Journal of Cancer Stem Cell Research (2017), 5:e1002
© 2017 Creative Commons. All rights reserved ISSN 2329-5872
DOI: 10.14343/JCSCR.2017.5e1002
http://cancerstemcellsresearch.com
Research Article Open Access
Chronic Myeloid Leukemia Stem Cell Line Production
Sibel Azizenur Ozturk1, Pinar Ayse Ercetin Ozdemir*, 1, Safiye Aktas1, Duygu Dursun1, and Mehmet Ali Ozcan1
1Dokuz Eylul University, Institute of Oncology, Izmir, TURKEY
*Corresponding Author: Pinar Ayse Ercetin Ozdemir, PhD, Dokuz Eylul University, Institute of Oncology, Izmir, TURKEY. E-mail: pinarercetin@gmail.com
Received: March 8, 2017; Revised: March 29, 2017;
Accepted: March 29, 2017
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 CD34+/CD38-/CD123+ and CD34+/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 CD34+, CD38+, CD34+/CD38- and CD34+/CD38-/CD123+) 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.
Keywords: Chronic myeloid leukemia, Cancer stem cells, K562 cell line, Leukemic stem cells, Imatinib resistant K562/IMA3 cell line.
INTRODUCTION

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell (HSC) malignancy which is characterized with proliferation of myeloid elements in every stage of differantiation, loss of adhesive characteristics, decrease of apoptosis and presence of BCR oncoprotein owing to specific reciprocal translocation, t(9;22) [1, 2]. CML is the first of cancer type defined with chromosomal abnormality, called as Philadelphia (Ph) chromosome at oncology [3].

Allogeneic HSC transplantation in the treatment of CML are considered as the only curative approach in the treatment of patients with CML, but are found difficultly suitable donor for treatment [4, 5]. In recent years, discovery of imatinib mesylate (Glivec, Gleevec, STI571) which is a tyrosine kinase inhibitor targeted at the management of CML is a revolution characteristic due to the significantly improved survival. However, the most important barrier against this and similar drugs is primary or secondary resistance developed adverse to treatment [6, 7].

Researches in recent years, it suggests that leukemic stem cell (LSC) is to be responsible for the initiation and progression of leukemia, for forming of a large number of differentiated cells community in CML and for resistance and relapse developing against to treatments [8, 9]. Therefore, many studies are still conducted for overcome the resistance problem, resulting in accumulation of primitive cells in blood and bone marrow. However, researches are concentrated on cancer stem cells (CSCs) for developing of treatments, prevent effectively cancer relapse [10, 11].

It is one of the current scientific topics that CSCs are understood of mechanisms of development and creating cancer and are targeted by multimodal treatment strategy. Also, current therapeutic strategies against cancer often has serious limitations to lead to treatment failures. The common cause of treatment failures in many malignancies, is the resistance to chemotherapy and radiotherapy. In addition, patients come face to face with the risk of recurrence and metastasis because of to be toxic to healthy tissues of non-selective many strategies against CSCs and not to eliminate CSCs of many treatments [12, 13, 14]. Therefore, elimination of CSCs are very important in the treatment of malignant diseases.

In CML, LSCs can be self-renew and can differentiate into relevant progenitor cells which can create mature hematopoietic cells as normal hematopoiesis. Due to be also their surface markers of this near similarity, to examine in vitro CML stem cells is difficult because of the challenges in separating with the HSCs [15, 16, 17]. Also there isn't production and marketing of cancer cell and CSC line in our country. There are foreign dependency on this issue and there aren't CML stem cell line in accessible source. Therefore in this project, we aimed that in vitro CML stem cell line produces in suitable quality and standard from both imatinib-sensitive and -resistant CML cell lines for future work. Thus, it can be started animal experiments and phase trials as a continuation of researches to be done with in vitro CSC line as a result of obtaining an significant biological material for development of new drugs and treatment options in a very important disease group, is the treatment problems in hematologic oncology.

