Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

Podsumowanie

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Streszczenie

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Wprowadzenie

Hematopoietic stem cells (HSCs) are a small population of cells residing in the bone marrow ensuring blood production and homeostasis throughout an organism's lifetime. HSCs mediate this process by giving rise to progenitors that in turn produce terminally differentiated mature blood cell lineages via several rounds of cell division and well-orchestrated differentiation steps¹. Importantly, HSCs produce their energy via anaerobic glycolysis. In contrast, more committed and active hematopoietic progenitors switch their metabolism toward mitochondrial metabolism^2,3,4. This distinct metabolic state is believed to protect the HSCs from cellular damage inflicted by reactive oxygen species (ROS) produced by active mitochondria, thereby maintaining their long-term in vivo function^5,6,7,8. Direct measurement of the HSC metabolic state is challenging and often low throughput due to their limited numbers. Here, we describe a flow cytometry-based assay for robust measurement of mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester (TMRM) fluorescence, and mitochondrial mass using a green fluorescent mitochondrial stain (Mitotracker Green) in HSCs. We have previously demonstrated that low ΔΨm is a bona-fide functional marker of highly purified HSCs⁹ and metabolic modulators capable of lowering ΔΨm enhance HSCs function^9,10. Here we propose use of our method on HSCs mitochondrial profiling as strategy to identify novel molecules capable of improving the HSCs' long-term blood reconstitution potential.

As an example, we demonstrate that this assay reliably measures the lowering of HSC ΔΨm upon exposure to vitamin B3 analog nicotinamide riboside (NR). Accordingly, in our recently published study we demonstrate that NR strongly ameliorates blood recovery posttransplant in both mouse and humanized mouse systems by directly improving hematopoietic stem and progenitor functions¹⁰. The capacity of such metabolic modulators is of great clinical value considering that a 25% death rate is linked to delay in blood and immune recovery in posttransplanted patients^11,12.

Moreover, we provide evidence that this methodology can be applied for the characterization of the metabolic profile and function of human T cells. In recent years, the development of adoptive cell therapy (ACT) using autologous tumor infiltrating lymphocytes (TILs) has become the most effective approach for certain types of advanced cancer with extremely unfavorable prognosis (e.g., metastatic melanoma, where >50% of patients respond to treatment and up to 24% of patients have complete regression)¹³. However, TILs harboring sufficient antitumor activity are difficult to generate¹⁴. The extensive proliferation and stimulation that TILs undergo during ex vivo expansion cause T cell exhaustion and senescence that dramatically impair T cell antitumor response¹⁵. Importantly, the TILs' antitumoral capacity is tightly linked to their metabolism^16,17 and approaches aimed to modulate metabolism through the inhibition of the PI3K/Akt pathway have produced encouraging results^18,19. For this reason, we compare the ΔΨm of T cells derived from peripheral blood mononuclear cells (PBMCs) and patient-derived TILs, and show that less differentiated PBMC-derived T cells have lower ΔΨm and mitochondrial mass as compared to terminally differentiated TILs.

We envision that this assay can be used to identify novel metabolic modulators that improve HSC and T cell function via the modulation of ΔΨm.

Protokół

All experiments described in the manuscript follow the guidelines of our institution and were carried out in accordance with Swiss law for animal experimentation (Authorization: VD3194) and for research involving human samples (Protocol: 235/14; CER-VD 2017-00490)

