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) in a non-CO2 37 &#176;C incubator overnight.</li>
		<li>Open the flux assay kit (Table of Materials) and separate the sensor cartridge (green; top) from the utility plate (transparent; bottom). Next, place the sensor cartridge upside down and adjacent to the utility plate.</li>
		<li>Using sterile water, fill the wells of the utility plate (200 &#181;L) and the chambers around the wells (400 &#181;L).</li>
		<li>Submerge the sensor cartridge into the utility plate making sure that the sensors are completely covered by water. Tap 3x to avoid the formation of bubbles.</li>
		<li>Place the cartridge submerged in the utility plate in a non-CO2 37 &#176;C incubator overnight. To prevent evaporation of the water, make sure that the incubator is properly humidified. Place an open beaker containing H2O as an extra precaution next to the cartridge-utility plate, particularly if using a regular oven.</li>
	</ol>
	</li>
	<li>Prepare the assay plate.
	<ol>
		<li>Sterilize the surface of biosafety cabinet class 2 using 70% ethanol. Open the assay plate under the hood.</li>
		<li>Add 40 &#181;L of commercially available 0.01% (w/v) poly-L-lysine (PLL) solution to each well of the assay plate under the hood.</li>
		<li>Cover the assay plate with the lid provided in the kit. Incubate the closed assay plate at room temperature (RT) under the hood for 1 h. 
		NOTE: The goal of the incubation is to let PLL coat the surface of the assay plate to facilitate adhesion of suspension cells to the surface of the assay plate.</li>
		<li>After 1 h incubation of the assay plate, remove the excess solution with a sterile vacuum-based aspirator and air dry the well under the hood. 
		NOTE: It takes ~30&#8722;60 min to air-dry the wells of the assay plate following the excessive PLL removal using the aspirator.</li>
	</ol>
	</li>
</ol>
2. Day of the Assay
<ol>
	<li>Prepare the cartridge.
	<ol>
		<li>Lift the sensor cartridge. Place it upside down in the tissue culture hood and discard the water from the utility plate.</li>
		<li>Fill the utility plate wells with 200 &#956;L of the pre-warmed calibrant.</li>
		<li>Fill the chambers around the wells with 400 &#956;L of the calibrant.</li>
		<li>Submerge the sensor cartridge into the utility plate, making sure that the sensors are completely covered by the calibrant. Tap 3x to avoid the formation of bubbles.</li>
		<li>Equilibrate the sensor cartridge submerged in the utility plate in a non-CO2 37 &#176;C incubator for 45&#8722;60 min.</li>
	</ol>
	</li>
	<li>Harvest murine bone marrow-derived LSK HSPCs.
	<ol>
		<li>To accommodate sufficient biological replicates, plan to use HSPCs derived from one mouse per well of the assay plate. 
		NOTE: This bone marrow-harvesting method provides ~50,000&#8722;80,000 LSK HSPCs from each mouse. This protocol to measure extracellular flux is optimized for ~70,000 LSK HSPCs per well of a 96 well-plate.</li>
		<li>Euthanize mice using CO2 overdose and cervical dislocation, following local IACUC approved methods.</li>
		<li>Sterilize the surface of dissecting tools and the bench using 70% ethanol.</li>
		<li>For each mouse, pre-fill one Petri dish with 1x phosphate-buffered saline (PBS) containing 2% heat inactivated fetal bovine serum (FBS) at RT. 
		NOTE: Do not use pre-chilled PBS as it will create clumps in the following steps.</li>
		<li>Spray 70% ethanol (v/v) on the entire euthanized mouse. Isolate all bones, including upper and lower limbs, hip bones, sternum, rib cage, and spine, from the mouse17,18. Pull out white matter from the spinal cord of the mouse as it can contaminate LSK cells. Place all bones in 1x PBS (+ 2% FBS), as they are being collected, in a Petri dish until further use.</li>
		<li>Invert a 50 mL conical tube (new each time) and use it to triturate bones submerged in 1x PBS (+ 2% FBS) in the Petri dish.</li>
		<li>Using a 10 mL serological pipette, pipette up and down (~10x) to uniformly flush out cells from bones, after crushing bones.</li>
		<li>Place a 40 &#181;m cell strainer on a 50 mL conical tube. Pre-wet the surface of the strainer by passing 1 mL of 1x PBS (+ 2% FBS).</li>
		<li>Harvest bone marrow-derived cell suspension from step 2.2.7 and pass it through the pre-wet surface of the cell strainer to remove bone debris.</li>
		<li>Repeat steps 2.2.6 through 2.2.9 until all bone materials turn white as a marker that most of bone marrow cells are collected in the 50 mL conical tube. 
