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 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.</li>
			<li>Compensation beads (if necessary)</li>
			<li>Suspension buffer: Hank&#39;s Balanced Salt Solution (HBSS) + 25 mM HEPES + 3% FCS</li>
			<li>Trypan blue and hemocytometer</li>
			<li>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.</li>
			<li>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&#39;s human research ethics committee for human welfare.</li>
		</ol>
		</li>
		<li>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 &#176;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.</li>
		<li>After washing with 5 mL staining buffer, resuspend the cells in 2 mL suspension buffer and determine the cell concentration. Place 5 &#181;L of cell suspension in a screw cap test tube. Add 95 &#181;L 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.</li>
		<li>Centrifuge the cells at 250 g for 10 min at 20 &#176;C, discard the supernatant and resuspend the cells in staining buffer at a concentration of up to 1 x 106 per 100 &#181;L.</li>
		<li>Block FcR by the use of 2.5 &#181;g Fc Block per 1 x 106 cells on ice for 10 - 15 min.</li>
		<li>Add the appropriate combination of 10 &#181;g mAb to stain 1 x 106 cells and incubate for 20 min at 4&#176; 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.</li>
		<li>Adjust the concentration to 20 x 106 cells/mL.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Sorting on the cell sorter
	<ol>
		<li>Prepare collection tubes from step 1.1.1.9.</li>
		<li>Set up a template that includes a bivariate plot to display forward scatter (FSC) and side scatter (SSC).</li>
		<li>Run the cells and adjust FSC/SSC to place the population of interest on scale</li>
		<li>Record the negative control tube.</li>
		<li>Run the single positive control tubes; record the data for each tube.</li>
		<li>Calculate compensation either manually or automatically with the function provided by the acquisition software.</li>
		<li>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).</li>
		<li>Once gates have been determined, the gates can be selected for sorting into external collection tubes.</li>
		<li>Proceed with sorting at 4 &#176;C until the required number of cells has been obtained</li>
		<li>Perform a post-sort analysis to determine the purity of the sorted cell populations (Figure 1E, 1F).</li>
	</ol>
	</li>
</ol>
2. Clonogenic Assay
<ol>
	<li>Preparation of reagents 
	Before starting the process, the following items need to be collected and prepared:
	<ol>
		<li>Pipettes, 100 mm plates</li>
		<li>Trypan blue and hemocytometer</li>
		<li>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 &#176;C to avoid repeated freeze-thaw.</li>
		<li>Prepare complete Iscove&#39;s Modified Dulbecco&#39;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).</li>
	</ol>
	</li>
	<li>Preparation of bone marrow cells
	<ol>
		<li>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).</li>
		<li>After centrifugation, remove the interface into a new 50 mL tube and add PBS to a final volume of 50 mL.</li>
		<li>After centrifugation with 250 g for 10 min at 10 &#176;C remove the supernatant and resuspend the cells in complete IMDM medium at approximately 0.5 x 106 cells/mL. Take 10 &#181;L of the cell suspension into a microtube, mix with the same volume of Trypan blue solution (0.4%) and count using a hemocytometer.</li>
		<li>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.</li>
		<li>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 &#181;L 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.</li>
		<li>Cells are cultured in a humidified 37 &#176;C incubator with 5% CO2 for 14 days.</li>
	</ol>
	</li>
	<li>Analysis of colonies
	<ol>
		<li>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&#39;s instructions for further colony subclassification.</li>
		<li>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 &#176;C, the cells are resuspended in PBS, counted, and processed for DNA extraction.</li>
	</ol>
	</li>
</ol>
3. Analysis of Chimerism
<ol>
	<li>DNA Isolation
	<ol>
		<li>Pellet the cells by centrifugation at 400 g for 10 min.</li>
		<li>Isolate DNA using a blood DNA extraction kit (QIAmp DNA Blood extraction kit). Pre-warm the elution buffer (AE) at 37 &#176;C to enhance the yield of eluted DNA.</li>
		<li>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.</li>
		<li>Dilute DNA with DNAase free H2O to 0.1 ng/&#181;L in a total volume of 50 &#181;L.</li>
	</ol>
	</li>
	<li>STR-Analysis
	<ol>
		<li>Set up the PCR reaction by mixing Reaction Mix, AmplTaq Gold DNA Polymerase and Profiler Plus Primer Set with 20 &#181;L genomic DNA (0.1 ng/&#181;L) 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.</li>
		<li>Perform PCR reaction using Eppendorf mastercycler gradient with the following conditions: 95 &#176;C for 11 min, 28 amplification cycles of 95 &#176;C for 1 min, 59 &#176;C for 1 min and 72 &#176;C for 1 min, followed by 60&#176; C for 45 min.</li>
		<li>Set up fragment analysis reaction by adding 12 &#181;L Hi-Di Formamid and 0.5 &#181;L GeneScan 500 ROX Size Standard (all Applied Biosystems) to 1 &#181;L of PCR product. At first and last positon an Allelic Ladder (Genotyper AmpFlSTR Blue, Green II and Yellow, Applied Biosystems) is needed.</li>
		<li>Start electrophoresis and acquisition with the electrophoresis instrument.</li>
		<li>Analyze Loci, alleles and peak heights of samples and controls with the software13.</li>
	</ol>
	</li>
</ol>

