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 a steam autoclave. Purchase needles, syringes, and plastic ware in single use sterile containers. Perform all animal and cell manipulations in a Class II, Type A2 Biological Safety Cabinet.</li>
		<li>Prepare BMNCs in a 14 mL round bottom tube containing 3 mL of HF2 (Hank&#39;s balanced salt solution supplemented with 2% fetal bovine serum).</li>
		<li>Euthanize C57Bl6 donor mice (positive for CD45 allele CD45.2), age typically 2-6 months, using a CO2 chamber.</li>
		<li>Sterilize the mouse carcass by spraying 70% ethanol over the entire body with a spray bottle. Peel the skin from the legs, from the pelvis down to the ankle.</li>
		<li>Remove the femora and tibiae from the carcass using sterile scissors (straight, sharp/sharp, 11.5 cm) and forceps (Graefe, serrated, 0.8-mm tip width). Trim attached muscles clearly.</li>
		<li>Cut the proximal and distal ends of the tibia with the scissors. Holding the tibia with forceps, insert a 27-gauge 3 mL syringe containing 2.5 mL of HF2 into the tibial marrow cavity at the distal end of the tibia (ankle). Flush the bone marrow cells from the tibia using gentle pressure into the 14 mL round bottom tube.
		<ol>
			<li>Repeat for the other tibia.</li>
		</ol>
		</li>
		<li>Cut the proximal and distal ends of the femur with scissors. Flush the femur marrow cavity by inserting a 20-gauge needle attached to a 3 mL syringe containing 2.5 mL of HF2 into the distal end of the femur marrow cavity and applying gentle pressure to the syringe.
		<ol>
			<li>Repeat for the other femur, collecting all bone marrow from both tibiae and femora in the same 14 mL round bottom tube.</li>
		</ol>
		</li>
		<li>Disperse the marrow mass by aspirating up and down several times with the same 20-gauge needle attached to the 3 mL syringe.</li>
	</ol>
	</li>
	<li>Antibody staining of BMNC
	<ol>
		<li>Count BMNC. Add 20 &#181;L of a 3% acetic acid solution to 20 &#181;L of the BMNC suspension generated in step 1.1.8. Then, count using a hemocytometer and an inverted microscope.</li>
		<li>Pellet the BMNC by gentle centrifugation at 450 x g for 5 min at 4 &#176;C. Resuspend the pellet with HF2 using 90 &#181;L per 107 cells and add 10 &#181;L of biotinylated anti-lineage antibody cocktail to the cell suspension.</li>
		<li>After incubating for 20 min at 4 &#176;C, wash the stained cells with 3 mL of phosphate-buffered saline (PBS).</li>
		<li>Resuspend the BMNC with 100 &#181;L of HF2 per 107 cells. Add 2 &#181;L of allophycocyanin (APC) conjugated anti-biotin antibody to the cell suspension, followed by incubation at 4 &#176;C for 20 min.</li>
		<li>Rinse the stained BMNC with 3 mL of PBS and resuspend the BMNC with HF2 supplemented with 0.2 &#181;g/mL of propidium iodide (PI) for flow cytometry cell sorting.</li>
	</ol>
	</li>
	<li>Flow cytometry cell sorting of BMNC
	<ol>
		<li>Calibrate flow cytometry cell sorter. Optimize all photomultiplier tubes (PMTs), scatter, and fluorescence parameters using unstained cells and set within the linear range of each detector.</li>
		<li>Prepare unstained, PI-stained, and APC labeled lineage cocktail cells in parallel from step 1.2. Analyze for spectral compensation.</li>
		<li>Discriminate doublet cells by sequential gating of FSC-H vs. SSC-H, 640-SSC-A vs. 640-SSC-H, and 640-SSC-S vs. 640-SSC-W.</li>
		<li>Exclude nonviable cells using PI excited at 561 nm and emission collected with a 614/20 band pass filter. Excite lineage APC labeled cells at 640 nm and collect emission with a 671/30 band pass filter.</li>
		<li>Record fluorescence values as voltage height and collect at least 100,000 events in list mode data files to visualize the populations to be sorted.</li>
		<li>Calculate spectral compensation after acquiring three samples of 10,000 cells for the following: unlabeled cells, PI labeled cells, and anti-lineage antibody cocktail conjugated with APC labeled cells.</li>
		<li>Set three regions to sort Lineage negative (LN), Lineage positive (LP) with dim fluorescence intensity (LP1), and LP population with bright fluorescence (LP2) into 5 mL tubes containing 0.5 mL of 10% FBS media.</li>
		<li>Sort cells using a one drop envelop and purify abort mode.</li>
		<li>Perform post sort analysis with 1,000 total cells on each sorted sample to confirm the purity and viability of the sorted cells.</li>
		<li>Collect sorted cells by centrifugation at 450 x g for 5 min at 4 &#176;C and resuspend cell pellet with 0.5 mL of HF2.</li>
		<li>Confirm sorted cell number with hemocytometer and Trypan blue. Adjust cell number with HF2 for transplantation. 