		NOTE: It often takes two 50 mL conical tubes per mouse to collect all bone marrow-derived cells.</li>
		<li>Centrifuge 50 mL conical tubes containing bone marrow-derived cells for 5 min at 500 x g and RT.</li>
		<li>Remove the supernatant. Resuspend bone marrow-derived cells (combine contents of both 50 mL tubes) in 5 mL of 1x PBS (+ 2% FBS) as a final volume and keep cells at RT.</li>
	</ol>
	</li>
	<li>Harvest mononucleated murine bone marrow cells.
	<ol>
		<li>Add 5 mL of density gradient medium (i.e., Ficoll) to a 15 mL conical tube. Then slowly add 5 mL of the bone marrow cell suspension. Make sure that cells remain as a layer above the density gradient medium.</li>
		<li>Centrifuge for 30 min at 500 x g and RT. Do not use a brake in the centrifuge. Make sure the centrifuge is at the lowest possible acceleration (e.g., 1 acceleration and 0 deceleration).</li>
		<li>Harvest the middle interface of mononucleated cells (white color) following centrifugation into a fresh 15 mL conical tube.</li>
		<li>Wash cells, harvested from density gradient medium, with 5 mL of 1x PBS (+ 2% FBS). Centrifuge for 5 min at 500 x g and 4 &#176;C. Remove the supernatant.</li>
		<li>Repeat step 2.3.4.</li>
		<li>Resuspend cell contents of the tube in 300 &#181;L of 1x PBS (+ 2% FBS). Aliquot 10 &#181;L of cell suspension for unstained or single-color control in a FACS tube.</li>
	</ol>
	</li>
	<li>Harvest LSK HSPCs from mononucleated murine bone marrow cells.
	<ol>
		<li>Make a cocktail of biotin-antibodies by mixing 3 &#181;L per sample of the following antibodies: Gr1, Cd8a, Cd5, B220, Ter119. Add 15 &#181;L of the biotin-antibody cocktail to 300 &#181;L of mononucleated bone marrow cells. 
		NOTE: Each antibody is used at 1:100 dilution.</li>
		<li>Incubate cells with the biotin-antibody cocktail for 30 min at 4 &#176;C with agitation to avoid cells clumping in the bottom of the tube.</li>
		<li>Add 10 mL of pre-chilled 1x PBS (+ 2% FBS) to cells mixed with the biotin-antibody cocktail.</li>
		<li>Centrifuge the tube for 5 min at 500 x g and 4 &#176;C. Discard the supernatant and resuspend the cell pellet in 400 &#181;L of 1x PBS (+ 2% FBS). Aliquot 10 &#181;L for streptavidin-single color control.</li>
		<li>Briefly vortex anti-biotin microbeads (Table of Materials) before use. Add 80 &#181;L of microbeads to each cell sample (of 400 &#181;L). Mix well and incubate for additional 20 min at 4 &#176;C, with agitation.</li>
		<li>Add 10 mL of pre-chilled 1x PBS (+ 2% FBS) to cells. Centrifuge the tube for 5 min at 500 x g and 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cell pellet in 1 mL of 1x PBS (+ 2% FBS). Store at 4 &#176;C while setting up magnetic separation unit.</li>
		<li>Place a column (Table of Materials) in the magnetic field of the magnetic assisted cell sorting (MACS) separator at 4 &#176;C. Prepare the column for magnetic separation by rinsing it with 3 mL of 1x PBS (+ 2% FBS) under the gravity flow at 4 &#176;C.</li>
		<li>Add the cell suspension from step 2.4.7 to the pre-wet column at 4 &#176;C. Allow the cells to pass through the column at 4 &#176;C and collect effluent in a 15 mL conical tube. 
