HEMATOPOETIK KÖK HÜCRE İZOLASYONUNDA ALDH ETKİNLİĞİNİN ÖNEMİ

Abstract

Amaç: Kan hücrelerine kolaylıkla farklılaşabilen hematopoetik kök hücrelerin (HKH) saflaştırmasında tercih edilen yöntem, flow sitometrik veya magnetik ayırım sistemi ile CD34⁺ hücrelerin seçimidir. İnsan kök hücrelerini tamamen fenotipik hücre yüzey belirteçlerine dayanmadan etkin olarak saptayan metodlara ihtiyaç vardır. Bu metodlardan biri retinoid metabolizması ve HKH’lerin siklofosfamid gibi alkilleyici ajanlara direncinde rol oynayan sitozolik aldehid dehidrogenaz (ALDH)’dır. Farklı fonksiyonları olan kordon kanından klinik uygulamalarda kullanılabilen en primitif insan hematopoetik kök ve progenitör hücrelerin daha yüksek saflıkta eldesinin CD34⁺CD38^- yüzey belirteçlerinin yanı sıra ALDH ile de işaretlenerek akım sitometri analizi sonrası ALDH⁺ hücrelerin diğer hücrelerden ayrılarak olabileceğinin belirlenmesi amaçlanmıştır. Hematopoetik hücre serilerine farklılaştırılma çalışmaları için sadece CD34⁺ hücrelerin seçilmesinin yeterli olmadığı ALDH⁺ ve CD38^- negatifliğin de analiz edilmesi amaçlanmıştır.

Gereç ve yöntemler: Biz çalışmamızda CD34 yüzey belirteci olan hücreleri kordon kanından magnetik nanopartiküller kullanarak ayırdıktan sonra flow sitometre cihazıyla, bu hücrelerin içerisinden ALDH etkinliği olup CD38 yüzey antijeni olmayan hücreleri saflaştırdık. Bu hücre gruplarını floresan mikroskobu ile görüntüledik.

Bulgular: Yaptığımız bu çalışmada kordon kanından saflaştırdığımız CD34⁺ hücrelerin ALDH-FITC işaretlenmeleri sonucunda aldığımız floresan mikroskop ve flow sitometrik (facs) analizinde CD34⁺ olan hücrelerin ALDH⁺ olduğu görüldü.

Sonuç: Bu çalışmamızın sonucunda kordon kanından hematopoetik kök hücre izolasyonunda ALDH⁺ hücre grubu CD34⁺CD38^- hücrelerden zengin olduğu ve hücre kültüründe koloni oluşturma özelliğine sahip olduğu görülmüştür. Kordon kanından hematopoetik kök hücre izolasyonunda yalnızca CD34⁺ olmasının yeterli olmadığı ALDH⁺ ve CD38^- negatifliğin de analiz edilmesi gerektiği düşünülmektedir.

Anahtar kelimeler: Hematopoetik kök hücre; kordon kanı; ALDH; CD34.

Keywords

• Hematopoetik kök hücre,kordon kanı,ALDH,CD34

IMPORTANCE OF ALDH ACTIVITY IN HEMATOPOIETIC STEM CELL ISOLATION

Abstract

Objective: The preferred method for the purification of hematopoietic stem cells (HSCs), which can easily differentiate into blood cells, is the selection of CD34⁺ cells by flow cytometry or magnetic discrimination. Methods are needed that effectively determine human stem cells without entirely relying on phenotypic cell surface markers. One of these methods is cytosolic aldehyde dehydrogenase (ALDH), which plays a role in retinoid metabolism and resistance of HSCs to alkylating agents such as cyclophosphamide. The prospective isolation of human hematopoietic stem and progenitor cells with different functions, as well as surface markers, are also recommended by ALDH and flow cytometry. In the above findings, ALDH⁺ cells can be isolated from other cells and the most primitive hematopoietic stem cells can be isolated and used in clinical applications.

Materials and methods: In our study, we separated the CD34 surface marker cells from cord blood using magnetic nanoparticles and then purified the cells with ALDH activity and non-CD38 surface antigen by flow cytometer. We visualized these cell groups with fluorescence microscopy.

Results: We observed that the majority of CD34⁺ cells were ALDH⁺ in fluorescence microscopy and flow cytometry (FACS) analysis of CD34⁺ cells purified from cord blood as a result of ALDH-FITC markings.

Conclusion: As a result of this study, it was found that the ALDH⁺ cells group is rich in CD34⁺ CD38^- cells in hematopoietic stem cells isolated from cord blood, and is capable of colony formation in cell culture. It is suggested that in hematopoietic stem cell isolation from cord blood, only CD34⁺ cells are not sufficient and ALDH⁺ and CD38^- negativity should also be analyzed.

Keywords: Hematopoietic stem cells; cord blood; ALDH; CD34.

