The long-term and short-term hematopoietic stem cell or HSC isolation method will help researchers better understand the cell renewal mechanisms and biological basis of heterogeneity in HSC compartment. A transcription factor, Hoxb5, which may be an exclusive marker of long-term HSCs, was identified. Based on this, a Hoxb5-reporter mice line was established and the long-term and the short-term HSCs were successfully isolated.
To begin the collection of the donor bone marrow cells, sterilize a mortar and pestle with 70%ethanol and let them dry completely. Equilibrate those with cell-staining buffer comprised of calcium and magnesium-free PBS-containing supplements. Put the bones isolated from the donor male Hoxb5-tri-mCherry mouse in the mortar and add three milliliters of the cell-staining buffer.
Brush the bones open with the pestle, then disaggregate the cell clumps by gentle pipetting and transfer the cell suspension through a 100-micron cell strainer into a 50-milliliter tube. Repeat the process with fresh cell-staining buffer until the solution becomes clear. Usually, repeating three times is enough to yield a clear solution.
After isolating the femurs and tibiae from the CD45.1.2 congenic mouse, cut both ends of the bones with sharp sterile scissors. Using a 23-gauge needle fitted with a five-milliliter syringe filled with ice-cold cell suspension buffer, flush the bone marrow out into a sterile cell culture dish containing cell suspension buffer. Disaggregate the cell clumps by gentle pipetting, then filter the cell suspension through a 40-micron cell strainer using a P1000 pipette.
For the gating setup, prepare the sample following the instructions provided in the text, and transfer 400 microliters of it to a round bottom polystyrene test tube with a 35-micron cell strainer snap cap. Prepare and add dead cell-straining reagent to the sample before the analysis, following the manufacturer's instructions. Then, turn on the flow cytometer and launch the analysis software following the manufacturer's instructions.
Then, press Load to begin acquiring the data. After excluding doublets, dead cells, and lineage-positive cells, gate the lineage-negative, the c-kit-positive, Sca-1-positive fraction. Next, gate out the Flk2-positive fraction.
Then gate Hoxb5-positive or Hoxb5-negative pHSCs in the CD150-positive, CD34-negative, or low fraction. Next, prepare a 1.5-millimeter low-protein binding tube with 600 microliters of calcium and magnesium-free PBS, supplemented with 10%heat-inactivated FBS. To perform the Hoxb5-positive or Hoxb5-negative pHSC sorting, set the prepared 1.5-milliliter low-protein binding tube on a sort collection device and sort the Hoxb5-positive or Hoxb5-negative pHSCs into the tube using the previously mentioned gating strategy.
In the first sorting, use the yield from the sorting precision mode. Set the 96-well plate with the previously prepared supporting cells on the automated cell deposition unit stage. Then, set the 1.5-milliliter low-protein binding tube to the loading port of the flow cytometer.
Sort the Hoxb5-positive or Hoxb5-negative pHSCs into the 96-well plate with the supporting cells using the previously mentioned gating strategy. Sort 10 Hoxb5-positive or Hoxb5-negative pHSCs to test their self-renewal capacities. The flow cytometry plots of the bone marrow analysis of the donor Hoxb5-tri-mCherry mice revealed approximately 20 to 25%of the cells in the pHSC fraction defined by lineage-negative, c-kit-positive, Sca-1-positive, CD150-positive, CD34-negative, or low, Flk2-negative were Hoxb5-positive pHSCs.
Shown here are the representative flow cytometry plots of the peripheral blood analysis of the recipient mice. Further, the peripheral blood analysis of the recipient mice after primary transplantation revealed that although Hoxb5-positive and Hoxb5-negative pHSCs presented similar donor chimerism four weeks after transplantation, continuous hematopoiesis was observed in only the Hoxb5-positive pHSC recipients. The Hoxb5-negative HSCs started losing the ability to produce hematopoietic cells eight weeks after transplantation.
In the secondary transplantation analysis, only the Hoxb5-positive pHSC recipients presented robust hematopoiesis. In contrast, donor cells were hardly observed in the Hoxb5-negative pHSC recipient mice, suggesting Hoxb5-negative pHSCs lost self-renewal ability within 16 weeks of primary transplantation. This protocol usually takes nine to 12 hours, so it is important to keep the sample at four degrees Celsius throughout the procedures as much as possible to maintain cell viability.
