Changes in stemness properties of human adenoid-derived mesenchymal stem cells during in vitro aging

Abstract

Mesenchymal stem cells (MSCs) have significant therapeutic potential in gene therapy. In vitro replicative senescence causes a decrease in the proliferation capacity of MSCs and changes in stem cell properties. In this study, adenoid tissue was focused as a new MSC source. The stem cell properties and the proliferation potential of adenoid-derived MSCs after the long-term in vitro replicative senescence were investigated. Adenoid-derived MSCs (A-MSCs) were cultured up to passage 20 and were analysed for cell morphology, proliferative capacity, differentiation potential, and surface marker expression. In addition, the expression profile of cell cycle, apoptosis, and senescence-related genes were evaluated. After in vitro replicative senescence, A-MSCs did not show any significant morphological differences. The proliferation potential of A-MSCs was rapid up to passage 16, and a reduction in the proliferation potential of senescent cells in vitro was observed depending on the passage number. The differentiation potential of late-passage A-MSCs was also reduced compared to early-passage cells. A-MSCs also provided significant closure at the 8th hour in early passages in terms of closure of the scratch area, while late passage A-MSCs exhibited a similar closure profile at the 24th hour. At the transcriptional level, the upregulation of the BAX gene and the downregulation of the p21 and p53 genes suggest that late-passage A-MSCs may not exhibit a senescence profile. In conclusion, A-MSCs have significant potential for clinical use due to the sustainability of MSC properties and their ability to proliferate and migrate with long-term culture.

Keywords

• Adenoid
• cell proliferation
• characterization
• mesenchymal stem cells
• senescence

References

1. Alcayaga-Miranda, F., Cuenca, J., Luz-Crawford, P., Aguila-Díaz, C., Fernandez, A., Figueroa, F. E., & Khoury, M. (2015). Characterization of menstrual stem cells: angiogenic effect, migration and hematopoietic stem cell support in comparison with bone marrow mesenchymal stem cells. Stem Cell Research & Therapy, 6, 1-14.
2. Alt, E. U., Senst, C., Murthy, S. N., Slakey, D. P., Dupin, C. L., Chaffin, A. E., ... & Izadpanah, R. (2012). Aging alters tissue resident mesenchymal stem cell properties. Stem Cell Research, 8(2), 215-225.
3. Amaral, J. D., Xavier, J. M., Steer, C. J., & Rodrigues, C. M. (2010). The role of p53 in apoptosis. Discovery Medicine, 9(45), 145-152.
4. Bagheri‐Mohammadi, S., Karimian, M., Alani, B., Verdi, J., Tehrani, R. M., & Noureddini, M. (2019). Stem cell‐based therapy for Parkinson’s disease with a focus on human endometrium‐derived mesenchymal stem cells. Journal of Cellular Physiology, 234(2), 1326-1335.
5. Banfi, A., Muraglia, A., Dozin, B., Mastrogiacomo, M., Cancedda, R., & Quarto, R. (2000). Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Experimental Hematology, 28(6), 707-715.
6. Baxter, M. A., Wynn, R. F., Jowitt, S. N., Wraith, J. E., Fairbairn, L. J., & Bellantuono, I. (2004). Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells, 22(5), 675-682.
7. Bonab, M. M., Alimoghaddam, K., Talebian, F., Ghaffari, S. H., Ghavamzadeh, A., & Nikbin, B. (2006). Aging of mesenchymal stem cell in vitro. BMC Cell Biology, 7, 1-7.
8. Bustos, M. L., Huleihel, L., Kapetanaki, M. G., Lino-Cardenas, C. L., Mroz, L., Ellis, B. M., ... & Rojas, M. (2014). Aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response. American Journal of Respiratory and Critical Care Medicine, 189(7), 787-798.

