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>
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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 ...

This method can help answer a few questions in the method of malignancy field about characterization of a cell that initiate a malignancy. The main advantage of this technique is that it provides a foundation for the functional analysis of these cancer stem cells. The implications of this technique extend to where the therapy of myelodysplastic syndromes or MDS, as it can aid in the identification of the disease initiating cells.To prepare the recipients for adoptive transfer, one week before the transplant provide sterile water supplemented with 100 milligrams per liter of ciprofloxacin to con genic recipient ANIMALS for seven days in a specific, pathogen free animal facility. On the seventh day, have a trained, certified cesium 17 operator set the cesium source instrument to deliver 70 centigrayS of total body gamma irradiation per minute. Place 10 mice in a custom-designed holder.Insert the holder into the irradiator chamber, deliver 9 grays total body irradiation in 13 minutes. Then return the animals to their cages. The next morning, spray 70 percent ethanol over an entire two to six moth old C57 black six mouse and use scissors to remove the skin from the pelvis to the ankles.Use forceps with the scissors to remove the femora and tibiae. Cleanly trim the muscles from the bones. Cut the proximal and distal ends for the tibiae and pick up one tibia with forceps.Insert a three milliliter syringe equipped with a 27 gage needle into the bone marrow cavity and flush the bone marrow into a 14 milliliter round bottom tube containing 2.5 milliliterS of Hanks buffered saline solution supplemented with 2%fetal bovine serum or HF2. When all the marrow has been collected, aspirate the tissue several times through the 20 gage needle tip to disperse the marrow mass into a single suspension for counting. To label the bone marrow nucleated cells, or BMNC, for flow sorting, collect the cells by centrifugalization and re suspend the pellet at a one times ten to the seventh BMNC per 90 microliters of HF2 concentration.Next, add ten microliters of a biotinylated, anti-lineage antibody cocktail to the cell suspension for 20 minute incubation at 4 degrees Celsius. Followed by a centrifuge wash in three milliliters of PBS. Re suspend the pellet in 100 microliters of HF2 per one times ten to the seventh cells.Secondarily label the cells with two microliters of APC conjugated anti biotin antibody. After 20 minuets at four degree Celsius, protected from light, wash the cells in three milliliters of fresh PBS. Re suspend the pellet in HF2 supplemented with 0.2 micrograms per milliliter of propidium iodide on ice.Now calibrate the flow cytometry cell sorter using unstained cells to optimize all of the photomultiplier tubes, scatter, and fluorescence parameters within the linear range of each detector. Acquire three samples of 10, 000 cells for the unlabeled, PI labeled and anti lineage APC labeled cells. At the end of the sort, analyze 1, 000 cells from each sample to confirm the purity and viability of the sorted cells.Spin down the samples in the centrifuge. Then re suspend the pellets in 0.5 milliliters of HF2 for counting. For adoptive transfer of the sorted cells, mix 100 microliters of two times the to the fifth wild-type BMNC from a con genic recipient animal in sterile HF2.With 100 microliters of two to five times ten to the fourth the sorted donor BMNC of interest in sterile HF2 in a micro centrifuge tube per recipient animal. Load one one milliliter syringe, equipped with a 28 gage per recipient with 200 microliters of cells. Then, place the recipient mice under a heat lamp to induce tail vein dilation.Deliver the cell mixtures to each lethally irradiated recipient animal via the tail vein. As stem cell self-renewal is confined to lineage negative wild-type BMNC, lineage negative, low lineage positive, and high lineage positive cells are sorted and transplanted into wild-type recipients to determine the self renewal capacity of each cell population. Adoptively transplanted donor trans genetic BMNC from MDS model animals can be distinguished ex vivo by their CD45.2 cell surface marker expression.By 16 weeks after transplantation of lineage negative BNMC, or unsorted BMNC from transgenic MDS donor animals, the transgenic cells out compete the wild-type cells as evidenced by an increasing percentage of CD45.2 positive donor cells, observed in the peripheral blood of the lethally irradiated recipient animals. Here cells from a leukemic transformation from a mouse transplanted with transgenic MDS cells are shown. Note the presence of numerous blasts and immature forms.While attempting the procedure, it is important to remember to limit the time of ex vivo manipulation, as it can place undue stress on the cells, resulting in cell death or functional loss. After it's development this technique paves the way for the researchers in the field program, for putting in malignancy to explore the source of the disease in genetically engineered mice.

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