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Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,DOI 10.1007/978-1-62703-128-8_10, © Springer Science+Business Media, LLC 2013

Chapter 10

Isolation and Characterization of Mouse Side Population Cells

Aysegul V. Ergen , Mira Jeong , Kuanyin K. Lin , Grant A. Challen , and Margaret A. Goodell

Abstract

The side population (SP) is a subpopulation of mouse bone marrow cells highly enriched for hematopoietic stem cell activity. The SP is identi fi ed using fl ow cytometry as a minor population that ef fi ciently ef fl uxes the DNA-binding dye Hoechst 33342 relative to the rest of the bone marrow. Phenotypic and functionally analysis has established SP cells as highly phenotypically homogeneous and functional active. In this chapter we describe a detailed protocol for the puri fi cation of murine bone marrow SP cells based on Hoechst dye ef fl ux in combination with the presence of HSC surface markers.

Key words: SP , Hoechst 33342 , Dye ef fl ux , Hematopoietic stem cell , Puri fi cation , Side population

The side population (SP) was fi rst found in murine hematopoietic stem cells (HSCs) in bone marrow by their ability to pump out fl uorescent DNA-binding dye Hoechst 33342 ( 1 ) . Hoechst 33342 binds to the AT-rich region of the DNA and emits primarily in the blue range (around 450 nm) and also has a weaker red emission (>675 nm) component. When these two emission wavelengths are detected and plotted against each other, the “side population” can be easily resolved (Fig. 1 ). In a dot plot of emission spectra they appear on the side of the staining pattern and constitute a discrete population of cells with an emission pro fi le that differs from that of the other cells (Fig. 1 ). This side population consists of highly enriched HSCs and comprises 0.02–0.15% of the whole mouse bone marrow cells depending on the age and gender. The frequency

1. Introduction

1.1. Side Population Cells

152 A.V. Ergen et al.

of SP cells steadily increases with age corresponding with the increasing proportion of HSCs in the bone marrow over time.

SP cells have been found in the hematopoietic tissues of various animal species including mice, monkeys, and humans ( 2 ) , in cell lines and primary cells from a variety of tissues and tumor types ( 3– 6 ) . SP cells are characterized by their high expression of multidrug-resistance ABC transporters such as transporter p- gly-coprotein (MDR1) and ABCG2, and these transporters are the major molecular mechanism of ef fl ux activity of the Hoechst 33342 dye and many types of chemotherapy agents ( 3, 7 ) . SP cells were shown to be sensitive to the ABC transporter protein inhibitor verapamil which reverses their phenotype ( 1, 8 ) . SP displayed elevated expression of ABCG2, and the ABCG2 knockout mouse was demonstrated to have a severe reduction in the SP ( 7, 9 ) . However, some non-SP cells were also found to express a detect-able level of ABCG2, indicating that ABCG2 expression is essential but not suf fi cient to characterize SP phenotype ( 7, 9, 10 ) . It is likely that multiple multidrug-resistance transporters contribute to the SP phenotype ( 11 ) .

Fig. 1. SP pro fi le of unenriched murine bone marrow sample. Flow cytometric pro fi le of SP population is visualized after staining bone marrow cells with 5 μ g/ml Hoechst 33342. Signals are displayed in a Hoechst Blue vs. Hoechast Red dot plot. The PMT voltages are adjusted until the majority of cells are at the upper right corner , whereas red blood cells and debris are at the lower left corner . SP cells (~0.02–0.05% of whole bone marrow) are very distinct and small subset of cells at the left side of the plot. PI positive cells (dead cells) are much brighter in the Hoechst Red channel.

