Name: analysis of hematopoietic stem progenitor cell metabolism
Uploaded: 2019-11-09T12:00:00
Description: Watch this Scientific Journal Video about Analysis of Hematopoietic Stem Progenitor Cell Metabolism at JoVE.com

This work is in part supported by the funding support from the National Institutes of Health (HL131645, CA016058), the St. Baldrick&#8217;s Foundation, and the Pelotonia Foundation.

This protocol was approved by Nationwide Children&#8217;s Hospital Animal Care and Use Committee (IACUC).
NOTE: The protocol is described in chronological order that spans over the period of two days. Use fresh reagents as described in the protocol below.
1. Preparation of Reagents on the Day Prior to the Assay
<ol>
	<li>Hydrate the sensor cartridge.
	<ol>
		<li>Incubate 5 mL of the calibrant (Table of Materials) ...

<ol>
	<li>Dharampuriya, P. 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=Tracking+the+origin,+development,+and+differentiation+of+hematopoietic+stem+cells.">Tracking the origin, development, and differentiation of hematopoietic stem cells.</a> Current Opinion in Cell Biology. 49, 108-115 (2017).</li><li>Wilkinson, A. C., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hematopoietic+stem+cell+diet.">The hematopoietic stem cell diet.</a> International Journal of Hematology. 107, 634-641 (2018).</li><li>Kohli, L., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+change:+the+metabolic+journey+of+hematopoietic+stem+cells.">Surviving change: the metabolic journey of hematopoietic stem cells.</a> Trends in Cell Biology. 24, 479-487 (2014).</li><li>Abdel-Wahab, O., Levine, R. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+regulation+of+hematopoietic+stem+cells+in+the+hypoxic+niche.">Metabolic regulation of hematopoietic stem cells in the hypoxic niche.</a> Cell Stem Cell. 9, 298-310 (2011).</li><li>Zhang, C. C., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+metabolic+properties+of+hematopoietic+stem+cells.">Hypoxia and metabolic properties of hematopoietic stem cells.</a> Antioxidants &amp; Redox Signaling. 20, 1891-1901 (2014).</li><li>Snoeck, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+hematopoietic+stem+cells.">Mitochondrial regulation of hematopoietic stem cells.</a> Current Opinion in Cell Biology. 49, 91-98 (2017).</li><li>Wu, 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=Multiparameter+metabolic+analysis+reveals+a+close+link+between+attenuated+mitochondrial+bioenergetic+function+and+enhanced+glycolysis+dependency+in+human+tumor+cells.">Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.</a> American Journal of Physiology-Cell Physiology. 292, 125-136 (2007).</li><li>Chen, 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=Enrichment+of+hematopoietic+stem+cells+with+SLAM+and+LSK+markers+for+the+detection+of+hematopoietic+stem+cell+function+in+normal+and+Trp53+null+mice.">Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice.</a> Experimental Hematology. 36, 1236-1243 (2008).</li><li>Jung, 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=Hematopoietic+stem+cells+regulate+mesenchymal+stromal+cell+induction+into+osteoblasts+thereby+participating+in+the+formation+of+the+stem+cell+niche.">Hematopoietic stem cells regulate mesenchymal stromal cell induction into osteoblasts thereby participating in the formation of the stem cell niche.</a> Stem Cells. 26, 2042-2051 (2008).</li><li>Masuda, S., Li, M., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-less+maintenance+of+HSCs+by+2i.">Niche-less maintenance of HSCs by 2i.</a> Cell Research. 23, 458-459 (2013).</li><li>Anderson, 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=Hematopoietic+stem+cells+develop+in+the+absence+of+endothelial+cadherin+5+expression.">Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression.</a> Blood. 126, 2811-2820 (2015).</li><li>Lo Celso, C., Scadden, 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> Journal of Visualized Experiment. (157), (2007).</li><li>Van Wyngene, L., Vandewalle, J., Libert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+basic+metabolic+pathways+in+microbial+sepsis:+therapeutic+targets+at+last.">Reprogramming of basic metabolic pathways in microbial sepsis: therapeutic targets at last.</a> EMBO Molecular Medicine. 10, (2018).</li><li>Olson, K. A., Schell, J. C., Rutter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyruvate+and+Metabolic+Flexibility:+Illuminating+a+Path+Toward+Selective+Cancer+Therapies.">Pyruvate and Metabolic Flexibility: Illuminating a Path Toward Selective Cancer Therapies.</a> Trends in Biochemical Sciences. 41, 219-230 (2016).</li><li>Halestrap, A. P., Wilson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+monocarboxylate+transporter+family--role+and+regulation.">The monocarboxylate transporter family--role and regulation.</a> IUBMB Life. 64, 109-119 (2012).</li><li>Kim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+Cancer+Energy+Metabolism:+Culprits+or+Bystanders.">Mitochondria in Cancer Energy Metabolism: Culprits or Bystanders.</a> Toxicological Research. 31, 323-330 (2015).</li><li>Fosslien, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+medicine--molecular+pathology+of+defective+oxidative+phosphorylation.">Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation.</a> Annals of Clinical &amp; Laboratory Science. 31 (1), 25-67 (2001).</li><li>Wick, A. N., Nakada, H. I., Wolfe, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+primary+metabolic+block+produced+by+2-deoxyglucose.">Localization of the primary metabolic block produced by 2-deoxyglucose.</a> The Journal of Biological Chemistry. 224 (2), 963-969 (1957).</li><li>Linnett, P. E., Beechey, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+the+ATP+synthetase+systems.">Inhibitors of the ATP synthetase systems.</a> Methods in Enzymology. 55, 472-518 (1979).</li><li>Rimmele, P., 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=Mitochondrial+metabolism+in+hematopoietic+stem+cells+requires+functional+FOXO3.">Mitochondrial metabolism in hematopoietic stem cells requires functional FOXO3.</a> EMBO Reports. 16, 1164-1176 (2015).</li><li>Riviere, I., Dunbar, C. E., Sadelain, M. <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+engineering+at+a+crossroads.">Hematopoietic stem cell engineering at a crossroads.</a> Blood. 119, 1107-1116 (2012).</li><li>Ito, K., Suda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+requirements+for+the+maintenance+of+self-renewing+stem+cells.">Metabolic requirements for the maintenance of self-renewing stem cells.</a> Nature Reviews Molecular Cell Biology. 15 (4), 243-256 (2014).</li></ol>

