Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Summary

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.

Abstract

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-Lineage^negSca1⁺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.

Introduction

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-proliferativ....

Protocol

This protocol was approved by Nationwide Children’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

1. Hydrate the sensor cartridge.
   1. Incubate 5 mL of the calibrant (Table of Materials) in a non-CO₂ 37 °C incubator overnight.
   2. Open the flux assay kit (Table of Materials) and separate the sens....

Results

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.

Discussion

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, .......

Materials

List of materials used in this article

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