Immortalization of Horse-Derived Fibroblast by Cell Cycle Regulator

PeerJ Life & Environment. 2024 Jan 26; 12: e16832.

Authors: Tani T.

Immortalized cells serve as a crucial research tool that capitalizes on their robust proliferative properties for functional investigations of an organism. Establishing an immortalized American miniature horse cell line could yield valuable insights into these animals' genetic and physiological characteristics and susceptibility to health issues. To date, immortalized small horse cells with normal karyotypes have not been established.

• A tissue sample was obtained from an American miniature horse. Muscle fragments were obtained from a syringe injected into the hind leg muscles and primary cells were established by explant culture in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-amphotericin mixture and 1% Zellshield at 37°C in a CO₂ incubator for 10 days.
• To immortalize American miniature horse fibroblasts, primary cells were transduced with a lentivirus composed of K4D-EGFP with or without TERT and SV40T. The efficiency of transduction was monitored via the expression of EGFP, which was confirmed 5 days after transduction.
• The established cells by K4D, K4D plus TERT, and SV40T were named as K4D, K4DT and SV40T cells respectively. To detect the introduced gene cassette, I performed genomic PCR using the genomic DNA obtained from the primary, K4D, K4DT, SV40T, and TERT cells derived from two individuals.
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• Under standard culture conditions, the primary cells exhibited a fibroblast-like morphology (Figs. 1A, B, and C). To confirm the cell type of the primary cells, primary antibodies against vimentin were used to stain the cells. As shown in Fig. 1D, the primary cells were found to be vimentin-positive. The primary American miniature horse fibroblasts were efficiently infected with the K4D-EGFP-expressing virus, resulting in a high percentage of green fluorescence-positive cells (Fig. 2A). The expression of TERT and SV40T was selected by multiple passages in the presence of blasticidin or puromycin. As shown in Fig. 2B, the expected size of CDK4-2A-CyclinD1 fragments in K4D and K4DT cells, TERT fragments in K4DT cells, and SV40T fragments in SV40T cells were detected. These results indicate that the target genes were successfully introduced into the transduced cells.

Fig. 1 Establishment of fibroblast from American miniature horse muscle fragments.

Fig. 2 Detection of K4D-EGFP expression and genomic integration of introduced genes cassette in American miniature horse-derived cell.

• As shown in Fig. 3A, sequential passages of primary, K4D, K4DT, SV40T, and TERT transduced cells derived from two individuals were carried out for 6-7 months. Although the primary and TERT cells of two individuals could not continue cell proliferation beyond 15 passages (25 PD value). Both K4D and SV40T cells stop cell proliferation around 65 PD and 80 PD.
• While both K4DT cells showed cell proliferation of more than 100 PD values. The doubling time at passages 10 was 32.1 h (miniature horse #1) and 34.4h (miniature horse #2) for primary when compared to K4D, K4DT, and SV40T cells significantly (Fig. 3B). Additionally, the BrdU incorporation rate for both primary cells was significantly lower when compared to K4D, K4DT, SV40T and TERT cells (Fig. 3C). To assess the degree of cellular senescence, SA-β-gal staining was performed at passage 15. Both primary and TERT cells almost stopped proliferating and stained positive for SA-β-gal. However, K4D, K4DT, and SV40T cells did not stain for SA-β-gal (Fig. 3D). These data showed that K4D, K4DT, and SV40T expressing cells from two individuals dramatically prolonged the proliferation up to 65 PD values of American miniature horse-derived cells. While both K4DT cells did not stop proliferation over 100 PD.

Fig. 3 Biological analysis of proliferation in American miniature horse-derived cells.

In this study, primary and immortalized fibroblast cell lines were successfully established through the combined expression of human-derived mutant cyclin-dependent kinase 4 (CDK4R24C), cyclin D1, and telomerase reverse transcriptase (TERT), although CDK4R24C and cyclin D1, SV40T and TERT did not result in successful immortalization.

Immortalized Human Cells

Reference

1. Tani T. (2024). "Immortalization of American miniature horse-derived fibroblast by cell cycle regulator with normal karyotype." PeerJ. 12: e16832.

For research use only. Not for any other purpose.