Here, we demonstrate the isolation of a maximum amount of pure and viable murine LSK HSPCs population as well as the measurement of their glycolysis and mitochondrial respiration with an extracellular flux analyzer. Specifically, the protocol overcomes the following technical issues for the use of LSK HSPCs : i) the low frequency of LSK HSPCs in murine bone marrow14, ii) low basal metabolic activity of LSK HSPCs26, iii) the fragility of LSK HSPCs27, ...

Since the lifespan of most mature blood cells is short, the homeostasis of blood relies on the self-renewal and differentiation of a long-lived but rare population of hematopoietic stem cells (HSPCs)1. HSPCs are quiescent, but they are quick to proliferate and undergo differentiation upon stimulation to sustain demands of the blood system. As each HSPC cellular state requires a unique bioenergetic demand, the metabolic changes are key drivers of HSPC fate decisions. Therefore, the loss of metabolic plasticity, by altering the equilibrium between quiescence, self-renewal, and differentiation of HSPCs, often leads to myelo- or lympho-proliferative disorders. Together, the understanding of metabolic regulation of HSPC development is critical to uncover mechanisms underlying hematologic malignancies2,3,4,5.
Mitochondrial respiration and glycolysis generate ATP to drive intracellular reactions and produce the building blocks necessary for macromolecule synthesis. Since HSPCs have low mitochondrial mass compared to differentiated cells6 and they sustain quiescence in hypoxic bone marrow niches, HSPCs primarily rely on glycolysis. Activation of HSPCs enhances their mitochondrial metabolism that leads to the loss of quiescence and their subsequent entry into the cell cycle. Such metabolic plasticity of HSPCs allows the maintenance of the HSPC pool throughout adult life6,7,8,9,10,11,12. Therefore, it is critical to investigate their metabolic activities, such as the oxygen consumption rate (OCR; index of oxidative phosphorylation) and the extracellular acidification rate (ECAR; index of glycolysis) to analyze the HSPC activation and the health status. Both the OCR and the ECAR can be measured simultaneously, in real time, using an extracellular flux analyzer. However, the current method requires large numbers of cells and is optimized for adherent cells13. Since HSPCs cannot be isolated in large quantities from mice14, require sorting to obtain a pure population, are non-adherent cells15, and cannot be cultured overnight without avoiding differentiation16, it has been difficult to measure the OCR and the ECAR of HSPCs. Here, we provide a set of clear, step-by-step instructions to accompany video-based tutorials on how to measure metabolic respiration and glycolysis of few thousands murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs.

