on opposite strands of the duplex while encircling dsDNA [L. D. Langston, M. E. O'Donnell, eLife 8, e46515 (2019)]. Two head-to-head T-Antigen helicases are long known to be loaded at the SV40 origin. We show here that T-Antigen tracks head (N-tier) first on ssDNA, opposite the direction proposed for decades. We also find that SV40 T-Antigen tracks directionally while encircling dsDNA and mainly tracks on one strand of the duplex in the same orientation as during ssDNA translocation. Further, two inward directed T-Antigen helicases on dsDNA are able to melt a 150-bp duplex. These findings explain the "rabbit ear" DNA loops observed at the SV40 origin by electron microscopy and reconfigure how the DNA loops emerge from the double hexamer relative to earlier models. Thus, the mechanism of DNA shearing by two opposing helicases is conserved in a eukaryotic viral helicase and may be widely used to initiate origin unwinding of dsDNA genomes.

A comprehensive study of p53 protein.

The protein p53 has been extensively investigated since it was found 43 years ago and has become a "guardian of the genome" that regulates the division of cells by preventing the growth of cells and dividing them, that is, inhibits the development of tumors. Initial proof of protein existence by researchers in the mid-1970s was found by altering and regulating the SV40 big T antigen termed the A protein. Researchers demonstrated how viruses play a role in cancer by employing viruses' ability to create T-antigens complex with viral tumors, which was discovered in 1979 following a viral analysis and cancer analog research. Researchers later in the year 1989 explained that in Murine Friend, a virus-caused erythroleukemia, commonly found that p53 was inactivated to suggest that p53 could be a "tumor suppressor gene." The TP53 gene, encoding p53, is one of human cancer's most frequently altered genes. The protein-regulated biological functions of all p53s include cell cycles, apoptosis, senescence, metabolism of the DNA, angiogenesis, cell differentiation, and immunological response. We tried to unfold the history of the p53 protein, which was discovered long back in 1979, that is, 43 years of research on p53, and how p53's function has been developed through time in this article.

Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells.

BACKGROUND: Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses. RESULTS: In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1ß, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated. CONCLUSIONS: In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening.

Reduced Expression of Prion Protein With Increased Interferon-ß Fail to Limit Creutzfeldt-Jakob Disease Agent Replication in Differentiating Neuronal Cells.

Immortalized uninfected septal (SEP) neurons proliferate but after physiological mitotic arrest they express differentiated neuronal characteristics including enhanced cell-to-cell membrane contacts and ≥ 8 fold increases in host prion protein (PrP). We compared proliferating uninfected and Creutzfeldt-Jakob Disease (CJD) agent infected cells with their arrested counterparts over 33 days by quantitative mRNA and protein blot analyses. Surprisingly, uninfected arrested cells increased interferon-ß (IFN-ß) mRNA by 2.5-8 fold; IFN-ß mRNA elevations were not previously associated with neuronal differentiation. SEP cells with high CJD infectivity titers produced a much larger 40-68-fold increase in IFN-ß mRNA, a classic host anti-viral response that is virucidal for RNA but not DNA viruses. High titers of CJD agent also induced dramatic decreases in host PrP, a protein needed for productive agent replication. Uninfected arrested cells produced large sustained 20-30-fold increases in PrP mRNA and protein, whereas CJD arrested cells showed only transient small 5-fold increases in PrP. A > 10-fold increase in infectivity, but not PrP misfolding, induced host PrP reductions that can limit CJD agent replication. In contrast to neuronal lineage cells, functionally distinct migratory microglia with high titers of CJD agent do not induce an IFN-ß mRNA response. Because they have 1/50th of PrP of an average brain cell, microglia would be unable to produce the many new infectious particles needed to induce a large IFN-ß response by host cells. Instead, microglia and related cells can be persistent reservoirs of infection and spread. Phase separations of agent-associated molecules in neurons, microglia and other cell types can yield new insights into the molecular structure, persistent, and evasive behavior of CJD-type agents.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (

Establishment of Down's syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT.

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.

BMP9 Can Induce Schwann Cells Expressing Simian Virus 40 T Antigen to Differentiate into Fat and Bone In Vivo and In Vitro.

