Oregon Stem Cell Center

Oregon Stem Cell Center Flow Cytometry Core

Hematopoietic Stem Cells

The hematopoietic stem cell (HSC) gives rise to all the lineages of the blood. One of the defining characteristics of HSCs, as well as other stem cells, is the ability to self-renew to form identical daughter cells or to undergo a commitment program to form differentiated cell types. A number of methods have been used to isolate HSCs from bone marrow.

Cell surface markers

One approach involves identification of hematopoietic stem cells (HSCs) based on the presence of cell surface molecules. HSCs are highly enriched in the population characterized by low or undetectable levels of the lineage markers found on mature hematopoietic cells (B220, CD3, CD4, CD5, CD8, Mac-1, GR-1, Ter119, and NK1.1) and by high levels of c-Kit and Sca-1. A cell population meeting this criteria is termed ‘KSL’. Lineage negative, c-Kit positive cells are selected for first, as in the left panel. Cells falling within this gate, 1.3% of the initial starting population, are further gated for positive Sca-1, as shown in the middle panel. The KSL cells shown in the box, 0.14% of the initial population, are sorted. Re-analysis of the KSL cells collected shows that the sorted population is pure.

Figure 1)

a) Whole bone marrow	b) Lin^-/lo c-Kit⁺ bone marrow cells	c) Post-sort purity check

Lineage PE	Sca-1 FITC	Sca-1 FITC

Figs. 1a – 1b are viewed through scatter, doublet discriminator, and live gates. The percentages shown are of the total starting population.	Fig. 1b is gated on c-Kit⁺ lin^-/lo cells from whole bone marrow	Post-sort analysis (Fig. 1c) is viewed through scatter and doublet discriminator gates. Cells lose fluorescence intensity going through the laser, shifting the population slightly leftward. Purity is 99.4%.

Dye-efflux

Another approach to isolating stem cell-enriched populations is based on the dye-efflux properties of hematopoietic stem cells. Due to high expression of transporters, primarily the ABCG2 transporter, HSCs rapidly efflux the DNA dye, Hoechst 33342. Efficient efflux can be identified on a UV 670nm / 425nm dot plot, with cells that efflux the dye referred to as ‘side population’ or SP cells. Fig. 2 shows dot plots, both taken of mouse bone marrow cells incubated with the Hoechst dye. The SP-defined cells are highlighted by the outlined area in the lower left quadrant in the panel on the left. The panel on the right shows cells incubated with 50 mM verapamil, which inhibits the ABCG2 transporter and effectively eliminates the SP population.

Figure 2)

	a)		b)

The results above were obtained using bone marrow cells from C57BL/6 mice. Bone marrow cells at a concentration of 106 /mL were incubated for 60 minutes at 37 degrees C in a media of Ca⁺⁺ and Mg⁺⁺-free Hanks, 2% FCS, 10 mM HEPES, and 1% penicillin with Hoechst 33342 at 5 micrograms/mL.