<table>
	<tbody>
		<tr>
			<td>0.01% (w/v) poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>Used for LSK attachment</td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Used for cell filtration after bone crushing</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-090-485</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse CD45R/B220 Clone RA3-6B2</td>
			<td>BD Biosciences</td>
			<td>553086</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse CD5 Clone 53-7.3</td>
			<td>BD Biosciences</td>
			<td>553019</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse CD8a Clone 53-6.7</td>
			<td>BD Biosciences</td>
			<td>553029</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse Ly-6G and Ly-6C Clone RB6-8C5</td>
			<td>BD Biosciences</td>
			<td>553125</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse TER-119/Erythroid Cells Clone TER-119</td>
			<td>BD Biosciences</td>
			<td>553672</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>CD117 (c-Kit) Monoclonal Antibody (2B8), APC</td>
			<td>eBioscience</td>
			<td>17-1171-83</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Falcon 15 ml Conical Centrifuge Tubes</td>
			<td>Falcon-Fischer Scientific</td>
			<td>14-959-53A</td>
			<td>Used in cell isolation</td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical Centrifuge Tubes</td>
			<td>Falcon-Fischer Scientific</td>
			<td>14-432-22</td>
			<td>Used in cell isolation</td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polypropylene Tubes</td>
			<td>Falcon-Fischer Scientific</td>
			<td>14-959-11A</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Neuromics</td>
			<td>FBS001-HI</td>
			<td>Used in FACS buffer</td>
		</tr>
		<tr>
			<td>Histopaque-1083</td>
			<td>Sigma</td>
			<td>10831</td>
			<td>Used for ficoll gradient separation</td>
		</tr>
		<tr>
			<td>L-glutamine 100x</td>
			<td>Fisher Scientific</td>
			<td>25-030-081</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Ly-6A/E (Sca-1) Monoclonal Antibody (D7), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-5981-82</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Murine Stem Cell Factor (SCF)</td>
			<td>PeproTech</td>
			<td>250-03-100UG</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>Murine Thrombopoietin (TPO)</td>
			<td>PeproTech</td>
			<td>315-14-100UG</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>PBS 1%</td>
			<td>Fisher Scientific</td>
			<td>SH3002802</td>
			<td>Used for FACS buffer</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Fisher Scientific</td>
			<td>15140122</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Fisher Scientific</td>
			<td>P1304MP</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Seahorse XFp Cell Culture Miniplate</td>
			<td>Agilent Technologies</td>
			<td>103025-100</td>
			<td>Used for LSK seeding</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>ThermoFisher</td>
			<td>11360070</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>Streptavidin eFluor 450 Conjugate</td>
			<td>eBioscience</td>
			<td>48-4317-82</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>XF Calibrant</td>
			<td>Agilent Technologies</td>
			<td>100840-000</td>
			<td>Used for cartridge equilibration</td>
		</tr>
		<tr>
			<td>XF media</td>
			<td>Agilent Technologies</td>
			<td>103575-100</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>XFp Glycolysis Stress Test Kit</td>
			<td>Agilent Technologies</td>
			<td>103017100</td>
			<td>Drugs for glycolysis stress test</td>
		</tr>
		<tr>
			<td>XFp Mitochondrial Stress Test Kit</td>
			<td>Agilent Technologies</td>
			<td>103010100</td>
			<td>Drugs for mitochondrial stress test</td>
		</tr>
		<tr>
			<td>XFp Sensor Cartridge</td>
			<td>Agilent Technologies</td>
			<td>103022-100</td>
			<td>Used for glycolysis and mitochondrial stress test</td>
		</tr>
	</tbody>
</table>

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.

Our extraction method allowed us to harvest up to ~80,000 LSK HSPCs per mouse. The viability and numbers of LSK cells were improved with our method, because we: (1) combined bone marrow from upper and lower limbs, hip bones, sternum, rib cage, and spine, (2) avoided using red cell lysis buffer that would have increased cell-death and clumping, (3) used the density gradient medium separation of mono-nucleated cells, and (4) avoided using pre-chilled buffer that would have caused the loss of cells-of-interest in clumps.</p...

Watch this Scientific Journal Video about Analysis of Hematopoietic Stem Progenitor Cell Metabolism at JoVE.com

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

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.

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

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