We gratefully acknowledge Hiroshi Kiyonari for the animal care and for providing recipient mice at RIKEN BDR, as well as Hitomi Oga, Kayoko Nagasaka, and Masaki Miyahashi for laboratory management at Kobe University. The authors also greatly appreciate the ongoing support for this work. Masanori Miyanishi was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17K07407 and JP20H03268, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Life Science Foundation of Japan, The Takeda Science Foundation, The Astellas Foundation for Research on Metabolic Disorders, and AMED-PRIME, AMED under Grant Number JP18gm6110020. Taro Sakamaki is supported by JSPS KAKENHI Grant Numbers JP21K20669 and JP22K16334 and was supported by the JSPS Core-to-Core Program and RIKEN Junior Research Associate Program. Katsuyuki Nishi was supported by JSPS Grant Number KAKENHI JP18J13408.

All the animal experiments described were approved by the RIKEN Center for Biosystems Dynamics Research.
1. Preconditioning of the recipient mice
<ol>
	<li>Prepare male C57BL/6 congenic mice aged 8-10 weeks old as recipient mice. The number of recipient mice depends on the experimental protocol. We typically prepare 10-20 mice for each condition.
	<ol>
		<li>Feed the mice with sterilized water supplemented with enrofloxacin (170 mg/L). As irradiated recipient mice are highly...

<ol>
	<li>Weissman, I. L., Shizuru, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+of+the+identification+and+isolation+of+hematopoietic+stem+cells,+and+their+capability+to+induce+donor-specific+transplantation+tolerance+and+treat+autoimmune+diseases.">The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases.</a> Blood. 112 (9), 3543-3553 (2008).</li><li>Majeti, R., Park, C. Y., Weissman, I. L. <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+a+hierarchy+of+multipotent+hematopoietic+progenitors+in+human+cord+blood.">Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood.</a> Cell Stem Cell. 1 (6), 635-645 (2007).</li><li>Spangrude, G. J., Heimfeld, S., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+mouse+hematopoietic+stem+cells.">Purification and characterization of mouse hematopoietic stem cells.</a> Science. 241 (4861), 58-62 (1988).</li><li>Ogawa, 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=Expression+and+function+of+c-kit+in+hemopoietic+progenitor+cells.">Expression and function of c-kit in hemopoietic progenitor cells.</a> Journal of Experimental Medicine. 174 (1), 63-71 (1991).</li><li>Ikuta, K., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+hematopoietic+stem+cells+express+mouse+c-kit+but+do+not+depend+on+steel+factor+for+their+generation.">Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.</a> Proceedings of the National Academy of Sciences of the United States of America. 89 (4), 1502-1506 (1992).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273 (5272), 242-245 (1996).</li><li>Christensen, J. L., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk-2+is+a+marker+in+hematopoietic+stem+cell+differentiation:+A+simple+method+to+isolate+long-term+stem+cells.">Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (25), 14541-14546 (2001).</li><li>Kiel, M. 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=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121 (7), 1109-1121 (2005).</li><li>Morrison, S. J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+repopulating+subset+of+hematopoietic+stem+cells+is+deterministic+and+isolatable+by+phenotype.">The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.</a> Immunity. 1 (8), 661-673 (1994).</li><li>Spangrude, G. J., Brooks, D. M., Tumas, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+repopulation+of+irradiated+mice+with+limiting+numbers+of+purified+hematopoietic+stem+cells:+In+vivo+expansion+of+stem+cell+phenotype+but+not+function.">Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: In vivo expansion of stem cell phenotype but not function.</a> Blood. 85 (4), 1006-1016 (1995).</li><li>Dykstra, B., Olthof, S., Schreuder, J., Ritsema, M., de Haan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+reveals+multiple+functional+defects+of+aged+murine+hematopoietic+stem+cells.">Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.</a> Journal of Experimental Medicine. 208 (13), 2691-2703 (2011).