Name: use of hematopoietic stem cell transplantation to assess the origin of myelodysplastic syndrome
Uploaded: 2018-10-03T14:30:00
Description: Watch this Scientific Journal Video about Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome at JoVE.com

This work was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health (grant numbers ZIA SC 010378 and BC 010983).

The animal procedures described in this article were approved by the National Cancer Institute at Bethesda Animal Care and Use Committee, and conform to the policies contained within The Public Health Service Policy on Humane Care and Use of Laboratory Animals, The Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals.
1. Cell Preparation
<ol>
	<li>Harvesting bone marrow nucleated cell (BMNC)
	<ol>
		<li>Use only sterile materials. Sterilize re-usable instruments using...

<ol>
	<li>Corey, S. 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=Myelodysplastic+syndromes:+the+complexity+of+stem-cell+diseases.">Myelodysplastic syndromes: the complexity of stem-cell diseases.</a> Nature Reviews Cancer. 7 (2), 118-129 (2007).</li><li>Garcia-Manero, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelodysplastic+syndromes:+2015+Update+on+diagnosis,+risk-stratification+and+management.">Myelodysplastic syndromes: 2015 Update on diagnosis, risk-stratification and management.</a> American Journal of Hematology. 90 (9), 831-841 (2015).</li><li>Heaney, M. L., Golde, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelodysplasia.">Myelodysplasia.</a> The New England Journal of Medicine. 340 (21), 1649-1660 (1999).</li><li>Nimer, S. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98+gene+fusions+and+hematopoietic+malignancies:+common+themes+and+new+biologic+insights.">NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights.</a> Blood. 118 (24), 6247-6257 (2011).</li><li>Lin, Y. W., Slape, C., Zhang, Z., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXD13+transgenic+mice+develop+a+highly+penetrant,+severe+myelodysplastic+syndrome+that+progresses+to+acute+leukemia.">NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia.</a> Blood. 106 (1), 287-295 (2005).</li><li>Block, M., Jacobson, L. O., Bethard, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preleukemic+acute+human+leukemia.">Preleukemic acute human leukemia.</a> Journal of the American Medical Association. 152 (11), 1018-1028 (1953).</li><li>Thanopoulou, E., 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=Engraftment+of+NOD/SCID-beta2+microglobulin+null+mice+with+multilineage+neoplastic+cells+from+patients+with+myelodysplastic+syndrome.">Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome.</a> Blood. 103 (11), 4285-4293 (2004).</li><li>Kerbauy, D. M., Lesnikov, V., Torok-Storb, B., Bryant, E., Deeg, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+distinct+clonal+MDS-derived+hematopoietic+precursors+in+NOD/SCID-beta2-microglobulin-deficient+mice+after+intramedullary+transplantation+of+hematopoietic+and+stromal+cells.">Engraftment of distinct clonal MDS-derived hematopoietic precursors in NOD/SCID-beta2-microglobulin-deficient mice after intramedullary transplantation of hematopoietic and stromal cells.</a> Blood. 104 (7), 2202-2203 (2004).</li><li>Benito, A. 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=NOD/SCID+mice+transplanted+with+marrow+from+patients+with+myelodysplastic+syndrome+(MDS)+show+long-term+propagation+of+normal+but+not+clonal+human+precursors.">NOD/SCID mice transplanted with marrow from patients with myelodysplastic syndrome (MDS) show long-term propagation of normal but not clonal human precursors.</a> Leukemia Research. 27 (5), 425-436 (2003).</li><li>Medyouf, H., 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=Myelodysplastic+cells+in+patients+reprogram+mesenchymal+stromal+cells+to+establish+a+transplantable+stem+cell+niche+disease+unit.">Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.</a> Cell Stem Cell. 14 (6), 824-837 (2014).</li><li>Chung, Y. J., Choi, C. W., Slape, C., Fry, T., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+a+myelodysplastic+syndrome+by+a+long-term+repopulating+hematopoietic+cell.">Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (37), 14088-14093 (2008).</li><li>Pietras, E. 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=Functionally+Distinct+Subsets+of+Lineage-Biased+Multipotent+Progenitors+Control+Blood+Production+in+Normal+and+Regenerative+Conditions.">Functionally Distinct Subsets of Lineage-Biased Multipotent Progenitors Control Blood Production in Normal and Regenerative Conditions.</a> Cell Stem Cell. 17 (1), 35-46 (2015).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Animal. 40 (5), 155-160 (2011).</li><li>Chung, Y. J., Fry, T. J., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloablative+hematopoietic+stem+cell+transplantation+improves+survival+but+is+not+curative+in+a+pre-clinical+model+of+myelodysplastic+syndrome.">Myeloablative hematopoietic stem cell transplantation improves survival but is not curative in a pre-clinical model of myelodysplastic syndrome.</a> PLoS One. 12 (9), e0185219 (2017).</li></ol>

Although MDS are a clonal hematopoietic stem cell disorder, the MDS &#34;stem&#34;, or initiating cells, have not yet been characterized. We previously demonstrated that MDS can be transplantable to WT mice using bone marrow from NHD13 mice by HSCT, characterized by macrocytic anemia, leukopenia, neutropenia, and morphologic evidence of dysplasia15. In addition, competitive repopulation assays identified a growth advantage of cells from the NHD13 MDS bone marrow. Taken together, these findings imp...