9. Čamernik, K., Mihelič, A., Mihalič, R., Presen, D. M., Janež, A., Trebše, R., ... & Zupan, J. (2019). Skeletal-muscle-derived mesenchymal stem/stromal cells from patients with osteoarthritis show superior biological properties compared to bone-derived cells. Stem Cell Research, 38, 101465.
10. Chang, Y. S., Oh, W., Choi, S. J., Sung, D. K., Kim, S. Y., Choi, E. Y., ... & Park, W. S. (2009). Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats. Cell Transplantation, 18(8), 869-886.
11. Cho, K. A., Lee, H. J., Jeong, H., Kim, M., Jung, S. Y., Park, H. S., ... & Kim, H. S. (2019). Tonsil-derived stem cells as a new source of adult stem cells. World Journal of Stem Cells, 11(8), 506.
12. Choi, J. S., Lee, B. J., Park, H. Y., Song, J. S., Shin, S. C., Lee, J. C., ... & Jung, J. S. (2015). Effects of donor age, long-term passage culture, and cryopreservation on tonsil-derived mesenchymal stem cells. Cellular Physiology and Biochemistry, 36(1), 85-99.
13. Choudhery, M. S., Badowski, M., Muise, A., Pierce, J., & Harris, D. T. (2014). Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation. Journal of Translational Medicine, 12, 1-14.
14. Deng, X., Zhang, S., Qing, Q., Wang, P., Ma, H., Ma, Q., ... & Lu, M. (2024). Distinct biological characteristics of mesenchymal stem cells separated from different components of human placenta. Biochemistry and Biophysics Reports, 39, 101739.
15. Donega, V., Nijboer, C. H., van Tilborg, G., Dijkhuizen, R. M., Kavelaars, A., & Heijnen, C. J. (2014). Intranasally administered mesenchymal stem cells promote a regenerative niche for repair of neonatal ischemic brain injury. Experimental Neurology, 261, 53-64.
16. Feng, X., Liu, J., Xu, Y., Zhu, J., Chen, W., Feng, B., ... & Cao, H. (2020). Molecular mechanism underlying the difference in proliferation between placenta‐derived and umbilical cord‐derived mesenchymal stem cells. Journal of Cellular Physiology, 235(10), 6779-6793.
17. Friedenstein, A. J., Gorskaja, J. F., & Kulagina, N. (1976). Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Experimental Hematology, 4(5), 267-274.
18. Gopalarethinam, J., Nair, A. P., Iyer, M., Vellingiri, B., & Subramaniam, M. D. (2023). Advantages of mesenchymal stem cell over the other stem cells. Acta Histochemica, 125(4), 152041.
19. Genovese, L., & Brendolan, A. (2016). Lymphoid tissue mesenchymal stromal cells in development and tissue remodeling. Stem Cells International, 2016(1), 8419104.
20. Gresham, R. C., Filler, A. C., Fok, S. W., Czachor, M., Schmier, N., Pearson, C., ... & Leach, J. K. (2024). Compliant substrates mitigate the senescence associated phenotype of stress induced mesenchymal stromal cells. Journal of Biomedical Materials Research Part A, 112(5), 770-780.
21. Gu, Y., Li, T., Ding, Y., Sun, L., Tu, T., Zhu, W., ... & Sun, X. (2016). Changes in mesenchymal stem cells following long-term culture in vitro. Molecular Medicine Reports, 13(6), 5207-5215.
22. Guo, F. F., Yu, B. Q., Chen, Y., He, J., Gu, Y., Wan, X., & Xiao, Z. A. (2023). Study on in vitro differentiation of human adenoid-derived mesenchymal stem cells into olfactory sensory neurons. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi= Chinese Journal of Otorhinolaryngology Head and Neck Surgery, 58(3), 233-239.
23. Hmadcha, A., Martin-Montalvo, A., Gauthier, B. R., Soria, B., & Capilla-Gonzalez, V. (2020). Therapeutic potential of mesenchymal stem cells for cancer therapy. Frontiers in bioengineering and biotechnology, 8, 43.
24. Hoffman, A. M., Paxson, J. A., Mazan, M. R., Davis, A. M., Tyagi, S., Murthy, S., & Ingenito, E. P. (2011). Lung-derived mesenchymal stromal cell post-transplantation survival, persistence, paracrine expression, and repair of elastase-injured lung. Stem Cells and Development, 20(10), 1779-1792.
25. Hoogduijn, M. J., Crop, M. J., Peeters, A. M. A., Van Osch, G. J. V. M., Balk, A. H. M. M., Ijzermans, J. N. M., ... & Baan, C. C. (2007). Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities. Stem Cells and Development, 16(4), 597-604.
26. Kern, S., Eichler, H., Stoeve, J., Klüter, H., & Bieback, K. (2006). Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, 24(5), 1294-1301.
27. Ledesma-Martínez, E., Mendoza-Núñez, V. M., & Santiago-Osorio, E. (2016). Mesenchymal stem cells derived from dental pulp: a review. Stem Cells International, 2016(1), 4709572.
