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Keywords:

  • cancer stem cells;
  • flow cytometry;
  • stem cell markers;
  • FACS;
  • molecular beacon

Abstract

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

In recent years, a special type of cancer cell—the cancer stem cell (CSC)—has been identified and characterized for different tumors. CSCs may be responsible for the recurrence of a tumor following a primarily successful therapy and are thought to bear a high metastatic potential. For the development of efficient treatment strategies, the establishment of reliable methods for the identification and effective isolation of CSCs is imperative. Similar to their stem cell counterparts in bone marrow or small intestine, different cluster of differentiation surface antigens have been characterized, thus enabling researchers to identify them within the tumor bulk and to determine their degree of differentiation. In addition, functional properties characteristic of stem cells can be measured. Side population analysis is based on the stem cell-specific activity of certain ATP-binding cassette transporter proteins, which are able to transport fluorescent dyes out of the cells. Furthermore, the stem cell-specific presence of aldehyde dehydrogenase isoform 1 can be used for CSC labeling. However, the flow cytometric analysis of these CSC functional features requires specific technical adjustments. This review focuses on the principles and strategies of the flow cytometric analysis of CSCs and provides an overview of current protocols as well as technical requirements and pitfalls. A special focus is set on side population analysis and analysis of ALDH activity. Flow cytometry-based sorting principles and future flow cytometric applications for CSC analysis are also discussed. © 2012 International Society for Advancement of Cytometry.

It is believed that the cellular structure of a tumor has a similar complexity as its nontumorous counterpart (1). According to the cancer stem cell hypothesis, a tumor consists of more differentiated highly proliferating cells, which constitute the tumor bulk and sustain tumor growth, and undifferentiated slow-cycling cells with self-renewing capacity, the so-called cancer stem cells (CSCs), (2–4). The origin of CSCs is still under debate (5, 6). Some investigators propagate that CSCs are derived from tissue stem cells with malignant changes (2, 7, 8), while others tend toward a reinitiation of stemness of differentiated cells by malignant transformation (9, 10). CSCs reside in special niches (11, 12) and have the potential to reconstitute a tumor after an otherwise successful therapy (13, 14). Furthermore, it has been suggested that CSCs might be responsible for the occurrence of distant metastases (15–17). CSCs have been identified in several solid tumors derived from breast (18), brain (19), prostate (20, 21), lung (22), ovarian (23), and liver tissue (24). CSCs are more resistant against common therapeutic approaches like chemo- (25) and radiotherapy (26, 27), probably due to their lower levels of reactive oxygen species (28), the expression of multidrug resistance proteins, and an increased DNA repair capacity (2, 3, 26). Thus, a targeted therapeutic intervention focused on CSCs might improve cancer therapy (29). The development of efficient treatment strategies therefore requires the establishment of reliable methods for the identification and effective isolation of CSCs (30–32).

Similar to hematopoietic stem cells or stem cells from other tissues (33), measurements of specific cluster of differentiation (CD) surface markers and stem cell-specific metabolic activities have been used for the characterization of CSCs. The list of CD markers found to be associated with CSCs is extensive and includes many markers used for the identification of nonmalignant hematopoietic or mesenchymal stem cells and other special markers like the B-cell marker CD20 for melanomas (34). Functional stem cell characteristics like ATP-binding cassette (ABC) transporter activity for side population (SP) analysis (35, 36) and aldehyde dehydrogenase isoform 1 (ALDH1) activity are common metabolic features to identify and analyze CSCs (36, 37).

Multiparametric flow cytometry is the method of choice for the analysis of CSCs (38). It allows the simultaneous analysis of different cellular features with high performance and reliability (39). Moreover, it enables the separation of living cells on the basis of marker expression or functional properties by fluorescence-activated cell sorting. A major advantage of this technique is its ability to isolate rare cells, which is a prerequisite of identifying small cell populations within the tumor bulk. Quantification is also possible and can be achieved either by the addition of count check beads to the sample or by volume-based flow cytometry (40, 41). However, a drawback of this technique is that tissue processing may cause artifacts and can potentially bias cell analysis (42). We present here an overview of current flow cytometric strategies and protocols to identify and analyze tumor stem cells. A special focus is set on the side population analysis and the analysis of aldehyde dehydrogenase (ALDH) activity, which are markers that provide information about stem cell-specific metabolic functions and can be used for their identification.

