Mustafa Ertan AY, Ezgi ÇOKAKLI, Murat ÇOKAKLI, Ümit KARAKAŞ, Özlem İZCİ AY, Hüseyin DURUKAN, Mehmet Emin ERDAL

Amniyotik sıvı hücrelerinde kök hücre pluripotensi belirteçlerinin ifadesi

Amaç: Amniyotik sıvı farklı seviyelerde kendini yenileyebilen hücre gruplarından oluşmaktadır ve bu hücrelerin kök hücre özellikleri henüz tam olarak karakterize edilmemiştir. Son yıllarda amniyotik sıvı hücreleri (AFC) rejenaratif tıp için umut veren kök hücre kaynağı olarak görülmektedir. AFC’ler transfeksiyonel ve kimyasal stratejiler ile indüklenmiş pluripotent kök hücre (iPSC)’ler oluşturmak için terminal olarak farklılaşmış hücrelerden daha kolay ve etkili bir şekilde yeniden programlanabilirler. Fakat AFC’lerin kök hücre potansiyelleri ve farklı hücre soylarına farklılaşma kapasitesi tam olarak anlaşılamamıştır. Bu çalışmada amniyotik sıvı hücrelerinde kök hücre pluripotensi belirteçlerinin ifadelerinin ve kök hücre karakterlerinin belirlenmesi amaçlanmıştır. Yöntem: Bu çalışmada, 17 amniyon sıvısından elde edilen AFC’lerde pluripotensi belirteçlerinin ifade edilip edilmediği geleneksel RT-PCR ile kalitatif olarak araştırılmıştır. Bulgular: Elde edilen sonuçlar, farklı hastalardan alınan AFC örneklerinde incelenen pluripotensi belirteçlerinden OCT3/OCT4, SOX2, KLF4, MYC, KIT, NANOG, DPPA3, DPPA5, FUT4, SALL4’ün ifade edildiğini, UTF1 için ise non-spesifik ifade olduğunu göstermektedir. Sonuç: Elde edilen bulgular doğrultusunda AFC’nin kendine özgü bir kök hücre profiline sahip olduğu ve pluripotensi belirteçlerinin yeniden programlama ve rejeneratif tıp uygulamaları için hücre seçiminde etkili şekilde kullanılabileceği düşünülmektedir.

Anahtar Kelimeler:

Amniyotik sıvı hücresi, pluripotensi belirteci, ekspresyon, rejeneratif tıp

Expression of stem-cell pluripotency markers in amniotic fluid cells

Aim: As being a heterogenous population but having different pools of self-renewing cells, amniotic fluid cells (AFCs) have been considered as promising cell sources for regenerative medicine owing to their multipotency and effective reprogramming capacity. AFCs can more easily and effectively be reprogrammed than terminally differentiated cells to generate induced pluripotent stem cells (iPSCs) by transfectional and chemical strategies. But stem cell dynamics and differentiation capacity of AFCs into multiple lineages were poorly understood. The aim of this study was to determine the expression and stem cell characteristics of stem cell pluripotency markers in amniotic fluid cells. Methods: In this study, the expression profile of pluripotency markers in AFCs taken from 17 patients were investigated by conventional RT-PCR by qualitatively. Results: The results showed that all the pluripotency markers (OCT3/OCT4, SOX2, KLF4, MYC, KIT, NANOG, DPPA3, DPPA5, FUT4, SALL4) were expressed in AFCs from different patients but UTF1 is shows a non-specific expression. Conclusion: Consequently, our results revealed that AFCs have a unique stem cell profile and pluripotency markers may be impactful when choosing cells for reprogramming and regenerative medicine applications.

Keywords:

