This work was in part supported by the French Association Laurette Fugain, LigueContre le Cancer (North Center), Fondation de France (Leukemia Committee), SIRIC Oncolille, and French National Cancer Institute.

We follow European standards for the use of animals for scientific purposes. This study was approved by the local Ethics committee (#3097-2015120414583482v3).
1. Sample preparation
<ol>
	<li>Collect bone marrow (2 mL) from de novo AML in tubes containing the anticoagulant ethylenediaminetetraacetic acid&#160;(EDTA) at a concentration of 1.8 mg per mL of bone marrow.</li>
	<li>Perform Ficoll separation, a density gradient separation to isolate mononuclear cells from red cells and granulocytes, as followed. Dilute bone marrow in 3 volumes of&#160;phosphate buffer saline (PBS: NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10mM, KH2PO4 1.8 mM). Carefully overlay 1 volume of Ficoll with 1 volume of diluted bone marrow. Spin 30 minutes at 300 x g without brake. Transfer the white buffy coat layer of mononucleated cells into a new sterile tube.</li>
	<li>Wash cells twice in 10 mL of PBS) and centrifuge at 300 x g for 5 min, in order to remove contaminating serum components.</li>
</ol>
2. Lysis of red blood cells (RBC)
<ol>
	<li>Add 2 mL of lysis buffer (ammonium chloride 0.8%) to the cell pellet, vortex gently and incubate at room temperature for 5 min. Centrifuge at 300 x g for 5 min. 
	Note: if necessary, repeat this step.</li>
	<li>Wash cells in PBS as previously described (1.3.).</li>
</ol>
3. Patient derived xenografts.
<ol>
	<li>Obtain blast cells from bone marrow after Ficoll separation and after lymphocytes depletion using immunomagnetic negative selection (CD3 for T-, and CD20 for B-lymphocytes). Follow the manufacturer&#39;s instructions.</li>
	<li>Inject 5 x 106 AML blast cells into the tail vein of unconditioned NSG mice. Use a restraining device to facilitate tail vein injection. Keep mice under conventional conditions and especially under specific-pathogen-free conditions at all times, by using individual ventilated cages and by manipulation under laminar flow hood. Every two weeks, take 60 &#181;L of blood by the submandibular collection method using a hematocrit capillary. Place the capillary into a 5 mL round bottom tube. Stop the bleed by applying gentle pressure using a small sterile gauze pad. Expulse blood with a clean syringe.&#160;</li>
	<li>Add 1ml of lysis buffer and incubate for 5min. Then centrifuge at 300 x g for 5min. Wash with PBS and centrifugre at 300 x g for 5 minutes. Remove supernatant, the add 100 &#181;L PBS with 10% bovine serum albumin (BSA) stain with 3 &#181;L anti-human CD45-FITC and 1 &#181;L anti-murine CD45-APC and incubate at 4&#176;C for 30 minutes.</li>
	<li>Wash cell pellet two times in PBS (2 mL) with 10% BSA and centrifuge at 300 x g for 5 min.</li>
	<li>Aliquot up to 1 x 106 cells per 100 &#181;L of PBS into flow cytometry tubes.</li>
	<li>Survey engraftment every two weeks by chimerism analysis of CD45 expression (human versus murine) using flow cytometry (Figure 2A and B).</li>
	<li>At sacrifice (peripheral blast count greater than 70% or severe clinical signs of sickness), collect mononuclear blast cells from crushed spleens and from bone marrow by flushing tibias and femurs with PBS as previously described13. Euthanize mice by cervical dislocation following international guidelines.&#160; &#160;</li>
