Purification of CD34+ Stem Cells from Myelodysplastic Syndrome with Ring Sideroblasts using Immunomagnetic Strategy Purification of CD34+ Stem Cells from Myelodysplastic Syndrome with Ring Sideroblasts using Immunomagnetic Strategy.

Background and Aim: While CD34+ stem cell purification can be easily achieved for most samples using magnetic activated cell sorting, it is a clue when working on diseases such as Myelodysplastic Syndromes (MDS) especially for the Ring Sideroblastic (RS) subcategories. High iron content of cells characterizing these patients induces a much lower purity due to contaminant cells. Methods: Herein, we developed an efficient and robust procedure to isolate highly purified CD34+ cells from MDS-RS using magnetic column. Flow cytometry analysis demonstrates the presence of erythroid and monocyte derived cells in samples purified from MDS-RS. Results: Precleaning column did magnetically retain these cells leading to a better purification percentage of the hematopoietic stem cells. Results have been confirmed by MGG and Perls staining. Conclusion: Malignant clonal cells purification is a clue when working on diseases such as Myelodysplastic Syndromes. We believe that these data may help for developing an alternative procedure for studying MDS-RS stem cells with a higher purity. Abbreviations:

is an accurate technic leading to well purified cells. Nevertheless, it requires to be equipped with a cell sorter and to apply some rules for a good achievement [1]. Moreover, this approach could be time consuming for a large sample. The second approach using magnetic bead separation technology has the advantages to be simple, robust and automatable. This immunomagnetic-beads (IMBs) cell sorting approach is usually giving a high percentage of purity, above 90-95% pure populations, but could be variable depending on the sample origin, the disease or the model used. Indeed, using IMBs separation remain challenging for the MDS-RS disease as multiple labs reported a lower purity level than those traditionally obtained with this technic [2,3].
We also observed this issue during our attempt to purified  [4] and human [5]. By this approach, the iron-loaded cells such as sideroblasts or siderocytes remained stick on the magnetic column due to their high level of magnetic metals. We hypothesized that it would be possible to purify a high percentage of HSC from MDS-RS using IMBs approach if the iron overloaded cells were first removed. Herein, we describe a simple and robust procedure for the purification of highly purified CD34+ HSC from human bone marrow samples with MDS-RS disease. By employing a magnetic column-based method followed by a classical magnetic bead-based selection, we were able to obtain a pure population of CD34+ HSC. Moreover, we provide evidence that this effective method for cell separation mainly retained erythroid cells which are highly positive for Perls staining.

Sample Collection and Patients Characteristics
Sternal bone marrow aspirations were obtained from 16 patients with MDS after informed consent during a diagnostic sampling and were preserved in a media containing heparin. This  [6]. In addition, bone marrow cells from 6 healthy controls (4 males, 2 females, median = 61 years, range = 31-74 years) were obtained during cardiac surgery after informed consent.

Morphological and Iron Assessment
Cells from different steps of purification were cytocentrifugated using Hettich Universal 16 (5 minutes, 45g) onto glass slides at

Statistical Analysis
After validation of the parametric distribution with Kolmogorov-Smirnoff test, paired or unpaired two-tailed Student t-tests were used to compare datasets. Comparison between MGG and Flow cytometry or clinical data and cells retained on precleaning column results were tested with Pearson correlation assay. P values < 0.05 were considered significant. After IMBs based purification, we determined the percentages of CD34+ cells using Flow Cytometry (FC) in the different samples.

CD34+ purity in MDS Patients
As summarized in (Figure 1), there was no difference (p=0.456) in percentage of CD34+ cells purity between control group reaching a median of 94.1% (range 85%-99%) and MDS-EB group with 95.5% (range 90%-97%). While CD34+ cells for these 2 last groups were purified with high percentages, the MDS-RS samples purity was lowering to a median value of 20.7% with percentages ranging from 5% to 59%. This significant differences between MDS-RS and

Clinical Comparison
As demonstrated in (Figures 3B & 3D), cells contaminating MDS-RS eluted fraction or retained on PcC were positive for Perls staining contrary to CD34+ from control samples ( Figure 3F).
We speculated that the percentage of cells retained on PcC might correlate with the quantity of RS on BM smears but no relationship between percentage of RS and percentage of cells retained on the columns was significantly observed in our small cohort (data not shown, n=5, p=0.056).

Discussion
Myelodysplastic syndromes are malignant diseases characterized by different hallmarks including ineffective erythropoiesis, severe anemia and iron accumulation for the MDS-RS subtype. Our experience on MDS-RS and previous studies from others described variable percentages of purified CD34+ cells using Immunomagnetic approach [2,3]. Herein, we provide evidence that purification of cells using IMBs-based approach for MDS-RS samples leads to the contamination of unwanted cells with high iron overload including a large majority of erythroid cells.
Our experiments also confirmed the results of Martin, et al. [4,5] about sideroblasts/siderocytes purification. Interestingly, if most cells retained on PcC were from the erythroblast compartment, we also purified granulocytes and monocytes/macrophages. The explanations for their purification could be a direct interaction with high iron content cells or the possibility that these cells contain themselves a high level of iron. As granulocytes and macrophages have phagocytic capabilities, it would not be surprising that their detection may be also related to their high iron content cell cleaning ability. Furthermore, macrophages are the bone marrow iron regulators and our approach probably selected the iron overloaded macrophages and discarded the others.
We also identified CD34+ cells among the retained cells. As MDS is a clonal disease, we may hypothesize that our approach also retained part of the abnormal progenitor proerythroblast population. Finally, comparison of Perls' prussian blue results realized on bone marrow smear with our FC data lead to a nonsignificant relationship. This difference could be due to the gradient separation method that induces siderocytes/sideroblasts loss [8] before the PcC step. To conclude, getting a high percentage of purified CD34+ cells with immunomagnetic methods on MDS-RS is impossible without using a precleaning column. We provide evidence that cells retained on the column are mainly expressing CD235a marker and are identified as erythroid cells by MGG. We believe that these data obtained from a small cohort of patients may help in developing further alternative processes for studying MDS-