İNSAN PERİFERİK KANINDAN ÇOK KÜÇÜK EMBRİYONİK (VSEL) KÖK HÜCRELERİN ELDE EDİLMESİ VE TANIMLANMASI

Yıl 2019, , 96 - 106, 19.06.2019

Serap Erdem Kuruca Dolay Damla Çelik Gülderen Demirel Dilşad Özerkan

https://doi.org/10.26650/IUITFD.2018.0001

Öz

Amaç: Kök hücre (KH) nakli malin kan hastalıkları, kemik iliği yetmezlikleri ve doğumsal kan hastalıklarının tedavisinde kullanılan yöntemdir. Bu amaçla, periferik kan projenitör hücreleri (PKPH) oldukça fazla kullanılmaktadır. Son yıllarda insan embriyonik kök hücrelerinin sahip olduğu pluripotent özelliği bulunan yeni KH türü tanımlanmıştır. Çok küçük embriyonik benzeri (VSEL) kök hücreler olarak adlandırılan bu hücrelerin, yetişkin bireyde periferik kanda (PK) bulunduğu düşünülmektedir. Bu çalışmada VSEL kök hücrelerinin periferik kan kaynağı kullanarak elde edilmesi ve tanımlanması amaçlanmıştır. Gereç ve Yöntem: Donörlerden alınan materyallerden lizis ve fikol gradient yöntemleri kullanılarak elde edilen mononükleer ve eritrosit katmanınlarından, VSEL hücreleri izole edilmiştir. Flov sitometri ve immünfloresan boyama ile NANOG, OCT4, SSEA-4 ve CXCR-4 embriyonal kök hücre belirteçlerinin varlığı incelenmiştir. Western blot yöntemiyle ise, NANOG ve OCT4 proteinlerinin varlığı araştırılmıştır. Bulgular: Flov sitometri sonuçlarına göre debris katmanında, VSEL belirteci taşıyan hücre sayısı CD45- popülasyonuna göre daha yüksek bulunmuştur. Elde edilen western ve immünfloresan sonuçlarına göre yüksek miktarda OCT4 ve NANOG ekspresyonu görülmüştür. Aynı zamanda bu proteinlerin hücre içinde hem sitoplazmada hem de çekirdekte bulunduğu gösterilmiştir. Sonuç: Pluripotent kök hücre belirteci olarak bilinen bu proteinlerin yetişkin periferik kanında yüksek miktarda ekspresyonunun, farklılaşmış dokularda nasıl görev yaptığı sorusunu akıllara getirmektedir. Bu bulgular; periferik kanda pluripotent belirteçler taşıyan yeni bir kök hücre populasyonu bulunduğu tezini güçlendirmekte ve bu konuda yapılacak ileriye dönük klinik çalışmalar içinde temel oluşturmaktadır.

Anahtar Kelimeler

Çok küçük embriyonik benzeri (VSEL) kök hücreler, Periferik kan, Pluripotent kök hücre markırları

CHARACTERIZATION AND ISOLATION OF VERY SMALL EMBRYONIC-LIKE (VSEL) STEM CELLS OBTAINED FROM HUMAN PERIPHERAL BLOOD

Öz

Objective: Stem cell transplantation is considered to be one of the available treatments for malign or hereditary blood diseases and bone marrow failure. Peripheral blood progenitor cells (PBPC) are widely used for this technique. Recently a new type of stem cell with a pluripotent potential has been identified. These cells, called very small embryonic-like (VSEL) stem cells, are thought to be found in peripheral blood (PB) in adult individuals. The aim of this study was to obtain and identify VSEL stem cells using a peripheral blood source. Material and Method: VSEL cells were isolated from mononuclear and erythrocyte layers obtained by using lysis and ficoll gradient methods from the materials taken from donors. The presence of NANOG, OCT4, SSEA-4 and CXCR-4 embryonal stem cell markers by flow cytometry and immunofluorescence staining was investigated. The presence of NANOG and OCT4 proteins was investigated using the Western blot method. Results: According to the flow cytometry results, the number of cells carrying the VSEL marker was higher in the debris layer than in the CD45-population. Western and immunofluorescence results showed high levels of OCT4 and NANOG expression. It was also determined that these proteins are present in both the cytoplasm and the nucleus. Conclusions: High expression of these proteins, known as pluripotent stem cell markers, in adult peripheral blood brings to mind the question of how they work in differentiated tissues. These findings strengthen the thesis of a new stem cell population that have pluripotent markers in peripheral blood and provide the basis for future clinical studies about this point.

