Turkiye Klinikleri Journal of Medical Sciences

.: ORIGINAL RESEARCH
Combination of Early and Late Growth Factors to Enrich Transplantable Cord Blood CD34+ Cells in Short Term Cultures
Kordon Kanı CD34+ Hücrelerinin Zenginleştirilmesi İçin Kısa Süreli Kültürlerde Erken ve Geç Etkili Büyüme Faktörlerinin Kombinasyonu
Faruk SÜZERGÖZa,b, Serap ERDEM KURUCAc
aDepartment of Immunology, İstanbul University, Institute for Experimental Medicine, cDepartment of Physiology, İstanbul University, Faculty of Medicine, İstanbul bDepartment of Biology, Harran University, Science&Art Faculty, Şanlıurfa
Turkiye Klinikleri J Med Sci. 2010;30(1):256-62
doi: 10.5336/medsci.2008-8861
Article Language: EN
Full Text
ABSTRACT
 Objective: Although umbilical cord blood (UCB) is a useful source of stem and progenitor cells for bone marrow transplantation, it is not possible to obtain sufficient number of transplantable cells from a single human umbilical cord for transplantation in adult patients. In this study, we combined early and late growth factors in short term cultures to enrich cord blood CD34+ hematopoietic progenitor cell count for adult patients. Material and Methods: Cord blood mononuclear cells (CBMC) harvested from 15 healthy volunteers, delivering in term, incubated in culture medium supplemented with interleukin-3 (IL-3), IL-6, IL-11, stem cell factor, flt3/flk2 ligand and thrombopoietin for 14 days. Starting cells and cultured cells (day 7 and 14 cells) were evaluated for; CD4, CD8, CD33, CD34, CD38 and HLA-DR by flow cytometry and seeded in semisolid methylcellulose culture for determining BFU-E, CFU-GM and CFU-GEMM colony forming abilities. Results: In the short-term cultures, total CBMC count significantly increased (p= 0.0001). Compared to starting cells, the expressions of CD34 (p= 0.002), CD33 (p= 0.0001), HLA-DR (p= 0.0001) and CD8 (p= 0.002) increased, but those of CD38 (p= 0.057) and CD4 (p= 0.0001) were suppressed on day 14 of cultures. There were an increase in generation of CFU-GM (p= 0.131) and CFU-GEMM (p= 0.134) colonies and a significant decrease in generation of BFU-E colonies (p= 0.0001). Conclusion: Ex vivo expansion of CBMC, under combination of the growth factors, contribute to CD34+ progenitor cell count and may help to reach sufficient amount of transplantable cells for adult patients. Suspension cultures may lead to elimination of graft-versus-host disease risk and protection of graft-versus-leukemia effect, through the decrease of CD4+ T cells and maintenance of CD8+ T lymphocytes, respectively.

Keywords: Cord blood stem cell transplantation; antigens, CD34; cell culture techniques
ÖZET
 Amaç: Göbek kordon kanı kök ve projenitör hücre açısından kemik iliği transplantasyonu için elverişli bir kaynak olmasına karşın tek bir insan göbek kordonundan erişkin hastalara nakil için yeterli sayıda hücre elde etme olanağı bulunmamaktadır. Çalışmamızda kordon kanı CD34+ hemopoetik projenitör hücreleri erişkin hastalara yetecek düzeyde çoğaltmak amacıyla erken ve geç etkili büyüme faktörlerini kısa süreli kültürlerde kombine ettik. Gereç ve Yöntemler: Miadında doğum yapan 15 sağlıklı gönüllüden kordon kanı mononükleer hücreleri alınarak interlökin-3 (IL-3), IL-6, IL-11, stem cell faktör, flt3/flk2 ligand and trombopoetin eklenen kültür medyumunda 14 gün süreyle inkübe edildi. Başlangıç hücreler ile kültürden alınan hücreler (7. ve 14. günler) flow sitometride CD4, CD8, CD33, CD34, CD38 ve HLA-DR yönünden analiz edildi ve aynı zamanda yarı katı metilselüloz kültürlere ekilerek BFU-E, CFU-GM ve CFU-GEMM kolonilerini meydana getirme kapasiteleri incelendi. Bulgular: Kısa süreli kültürlerde total kordon kanı mononükleer hücre sayısında anlamlı artış gözlendi (p= 0.0001). Başlangıç hücreleri ile karşılaştırıldığında, kültürlerin 14. gününde hücrelerde CD34 (p= 0.002), CD33 (p= 0.0001) HLA-DR (p= 0.0001) ve CD8 (p= 0.002) ekspresyonlarında artışla birlikte CD38 (p= 0.057) ve CD4 (p= 0.0001) ekspresyonunda baskılanma gözlendi. CFU-GM (p= 0.131) ve CFU-GEMM (p= 0.134) kolonilerini oluşturma kapasitelerinde artış ve BFU-E (p= 0.0001) koloni oluşturma kapasitelerinde anlamlı düşüş gözlendi. Sonuç: Büyüme faktörlerinin kombinasyonu altında kordon kanı mononükleer hücrelerinin vücut dışı ortamda çoğaltılması, erişkin hastalara yetecek miktarda nakledilebilir CD34+ projenitör hücre sayısına ulaşmaya yardımcı olabilecektir. Süspansiyon kültürü sonrası CD4+ T hücre sayısındaki azalma graft-versus-host hastalığı riskinin ortadan kaldırılmasına yardımcı olabilecek ve CD8+ T lenfositlerinin yaşamlarını sürdürmeleri ile de graftın anti-lösemik etkisi korunabilecektir.

Anahtar Kelimeler: Kordon kanı kök hücre nakli; antijenler, CD34; hücre kültürü teknikleri

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