4.1. Sample preparation
The UCB cells originally isolated from healthy donors were obtained from Eurocord Slovakia s.r.o. – Slovak cord blood bank. Cells were thawed as previously described [26, 27] with addition of 100 µg/ml DNase I (Sigma-Aldrich, St.Louis, Missouri, United States) to the RPMI (Gibco, Thermo Fischer Scientific, Waltham, Massachussets, United States) during thawing in order to prevent formation of clamps. After thawing, centrifugation and counting of cells using 0.4 % Trypan blue solution (Gibco, Thermo Fischer Scientific), cells were incubated in complete media (89 % RPMI, 10 % FBS, 1% ATB) for 30 min in at 37°C at 5% CO2. Then cells were spun down at 175 g/15 min, cell pellet was resuspended in 5 ml of complete media, and cell suspension was filtrated through 100 µm cell strainer (BD biosciences, San Jose, California, United States) to prevent formation of clogs during cell sorting. The final viability of cells in all experiments reached at least 95%.
4.2. Cell sorting
Sample pellets were resuspended in 400 µl of PBS containing 1% BSA (Sigma-Aldrich) with FC blocking (BD biosciences) in concentration 1:100 and incubated for 10min at RT. For compensation, single-color stained controls and unstained control were prepared by adding 10 µl of cell solution to 30 µl of 1% BSA PBS. The rest of cell solution was used for cell sorting. For immunophenotyping of different cell population following antibody conjugates were used: CD34-APC (Miltenyi Biotes, Bergisch-Gladbach, Germany), CD45-V450, CD38-PeCy7, CD45RA-FITC, CD90-PE and Lineage cocktail (CD56, CD8, CD3 and CD235a-PeCy5) (all BD biosciences). The cells were then incubated for 60 min in fridge. Next, 500µl of 1% BSA PBS was added to each tube and samples were centrifuged for 10 min/800 rpm. Sample was resuspended in 400 µl of 1 % BSA PBS and compensation controls in 200 µl. Finally, 2 µl and 1 µl of 7AAD were added to the sample and Lineage compensation tube, respectively.
Various subpopulations of HSPC were then sorted using BD FACSAria Special research order product (BD biosciences) into separate tubes with 200 µl of complete media according to the expression of different surface antigens:
HSPC: CD45+ CD45RA- CD34+
Progenitors: CD45+ CD45RA- CD34+ CD38+
HSC/MPP: CD45+ CD45RA- CD34+ CD38-
HSC: CD45+ CD45RA- CD34+ CD38- CD90+
MPP: CD45+ CD45RA- CD34+ CD38- CD90-
Purity of cell sorting was at least 95% in each one of the experiments.
4.3. Cell expansion
The sorted cell subpopulations were applied into wells on 24 well plates in volume 1.5 ml of complete expansion media (CEM). CEM for one well contained 1 ml of MEM alpha EAGLE with UltraGlutamine I, deoxyribonucleoside, and ribonucleosides (αMEM) + 0.5 ml IMDM with HEPES w/L-Glutamine (both LONZA) with 10% of FBS (Fetal Bovine Serum, HYCLONE) with 1% of ATB/ATM (Antibiotic/Antimycotic Solution, PAA) containing stem cell factor (25 ng/ml), Flt-3L (25 ng/ml), and trombopoetin (25 ng/ml) (all three R&D Systems). Mesenchymal stem cells (MSC) from umbilical cord in amount of 2x104/well/1ml αMEM as a “feeder” cells were added into the wells 48 h before set the expansion. Optimal concentration of starting cells for every CD34+ subpopulation was 1500 cells/well. The maximum level of expansion achieved was ~ 1,000x multiplication of starting culture. Cells were checked visually in microscope in intervals of every 2 - 4 days and in day 8th of expansion were expanded cells harvested and transferred in fresh CEM into the wells with new MSC feeder. The optimal time for harvesting was defined before the reaching “plateau” phase during 10 - 12 days of expansion, when the multiplication is sufficient and the cells still contain the optimal amount of RNA. The flow cytometry of sorted and expanded CD34+ cells showed that expansion resulted in approximately 10 % of CD34+ cells and 90 % of CD34- cells.
4.4. Fusion gene and transcript detection
The RT-qPCR was performed as described previously . However, the method of cDNA synthesis was dependent on the amount of cells and thus RNA available for the screening. For standard reverse transcription (RT) reaction, 1 µg of total RNA was used. All “no template controls” (NTC) were negative (0/3) in all RT-qPCR assays. In case of expanded hematopoietic stem/progenitor cell subpopulations yielding often a limited amount of cells and RNA, an amplification of cDNA has been introduced. Using this procedure based on Smart-seq2  a controlled amplification of mRNA-derived cDNA (from 100- up to 140,000-fold) has been obtained.
For FISH analysis, dried slides were washed in KCl (0.55 g/100 ml; pH = 6.8; 10 min; room temperature (RT)) and placed in fixation solution (methanol 75% and 25% acetic acid; 10 min; RT) and dried. Next step was degreasing in xylene (15 min; RT) and dehydration in 96% ethanol (15 min; RT) and repeat washing in KCl and fixation step. The slides were dried in flow box. Next ready to use or diluted probes were applied in volume of 3 µl(). Slides were covered by cover slips and sealed by Fixogum (Marabu, Bietigheim-Bissingen, Germany) and applied into ThermoBlock on 75°C (CytoCell Aquarius, Cambridge, United Kingdom) for 5 min. The samples were put into humid chamber in incubator on 37°C overnight. The cover slips were removed and the slides were washed in 0.4 x SSC (60 s; 72°C) and immediately in 2 x SSCT (30 s; RT). Next the slides were dehydrated in ethanol row 50%, 70% and 96% for 2 min in every concentration without drying. After this step the slides were dried in flow in dark. We applied 3 µl DAPI in concentration 0.125 µg/ml containing antifade reagent (manufacturer, city, country). The slides were analyzed using fluorescent microscopy in spectrum DAPI, spectrum green, spectrum orange, and spectrum red. We analyzed 200 – 1,000 cells depending on the quality of the nuclei.
4.5. Statistical analysis
Analysis of variance (ANOVA), Fisher exact test and LSD was applied using Statistica software (Dell software, Round Rock, Texas, United States). The results were considered significantly different at p < 0.05.