</li><li>Grover, 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=Single-cell+RNA+sequencing+reveals+molecular+and+functional+platelet+bias+of+aged+haematopoietic+stem+cells.">Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.</a> Nature Communications. 7, 11075 (2016).</li><li>Kataoka, K., 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=Evi1+is+essential+for+hematopoietic+stem+cell+self-renewal,+and+its+expression+marks+hematopoietic+cells+with+long-term+multilineage+repopulating+activity.">Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.</a> Journal of Experimental Medicine. 208 (12), 2403-2416 (2011).</li><li>Gazit, R., 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=Fgd5+identifies+hematopoietic+stem+cells+in+the+murine+bone+marrow.">Fgd5 identifies hematopoietic stem cells in the murine bone marrow.</a> Journal of Experimental Medicine. 211 (7), 1315-1331 (2014).</li><li>Acar, 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=Deep+imaging+of+bone+marrow+shows+non-dividing+stem+cells+are+mainly+perisinusoidal.">Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.</a> Nature. 526 (7571), 126-130 (2015).</li><li>Chen, J. Y., 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=Hoxb5+marks+long-term+haematopoietic+stem+cells+and+reveals+a+homogenous+perivascular+niche.">Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.</a> Nature. 530 (7589), 223-227 (2016).</li><li>Ema, 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=Quantification+of+self-renewal+capacity+in+single+hematopoietic+stem+cells+from+normal+and+Lnk-deficient+mice.">Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice.</a> Developmental Cell. 8 (6), 907-914 (2005).</li><li>Morita, Y., Ema, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+and+hierarchy+within+the+most+primitive+hematopoietic+stem+cell+compartment.">Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.</a> Journal of Experimental Medicine. 207 (6), 1173-1182 (2010).</li><li>Yamamoto, R., 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=Clonal+analysis+unveils+self-renewing+lineage-restricted+progenitors+generated+directly+from+hematopoietic+stem+cells.">Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.</a> Cell. 154 (5), 1112-1126 (2013).</li><li>Fathman, J. 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=Upregulation+of+CD11A+on+hematopoietic+stem+cells+denotes+the+loss+of+long-term+reconstitution+potential.">Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential.</a> Stem Cell Reports. 3 (5), 707-715 (2014).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Haas, S., Trumpp, A., Milsom, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+hematopoietic+stem+cell+heterogeneity.">Causes and consequences of hematopoietic stem cell heterogeneity.</a> Cell Stem Cell. 22 (5), 627-638 (2018).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+heterogeneity:+Subtypes,+not+unpredictable+behavior.">Hematopoietic stem cell heterogeneity: Subtypes, not unpredictable behavior.</a> Cell Stem Cell. 6 (3), 203-207 (2010).</li><li>Muller-Sieburg, C. E., Sieburg, H. B., Bernitz, J. M., Cattarossi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+heterogeneity:+Implications+for+aging+and+regenerative+medicine.">Stem cell heterogeneity: Implications for aging and regenerative medicine.</a> Blood. 119 (17), 3900-3907 (2012).</li><li>Duran-Struuck, R., Dysko, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+bone+marrow+transplantation+(BMT):+Providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): Providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> Journal of the American Association for Laboratory Animal Science. 48 (1), 11-22 (2009).</li><li>Nishi, K., 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=Identification+of+the+minimum+requirements+for+successful+haematopoietic+stem+cell+transplantation.">Identification of the minimum requirements for successful haematopoietic stem cell transplantation.</a> British Journal of Haematology. 196 (3), 711-723 (2022).</li><li>Sakamaki, 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=Hoxb5+defines+the+heterogeneity+of+self-renewal+capacity+in+the+hematopoietic+stem+cell+compartment.">Hoxb5 defines the heterogeneity of self-renewal capacity in the hematopoietic stem cell compartment.</a> Biochemical and Biophysical Research Communications. 539, 34-41 (2021).</li></ol>