	<li>If cell pellet contains RBC, perform lysis of red blood cells with lysis buffer (Step 2.1).</li>
	<li>Wash cells pellet two times in PBS (2 mL) with 10% BSA and centrifuge at 300 x g for 5 minutes.</li>
	<li>Collect cells and aliquot up to 1 x 106 cells per 100 &#181;L of PBS into flow cytometry tubes.</li>
</ol>
4. Staining
<ol>
	<li>Add conjugated antibodies (see Step 4.2) and vortex. Incubate cells for 15 minutes in the dark at room temperature.</li>
	<li>Use the following panel for human primary or PDX samples consisting of 10 &#181;L of anti-CD36-FITC, 5 &#181;L of anti-CD19-ECD, 5 &#181;L of anti-CD33-PC5.5, 5 &#181;L of anti-CD90-APC, 5 &#181;L of anti-CD34-AA700, 5 &#181;L of anti-CD45RA-APC-H7, 5 &#181;L of anti-CD38-Pacific Blue , 5 &#181;L of anti-CD123-PC7 and 2.5 &#181;L of anti-CD45-KO.</li>
	<li>Remove unbound antibodies by washing the cells in 2 mL of PBS by centrifugation at 300 x g for 5 min.</li>
	<li>Re-suspend cells in 450 &#181;L of PBS for final flow cytometric analysis.</li>
</ol>
5. Gating strategy for flow cytometry analysis
<ol>
	<li>Perform data acquisition (at least 500,000 cells) on a flow cytometer equipped with red, blue and violet lasers. Verify cytometer settings every day for 1) optical alignment, 2) fluidic system, 3) optical sensitivity, and 4) standardization using fluorospheres</li>
	<li>Follow the sequential gating strategy to define CD38 expression (Figure 1).
	<ol>
		<li>Use cells from normal bone marrow samples to define hematogones or plasma cells which will serve as positive control &#160;for CD38 expression. 
		Note: this gate is especially important to assess as subsequent putative LSC populations depend on its definition.</li>
		<li>Define physiologic precursors cells (normal blast cells) by CD45dim/SSC (side scatter) low population criteria (Figure 1A). Within this blast population, define hematogones, which display a CD38++CD19+ phenotype (Figure 1B) and finally, use CD36 and CD33 expression to define myeloblasts, monoblasts, and erythroblasts (Figure 1C). Determine CD34 expression on hematogones as shown in (Figure 1E). Assess several subpopulations of precursors within the blast cells defined as P6-P10 (Figure 1D). Separate the CD34 compartment of blast cells into P6, P7, P8 progenitor subpopulations based on preset CD38 gates. Ensure that the P8 compartment contains the blasts that have the same CD38 intensity as the hematogones, that P6 includes cells which are CD38-based on the CD38 fluorescence minus one (FMO) control and that P7 cells are in between the two extremes with regard to CD38 expression (Figure 1D).</li>
	</ol>
	</li>
	<li>From the CD34+38- (P6) gated cells, separate HSC from putative LSC using CD90 and CD45RA expression (Figure 1F). 
	Note: the HSC phenotype is CD34+CD38-CD90+CD45RA- and the multipotent progenitors (MPP) are defined as CD34+CD38-CD90-CD45RA-. The putative LSC phenotype is CD34+CD38-CD90dimCD45RA+ and the more immature downstream population are the lymphoid-primed progenitors (LMPP) defined as CD34+CD38-CD90-CD45RA+.</li>
</ol>
6. MRD monitoring by RT-PCR
<ol>
	<li>Monitor MRD levels by RT-PCR as described previously14.</li>
</ol>