MATERIALS AND METHODS
Cell Culture

The Ph chromosome-positive K562 human CML cell and K562/IMA-3 (Imatinib-resistant version of K562) cell lines were provided by Professor Yusuf Baran. The cells were cultured in RPMI-1640 growth medium (Biochrom F1215) containing 20% fetal bovine serum (Biochrom S0113) and 1% penicillin-streptomycin (Biochrom A213) at 37°C in 5% CO2. Medium was refreshed every 3 days. The cell suspension was taken from tissue culture flasks into a sterile falcon tube and was centrifuged for 10 min at 1000 rpm. The supernatant was removed and the pellet was washed with 2 ml of phosphate buffered saline (PBS). The cells were recentrifuged for 10 min at 1000 rpm. The cells were resuspended in 15 ml of RPMI-1640 medium and transferred into sterile culture flasks. Additionally, the leukemic stem cells were cultured with the same conditions mentioned above in four different media: RPMI-1640 medium, differentiated medium (Celprogen), undifferentiated medium (Celprogen) and 10% CD34+ expansion supplement (StemSpam, StemCell) + leukemic stem cell medium (Celprogen). For trypan blue exclusion analysis, cells were counted using a hemocytometer in the presence of trypan blue solution at a 1:1 ratio (v/v) (Sigma). For alkaline phosphatase analysis, LSCs were fixed in 4% paraformaldehit (with PBS) for 1 minute, washed with rinse buffer, added methanol, quickly dried out, stained using the Alkaline Phosphatase Detection Kit (Millipore, Miltenyi Biotec) and incubated in the dark for 15 minutes at room temperature. It was applied mycoplasma detection elisa, pcr kit (Roche) procedure for detection of LSCs contaminated with mycoplasma.

CD34+/CD38-/CD123+ LSC isolation by FACS

For isolation of the CD34+/CD38-/CD123+ LSC subpopulations by FACS (Fluorescence activated cell sorting) analysis, cultured K562 and K562/IMA-3 CML cells were resuspended in PBS containing 0.1% sodium azide and were stained with FITC-conjugated CD34, PE- conjugated CD38 and APC- conjugated CD123 on ice for 30 min. Cells were then washed with PBS and analysis was performed on a FACS Calibur flow cytometer using the Cell Quest software (BD Biosciences). Gates were drawn to exclude nonviable cells and debris. Thus, collected CD34+/CD38-/CD123+ LSCs were cultured in the mentioned above in four different media.

CD34+/CD38-/CD123+ LSC isolation by MACS (mmagnetics cell sorting)

Cultured K562 and K562/IMA-3 CML cells were subjected to CD34+/CD38- cell selection by magnetic-activated cell sorting (MACS) technology, according to manufacturer's instruction (CD34 DynaBead; Invitrogen) (CD38 MicroBeads; Miltenyi Biotec). Both CD34+ and CD38- cell fractions were analyzed with positive and negative selection methods, respectively. Firstly, cells were subjected the isolation of CD38-negative cell populations by negative selection. Firstly, negative selection were subjected to sorting CD38- cell subpopulations by negative selection Briefly, the cells were washed with PBS, re-suspended in magnetic separation buffer, incubated with CD38-biotin antibody for 10 min at 4°C. Then the cells were re-suspended in magnetic separation buffer and incubated with anti-biotin MicroBead for 15 min at 4°C. The tubes with the cell suspension were placed in a magnet. Magnetically tagged CD38+ cells migrat-ed toward the tube wall on the magnet side, leaving the untagged CD38- cells in suspension. Then, the un-tagged CD38- cells were subsequently labeled with CD34-biotin antibody and incubated with to sort the CD34+ cellsfor 30 min at 4 °C. The tubes with the cell suspension were then placed in a magnet. While the untagged CD34- cells left in suspension, magnetically tagged CD34+ cells migrated toward the tube wall on the magnet side and added DetachaBead on tagged cells to separate from microbeads. Thus, CD34+/CD38- LSCs were collected and cultured the mentioned above in four different media.

Tumorigenicity (Xenotransplant) experiments

This study was approved by the animal experiments local ethics committee of Dokuz Eylul University Medical School. Xenotransplant experiments of K562 and K562/IMA-3 LSCs were studied in the laboratory of Department of Laboratory Animal Science, Dokuz Eylul University, İzmir, TURKEY. After cells were resuspended in PBS at a concentration of 1 × 106 cells in 0.2 ml, female 21-week-old athymic nude mice (Kobay Experimental Animals Laboratory A.Ş., Ankara, Turkey) were injected subcutaneously at a single site. The animals were examined for tumor formation for up to 2 week. When solid tumors developed in animals, they were killed.

