Targeted Metabolomics on Rare Primary Cells

The main goal of my lab is to maintain a healthy metabolomic stem cell pull up in aging to avoid hematological malignancies, and we do so by performing dietary interventions. So, in particular, we're interested to understand how dietary-derived metabolites regulate stemness, and also intracellular metabolites, how they regulate the stem cell fate. Hemato-based stem cells is a very rare sub population, meaning that we always face the challenge that we have not enough material to perform our experiments.

But now with the establishment of single-cell methods and also with low input methods, we have achieved a new knowledge on how these stem cells are regulated. Hematopoietic stem cells is a very rare sub population and on top they're very tiny. Now, with the methods that we have developed such, for example, low input metabolomics based on mass spec, we now can detect with very low number of cells, hundreds of metabolites.

This now allowed us to understand what is the role of certain metabolites for stem cell regulation. In the past, metabolomics analysis of rare primary cells required many mice and days of work and sample preparation. With our new protocol we need fewer mice and less time.

Additionally, we have reduced the number of manual steps, so the whole protocol becomes more robust. For the future we have planned to adapt our low input protocol to enable additional analysis in rare primary cells, for example, non-targeted metabolomics and lipidomics. To begin, place a 70 micron cell strainer on top of a 50 milliliter centrifuge tube and wet it with PBS.

After isolating the spleen from a mouse to generate a single-cell suspension, pass the spleen through the mesh using the thumb rests of a syringe plunger and 30 milliliters of cold PBS. Centrifuge the filtrate at 300G for five minutes, and remove the supernatant. Re-suspend the pellet in one milliliter of ammonium-chloride-potassium lysis buffer, and incubate for two minutes at room temperature.

After lysis, add 50 milliliters of cold PBS into the tube. Then centrifuge and discard the supernatant before re-suspending the pellet in the freshly prepared antibody cocktail. Transfer the cell suspension to a five milliliter fax tube and incubate for 30 minutes at four degrees Celsius in the dark.

After incubation, add three milliliters of cold PBS into the tube, then centrifuge and discard the supernatant before re-suspending the pellet in one milliliter of cold fax re-suspension buffer. Filter the cells through a filter cap fax tube. To begin, add 30 grams of sodium chloride in six liters of deionized water in a sheath fluid tank, and shake the tank to dissolve the salt.

Then connect the tank to a cell sorter. Turn on the cell sorter. Start the fluidics system and wait for the fluidics to equilibrate.

Set up drop delay. To prepare yeast extract stock solution, re-suspend one aliquot of carbon 13 yeast extract in 7.5 milliliters of ultrapure water and 2.5 milliliters of methanol. Then to prepare the extraction buffer, add 10 microliters of carbon 13 yeast extract stock solution to 10 milliliters of acetonitrile and mix it well.

Add 25 microliters of the extraction buffer to each well of a 96-well PCR plate and cover the wells with a lid. Place the plate on the cell sorter to collect the sorted cells. Sort 5, 000 events each of Alexa Fluor 647 positive and PE negative populations into the first four wells.

Then sort 5, 000 events each of the PE positive and Alexa Fluor 647 negative populations into the next four wells. Next, for hematopoietic stem cells, sort 5, 000 events of the PE-Cy7 low PE positive, APC-Cy7, Brilliant Violet 605 positive, and Brilliant Violet 421 negative population into the first well, followed by sorting 5, 000 events of the PE-Cy7 low, PE positive, APC-Cy7 positive, Brilliant Violet 421 positive, and Brilliant Violet 605 negative population into the next well. For debris samples, select events too small to represent intact cells based on forward and sideward scatter signals.

Next, turn on the LC-MS instrument, place the sample plate in the auto sampler of the instrument. Open the LC-MS compatible software and prepare the instrument for analysis. Run at least four process blank or pool samples to equilibrate the column and an LC test mix to check chromatographic performance.

Confirm that the initial and maximum back pressure is less than 150 and 400 bar respectively. Then ensure that retention times fall within a one minute window. Next, verify that the valley between leucine and isoleucine in the positive 132 to 86 transition is less than 20%of the peak height.

Finally, ensure that the difference in retention time AMP to ADP is 1.5 to 1.9 minutes, and ADP to ATP is 1.1 to 1.5 minutes. After confirming experimental parameters, set up a run with a process blank or a pool sample to minimize carryover from the LC test mix, followed by the test samples in randomized order. Fax sorting enables the isolation of pure populations of different cell types from the same cell suspension.

Metabolic differences among cell types from the same tissue were preserved using fax cell CMS protocol. For hematopoietic stem and multipotent progenitor cells, cells from six mice were required to generate six samples, leading to a larger variability within each group compared to B and T cells, which were sorted from the same mirroring spleen. Debris samples representing process blank control samples were clearly separated from samples containing cell extracts.

A heat map showing information on relative levels of metabolites across different cell types is shown here.