<ol>
	<li>Shlush, L. I., 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=Tracing+the+origins+of+relapse+in+acute+myeloid+leukaemia+to+stem+cells.">Tracing the origins of relapse in acute myeloid leukaemia to stem cells.</a> Nature. , (2017).</li><li>Terwijn, 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=Leukemic+stem+cell+frequency:+a+strong+biomarker+for+clinical+outcome+in+acute+myeloid+leukemia.">Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.</a> PLoS One. 9 (9), e107587 (2014).</li><li>Jordan, C. T., 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+interleukin-3+receptor+alpha+chain+is+a+unique+marker+for+human+acute+myelogenous+leukemia+stem+cells.">The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.</a> Leukemia. 14 (10), 1777-1784 (2000).</li><li>van Rhenen, 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=The+novel+AML+stem+cell+associated+antigen+CLL-1+aids+in+discrimination+between+normal+and+leukemic+stem+cells.">The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.</a> Blood. 110 (7), 2659-2666 (2007).</li><li>Bonardi, 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=A+proteomics+and+transcriptomics+approach+to+identify+leukemic+stem+cell+(LSC)+markers.">A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.</a> Mol Cell Proteomics. 12 (3), 626-637 (2013).</li><li>Kikushige, Y., Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+TIM-3+as+a+Leukemic+Stem+Cell+Surface+Molecule+in+Primary+Acute+Myeloid+Leukemia.">Identification of TIM-3 as a Leukemic Stem Cell Surface Molecule in Primary Acute Myeloid Leukemia.</a> Oncology. 89, 28-32 (2015).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nat Med. 3 (7), 730-737 (1997).</li><li>Kersten, B., 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=CD45RA,+a+specific+marker+for+leukaemia+stem+cell+sub-populations+in+acute+myeloid+leukaemia.">CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.</a> Brit J Haematol. 173 (2), 219-235 (2016).</li><li>Goardon, N., 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=Coexistence+of+LMPP-like+and+GMP-like+leukemia+stem+cells+in+acute+myeloid+leukemia.">Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia.</a> Cancer Cell. 19 (1), 138-152 (2011).</li><li>Ng, S. W., 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+17-gene+stemness+score+for+rapid+determination+of+risk+in+acute+leukaemia.">A 17-gene stemness score for rapid determination of risk in acute leukaemia.</a> Nature. 540 (7633), 433-437 (2016).</li><li>Shultz, L. D., 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=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J. Immunol. 174 (10), 6477-6489 (2005).</li><li>Roloff, G. W., Lai, C., Hourigan, C. S., Dillon, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Advances+in+the+Measurement+of+Residual+Disease+in+Acute+Myeloid+Leukemia.">Technical Advances in the Measurement of Residual Disease in Acute Myeloid Leukemia.</a> J Clin Med. 6 (9), (2017).</li><li>Swamydas, S., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Purification+and+Labeling+of+Mouse+Bone+Marrow+Neutrophils+for+Functional+Studies+and+Adoptive+Transfer+Experiments.">Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments.</a> J Vis Exp. (77), e50586 (2013).</li><li>Gabert, 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=Standardization+and+quality+control+studies+of+'real-time'+quantitative+reverse+transcriptase+polymerase+chain+reaction+of+fusion+gene+transcripts+for+residual+disease+detection+in+leukemia+-+a+Europe+Against+Cancer+program.">Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.</a> Leukemia. 17 (12), 2318-2357 (2003).</li></ol>

