Detection of microRNAs in CD34+ Microparticles of Cord Blood Units

Introduction:Micropartices (MPs) are small vesicles covered by a bilayered membrane which can serve as cell-to-cell shuttles for bioactive molecules such as lipids, growth factors, microRNAs. Umbilical Cord Blood Transplant Units (UCB) has been established as a source of Hematopoietic Stem Cells (HSC) in allogeneic transplantation. Although that UCB has unique advantages of easy procurement, low risk of transmitting infections, absence of risk to donors, immediate availability, greater tolerance of HLA disparity and lower-than-expected incidence of severe graft-versus-host disease monitoring of certain parameters on UCB transplantation remains a challenging issue. MicroRNAs (miRNAs) are short non-coding RNA chains that regulate gene expression displaying important regulatory roles in many cellular processes, including differentiation, cell replication and regeneration. We have reported herein monitoring of miRNAs containing CD34+ microparticles (MPs) as a candidate parameter of the UCBs.

Methods: Cord blood units (CBUs) (n=37) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controlled rate freezer (IceCube, Sy-lab). Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with a hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining were assessed. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V (AnnV+) and CD34 antibody by flow cytometry. Electron microscope photographs were obtained, after the appropriate preparation for TEM. CD34+ specific MPs as well as CD34+ cells isolation with immunomagnetic beads has been performed through MiniMACS columns (Miltenyi). Total RNA, including miRNA, was extracted from CD34+ and MPs using the miRNeasy Micro kit and miRNeasy Serum/Plasma kit (Qiagen), respectively. Samples' concentration was assessed by Qubit fluorimeter (Thermo Fisher scientific) and cDNA synthesis was achieved using miScript II RT kit (Qiagen). Quantitative Real Time PCR was performed to assess the presence of selected miRNAs in CD34+ and MPs in five independent donor samples (miScript SYBR Green PCR kit, Qiagen). Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables.

Results: The AnnV+/CD34+MPs during post-processing (51,38±37,87) decreased compared to the number of pre-processing AnnV+/CD34+MPs (57,7±51,2). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p<0,000, Spearman's rho =0,696) whereas the number of post-processing CD34 cell per unit was not statistically significant.

The number of post-processing AnnV+/CD34+MPs has positive correlation to the number of BFUe pre- and post-processing (p<0,037;r=0,344 and p<0,003;r=0,474 respectively). The post-processing AnnV+/CD34+MPs has positive correlation to the number of CFU-GM pre- and post-processing (p<0,015;r=0,396 and p<0,036;r=0,347 respectively).

Furthermore molecular analysis of post-processing CD34+ cells and MPs was performed for the presence of microRNA 106b, 221, 224, 517c, 518a, 519d, 520h. miRNAs 517c and 520h were detected in low levels. miRNAs 106b, 221, 224, 518a, 519d were very low or undetectable. Interestingly a different profile was observed in cell lines suggesting a line specificity of the selected miRNAs.

Conclusions: Post-processing stem cell derived MPs were correlated with CFU progenitor cells (BFUe and CFU-GM). Molecular analysis confirmed the identification of line-specific miRNAs in CD34+ cells and MPs from post- processed cord blood samples. Thus miRNAs in stem cell-derived MPs could be candidate biomarkers in cord blood units.