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,

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 ch....

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

List of materials used in this article

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