The authors have nothing to disclose.

An accurate and reproducible assessment of membrane or cytoplasmic markers by flow cytometry requires that instrument settings are verified every day for optical alignment, proper functioning of the fluidic system,&#160; optical sensitivity, and standardization using calibration beads.
Detection of blast cell populations in AML using CD90 and CD45RA by multi-parametric flow cytometry analysis is a relatively simple and reliable method. A critical step in this analysis is an accurate assessment...

Acute myeloid leukemia (AML) is the most common cause of leukemia-associated mortality. Initially, AML is a clonal disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of blast cells, and reduced production of functional hematopoietic elements. Recently, clonal heterogeneity of the blast cell population has been established essentially by using next generation sequencing (NGS) strategies and showing the existence of intra-clonal evolution of tumor cells1.
Heterogeneity extends to the presence of leukemic stem cells (LSC). It is hypothesized that this dynamic cell compartment evolves to overcome various selection pressures imposed upon during leukemia progression and treatment. Therefore LSC are supposed to be resistant to current chemotherapeutic regimens and mediate disease relapse, with profound clinical implications2. Various markers have been described to characterize LSC like CD1233, CLL-14, CD975 or TIM-36. The CD34+CD38- compartment of blast cells is considered to be enriched for LSC7 but also includes normal HSC. CD90 and CD45RA expression applied to the CD34+CD38- (P6) compartment (Figure 1) permits to segregate several stages of normal and malignant hematopoietic precursors8. Specifically, normal CD34+CD38-CD90+CD45RA- HSCs, multipotent progenitors (MPP) like CD34+CD38-CD90dimCD45RA- cells and CD34+CD38-CD90-CD45RA+ lymphoid-primed multipotent progenitor (LMPP) cells can be determined in the majority of AML cases8,9. The mean fluorescence intensity (MFI) of CD38 is particularly important to be considered within the CD34+ cell compartment. Because CD38 intensity defines three new cell compartments thereof: CD38 negative (P6), CD38 dim (P7) and CD38 bright (P8) (Figure 1). The MFI of CD38 may be determined using either hematogones and/or plasma cells as an internal positive control as these cells strongly express CD38.
The immunodeficient NOD/SCID/IL2R&#947;cnull (NSG) mouse model is widely used for engraftment of normal and malignant human hematopoietic cells10,11. Serial xenotransplantation assays are used to experimentally validate LSC or HSC function. These studies have been extensively analyzed by large scale sequencing approaches. Nevertheless, less is known about the LSC and HSC immunophenotype of AML blast population engrafted into NSG mice compared to its primary AML (Figure 2).
The numbers of LSC are inherently low thus, identification and quantification of LSC are challenging and the method described here may be used as a flow cytometric assay at diagnosis and at clinical follow up to evaluate chemotherapy response (as residual LSC may cause AML relapse). The presence of LSC may indicate positive minimal residual disease (MRD). To date,&#160; MRD monitoring in AML mostly rely on molecular methods (i.e., RT-PCR, NGS)10,12. However, in this protocol we detect simultaneous protein expression of several HSC/LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry2,8,9. This combination of antibodies may be applied in any standard flow cytometry laboratory and this flow MRD assay is particularly interesting when MRD by real-time polymerase chain reaction (RT-PCR) is not possible, i.e., if&#160; leukemia specific molecular markers are not detectable in the diagnostic AML sample. Furthermore, this protocol, is complementary to the more sensitive molecular MRD techniques, as it aims to detect and quantify the abnormal hematopoietic progenitor cells which represents a functional cell characteristic (Figure 3).
We show how to quantify three hematopoietic progenitor populations and the putative LSC compartment with different degree of maturation by flow cytometry with antibodies available in the majority of clinical laboratories. Furthermore, we confirmed the presence of these cell compartments in corresponding patient-derived-xenografts (PDX) and at treatment follow up.

