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Establishment and Characterization of hTERT Immortalized Hutchinson–Gilford Progeria Fibroblast Cell Lines

Lin, Haihuan, Mensch, Juliane, Haschke, Maria, Jäger, Kathrin, Köttgen, Brigitte, Dernedde, Jens, Orsó, Evelyn, Walter, Michael·2022·Cells
DOI 10.3390/cells11182784PMC PMC9497314
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Abstract

Hutchinson–Gilford progeria syndrome (HGPS) is a rare premature aging syndrome caused by a dominant mutation in the LMNA gene. Previous research has shown that the ectopic expression of the catalytic subunit of telomerase (hTERT) can elongate the telomeres of the patients’ fibroblasts. Here, we established five immortalized HGP fibroblast cell lines using retroviral infection with the catalytic subunit of hTERT. Immortalization enhanced the proliferative life span by at least 50 population doublings (PDs). The number of cells with typical senescence signs was reduced by 63 + 17%. Furthermore, the growth increase and phenotype improvement occurred with a lag phase of 50–100 days and was not dependent on the degree of telomere elongation. The initial telomeric stabilization after hTERT infection and relatively low amounts of hTERT mRNA were sufficient for the phenotype improvement but the retroviral infection procedure was associated with transient cell stress. Our data have implications for therapeutic strategies in HGP and other premature aging syndromes.

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Figure 1 The growth curves of control (N) and HGP fibroblasts. Primary fibroblasts in black, immortalized fibroblasts in red, negative control cells (infected with empty vector) in green. L, low PD; M, medium PD. H, high PD.
Figure 1 The growth curves of control (N) and HGP fibroblasts. Primary fibroblasts in black, immortalized fibroblasts in red, negative control cells (infected with empty vector) in green. L, low PD; M, medium PD. H, high PD.CC BY · PubMed Central
Figure 2 The average telomere length of control (CON) and HGP cells before and after immortalization. TL as T/S ratio (ratio of the content of telomere (T) to a single-copy gene (S)). HGP (n = 5) and control (n = 5) cells at L, M, and H PD. The data are presented as the mean ± SD and analyzed using Mann–Whitney-U-Test. * p ≤ 0.05, Bonferroni correction p < 0.016 (hTERT cells vs. primary cells at the corresponding PD class). L, low PD; M, medium PD. H, high PD.
Figure 2 The average telomere length of control (CON) and HGP cells before and after immortalization. TL as T/S ratio (ratio of the content of telomere (T) to a single-copy gene (S)). HGP (n = 5) and control (n = 5) cells at L, M, and H PD. The data are presented as the mean ± SD and analyzed using Mann–Whitney-U-Test. * p ≤ 0.05, Bonferroni correction p < 0.016 (hTERT cells vs. primary cells at the corresponding PD class). L, low PD; M, medium PD. H, high PD.CC BY · PubMed Central
Figure 3 Average absolute and relative hTERT mRNA (fold-change) expression of immortalized fibroblasts. The absolute and relative mRNA expression of each cell line is shown for control and HGP fibroblasts at L, M, and H PD, as described in Materials and Methods. The data are presented as the mean ± SD and analyzed using Mann–Whitney-U-Test. There was no significant difference (HGP vs. CON at the corresponding PD class). L, low PD; M, medium PD. H, high PD.
Figure 3 Average absolute and relative hTERT mRNA (fold-change) expression of immortalized fibroblasts. The absolute and relative mRNA expression of each cell line is shown for control and HGP fibroblasts at L, M, and H PD, as described in Materials and Methods. The data are presented as the mean ± SD and analyzed using Mann–Whitney-U-Test. There was no significant difference (HGP vs. CON at the corresponding PD class). L, low PD; M, medium PD. H, high PD.CC BY · PubMed Central
Figure 4 Scatter plot for the absolute and relative mRNA (fold-change) vs. telomere length (T/S Ratio) of primary and hTERT control (black) and HGP (red) cells. The absolute and relative mRNA expression was calculated as described in Materials and Methods. Spearmans rho and p values are presented. T/S ratio represents the ratio of the content of telomere (T) to a single-copy gene (S) and represents the overall relative telomere length.
Figure 4 Scatter plot for the absolute and relative mRNA (fold-change) vs. telomere length (T/S Ratio) of primary and hTERT control (black) and HGP (red) cells. The absolute and relative mRNA expression was calculated as described in Materials and Methods. Spearmans rho and p values are presented. T/S ratio represents the ratio of the content of telomere (T) to a single-copy gene (S) and represents the overall relative telomere length.CC BY · PubMed Central
Figure 5 Average SA-β-gal activity (4-MU fluorescence) of healthy control (CON) and HGP primary cells (black) and immortalized cells (red) in different PDs. The intracellular stress levels of immortalized HGP (n = 5) and control (n = 5) cells (from L to H PDs) were compared with their primary cells, respectively. The data are presented as the mean ± SD and analyzed using Mann–Whitney-U-Test. * p ≤ 0.05, Bonferroni correction p < 0.016 (hTERT vs. primary cells at the corresponding PD class). L, low PD; M, medium PD. H, high PD.
Figure 5 Average SA-β-gal activity (4-MU fluorescence) of healthy control (CON) and HGP primary cells (black) and immortalized cells (red) in different PDs. The intracellular stress levels of immortalized HGP (n = 5) and control (n = 5) cells (from L to H PDs) were compared with their primary cells, respectively. The data are presented as the mean ± SD and analyzed using Mann–Whitney-U-Test. * p ≤ 0.05, Bonferroni correction p < 0.016 (hTERT vs. primary cells at the corresponding PD class). L, low PD; M, medium PD. H, high PD.CC BY · PubMed Central

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Cites

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Metadata

Authors
Lin, H., Mensch, J., Haschke, M., Jäger, K., Köttgen, B., Dernedde, J., Orsó, E., & Walter, M.
Venue
Cells
Publisher
MDPI AG
Published
2022-09-06
Volume
11
Issue
18
Pages
2784
ISSN
2073-4409
DOI
10.3390/cells11182784
PMC
PMC9497314
Cited by
11
References
6
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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CoChat. (2026). Citation check: Establishment and Characterization of hTERT Immortalized Hutchinson–Gilford Progeria Fibroblast Cell Lines. https://app.cochat.ai/papers/10.3390/cells11182784
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Lin, H., Mensch, J., Haschke, M., Jäger, K., Köttgen, B., Dernedde, J., Orsó, E., & Walter, M. (2022). Establishment and Characterization of hTERT Immortalized Hutchinson–Gilford Progeria Fibroblast Cell Lines. Cells. https://doi.org/10.3390/cells11182784