28. Li, C. H., Zhao, J., Zhang, H. Y., & Wang, B. (2023). Banking of perinatal mesenchymal stem/stromal cells for stem cell-based personalized medicine over lifetime: Matters arising. World Journal of Stem Cells, 15(4), 105.
29. Liu, M., Lei, H., Dong, P., Fu, X., Yang, Z., Yang, Y., ... & Xiao, R. (2017). Adipose-derived mesenchymal stem cells from the elderly exhibit decreased migration and differentiation abilities with senescent properties. Cell Transplantation, 26(9), 1505-1519.
30. Mazini, L., Rochette, L., Amine, M., & Malka, G. (2019). Regenerative capacity of adipose derived stem cells (ADSCs), comparison with mesenchymal stem cells (MSCs). International Journal of Molecular Sciences, 20(10), 2523.
31. Melnik, S., Werth, N., Boeuf, S., Hahn, E. M., Gotterbarm, T., Anton, M., & Richter, W. (2019). Impact of c-Myc expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells. Stem Cell Research & Therapy, 10, 1-18.
32. Mueller, S. N., & Germain, R. N. (2009). Stromal cell contributions to the homeostasis and functionality of the immune system. Nature Reviews Immunology, 9(9), 618-629.
33. Naaldijk, Y., Johnson, A. A., Ishak, S., Meisel, H. J., Hohaus, C., & Stolzing, A. (2015). Migrational changes of mesenchymal stem cells in response to cytokines, growth factors, hypoxia, and aging. Experimental Cell Research, 338(1), 97-104.
34. Noronha‐Matos, J. B., & Correia‐de‐Sá, P. (2016). Mesenchymal stem cells ageing: targeting the “purinome” to promote osteogenic differentiation and bone repair. Journal of Cellular Physiology, 231(9), 1852-1861.
35. Oh, S. Y., Choi, Y. M., Kim, H. Y., Park, Y. S., Jung, S. C., Park, J. W., ... & Jo, I. (2019). Application of tonsil-derived mesenchymal stem cells in tissue regeneration: concise review. Stem Cells, 37(10), 1252-1260.
36. Rajput, S. N., Naeem, B. K., Ali, A., Salim, A., & Khan, I. (2024). Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine. World Journal of Stem Cells, 16(4), 410.
37. Rochette, P. J., & Brash, D. E. (2008). Progressive apoptosis resistance prior to senescence and control by the anti-apoptotic protein BCL-xL. Mechanisms of Ageing and Development, 129(4), 207-214.
38. Ryu, K. H., Cho, K. A., Park, H. S., Kim, J. Y., Woo, S. Y., Jo, I., ... & Kim, H. S. (2012). Tonsil-derived mesenchymal stromal cells: evaluation of biologic, immunologic and genetic factors for successful banking. Cytotherapy, 14(10), 1193-1202.
39. Shin, S. C., Seo, Y., Park, H. Y., Jung, D. W., Shin, T. H., Son, H., ... & Lee, B. J. (2018). Regenerative potential of tonsil mesenchymal stem cells on surgical cutaneous defect. Cell Death & Disease, 9(2), 183.
40. Stolzing, A., Jones, E., Mcgonagle, D., & Scutt, A. (2008). Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mechanisms of Ageing and Development, 129(3), 163-173.
41. Wang, B., Liu, X. M., Liu, Z. N., Wang, Y., Han, X., Lian, A. B., ... & Liu, J. Y. (2020). Human hair follicle-derived mesenchymal stem cells: Isolation, expansion, and differentiation. World Journal of Stem Cells, 12(6), 462.
42. Wang, C., Xie, T., Li, X., Lu, X., Xiao, C., Liu, P., ... & Zhang, B. (2024). Effect of in vivo culture conditions on the proliferation and differentiation of rat adipose-derived stromal cells. Mechanisms of Ageing and Development, 219, 111935.
43. Wu, G., Pan, M., Wang, X., Wen, J., Cao, S., Li, Z., ... & Guo, J. (2015). Osteogenesis of peripheral blood mesenchymal stem cells in self assembling peptide nanofiber for healing critical size calvarial bony defect. Scientific Reports, 5(1), 16681.
44. Yang, Y. H. K. (2018). Aging of mesenchymal stem cells: Implication in regenerative medicine. Regenerative Therapy, 9, 120-122.
45. Yang, Y. H. K., Ogando, C. R., Wang See, C., Chang, T. Y., & Barabino, G. A. (2018). Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem Cell Research & Therapy, 9, 1-14.
46. Yuce, M., & Albayrak, E. (2022). Hyperthermia‐stimulated tonsil‐mesenchymal stromal cells suppress hematological cancer cells through downregulation of IL‐6. Journal of Cellular Biochemistry, 123(12), 1966-1979.
47. Zhou, S., Greenberger, J. S., Epperly, M. W., Goff, J. P., Adler, C., LeBoff, M. S., & Glowacki, J. (2008). Age‐related intrinsic changes in human bone‐marrow‐derived mesenchymal stem cells and their differentiation to osteoblasts. Aging Cell, 7(3), 335-343.
48. Zhu, D., Barabadi, M., McDonald, C., Kusuma, G., Inocencio, I. M., & Lim, R. (2024). Implications of maternal-fetal health on perinatal stem cell banking. Gene Therapy, 31(3), 65-73.