Flow Cytometry Analysis

For DNA ploidy analysis by flow cytometry, cells were then suspended in PBS containing propidium iodide and RNase to stain nuclear DNA for 30 min at room temperature. The distribution of cells in the different phases of the cell cycle was analyzed from DNA histograms. Cell cycle analysis was done on CD34+ and CD34- cells from K562 and K562/IMA-3. For CD analysis by flow cytometry, staining protocols were performed according to the manufacturers' instructions. Briefly, directly conjugated monoclonal antibodies (mAbs) against CD34-fluorescein isothiocyanate (FITC) (eBioscience), CD38-pyhcoerythryrin (PE) (eBioscience), CD123-allophycocyanin (APC) (eBioscience) and appropriate IgG isotype controls were used to stain CML cells and CML LSCs, and then incubated for 15 min at room temperature in the dark. Both cell cycle analysis and CD panel analyzes was performed on an Epics XL (Beckman Coulter) and analyzed with Expo 32 ADIC XL 4 Color software.

Quantitative Real-Time PCR Analysis

Total RNA was isolated from LSCs using the High Pure RNA isolation kit (Roche Diagnostics GmbH, Germany) according to the manufacturer's instructions. BCR/ABL translocation and Telomerase mRNA levels in LSCs were measured by using a qRT-PCR method. Primers and probes were designed with the Universal Probe Library Assay Design Center (https://www.universalprobelibrary.com). For each reaction the complementary DNA (cDNA) equivalent was amplified for 35 cycles using cycle parameters of 94°C for 30 seconds, 60°C for 60 seconds, and 72°C for 60 seconds. All PCR reactions were performed in the LightCycler® 480 instrument (Roche Diagnostics, Germany). Glyceraldehyde 3-phosphate dehydrogenase was used as the housekeeping gene in order to normalize expression levels. Each sample was tested in triplicate.

RESULTS
Comparing of the Efficiency of the FACS and MACS Cell Isolation Method

K562 and K562/IMA-3 cells with cultured were reached a certain number. Before at Research and Training Laboratory (AREL) of Medicine Faculty, Ege University, CD34+/CD38-/CD123+ LSCs were isolated by FACS (Fluorescence-activated cell sorting) method. Then at Basic Oncology Laboratory of Dokuz Eylül University, CD34+/CD38- LSCs were isolated with the MACS (Magnetic-activated cell sorting) method.

In the chart below, it is presented comparatively the percentage of CD34+ population of K562 and K562/IMA-3 LSCs with FACS and MACS methods. For K562 cell line, isolation of LSCs with MACS method was 4.7 times more efficient than isolation of LSCs with FACS method. For K562/IMA-3 cell line, it seen that isolation of LSCs with MACS procedure was 1,3 times more efficient than FACS method (Figure 1).



Figure 1. Percentage rates according to the FACS and MACS methods of CD34+ leukemic stem cells, be isolated from K562 and K562/IMA-3 cell lines. This experiment could only be repeated twice because of experimental limitations (error bars could not be made because obtaining and production of cancer stem cells were difficult due to time, cost and applicability of the experiment).



As shown in Figure 2, rate of CD34+/CD38-/CD123+ LSCs, isolated from K562 cells with FACS and MACS method was obtained almost equal. In K562/IMA-3 LSCs, the efficiency of FACS method according to the MACS method was found 3,1-fold higher.



Figure 2. Percentage rates according to the FACS and MACS methods of CD34+/CD38-/CD123+ leukemic stem cells, be isolated from K562 and K562/IMA-3 cell lines. This experiment could only be repeated twice because of experimental limitations (error bars could not be made because obtaining and production of cancer stem cells were difficult due to time, cost and applicability of the experiment).



Cell cycle analysis

Cell cycle analysis was made by flow cytometry in both K562 and K562/IMA-3 cell lines. DI was found 2.03 (4N) in K562 CD34- cells, while DI was found ≤1 (2N) in the K562 CD34+ cells (Figure 3). DI was found 1(2N) in K562/IMA-3 CD34- and CD34+ cells (Figure 4).



