Characterization and comparison of mesenchymal stem cells derived from rat perirenal and periovarian adipose tissue

Öz

Purpose: In rats, adipose tissue with white unilocular features is found in the perigonadal region. Adipose tissue contains adipocytes, adipocyte progenitor cells, mesenchymal stem cells, fibroblasts, endothelial cells, pericytes, and immune cells. Mesenchymal stem cells are multipotent stem cells and have been isolated from many tissues, including adipose tissue and connective tissue. Our aim in this study is to isolate mesenchymal stem cells from perirenal adipose tissue and periovarian adipose tissue and compare their features with each other. Materials and methods: Adipose tissues around the ovaries and kidneys of two 6-week-old Wistar Albino rats were used. Adipose tissue was cut into small pieces, and explant cell culture was established. Flow cytometry analysis was performed at the third passage (P3) for the characterization of isolated and produced mesenchymal stem cells. CD29, CD54, CD90, CD45, and MHC Class II were used as stem cell markers. mRNA expression changes of CDK6, Cyclin-D1, p21, caspase-3, caspase-8, and caspase-9 genes involved in cell cycle regulation and apoptosis were examined in cell groups. Results: Adherent cells that proliferated in explant cell culture were isolated and multiplied. In the flow cytometry analysis, it was observed that the cells expressed CD29, CD54, and CD90 markers, but did not express the cell surface antigens CD45 and MHC Class II. In the analyses performed with perirenal adipose tissue and periovarian adipose tissue-derived mesenchymal stem cells, no statistically significant difference was found in the expression of genes involved in the regulation of the cell cycle and apoptosis. Conclusion: The use of adipose tissue-derived mesenchymal stem cells from different regions, which have similar molecular properties to periovarian adipose tissue-derived mesenchymal stem cells, in ovarian failures may result in better development of follicles and the acquisition of quality oocytes.

Anahtar Kelimeler

• Adipose tissue
• cell culture
• infertility
• ovary
• mesenchymal stem cell

Sıçan perirenal ve perioveryan adipoz doku kaynaklı mezenkimal kök hücrelerin karakterizasyonu ve birbirleriyle karşılaştırılması

Öz

Amaç: Sıçanlarda, perigonodal bölgede beyaz unilokuler özellikte olan adipoz doku bulunur. Adipoz doku, adipositler yanında adiposit progenitör hücreler, mezenkimal kök hücreler, fibroblastlar, endotel hücreleri, perisitler ve immün hücreleri içerir. Mezenkimal kök hücreler multipotent özellikte olan kök hücrelerdir ve şimdiye kadar adipoz doku dahil olmak üzere bağ dokusu içeren birçok dokudan izole edilmiştirler. Bu çalışmadaki amacımız perirenal adipoz dokudan ve perioveryan adipoz dokudan mezenkimal kök hücreleri izole ederek özelliklerini birbirleriyle karşılaştırmaktır. Gereç ve yöntem: İki adet 6 haftalık Wistar Albino tipi sıçanın ovaryum ve böbrek çevresindeki adipoz dokuları çıkarıldı. Adipoz doku küçük parçalara ayrılarak eksplant hücre kültürü oluşturuldu. 826 İzole edilip üretilen mezenkimal kök hücrelerin karakterizasyonu için üçüncü pasajda (P3) flow sitometri analizi yapıldı. Kök hücre belirteçleri olarak CD29, CD54, CD90, CD45 ve MHC Class II kullanıldı. Hücre döngüsünün regülasyonunda ve apoptozda rol alan genlerde CDK6, Cyclin-D1, p21, caspase-3, caspase-8, caspase-9 genlerinin hücre gruplarındaki mRNA ekspresyon değişimleri incelendi. Bulgular: Eksplant hücre kültüründe prolifere olan adherent özellikteki hücreler izole edildi ve çoğaltıldı. Yapılan flow sitometri analizinde hücrelerin CD29, CD54 ve CD90 belirteçlerini eksprese ettikleri, buna karşılık hücre yüzey antijenleri olan CD45 ve MHC Class II’yi eksprese etmedikleri gözlemlenmiştir. Perirenal adipoz doku ve perioveryan adipoz doku kökenli mezenkimal kök hücrelerle yapılan analizlerde hücre döngüsünün regülasyonunda ve apoptozda rol alan genlerin ekspresyonlarında istatistiksel olarak bir farklılık bulunamadı. Sonuç: Perioveryan adipoz doku kaynaklı mezenkimal kök hücrelere benzer moleküler özelliklere sahip, farklı bölgelerdeki adipoz doku kökenli mezenkimal kök hücrelerin overyan yetmezliklerde kullanılması foliküllerin daha iyi gelişim göstermesine ve kaliteli oositlerin elde edilmesine neden olabilir.