Keywords

• Hematopoietic stem cells,cord blood,ALDH,CD34

References

1. 1. Akel S., Perrow S.C., Laughlin J.M., Ruscett W. Neutralization of Aytocrine Transforming Growthβ Factor-βin Human Cord Blood CD34+ CD38-Lin- Cell Promotes Stem Cell Factor-Mediated Erythropoetin-Independed Early Erythroid Progenitor Development and Reduces Terminal Differentiation.Stem Cells 2003;21:557-67.
2. 2. Storms R.W., Green P.D., Saford K.M., Niedzwiecki D., Cogle C.R., Colvin O.M., Chao N.J., Rice H.E., Smith C.A. Distinct hematopoietic progenitor compartments are delinated by the expression of aldehyde dehydrogenase and CD34. Blood 2005; 106: 95-102.
3. 3. Jones R.J., Zuehlsdorf M., Rowley S.D., Hilton J., Santos G.W., Sensenbrenner L.L., Colvin O.M. Variability in 4- hydroperoxycyclophosphamide activity during clinical purging for autologous bone marrow transplantation. Blood 1987; 70: 1490-4.
4. 4. Fallon P., Gentry T., Balber A.E., Boulware D., Janssen W.E., Smilee R., Storms R.W., Smith C. Mobilized peripheral blood SSClo ALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation. Br J Haematol 2003; 122: 99- 108.
5. 5. Lacombe C., Mayeux P. Biology of erythropoietin Haematologica 1998; 83:724-32.
6. 6. Yalcintepe L., Albeniz I., Cinar S., Tiryaki D., Bermek E., Lee H.C. Nuclear CD38 in retinoic acid-induced HL-60 cells. Exp Cell Res 2005;303(1):14-21.
7. 7. Russo J., Hilton J., Colvin O. The role of aldehyde dehydrogenase isozymes in cellular resistance to the alkylating agent cyclophosphamide. In: Enyzymology and Molecular Biology of Carbonyl Metabolism. H.Weiner & T. Flynn, editors. p.65. New York.
8. 8. Hess D.A., Meyerrose T.E., Wirthin L., Craft T.P., Herrbrich P.E., Creer M.H., Nolta J.A. Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity. Blood. 2004; 104: 1648-55.

9. 9. Williams O., Demirer T., Lilleby K., Buckner C.D. Bensinger W.I. Tempo of hematologic recovery correlates with peripheral blood CD34+ cell level in patients undergoing stem cell mobilization. J Clin Apher. 1998; 13: 1-6.
10. 10. Lagasse E., Connors H., Al-Dhalimy M., Reitsma M., Dohse M., Osborne L., Wang X., Finegold M., Weissman I.L., Grompe M. Purified hematopoietic stem cells can differantiate into hepatocytes in vivo. Nat Med. 2000; 6: 1229-34.
11. 11. Armstrong L., Stojkovic M., Dimmick I., Ahmad S., Stojkovic P., Hole N., Lako M. Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells. 2004; 22: 1142-51.
12. 12. Gacar G. Mezenkimal kök hücrelerin karakterizas-yonunda flow sitometri. Temel kök hücre teknikleri ve moleküler biyolojide uygulamaları kursu. 2010.
13. 13. Dunphy C.H. Applications of Flow Cytometry and immunohistochemistry to Diagnostic Hematopathology. Arch. Pathol. Lab. Med 2004;128:9,1004-22.
14. 14. Laane E., Tani E., Bjorklund E., Elmberger G., Everaus H., Skoog L., Porwit-MacDonald A. Flow cytometric immunophenotyping including Bcl-2 detection on fine needle aspirates in the diagnosis of reactive lymphadenopathy and non-Hodgkin's lymphoma. Cytometry Part B Clinical Cytometry 2005;64:34-42.
15. 15. Luider J., Cyfra M., Johnson P., Auer, I. Impact of the New Beckman Coulter Cytomics FC 500 5-Color Flow Cytometer on a Regional Flow Cytometry Clinical Laboratory Service. Laboratory Hematology 2004;10:102-8.
16. 16. Stewart C.C., Woodrıng M.L., Podniesinski E., Gray E. Flow Cytometer in the Infrared: Inexpensive Modifications to a Commercial Instrument. International Society for Analytical Cytology 2005; 67:104-11.
17. 17. Albenız I, Türker-Şener L, Baş A, Kalelıoğlu I, Nurten R. Isolation of hematopoietic stem cells and the effect of CD38 expression during the early erythroid progenitor cell development process. Oncol Lett 2012 ;3(1):55-60.
18. 18. Storms R.W., Green P.D., Saford K.M., Niedzwiecki D., Cogle C.R., Colvin O.M., Chao N.J., Rice H.E., Smith C.A. Distinct hematopoietic progenitor compartments are delinated by the expression of aldehyde dehydrogenase and CD34. Blood 2005; 106: 95-102.
19. 19. Armstrong L., Stojkovic M., Dimmick I., Ahmad S., Stojkovic P., Hole N., Lako M. Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells 2004; 22: 1142-51. 20. Mitchell S., Cairo and John E. Wagner Placental and/or Umbilical Cord Blood: An Alternative Source of Hematopoietic Stem Cells for Transplantation. Blood The American Society of Hematology 1997; 90:12.
20. 21. Moreb J.S. Aldehyde dehydrogenase as a marker for stem cells. Curr Stem Cell Res Ther. 2008; 3(4):237-46.
21. 22. David M. Putman, Tyler T. Cooper, Stephen E. Sherman, Ayesh K. Seneviratne, Mark Hewitt, Gillian I. Bell, David A. Hess, Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization, Stem cells translational medicine, 2017; DOI: 10.1002/sctm.16-0472.