Traditionally, cell surface marker-defined HSCs have been prepared to study the functions of HSCs, such as self-renewal capacity and multi-potency19,20,21. However, the immunophenotypically defined (Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;) HSC fraction contains two discrete HSC populations: LT-HSCs and ST-HSCs9<...

Hematopoietic stem cells (HSCs), which possess self-renewal capacity and multipotency, reside at the apex of the hematopoietic hierarchy1,2. In 1988, Weissman and colleagues demonstrated for the first time that the isolation of mouse HSCs could be achieved using flow cytometry3. Subsequently, a fraction defined by a combination of cell surface markers, Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;, was reported to contain all HSCs in mice4,5,6,7,8.
Immunophenotypically defined (Lineage&#8722;c-Kit+Sca-1+CD150+CD34&#8722;/loFlk2&#8722;) HSCs (hereafter, pHSCs) were previously considered functionally homogeneous. However, recent single-cell analyses have revealed that pHSCs still exhibit heterogeneity with respect to their self-renewal capacity9,10 and multipotency11,12. Specifically, two populations seem to exist in the pHSC fraction with regard to their self-renewal capacity: long-term hematopoietic stem cells (LT-HSCs), which have continuous self-renewal capacity, and short-term hematopoietic stem cells (ST-HSCs), which have transient self-renewal capacity9,10.
To date, the molecular mechanisms of self-renewal capacity that distinguish LT-HSCs and ST-HSCs remain poorly understood. It is crucial to isolate both cell populations based on their self-renewal capacities and to discover underlying molecular mechanisms. Several reporter systems have also been introduced to purify LT-HSCs13,14,15; however, the LT-HSC purity defined by each reporter system is variable, and exclusive LT-HSC purification has not been achieved to date.
Therefore, developing an isolation system for LT-HSCs and ST-HSCs will accelerate research regarding self-renewal capacity in the pHSC fraction. In the isolation of LT-HSCs and ST-HSCs, a study using multi-step, unbiased screening identified a single gene, Hoxb5, that is heterogeneously expressed in the pHSC fraction16. Additionally, bone marrow analysis of the Hoxb5 reporter mice revealed that approximately 20%-25% of the pHSC fraction consists of Hoxb5pos cells. A competitive transplantation assay using Hoxb5pos pHSCs and Hoxb5neg pHSCs revealed that only Hoxb5pos pHSCs possess long-term self-renewal capacity, while Hoxb5neg pHSCs lose their self-renewal capacity within a short period, indicating that Hoxb5 identifies LT-HSCs in the pHSC fraction16.
Here, we demonstrate a step-by-step protocol to isolate LT-HSCs and ST-HSCs using the Hoxb5 reporter system. In addition, we present a competitive transplantation assay to assess the self-renewal capacity of Hoxb5pos/neg pHSCs (Figure 1). This Hoxb5 reporter system allows us to prospectively isolate LT-HSCs and ST-HSCs and contributes to the understanding of LT-HSC-specific characteristics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL Strip of 8 Tubes, Dome Cap</td>
			<td>SSIbio</td>
			<td>3230-00</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA pH 8.0</td>
			<td>Iinvtrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>30G insulin syringe</td>
			<td>BD</td>
			<td>326668</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>FALCON</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>7-AAD Viability Staining Solution</td>
			<td>BioLegend</td>
			<td>420404</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-Bottom</td>
			<td>FALCON</td>
			<td>351177</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-APC-MicroBeads</td>
			<td>Milteny biotec</td>
			<td>130-090-855</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator with trap flask</td>
			<td>Biosan</td>
			<td>FTA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-Alexa Fluor 700 (RA3-6B2)</td>
			<td>BioLegend</td>
			<td>103232</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-Biotin (RA3-6B2)</td>
			<td>BioLegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-BV786 (RA3-6B2)</td>
			<td>BD Biosciences</td>
			<td>563894</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.CD45.1 congenic mice&#160;</td>
			<td>Sankyo Labo Service</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 10%</td>
			<td>BAYER</td>
			<td>341106546</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Aria II special order system&#160;</td>
			<td>BD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant stain buffer</td>
			<td>BD</td>
			<td>566349</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-Alexa Fluor 700 (M1/70)</td>
			<td>BioLegend</td>
			<td>101222</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-Biotin (M1/70)</td>
			<td>BioLegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BUV395 (M1/70)</td>
			<td>BD Biosciences</td>
			<td>563553</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BV711 (M1/70)</td>
			<td>BD Biosciences</td>
			<td>563168</td>
			<td></td>
		</tr>
		<tr>
			<td>CD127-Alexa Fluor 700 (A7R34)</td>
			<td>Invitrogen</td>
			<td>56-1271-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD150-BV421 (TC15-12F12.2)</td>
			<td>BioLegend</td>
			<td>115943</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16/CD32-Alexa Fluor 700 (93)</td>
			<td>Invitrogen</td>