Anahtar Kelimeler

Very Small Embryonic-like (VSEL) stem cells, Peripheral blood, Pluripotent stem cell markers

Kaynakça

• 1. Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M, Ratajcsak J. et al, A population of very embryonic-like (VSEL) CXCR4+ SSEA-1+ Oct-4+ stem cells identified in adult bone marrow. Leukemia 2006;20:857-69.
• 2. Zuba-Surma EK, Kucia M, Ratajczak J, Patajczak MZ. “Small stem cells” in adult tissues: Very small embryonic-like stem cells (VSELs) Stand up. Cytometry 2009;75(1):4-13.
• 3. Kucia M, Wysoczynski M, Wu W, Zuba-Surma EK, Ratajczak J, Ratajczak MZ. Evidence that very small embryonic-like stem cells are mobilized into peripheral blood. Stem Cells 2008;26:2083-92.
• 4. Loos H, Blok-Schut B, Van Doorn R, Hoksbergen R, Riviere AB, Meerhof L. A method for the recognition and separation of human blood monocytes on density gradient. Blood 1976;48:731-42.
• 5. Bhartiya D, Shaikh A, Nagvenkar P, Kasiviswananthan S, Pethe Prasad, Pawani H, et al. Very small embryoniclike stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy. Stem Cells Dev 2012;21(1):1-6.
• 6. Chang Y, Tien K, Wen C, Hsieh T, Hwang S. Recovery of CD45(-)/Lin(-)/SSEA-4(+) very small embryonic-like stem cells by cord blood bank standard operating procedures. Cytotherapy 2014;16(4):560-5.
• 7. Sovalat H, Scrofani M, Eidenschenk A, Pasquet S, Rimelen V, Henon P. Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34+/CD133+/CXCR4+/ Lin-CD45- cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells. Exp Hematol 2011;39:495-505.
• 8. Ratajczak MZ, Zuba-Surma E, Wojakowski W, Kucia M, Ratajczak J. Bone marrow – Home of versatile stem cells. Transfus Med Hemother 2008;35:248-59.
• 9. Kucia M, Ratajczak J, Ratajczak MZ. Are bona marrow stem cells plastic or heterogenous – That is the question. Exp Hematol 2005;33:613-23.
• 10. Ratajczak MZ, Zuba-Surma E, Wojaskowski W, Suszynzka M, Liu R, Ratajczak J, et al. Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate. Leukemia 2014;28:473-84.
• 11. Parker GC. Very small embryonic-like stem cells: A scientific debate? Stem Cells Dev 2014;23(7):687-8.
• 12. Kassmer SH, Krause DS. Very small embryonic-like stem cells: Biology and function of these potential endogenous pluripotent stem cells in adult tissues. Mol Reprod Dev 2013;80:677-90.
• 13. Heider A, Danova-Alt R, Egger D, Cross M, Alt R. Murine and human very small embryonic-like cells: A perspective. Cytometry 2012;83A:72-5.
• 14. Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M, Moldenhawer S, Zuba-Surma E. et al. Morphological and molecular characterization of novel population of CXCR4+/ SSEA-4+/Oct-4+ very small embryonic-like cells purified from human cord blood – preliminary report. Leukemia 2007;21;297-303.
• 15. Zangrossi S, Marabese M, Broggini M, Giordano R, D’Erasmo M, Montelatici E. et al. Oct-4 expression in adult human differentiated cells challenges its role as a pure stem cell marker. Stem Cells 2007;25:1675-80.
• 16. Lee J, Kim HK, Rho J, Han Y, Kim J. Human Oct4 isoforms differ in their abilitiy to confer self-renewal. J Biol Chem 2006;281:33554-65.
• 17. Caufmann G, Liebaers I, Steirteghem AV, Velde HVD. POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embriyos. Stem Cells 2006;24;2685-91.
• 18. Liedtke S, Enczmann J, Walclawczyk S, Wernet P, Kögler G. Oct4 and its pseudogenes confuse stem cell research. Cell Stem Cell 2007;1:364-6.
• 19. Tai M, Chang C, Olson LK, Trosko JE. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 2005;26(2):495502.