We thank Nobuyuki Onai, Hitoshi Takizawa and Markus Manz for precious advice. This work was funded by Swiss National Science Foundation, Swiss Cancer League and Fondazione Ticinese per la RIcerca sul Cancro.

1. Preparation of Cell Suspension from Bone Marrow
<ol>
 <li>Euthanize the mouse and place the animal in a stainless pan and spray 70% ethanol onto its abdomen and back. Collect femur and tibia from hind legs, and spinal column.</li>
 <li>Accurately remove all soft tissue residues from the spinal column with a sharp tip scissors and from legs bones with a gauze. Store bones in a 50 mL Falcon tube with 30 mL of RPMI medium containing 10% heat inactivated fetal bovine serum (FBS), supplemented with 50 &mu;M &beta;-mercap...

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	<li>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=Stem+cells:+units+of+development,+units+of+regeneration,+and+units+in+evolution.">Stem cells: units of development, units of regeneration, and units in evolution.</a> Cell. 100, 157-168 (2000).</li><li>Calvi, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425, 841-846 (2003).</li><li>Zhang, J. <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+haematopoietic+stem+cell+niche+and+control+of+the+niche+size.">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425, 836-841 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., 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+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, 1109-1121 (2005).</li><li>Arai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie2/angiopoietin-1+signaling+regulates+hematopoietic+stem+cell+quiescence+in+the+bone+marrow+niche.">Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche.</a> Cell. 118, 149-161 (2004).</li><li>Essers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo.">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458, 904-908 (2009).</li><li>Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+on+hematopoietic+progenitor+cells+stimulate+innate+immune+system+replenishment.">Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment.</a> Immunity. 24, 801-812 (2006).</li><li>Wilson, A. <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+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135, 1118-1129 (2008).</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, 242-245 (1996).</li><li>Lo Celso, C., Scadden, D., D, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Transplantation+of+Hematopoietic+Stem+Cells+(HSCs.">Isolation and Transplantation of Hematopoietic Stem Cells (HSCs.</a> J. 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The method described here enables rapid and accurate analysis of hematopoiesis in individual mice (Figure 1). This analysis in various experimental settings, including murine models of inflammation, autoimmunity, immunodeficiency, degenerative diseases, metabolic disorders and cancer, allows addressing the impact of pathological conditions on hematopoiesis. Figure 3 shows the analysis of cell cycling activity on electronically gated LKS CD34- cells from healthy mice...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Gibco</td>
 <td>42401</td>
 </tr>
 <tr>
 <td>MEM NEAA 100X</td>
 <td>Gibco</td>
 <td>11140</td>
 </tr>
 <tr>
 <td>Sodium Pyruvate</td>
 <td>Gibco</td>
 <td>11360</td>
 </tr>
 <tr>
 <td>PenStrep</td>
 <td>Gibco</td>
 <td>15070</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>20012</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000</td>
 </tr>
 <tr>
 <td>Cell Strainer 40 &mu;m</td>
 <td>BD Falcon</td>
 <td>352340</td>
 </tr>
 <tr>
 <td>7-AAD Staining solution</td>
 <td>BD Pharmingen</td>
 <td>559925</td>
 </tr>
 <tr>
 <td>Lyse/Fix buffer</td>
 <td>BD Pharmingen</td>
 <td>558049</td>
 </tr>
 <tr>
 <td>Perm buffer III</td>
 <td>BD Pharmingen</td>
 <td>558050</td>
 </tr>
 <tr>
 <td>Ki-67</td>
 <td>BD Pharmingen</td>
 <td>556026</td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Invitrogen</td>
 <td>D21490</td>
 </tr>
 <tr>
 <td>CD4 (GK1.5)</td>
 <td>eBioscience</td>
 <td>150041 </td>
 </tr>
 <tr>
 <td>CD8 (53-6.7)</td>
 <td>eBioscience</td>
 <td>150081 </td>
 </tr>
 <tr>
 <td>CD3 (145-2C11)</td>
 <td>eBioscience</td>
 <td>150031 </td>
 </tr>
 <tr>
 <td>CD45R (RA3-6B2)</td>
 <td>eBioscience</td>
 <td>150452 </td>
 </tr>
 <tr>
 <td>CD19 (6D5)</td>
 <td>eBioscience</td>
 <td>150193</td>
 </tr>
 <tr>
 <td>Gr1 (RB6-8C5) </td>
 <td>eBioscience</td>
 <td>155931 </td>
 </tr>
 <tr>
 <td>Tre119 (TER-119)</td>
 <td>eBioscience</td>
 <td>155921 </td>
 </tr>
 <tr>
 <td>NK-1.1 (PK136)</td>
 <td>eBioscience</td>
 <td>455941 </td>
 </tr>
 <tr>
 <td>c-Kit (2B8)</td>
 <td>eBioscience</td>
 <td>171171 </td>
 </tr>
 <tr>
 <td>Sca-1 (D7)</td>
 <td>eBioscience</td>
 <td>135981 </td>
 </tr>
 <tr>
 <td>CD34 (RAM34)</td>
 <td>eBioscience</td>
 <td>110341 </td>
 </tr>
 <tr>
 <td>Fc&gamma;R (2.4G2)</td>
 <td>eBioscience</td>
 <td>553145</td>
 </tr>
 <tr>
 <td>Anti-APC MicroBeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-090-855</td>
 </tr>
 <tr>
 <td>LS Columns </td>
 <td>Miltenyi Biotec</td>
 <td>130-042-401</td>
 </tr>
 </tbody>
</table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

Research

JoVE Journal

Biology

18.5K vues.  Bellinzona (Switzerland). Une méthode pour analyser la distribution des progéniteurs hématopoïétiques dans la moelle osseuse par cytométrie en flux ainsi que d'isoler efficacement hautement purifiées de cellules souches hématopoïétiques (CSH) est décrite. La procédure d'isolement est essentiellement basée sur l'enrichissement magnétique des cellules + c-kit et de tri cellulaire pour purifier CSH pour des études cellulaires et moléculaires.