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;-mercaptoethanol, 100 &mu;M MEM Non-Essential Amino Acids, 1 mM MEM Sodium Pyruvate, 2 mM L-glutamine, 100 &mu;g/ml streptomycin and 100 U/ml penicillin.</li>
 <li>Cover the bottom of a previously sterilized mortar with sterile filters.</li>
 <li>Transfer the bones into the mortar and cover them with a layer of filters in order to assemble a friction surface.</li>
 <li>Prepare a 50 mL Falcon tube provided with a Falcon filter.</li>
 <li>Smash the bones with the help of a pestle until obtaining a homogeneous cell suspension.</li>
 <li>Transfer the suspension into the tube.</li>
 <li>Add to the mortar 5 mL of fresh RPMI complete medium to wash and harvest the remaining cells.</li>
 <li>Repeat the last two points until the homogenized bones appear clear.</li>
 <li>Transfer and filter in a new tube the cell suspension in order to eliminate the remaining tissue debris.</li>
 <li>Centrifuge the tube at 1,500 rpm for 5 min and carefully remove the supernatant by aspiration. Make sure not to disturb the cell pellet.</li>
</ol>
2. Phenotypic Analysis
<ol>
 <li>Resuspend and wash cells of individual mice in 15 mL of PBS and 2% FBS.</li>
 <li>Centrifuge the tube at 1,500 rpm for 5 min and carefully remove the supernatant by aspiration. Make sure not to disturb the pellet.</li>
 <li>Suspend the pellet in 5 mL PBS and 2% FBS and count cells.</li>
 <li>Transfer 1&times;106 cells/well in a 96 wells round bottom plate.</li>
 <li>Centrifuge the plate at 1,500 rpm for 5 min.</li>
 <li>Prepare a mix of mAbs directed against the following surface markers. Dilute them as indicated in PBS and 2% FBS to stain the cells.
 <ul type="disc">
 <li>CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 conjugated to PE-Cy5 1:200</li>
 <li>c-Kit conjugated to APC 1:100</li>
 <li>Sca1 conjugated to biotin 1:50</li>
 <li>CD34 conjugated to FITC 1:50</li>
 <li>Fc&gamma;R conjugated to PE 1:100</li>
 <li>IL-7R conjugated to PE-Cy7 1:100</li>
 </ul>
 </li>
 <li>Discard the supernatant and add 50 &mu;L of mAbs mix to each well. Dissociate into single cells by pipetting.</li>
 <li>Incubate cells for 20 min in the dark at 4 &deg;C.</li>
 <li>Wash cells twice with 150 &mu;L of PBS and 2% FBS.</li>
 <li>Centrifuge the plate at 1,500 rpm for 5 min and discard the supernatant by inverting the plate.</li>
 <li>Dilute streptavidin in PBS and 2% FBS as indicated to stain the cells.
 <ul type="disc">
 <li>Streptavidin conjugated to APC-Cy7 1:300</li>
 </ul>
 </li>
 <li>Add 50 &mu;L of the solution to each well. Dissociate into single cell suspension by pipetting.</li>
 <li>Incubate cells for 10 min in the dark at 4 &deg;C.</li>
 <li>Wash cells twice with 150 &mu;L of PBS and 2% FBS.</li>
 <li>Centrifuge the plate at 1,500 rpm for 5 min and discard the supernatant.</li>
 <li>Resuspend cells in 100 &mu;L of PBS and 2% FBS.</li>
 <li>Acquire samples at FACS.</li>
</ol>
3. Magnetic Enrichment of c-Kit+ Cells
<ol>
 <li>Prepare a buffer solution containing PBS, pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA.</li>
 <li>Resuspend the cell pellet of bone marrows derived from at least 10 mice in 1 mL of buffer solution containing c-Kit mAb conjugated to APC diluted 1:50.</li>
 <li>Incubate cells for 20 min in the dark at 4 &deg;C.</li>
 <li>Wash two times the cells with 40 mL of buffer solution.</li>
 <li>Centrifuge the tube at 1,500 rpm for 5 min and carefully remove the supernatant by aspiration. Make sure not to disturb the pellet.</li>
 <li>Resuspend cell pellet and add 50 &mu;l of Anti-APC MicroBeads for each euthanized mouse directly to the pellet.</li>
