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

Summary

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

Abstract

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Introduction

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available curative approach for a variety of inborn genetic disorders of the hematopoietic system, achieving disease-free survival rates of more than 90% for otherwise highly compromised and life-limited patients¹. The efficacy of this important therapeutic tool has been optimized by limiting the toxicity of pre-and post-transplant regimens², but also by interventions aimed at sustaining stable graft function, which is quantified by close monitoring of donor-derived cells^3,4,5.

In general, complete chimerism (CC) implies the total replacement of the lymphohematopoietic compartment by donor-derived cells, whereas the term mixed chimerism (MC) is used when donor- and recipient-derived cells are simultaneously present in various proportions. Split chimerism (SC) denotes the coexistence of mixed chimerism observed in single-cell lineages, such as in the erythroid compartment. Prompt determination of chimerism status following HSCT is critical, as it may help identify patients susceptible for disease relapse and initiate subsequent immunomodulatory strategies, such as donor lymphocyte infusions or reduction of immunosuppressive therapies⁶.

Several methods have been developed for monitoring engraftment after HSCT. Isotyping of immunoglobulins and analysis of cytogenetics have poor sensitivity and are limited in their ability to detect polymorphism^7,8. The introduction of fluorescent in situ hybridization (FISH) can enhance sensitivity in chimerism monitoring after HSCT, but is restricted to sex-mismatched allografts⁹. Currently, polymerase chain reaction (PCR) is the most widespread method used to detect chimerism and is based on conventional agarose-acrylamide gel electrophoresis of variable number tandem repeats (VNTRs) or short tandem repeats (STRs). Routinely used quantitative PCR is able to detect an extremely small proportion of residual donor cells following HSCT. The major limitation of the studies so far is that MC detection is almost exclusively limited to the presence of nucleated cells, rather than mature erythrocytes, namely cells that are functionally crucial for patients affected by hemoglobinopathies. In patients with different blood groups, it is worth remembering that cytofluorometric analysis is able to identify chimerism in red blood cells by utilizing monoclonal antibodies directed towards the erythrocyte antigens ABO and C, c, D, E, and e^10,11. A different, but very interesting means of assessing chimerism in the erythroid lineage is the combination of flow cytometric sorting of erythroid progenitors and selection of various erythroid progenitor types by culturing in clonogenic assays, followed by analysis of STR¹². This approach is able to quantify relative proportions of donor-versus-recipient chimerism in the erythroid compartment and may be utilized in the strategy to sustain the bone marrow graft.

Protocol

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.

Results

Separation of lymphohematopoietic progenitors by FACS cell sorting

We here demonstrate results from sorting the necessary cell populations for downstream STR analysis. Bone marrow cells were stained with V450-conjugated anti-CD45, FITC-conjugated anti-CD36 and APC-conjugated anti-CD34. The population of interest is the megakaryocyte erythroid progenitors (MEP), nucleated cells responsible for the development of ...

Discussion

The objective of the current study is to provide the audience a combination of two approaches for analyzing donor/recipient chimerism in erythroid progenitors following HSCT in patients treated for hemoglobinopathies: 1.) fluorescence-activated cell sorting of hematopoietic progenitor cells in bone marrow samples followed by analysis of short tandem repeats and 2.) colony-forming unit growing of bone marrow cells, classification of colonies in various progenitor types followed by analysis of short tandem repeats. The nov...

Materials

Name	Company	Catalog Number	Comments
Ficoll-Paque	GE Healthcare	GE17-1440-02	Remove RBC
50 mL conical tubes	Falcon	14-432-22	Sample preparation
12 x 75 mm flow tubes	Falcon	352002	FACS sorting
Phosphate buffered saline	Gibco	10010023	PBS
Fetal calf serum	Invitrogen Inc.	16000-044	FCS (heat-inactivated)
CD34 APC	BD Bioscience	561209	FACS-Ab
CD36 FITC	BD Bioscience	555454	FACS-Ab
CD45 V450	BD Bioscience	642275	FACS-Ab
Trypan blue	Gibco	15250061
Hemocytometer	Invitrogen Inc.	C10227	Automatic cell counting
Hank’s Balanced Salt Solution	Gibco	14025092	Suspension buffer in FACS analysis
HEPES	Gibco	15630080	Component of suspension buffer
FcR	BD Bioscience	564220	Block FCR
FACS Aria I	BD Bioscience	23-11539-00	FACS Sorter
Recombinant  human erythropoietin	Affimetrix eBioscience	14-8992-80	EPO
Isocove’s Modified Dulbecco’s Medium	Gibco	12440053	IMDM
L-Glutamine	Invitrogen	25030-081	Component of Culture Medium
CD34+ magnetic beads	Milteny Biotech	130-046-702	CD34+ purification
Recombinant  human G-CSF	Gibco	PHC2031	CFU-Assay
Recombinant  human SCF	Gibco	CTP2113	CFU-Assay
Recombinant  human GM-CSF	Gibco	PHC2015	CFU-Assay
Recombinant  human IL-3	BD Bioscience	554604	CFU-Assay
Recombinant  human IL-6	BD Bioscience	550071	CFU-Assay
Methocult H4434 Medium	Stemcell Technologies	4444	CFU-Assay
QiAmp DNA Blood extraction kit	Qiagen	51306	DNA Isolation
Nanodrop ND-1000 spectra photometer	Thermo Scientific	ND 1000	DNA Quantification
DNAase free H2O	Thermo Scientific	FEREN0521	DNA Preparation
AmplTaq Gold DNA Polymerase	Applied Bioscience	N8080240	PCR
Eppendorf mastercycler gradient	Eppendorf	6321000019	PCR
Hi-Di Formamid	Applied Bioscience	4311320	PCR
GeneScan 500 ROX Size Standard	Applied Bioscience	4310361	PCR
3130 Genetic Analyzer	Applied Bioscience	313001R	PCR