1. Hematopoietic Stem Cell Extraction

1. Purchase wild type C57BL6/J mice and keep them in the animal house for at least a week to reduce transport-associated stress.
2. On the day of the experiment, euthanize the mouse using CO₂ asphyxiation.
3. Spray the mouse with 70% ethanol to sterilize the fur and cut open the mouse at the belly using standard surgical tools, such as dissection scissors and forceps, to cut the femur and tibia bones from the hind legs.
4. Remove the muscles attached to the femur, tibia, and the pelvis using a soft paper towel and place the cleaned bones in a 50 mL tube containing PBS with 1 mM EDTA (buffer) on ice.
5. Spray a mortar and pestle with 70% ethanol and place it in a cell culture hood. Sterilize it with UV for 30 min. Post sterilization rinse the mortar and pestle with buffer to remove traces of ethanol.
6. Put the clean bones with some buffer (~10 mL) in the mortar and gently crush them to get the bone marrow out in suspension. Now, collect the cell suspension and pass it through a 70 µm cell strainer into a 50 mL tube to get a single cell suspension.
7. Repeat step 1.6 until all the bone marrow has been extracted and the bone debris has turned white.
8. Place the 50 mL tube(s) containing the bone marrow single cell suspension on a centrifuge. Run the centrifuge at 300 x g for 10 min at 4 °C to pellet the cells.
9. Meanwhile, prepare 10 mL of 1x RBC lysis buffer in autoclaved distilled water. Filter the solution through a 0.22 µm filter.
10. Collect the sample tube from the centrifuge and decant the supernatant. Pipette the 1x RBC lysis buffer (Table of Materials) on the cell pellet. Dislodge the pellet and prepare a homogenous solution by pipetting up and down a few times. Allow the tube to be at room temperature for 1–2 min for the RBC lysis to occur. Stop the lysis process by filling up the tube with the buffer.
11. Place the tube on a centrifuge and spin at 300 x g for 5 min at 4 °C. Collect the tube from the centrifuge and decant the supernatant. Resuspend the pellet by adding 10 mL of buffer and filter the solution into a new 50 mL tube via a 70 µm cell strainer to remove the debris due to RBC lysis.
12. Centrifuge the tube at 300 x g for 5 min at 4 °C. Collect the tube from the centrifuge and decant the supernatant. Resuspend the pellet in 500 µL of buffer.
13. Remove a 100 µL aliquot and keep in a separate 1.5 mL tube. Add 50 µL of biotin lineage depletion antibody cocktail from the progenitor enrichment kit (Table of Materials) to the remaining 450 µL of cell suspension. Incubate at 4 °C on a shaker for 15 min.
14. Add 15 mL of buffer and centrifuge the tube at 300 x g for 5 min at 4 °C. Collect the tube from the centrifuge and decant the supernatant. Resuspend the pellet in 460 µL of buffer. Remove a 10 µL aliquot and keep in a separate 1.5 mL tube.
15. Add 50 µL of streptavidin magnetic beads from the progenitor enrichment kit (Table of Materials), to the remaining 450 µL cell suspension. Incubate at 4 °C on a shaker for 15 min.
16. Add 15 mL of buffer and centrifuge the tube at 300 x g for 5 min at 4 °C. Collect the tube from the centrifuge and decant the supernatant. Resuspend the pellet in 5 mL of buffer and transfer the solution to a 15 mL tube.
17. Take the tube to an automated cell separator (Table of Materials). Run a wash program to rinse and prime the tubing of the cell separator. Place the sample and two collection tubes on the tube holder. Perform separation using the "Deplete" program. Collect the positive and the negative fractions from the automated cell separator once the run has ended.
    NOTE: In the absence of an automatic cell separator the users can use manual magnetic columns and corresponding magnets, per the user manual. The users should keep in mind that the process of manual separation is slower than the automated one. Also, the manual columns are more prone to clogging. Therefore, users are advised to dilute the sample and load on the column slowly.
18. Discard the positive fraction. Fill the negative fraction tube with buffer. Centrifuge the tube at 300 x g for 5 min at 4 °C.
19. Meanwhile, prepare the antibody mix in 1 mL of final volume solution and the single-color controls in 200 µL of final volume solution as described in Table of Materials and Table 1.
    NOTE: If the TMRM and the green fluorescent mitochondrial stain are to be combined with stem cell marker staining, then replace CD150-PE with CD150-PEcy5 and Streptavidin-Tx red with Streptavidin-Pac Orange.
20. Collect the sample tube from the centrifuge and decant the supernatant. Resuspend the pellet in 1 mL of antibody mix. Add 10 µL of cells (from step 1.13) in each of the single-color control tubes (except lineage). Add 10 µL of cells (from step 1.14) in the lineage single color tube.
21. Incubate the sample and single-color control tubes at 4 °C on a shaker for 45 min. Cover the ice bucket with a lid or aluminum foil.
22. Fill all tubes with buffer and centrifuge at 300 x g for 5 min at 4 °C. Discard the supernatant and resuspend the sample in 1 mL of buffer and single-color controls in 200 µL of buffer.
23. Transfer the sample and the single-color controls to 5 mL filter top FACS tubes.
24. Take the tubes to the sorting machine and sort the HSC population (gating strategy in Figure 1A) in 1.5 mL tubes containing 400 µL of stem cell expansion medium.