15310 Mouse Side Population Cells

The puri fi cation of HSCs has been substantially improved by the application of fl ow cytometry with combinatorial SP staining and surface marker staining. The SP assay is a novel method by which rare HSC populations in the bone marrow can be identi fi ed without surface markers. Subsequent multiparameter fl ow cytometric anal-ysis of mouse bone marrow SP cells showed that approximately 95% expressed HSC surface markers and exhibited the highest hematopoietic repopulating activity. HSC activity is determined by quantifying the long-term repopulation of the transplanted cells to the peripheral blood. First it was found that HSC activity resides in cells that express c-kit (K) and Sca-1 (S) and do not express any of several surface markers found on different more mature blood cells (lineage negative , L). KSL is the canonical cell surface marker cocktail that is used to enrich for HSCs for more than a decade. However, it is still a very heterogeneous population that includes lineage-primed multipotent progenitors as well as short-term HSCs and long-term HSCs. Additionally, several alternative or improved HSC enrichment approaches have been developed. More studies have identi fi ed a number of additional HSC cell surface antigens including Thy1.1 ( 12 ) , CD34 ( 13 ) , Flk-2 ( 14 ) , the Tie-2 ( 15 ) , endoglin ( 16 ) , Epcr ( 17 ) , and CD150 ( 18 ) . Cells within the SP are very similar in terms of expression of canonical stem cell markers. SP highly overlaps with HSCs isolated via classical cell surface marker schemes KSL-Thy1 lo CD34 − Flk2 − or EPCR + CD48 − . Unlike all other markers, CD150 shows a bimodal distribution on the SP (Fig. 2 ) ( 19 ) . While both CD150 + and CD150 − cells from the SP are functional HSC ( 19 ) , the CD150 + subset has greater long-term self-renewal and engraftment potential, but a myeloid-biased lineage differentiation output, and thus may be selected if the most homogeneous and most potent HSC population is desired. We typically purify HSCs by the phenotype of SP + KSL + CD150 (called SP KSL CD150 + ) (Fig. 2 ).

Recent studies have identi fi ed new HSC subtypes with distinct functional properties within previously characterized populations. Our group showed HSCs from different regions of the SP, desig-nated as lower SP and upper SP, possess different functional poten-tials. Lower SP cells predominantly generated myeloid cells with great self-renewal potential, whereas upper SP cells were much more effective at generating lymphoid cells ( 19 ) . Other groups reported similar fi ndings using different enrichment strategies. The Eaves group used combinations of CD150, EPCR, CD48, and CD45, for the enrichment of HSCs, and they showed lymphoid vs. myeloid patterns associated with the absence or presence of CD150 ( 20 ) . They showed that HSC with higher repopulating activity and strong myeloid bias are enriched in the CD150 + subset of EPCR + CD48 − CD45 + bone marrow cells, whereas those in the CD150 − subset have limited self-renewal activity and a lymphoid

1.2. SP Cells and HSC Surface Markers

1.3. Functional Characterization of SP Cells

154 A.V. Ergen et al.

differentiation bias. Both the Nakauchi and Rossi groups also detected the same lineage bias associated with CD150 expression ( 21, 22 ) . They demonstrated that CD150 high subsets of CD34 − KSL exhibit the highest long-term HSC activity correlating with persis-tent myelopoiesis and CD150 high HSCs can give rise to CD150 high as well as to CD150 low and CD150 neg HSCs, but CD150 low and CD150 neg HSCs fail to give rise to CD150 high cells, suggesting that CD150 high HSCs reside at the top of the HSC hierarchy.

Usually, only a few thousand HSCs can be obtained from one mouse, and even this small population seems very heterogeneous and the cells differ in their functional properties when assayed on an individual level. Therefore, to ensure successful puri fi cation of

Fig. 2. The SP KSL CD150+ (SP, c-Kit + , Sca-1 + , Lin − , CD150 + ) cells. SP cells are co-stained with Sca-1, c-Kit, CD150 and lineage marker antibodies in order to exclude low level contamination of progenitor cells. This sample was pre-enriched with Sca-1 antibody using magnetic sorting.