Amniotic fluid cells, pluripotency markers, expression, regenerative medicine,

PDF

___

1. Roubelakis MG, Trohatou O, Anagnou NP. Amniotic fluid and amniotic membrane stem cells: marker discovery. Stem Cells International 2012, 107836.
2. Gholizadeh-Ghalehaziz S, Farahzadi R, Fathi E, Pashaiasl M. A mini overview of isolation, characterization and application of amniotic fluid stem cells. International Journal of Stem Cells 2015; 8(2): 115-120.
3. Klemmt PA, Vafaizadeh V, Groner B. The potential of amniotic fluid stem cells for cellular therapy and tissue engineering. Expert Opin Biol Ther 2011; 11(10): 1297-1314.
4. Galende E, Karakikes I, Edelman L, Desnick AJ, Kerenyi T, Khoueiry G, Lafferty J, McGinn JT, Brodman M, Fuster V, Hajjar R, Polgar K. Amniotic fluid cells are more efficiently reprogrammed to pluripotency than adult cells. Cellular Reprograming 2010; 12(2): 117-125.
5. Roubelakis MG, Pappa KI, Bitsika V, Zagoura D, Vlahou A, Papadaki HA, Antsaklis A, Anagnou NP. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev 2007; 16(6): 931-952.
6. Li C, Zhou J, Shi G, Ma Y, Yang Y, Gu J, Yu H, Jin S, Wei Z, Chen F, Jin Y. Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells. Human Molecular Genetics 2009; 18(22): 4340-4349.
7. Ditadi A, Coppi P, Picone O, Gautreau L, Smati R, Six E, Bonhomme D, Ezine S, Frydman R, Cavazzana-Calvo M, Andre-Schmutz I. Human and murine amniotic fluid c-Kit+/Lin- cells display hematopoietic activity. Blood 2017; 113(17): 3953-3960.
8. Bossolasco P, Montemurro T, Cova L, Zangrossi S, Calzarossa C, Buiatiotis S, Soligo D, Bosari S, Silani V, Deliliers LG, Rebulla P, Lazzari L. Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential. Cell Research 2006; 16: 329-336.
9. Kang JH, Park HJ, Jung YW, Shim SH, Sung SR, Park JE, Cha DH, Ahn EH. Comparative transriptome analysis of cell-free fetal RNA from amniotic fluid an RNA from amniocytes in uncomplicated pregnancies. Plos One 2015;10(7):1-13.
10. Chen L., Daley GQ. Molecular basis of pluripotency. Human Molecular Genetics 2008; 17(1): 23-27.
11. Liu X, Huang J, Chen T, Wang Y, Xin S, Li J, Pei G, Kang J. Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Cell Research 2008; 18: 1177-1189.
12. Zhao W, Ji X, Zhang F, Li L, Ma L. Embryonic stem cell markers. Molecules 2012; 17: 6196-6236.
13. Pazhanisamy S. Adult stem cells and embryonic stem cell markers. Mater Methods 2013; 3: 200.
14. Feng C, Jia YD, Zhao XY. Pluripotency of induced pluripotent stem cells. Genomics Proteomics Bioinformatics 2013; 11: 299-303.
15. Ramirez JM, Gerbal-Chaloin S, Milhavet O, Qiang B, Becker F, Assou S, Lemaitre JM, Hamamah S, De Vos J. Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogenity – all markers are not equal. Stem Cells 2011; 29:1469-1474.
16. Maguire CT, Demarest BL, Hill JT, Palmer JD, Brothman AR, Yost HJ, Condic ML. Genome-wide analysis reveals the unique stem cell identity of human amniocytes. PloS One 2013; 8(1): 1-16.
17. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162(1): 156-159.
18. Kunsaki SM, Freedman DA, Fauza DO. Fetal tracheal reconstruction with cartiaginous grafts engineered from mesenchymal amniocytes. Journal of Pediatric Surgery 2006; 41: 675-682.
19. Gekas J, Walther G, Skuk D, Bujold E, Harvey I, Franc O, Bertrand F. In-vitro and in-vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage. Clinical Experimental Medicine 2010; 10: 1-6.
20. Ge X, Wang IE, Toma I, Sebastiano V, Liu J, Butte MJ, Pera RAR, Yang PC. Human mesenchymal stem cell-derived induced pluripotent sttem cells may generate a universal source of cardiac cells. Stem Cells and Development 2012; 21(15): 2798-2808.
21. Anchan RM, Quaas P, Gerami-Naini B, Bartake H, Griffin A, Zhou Y, Eaton JL, George LL, Naber C, Turbe-Doan A, Park JP, Hornstein MD, Maas RL. Amniocytes can serve a dual function as a source of iPS cells and feeder layers. Human Molecular Genetics 2011; 20(5): 962-974.
22. Kunisaki SM, Armant M, Kao SG, Stevenson K, Kim H, Fauza DO. Tissue engineering from human mesenchymal amniocytes: a prelude to clinical trials. Journal of Pediatric Surgery 2007; 42: 974-980.
23. Kaviani A, Guleserian K, Perry TE, Jenningd RW, Ziegler MM, Fauza DO. Fetal tissue engineering from amniotic fluid. Journal of the American College of Surgeons 2003; 196: 592-597.