Figure 3. Respectively, histograms showing cycle phase of K562 CD34- and CD34+ cells histograms.





Figure 4. Respectively, histograms showing cycle phase of K562/IMA-3 CD34- and CD34+ cells histograms.



K562 and K562/IMA-3 leukemic stem cell cultures

Isolated LSCs from K562 and K562/IMA-3 lines (RPMI-1640 medium, Differentiated medium, Undifferentiated medium and Leukemic stem cell-expanded medium) were cultured in four different media and were observed their behavior. In RPMI-1640 medium, it was seem that majority of K562 and K562/IMA-3 LSCs proliferated after differentiated to leukemia cells (Figure 5).



Figure 5. Respectively, morphological image of K562 and K562/IMA-3 leukemic stem cells in RPMI-1640 culture medium (10X).



In differentiated medium, it was seem that majority of K562 and K562/IMA-3 LSCs proliferated after differentiated to leukemia cells (Figure 6).



Figure 6. Respectively, morphological image of K562 and K562/IMA-3 leukemic stem cells in differentiated culture medium (10X).



In undifferentiated medium, it was seem that majority of K562 and K562/IMA-3 LSCs maintained their phenotypes as undifferentiated leukemia cells, but unreplication and could be cultured about 2 wk (Figure 7).



Figure 7. Respectively, morphological image of (10X) K562 and (20X) K562/IMA-3 leukemic stem cells in undifferentiated culture medium.



In leukemic stem cell-expanded medium, it was seem that majority of K562 and K562/IMA-3 LSCs proliferated without undifferentiated leukemia cells (Figure 8).



Figure 8.

Respectively, morphological image of K562 and K562/IMA-3 leukemic stem cells in leukemic stem cell-expanded culture medium (10X).



CD Panel analysis

It was investigated with CD panels' analysis in Flow cytometry whether proliferated maintaining the stem cell characteristics of the majority of LSCs. For proliferated maintaining the stem cell characteristics, the K562 and the K562/IMA-3 LSCs was cultured in only leukemic stem cell-expanded medium.

CD panel of the produced cells was collectively shown with graphic in Figure 9: Rate of CD34+; CD38+; CD34+/CD38- and CD34+/CD38-/CD123+ cell population was separately analyzed in K562 and K562/IMA-3 cells; isolated K562 and K562/IMA-3 LSCs; and K562 and K562/IMA-3 LSCs that were exposed in differentiated medium. In K562 cell line, CD34+/CD38-, CD34+/CD38-/CD123+ and all cell populations was below 1%. CD34+/CD38- cell population was seen very high levels in also both the LSCs besides being the highest level in K562/IMA-3 LSCs. Moreover, the highest level of CD34+, CD34+/CD38- and CD34+/CD38-/CD123+ cell populations is observed in K562/IMA-3 LSCs. CD34+, CD34+/CD38- and CD34+/CD38-/CD123+ population rate of K562/IMA-3 LSCs was seen to be respectively, 1.3-fold, 1.7-fold and 4.5-fold more according to K562 LSCs. CD34+, CD34+/CD38- and CD34+/CD38-/CD123+ population rate of K562/IMA-3 LSCs was seen to be respectively, 4.9-fold, 5.2-fold and 12.7-fold more according to differentiated K562/IMA-3 LSCs. CD34+, CD34+/CD38- and CD34+/CD38-/CD123+ population rate of K562/IMA-3 LSCs was seen to be respectively, 5.2-fold, 4.5-fold and 3.1-fold more according to K562/IMA-3 cells. CD38+ cell population, which is known as the normal hematopoietic progenitor cell (HPC) marker was seen high levels in also K562/IMA-3 LSCs besides being the highest level in K562 LSCs. CD38 + cell population ratio of K562 LSCs was seen to be 1.6-fold more than K562/IMA-3 LSCs, 7.2-fold more than differentiated K562/IMA-3 LSCs, 10.7-fold more than differentiated K562 LSCs, 86-fold more than K562/IMA-3 cells, and 86-fold more than K562 cells (Figure 9).