CELL SURFACE MARKER ANALYSIS

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

Similar to the CD of the hematopoietic lineages and other tissues (33, 43, 44), stem cells of solid tumors have been found to be identifiable by a set of cell surface markers (4, 6, 45–47). Different markers which have been successfully used in hematopoietic and mesenchymal stem cell analysis like CD34, CD133 (prominin-I), CD44, CD90 (Thy-1), CD105 (endoglin), and CD117 (c-kit) are also suitable for CSC characterization. An overview of markers used for the identification of CSCs in different tumors is given in Table 1. The expression of distinct markers varies among different tumors. While CD133 has been effectively used as a single marker for the identification of CSCs in colorectal cancer (48) or brain tumors (49, 50), a set of markers is necessary to identify breast CSCs, usually consisting of the combination of lineage markers (CD3, CD14, CD16, CD19, CD20, and CD56) with the hyaluronan receptor and cell adhesion molecule CD44 and the glycosyl phosphatidylinositol-anchored surface molecule CD24 (18, 51). Other marker combinations may be more useful for the analysis of other tumor types like CD133, alpha-2-beta-1 integrin, and CD44 for prostate CSCs (20, 52).

Table 1. Overview of cancer stem cell markers suitable for flow cytometric analysis
 Putative marker patterns for cancer stem cells (CSCs)ReferencesAnnotation
  • a

    lin = lineage marker CD3, CD14, CD16, CD19, CD20, and CD56.

Acute myelogenic leukemia (AML)CD34+/CD38(53, 54) 
CD90+(55)High risk AML
Acute lymphoblastic leukemiaCD34+/CD38/CD19+(56) 
Bone sarcomaStro-1+/CD105+/CD44+(57) 
Brain tumorCD133+(50, 58) 
Breast cancerESA+/CD44+/CD24−/low/lina)(18)Further enrichment by selection of ESA+ cells
CD90low/CD44+(59)Cells localized at the invasive front
CD44+/CD24−/low/ALDH1high(51) 
Colon cancerCD133+(48, 60) 
ESAhigh/CD44+/(CD166+)(61) 
CD133+/CD44+(62, 63) 
CD133+/CD24+(9) 
Colon cancer (metastatic)CD133+/CD44low/CD24+(64)Two subsets of tumor-initiating cells identified
CD133/CD44+/CD24
Endometrial cancerCD133+(65) 
SP+(66) 
Gall bladder cancerCD133+/CD44+(67) 
Gastric cancerCD44+(68) 
Liver cancerCD133+/CD44+(69) 
CD90+(70) 
EpCAM+(71) 
CD133+(24, 72) 
Metastatic melanomaCD20+(34) 
Ovarian cancerCD133+/ALDH+(73) 
CD44+/CD117+(74) 
Pancreatic cancerCD44+/CD24+/ESA+(75) 
Prostate cancerCD44+2β1hi/CD133+(20) 
CD44+/CD24(76) 
SP+(77) 
Renal cell carcinomaCD105+/(CD133/CD24)(78) 
Head and neck cancerCD44+(79)No further enrichment by endothelial-specific antigen or CD24

A number of new putative markers are under investigation and considerable effort is currently being directed toward the identification of additional ones. CD176 (Thomsen-Friedenreich, core 1 antigen) is under consideration for breast, liver, and lung carcinoma. This novel carbohydrate marker is coexpressed in a subset of CD44 or CD133 positive cells and may mark a subpopulation of breast CSCs (80).

According to Fábian et al. (4), one question remains unanswered: how reliably do these markers define CSCs? In contrast to normal stem cells, CSCs may have accumulated mutations in key signaling networks which alter pathways like Wnt (15, 81), Notch (66, 82), and Hedgehog (83). This causes a deregulation in terms of apoptosis, cell cycle, repair or other functional features (84). Genetic lesions can also trigger a switch of the tumor phenotype. The “phenotype instability,” where cancer cells can switch their phenotypes in response to microenvironmental cues, has been described in detail for melanoma by Hoek and Goding in 2010 (85). Both genetic and epigenetic (86) alterations can be responsible for the different phenotypes of otherwise histologically identical cancer types (87). Lambert et al. reported a continuous change of the hematopoietic stem cell phenotype over time, while progressing through the cell cycle (88). In other words, the cellular phenotype of stem cells can vary or change during the development and growth of tumors in vivo and under different conditions in cell culture (89).

