SV40LT mouse fibroblast line retains feeder function without detected tumors

Researchers have established and characterized a mouse embryonic fibroblast line, designated SV40-CMEF, that sustained proliferation beyond the limited working life of primary cells, supported self-renewal of mouse embryonic stem cells and retained adipogenic, osteogenic and chondrogenic differentiation potential. The line was generated from C57BL/6J mouse embryonic fibroblasts by transfection with a plasmid carrying the simian vacuolating virus 40 large T antigen, or SV40LT. Although SV40-CMEF accumulated extensive chromosomal numerical abnormalities, the cells did not form colonies in soft agar or tumors in immunodeficient mice during the study’s three-month observation period. The experiments characterize a cell culture resource; they do not constitute evidence of a clinical benefit.

Mouse embryonic fibroblasts are widely used as feeder layers for pluripotent stem-cell cultures because they create a supportive cellular environment and secrete growth factors and cytokines associated with stem-cell maintenance. Primary fibroblasts have limited proliferative capacity and commonly enter replicative senescence after approximately five passages. Repeated preparation from mouse embryos introduces batch-to-batch variation, increases animal use and can limit the reproducibility and scalability of experiments. The investigators therefore evaluated SV40-CMEF across several dimensions: persistence of the SV40LT transgene, escape from senescence, proliferative capacity, fibroblast identity, genetic authentication, karyotype, tumorigenicity, feeder function, differentiation potential and genome-wide gene expression.

The immortalization procedure began with passage-3 primary fibroblasts isolated from C57BL/6J embryos at postcoitus day 13.5. The cells were maintained in Dulbecco’s modified Eagle medium supplemented with fetal bovine serum and nonessential amino acids, then transfected with the pLenti CMV/TO SV40LT-GFP plasmid. Immortalized cells emerged through continuous passaging without drug selection, and the resulting polyclonal population was named SV40-CMEF. The line is publicly accessible through the China Center for Type Culture Collection and the National Collection of Authenticated Cell Cultures. Routine mycoplasma testing was performed during long-term culture, and PCR was repeated after the cells were deposited to confirm that the deposited material remained mycoplasma-free.

Molecular analyses confirmed that the SV40LT construct persisted during prolonged culture. Gene-specific PCR detected the transgene, and Western blotting identified the SV40 large T antigen protein at the expected molecular weight. The plasmid also carried an EGFP reporter, and flow cytometry detected EGFP in 95% of the established cells. Quantitative PCR estimated approximately four SV40LT copies per mouse genome at low passage, defined as no more than 25 passages, and five copies per genome at high passage, defined as at least 75 passages, supporting genomic integration. PCR also amplified a roughly 3.4-kilobase DNA fragment carrying the full-length SV40LT-IRES2-EGFP cassette, consistent with physical linkage between the transgene and reporter in the polyclonal SV40-CMEF population.

SV40-CMEF also lacked the senescence phenotype observed in late-passage primary fibroblasts. Senescence-associated beta-galactosidase staining produced the characteristic blue signal in primary cells but not in low- or high-passage SV40-CMEF. Real-time confluence measurements, crystal violet assays and EdU incorporation indicated faster and sustained proliferation in the immortalized line. Its morphology was more uniform than the heterogeneous spindle-shaped appearance of primary fibroblasts. Cell-cycle analysis was complicated by extensive aneuploidy, so the researchers generated DNA-content profiles after doublet discrimination and used EdU incorporation as an orthogonal measure of DNA synthesis. Together, these observations showed that the immortalized cells escaped replicative senescence and displayed enhanced proliferative capacity under the tested culture conditions.

The cells retained several molecular features associated with fibroblast identity. Immunofluorescence showed filamentous vimentin and cytosolic alpha-smooth muscle actin and S100A4 in both primary and immortalized fibroblasts, while RT-PCR and Western blotting supported persistent expression of the corresponding genes and proteins at low and high passage. Short tandem repeat profiling showed extremely high similarity between SV40-CMEF and the parental C57BL/6J cells, supporting their origin and arguing against cross-contamination. Karyotype analysis, however, revealed substantial numerical abnormalities. Among 100 metaphase spreads, low-passage cells ranged from 68 to 166 chromosomes, with a modal number of 78, whereas high-passage cells ranged from 59 to 142 chromosomes, with a modal number of 72. These measurements indicate pronounced chromosomal instability and a lower modal chromosome number in the later culture window, although the analysis did not characterize structural chromosome abnormalities.