<ol><li>Angelucci, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hematopoietic+stem+cell+transplantation+in+thalassemia+major+and+sickle+cell+disease%3A+indications+and+management+recommendations+from+an+international+expert+panel%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel.</a> Haematologica. 99, 811-820 (2014).</li>
<li>Lucarelli, G., Isgro, A., Sodani, P., Gaziev, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hematopoietic+stem+cell+transplantation+in+thalassemia+and+sickle+cell+anemia%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Med%22%5BJournal%5D)">Hematopoietic stem cell transplantation in thalassemia and sickle cell anemia.</a> Cold Spring Harb Perspect Med. 2, a011825(2012).</li>
<li>Andreani, M., Testi, M., Lucarelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mixed+chimerism+in+haemoglobinopathies%3A+from+risk+of+graft+rejection+to+immune+tolerance%5BTitle%5D)+AND+%22Tissue+Antigens%22%5BJournal%5D)">Mixed chimerism in haemoglobinopathies: from risk of graft rejection to immune tolerance.</a> Tissue Antigens. 83, 137-146 (2014).</li>
<li>Andreani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistence+of+mixed+chimerism+in+patients+transplanted+for+the+treatment+of+thalassemia%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia.</a> Blood. 87, 3494-3499 (1996).</li>
<li>Andreani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+survival+of+ex-thalassemic+patients+with+persistent+mixed+chimerism+after+bone+marrow+transplantation%5BTitle%5D)+AND+%22Bone+Marrow+Transplant%22%5BJournal%5D)">Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation.</a> Bone Marrow Transplant. 25, 401-404 (2000).</li>
<li>Kumar, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pilot+study+of+prophylactic+ex+vivo+costimulated+donor+leukocyte+infusion+after+reduced-intensity+conditioned+allogeneic+stem+cell+transplantation%5BTitle%5D)+AND+%22Biol+Blood+Marrow+Transplant%22%5BJournal%5D)">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/pubmed?term=((Cytogenetic+follow-up+studies+of+recipients+of+T-cell+depleted+allogeneic+bone+marrow%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">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/pubmed?term=((Hemopoietic+chimerism+following+stem+cell+transplantation%5BTitle%5D)+AND+%22Transfus+Apher+Sci%22%5BJournal%5D)">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/pubmed?term=((A+multicenter+investigation+with+interphase+fluorescence+in+situ+hybridization+using+X-+and+Y-chromosome+probes%5BTitle%5D)+AND+%22Am+J+Med+Genet%22%5BJournal%5D)">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/pubmed?term=((Quantitatively+different+red+cell%2Fnucleated+cell+chimerism+in+patients+with+long-term%2C+persistent+hematopoietic+mixed+chimerism+after+bone+marrow+transplantation+for+thalassemia+major+or+sickle+cell+disease%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">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/pubmed?term=((Split+chimerism+between+nucleated+and+red+blood+cells+after+bone+marrow+transplantation+for+haemoglobinopathies%5BTitle%5D)+AND+%22Chimerism%22%5BJournal%5D)">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/pubmed?term=((Successful+management+of+mixed+chimerism+after+bone+marrow+transplant+in+beta-thalassemia+major%5BTitle%5D)+AND+%22Am+J+Hematol%22%5BJournal%5D)">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/pubmed?term=((Automated+DNA+profiling+employing+multiplex+amplification+of+short+tandem+repeat+loci%5BTitle%5D)+AND+%22PCR+Methods+Appl%22%5BJournal%5D)">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/pubmed?term=((The+importance+of+erythroid+expansion+in+determining+the+extent+of+apoptosis+in+erythroid+precursors+in+patients+with+beta-thalassemia+major%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((In+vivo+selection+of+genetically+modified+erythroblastic+progenitors+leads+to+long-term+correction+of+beta-thalassemia%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U.S.A%22%5BJournal%5D)">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 authors have nothing to disclose.