		NOTE: A wide spectrum of BMNC populations can be isolated for transplantation using additional combinations of fluorochrome-conjugated antibodies in step 1.2.2 above.</li>
	</ol>
	</li>
</ol>
2. Recipient Mice Preparation and Hematopoietic Stem Cell Transplantation (HSCT)
<ol>
	<li>Preparation of transplant recipients
	<ol>
		<li>Maintain veterinary and husbandry care of congenic recipient mice (B6-LY5.1/Cr, CD45.1) in a specific pathogen free (SPF) animal facility.</li>
		<li>For prophylactic decontamination of the gastrointestinal track, provide sterile water containing 100 mg/L of ciprofloxacin to the recipients for 7 days prior to lethal dose (9 Gy) irradiation, and maintain for 14 days following irradiation.</li>
		<li>Provide a lethal dose (9 Gy) irradiation from a Cs source as follows. Use a trained, certified Cs operator. Set the Cs source instrument to deliver 70 cGy/min total body gamma irradiation. Place 10 mice in a custom-designed holder and insert the holder into the irradiator chamber. Deliver 9 Gy total body irradiation in 13 min and return the mice to their cages.</li>
	</ol>
	</li>
	<li>Hematopoietic stem cell transplantation
	<ol>
		<li>Mix wild type (WT) BMNC from a congenic recipient mouse (B6-LY5.1/Cr, CD45.1) at a cell dose of 2 x 105 cells per mouse with 2 to 5 x 104 cells of the purified test BMNC prepared with flow cytometry sorting as described in 1.3 above. Mix cells in the same syringe. 
		NOTE: The WT BMNC provide a radiation sparing-effect to the recipient mouse and allow for the survival of the recipient mouse if the test cells do not support hematopoiesis. The WT BMNC used in this type of experiment express the CD45.1 allele, and thus can be distinguished from the test cells using antibodies which discriminate between CD45.1 and CD45.2. The WT BM cells are referred to as &#34;helper cells&#34;, &#34;radiation-sparing cells&#34;, or &#34;competitor cells&#34;.</li>
		<li>Adjust the cell mixture volume to 200 &#181;L per recipient using sterile HF2 for facilitating the injection.</li>
		<li>Transplant the cell mixtures to the lethally irradiated recipient mice via intravenous tail vein injection using a 1 mL syringe and 28-gauge needle, within 24 h&#160;of irradiation. Place the mouse tail under a heat lamp, as heat leads to tail vein dilation. 
		NOTE: Euthanize all the recipient mice at the end of the study and evaluate the disease progression by necropsy, histology, and flow cytometry.</li>
	</ol>
	</li>
</ol>
3. Engraftment Assay using Flow Cytometry
<ol>
	<li>To evaluate donor cell engraftment, collect peripheral blood (PB) from the recipient mice at 6th week, 12th week, and 16th week after transplantation.</li>
	<li>Warm the recipients in the cage with heat lamp for about 1 min and place mouse into a restrainer.</li>
	<li>Cut the tail vein with a scalpel (blade 10, #3 scalpel handle) and collect 100 &#181;L of PB per recipient using a microcapillary tube containing EDTA as an anticoagulant.</li>
	<li>Divide the collected PB into two equal aliquots by placing 50 &#181;L in a sterile microcentrifuge tube. Use one aliquot for an automated complete blood count (CBC) (performed at the NCI Histopathology Core) and use the second aliquot for antibody staining and flow cytometry.</li>
	<li>To detect donor engraftment by flow cytometry, lyse the red blood cells (RBC) by adding 1 mL of hypotonic RBC lysis buffer (8.29 g/L of NH4Cl, 1 g/L of KHCO3, 0.037 g/L of Na2EDTA, pH 7.2) to 50 &#181;L of the collected PB. Vortex the sample to mix and incubate 10 min at room temperature.</li>
	<li>Centrifuge the lysed PB at 4,600 x g for 1.5 min. Carefully aspirate the supernatant and discard.</li>
	<li>Partially disrupt the cell pellet by gently tapping or &#34;flicking&#34; the bottom of the tube. Add 1.3 mL of PBS into the pellet located in tube and centrifuge at 4,600 x g for 1.5 min. Carefully aspirate and discard the supernatant.</li>
	<li>Disrupt the cell pellet by tapping or flicking, and add 200 &#181;L of HF2 supplemented with 5% rat serum to the cell pellet.</li>
	<li>Add anti-CD45.2 antibody (1 &#181;L) conjugated with allophycocyanin (APC) to the cell suspension. Incubate at 4 &#176;C for at least 30 min, and briefly vortex the mixture.</li>
	<li>After the staining, wash the cells twice as described above, and resuspend with 400 &#181;L of HF2 supplemented with 1 &#181;g/mL of PI.</li>
	<li>Detect engraftment using a 4-color flow cytometer. Obtain additional information on cell types within the peripheral blood by adding additional antibodies to detect specific cell types, such as B or T lymphocytes.</li>
	<li>Record serial engraftment data using a spread sheet to allow for further analysis of data.</li>
</ol>