In our previous study, we constructed Schwann cells (SCs) that stably express Simian virus 40 T antigen (SV40T-SCs). SV40T-SCs functions and markers are similar to those of neural crest cells. There we used bone morphogenetic protein 9 (BMP9) to induce SV40T-SCs differentiation in vitro and in vivo and study possible related mechanism. SV40T-SCs differentiation was induced by BMP9 conditioned medium. The lipogenic differentiation of SV40T-SCs was assessed by Oil Red O staining. Alizarin red and Alcian blue staining, and alkaline phosphatase (ALP) assays were used to evaluate the SV40T-SCs osteogenic differentiation. The expression of adipocyte differentiation (c/EBPα and c/EBPß) and osteoblast differentiation markers (OSX and RUNX2) were detected by quantitative polymerase chain reaction (qPCR). To study possible mechanism related to SV40T-SCs differentiation, the P53 and E2F1 activity were assessed by luciferase reporter plasmid, and Slug and E-cadherin expression by qPCR. In vivo, SV40T-SCs infected by Ad-BMP9 or Ad-GFP were injected under the skin of nude mice. After 4-6 W, the mice were euthanized and subcutaneously mass formed at injecting sites was collected for pathological analysis. After SV40T-SCs were cultured in BMP9 conditioned medium, lipid droplets were formed in the cytoplasm of these cells. Alizarin red and Alcian blue staining were positive, and ALP activity of SV40T-SCs increased significantly. The expression of adipocyte differentiation (c/EBPα and c/EBPß) and osteoblast differentiation markers (OSX and RUNX2) in SV40T-SCs was upregulated by BMP9. SV40T significantly increased Slug expression and decreased E-cadherin expression. SV40T-SCs infected with Ad-BMP9 were able to differentiate into adipose tissue and form a small bone matrix under the nude mice skin. SV40T-SCs have the ability to differentiate into adipocytes and osteoblasts in vivo and in vitro. SV40T can upregulate the Slug expression and downregulate the E-cadherin expression to produce endothelial-to-mesenchymal transition (EMT). The multidirectional differentiation ability of SV40T-SCs may be related to EMT.

Immortalization of primary sheep embryo kidney cells.

Sheep primary epithelial cells are short-lived in cell culture systems. For long-term in vitro studies, primary cells need to be immortalized. This study aims to establish and characterize T immortalized sheep embryo kidney cells (TISEKC). In this study, we used fetal lamb kidneys to derive primary cultures of epithelial cells. We subsequently immortalized these cells using the large T SV40 antigen to generate crude TISEKC and isolate TISEKC clones. Among numerous clones of immortalized cells, the selected TISEKC-5 maintained active division and cell growth over 20 passages but lacked expression of the oncogenic large T SV40 antigen. Morphologically, TISEKC-5 maintained their epithelial aspect similar to the parental primary epithelial cells. However, their growth properties showed quite different patterns. Crude TISEKC, as well as the clones of TISEKC proliferated highly in culture compared to the parental primary cells. In the early passages, immortalized cells showed heterogeneous polyploidy but in the late passages the karyotype of immortalized cells became progressively stable, identical to that of the primary cells, because the TISEKC-5 cell line has lost the large SV40 T antigen expression, this cell line is a valuable tool for veterinary sciences and biotechnological productions.

Establishment of a Stable ß-Casein Protein-Secreted Laoshan Dairy Goat Mammary Epithelial Cell Line.

Mammary epithelial cells are widely used as models in mastitis research and as tools for mammalian bioreactors; however, the short lifespan of these cells limits their utility. Several mammal epithelial cell line models have been established; however, the secretion capacity and the bacterial sensitivity of these lines have not been effectively evaluated. In this study, a stable immortalized goat mammary epithelial cell (GMEC) line was constructed by transfection with the SV40 gene. The monoclonal cells were then passaged through more than 50 generations after puromycin selection. The GMEC line was evaluated by reverse transcriptase polymerase chain reaction, the cell cycle, karyotype analysis, detection of apoptosis, Western blotting, and ß-casein (CSN2) inducible assays. The GMEC line had a strong proliferation capacity relative to the primary GMECs. GMECs had the same karyotype as the primary cells. The GMEC lines maintained basic biological properties and had estrogen, prolactin, and progesterone receptors as same the primary cells. Additionally, the cells and the cell line could synthesize and secrete ß-casein proteins. Finally, the rate of apoptosis of the transfected cells suggested that the cell line could provide a useful tool for signal research and mammary gland bioreactors.

Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region.

The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 107 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 107 TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.

Checkpoint inhibitor blockade and epigenetic reprogrammability in CD8+ T-cell activation and exhaustion.

CD8+ T-cell exhaustion is a dysfunctional state that is regulated through the expression of inhibitory checkpoint receptor genes including the cytotoxic T-lymphocyte-associated antigen 4, programmed death 1, and DNA methylation of effector genes interferon-γ, perforin, and granzyme B. Different strategies have been used to reverse T-cell exhaustion, which is an adverse event of checkpoint inhibitor blockade. Here, we present the mechanisms by which DNA methyltransferase inhibitors and Simian virus 40 large T antigen through viral mimicry can promote the reversion of exhausted CD8+ T cells. We examine how these pharmacological strategies can work together to improve the clinical efficacy of immunotherapies.

Immortalization of porcine hepatocytes with a α-1,3-galactosyltransferase knockout background.

BACKGROUND: In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in "bridging" liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT-KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild-type and GalT-KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. METHODS: Primary hepatocytes extracted from GalT-KO and wild-type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT-KO porcine hepatocytes (GalT-KO-hep) and wild-type porcine hepatocytes (WT). Hepatocyte biomarkers and function-related genes were assessed by immunofluorescence, periodic acid-Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT-PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement-dependent cytotoxicity (CDC) assay was performed with GalT-KO-hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK-8 assay. RESULTS: GalT-KO and wild-type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT-KO-hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT-KO-hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. CONCLUSIONS: Two types of immortalized cell lines of porcine hepatocytes with GalT-KO and wild-type backgrounds were successfully established. GalT-KO-hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.