15510 Mouse Side Population Cells

the most homogenous HSC based on the SP, it is optimal to use the SP in combination with conventional cell surface marker staining methods. Furthermore, because HSC are present at such a low proportion in the bone marrow, the highest purities are achieved by fi rst enriching for stem cells using magnetic enrich-ment for cells expressing a stem cell marker (e.g., Sca1 + or c-Kit + ) or lack of differentiation markers (lineage depletion). In the following sections, we introduce techniques for the puri fi cation of highly homogeneous long-term HSCs by combining both SP and HSC surface marker staining methods.

1. C57Bl/6(B6) mice, 8–10 weeks of age (see Note 1). 2. DMEM+: Dulbecco’s Modi fi ed Eagle’s Medium (DMEM)

with high glucose (Cat. No. 11965-092, Gibco Invitrogen) supplemented with penicillin/streptomycin (Cat. No. 15140-122, Gibco Invitrogen), 10 mM HEPES (Cat. No. 15630-080, Gibco Invitrogen), and 2% fetal bovine serum (FBS).

3. HBSS+: Hank’s balanced salt solution (HBSS, Cat. No. 14170-112, Gibco Invitrogen) supplemented with 10 mM HEPES (Cat. No. 15630-080, Gibco Invitrogen) and 2% FBS.

4. Hoechst 33342 powder (Cat. No. B2261, Sigma) is dissolved in distilled water and fi lter sterilized at 1 mg/ml concentration which makes 200× stock and frozen at −20°C. One whole bottle of powder is used to make ~500 ml of Hoechst stock solution at once, and frozen in small (~1 ml) aliquots. Thawed Hoechst powder may be less reliable after re-freezing, possibly due to acquisition of water.

5. Red blood cell lysis buffer (D-5001, Gentra). 6. Verapamil (Cat. No. V-4629, Sigma) is dissolved in 95% ethanol

as a 5 mM 100× stock. Stored at −20°C in 100 μ l aliquots. 7. Dissecting tools, scissors, and forceps. 8. 18-G and 27-G needles. 9. 40 μ m Cell strainers (Cat. No 22363547, Fisher). 10. 15 and 50 ml Conical polypropylene centrifuge tubes, sterile

(Falcon). 11. 10-cm Tissue culture dishes. 12. Refrigerated centrifuge. 13. 250 ml Polypropylene tubes (Cat. No 430776, Corning). 14. Circulating water bath at exactly 37°C.

2. Materials

2.1. Isolation of Bone Marrow SP Cells

156 A.V. Ergen et al.

15. Biotinylated Sca-1 antibody (Cat. No 553334, BD Pharmingen).

16. Anti-biotin magnetic microbeads from Miltenyi Biotech (Cat. No. 130-090-485).

17. Magnetic separation machine: autoMACS. 18. Monoclonal Antibodies (Table 1 ). 19. Propidium iodide (Cat. No. P-4170, Sigma) is dissolved at

200 μ g/ml in PBS as 100× stock and covered with aluminum foil and kept in 4°C fridge. Final concentration of PI in HBSS+ should be 2 μ g/ml.

20. Flow/sorting equipment with UV laser capable of excitation at 350 nm and detection with 450/20 and 675LP optical fi lters.

HSCs have the ability to ef fl ux Hoechst dye which appears as the side population in FACS (Fig. 1 ) after staining with Hoechst 33342. Reproducible SP staining is dependent on many parame-ters such as Hoechst concentration, cell number, staining tempera-ture, and time. A proper Hoechst staining will yield an SP population comprising 0.02–0.05% (Fig. 1 ) of whole bone mar-row cells from ~8-week-old C57Bl/6 mice (see Note 2). To increase the yield, a magnetic-based enrichment of progenitor cells using a canonical cell surface marker (Sca-1 or c-Kit) can be per-formed prior to FACS. Thus, an enrichment protocol, which

