Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

1. Isolation of Hematopoietic Bone Marrow Cells by Multi-parameter Fluorescence-activated Cell Sorting

1. Sample staining
   1. Before starting the process, the following items need to be collected and prepared:
      1. Gradient media for the preparation of mononuclear cells (GM), 50 mL conical tubes
      2. 12 x 75 mm flow tubes for staining cells
      3. Staining buffer: phosphate buffered saline (PBS) + 3% fetal calf serum (FCS)
      4. Pipettes
      5. FC block and staining antibodies (CD34 APC, CD36 FITC, CD45 V450, BD Biosciences). With this fluorochrome combination there is negligible spillover into other channels, but any other suitable fluorochrome combination is also possible.
      6. Compensation beads (if necessary)
      7. Suspension buffer: Hank's Balanced Salt Solution (HBSS) + 25 mM HEPES + 3% FCS
      8. Trypan blue and hemocytometer
      9. Collection tubes: any rich medium with high serum tubes (containing 1 mL FCS + 25 mM HEPES) can be used for the collection of sorted cells.
      10. Bone marrow sample: informed consent for bone marrow biopsy from the iliac crest of the back of the hip bone is typically required. The bone marrow sample is than kept in heparinized syringes at RT until staining. The following protocol steps are performed in compliance with the guidelines of the institution's human research ethics committee for human welfare.
   2. Generate a single cell suspension of the bone marrow sample. Add 3 mL of GM to the centrifuge tube. Carefully layer the 4 mL of the single cell suspension onto the GM solution. Centrifuge at 550 g for 30 min at 20 °Celsius (C) (brake is turned off). Draw of the upper layer containing plasma and platelets using a sterile pipette, leaving the mononuclear cell layer undisturbed at the interface. Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette.
   3. After washing with 5 mL staining buffer, resuspend the cells in 2 mL suspension buffer and determine the cell concentration. Place 5 µL of cell suspension in a screw cap test tube. Add 95 µL of 0.2% Trypan blue stain. Mix thoroughly. Allow to stand for 5 min at room temperature. Fill a hemocytometer as for cell counting. Under a microscope, observe if non-viable are stained and count the viable cells.
   4. Centrifuge the cells at 250 g for 10 min at 20 °C, discard the supernatant and resuspend the cells in staining buffer at a concentration of up to 1 x 10⁶ per 100 µL.
   5. Block FcR by the use of 2.5 µg Fc Block per 1 x 10⁶ cells on ice for 10 – 15 min.
   6. Add the appropriate combination of 10 µg mAb to stain 1 x 10⁶ cells and incubate for 20 min at 4° C in the dark, followed by a washing step with 3 mL staining buffer. For correct compensation, small aliquots of the cells (or preferably compensation beads) are stained with single antibodies each; one aliquot remaining unstained.
   7. Adjust the concentration to 20 x 10⁶ cells/mL.
   8. Set up and optimize the cell sorter. The process of setting up a flow cytometer and software is standardized with a detailed instruction provided by the company and needs to be performed by appropriately trained personnel.
2. Sorting on the cell sorter
   1. Prepare collection tubes from step 1.1.1.9.
   2. Set up a template that includes a bivariate plot to display forward scatter (FSC) and side scatter (SSC).
   3. Run the cells and adjust FSC/SSC to place the population of interest on scale
   4. Record the negative control tube.
   5. Run the single positive control tubes; record the data for each tube.
   6. Calculate compensation either manually or automatically with the function provided by the acquisition software.
   7. Acquire the experimental sample and gate them on a bivariate FSC/SSC dot plot (Figure 1A) to include both lymphocytic and myeloid cells. Exclude doublets and aggregates by comparing the different signals of FSC (i.e., height, width, and area) (Figure 1B). Visualize the singlet cells on a bivariate dot plot displaying CD45 and CD36 (Figure 1C). Events positive for CD45 are leucocytes (including their progenitors), those negative for CD45 and positive for CD36 are erythroid progenitors. Use gating tools to define CD36+ (if desired, these cells can be further divided in CD36 high and low expressing cells) and CD45+ cells. Visualize CD45+ events in another dot plot displaying CD34 and SSC parameters. Use gating tools to define CD34+ cells (leukocyte progenitors) and SSC high cells (myeloid cells at various stages of differentiation).
   8. Once gates have been determined, the gates can be selected for sorting into external collection tubes.
   9. Proceed with sorting at 4 °C until the required number of cells has been obtained
   10. Perform a post-sort analysis to determine the purity of the sorted cell populations (Figure 1E, 1F).

