The Effect of Antioxidant Culture Conditions and Isolation Methods in Obtaining of Mesenchymal Stem Cells from The Wharton's Jelly of Human Umbilical Cord

Year 2021, , 43 - 60, 06.04.2021

Tugba Şan Uğur Bora Özge Sayın Mehmet Güneş Deniz Öztekin , Bekir Ergür Kemal Baysal Pınar Akan

https://doi.org/10.5505/deutfd.2021.27132

Abstract

INTRODUCTION: Obtaining and growing human stem cells are the first stages of regenerative medicine applications.During the cell proliferation for therapeutic purposes, MSCs rapidly undergo premature aging, possibly involving oxidative stress.Bone marrow mesenchymal stem cells(BM-MSCs) are the most clinically used cells.It's suggested that Wharton-jelly mesenchymal stem cells(WJ-MSC) which are located around the umbilical cord may be more useful than BM-MSCs due to their non-invasive acquisition,not expressing proteins cause tissue rejection,and their immunosuppressive properties.It was aimed to compare the efficiency of the isolation methods used in the reproduction of WJ-MSCs and to determine the effect of anti-oxidative culture conditions on the cell viability and expression of cell-surface antigens.
METHODS: Postnatal cord samples were taken from 17 healthy pregnant women who met the inclusion criteria. Stem cells were obtained from Wharton jelly using enzymatic and explant methods, and cell viability and proliferation capacities were compared.
RESULTS: The differentiation capacity of stem cells into osteoblasts, chondrocytes and adipocytes was demonstrated immunohistochemically. During the proliferation process, it was determined by flow cytometry that CD44, CD73, CD90, CD105 surface antigen expressions were expressed and unchanged until the fourth passage.The effect of culture conditions provided by addition of anti-oxidant molecules N-acetyl cysteine and ascorbic acid on cell viability and cell surface antigen expressions were demonstrated.
DISCUSSION AND CONCLUSION: In conclusion, it was thought that the explant method in the isolation of MSCs from wharton jelly was more advantageous than the enzymatic method due to the total number of cells obtained in a shorter time period, without significantly changing the surface antigen expressions.

Keywords

Wharton jelly, isolation methods, antioxidants, mesenchymal stem cells, cell viability

Project Number

2017.KB.SAG.028

İnsan Göbek Kordonu Wharton Jölesinden Mezenkimal Kök Hücrelerin Eldesinde İzolasyon Yöntemlerinin Ve Antioksidatif Kültür Koşullarının Etkisi

Abstract

GİRİŞ ve AMAÇ: Günümüzde, insan kök hücrelerin eldesi ve kültür ortamında çoğaltılması rejeneratif tıp uygulamalarının ilk aşamalarından biridir. Ancak terapötik amaçlar için in vitro hücre çoğaltmada, MKH'lerin hızla muhtemelen oksidatif stresi içeren erken yaşlanmaya girmeleri önemli bir sorundur. Günümüzde üzerinde en fazla çalışılmış ve klinikte tedavide kullanılan hücreler kemik iliği mezenkimal kök hücreleri (Kİ-MKH) olmakla birlikte, insan perinatal dokularında tespit edilmiş mezankimal kök hücreler giderek önem kazanmaktadır. Göbek kordonu çevresinde yer alan Wharton jölesi mezenkimal kök hücrelerinin (WJ-MKH) invaziv olmayan şekilde elde edilmeleri, doku reddine yol açan proteinleri eksprese etmemeleri ve immünosupresif özellikleri nedeni ile Kİ-MKH’lerinden daha kullanışlı olabilecekleri ileri sürülmektedir. Çalışmamız ile WJ-MKH’inin eldesi ve çoğaltılmasında kullanılan izolasyon yöntemlerinin etkinliğinin karşılaştırılması ve anti-oksidatif kültür koşullarının hücre canlılığı ve kök hücrelere özgü yüzey antijen ekspresyonlarına etkisinin belirlenmesi amaçlandı.
YÖNTEM ve GEREÇLER: Etik Kurul onayı alındıktan sonra gönüllü onam alınmış, çalışmaya dahil etme kriterlerine uyan toplam 17 sağlıklı gebeden doğum sonrası kordon örnekleri alındı. Wharton jölelesinden enzimatik ve eksplant yöntemi ile kök hücreler elde edilerek, hücre canlılıkları ve proliferasyon kapasiteleri karşılaştırıldı. Kök hücrelerin osteoblast, kondrosit ve adipositlere farklılaşma kapasiteleri immunohistokimyasal olarak gösterildi.
BULGULAR: Hücrelerin çoğaltılması sürecinde CD44, CD73, CD90, CD105 yüzey antijen ifadelerinin dördüncü pasaja kadar anlamlı değişmeden eksprese edildiği akış sitometrik olarak belirlendi. Anti oksidan moleküller N-asetil sistein ve askorbik asit ilavesi ile sağlanan kültür koşullarının hücre canlılığı ve hücre yüzey antijen ekspresyonları üzerine etkisi gösterildi.
TARTIŞMA ve SONUÇ: Sonuç olarak wharton jölesinden MKH’lerin izolasyonunda eksplant yönteminin yüzey antijen ifadeleri anlamlı değişmeksizin, daha kısa zaman diliminde elde edilen toplam hücre sayısı nedeni ile enzimatik yönteme göre daha avantajlı olduğu düşünüldü.