3. Methods

3.1. Harvesting Bone Marrow Cells

Table 1 Monoclonal antibody list for puri fi cation of SP KSL CD150 +

Antibody Clone Conjugate Dilution Company

Mac-1 M1/70 PE-Cy5 1:100 eBioscience

Gr-1 RB6-8C5 PE-Cy5 1:100 eBioscience

B220 RA3-6B2 PE-Cy5 1:100 eBioscience

Ter119 TER119 PE-Cy5 1:100 eBioscience

CD4 RM4-5 PE-Cy5 1:100 eBioscience

CD8 53-6.7 PE-Cy5 1:100 eBioscience

Sca-1 E13-161.7 Biotin 1:100 BD Pharmingen

c-Kit 2B8 AlexaFluor-750 1:100 eBioscience

CD150 TC15-12F12.2 PE 1:100 BioLegend

15710 Mouse Side Population Cells

provides tenfold enrichment for bone marrow SP (Fig. 2 ) is also provided. It is generally expected that 3,000–5,000 HSCs can be puri fi ed from one 10–12-week-old mouse. Moreover, it is impor-tant to note that the percentage of bone marrow HSCs increases with age, and this is also re fl ected in the percentage of SP cells. One may expect to obtain up to 10,000 from a mouse ~1-year-old (however, these HSC are of lower quality in terms of function).

1. Warm-up DMEM+ medium in a 37°C water bath. It is critical to have the temperature of water bath exactly 37°C.

2. Euthanize C57Bl/6 mice of 8–10 weeks of age. Dissect out femora and tibiae from mice and remove all the muscle and connective tissue from the bone using scissors and forceps. Additional bones can be used as desired, such as the hips which have additional bone marrow. Place the bones in ice-cold HBSS+. Keep them on ice throughout the process.

3. Trim the ends of the each bone and fl ush out the bone marrow into a sterile tissue culture dish using a syringe (5–10 ml) with a 27-G needle that is fi lled with ice-cold HBSS+. Flush from both ends to ensure all the marrow is removed. Bones should be very pale after fl ushing of the bone marrow. Crushing of all the bones with mortar and pestle can also be used to isolate bone marrow cells which results in more cells. Spines and other bones can also be used to collect more bone marrow cells.

4. Change the needle to 18-G and pass bone marrow-media mixture through 18-G needle several times in order to make a single cell suspension, while trying to avoid excessive bubble formation which reduces cell viability.

5. Filter cells through a 70 μ m cell strainer into a 50 ml falcon tube.

6. Count nucleated cells (see Note 3). In order to avoid counting red blood cells (RBC), an aliquot of the bone marrow cell suspension can be removed and mixed with RBC lysis buffer for counting. Take out 5 μ l from bone marrow suspension and mix it with 95 μ l of RBC lysis buffer, vortex thoroughly, and take 10 μ l to count cells using a hemacytometer. Do not use RBC lysis for the whole bone marrow suspension. This proce-dure generally yields an average of 40–70 million nucleated cells per C57Bl/6 mouse (2 femur and 2 tibias).

7. Spin down the cells in a refrigerated centrifuge (1,050 × g , for 6 min at 4°C).

8. Remove supernatant. Resuspend cell pellet at 10 6 cells/ml in pre-warmed DMEM+. Polypropylene tubes must be used while staining with Hoechst to avoid retention of cells in tubes. For large volumes, staining in 250 ml polypropylene tubes is the most convenient method.

158 A.V. Ergen et al.

1. Add Hoechst to a fi nal concentration of 5 μ g/ml. 2. Incubate cells in a circulating 37°C water bath for exactly

90 min (see Note 4). 3. Spin down the cells in a centrifuge at 1,050 × g for 6 min at 4°C

and remove the supernatant. Resuspend cells at 10 8 cells/ml in ice-cold HBSS+.

4. In order to ensure optimal HSC puri fi cation, cells should be co-stained with antibodies such as Sca-1, c-Kit, CD150, and lineage markers. Antibodies are added at concentrations deter-mined by standard antibody titration procedures or as recom-mended by the manufacturer (e.g., Becton Dickinson/Pharmingen). All staining and centrifugation should be performed at 4°C.

