This protocol permits the expansion and genetic manipulation of functional mouse hematopoietic stem cells, allowing a deeper understanding of hematopoietic stem cell biology and hematopoiesis. The advantage of this technique is that it can support hematopoietic stem cells in long-term ex vivo culture and permit genetic manipulation to interrogate the genetics of hematopoiesis. This culture system provides a platform technology for various studies into hematopoietic stem cell biology and hematopoiesis, including genetic, molecular, and biochemical studies.
The troubleshooting table lists the causes and solutions for the common struggles encountered with this technique. It's much easier and more effective to show how to correctly extract bone marrow cells and perform complete media changes rather than read about it. To begin hematopoietic stem cell, or HSC, extraction, use lint-free delicate task wipes to clean the bones freshly isolated from properly euthanized mice.
Remove muscles and spinal cord from the bones before adding them to a mortar containing approximately three milliliters of PBS. Using a pestle, crush the bones without grinding them to minimize shearing forces. Then, using a 19-gauge needle attached to a 5-milliliter syringe, break up the large bone marrow fragments released into the PBS and transfer the suspension through a 70-micron filter into a 50-milliliter conical tube.
Once the suspension has been transferred, repeat the process with fresh PBS until the bones are bleached. Aim for an end volume of approximately 30 milliliters per mouse and 50 milliliters for two mice to bleach the bones until no more marrow is visible. Prepare for column enrichment by placing a magnetic filtration column into the magnet of a magnetic column separator with a 50-micron filter on top and a 15 milliliter conical tube below.
Next, after treating the cells with allophycocyanin anti-c-Kit antibody and allophycocyanin magnetic microbeads, run three milliliters of sterile PBS through the 50-micron filter and the filtration column. Once the PBS has run through, pass the cell suspension through the column, followed by three washes of three milliliters of cold PBS each time. After each wash, wait for the column to stop dripping before adding PBS for the next wash.
Next, remove the column from the magnet and place it on top of a fresh 15-milliliter tube and add five milliliters of cold PBS into the column before fitting the column plunger onto it and eluting the cells by pushing the plunger. Follow the protocol described in the text to prepare enough HSC medium for the desired number of cells or wells to seed 0.5 to 1 million cells per milliliter. Then spin down the c-Kit-enriched hematopoietic stem and progenitor cells, or HSPCs, and resuspend the pellet in the freshly prepared HSC medium at the desired cell density.
Transfer the cells to fibronectin-coated or negative surface charged plates, maintaining appropriate volume. Place the cells in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. After gently removing the plate from the tissue culture incubator without disturbing the cell cultures, use a pipette or vacuum pump to slowly aspirate approximately 90 to 95%of the medium from the liquid meniscus of each well.
Avoid drawing up medium from the base of the well. Then add one milliliter of fresh medium prewarmed to 37 degrees Celsius into the well. Return the plate to the tissue culture incubator, and change media every two to three days until the experiment requires.
After resuspending the cells in nucleofection buffer, immediately transfer the cell suspension into the PCR tube containing the complexed ribonucleoprotein, and gently mix by pipetting up and down slowly. Now transfer the mixture into an electroporation cuvette of appropriate volume very slowly, maintaining a continuous fluid motion to avoid forming air bubbles inside the cuvette. Then, on the electroporator, select the position of the wells being electroporated.
Using the touchscreen, select the cell type program as CD34-positive human or type in the pulse code EO100, and then press the OK button. Transfer the cuvette to the electroporator and press the Start button on the touchscreen to initiate electroporation. Immediately after electroporation, add five times the reaction volume of the culture medium to the cuvette.
Now gently transfer the cells to the prepared plate, and place the plate back in the tissue culture incubator. After performing a medium change as demonstrated previously, collect 10 microliters of the cells and count them using Turk's solution at a dilution of 1:2 with a hemocytometer. Replate the required dose of cells in fibronectin-coated plates for transduction, and separately, plate the un-transduced negative control cells.
Then add the lentiviral vector to each well containing cells, maintaining a dose of 20 transduction units per cell to achieve around 30%transduction efficiency. However, determine the lentiviral vector dose empirically depending on the experimental requirements. Finally, return the cells to the tissue culture incubator for six hours.
Perform a medium change as described previously. The fluorescence-activated cell sorting of the c-Kit-enriched HSCs yielded approximately 0.2%of the cells that were CD150-positive, CD34-negative, c-Kit-positive, Sca1-positive, and lineage-negative. After four weeks of HSPC culture, the CD201-positive, CD150-positive, c-Kit-positive, Sca1-positive, and lineage-negative fractions was found to be about 10%Following transduction with a GFP-expressing lentiviral vector, the GFP-positive cells were approximately 30%When confluent, the expected density of the cultures was around 2 million cells per milliliter.
Approximately 50%cell death was observed within the first 24 to 48 hours before the first medium change was performed. After one week, however, cell numbers returned to 80 to 100%of the seeded cell number. Accurate media changes using fresh and prewarmed media are critical for the long-term stability of this HSC culture method.
Since the original method was published in 2019, the field has begun to use it in different ways to study HSC biology.