Figure 9. Collective CD panel of K562 and K562/IMA-3 cells; K562 and K562/IMA-3 leukemic stem cells; differentiated K562 and K562/IMA-3 leukemia stem cells.



Xenotransplantation experiments

Produced LSCs was confirmed by xenotransplantation experiments whether maintaining the stem cell characteristics. For this purpose, K562 and K562/IMA-3 CD34+/CD38- LSCs were transplanted into immuno-deficient NUDE mice. Also, K562 and K562/IMA-3 CD34+/CD38+ non-stem cells were transplanted into immuno-deficient NUDE mice. It was seen that the K562 and K562/IMA-3 CD34+/CD38- LSCs created tumor after about 2 hf. K562/IMA-3 LSCs to had the ability to create a faster and larger tumor according to K562 LSCs. However it was observed that K562 CD34+/CD38+ cells couldn't create tumor while the K562/IMA-3 CD34+/CD38+ cells caused a small tumor formation (Figure 10).



Figure 10. Xenotransplantation into immunedeficit-NUDE mice of the K562 and K562/IMA-3 leukemic stem cells, respectively. Tumor seen in the neck region of mice was developed in about 2–3 weeks.



Quantitative Real Time PCR Assay
Determination of the translocation presence with measurement of mRNA level of BCR-ABL

Quantity of BCR-ABL mRNA of K562 and K562/IMA-3 LSCs were observed respectively, 1.8 and 1.6 times lower than K562 and K562/IMA-3 cells besides protected of levels of BCR/ABL expression (Figure 11).



Figure 11. BCR-ABL mRNA levels of K562 and K562/IMA-3 cells, and K562 and K562/IMA-3 stem cells.



Measurement of telomerase RNA levels

The mRNA levels of telomerase activity in order to determine the proliferation ability of leukemic stem cells were measured with Quantitative Real Time PCR experiments. Telomerase activity of K562/IMA-3 leukemic stem cells were determined 2.8 times more active compared to K563/IMA-3 cells, while telomerase activity of K562 leukemic stem cells was observed 1.8-fold more reduction compared to K562 cells (Figure 12).



Figure 12. Telomerase mRNA levels of K562 and K562/IMA-3; K562 and K562/IMA-3 leukemic stem cells.



Alkaline Phosphatase Assay

Red colonies in undifferentiated stem cells is seen with alkaline phosphatase assay. Thus, undifferentiated stem cells is confirmed. Colonies painted with red were seen after experiment in K562 and K562/IMA-3 leukemic stem cells (Figure 13 and 14).



Figure 13. Appearance of K562 leukemic stem cell with alkaline phosphatase staining.





Figure 14. Appearance of K562/IMA-3 leukemic stem cell with alkaline phosphatase staining.



Mycoplasma Contamination Demonstration

Produced LSCs were shown that there is no contamination with mycoplasma testing.

DISCUSSION

The number of laboratories using as a research tool to CSCs is increasing rapidly in worldwide. However, there are still many challenges that must be solved. It is being experienced also the problems in their recognition due to similarities with normal stem cells of CSCs. Therefore, it can be targeted directly the destruction of CSCs with the identification of differences between CSCs and normal stem cells. First of all, there is a need to stable CSC line for resolving the surprising potential of CSCs. Recognition of problems that may be occurring for be obtaining of this stability will provide significant developments of us in terms of understanding of the differentiation mechanism. Existing protocols used for CSCs are absent or are under of optimal despite major efforts due to be of spontaneous differentiations. For elimination of CSCs and the development of different therapeutic strategies, the first thing to be done is determination of the specific culture conditions, proliferating unchanged of CSC features and preventing of differentiation potentials of CSCs. Therefore, the overall aim of our study was practical to develop a powerful protocol against differentiation mechanisms to many tumor serials of CSCs in the culture medium and to create in vitro CSC line. Thus, difficulties that may occur in forming of CSC line were that can be brought into the agenda and controversial points were that can be unfold. Also, we believe that CML cell lines provided an additional advantage for our study due to being a malignant disorder of pluripotent HSCs.