5. When samples are ready for fl uorescent activated cell sorting (FACS), resuspend cells in cold HBSS+ with 2 μ g/ml propidium iodide (PI) to distinguish and eliminate dead cells.

Hoechst-stained cells can be enriched for progenitors by using biotinylated Sca-1 or c-Kit antibodies. This will increase the yield, increase the purity, and decrease the time required for sorting.

1. For Sca-1 enrichment, add biotinylated Sca-1 antibody to the cell suspension at 1/100 dilution, and incubate on ice for 15 min. Alternatively, biotinylated c-Kit antibody can also be used for the enrichment.

2. Wash out unbound antibodies with tenfold volume of ice-cold HBSS+.

3. Centrifuge the cells for 6 min at 1,050 × g , at 4°C and remove the supernatant.

4. Resuspend cells at 10 8 cells/ml in ice-cold HBSS+. 5. Label cells with magnetic beads. We typically use anti-biotin

magnetic microbeads from Miltenyi Biotech, but alternatives are also effective. Incubate cells with 20% volume of micro-beads and place at 4°C in the fridge for 15 min. It is recom-mended to incubate cells in the fridge instead of on ice, because of the low binding ef fi ciency of microbeads on ice.

6. Wash the cells with a tenfold volume of ice-cold HBSS+. 7. Centrifuge the cells for 6 min at 1,050 × g , at 4°C and remove

the supernatant. 8. Resuspend cells at 2 × 10 8 cells/ml in ice-cold HBSS+. 9. Load the cells into autoMACS (or alternative) column. 10. Take the positive fraction from the autoMACS column and

wash with ice-cold HBSS+. 11. Centrifuge the cells for 6 min at 1,050 × g , at 4°C and remove

the supernatant.

3.2. Hoechst Staining

3.3. Magnetic Enrichment of Hoechst-Stained Cells and Antibody Staining

15910 Mouse Side Population Cells

12. Resuspend cells at 1 × 10 8 cells/ml in ice-cold HBSS+. 13. Prepare the stem and lineage marker antibody cocktail. Mix

monoclonal antibodies of anti-mouse c-Kit, anti-mouse CD150, lineage markers of anti-mouse CD4, anti-mouse CD8a, anti-mouse B220, anti-mouse Gr-1, anti-mouse Mac-1, and anti-mouse Ter-119 at 1/100 dilution. Use streptavidin conjugated fl uorescently labeled antibody to verify magnetic enrichment.

Analysis of SP cells has been performed on a variety of instruments, but we have had the most experience with cytometers from either BD (Aria) or Cytomation (MoFlo). In order to view the SP popu-lation, an ultraviolet laser is needed to excite the Hoechst 33342 dye and PI. A violet laser has also been used with good results ( 23 ) . Excitation of the Hoechst dye occurs at 350 nm and the emission of Hoechst dye is measured with Hoechst Blue and Hoechst Red detectors. Ideally, lasers with 100 mW of power give the best results, but lasers with lower power have been used successfully. Hoechst Blue is measured with a 450/20 band pass (BP) fi lter and red is measured with a 675 edge fi lter long pass (EFLP; Omega Optical, Brattleboro VT) fi lter. Emission wavelengths are separated with a 610 dichroic mirror short pass (DMSP). Fluorescence of PI is also measured with the 675EFLP fi lter, when excited with 350 nm. Although other fi lter sets similar to these ones works fi ne, these give better resolution of the SP.

1. Samples stained with Hoechst are placed on the cytometer and kept cold by a chilling apparatus if possible.

2. First, Hoechst fl uorescence is displayed with Hoechst Blue (450BP fi lter) on the vertical axis vs. Hoechst Red on the hori-zontal axis, both in linear mode. Voltage adjustments are made so that red blood cells can be viewed in the lower left corner (they have no nuclei so uptake of the DNA-binding Hoechst dye is minimal) and dead cells which are stained brightly with PI are seen against on the far right in a vertical line. The majority of the cells can be viewed in the center or in the upper right quarter (Fig. 1 ). A major GO-G1 population with S-G2M cells going toward the upper right corner can also be detected.