Anahtar Kelimeler

• Adipoz doku
• hücre kültürü
• infertilite
• ovaryum
• mezenkimal kök hücre

Kaynakça

1. Ross MH, Pawlina W. Histology. Lippincott Williams & Wilkins; 2006.
2. Junqueira LCU, Carneiro J. Basic histology: text & atlas. Vol 3: McGraw-Hill New York; 2005.
3. Kierszenbaum AL, Tres L. Histology and cell biology: an introduction to pathology. (No Title). 2003.
4. Ünal MS, Tan S, Seçme M. Changes in structure during the corpus luteum's formation. Pam Med J. 2024;17(2):285-301. doi:10.31362/patd.1383988
5. Unal MS, Secme M. Does the ovarian surface epithelium differentiate into primordial follicle and primary follicle precursor structures? Cukurova Med J. 2022;47(3):1256-1262. doi:10.17826/cumj.1134852
6. Niyaz M, Gürpinar ÖA, Günaydin S, Onur MA. Isolation, culturing and characterization of rat adipose tissue-derived mesenchymal stem cells: a simple technique. Turkish Journal of Biology. 2012;36(6):658-664. doi:10.3906/biy-1109-31
7. Çil N, Yaka M, Ünal MS, et al. Adipose derived mesenchymal stem cell treatment in experimental asherman syndrome induced rats. Mol Biol Rep. 2020;47(6):4541-4552. doi:10.1007/s11033-020-05505-4
8. Kus G, Oztopcu-Vatan P, Uyar R, et al. Cytotoxic and apoptotic functions of licofelone on rat glioma cells. Acta Biol Hung. 2013;64(4):438-452. doi:10.1556/ABiol.64.2013.4.4

