Immortalization of American miniature horse-derived fibroblast by cell cycle regulator with normal karyotype

The primary cell of American miniature horse

A tissue sample was obtained from an American miniature horse that had died of natural causes in a private Zoo (World Ranch, Osaka, Japan). The 15-year-old male and 13-year-old male individuals were registered by The American Miniature Horse Association as a pet. 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; Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin-amphotericin mixture and 1% Zellshield (Minerva Biolabs, Berlin, Germany) at 37 °C in a CO₂ incubator for 10 days. After several passages by 0.25% trypsin-1 mM EDTA solution, the primary cells were suspended in CultureSure cell freezing medium in a liquid nitrogen tank until use. To confirm mycoplasma contamination, established cells were subjected to PCR Mycoplasma Detection kit (Takara Bio, Shiga, Japan).

Preparation of recombinant viruses for gene transfer into primary cells

To prepare the lentivirus for gene transfer into primary cells, we obtained all relevant plasmids from Dr. Hiroyuki Miyoshi (RIKEN BioResource Center, Tsukuba, Japan) or Addgene (Watertown, MA, USA). We used the expression plasmids CSII-CMV-CDK4R24C-P2A-Cyclin D1-T2A-EGFP, pLV-CMV-TERT-PGK-Bsd (Vectorbuilder, Chicago, IL, USA), and pLV-CMV-SV40T-PGK-Puro (Vectorbuilder). We introduced the packaging plasmids psPax2 (Addgene #12260) and pMD2.G (Addgene#12259) into 293T cells (RCB5708; RIKEN BioResource Center, Kyoto, Japan) using lipofection. The virus supernatant was recovered after 48 h transfection and filtered using a 0.45 mm disk filter (Merck Millipore, Burlington, MA, USA) in accordance with the Addgene lentivirus protocol (https://www.Addgene.org/protocols/lentivirus-production/). The primary cells were incubated with the lentivirus at a multiplicity of infection (MOI) of five for each virus, supplemented with 8 μg/ml of polybrene (Sigma) for 24 h. To estimate the efficiency of virus infection, enhanced green fluorescent protein-expressing cells (K4D-EGFP) were used as a control. Infected cells of TERT and SV40T lentivirus were selected using culture containing 500 ug/ml hygromycin or 10ug/ml blasticidin for 5–7 days.

Population doubulings and cell doubling time assay

Primary and established cells were seeded in 24-well plates at a density of 1.0 × 10^4 cells/well, the cells were cultured when one well of the six-well plate became confluent. Cell passages were repeated until they could no longer continue. The population doubling level (PDL) was calculated using the formula log2 (A/B), where A is the number of cells harvested in a passage and B is the number of cells seeded. The results of the PDL assay were plotted as the average of three consecutive samples. Cell doubling time were calculated using the formula T × (ln2)]/[ln (Xe/Xb)], where T is the incubation time, Xb is the number of cells at the beginning of the time incubation, Xe is the number of cells at the end of the incubation time, and ln is the Napierian logarithm.

BrdU incorporation assay

To detect the cell ratio of proliferating cells based on the measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis, 10 μM of BrdU (Cayman, Ann Arbor, MI, USA) incorporated for 3 h at 37 °C in a CO₂ incubator. After several wash in PBS, fix and permeabilized by 4% PFA and PBS containing 0.1% Triton-X-100 (PBT). Then, cells were incubated overnight with a rabbit polyclonal anti-Brdu antibody (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA) in PBS-1%BSA at 4 °C. After several washes in PBS containing 0.1% Tween20, they were incubated for 60 min at room temperature with a secondary antibody: an Alexa-Fluor-594-conjugated goat anti-rabbit IgG antibody (1:1,000; Thermo Fisher Scientific, Waltham, MA, USA). The cells were washed several times in PBS, mounted on a glass slide with the ProLong Gold Antifade Reagent and 10 μg/ml Hoechst 33342 solution, and were examined under a fluorescence microscope EVOS M5000 (Thermo Fisher Scientific, Waltham, MA, USA). BrdU incorporation was quantified by counting at least 5,000 nuclei in each established cell.