3. In order to obtain an SP pro fi le similar to the one shown in Fig. 1 , a sample gate is drawn to exclude red blood cells and dead cells. 50,000–100,000 events should be collected within this sample gate for an unenriched bone marrow sample. The SP region should be similar to that shown in Fig. 1 . The SP prevalence is around 0.01–0.05% of an unenriched whole bone marrow in the mouse (see Note 5).

4. SP cells are highly enriched for HSCs in mouse bone marrow. With a proper Hoechst staining, 60–80% of them are lineage

3.4. FACS Analysis for Hoechst SP Cells

160 A.V. Ergen et al.

negative and Sca-1 and c-Kit positive. These cells can be further separated for CD150 expression which marks for myeloid-biased HSC population. In young mice, 25–40% of SPKSL cells express CD150, whereas in the old mice this percentage is increased to 60–85%. In order to con fi rm proper SP staining, verapamil can be included in a separate control sample (the bulk of the SP should be eliminated when Hoechst staining in the presence of verapamil) (see Note 6).

1. This protocol is fi rst developed for mouse HSC from bone marrow of C57Bl/6 mice, thus optimization may be required for other strains and tissues. We recommend using C57Bl/6 bone marrow fi rst to establish the protocol before establishing Hoechst staining on other species or tissues.

2. Staining conditions are critical. Thus, the staining protocol should be followed precisely, otherwise it will result in low-quality of Hoechst stain, hence a decrease in the purity of HSCs. The concentration of Hoechst dye, numbers of the cells, staining temperature, and time are all parameters which have an effect on the SP pro fi le. It is also very important to keep cells at 4°C after Hoechst staining to prevent further Hoechst ef fl ux. Ficoll or other extended higher temperature procedures should not be performed after the Hoechst staining. The increased temperature will allow other bone marrow cells to ef fl ux Hoechst dye, resulting in an arti fi cially increased percentage of SP which is contaminated with non-stem cells.

3. A precise cell count of the bone marrow suspension is critical for a successful Hoechst staining, so counting correctly is important. As mentioned in Note 2, incorrect counting may result in low-quality staining and low purity.

4. Temperature and time are crucial for Hoechst staining, so DMEM+ should be warmed up and tubes should be fully immersed in the water bath in order to maintain the tempera-ture of cells at 37°C. Tubes should be mixed periodically during incubation to ensure equal exposure of the cells to the dye.

5. A very high proportion of SP cells (>0.05%) in normal mouse bone marrow may indicate poor staining and contamination of the population with non-stem cells. This problem can also be identi fi ed by identifying the presence of a large number of cells within the SP population that are Lineage + or do not express canonical HSC cell surface markers Sca-1 and c-Kit. In a proper

4. Notes

16110 Mouse Side Population Cells

Hoechst staining, 75–95% of the SP cells will have the canonical HSC cell surface markers (Sca-1 and c-Kit positive, lineage negative, CD34-negative/low, Flk2-negative). Sorting on at least two of these surface markers in addition to Hoechst staining will ensure the highest purity. We typically sort on SP and Kit+, Sca+, and Lineage-neg.

6. Con fi rmation of SP cells can be made by including a Verapamil-treated control which blocks the SP phenotype. Verapamil (Sigma, 100× stock made in 95% EtOH) is used at 50 μ M fi nal concentration and is added throughout the entire Hoechst staining. Verapamil treatment will result in the absence of SP which con fi rms SP identity.

Acknowledgments

The authors are supported by grants from the NIH, the Ellison Foundation, and the American Heart Association. M.J. was supported by University of Science and Technology though UST Post-Doc Research Program. G.A.C. is a scholar of the American Society of Hematology.

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