2. Ex Vivo Culture of Hematopoietic Stem Cells

1. Collect tubes containing sorted cells (see section 1 for cell extraction). Centrifuge the tubes at 300 x g for 5 min at 4 °C. Gently remove most of the supernatant without dislodging the pellet and leave 50–80 µL on top of the cell pellet. This minimizes cell loss.
2. Resuspend the cell pellet in stem cell expansion medium to a final volume dependent on the number of conditions to be tested (count for 100 µL per well/condition of culture).
3. Prepare a 2x culture medium containing stem cell expansion medium, stem cell factor (200 ng/mL), FLT3 ligand (4 ng/mL) and pen-strep antibiotics (1%) (2x basal medium; Table of Materials).
4. Take a sterile tissue culture treated 96 U-bottom well plate (Table of Materials) and identify the wells where the cells will be cultured (plate design in Figure 1B).
   NOTE: Users are advised to avoid marginal wells, as they are more susceptible to evaporation.
5. Put 100 µL of 2x basal medium previously prepared in step 2.3 in these wells. In the NR marked well add 2 µL of a 100x NR solution (Table of Materials). Replenish NR every 24 h.
   NOTE: Replenishment is specific to NR. Other metabolic modulators may or may not need replenishment.
6. Seed 100 µL of cells prepared in step 2.2 on top of the wells containing 2x basal medium. In this experiment the number of HSCs seeded per well were between 800–1,000 cells.
7. Prepare five extra wells containing 2x basal medium. In each of these add 100,000 whole bone marrow cells (from step 1.13) resuspended in 100 µL of stem cell expansion medium to be used as staining controls for post culture flow cytometry analysis.
   NOTE: If users want to combine stem cell markers and mitochondrial markers for post culture analysis it is recommended to additionally sort the progenitor population (either Ckit+ cells or LKSCD150- cells). Seed these sorted progenitors in the staining control wells for post culture flow cytometry analysis. Prepare one well per single stain color.
8. Put 200 µL of autoclaved water in all surrounding wells to reduce evaporation from wells containing cells. Leave the plate undisturbed in an incubator at 37 °C and 5% CO₂ for the duration of the culture period (72 h). Remove the plate to replenish NR every 24 h and place it back in the incubator.

3. Measurement of Mitochondrial Mass and Membrane Potential

1. At the end of the culture period prepare a 100x solution of TMRM (20 µM) and Mitotracker green (10 µM) (green fluorescent stain) in stem cell expansion medium (Table of Materials).
2. Add 2 µL of 100x TMRM solution and 2 µL of 100x green fluorescent stain solution in each of the test wells. Add 2 µL of 100x TMRM in the TMRM control well. Add 2 µL of 100x green fluorescent stain in the Mitotracker control well. Place the plate back in an incubator at 37 °C and 5% CO₂ for 45 min. Cover the top of the plate with aluminum foil.
   NOTE: An additional control with Verapamil (ABC pump inhibitor) can be prepared if ABC pump mediated dye efflux needs to be tested. For this, add 50 µM Verapamil in one of the test wells 1 h before staining for TMRM and green fluorescent stain.
3. Remove the plate from the incubator and centrifuge it at 300 x g for 5 min. Invert the plate to remove the supernatant. Add 200 µL of standard FACS buffer (PBS-1 mM EDTA-P/S-2% FBS), centrifuge the plate at 300 x g for 5 min. Remove the supernatant. Repeat this washing step 3x. The users must ensure that the plate is always covered with foil, to provide minimal exposure to direct light.
   NOTE: If users need to combine the mitochondrial staining with stem cell staining, the sample will need to be incubated with an antibody mix of all stem cell markers and the single-color controls will need to be stained with individual antibodies separately at 4 °C for 30–45 min.
   NOTE: At all steps users must keep the plate covered with aluminum foil. Users must note that this additional staining step and subsequent washing steps can result in additional cell loss.
4. Resuspend the cells in 200 µL of FACS buffer and transfer to FACS tubes.
5. Run the samples on the flow cytometer (see Figure 1). Single color tubes containing WBM include: (1) Unstained; (2) DAPI; (3) TMRM (PE); (4) green fluorescent stain (FITC); (5) Full stain (PE and FITC).
6. First acquire the single-color controls to set up the machine. Use the running software on the machine to calculate the compensation. Once compensation has been applied, acquire the HSC sample and record as many events as possible.
   NOTE: If the stem cell and mitochondrial markers are combined, the users need to be particularly careful with compensation between TMRM (PE), CD150 (PE-Cy5), and Sca-1 (APC). Also, the samples should be run immediately post staining.
7. Export the FACS files from the cytometer and analyze the data on an analysis software (Table of Materials).
   1. For the analysis, open the file on the analysis software. Using FSC-A and SSC-A gating identify the cell population. Identify singlets in the next gates before plotting the DAPI negative fraction (live cells). In the live cell gate make a contour plot in the TMRM and green fluorescent stain channel to measure ΔΨm and mass, respectively (Figure 2A). Export the mean fluorescence intensity (MFI) of these two channels in the live cell gate.
   2. The TMRM low gate is set based on the shoulder population in the TMRM channel. The TMRM single-color control can be used to identify this shoulder population to set the gate. Export the proportion of live of cells in the TMRM low gate in your control and test samples for plotting.

