Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones

- Hematopoietic stem and progenitor cell or HSPC, can self-renew and differentiate into various types of blood cells. HSPCs gradually accumulate DNA mutations during a lifespan, which can contribute to leukemia. To analyze such mutations, HSPCs are generated usually from the bone marrow.

Begin by adding fluorophore-conjugated antibodies to mononuclear cells-- adult stem cells isolated from the bone marrow-- and incubating them for the desired duration. The antibodies bind to specific cell surface markers such as CD34. Now, load the mixture in the fluorescence-activated cell cytometry or FACS machine.

The fluorophore-conjugated antibodies fluoresce when hit by the laser. Individual cell droplets pass through the detector that measures the fluorescence intensity, based on which the drop possesses an electric charge. In the presence of electromagnetic fields, positively charged cells move to the negative container and vice versa.

Now, grow the FACS-sorted CD34-positive HSPCs in an appropriate medium for the desired duration until clones are visible. Next, dislodge the clones by vigorous pipetting and centrifuge to pellet the cells. Store the pellet containing clonal HSPCs at minus 20 degrees Celsius for further analysis. In the following protocol, we will generate HSPC clones from the mononuclear cells.

- Begin this procedure with preparation of sample material and staining as described in the text protocol. Then, prepare 25 milliliters of hematopoietic stem and progenitor cell, or HSPC, culture medium. Fill a 384-well cell culture plate with 75 microliters of HSPC culture medium in each well. To prevent evaporation of the medium in the outer wells, fill the outer wells with 75 microliters of sterile water or PBS and do not use these wells for cell sorting.

Correct sorting of viable signal HSPCs is the most critical step in this protocol. Set gates for the HSPC sorting based on an unstained control in 10,000 cells from the stain sample. Gate single cells by drawing a gate around the linear forward scatter height versus forward scatter area fraction.

Use the unstained control fraction to draw a gate for the lineage fraction. Draw gates for CD34 positive cells, and further characterize this subset by setting a specific gate for CD38 negative, CD45RA negative cells. Load the 384-well plate on the FACS machine and sort single cells. If applicable to the FACS machine, toggle on the option to keep index sorting data to enable retracing of the sorted cells.

To culture singly sorted HSCs, wrap the 384-well culture plate with lid in transparent polyethylene wrap. Transfer the 384-well plate to a humidified 37 degrees Celsius incubator with 5% carbon dioxide and keep it in the incubator for three to four weeks until visible clones appear. After four weeks of culturing, determine which wells have a confluency of 30% or higher.

For each clone, prefill 1.5 millimeter microtubes with 1 milliliter of 1% BSA in PBS and label the tube according to the corresponding well. Pre-wet a pipette tip with 1% BSA in PBS to minimize the number of cells sticking to the pipette tip. Fiercely pipette the medium up and down at least five times while scraping the bottom of the well with a 200-microliter pipette set at 75 microliters.

Collect the cell suspension in the labeled microtube that corresponds to the well. Repeat pipetting in the well with up to 75 microliters of fresh 1% BSA and PBS to ensure maximum uptake of cells. Clonally cultured cells can stick to the bottom of a well. Inspect the wells using a standard inverted light microscope to ensure whether all cells have been collected.

If all wells with greater than 30% confluency have been harvested, place the 384-well plate back in the incubator. Clonal cultures can proliferate for up to five weeks. Spin down the cell suspension for five minutes at 350 times g. A small pellet should be visible. Carefully remove all but about five microliters of the supernatant. Cell pellets can be frozen at minus 20 degrees Celsius and stored for multiple months before DNA isolation.