The first step of forming of CSC line is to obtain the required number of CSCs by separation system which works high efficiency. In recent years, the use of FACS and MACS systems for cell sort has increased intensively. The most important reason is easy and fast application. Therefore, in our study, it was isolated by FACS and MACS methods the stem cells of K562 and K562/IMA-3 lines. Also, these two methods were confirmed to each other and compared results.

CD34 marker indicates that the cells have both hematopoietic and stemness features. Therefore, in terms of efficiency of FACS and MACS methods, it was compared rates of CD34+ population in both cell lines. CD34+ population isolated by MACS method was found higher rates than isolated by FACS method in both of K562 and K562/IMA-3 leukemic cells (respectively 4.7 and 1.3 times higher). In addition, it was examined also the rates of CD34+/CD38-/CD123+ LSCs, purified from both cell lines. CD34+/CD38-/CD123+ LSC population with FACS method was isolated directly from the K562 and K562/IMA-3 cell lines. In the MACS method, CD34+/CD38-/CD123+ subpopulation rates were determined by flow cytometry after CD34+/CD38- LSCs isolated. Thus, by the flow cytometric method, it was provided to confirmation of the MACS for CD34+/CD38- subpopulation and of the FACS for CD34+/CD38-/CD123+ subpopulation. In our study, CD34+/CD38-/CD123+ LSCs isolated from K562 cells were seen in almost equal proportions by FACS and MACS method. However, we observed that the rate of CD34+/CD38-/CD123+ LSCs isolated from K562/IMA-3 cells was 3.1 times more efficient of FACS method according to MACS method.

It brings to mind that above situations may have resulted from some disruptions while sort by MACS and FACS. Namely, the results were direct for the FACS method and indirect for the MACS method. CD34+/CD38-/CD123+ cells were isolated by FACS system and then the information about of CD34+ cell ratios were taken from the device data at the same time. Whereas, CD34+/CD38- cells were isolated firstly by MACS method, then rates of CD34+/CD38-/CD123+ cells in these cells were checked by flow cytometric method. For the MACS system, it was performed purification of first the CD38- cells with negative selection and then the CD34+ cells with positive selection, or solely CD34+ cells were isolated with positive selection. Therefore, HPCs can be isolated also with a cause like affinity problems to CD38+ cells of the magnetic beads. Also, plucked from cells of magnetic beads after positive selection can cause additional stress and damage of cells. Such problems in the MACS system may be the cause of yield loss in results of cell isolation. On the other hand, for the FACS method, it should not be ignored that above results could arise from errors gating or from problems binding to the cell of fluorochrome-conjugated antibodies. In addition, it must be at the appropriate concentration for isolation by FACS system of the cells in the best way. Too concentrated of or too diluted of the cells to be studied can cause problems. However, it is not an ideal concentration for the sort. Simply put, due to accidental falls of the cells, concentrated much will cause lower collection. For example; when two cells are very close together will be rejected this cells by the device to ensure purity. Such problems, may come to mind may be the cause of yield loss in results of cell isolation, made with the FACS system. However, both methods in CD34+/CD38-/CD123+ cells were confirmed due to come out almost the same results in K562 LSCs. Considering above-discussed disadvantages we observed yield reduction for unknown reasons in both methods.

In our study, cell cycle analysis was performed with flow cytometry in both cell lines. For division phase and amounts of cells can be determined with flow cytometry, DNA Index (DI) reflects the presence of major DNA content changes during cell culture. In this way, information about the growth rate of the cells is collected. While diploid cells are located in the G0/G1 phase, DNA content of cells in the S (synthesis) phase are between tetraploid and diploid cells. Cells in the G2 and mitosis are seen as tetraploid because of they carry the DNA amount of 4N. In our study, while it was observed diploidy in K562/IMA-3 CD34+ and CD34- cells, it was observed DI 2.03 (tetraploidy, 4N) in the K562 CD34- cells and DI ≤1 (2N) in a part of the K562 CD34 + cells. Tetraploidy indicated that K562 CD34- cells were in mitosis phase. As expected, this are a sign of that they are not stem cells. K562 CD34+ and K562/IMA-3 CD34+ cells was observed in diploidy. As expected in stemness feature, this diploidy in CD34+ cells can be considered as a symptom of G0 (silent) phase. However for the characterization of K562/IMA-3 CD34- cells, it suggest that further testing needs to be done.