Wyniki

In Figure 1 we show the gating strategy for the isolation of hematopoietic stem cells from the mouse bone marrow and the layout of the plate for their ex vivo culture. Figure 1A shows the identification of the lymphocyte fraction in the SSC-A/FSC-A plot. Doublets were removed in the singlet gate followed by identification of live cells by the absence of DAPI signal. The LKS population, defined by lineage- Sca1⁺cKit⁺, was id...

Dyskusje

A tight regulation of HSC function is important to maintain stable hematopoiesis during an organism's lifetime. Like various other cell types in the body, a key component that contributes to the regulation of HSC function is cellular metabolism. Previous studies from our lab⁹ and others^2,3 have implicated the importance of mitochondria in maintaining a distinct metabolic state in HSCs. Due to the extremely low number of HSCs isolated f...

Materiały

Name	Company	Catalog Number	Comments
5 mL FACS tubes	Falcon	352235	Sample preparation
96-U bottom plate	Corning	3799	Cell culture
AutoMACS pro separator	Miltenyi Biotec		Automatic Cell separation
BD FACS AriaIII	Becton and Dickinson		Cell sorting
BD IMag mouse hematopoietic progenitor cell enrichment kit	BD	558451	Lineage depletion
BD LSRII	Becton and Dickinson		FACS acquisition machine
BD-DIVA	Becton and Dickinson		Acquisition software
CD150 PE	Biolegend	115904	Antibody staining mix
CD150 PE-Cy5	Biolegend	115912	Antibody staining mix
CD48 PB	Biolegend	103418	Antibody staining mix
Centrifuge- 5810R	Eppendorf		Centrifugation
Ckit PeCy7	Biolegend	105814	Antibody staining mix
Flow jo	FlowJo LLC		FACS Analysis software
GraphPad-Prism	GraphPad		Plotting data into graphs
Mitotracker Green	Invitrogen	M7514	Green-fluorescent mitocondrial stain to measure mitochondrial mass; working concentration = 100 nM; stock concentration = 1 mM
Nicotinamide Riboside (NR)	Custom synthesized in house		Metabolic modulator; working concentration = 500 µM; stock concentration = 50 mM
PBS	CHUV	1000324	Buffer preparation; working concentration = 1x; stock concentration = 1x
Pen-Strep (P/S)	Life technologies	15140122	Ex vivo culture; working concentration = 1x; stock concentration = 1x
RBC Lysis buffer	Biolegend	420301	Lysing Red blood cells; working concentration = 1x; stock concentration = 10x
Recombinant Mouse Flt-3 Ligand (FLT3)	RnD	427-FL-005/CF	Ex vivo culture; working concentration = 2 ng/mL; stock concentration = 10 µg/mL
Recombinant mouse stem cell factor (SCF)	RnD	455-MC-010/CF	Ex vivo culture; working concentration = 100 ng/mL; stock concentration = 50 µg/mL
Sca1 APC	Thermo Fisher Scientific	17-5981-82	Antibody staining mix
StemlineII Hematopoietic Stem Cell Expansion Medium	SIGMA	S0192	Ex vivo culture
Streptavidin Pac orange	Life Technologies	S32365	Antibody staining mix
Streptavidin Tx red	Life Technologies	S872	Antibody staining mix
TMRM	Invitrogen	T668	Staining mitochondrial membrane potential; working concentration = 200 nM; stock concentration = 10 mM
Ultra pure EDTA	Invitrogen	15575-038	Buffer preparation; working concentration = 0.5 M; stock concentration = 1 mM