Senescence-associated $\mathrm{\beta }$-galactosidase staining assay (SA- $\mathrm{\beta }$-gal)

To detect cellular senescence, SA-β-Gal staining was carried out at passage 15, with Cellular Senescence Detection Kit (CELL BIOLABS, San Diego, CA, USA). Cells were fixed at RT for 10 min, washed with PBS, and stained in β-galactosidase staining solution containing X-gal (pH 6) at 37 °C overnight in dry incubator. Stained cells were monitored under bright field microscopy.

DNA damage-associated $\mathrm{\gamma }$H2AX staining

To detect the DNA damage, the cells were immune-stained with a monoclonal anti-γ H2AX antibody (1:1,000; Abcam, Cambridge, UK). Counting of gamma H2AX foci in a cell was quantified by counting at least 50 nuclei in each established cell by a confocal microscopy FV-3000 (Olympus, Tokyo, Japan).

ATP measurement

Established cells were washed several times with PBS. The intracellular ATP content in 1.0 × 10⁴ cells was quantified using Cell Titer-Glo Luminescent cell viability assay (Promega, Tokyo, Japan).

Immunostaining

To identify the type of cells established, primary cells were immunostained with anti-vimentin, cytokeratin and desmin antibodies. Immunofluorescence staining of the cells on coverslips was performed as BrdU incorporation assay using anti-vimentin (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA), anti-cytokeratin (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA) and anti-desmin (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA). As a negative control, all cells were incubated with nonimmune rabbit serum instead of rabbit polyclonal primary antibodies.

Telomerase repeated amplification qPCR (TRAP-qPCR) assay

Telomerase activity was measured by TRAP-qPCR as previously with slight modification (Pinto et al., 2021). In brief, quantitative Real-Time PCR was performed using Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) in a final reaction volume of 10 μl. The 293T cells standard curve were used as Relative Telomerase Activity (RTA) quantification.

F-actin staining

To detect of cell morphology, cytoskeleton, cell motility, and polarity, the cells were stained with a rhodamine X conjugated-phalloidin and Hoechst 33342. In brief, fix and permeabilized by 4% PFA and PBT were incubated for 60 min at room temperature with 0.1% of rhodamine X conjugated-phalloidin solution.

Chromosome analysis

To determine the chromosomal normality, the chromosomes of the established cells were counted using the Giemsa staining method. In brief, cells were treated with 50 ng/ml demecolcine solution for 2 h to arrest the cell cycle at metaphase, after trypsinization, then we treated the cells with a hypotonic solution of 0.075 M potassium chloride and fixed using Carnoy’s fixative (75% methanol/25% acetic acid). Post-fixation, the cells were stained with Giemsa solution. We analyzed the detailed chromosomal patterns of 20 mitotic cells.

Detection of inserted gene cassettes by genomic polymerase chain reaction (PCR)

Genomic DNAs was extracted using NaOH-base extraction method. In brief, 1.0 × 10⁴ cells were heated in 90 μl of 50 mM NaOH solution at 100 °C for 10 min and neutralized with 10 μl of 1 M Tris-HCl solution at pH 7.5. Genomic DNAs were subjected to genomic PCR by KOD one enzyme (Toyobo, Osaka, Japan) according to manufacture (2 min of pre denaturation at 94 °C, 40 cycles of 10 s at 98 °C and 20 s at 68 °C). For detection of introduced gene cassettes as well as 12s rRNA gene as a positive control. The sequences of the CDK4R24C-2A-cyclin D1 primers were

5′-GCTGGAGATGCTCACCTTCA-3′ and 5′-TCCAGGTGGCCACGATCTTTC-3′, TERT 5′-ACTACCGCGAGGTGCTG-3′ and 5′-ACCGTGTTGGGCAGGTA-3′, SV40T 5′-TTCTTACTCCACACAGGCATAG-3′ and 5′-ACTCCAGCCATCCATTCTTC-3′, 12srRNA 5′-CAAACTGGGATTAGATACCC-3′ and 5′-CAGGAAACAGCTATGACC-3′. The PCR products were detected the in 1.8% agarose gel electrophoresis with ethidium bromide.