2. Clonogenic Assay

1. Preparation of reagents
   Before starting the process, the following items need to be collected and prepared:
   1. Pipettes, 100 mm plates
   2. Trypan blue and hemocytometer
   3. Reconstitute human recombinant erythropoietin (EPO) at 500 U/mL in sterile PBS containing at least 0.1% human serum albumin. Divide this solution into small aliquots and keep at -20 °C to avoid repeated freeze-thaw.
   4. Prepare complete Iscove's Modified Dulbecco's Medium (IMDM) with final concentrations of 5% FCS, 2 mM Glutamine, 100 units/mL Penicillin/Streptomycin (PS). Add human recombinant erythropoietin (EPO) at different concentrations in separate wells to the complete IMDM medium: 3 units EPO/well, 6 units EPO/well and 12 EPO units/well (1 x, 2 x, 4 x EPO respectively).
2. Preparation of bone marrow cells
   1. 10 mL of the bone marrow sample diluted with 20 mL PBS are slowly layered on top of 15 mL density gradient medium in a 50 mL conical tube, maintaining clear interface between the two phases and under sterile conditions. Then centrifuge the 15 mL conical tubes at 550 x g for 30 min at room temperature (RT) with acceleration set as 9 and deceleration set as 0 (or Brake OFF).
   2. After centrifugation, remove the interface into a new 50 mL tube and add PBS to a final volume of 50 mL.
   3. After centrifugation with 250 g for 10 min at 10 °C remove the supernatant and resuspend the cells in complete IMDM medium at approximately 0.5 x 10⁶ cells/mL. Take 10 µL of the cell suspension into a microtube, mix with the same volume of Trypan blue solution (0.4%) and count using a hemocytometer.
   4. Optional: CD34+ cells are enriched by magnetic cell sorting with Milteny CD34 beads according to the instructions given by the manufacture. The main advantage of this enrichment step is to detect the quality of hematopoietic stem cells, which are grown on a semi-solid matrix added with 50 ng/mL stem cell factor (SCF), 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL interleukin-3 (IL-3), 20 ng/mL interleukin-6 (IL-6), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), and 3 different concentrations of EPO as in 1.4.
   5. Dilute the cells to 0.1 – 0.15 x 10⁶ mononuclear cells/mL or 2 x 10³ CD34⁺ cells/mL by adding complete IMDM medium supplemented with EPO and transfer 300 µL to 3 mL Methocult H4434 medium. After agitation and a short incubation for 5 min plate three times 1,1 mL on a 35 mm dish. Two of these culture dishes together with a third – filled with water – are placed in 100 mm plates.
   6. Cells are cultured in a humidified 37 °C incubator with 5% CO₂ for 14 days.
3. Analysis of colonies
   1. Colonies are scored according to their morphology with an inverted microscope at 40X magnification in a culture dish marked with a scoring grid. For the purposes of our assay, the colony forming units (CFU) are classified into 4 categories: multipotential progenitor cells (CFU-GEMM), granulocyte-macrophage progenitor cells (CFU-GM), burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E). See manufacturer's instructions for further colony subclassification.
   2. For further analysis, cells from the CFU assay plate are recovered separately according to the 4 categories by suspending in 4 mL room temperature PBS containing 2% FCS/IMDM. After centrifugation at 400 g for 10 min at 4 °C, the cells are resuspended in PBS, counted, and processed for DNA extraction.

3. Analysis of Chimerism

1. DNA Isolation
   1. Pellet the cells by centrifugation at 400 g for 10 min.
   2. Isolate DNA using a blood DNA extraction kit (QIAmp DNA Blood extraction kit). Pre-warm the elution buffer (AE) at 37 °C to enhance the yield of eluted DNA.
   3. Measure the DNA concentration with Nanodrop ND-1000 spectra photometer. Samples with OD 260/280 between 1.8 – 2.0 are used for further analysis.
   4. Dilute DNA with DNAase free H₂O to 0.1 ng/µL in a total volume of 50 µL.
2. STR-Analysis
   1. Set up the PCR reaction by mixing Reaction Mix, AmplTaq Gold DNA Polymerase and Profiler Plus Primer Set with 20 µL genomic DNA (0.1 ng/µL) according to the manual (The primer kit contains the unlabeled primers in buffer to amplify the STR loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820, and the gender marker amelogenin). Include nuclease-free water as negative control and 9947A as positive control. Pre-transplant DNA from both donor and recipient are also included.
   2. Perform PCR reaction using Eppendorf mastercycler gradient with the following conditions: 95 °C for 11 min, 28 amplification cycles of 95 °C for 1 min, 59 °C for 1 min and 72 °C for 1 min, followed by 60° C for 45 min.
   3. Set up fragment analysis reaction by adding 12 µL Hi-Di Formamid and 0.5 µL GeneScan 500 ROX Size Standard (all Applied Biosystems) to 1 µL of PCR product. At first and last positon an Allelic Ladder (Genotyper AmpFlSTR Blue, Green II and Yellow, Applied Biosystems) is needed.
   4. Start electrophoresis and acquisition with the electrophoresis instrument.
   5. Analyze Loci, alleles and peak heights of samples and controls with the software¹³.