Keywords

wharton jeli, izolasyon yöntemleri, antioksidanlar, mezenkimal kök hücre, hücre canlılığı

Supporting Institution

DEÜ BAP

Project Number

2017.KB.SAG.028

Thanks

Bu araştırma Dokuz Eylül Üniversitesi Bilimsel Araştırma Projeleri Şube Müdürlüğü tarafından 2017.KB.SAG.028 kodu ile desteklenmiştir.

References

• Bianco P, Robey PG, Simmons PJ. Mesenchymal Stem Cells: Revisiting History, Concepts, and Assays. Cell Stem Cell. 2008;2:313–19.
• Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-47.
• Gilbert DM. The future of human embryonic stem cell research: Addressing ethical conflict with responsible scientific research. Med Sci Monit. 2004;10:RA99-103.
• Friedenstein AJ, Chailakhjan RK, Lalykina LK. The Development of Fibroblast Colonies in Marrow and Spleen Cells. Cell Tissue Kinet. 1970;3:393-403.
• Prockop DJ. Repair of tissues by adult stem/progenitor cells (MSCs): Controversies, myths, and changing paradigms. Mol Ther. 2009;17:939-46.
• Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–47.
• Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: Paradoxes of passaging. Exp Hematol. 2004;32:414-25.
• Chen G, Yue A, Ruan Z, Yin Y, Wang R, Ren Y, et al. Comparison of biological characteristics of mesenchymal stem cells derived from maternal-origin placenta and Wharton’s jelly. Stem Cell Res Ther. 2015;6:228.
• Kalaszczynska I, Ferdyn K. Wharton’s jelly derived mesenchymal stem cells: Future of regenerative medicine? Recent findings and clinical significance. BioMed Res Int. 2015;2015:430847.
• Liau LL, Ruszymah BHI, Ng MH, Law JX. Characteristics and clinical applications of Wharton’s jelly-derived mesenchymal stromal cells. Curr Res Transl Med. 2020;68:5–16.
• Bieback K, Brinkmann I. Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy. World J Stem Cells. 2010;2: 81–92.
• Zhang Y, Tao H, Gu T, Zhou M, Jia Z, Jiang G, et al. The effects of human Wharton’s jelly cell transplantation on the intervertebral disc in a canine disc degeneration model. Stem Cell Res Ther. 2015;6:154.
• Paldino E, Cenciarelli C, Giampaolo A, Milazzo L, Pescatori M, Hassan HJ, et al. Induction of Dopaminergic Neurons From Human Wharton’s Jelly Mesenchymal Stem Cell by Forskolin. J Cell Physiol. 2014;229(2):232-44.
• Zhang L, Tan X, Dong C, Zou L, Zhao H, Zhang X, et al. In vitro differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs), derived from Wharton’s jelly, into choline acetyltransferase (ChAT)-positive cells. Int J Dev Neurosci. 2012;30:471-7.
• Liang J, Wu S, Zhao H, Li S lei, Liu Z xin, Wu J, et al. Human umbilical cord mesenchymal stem cells derived from Wharton’s jelly differentiate into cholinergic-like neurons in vitro. Neurosci Lett. 2013;532:59-63.
• Fu Y-S, Cheng Y-C, Lin M-YA, Cheng H, Chu P-M, Chou S-C, et al. Conversion of Human Umbilical Cord Mesenchymal Stem Cells in Wharton’s Jelly to Dopaminergic Neurons In Vitro: Potential Therapeutic Application for Parkinsonism. Stem Cells. 2006;24(1):115-124.
