Creation and characterization of human mesenchymal stromal cell cultures with prolonged proliferative potential for the tasks of regenerative medicine

The study of molecular mechanisms of regeneration requires convenient models for in vitro and in vivo studies. In vitro cell cultures can fulfill such a function. However, their rapid aging and loss of initial specific properties in culture is a significant limitation of their use. Telomerase expression can help to overcome these limitations: it can prolong proliferative activity and stabilize the initial properties of a primary cell culture. Here, we created and studied the properties of human adipose tissue multipotent mesenchymal stromal cell (MSC) cultures that overexpress the catalytic protein subunit of human telomerase ( hTERT). We found that these MSC cultures were able to proliferate up to 38–63 PD, kept sensitivity to noradrenaline, serotonin, glutamate, γ-aminobutyric acid, parathyroid hormone, angiotensin II and histamine until at least 26 PD, retained MSC-specific immunophenotype until at least 36 PD, and preserved the ability to adipogenic, osteogenic and chondrogenic differentiation until at least 39 PD. Moreover, overexpression of hTERT in MSC cultures stabilized the qualitative and quantitative composition of their secretome at long-term passaging (at least up to 30 PD). The obtained results allow us to consider telomerase hyperexpression as a promising approach to obtaining MSC cultures with prolonged proliferative activity, which can be used as a stable and convenient object for fundamental and applied studies in the field of regenerative medicine.

1. Varier P, Raju G, Madhusudanan P, Jerard C, Shankarappa SA. A Brief Review of In Vitro Models for Injury and Regeneration in the Peripheral Nervous System. Int J Mol Sci. 2022;23(2):816. DOI: 10.3390/ijms23020816

2. Karagyaur M, Primak A, Efimenko A, Skryabina M, Tkachuk V. The Power of Gene Technologies: 1001 Ways to Create a Cell Model. Cells. 2022;11(20):3235. DOI: 10.3390/cells11203235

3. Voloshin N, Tyurin-Kuzmin P, Karagyaur M, Akopyan Z, Kulebyakin K. Practical Use of Immortalized Cells in Medicine: Current Advances and Future Perspectives. Int J Mol Sci. 2023;24(16):12716. DOI: 10.3390/ijms241612716

4. Lenz LS, Wink MR. The other side of the coin: mesenchymal stromal cell immortalization beyond evasion of senescence. Hum Cell. 2023;36(5):1593–1603. DOI: 10.1007/s13577-023-00925-3

5. Basalova N, Illarionova M, Skryabina M, Vigovskiy M, Tolstoluzhinskaya A, Primak A, et al. Advances and Obstacles in Using CRISPR/Cas9 Technology for Non-Coding RNA Gene Knockout in Human Mesenchymal Stromal Cells. Noncoding RNA. 2023;9(5):49. DOI: 10.3390/ncrna9050049

6. Hahn WC. Immortalization and transformation of human cells. Mol Cells. 2002;13(3):351–361, DOI: 10.1016/S1016-8478(23)15045-X

7. Meltzer PS, Guan XY, Trent JM. Telomere capture stabilizes chromosome breakage. Nat Genet. 1993;4(3):252–255. DOI: 10.1038/ng0793-252

8. Richter M, Piwocka O, Musielak M, Piotrowski I, Suchorska WM, Trzeciak T. From Donor to the Lab: A Fascinating Journey of Primary Cell Lines. Front Cell Dev Biol. 2021;9:711381. DOI: 10.3389/fcell.2021.711381

9. Longo PA, Kavran JM, Kim MS, Leahy DJ. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 2013;529:227–240. DOI: 10.1016/B978-0-12-418687-3.00018-5

10. Tyurin-Kuzmin PA, Karagyaur MN, Kulebyakin KY, Dyikanov DT, Chechekhin VI, Ivanova AM, et al. Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells. Int J Mol Sci. 2020;21(12):4442. DOI: 10.3390/ijms21124442

11. Böcker W, Yin Z, Drosse I, Haasters F, Rossmann O, Wierer M, et al. Introducing a singlecell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer. J Cell Mol Med. 2008;12(4):1347–1359. DOI: 10.1111/j.1582-4934.2008.00299.x

12. Jun ES, Lee TH, Cho HH, Suh SY, Jung JS. Expression of telomerase extends longevity and enhances differentiation in human adipose tissue-derived stromal cells. Cell Physiol Biochem. 2004;14(4–6):261–268. DOI: 10.1159/000080335

13. Kulebyakin K, Tyurin-Kuzmin P, Efimenko A, Voloshin N, Kartoshkin A, Karagyaur M, et al. Decreased Insulin Sensitivity in Telomerase-Immortalized Mesenchymal Stem Cells Affects Efficacy and Outcome of Adipogenic Differentiation in vitro. Front Cell Dev Biol. 2021;9:662078. DOI: 10.3389/fcell.2021.662078

14. Ozkinay C, Mitelman F. A simple trypsin-Giemsa technique producing simultaneous Gand C-banding in human chromosomes. Hereditas. 1979;90(1):1–4. DOI: 10.1111/j.1601-5223.1979.tb01287.x

15. An International System for Human Cytogenomic Nomenclature. Eds. McGowan-Jordan J, Hastings RS, Moore S. S.Karger AG, Basel (Switzerland), 2020. DOI: 10.1159/isbn.978-3-318-06867-2

16. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells. The International Society for Cellular Therapy Position Statement. Cytotherapy. 2006;8:315–317. DOI: 10.1080/14653240600855905

17. Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissuederived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013;15(6):641–648. DOI: 10.1016/j.jcyt.2013.02.006

18. Viswanathan S., Shi Y., Galipeau J., Krampera M., Leblanc K., Martin I. et al. Mesen-chymal Stem versus Stromal Cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell Committee Position Statement on Nomenclature. Cytother. 2019;21(10):1019–1024. DOI: 10.1016/j.jcyt.2019.08.002

19. Zietzer A, Hosen MR, Goody PR, Werner N, Nickenig G, Jansen F. HnRNPU regulates intra-and intercellular microRNA trafficking in a sequence specific manner, Europ Heart J. 2020;41(Suppl. 2):ehaa946.3611. DOI: 10.1093/ehjci/ehaa946.3611

20. Zietzer A, Hosen MR, Wang H, Goody PR, Sylvester M, Latz E, et al. The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles. J Extracell Vesicles. 2020;9(1):1786967. DOI: 10.1080/20013078.2020.1786967

21. Karagyaur M, Primak A, Efimenko A, Skryabina M, Tkachuk V. The Power of Gene Technologies: 1001 Ways to Create a Cell Model. Cells. 2022;11(20):3235. DOI: 10.3390/cells11203235

22. Chen J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harb Perspect Med. 2016;6(3):a026104. DOI: 10.1101/cshperspect.a026104

23. Engeland K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ. 2022;29(5):946–960. DOI: 10.1038/s41418-022-00988-z

24. Simon M, Köster G, Menon AG, Schramm J. Functional evidence for a role of combined CDKN2A (p16-p14(ARF))/CDKN2B (p15) gene inactivation in malignant gliomas. Acta Neuropathol. 1999;98(5):444–452. DOI: 10.1007/s004010051107

25. Toouli CD, Huschtscha LI, Neumann AA, Noble JR, Colgin LM, Hukku B, Reddel RR. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene. 2002;21(1):128–139. DOI: 10.1038/sj.onc.1205014

26. Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet. 1999;21(1):111–114. DOI: 10.1038/5056

27. Tyurin-Kuzmin PA, Karagyaur MN, Kulebyakin KY, Dyikanov DT, Chechekhin VI, Ivanova AM, et al. Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells. Int J Mol Sci. 2020;21(12):4442. DOI: 10.3390/ijms21124442

28. Vorontsova MV, Kulebyakin KY, Makazan NV, Sozaeva LS, Tyurin-Kuzmin PA. Parathyroid hormone in the regulation of bone growth and resorption processes in norm and pathology. Bulletin of the Russian Academy of Medical Sciences. 2021;76(5):506-517.

29. Kulebyakin K, Tyurin-Kuzmin P, Efimenko A, Voloshin N, Kartoshkin A, Karagyaur M, et al. Decreased Insulin Sensitivity in Telomerase-Immortalized Mesenchymal Stem Cells Affects Efficacy and Outcome of Adipogenic Differentiation in vitro. Front Cell Dev Biol. 2021;9:662078. DOI: 10.3389/fcell.2021.662078

30. Tyurin-Kuzmin PA, Chechekhin VI, Ivanova AM, Dyikanov DT, Sysoeva VY, Kalini-na NI, Tkachuk VA. Noradrenaline Sensitivity Is Severely Impaired in Immortalized Adipose-Derived Mesenchymal Stem Cell Line. Int J Mol Sci. 2018;19(12):3712. DOI: 10.3390/ijms19123712

31. Cai Y, Laustsen A, Zhou Y, Sun C, Anderson MV, Li S, et al. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors. Elife. 2016;5:e12213. DOI: 10.7554/eLife.12213

32. Kane NM, Nowrouzi A, Mukherjee S, Blundell MP, Greig JA, Lee WK, et al. Lentivirus-mediated Reprogramming of Somatic Cells in the Absence of Transgenic Transcription Factors. Mol Ther. 2010;18(12):2139–2145. DOI: 10.1038/mt.2010.231

33. Barsov EV. Telomerase and primary T cells: biology and immortalization for adoptive immunotherapy. Immunotherapy. 2011;3(3):407–421. DOI: 10.2217/imt.10.107

34. Dos Santos A, Lyu N, Balayan A, Knight R, Zhuo KS, Sun Y, et al. Generation of Functional Immortalized Human Corneal Stromal Stem Cells. Int J Mol Sci. 2022;23(21):13399. DOI: 10.3390/ijms232113399

35. Kalinina N, Kharlampieva D, Loguinova M, Butenko I, Pobeguts O, Efimenko A, et al. Characterization of secretomes provides evidence for adipose-derived mesenchymal stromal cells subtypes. Stem Cell Res Ther. 2015;6:221. DOI: 10.1186/s13287-015-0209-8

36. Carvalho MM, Teixeira FG, Reis RL, Sousa N, Salgado AJ. Mesenchymal stem cells in the umbilical cord: phenotypic characterization, secretome and applications in central nervous system regenerative medicine. Curr Stem Cell Res Ther. 2011;6(3):221–228. DOI: 10.2174/157488811796575332