 <li>Mix well and incubate for 20 min in the dark at 4 &deg;C.</li>
 <li>Wash cells by adding 40 mL of buffer solution and centrifuge at 1,500 rpm for 5 min. Aspirate supernatant completely.</li>
 <li>Resuspend cell pellet in multiple of 4 mL of buffer solution every three mice euthanized.</li>
 <li>Place MACS LS columns in the magnetic field of a suitable MACS Separator. Use 1 column every three mice euthanized.</li>
 <li>Prepare columns by rinsing with 4 mL of buffer solution.</li>
 <li>Apply cell suspension onto the columns.</li>
 <li>Collect unlabeled cells that pass through and wash column with 4 mL of buffer solution. Perform washing steps by adding 4 mL of buffer solution three times. Add new buffer when the column reservoir is emptied.</li>
 <li>Remove columns from the separator and place each one on top of 15 mL falcon tubes.</li>
 <li>Pipette 4 mL of buffer solution onto each column. Immediately flush out the magnetically labeled cells by pushing the plunger into the column.</li>
 <li>Centrifuge the tubes at 1,500 rpm for 5 min and carefully remove the supernatant by aspiration.</li>
 <li>Resuspend each pellet with 1 mL of buffer solution and group pellets in a single 15 mL falcon tube.</li>
</ol>
4. Sorting of Hematopoietic Stem Cells and Lineage committed Progenitors
<ol>
 <li>Prepare a mix of mAbs directed against the following surface markers. Dilute them as indicated in 500 &mu;L PBS and 2% FBS to stain the cells.
 <ul type="disc">
 <li>CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 conjugated to PE-Cy5 1:200</li>
 <li>c-Kit conjugated to APC 1:100</li>
 <li>Sca-1 conjugated to biotin 1:50</li>
 <li>CD34 conjugated to FITC 1:50</li>
 <li>Fc&gamma;R conjugated to PE 1:100</li>
 </ul>
 </li>
 <li>Centrifuge the tube from the previous section at 1,500 rpm for 5 min and carefully remove the supernatant by aspiration.</li>
 <li>Add the mAbs mix to the pellet. Dissociate into single cell suspension by pipetting.</li>
 <li>Incubate cells for 20 min in the dark at 4 &deg;C.</li>
 <li>Wash two times the cells with 14mL of PBS and 2% FBS.</li>
 <li>Centrifuge the tube at 1,500 rpm for 5 min and discard the supernatant.</li>
 <li>Dilute streptavidin as indicated in 500 &mu;L PBS and 2% FBS to stain the cells.
 <ul type="disc">
 <li>Streptavidin conjugated to APC-Cy7 1:300</li>
 </ul>
 </li>
 <li>Add the solution to the tube. Dissociate into single cell suspension by pipetting.</li>
 <li>Incubate cells for 10 min in the dark at 4 &deg;C.</li>
 <li>Wash two times the cells with 14 mL of PBS and 2% FBS.</li>
 <li>Centrifuge the tube at 1,500 rpm for 5 min and discard the supernatant.</li>
 <li>Resuspend cells in 500 &mu;L of PBS and 2% FBS.</li>
 <li>Transfer and filter the cell suspension in a new 5 mL tube in order to eliminate cellular aggregates that can interfere with the sorting procedure.</li>
 <li>Add 5 &mu;L of 7-AAD (use it diluted 1:100) to the cell suspension in order to exclude 7-AAD+ dead cells from the sorted populations.</li>
 <li>Sort cells with a BD FACSAria according to the following phenotypes:
 <ul type="disc">
 <li>LKS cells: lin- c-Kit+ Sca-1+</li>
 <li>LT-HSCs: LKS CD34-</li>
 <li>ST-HSCs: LKS CD34+</li>
 <li>GMPs: lin- c-Kit+ Sca-1low Fc&gamma;Rhigh CD34+</li>
 <li>MEPs: lin- c-Kit+ Sca-1low Fc&gamma;Rlow CD34-</li>
 <li>CMPs: lin- c-Kit+ Sca-1low Fc&gamma;Rlow CD34+</li>
 </ul>
 </li>
</ol>
5. Representative Results
