Name: detection of residual donor erythroid progenitor cells after hematopoietic stem cell transplantation for patients with hemoglobinopathies
Uploaded: 2017-09-06T13:00:00
Description: Watch this Scientific Journal Video about Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies at JoVE.com

This work was supported by the Kinderkrebshilfe Regenbogen S&#252;dtirol.

1. Isolation of Hematopoietic Bone Marrow Cells by Multi-parameter Fluorescence-activated Cell Sorting
<ol>
	<li>Sample staining
	<ol>
		<li>Before starting the process, the following items need to be collected and prepared:
		<ol>
			<li>Gradient media for the preparation of mononuclear cells (GM), 50 mL conical tubes</li>
			<li>12 x 75 mm flow tubes for staining cells</li>
			<li>Staining buffer: phosphate buffered saline (PBS) + 3% fetal calf serum (FCS)</li>
			<li>Pipettes</li>
			<li>FC block an...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+study+of+prophylactic+ex+vivo+costimulated+donor+leukocyte+infusion+after+reduced-intensity+conditioned+allogeneic+stem+cell+transplantation.">Pilot study of prophylactic ex vivo costimulated donor leukocyte infusion after reduced-intensity conditioned allogeneic stem cell transplantation.</a> Biol Blood Marrow Transplant. 19, 1094-1101 (2013).</li><li>Lawler, S. D., Harris, H., Millar, J., Barrett, A., Powles, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+follow-up+studies+of+recipients+of+T-cell+depleted+allogeneic+bone+marrow.">Cytogenetic follow-up studies of recipients of T-cell depleted allogeneic bone marrow.</a> Br J Haematol. 65, 143-150 (1987).</li><li>McCann, S. R., Crampe, M., Molloy, K., Lawler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemopoietic+chimerism+following+stem+cell+transplantation.">Hemopoietic chimerism following stem cell transplantation.</a> Transfus Apher Sci. 32, 55-61 (2005).</li><li>Dewald, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicenter+investigation+with+interphase+fluorescence+in+situ+hybridization+using+X-+and+Y-chromosome+probes.">A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.</a> Am J Med Genet. 76, 318-326 (1998).</li><li>Andreani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitatively+different+red+cell/nucleated+cell+chimerism+in+patients+with+long-term,+persistent+hematopoietic+mixed+chimerism+after+bone+marrow+transplantation+for+thalassemia+major+or+sickle+cell+disease.">Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease.</a> Haematologica. 96, 128-133 (2011).</li><li>Andreani, M., Testi, M., Battarra, M., Lucarelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+chimerism+between+nucleated+and+red+blood+cells+after+bone+marrow+transplantation+for+haemoglobinopathies.">Split chimerism between nucleated and red blood cells after bone marrow transplantation for haemoglobinopathies.</a> Chimerism. 2, 21-22 (2011).</li><li>Kropshofer, G., Sopper, S., Steurer, M., Schwinger, W., Crazzolara, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+management+of+mixed+chimerism+after+bone+marrow+transplant+in+beta-thalassemia+major.">Successful management of mixed chimerism after bone marrow transplant in beta-thalassemia major.</a> Am J Hematol. 91, E357-E358 (2016).</li><li>Kimpton, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+DNA+profiling+employing+multiplex+amplification+of+short+tandem+repeat+loci.">Automated DNA profiling employing multiplex amplification of short tandem repeat loci.</a> PCR Methods Appl. 3, 13-22 (1993).</li><li>Centis, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+erythroid+expansion+in+determining+the+extent+of+apoptosis+in+erythroid+precursors+in+patients+with+beta-thalassemia+major.">The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.</a> Blood. 96, 3624-3629 (2000).</li><li>Miccio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+selection+of+genetically+modified+erythroblastic+progenitors+leads+to+long-term+correction+of+beta-thalassemia.">In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia.</a> Proc Natl Acad Sci U.S.A. 105, 10547-10552 (2008).</li></ol>

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...

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 cells3,4,5.
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 polymorphism7,8. 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 e10,11. 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.

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detection of residual donor erythroid progenitor cells after hematopoietic stem cell transplantation for patients with hemoglobinopathies

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.

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 ...

Watch this Scientific Journal Video about Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies at JoVE.com

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

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.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

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