LSCs isolated from K562 and K562/IMA-3 lines are cultured by four different media (RPMI-1640 medium, differentiated medium, non-differentiated medium and leukemic stem cells expanded medium) and behaviors of LSCs were observed as result of exposure to these four media. Thus the most suitable medium for formation of CML stem cell line were selected. For we thought to be an additional contribution to LSCs, these media were cultivated on 25 cm2 culture flasks, coated with extracellular matrix. It were found that proliferated after differentiated to leukemic cells of K562 and K562/IMA-3 LSCs in RPMI-1640 and differentiated medium. The feasibility of passages of LSCs in these mediums was bad. In the undifferentiated medium, it was observed that both the LSCs retained their phenotype from non-proliferation. However, the viability of LSCs could be continued for about two weeks. In our study, we were found that LSCs were very sensitive to type and quality of used the serum and were great importance of the properties of medium for their maintenance. The medium used for the culturation of LSCs needed to be different from the medium used in the culture of any cells. In our study, we added “CD34+ expansion supplements” into “leukemic stem cell medium”, developed for LSCs. Our aim was to ensure the undifferentiation after many passaging. As a result of cultivation, we observed that the majority of LSCs proliferated without differentiation.

In our study, it was cultured that K562 and K562/IMA-3 LSCs, produced on expanded medium for the LSCs; differentiated LSCs of K562 and K562/IMA-3 because of exposure to differentiated medium; K562 and K562/IMA-3 cells. Then these cells were investigated the CD panel by flow cytometry to compare the amount of CD34+; CD38+; CD34+/CD38- and CD34+/CD38/CD123+ populations. Thus it was performed by the CD panel a confirmation of MACS analysis with the determination of CD34+/CD38- cell populations; a confirmation of FACS analysis with the determination of CD34+/CD38-/CD123+ cell populations. Ratio of CD34+/CD38- population, known as LSC marker were seen to be higher levels in both of LSCs, as well as particularly to be the most in K562/IMA-3 LSCs. Thus it was understood that the CD34+/CD38- protects marker characterization of LSC as indicated in many literature. The CD34+/CD38-/CD123+ population, known as LSC marker were observed the highest rate in K562/IMA-3 LSCs. This population was seen 4.5-fold higher rate in K562/IMA-3 LSCs according to K562 LSCs. Also, it is understood that this difference is based on the CD123+ population owing to be 2.4 times more of CD34+/CD38- population compared to the CD34+/CD38-/CD123+ population in the K562 LSCs. Therefore, it was suggested that CD123 showed a strong marker characteristic for the K562/IMA-3 LSCs and but was insufficient of marker feature for the K562 LSCs. In addition, such as to be seen in previous CD34+/CD38- and CD34+/CD38-/CD123+ populations, CD34+ cells, are the other cell population in the CD panel, was observed the highest rates in K562/IMA-3 LSCs.

At the end of our study, it were shown that K562 cells contained 0.4% of LSCs and K56/IMA-3 cells contained 5.3% of LSCs. All populations in K562 cell line were less than 1%. In our study, CD34+/CD38- and CD34+/CD38-/CD123+ cells populations, are LSC markers were seen in very less rates than 1% in the K562 cell line. This rate may be an indicator of rate of CSC population as reported in many studies. Again, the presence of more stem cell in the K562/IMA-3 cells are also compatible with the resistance of these cells. In differentiated LSC, rate of CD34+/CD38- and CD34+/CD38/CD123+ cell population were below of 5%. This shows that LSCs disappeared stemness characters due to be differentiated.