Identification of the species of animal

To confirm animal species for avoid cell contamination, mitochondrial 12S ribosomal RNA gene was amplified by PCR and sequenced as previously reported (Yang et al., 2014). In brief, isolation of genomic DNA were isolated from cultured cells by Template Prepper for DNA kit (Nippongene, Tokyo, Japan). A total of 10–100 ng of DNA were used for PCR amplification and 12S rRNA gene was amplified the following primers;

5′-TGTAAAACGACGGCCAGT CAAACTGGGATTAGATACCC-3′ and

5′-CAGGAAACAGCTATGACCGAGGGTGACGGGCGGTGTGT-3′ in KOD One PCR enzyme according to manufacture. The PCR product was direct sequenced by M13 forward or reverse primer and analyzed via Standard Nucleotide BLAST searches at the NCBI website.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). All the data shown are the mean ± SEM from at least six biological replicates. Significance was assessed compared with the primary cells as controls using One-way ANOVA with post-hoc Dunnett’s test. A p-value less than 0.05 was considered statistically significant.

Cell culture and morphology of established cell lines derived from American miniature horses

Primary cells were isolated from muscle tissue fragments obtained via syringe biopsy from two male American miniature horses (Fig. 1A). Under standard culture conditions, the primary cells exhibited a fibroblast-like morphology (Figs. 1B and 1C). 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. 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 primary American miniature horse fibroblasts were efficiently infected with 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.

The established cells by K4D, K4D plus TERT, and SV40T were named as K4D, K4DT and SV40T cells respectively. To detect 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. As shown in Fig. 2B, 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.

K4D, K4DT and SV40T immortalized cells of American miniature horse-derived cells are free from cellular senescence

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 of 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 were 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 were 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.

Biological characterization of established American miniature horse-derived cells

For the purpose of comparing the biological characteristics of the established cells from two individuals, I conducted an analysis of ATP content, γ-H2AX immunostaining, and telomerase activity for miniature horse#1. The ATP contents of the established cells were found to be almost equivalent (Fig. 4A). Figures 4B and 4C displayed that SV40T cells had a higher number of γ-H2AX foci in a cell significantly when compared to primary, K4D, K4DT and TERT cells, which resulted in a greater accumulation of DNA damage under these culture conditions. Finally, I compared the telomerase activity, and the results presented in Fig. 4D demonstrated that K4DT and TERT cells exhibited enzymatic activity for extending the telomere sequence through TERT enzyme activity significantly.

Karyotype and F-actin distribution of primary and K4D American miniature horse-derived cells

To further investigate F-actin distribution and the karyotype of primary and K4DT American miniature horse #1-derived cells were analyzed. Utilizing phalloidin and Hoechst33342 labeling, I examined the distribution of F-actin in the primary, K4D, and K4DT cells. Figure 5A displayed that none of the cell types’ F-actin distribution or structure. Next, I analyzed twenty metaphase plates for primary (passage 5 and 6), K4D (passage 16 and 11), and SV40T (passage 21 and 24) from two individuals. As illustrated in Figs. 5B and 5C, over 70% of primary, K4D, K4DT and TERT cells exhibited normal karyotypes (2n = 64) of both individuals. However, all SV40T cells of both individuals had abnormal chromosome numbers ranging from 69 to 88, with an average chromosome number of 77.33 and 78.1, respectively. These findings demonstrate that K4D, K4DT and TERT cells have a similar chromosome number and karyotype as the primary cells, whereas SV40T cells differ from the primary cells.

Identification of the species of origin by mitochondrial 12s rRNA

The amplified fragment of mitochondrial 12S rRNA from K4DT cells derived from two individuals was sequenced using universal oligodeoxynucleotide primers with M13 sequence, and the resulting sequence was determined by searching the NCBI database, as previously reported (Yang et al., 2014). The sequence demonstrated complete homology with the 12S rRNA genes in Equus caballus (MN187575.1; Figs. 1S and 2S). This revealed that both cell lines derived from two individuals had no cross-contamination with other species. Furthermore, this study demonstrated that the established cells were free of mycoplasma contamination using commercial mycoplasma detection kit, although the corresponding data was not presented.