<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. D. <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.">Myelodysplastic syndromes.</a> Blood. 111 (10), 4841-4851 (2008).</li><li>Aul, C., Giagounidis, A., Germing, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+features+of+myelodysplastic+syndromes:+results+from+regional+cancer+surveys+and+hospital-based+statistics.">Epidemiological features of myelodysplastic syndromes: results from regional cancer surveys and hospital-based statistics.</a> International Journal of Hematology. 73 (4), 405-410 (2001).</li><li>Pedersen-Bjergaard, J., Christiansen, D. H., Desta, F., Andersen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+genetic+pathways+and+cooperating+genetic+abnormalities+in+the+pathogenesis+of+therapy-related+myelodysplasia+and+acute+myeloid+leukemia.">Alternative genetic pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia.</a> Leukemia. 20 (11), 1943-1949 (2006).</li><li>Uy, G. L., 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=Dynamic+changes+in+the+clonal+structure+of+MDS+and+AML+in+response+to+epigenetic+therapy.">Dynamic changes in the clonal structure of MDS and AML in response to epigenetic therapy.</a> Leukemia. 31 (4), 872-881 (2017).</li><li>Gough, S. M., Slape, C. I., 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+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>

We have nothing to disclose.

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 imply the existence of an MDS stem or initiating cell. Additional experiments are now in progress, aimed at refining the immunophenotypic characteristics of an MDS initiating cell15 using more defined stem and progenitor cell markers (CD150, CD48, c-Kit and Sca-1)16 combined with the lineage marker staining method described in this protocol.
Identifying MDS initiating cells using NHD13 mice involves extensive flow cytometry sorting procedure. The ex vivo manipulation of primary BMNCs places stress on the cells, potentially resulting in cell death or functional loss. Therefore, efforts should be made to reduce the time of ex vivo manipulation including the time of flow cytometry sorting. The maker and model of flow cytometry instruments and flow dynamics are additional variables that may affect cell viability after sorting. The HSCT procedure requires cell infusion through a blood vessel. Although tail vein injection may be a more challenging technique than retro-orbital injection, in our experience, tail vein injection can be performed more rapidly than retro-orbital injection as the recipient mice are not anesthetized for the tail vein injections. Limitation of injection volume (maximum 150 &#181;L) is another issue in retro-orbital injection17, as a higher cell concentration may lead to additional loss of cells in the course of cell preparation, such as shearing of cells on the wall of syringes and needles.
Although these experiments are focused on the NHD13 model for MDS, the HSCT sorting and transplant approaches described here can be used for any genetically engineered mouse. In addition to determining the characteristics of self-renewing hematopoietic cells, this transplant approach can be used for other purposes. For instance, if one desires to obtain a large cohort of mice with MDS bone marrow to assess the efficacy of treatment with a small molecule, a large cohort (30+ mice) can be generated by transplanting WT mice with MDS BM. Alternatively, the HSCT strategy can be reversed. In an attempt to cure mice of MDS, NHD13 mice with MDS can be transplanted with WT BM, and the success of the transplant can be monitored by assessing the proportion of hematopoietic cells that express CD45.1 (which marks WT BM) as opposed to CD45.2 (which marks MDS BM)18.