9. Sepehri B, Darbani R, Mesgari-Abbasi M, et al. The effects of short-time air pollution, SO₂, and ozone on biochemical, histo-pathological, oxidative stress, and carcinogenesis-related gene expressions in the liver of rats. Hum Exp Toxicol. 2024;43:9603271241263569. doi:10.1177/09603271241263569
10. Ashrafi M, Bathaie SZ, Abroun S. High expression of Cyclin D1 and p21 in N-nitroso-N-methylurea-induced breast cancer in Wistar albino female rats. Cell J. 2012;14(3):193-202.
11. Liu M, Li M, Liu J, et al. Elevated urinary urea by high-protein diet could be one of the inducements of bladder disorders. J Transl Med. 2016;14:53. doi:10.1186/s12967-016-0809-9
12. Baglioni S, Francalanci M, Squecco R, et al. Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J. 2009;23(10):3494-3505. doi:10.1096/fj.08-126946
13. Hendawy H, Kaneda M, Metwally E, Shimada K, Tanaka T, Tanaka R. A Comparative Study of the Effect of Anatomical Site on Multiple Differentiation of Adipose-Derived Stem Cells in Rats. Cells. 2021;10(9):2469. doi:10.3390/cells10092469
14. Balducci L, Alessandri G. Isolation, Expansion, and Immortalization of Human Adipose-Derived Mesenchymal Stromal Cells from Biopsies and Liposuction Specimens. Methods Mol Biol. 2016;1416:259-274. doi:10.1007/978-1-4939-3584-0_15
15. Sherman LS, Condé Green A, Kotamarti VS, Lee ES, Rameshwar P. Enzyme-Free Isolation of Adipose-Derived Mesenchymal Stem Cells. Methods Mol Biol. 2018;1842:203-206. doi:10.1007/978-1-4939-8697-2_14
16. Li J, Li H, Tian W. Isolation of Murine Adipose-Derived Stromal/Stem Cells Using an Explant Culture Method. Methods Mol Biol. 2018;1773:167-171. doi:10.1007/978-1-4939-7799-4_14
17. Francis MP, Sachs PC, Elmore LW, Holt SE. Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction. Organogenesis. 2010;6(1):11-14. doi:10.4161/org.6.1.10019
18. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12):4279-4295. doi:10.1091/mbc.e02-02-0105
19. Dipali SS, Ferreira CR, Zhou LT, Pritchard MT, Duncan FE. Histologic analysis and lipid profiling reveal reproductive age-associated changes in peri-ovarian adipose tissue. Reprod Biol Endocrinol. 2019;17(1):46. doi:10.1186/s12958-019-0487-6
20. Yang L, Chen L, Lu X, et al. Peri-ovarian adipose tissue contributes to intraovarian control during folliculogenesis in mice. Reproduction. 2018;156(2):133-144. doi:10.1530/REP-18-0120
21. Zhu M, Shen Q, Li X, Kang J. Removal of peri-ovarian adipose tissue affects follicular development and lipid metabolism†. Biol Reprod. 2020;103(6):1199-1208. doi:10.1093/biolre/ioaa144
22. Wagner IV, Sahlin L, Savchuk I, Klöting N, Svechnikov K, Söder O. Adipose Tissue is a Potential Source of Hyperandrogenism in Obese Female Rats. Obesity (Silver Spring). 2018;26(7):1161-1167. doi:10.1002/oby.22198
23. Cousin B, Cinti S, Morroni M, et al. Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization. J Cell Sci. 1992;103(4):931-942. doi:10.1242/jcs.103.4.931
24. Zhang L, An G, Wu S, et al. Long-term intermittent cold exposure affects peri-ovarian adipose tissue and ovarian microenvironment in rats. J Ovarian Res. 2021;14(1):107. doi:10.1186/s13048-021-00851-8
25. Nteeba J, Ortinau LC, Perfield JW 2nd, Keating AF. Diet-induced obesity alters immune cell infiltration and expression of inflammatory cytokine genes in mouse ovarian and peri-ovarian adipose depot tissues. Mol Reprod Dev. 2013;80(11):948-958. doi:10.1002/mrd.22231
26. Grzesiak M, Burzawa G, Kurowska P, et al. Altered vitamin D3 metabolism in the ovary and periovarian adipose tissue of rats with letrozole-induced PCOS. Histochem Cell Biol. 2021;155(1):101-116. doi:10.1007/s00418-020-01928-z
27. Borges CC, Bringhenti I, Aguila MB, Mandarim de Lacerda CA. Vitamin D restriction enhances periovarian adipose tissue inflammation in a model of menopause. Climacteric. 2020;23(1):99-104. doi:10.1080/13697137.2019.1597841
28. Zuccaro A, Zapatería B, Sánchez Alonso MG, et al. Pleiotrophin Deficiency Induces Browning of Periovarian Adipose Tissue and Protects against High-Fat Diet-Induced Hepatic Steatosis. Int J Mol Sci. 2021;22(17):9261. doi:10.3390/ijms22179261
29. El Husseiny HM, Kaneda M, Mady EA, Yoshida T, Doghish AS, Tanaka R. Impact of Adipose Tissue Depot Harvesting Site on the Multilineage Induction Capacity of Male Rat Adipose-Derived Mesenchymal Stem Cells: An In Vitro Study. Int J Mol Sci. 2023;24(8):7513. doi:10.3390/ijms24087513
30. Luukkaa V, Savontaus E, Rouru J, et al. Effects of estrous cycle and steroid replacement on the expression of leptin and uncoupling proteins in adipose tissue in the rat. Gynecol Endocrinol. 2001;15(2):103-112. doi:10.1080/gye.15.2.103.112
31. Hendawy H, Kaneda M, Yoshida T, et al. Heterogeneity of adipose stromal vascular fraction cells from the different harvesting sites in rats. Anat Rec (Hoboken). 2022;315(4):789-802. doi:10.1002/ar.24915