• Orazızadeh M, Hashemıtabar M, Bahramzadeh S, Dehbashı Fn, Saremy S. Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin. Biomed Rep. 2015;3:304-8.
• Kozubenko N, Turnovcova K, Kapcalova M, Butenko O, Anderova M, Rusnakova V, et al. Analysis of in vitro and in vivo characteristics of human embryonic stem cell-derived neural precursors. Cell Transplant. 2010;19:471-86.
• Choo KB, Tai L, Hymavathee KS, Wong CY, Nguyen PNN, Huang CJ, et al. Oxidative stress-induced premature senescence in Wharton’s jelly-derived mesenchymal stem cells. Int J Med Sci. 2014;11:1201-7.
• Jain AK, Singh D, Dubey K, Maurya R, Mittal S, Pandey AK. Models and Methods for In Vitro Toxicity. In: In Vitro Toxicology. 2018;3:45-65.
• Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal.2011;9:12.
• 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;8:315-17.
• Marcus AJ, Woodbury D. Fetal stem cells from extra-embryonic tissues: Do not discard: Stem Cells Review Series. J Cell Mol Med. 2008;12:730–42.
• Kelly SS, Sola CBS, De Lima M, Shpall E. Ex vivo expansion of cord blood. Bone Marrow Transplant. 2009;44:673-81.
• Taghizadeh RR, Cetrulo KJ, Cetrulo CL. Wharton’s Jelly stem cells: Future clinical applications. Placenta. 2011;32:311-315.
• Salehinejad P, Banu Alitheen N, Ali AM, Omar AR, Mohit M, Janzamin E, et al. Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly. Vitr Cell Dev Biol Anim. 2012;48:75-83.
• Lim J, Razi ZRM, Law J, Nawi AM, Idrus RBH, Ng MH. MSCs can be differentially isolated from maternal, middle and fetal segments of the human umbilical cord. Cytotherapy. 2016;18:1493-1502.
• Tang J, Wang X, Tan K, Zhu H, Zhang Y, Ouyang W, et al. Injury-induced fetal reprogramming imparts multipotency and reparative properties to pericardial adipose stem cells. Stem Cell Res Ther. 2018;9:218.
• Sousa BR, Parreira RC, Fonseca EA, Amaya MJ, Tonelli FMP, Lacerda SMSN, et al. Human adult stem cells from diverse origins: An overview from multiparametric immunophenotyping to clinical applications. Cytometry Part A. 2014;85:43-77.
• Tan K, Zhu H, Zhang J, Ouyang W, Tang J, Zhang Y, et al. CD73 expression on mesenchymal stem cells dictates the reparative properties via its anti-inflammatory activity. Stem Cells Int. 2019;2019:1-12.
• Guo YL, Chakraborty S, Rajan SS, Wang R, Huang F. Effects of oxidative stress on mouse embryonic stem cell proliferation, apoptosis, senescence, and self-renewal. Stem Cells Dev. 2010;19:1321–31.
• Tan DQ, Suda T. Reactive Oxygen Species and Mitochondrial Homeostasis as Regulators of Stem Cell Fate and Function. Antioxid Redox Signal. 2018;29:149-168.
• Lin TM, Tsai JL, Lin SD, Lai CS, Chang CC. Accelerated growth and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants. Stem Cells Dev. 2005;14:92-102.