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 from NHD13 bone marrow into lineage-negative, low lineage positive, and high lineage-positive cells. These sorted cells were then transplanted into WT recipients to determine self-renewal capacity.
Figure 2 shows results from a separate experiment, in which lineage negative cells or unsorted cells from NHD13 donors with MDS were transplanted into lethally irradiated WT recipients. The NHD13 donor cells expressed the CD45.2 cell surface marker, whereas the WT competitor cells (see section 2.2.1 above) expressed the CD45.1 cell surface marker. Figure 3 shows serial engraftment analysis of either unsorted NHD13 BMNC, or lineage-negative NHD13 BMNC. After approximately 16 weeks, the NHD13 cells outcompete the WT cells, as shown by the increasing percentage of CD45.2+ cells in the peripheral blood. Figure 4 shows an example of leukemic transformation from a mouse transplanted with NHD13 MDS cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58140/58140fig1.jpg" /> 
Figure 1: Flow cytometry sorting strategy of BMNC stained with lineage antibody cocktail. Each rectangular box on the dot plot represents the cell population sorted for HSCT to evaluate cell function. R4, lineage negative; R5, low lineage positive; R6, high lineage positive. <a href="https://www.jove.com/files/ftp_upload/58140/58140fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58140/58140fig2.jpg" /> 
Figure 2: Representative FACS profiles for engraftment assay using donor specific anti-CD45.2 antibody. BMNC recipient was transplanted with 1 X 106 NHD13 BMNC (CD45.2+) and LNBM recipient with 5 X 104 NHD13 lineage negative BM cells (CD45.2+). In each case, 2 x 105 WT BMNC (CD45.1+) were used as competitor cells. Numbers in the upper and lower boxes represent CD45.2 positive (derived from NHD13 donor) and CD45.2 negative (derived from WT competitor cells), respectively, at post-transplantation 6 week and 24 week. <a href="https://www.jove.com/files/ftp_upload/58140/58140fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58140/58140fig3.jpg" /> 
Figure 3: Engraftment kinetics of individual recipients after transplantation. Each line on the graph shows serial engraftment assays of an individual recipient mouse. BMNC recipients were transplanted with 1 X 106 cells of NHD13 whole BMNC, and LNBM recipients were transplanted with 5 X 104 of NHD13 lineage negative BM after sorting. NHD indicates NHD13 donor. BM, BMNC recipient mice; LNBM, lineage negative BM recipient; PBMC, peripheral blood mononucleated cell. In all cases, the NHD13 donor cells gradually outcompete the WT cells. <a href="https://www.jove.com/files/ftp_upload/58140/58140fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58140/58140fig4.jpg" /> 
Figure 4: May-Gr&#252;nwald Giemsa (MGG) staining of bone marrow (BM) from MDS recipient that progressed to AML. Note the presence of numerous blasts and immature forms. Arrows indicate blasts, and arrowheads indicate immature forms. Original 400X. <a href="https://www.jove.com/files/ftp_upload/58140/58140fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

use-hematopoietic-stem-cell-transplantation-to-assess-origin

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.7K Views.  Center for Cancer Research, National Cancer Institute, National Institutes of Health. 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.

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

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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 ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

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 ...

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.

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Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

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

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Chapters in this video

An Efficient Method to Obtain Dedifferentiated Fat Cells

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

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

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

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Analysis of Hematopoietic Stem Progenitor Cell Metabolism