Chronic Myeloid Leukemia Stem Cell Line Production

Abstract

Chronic myeloid leukemia (CML), characterized with BCR/ABL fusion protein is a clonal disorder developing results in malignant transformation of pluripotent hematopoietic stem cells. The aim of this project is to produce in vitro CML leukemic stem cell (LSC) line in appropriate quality and standard for future work by developing a strong protocol against the differentiation mechanism to many tumors series of its in culture medium of CML LSC. In this study, behaviors of LSCs were observed by exposure to different culture conditions after isolation of LSC subpopulations of CD34C/CD38-/CD123C and CD34C/CD38-in K562 (imatinib-sensitive) and K562/IMA-3 (imatinib-resistant) human CML cells by FACS and MACS methods. LSCs were analized cell cycle by flow cytometry; potential of formating tumor by tumorigenicity; expression levels of telomerase activity and BCR/ABL by quantitative real-time PCR; differentiation by alkaline phosphatase (AF) staining; and presence of infectious agents by mycoplasma detection. Also, CD panel (subpopulations of CD34C, CD38C, CD34C/CD38-and CD34C/CD38-/CD123C) were analyzed by flow cytometry in CML cells, LSCs and differentiated LSCs. We found that K562 and K562/IMA-3 cells contained LSCs in 0,4% and 5,3% ratios, respectively. It was observed that LSCs maintained the phenotype without proliferation in dedifferentiated medium, and that the majority of cells proliferated as LSCs in LSC expanded medium. However, it was observed that LSCs proliferated by transforming into leukemia cells in differentiated and serum mediums. LSCs of both cell lines generated tumors as well as causing to faster tumorigenicity of K562/IMA-3 LSCs in athymic nude mice. LSCs of both cell lines were found AP positive. Telomerase activity was found to increased in K562/IMA-3 LSCs, while to decreased in K562 LSCs. BCR/ABL expression was maintained although decreased in LSCs of both cell lines. Mycoplasma contamination was not seen in LSCs. The highest levels of all populations in CD panel were seen in LSCs. In result of this study, the production of CML LSC line was found to be possible, but to be difficult.