<table><tbody><tr><td>Pancoll</td><td>PAN-Biotech</td><td>P04-60500</td><td></td></tr><tr><td>RBC Lysis Buffer (10x)</td><td>Ozyme</td><td>BLE420301</td><td>RBC Lysis solution A : 8.29 g of NH&lt;sub&gt;4&lt;/sub&gt;Cl + 0.037 g of Na&lt;sub&gt;2&lt;/sub&gt;EDTA in 100 mL of sterile H&lt;sub&gt;2&lt;/sub&gt;O and RBC Lysis solution B: 1 g of KHCO&lt;sub&gt;3&lt;/sub&gt; in 100 mL of sterile H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>NOD/SCID/IL2R&amp;gamma;cnull&amp;nbsp; (NSG)&amp;nbsp; mice</td><td>JACKSON LABORATORY</td><td>Stock No: 005557</td><td></td></tr><tr><td>PBS</td><td>Gibco by life technologies</td><td>14190-144</td><td></td></tr><tr><td>AutoMACS Running Buffer &amp;ndash; MACS Separation Buffer</td><td>MACS Buffer Miltenybiotec</td><td>130-091-221</td><td></td></tr><tr><td>Anti-CD36-FITC (clone FA6-152, Iotest)</td><td>Beckman Coulter</td><td>PN IM0766U</td><td></td></tr><tr><td>Anti-CD33-PC5.5 (clone D3HL60.251, Iotest)</td><td>Beckman Coulter</td><td>B36289</td><td></td></tr><tr><td>Anti-CD90-APC (clone 5E10)</td><td>Biolegend</td><td>328114</td><td></td></tr><tr><td>Anti-CD19-ECD (clone J3-119, Iotest)</td><td>Beckman Coulter</td><td>A07770</td><td></td></tr><tr><td>Anti-CD34-AA700 (clone 581, Iotest)</td><td>Beckman Coulter</td><td>B92417</td><td></td></tr><tr><td>Anti-CD45RA-APC-H7 (clone HI100)&amp;nbsp;</td><td>Beckton Dickinson</td><td>560674</td><td></td></tr><tr><td>Anti-CD38-Pacific Blue (clone LSI98-4-3, Iotest)</td><td>Beckman Coulter</td><td>B92396</td><td></td></tr><tr><td>Anti-CD45-KO (clone J.33, Iotest)</td><td>Beckman Coulter</td><td>B36294</td><td></td></tr><tr><td>Anti-CD3-PE</td><td>Beckman Coulter</td><td>IM1451</td><td></td></tr><tr><td>Anti-CD20-PE</td><td>Beckman Coulter</td><td>A07747</td><td></td></tr><tr><td>Anti-CD123-PC7</td><td>Beckman Coulter</td><td>B13647</td><td></td></tr><tr><td>Flow-Set</td><td>Beckman Coulter</td><td>A63492</td><td></td></tr><tr><td>Flow-Check</td><td>Beckman Coulter</td><td>A63493</td><td></td></tr><tr><td>Navios</td><td>Beckman Coulter</td><td>DS-14644A</td><td></td></tr><tr><td>LSR Fortessa X20</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>CST BD</td><td>BD Biosciences</td><td>655051</td><td></td></tr><tr><td>FacsFlow&amp;nbsp;</td><td>BD Biosciences</td><td>336911</td><td></td></tr><tr><td>Anti-hCD45-FITC</td><td>Biolegend</td><td>BLE304006</td><td></td></tr><tr><td>Anti-mCD45-APC</td><td>Biolegend</td><td>BLE103112</td><td></td></tr><tr><td>EasySep human PE selection kit</td><td>Stem Cell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep&amp;nbsp; magnet&amp;nbsp;</td><td>Stem Cell Technologies</td><td>18551</td><td></td></tr></tbody></table>

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.

Here we present a method to determine progenitor populations in normal and malignant human bone marrow samples. We collected bone marrow and spleen from a successful PDX and performed multiparametric flow cytometry as described above. We compared several subpopulations of blast cells between the diagnostic patient sample and the corresponding PDX and particularly, the CD34+CD38- progenitor compartment, notably enriched in putative LSC. We have found that the CD34+CD38- blast fraction was higher in PDX compared to the dia...

Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

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

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 ...

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Cancer Research

13.9K Views.  CHU Lille. Duidelijk naar voren komt een protocol voor het detecteren van gelijktijdige uitdrukking van leukemie stamcel merkers op primaire acute myeloïde leukemie cellen door stroom cytometry. We laten zien hoe te kwantificeren drie voorlopercellen populaties en een vermeende LSC bevolking met de toenemende mate van rijping. We hebben bevestigd dat de aanwezigheid van deze populaties in overeenkomstige patiënt-afgeleid-xenografts.

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

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

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 ...

Developmental Biology

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

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.

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 ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

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 ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Estimating LSC Frequency in Zebrafish T-ALL Using Cell Proliferation Dye Sorting

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.
Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.
Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Author Spotlight: Identification and Isolation of Quiescent Leukemia Stem Cells from Zebrafish T-ALL

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Medicine

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

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

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