		NOTE: The fraction with unlabeled cells in such effluent represents the enriched lineage negative cells.</li>
		<li>Wash column with 3 mL of 1x PBS (+ 2% FBS) at 4 &#176;C. Repeat 3x. Collect the flow-through and keep it at 4 &#176;C. Count the eluted viable cells by trypan blue exclusion using a hemocytometer.</li>
		<li>Centrifuge the 15 mL conical tube containing the flow-through for 5 min at 500 x g and 4 &#176;C. Discard the supernatant. Resuspend cells in 0.5 mL of 1x PBS (+ 2% FBS) and transfer the contents to a FACS tube.</li>
		<li>Add 24 &#181;L of the LSK antibody cocktail to each 107 cells. The antibody cocktail contains equal concentration of 450-streptavidin antibody, PE-CY7-Sca1 antibody, and APC-c-Kit antibody.</li>
		<li>Incubate for 1 h at 4 &#176;C with agitation under dark (covered with tin foil).</li>
		<li>Add 3 mL of 1x PBS (+ 2% FBS) to the FACS tube. Centrifuge for 5 min at 500 x g and 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend antibody-labelled cells in 1 mL of 1x PBS (+ 2% FBS). Add 1 &#181;L of 1 mg/mL propidium iodide to cell suspension just before sorting.</li>
		<li>Filter contents of the FACS tube using a 40 &#181;m strainer right before sorting LSK cells.</li>
		<li>Collect LSK cells, via FACS sorting, into 1.5 mL tube containing 0.5 mL of complete media supplemented with 2 mM glutamine, 3 mg/mL glucose, 1 mM pyruvate, 1x thrombopoietin (TPO), 1x stem cell factor (SCF), 0.5x penicillin/streptomycin (P/S), pH 7.4 (Table 1).</li>
	</ol>
	</li>
</ol>
3. Mitochondrial Respiration and Glycolysis Assays of LSK HSPCs
<ol>
	<li>LSK seeding in the assay plate
	<ol>
		<li>Centrifuge LSK cells from step 2.4.17 for 5 min at 500 x g and RT. Discard the supernatant.</li>
		<li>Resuspend cells in complete media to a final concentration of at least 70,000 cells/40 &#181;L.</li>
		<li>Seed contents of 40 &#181;L media (containing 70,000 cells) in the PLL-coated 8-well plate from step 1.2.4. Leave all corner wells empty.</li>
		<li>Centrifuge for 1 min at 450 x g and RT. Do not apply the brake. Make sure that cells are attached to the well bottom using the inverted microscope.</li>
		<li>Add 135 &#181;L of complete media to the cells in each well for a final volume of 175 &#181;L. Add 175 &#181;L of complete media to the 2 corners of the plate as blanks.</li>
		<li>Incubate the cells in the non-CO2 incubator for 2 h at 37 &#176;C.</li>
		<li>Set up a program to add drugs to each well of the well plate in the analyzer using a metric described in Table 2 (mitochondrial stress test) and Table 3 (glycolysis stress test). 
		NOTE: While cells are incubating, turn on the instrument and make sure it is at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Mitochondrial stress test
	<ol>
		<li>Prepare 45 &#181;M stock solutions of oligomycin, 50 &#181;M carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP) and 25 &#181;M stock solutions of rotenone/antimycin A. To prepare 45 &#181;M oligomycin stock solution, dissolve the contents of the commercially available pouch in 280 &#181;L of the complete media. To prepare 50 &#181;M FCCP stock solution, dissolve the contents of the commercially available pouch in 288 &#181;L of the complete media. To prepare 25 &#181;M rotenone/antimycin A stock solution, dissolve the contents of the commercially available pouch in 216 &#181;L of the complete media.</li>
		<li>Take the sensor cartridge in the utility plate out from the incubator and load its ports (A, B, and C) such that each well would have final concentration of 2 &#181;M oligomycin, 1.5 &#181;M FCCP, and 0.5 &#181;M of rotenone/antimycin A as needed, following dilution metrics described in Table 4 (for oxygen consumption rate).</li>
		<li>Remove the lid from the sensor cartridge assembled in the utility plate and place it on the instrument tray. Start the calibration that will take 20 min.</li>
		<li>After the calibration, remove the utility plate and substitute it with assay plate containing LSK cells, which are now adhered to the bottom of the well.</li>
		<li>Press Continue to start the program described in Table 2. After the completion of the program, retrieve the data and analyze them using the Wave Desktop software. 
		NOTE: The data generated from the extracellular flux assays can be plotted using the dot plot from the Wave Desktop software or exported to web-based statistics program.</li>
	</ol>
	</li>
	<li>Glycolysis stress test
	<ol>
		<li>Reconstitute glucose in 300 &#181;L (100 mM), oligomycin in 288 &#181;L (50 &#181;M), 2-D glucose (2-DG) in 300 &#181;L (500 mM) of complete media from the commercially available pouch.</li>
		<li>Gently pipette up and down (~10x) to solubilize the compounds. Vortex the 2-DG for approximately 1 min to ensure proper dissolution into media.</li>
		<li>Remove the sensor cartridge out from the incubator. Load ports A through C following dilution metrics described in the Table 5 to obtain a final concentration of 10 mM glucose, 2 &#181;M oligomycin, and 50 mM 2-DG.</li>
		<li>Repeat steps 3.2.3 and 3.2.4.</li>
		<li>Press Continue to start the program described in Table 3. After the completion of the program, retrieve the data and analyze them using the Wave Desktop software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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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6.7K Views.  Nationwide Children's Hospital. 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.

Video: Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

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

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...