CD38+ cells are positive in normal HPC and was seen in the highest percentage in LSCs, in particularly K562 LSCs. It was found that the CD38+ cell population in K562 LSCs is 1.6 times more than K562/IMA-3 LSCs; 7.2 times more than differentiated K562/IMA-3 LSCs; 10.7 times more than differentiated K562 LSCs; 86 times more than K562/IMA-3 cells; and 86 times more than K562 cells. In our study, the CD38+ population were seen in very small rates in differentiated LSCs, and almost never seen in leukemic cells. Contrary to expectations in our study, that CD38+ population is seen in quite high rates in LSCs contradicts with knowledge that CD38 isn't expressed in LSCs as indicated in many of previous studies. This may be an indication of not pure of isolated LSCs. At the same time, even if is cancerous, it can also mean that CSC is similar with the biology of normal HPC in terms of stemness features. In addition, it could also be a high indication of the possibility of development from normal stem cells of tumor-initiating cells. However, it might be considered that CSCs unroll also difficulties in the separation from normal stem cells. When considering these results, it is clear that require to be made of further research about the proliferation in LSC population of the CD38, is HPC marker, and that need to be found of more effective markers for isolation of CSC.

In our study, the CD34+/CD38- LSCs, are proliferated in LSCs expanding medium was subcutaneously xenotransplanted into NUDE mouse. It was shown to have the ability to form tumors in mice of both LSCs, including faster and larger the K562/IMA-3 LSCs. Thus, it was provided the confirmation in terms of features of CSC of the LSCs isolated from two cell line. In addition, it was understood that the CD34+/CD38+ K562 cells haven't the ability of tumor initiation due to not form tumor in NUDE mouse and it was seen that CD34+/CD38+ K562/IMA-3 cells have the ability of tumor initiation due to create a small tumor in NUDE mouse. It is clear that this situation need to enlighten with further researches.

In this study, to determined that is specific to CML cells of isolated LSCs, BCR/ABL expression levels were measured and compared. It was found that BCR-ABL expression amounts were preserved although were reduced in both the K562 and K562/IMA-3 LSCs (respectively, 1.8 and 1.6-fold lower) according to K562 and K562/IMA-3 cells. It may be thought that this reduction in BCR/ABL expression levels occurred result to be located in G0 phase of LSCs. Even so, this situation should be clarified with more detailed researches in CML stem cells.

Telomeric structures which locate at the ends of the chromosomes and protect the chromosome integrity are slightly reduced after each cell cycle in eukaryotic cells. Cells stop proliferation when this shortening comes to a critical length. Telomerase is active in germ cells, stem cells and cancer cells and increases the capacity to divide of the cells via elongating the telomeric structure. In addition, determination of telomerase activity is important for the evaluation of the capacity of cancer cell division [18]. In the present study, we evaluated the telomerase enzyme activity by measuring the amount of mRNA. While telomerase activity in the K562/IMA-3 LSC was seen as increased compared to K562/IMA-3 cells (2.8 fold higher), telomerase activity in K562 LSCs was seen as decreased compared to K562 cells (1.8 fold lower). These results is important to understand the division and the mortality capacity of both LSCs. As expected, it was found that the K562/IMA-3 LSCs have higher capacity of division and immortality compared to the K562/IMA-3 cells, was isolated them. However, it were found that the K562 LSCs have less capacity of division and immortality compared to the K562 cells, were isolated them. Although they have the stemness characterized in both LSCs, the K562/IMA-3 LSCs were found more advantageous for production of CML stem cell line. For reduced telomerase activity in K562 LSCs, it may require to do further investigations in terms of determining whether relationship with the G0 phase, is quiescent of the cell.

Positive staining with alkaline phosphatase indicates the presence of undifferentiated cells. Therefore, in our study, it was confirmed proliferation without differentiation of LSCs due to the positive staining with alkaline phosphatase of isolated LSCs.

It must be evaluated in terms of contamination for continue of cell cultures. In the present study, we applied mycoplasma test to produced LSCs and showed to not be contamination.

CONCLUSIONS

Our research is a preliminary study for further researches. In this respect, for production of CML stem cell line, that challenges may arise were brought on the agenda and controversial points were revealed for production of CML stem cell line. As a result, it was determined to be possible, but to be difficult of in vitro production of CML LSCs. Result of this study, we believe that can be passed to primary culture and further studies. However, it is obvious that the topics listed above needs to be illuminated with further researches.

ACKNOWLEDGMENTS

This study was supported by the Dokuz Eylul University Scientific Research Projects Coordination Unit (Project No: 2013.KB.SAG.030).

CONFLICT OF INTEREST

Authors declare no conflict of interest.

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