			<td>56-0161-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34-Alexa Fluor 647 (RAM34)</td>
			<td>BD Biosciences</td>
			<td>560230</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34-FITC (RAM34)</td>
			<td>Invitrogen</td>
			<td>11034185</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-Alexa Fluor 700 (17A2)</td>
			<td>BioLegend</td>
			<td>100216</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3&#949; -Biotin (145-2C11)</td>
			<td>BioLegend</td>
			<td>100304</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3&#949; -BV421 (145-2C11)</td>
			<td>BioLegend</td>
			<td>100341</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.1/CD45.2 congenic mice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in our Laboratory</td>
		</tr>
		<tr>
			<td>CD45.1-FITC (A20)</td>
			<td>BD Biosciences</td>
			<td>553775</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2-PE (104)</td>
			<td>BD Biosciences</td>
			<td>560695</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-Alexa Fluor 700 (GK1.5)</td>
			<td>BioLegend</td>
			<td>100430</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-Biotin (GK1.5)</td>
			<td>BioLegend</td>
			<td>100404</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a-Alexa Fluor 700 (53-6.7)</td>
			<td>BioLegend</td>
			<td>100730</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8a-Biotin (53-6.7)</td>
			<td>BioLegend</td>
			<td>100704</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube 15ml</td>
			<td>NICHIRYO</td>
			<td>00-ETS-CT-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube 50ml</td>
			<td>NICHIRYO</td>
			<td>00-ETS-CT-50</td>
			<td></td>
		</tr>
		<tr>
			<td>c-Kit-APC-eFluor780 (2B8)</td>
			<td>Invitrogen</td>
			<td>47117182</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (-) without Ca and Mg, liquid&#160;</td>
			<td>Nacalai</td>
			<td>14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Flk2-PerCP-eFluor710 (A2F10)</td>
			<td>eBioscience</td>
			<td>46135182</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo version 10</td>
			<td>BD Biosciences&#160;</td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
			<td></td>
		</tr>
		<tr>
			<td>Gmmacell 40 Exactor</td>
			<td>Best theratronics</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr-1-Alexa Fluor 700 (RB6-8C5)</td>
			<td>BioLegend</td>
			<td>108422</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr-1-Biotin (RB6-8C5)</td>
			<td>BioLegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoxb5-tri-mCherry mice (C57BL/6J background)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in our Laboratory</td>
		</tr>
		<tr>
			<td>IgG from rat serum, technical grade, &#62;=80% (SDS-PAGE), buffered aqueous solution</td>
			<td>Sigma-Aldrich</td>
			<td>I8015-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Pfizer</td>
			<td>4987-114-13340-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes S200</td>
			<td>NIPPON PAPER CRECIA&#160;</td>
			<td>6-6689-01</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Milteny biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis buffer&#160;</td>
			<td>BD</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS&#160; MultiStand</td>
			<td>Milteny biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate for Tissue Culture (For Adhesion Cell) 6Well</td>
			<td>IWAKI</td>
			<td>3810-006</td>
			<td></td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Milteny biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Pie Cages</td>
			<td>Natsume Seisakusho</td>
			<td>KN-331</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose refrigerated Centrifuge</td>
			<td>TOMY</td>
			<td>EX-125</td>
			<td></td>
		</tr>
		<tr>
			<td>NARCOBIT-E (II)</td>
			<td>Natsume Seisakusho</td>
			<td>KN-1071-I</td>
			<td></td>
		</tr>
		<tr>
			<td>NK-1.1-PerCP-Cy5.5 (PK136)</td>
			<td>BioLegend</td>
			<td>108728</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution</td>
			<td>nacalai</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcelain Mortar &#966;120mm with Pestle</td>
			<td>Asone</td>
			<td>6-549-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 1.5 mL&#160;</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Sca-I-BUV395 (D7)</td>
			<td>BD Biosciences</td>
			<td>563990</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel scalpel blade</td>
			<td>FastGene</td>
			<td>FG-B2010</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-BUV737</td>
			<td>BD Biosciences</td>
			<td>612775</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX-red</td>
			<td>Invitrogen</td>
			<td>S34859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tailveiner Restrainer for Mice standard</td>
			<td>Braintree</td>
			<td>TV-150 STD</td>
			<td></td>
		</tr>
		<tr>
			<td>TCRb-BV421 (H57-597)</td>
			<td>BioLegend</td>
			<td>109230</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter-119-Alexa Fluor 700 (TER-119)</td>
			<td>BioLegend</td>
			<td>116220</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter-119-Biotin (TER-119)</td>
			<td>BioLegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Terumo 5ml Concentric Luer-Slip Syringe</td>
			<td>TERUMO</td>
			<td>SS-05LZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Terumo Hypodermic Needle 23G x 1</td>
			<td>TERUMO</td>
			<td>NN-2325-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation method for long-term and short-term hematopoietic stem cells