Tumorigenicity testing distinguished the line’s proliferative behavior from detectable malignant transformation. In soft agar, Lewis lung carcinoma cells formed colonies, while primary MEFs and SV40-CMEF did not. For the in vivo test, five BALB/c nude mice per group received subcutaneous injections of either SV40-CMEF or the positive-control carcinoma line. Control cells produced palpable tumors within two to three days and reached the institutionally permitted size limit after about two weeks. No tumor was detected in mice receiving SV40-CMEF during three months of monitoring. The observation applies to the tested population, injected dose and monitoring period; it does not establish that the line can never become tumorigenic under other conditions. Within this assay, however, extensive aneuploidy alone was not sufficient to produce detectable tumors.

Functional testing showed that irradiated SV40-CMEF could serve as a feeder layer for R1/E mouse embryonic stem cells. The immortalized feeder monolayers supported compact, dome-shaped R1/E colonies, and immunofluorescence detected the pluripotency-associated markers Oct-4A, Sox2 and Nanog. Colony-forming efficiency and the proportion of alkaline-phosphatase-positive undifferentiated colonies were similar on primary and immortalized feeders, with no statistically significant difference reported. The stem-cell assay therefore demonstrated feeder-layer function and maintenance of an undifferentiated colony phenotype in this particular mouse embryonic stem-cell system. It was not a test of every stem-cell platform, including human pluripotent stem-cell cultures.

SV40-CMEF also retained the tri-lineage differentiation potential associated with primary MEFs. After nine days of adipogenic induction, cells stained strongly with Oil Red O, indicating lipid-droplet accumulation, while uninduced controls did not. Osteogenic induction produced positive Alizarin Red S staining after seven days, consistent with mineralized-matrix deposition, again unlike the controls. In chondrogenic conditions, both induced cells and a serum-free control stained with Alcian blue, showing that the basal condition itself supported some proteoglycan accumulation. The induced group had significantly greater staining intensity and significant increases in Sox9 and Acan expression; Col2a1 showed an upward trend that did not reach statistical significance at the measured time point. The combined staining and gene-expression results were interpreted as evidence of retained adipogenic, osteogenic and chondrogenic potential under the specified induction conditions.

RNA sequencing demonstrated that this functional preservation occurred amid extensive transcriptomic remodeling. In comparisons with primary C57BL/6J MEFs, principal component analysis separated the two cell types along an axis explaining 79.7% of the variance, and thousands of genes met the study’s differential-expression criteria of an absolute log2 fold change of at least 1 and a false discovery rate below 0.05. The immortalized cells clustered more tightly than primary cells, suggesting reduced transcriptional heterogeneity. Nevertheless, selected modules relevant to feeder activity and differentiation were retained or increased. These included Il6st, the Bmp4-Smad1/Smad5 axis and differentiation-associated transcription factors Runx2, Pparg and Twist1. Proliferation- and survival-associated pathways such as PI3K-Akt, MAPK and Ras signaling were enriched, while gene-set analysis highlighted cell cycle, DNA replication and mitotic cytokinesis. The stem-cell pluripotency-signaling pathway was also enriched, with Wnt7a, Tcf7 and Acvr2b among the upregulated genes associated with that result. These molecular associations offer possible explanations for the observed feeder function, but they do not establish which factors are causally responsible.

The study’s principal limitations concern the line’s genetic and biological stability. The authors noted that SV40-CMEF had exceeded 300 population doublings and 105 passages, but said more detailed work was required on p53 inactivation, telomere length and telomerase activity before the precise mechanism underlying its long-term proliferation was fully resolved. Structural chromosomal abnormalities were not assessed using fluorescence in situ hybridization, spectral karyotyping or sequencing-based structural-variant analysis. Because the immortalized population is heterogeneous, individual subclones may differ in SV40LT integration sites, proliferative lifespan and ability to support embryonic stem-cell self-renewal; the authors said they were beginning to isolate single-cell clones by limiting dilution. The study therefore presents a publicly accessible and functionally characterized feeder-cell resource, while further genomic and clonal analysis will be needed to define its reproducibility and molecular stability.

Subject of Research: Characterization of an SV40LT-immortalized mouse embryonic fibroblast line for feeder-cell and differentiation research

Article Title: Establishment and characterization of immortalized mouse embryonic fibroblasts by SV40 large T antigen

Article References: Li, X., Shao, T., Xu, L., Zheng, L., & Chen, Y.-L. (2026). Establishment and characterization of immortalized mouse embryonic fibroblasts by SV40 large T antigen. Frontiers in Cell and Developmental Biology, 14, Article 1936956. https://doi.org/10.3389/fcell.2026.1936956

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1936956

Keywords: SV40 large T antigen, mouse embryonic fibroblasts, cell immortalization, stem-cell feeders, mouse embryonic stem cells, tri-lineage differentiation, aneuploidy, tumorigenicity testing, RNA sequencing, C57BL/6J mice, Establishment, characterization