Myelodysplastic syndromes (MDS) represent a diverse set of clonal blood disorders characterized by ineffective hematopoiesis, morphologic evidence of dysplasia, and a propensity for transformation to acute myeloid leukemia (AML)1,2,3,4. Ineffective hematopoiesis is recognized as a maturation arrest in bone marrow, and results in peripheral blood cytopenias despite a hypercellular bone marrow1,3. The incidence of MDS has been variously estimated as 2-12 cases per 100,000 persons annually in the United States, and the incidence of MDS increases with age, making this an important condition to understand given the aging U.S. population3,5. Although most cases of MDS have no clear etiology, some cases of MDS are thought to be due to exposure to known genotoxic agents, including solvents such as benzene, and cancer chemotherapy6.
MDS patients typically have acquired mutations in the MDS cells7. Although relatively uncommon, a number of MDS patients have acquired balanced chromosomal translocations involving genes such as NUP98, EVI1, RUNX1, and MLL (http://cgap.nci.nih.gov/Chromosomes/Mitelman). Our laboratory has a long-standing interest in chromosome translocations, which involve the NUP98 gene8. Transgenic mice that express a NUP98-HOXD13 (NHD13) transgene regulated by the Vav1 promoter and enhancer elements display all of the key features of MDS, including peripheral blood cytopenias, morphologic evidence of dysplasia, and transformation to AML9.
Although MDS have been recognized for over 60 years10, and are considered to be a clonal stem cell disorder, efforts to engraft human MDS cell in immunodeficient mice have been largely unsuccessful, because the MDS cells engraft poorly11,12,13,14 and the mice do not develop clinical disease. In an effort to identify which hematopoietic cells can transmit MDS, we turned to the NHD13 model, and showed that we could engraft MDS as a disease entity that showed all of the cardinal features of human MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML15. In this report, we present the technical details of these experiments, as well as approaches to further fractionate hematopoietic stem and precursor cells (HSPC), in an effort to identify MDS-initiating cells.

<table>
	<tbody>
		<tr>
			<td>14 mL round bottom tube</td>
			<td>Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Lonza</td>
			<td>10-527F</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD45.2 antibody</td>
			<td>Southern Biotech</td>
			<td>1800-15</td>
			<td>LOT# A077-T044O</td>
		</tr>
		<tr>
			<td>3 mL Syringe</td>
			<td>Monoject</td>
			<td>8881513934</td>
			<td></td>
		</tr>
		<tr>
			<td>27-G needle</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>20-G needle</td>
			<td>BD</td>
			<td>305176</td>
			<td></td>
		</tr>
		<tr>
			<td>Lineage Cocktail</td>
			<td>Miltenyi</td>
			<td>130-090-858</td>
			<td>LOT# 5170418221</td>
		</tr>
		<tr>
			<td>Anti-Biotin antibodies</td>
			<td>Miltenyi</td>
			<td>130-113-288</td>
			<td>LOT# 5171109046</td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>Excelint</td>
			<td>26027</td>
			<td>Insulin Syringe</td>
		</tr>
		<tr>
			<td>Heating Lamp</td>
			<td>Thermo Fisher Scientific</td>
			<td>E70001901</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Cytec</td>
			<td>FACScan</td>
			<td>2 lasers, 5 color detectors</td>
		</tr>
		<tr>
			<td>FACS sorting instrument</td>
			<td>Beckman Coulter</td>
			<td>MOFLO ASTRIOS</td>
			<td>5 lasers, 23 parameters, 6 population sorting simulteneously</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma Irradiator</td>
			<td>Best Theratronics</td>
			<td>Gammacell 40</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tube</td>
			<td>RAM scientific</td>
			<td>76011</td>
			<td></td>
		</tr>
		<tr>
			<td>Recipient mice</td>
			<td>Charles River</td>
			<td>B6-LY5.1/Cr, CD45.1</td>
			<td></td>
		</tr>
		<tr>
			<td>NUP98-HOXD13 mice</td>
			<td>n/a</td>
			<td>C57Bl/6, CD45.2</td>
			<td>Colony maintained at NIH</td>
		</tr>
		<tr>
			<td>5 mL round bottom tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 show representative figures for results of several experiments. Figure 1 shows a representative flow cytometry sorting experiment. During normal hematopoietic differentiation, as cells become committed to a specific hematopoietic lineage, they acquire lineage-defining cell surface markers and lose the potential for self-renewal. Therefore, in wild-type mice, stem cell self-renewal is confined to lineage-negative BMNC. In this experiment, we sorted BMNC fro...

Watch this Scientific Journal Video about Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome at JoVE.com

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

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 ...

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