Identification of Mesenchymal Stem Cells and Proliferation Properties

Abstract

Mesenchymal stem cells (MSCs) are undifferentiated cells that are highly abundant in animals and humans and have therapeutic efficacy. The aim of this study was to determine the MSC characteristics of passage 3 cells isolated from adipose tissue, immunolocalization of Ki-67 antibody, and to evaluate proliferation by cell growth analysis. Passage 3 cells were differentiated into adipocytes, osteoblasts and chondroblasts and stained with Oil Red O, Alizarin Red and Alcian Blue techniques. MSCs characterization was identified by stem cell surface markers positive expression of CD 90 and CD 105 negative expression of CD 45 and CD 11b. As a result; fat tissue origin passage in 3 cells have been shown that immunophenotypic characterization, differentiation in osteogenic, chondrogenic and adipogenic aspects, preserving optimal cellular hemostasis by Ki-67 positive immunpositive cells, PDT findings and therapeutically healthy mesenchymal stem cell proliferation.

Keywords

• Stem Cell Differentiation
• Immunophenotyping
• Ki-67
• Analyse of Cell Development

Mezenkimal Kök Hücrelerin Tanımlanması ve Proliferasyon Özellikleri

Abstract

Mezenkimal kök hücreler (MKH), hayvanlar ve insanlarda yüksek oranda bulunan ve terapötik etkinliğe sahip farklılaşmamış hücrelerdir. Bu çalışmada, yağ dokudan izole edilen pasaj 3 hücrelerinde, MKH özelliklerinin tanımlanması, Ki-67 antikorunun immunolokalizasyonu, hücre gelişim analizi ile proliferasyonun değerlendirilmesi amaçlandı. Pasaj 3 hücreleri adiposit, osteoblast ve kondroblastlara farklılaştırılarak Oil Red O, Alizarin Red ve Alcian Blue teknikleri ile boyandı. MKH karakterizasyonu; kök hücre yüzey işaretleyicilerinden CD 90 ve CD 105 ile pozitif; CD 45 ve CD 11b ile negatif ekspresyonu tanımlandı. Sonuç olarak çalışmada; yağ doku kökenli pasaj 3 hücrelerde; immünofenotipik karakterizasyonun, osteojenik, kondrojenik ve adipojenik yönde farklılaşmanın, Ki-67 immunpozitif hücrelerde, PDT bulgularında optimal hücresel hemostazisin korunduğu ve terapötik açıdan sağlıklı mezenkimal kök hücre proliferasyonunun gerçekleştiği gösterilmiştir.

Keywords

• Kök Hücre Farklılaşması
• İmmunofenotiplendirme
• Ki-67
• Hücre Gelişim Analizi

References

1. Referans 1. Spees JL, Lee RH, Gregory CA. Mechanisms of mesenchymal stem/stromal cell function. Stem Cell Res Ther. 2016;7(1):125.
2. Referans 2. Zakrzewski W, Dobrzyński M, Szymonowicz M, et al. Stem cells: past, present, and future. Stem Cell Res Ther. 2019;10(1):68.
3. Referans 3. Murray PE, Hargreaves KM. Regenerative Endodontics: A Review of Current Status and a Call for Action. J Endod. 2007;33:377-390.
4. Referans 4. Gardner RL. Stem Cells: Potency, plasticity and public perception. J Anat. 2002;200:277-282.
5. Referans 5. Friedenstein AJ, Gorskaja JF, Kulagina NN, et al. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976;4(5):267-274.
6. Referans 6. Caplan AI. Mesenchymal stem cells. Journal of orthopaedic research: J Orthop Res 1991;9(5):641-650.
7. Referans 7. Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211-228.
8. Referans 8. Rosen ED, Hsu CH, Wang X, et al. EBP alpha induces adipogenesis through PPARgamma: A unified pathway. Genes Dev. 2002;16(1):22-26.