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.

<table><tbody><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>GE17-1440-02&amp;nbsp;</td><td>Remove RBC</td></tr><tr><td>50 mL conical tubes</td><td>Falcon</td><td>14-432-22</td><td>Sample preparation</td></tr><tr><td>12 x 75 mm flow tubes</td><td>Falcon</td><td>352002</td><td>FACS sorting</td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010023</td><td>PBS</td></tr><tr><td>Fetal calf serum</td><td>Invitrogen Inc.</td><td>16000-044</td><td>FCS (heat-inactivated)</td></tr><tr><td>CD34 APC</td><td>BD Bioscience</td><td>561209</td><td>FACS-Ab</td></tr><tr><td>CD36 FITC</td><td>BD Bioscience</td><td>555454</td><td>FACS-Ab</td></tr><tr><td>CD45 V450</td><td>BD Bioscience</td><td>642275</td><td>FACS-Ab</td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Hemocytometer</td><td>Invitrogen Inc.</td><td>C10227</td><td>Automatic cell counting</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Gibco</td><td>14025092</td><td>Suspension buffer in FACS analysis</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td>Component of suspension buffer</td></tr><tr><td>FcR</td><td>BD Bioscience</td><td>564220</td><td>Block FCR</td></tr><tr><td>FACS Aria I</td><td>BD Bioscience</td><td>23-11539-00</td><td>FACS Sorter</td></tr><tr><td>Recombinant&amp;nbsp; human erythropoietin</td><td>Affimetrix eBioscience</td><td>14-8992-80</td><td>EPO</td></tr><tr><td>Isocove&amp;rsquo;s Modified Dulbecco&amp;rsquo;s Medium</td><td>Gibco</td><td>12440053</td><td>IMDM</td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030-081</td><td>Component of Culture Medium</td></tr><tr><td>CD34+ magnetic beads</td><td>Milteny Biotech</td><td>130-046-702</td><td>CD34+ purification</td></tr><tr><td>Recombinant&amp;nbsp; human G-CSF</td><td>Gibco</td><td>PHC2031</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human SCF</td><td>Gibco</td><td>CTP2113</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human GM-CSF</td><td>Gibco</td><td>PHC2015</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human IL-3</td><td>BD Bioscience</td><td>554604</td><td>CFU-Assay</td></tr><tr><td>Recombinant&amp;nbsp; human IL-6</td><td>BD Bioscience</td><td>550071</td><td>CFU-Assay</td></tr><tr><td>Methocult H4434 Medium</td><td>Stemcell Technologies</td><td>4444</td><td>CFU-Assay</td></tr><tr><td>QiAmp DNA Blood extraction kit</td><td>Qiagen</td><td>51306</td><td>DNA Isolation</td></tr><tr><td>Nanodrop ND-1000 spectra photometer</td><td>Thermo Scientific</td><td>ND 1000</td><td>DNA Quantification</td></tr><tr><td>DNAase free H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Scientific</td><td>FEREN0521</td><td>DNA Preparation</td></tr><tr><td>AmplTaq Gold DNA Polymerase</td><td>Applied Bioscience</td><td>N8080240</td><td>PCR</td></tr><tr><td>Eppendorf mastercycler gradient</td><td>Eppendorf</td><td>6321000019</td><td>PCR&amp;nbsp;</td></tr><tr><td>Hi-Di Formamid</td><td>Applied Bioscience</td><td>4311320</td><td>PCR</td></tr><tr><td>GeneScan 500 ROX Size Standard</td><td>Applied Bioscience</td><td>4310361</td><td>PCR</td></tr><tr><td>3130 Genetic Analyzer</td><td>Applied Bioscience</td><td>313001R</td><td>PCR</td></tr></tbody></table>

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 ...

detection-residual-donor-erythroid-progenitor-cells-after

Research

JoVE Journal

Developmental Biology

7.3K Views.  Medical University Innsbruck. Kwantificering van donor-afgeleide cellen is vereist voor het controleren van engraftment na stamceltransplantatie bij patiënten met Hemoglobinopathieën. Een combinatie van stroom cytometry gebaseerde cel sorteren, kolonie vorming assay, en na diverse analyses van korte tandem herhalingen kunnen worden gebruikt voor het beoordelen van de proliferatie en differentiatie van de progenitoren in het compartiment van de erythroid.

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

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Cancer Research

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Medicine

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Biology

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Bioengineering

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),&#160;defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...

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

Samenvatting

Meer video's verkennen