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

Previously, self-renewal capacity has been measured using competitive transplantation assays, in which donor HSCs are thought to retain their self-renewal capacity only if multi-lineage donor cells in the recipient peripheral blood are observed17. In addition, several reports define LT-HSCs as cells that continue to produce peripheral blood cells several months after the second bone marrow transplantation10,18. Therefore, in order to compa...

Watch this Scientific Journal Video about Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells at JoVE.com

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

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

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

The long-term and short-term hematopoietic stem cell or HSC isolation method will help researchers better understand the cell renewal mechanisms and biological basis of heterogeneity in HSC compartment. A transcription factor, Hoxb5, which may be an exclusive marker of long-term HSCs, was identified. Based on this, a Hoxb5-reporter mice line was established and the long-term and the short-term HSCs were successfully isolated.To begin the collection of the donor bone marrow cells, sterilize a mortar and pestle with 70%ethanol and let them dry completely. Equilibrate those with cell-staining buffer comprised of calcium and magnesium-free PBS-containing supplements. Put the bones isolated from the donor male Hoxb5-tri-mCherry mouse in the mortar and add three milliliters of the cell-staining buffer.Brush the bones open with the pestle, then disaggregate the cell clumps by gentle pipetting and transfer the cell suspension through a 100-micron cell strainer into a 50-milliliter tube. Repeat the process with fresh cell-staining buffer until the solution becomes clear. Usually, repeating three times is enough to yield a clear solution.After isolating the femurs and tibiae from the CD45.1.2 congenic mouse, cut both ends of the bones with sharp sterile scissors. Using a 23-gauge needle fitted with a five-milliliter syringe filled with ice-cold cell suspension buffer, flush the bone marrow out into a sterile cell culture dish containing cell suspension buffer. Disaggregate the cell clumps by gentle pipetting, then filter the cell suspension through a 40-micron cell strainer using a P1000 pipette.For the gating setup, prepare the sample following the instructions provided in the text, and transfer 400 microliters of it to a round bottom polystyrene test tube with a 35-micron cell strainer snap cap. Prepare and add dead cell-straining reagent to the sample before the analysis, following the manufacturer's instructions. Then, turn on the flow cytometer and launch the analysis software following the manufacturer's instructions.Then, press Load to begin acquiring the data. After excluding doublets, dead cells, and lineage-positive cells, gate the lineage-negative, the c-kit-positive, Sca-1-positive fraction. Next, gate out the Flk2-positive fraction.Then gate Hoxb5-positive or Hoxb5-negative pHSCs in the CD150-positive, CD34-negative, or low fraction. Next, prepare a 1.5-millimeter low-protein binding tube with 600 microliters of calcium and magnesium-free PBS, supplemented with 10%heat-inactivated FBS. To perform the Hoxb5-positive or Hoxb5-negative pHSC sorting, set the prepared 1.5-milliliter low-protein binding tube on a sort collection device and sort the Hoxb5-positive or Hoxb5-negative pHSCs into the tube using the previously mentioned gating strategy.In the first sorting, use the yield from the sorting precision mode. Set the 96-well plate with the previously prepared supporting cells on the automated cell deposition unit stage. Then, set the 1.5-milliliter low-protein binding tube to the loading port of the flow cytometer.Sort the Hoxb5-positive or Hoxb5-negative pHSCs into the 96-well plate with the supporting cells using the previously mentioned gating strategy. Sort 10 Hoxb5-positive or Hoxb5-negative pHSCs to test their self-renewal capacities. The flow cytometry plots of the bone marrow analysis of the donor Hoxb5-tri-mCherry mice revealed approximately 20 to 25%of the cells in the pHSC fraction defined by lineage-negative, c-kit-positive, Sca-1-positive, CD150-positive, CD34-negative, or low, Flk2-negative were Hoxb5-positive pHSCs.Shown here are the representative flow cytometry plots of the peripheral blood analysis of the recipient mice. Further, the peripheral blood analysis of the recipient mice after primary transplantation revealed that although Hoxb5-positive and Hoxb5-negative pHSCs presented similar donor chimerism four weeks after transplantation, continuous hematopoiesis was observed in only the Hoxb5-positive pHSC recipients. The Hoxb5-negative HSCs started losing the ability to produce hematopoietic cells eight weeks after transplantation.In the secondary transplantation analysis, only the Hoxb5-positive pHSC recipients presented robust hematopoiesis. In contrast, donor cells were hardly observed in the Hoxb5-negative pHSC recipient mice, suggesting Hoxb5-negative pHSCs lost self-renewal ability within 16 weeks of primary transplantation. This protocol usually takes nine to 12 hours, so it is important to keep the sample at four degrees Celsius throughout the procedures as much as possible to maintain cell viability.

isolation-method-for-long-term-and-short-term-hematopoietic-stem-cells

Research

JoVE Journal

Biology

1.4K ビュー.  Kobe University Graduate School of Medicine. 長期および短期の造血幹細胞またはHSC単離法は、研究者がHSCコンパートメントにおける細胞再生メカニズムと不均一性の生物学的基盤をよりよく理解するのに役立ちます。長期造血幹細胞の排他的マーカーである可能性のある転写因子Hoxb5が同定されました。これに基づいて、Hoxb5レポーターマウス系統を確立し、長期および短期のHSCを正常に分離しました。ドナー...