9. Referans 9. Bourin P, Bunnell BA, Casteilla L, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics (IFATS) and Science and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013;15(6):641-648.
10. Referans 10. Liu L, Cheung TH, Charville GW, et al. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat. Protoc. 2015;10:1612 -1624.
11. Referans 11. Marx C, Silveira M, Nardi N, et al. Adipose-Derived Stem Cells in Veterinary Medicine: Characterization and Therapeutic Applications. Stem Cells Dev. 2015;24(7):803-813.
12. Referans 12. Pelttari K, Steck E, Richer W, et al. The use of mesenchymal stem cells for chondrogenesis; Injury. Int. J. Care Injured. 2008;39S1: 58-65.
13. Referans 13. Fraser JK, Wulur I, Alfonso Z, et al. Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol. 2006;24: 150-154.
14. Referans 14. Gimble JM, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy. 2003;5(5): 362-369.
15. Referans 15. Cooper GM. The Cell: A Molecular Approach. 2nd ed. United States, USA: Sunderland (MA): Sinauer Associates; 2000.
16. Referans 16. Sobecki M, Mrouj K, Colinge J, et al. Cell cycle regulation accounts for variability in Ki-67 expression levels. Cancer Res. 2017;10.
17. Referans 17. Landberg G, Tan EM, Roos G. Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle. Exp Cell Res. 1990;187:111-118.
18. Referans 18. Gronthos S, Franklin DM, Leddy HA, et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol. 2001;189(1):54–63.
19. Referans 19. Özen A., Gül Sancak İ., Caylan A., Özgenç Ö. 2016. ‘’Isolation of adipose tissue-derived stem cells’’.Turk J Vet Anim Sci. 40: 137-141.
20. Referans 20. Eslaminejad B., Fallah N. Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow-derived mesenchymal stem cells Vet Res Forum. 2013; 4 (2):69-76.
21. Referans 21. Pınarbaşı E. Apopitozis (Programlı Hücre Ölümü). Ed.: Yıldırım A, Bardakçı F, Karataş M, Tanyolaç B, Moleküler Biyoloji, 2. Baskı, Nobel Yayın Dağıtım, 425-470, 2010.
22. Referans 22. Chieregato K, Castegnaro S, Madeo D, et al. Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue. Cytotherapy. 2011;13(8):933-943.
23. Referans 23. Zheng H, Ziyou Yu Z, Deng M, et al. Fat extract improves fat graft survival via proangiogenic, anti-apoptotic and pro-proliferative activities. Stem Cell Res Ther. 2019;10:174.
24. Referans 24. Kim HJ. Stem cell potential in Parkinson’s disease and molecular factors for the generation of dopamine neurons. Biochim Biophys Acta. 2010; 1812(1):1-11.
25. Referans 25. Hass R, Kasper C, Böhm S, et al. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. J Cell Commun Signal. 2011;9(12):1-14.
26. Referans 26. Varga I, Miko M, Oravcová L et al. Ultra-structural morphology of long-term cultivated white adipose tissue-derived stem cells. Cell Tissue Bank. 2015;16(4):639-647.
27. Referans 27. Osnes-Ringen Ø, Berg KH, Moe MC et al. Cell death pattern in lens epithelium of cataract patients. Acta Ophthalmol. 2016; 94(5): 514–520.
28. Referans 28. Ross W and Hall PA. Ki-67 from antibody to molecule to understanding? Clin. Mol. Pathol.1995;48:113-117.
29. Referans 29. Heidari B, Shirazi A, Akhondi MM., et al. Comparison of proliferative and multilineage differentiation potential of sheep mesenchymal stem cells derived from bone marrow, liver, and adipose tissue. Avicenna J Med Biotechnol. 2013;5(2):104-117.
30. Referans 30. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8: 315-317.
31. Referans 31. Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, Ma K, Zhou C. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem Cells Dev. 2008;17: 761–773.