Figure 1 shows and example of the electronic gating procedure to score common lymphoid progenitors (CLPs) as Lin-/c-Kitlo/interleukin (IL)-7R+ cells; Lin-/c-Kit+/Sca-1+ (LKS) and Lin-/c-Kit+/Sca-1lo (c-Kit+) cells; from the c-Kit+ gate, common myeloid progenitors (CMPs) are identified as Fc&gamma;RloCD34+ cells, granulocyte/monocyte progenitors (GMPs) as Fc&gamma;RhiCD34+ cells and megakaryocyte/erythrocyte progenitors (MEPs) as Fc&gamma;RloCD34- cells; from the LKS gate, LT-HSC and ST-HSC are identified as CD34- and CD34+ cells, respectively. This bone marrow cell suspension can be enriched for c-Kit+ cells with magnetic beads (Fig. 2); LT-HSC and ST-HSC can then be sorted according to CD34 expression as well as other progenitors according to the phenotype described above.
HSCs can be analyzed in live imaging confocal microscopy as shown in Figure 4, where CD34- cells were stained with the nucleotide-binding compound quinacrine11 as well as nuclear red (DRAQ5). In HSCs, but not GMPs, quinacrine-positive vesicles are visible in the cytoplasm12. Moreover, sorted HSCs can be used in functional experiments as demonstrated in figure 5, which shows the increase in cytosolic Ca2+ in LT-HSC by activation of purinergic P2 receptors upon exposure to adenosine-triphosphate (ATP) and ionomycin12.
<img alt="Figure 1" src="/files/ftp_upload/3736/3736fig1.jpg" /> 
Figure 1. Representative dot plot analysis of Lin- BM cells. Electronic gating procedure at flow cytometer to identify and score hematopoietic progenitors and HSCs from bone marrow cell suspension stained as described in the protocol text.
<img alt="Figure 2" src="/files/ftp_upload/3736/3736fig2.jpg" /> 
Figure 2. A scheme for selective enrichment of c-Kit+ cells from bone marrow.
<img alt="Figure 3" src="/files/ftp_upload/3736/3736fig3.jpg" /> 
Figure 3. Cell cycle analysis of electronically gated LKS CD34- HSCs in healthy controls and mice with IBD stained with Ki-67 antibody and DAPI. Cells were fixed and permeabilized with Lyse/Fix and Perm buffers followed by staining with FITC conjugated Ki-67 and DAPI at 1 &mu;g/ml. Cycling cells are barely if at all detectable in the LKS CD34- HSCs compartment of healthy controls12.
<img alt="Figure 4" src="/files/ftp_upload/3736/3736fig4.jpg" /> 
Figure 4. Live imaging confocal microscopy of FACS sorted CD34- HSCs and GMPs stained with the nucleotide-binding compound quinacrine and nuclear red (DRAQ5). The small quadrants show quinacrine positive vesicles detected in the cytoplasm of HSCs but not GMPs.
<img alt="Figure 5" src="/files/ftp_upload/3736/3736fig5.jpg" /> 
Figure 5. Cytosolic Ca2+ elevations in sorted HSCs loaded with Fura-2 and stimulated with ATP and ionomycin. Traces from single cells are shown. All cells were responsive to extracellular ATP showing prominent increase in cytosolic Ca2+ after addition of ATP.

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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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No conflicts of interest declared.

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

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Biology

18.6K vistas.  Bellinzona (Switzerland). Un método para analizar la distribución de progenitores hematopoyéticos de médula ósea en citometría de flujo, así como para aislar eficazmente altamente purificadas células madre hematopoyéticas (HSC) se describe. El procedimiento de aislamiento se basa esencialmente en el enriquecimiento magnética de c-kit + células y células de clasificación para purificar las CMH para los estudios celulares y moleculares.

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...