There are also several experimental circumstances influencing marker expression and detectability (90). Immunophenotyping depends on tumor growth conditions and on external influences during the separation and culture of the cells (42). Some cell surface markers are sensitive to enzymatic digestion, which becomes a requirement when a solid tumor sample needs to be analyzed and sorted by flow cytometry or if the cells grow in an adherent manner in cell culture (81). Thus, optimization of the isolation, culture conditions, and flow cytometric analysis is necessary to avoid measurement artifacts due to experimental conditions (33). For example, the choice of protease for limited proteolysis during cell detachment (e.g., trypsin versus papain) may have a profound effect on the preservation of the antigenicity of individual cell surface markers (91). As an alternative to trypsin, tissue dissociation can be achieved using a mixture of hyaluronidase, DNAse, and collagenases (92). Although this preparation also contains a proteolytic enzyme, collagenase mainly digests extracellular matrix molecules containing unique structural motifs not commonly present in most cell surface epitopes relevant to the identification or sorting of CSCs (93). With respect to cultured primary tumor cells and tumor cell lines which contain subpopulations of CSCs (22, 63, 66), a detachment by the Ca2+-chelating agent EDTA can serve as a viable alternative to trypsinization. Indeed, in our laboratory, a protocol involving a washing step with ice-cold Ca2+-free phosphate-buffered saline (PBS; to induce cell rounding), followed by incubation with 2 mM EDTA in PBS (∼5 min) and pipetting-based dissociation in full media has proved successful for several mammalian cell lines (Götte and Greve, unpublished). As prolonged incubations in EDTA solution carry the risk of cellular toxicity, incubation times and the impact on cell viability need to be carefully evaluated for each individual cellline. It should be noted that this method may not be applicable to particularly sensitive cells, e.g. primary endothelial cells. Moreover, this method is not applicable for the analysis of tissue samples.

However, as there is very little consensus concerning the stem cell surface markers for many tissue types, the identification of markers of universal stemness based on intrinsic properties instead of the variable surface marker phenotype would be of great value for the stem cell research field (37).

SIDE POPULATION ANALYSIS

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

A unique characteristic of stem cells is their high expression of ABC transporter protein family members, which use ATP to translocate various substrates across membranes (94–96). Many different transporters have been described and are assigned to seven subfamilies (A–G). Important members of this family are ABCB1 (multidrug resistance protein 1, MDR1), ABCC1, ABCF2, ABCB2, ABCC7, and ABCA5, which are upregulated in the SP of different cells and may vary from tumor to tumor (94). The most important one may be ABC subfamily G member 2 (ABCG2) (36, 95, 96). In a study by Patrawala et al., ABCG2-negative and positive cells showed similar tumorigenicity; however, the ABCG2-expressing cells were mainly fast-cycling tumor progenitors, while the ABCG2-negative population was identified as being primitive stem-like cancer cells (97).

Under normal circumstances, drug effluxing pumps are responsible for the rapid elimination of metabolic products, toxic compounds or drugs. In this respect, ABC transporter proteins have a protective potential and warrant a higher chemoresistance of normal stem cells as well as CSCs in comparison to differentiated cells and tumor bulk (98–100). This has been analyzed in studies with glioblastoma (101), colon carcinoma (102), breast cancer (103), and other cancer types (104, 105).

The high expression of ABC transporter proteins in CSCs enables a flow cytometric identification of these cells as a “side population” (106, 107). SP analysis has been performed with numerous cancer cell lines and tumor material of different origins including breast (107), glioma (108), leukemia (109), colorectal cancer (110), and endometrial carcinoma (66). When a SP is identified, it is not clear if it represents the entire pool of CSCs in a tumor or just a part of it (4). In some cases, this problem might be related to inhomogeneity in the staining protocols (dye concentrations, cell densities, incubation times, etc.) (111) or different gating strategies (112). Variability of individual cell line-specific properties can also influence the results (112). Thus, a careful adjustment of current measuring protocols is indicated.

In flow cytometric analysis, SP cells usually form a distinct small cell population (typically <0.1%) showing little or no fluorescence with vital DNA dyes like Hoechst 33342. Vital dyes are effectively eliminated from ABC transporter protein-expressing cells. In 2005, Goodell described in detail the staining and measuring technique (113). Apart from Hoechst 33342, other vital dyes may also be useable like SYTO-13 or rhodamine 123 (Rh123) which was first introduced into stem cell research by Udomsakdi et al. in 1991 (114). It is described to be less toxic and excitable with a common 488 nm argon laser (115). Thus, Rh123 may be more useful to sort SP cells for further cultivation. A dual-dye efflux strategy using Hoechst 33342 and Rh123 has been described for isolating cells with the highest hematopoietic stem cell activity (116). However, ABCG2 varies in substrate specificity—cells with a point mutation at the R482 position are more resistant to drugs and have a much higher capacity to exclude Rh123 (117, 118), a circumstance that limits the applicability of Rh123. SP analysis has recently been performed with DyeCycle Violet, a cell-permeable DNA-binding dye which is excitable with a laser diode in the violet wavelength range (∼395–410 nm) (119, 120). Figure 1 illustrates a SP staining protocol with Hoechst 33342 which is successfully used in our laboratory.

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Figure 1. Example of a useable SP staining protocol. Suspend 1 × 106 cells in 1 ml DMEM containing 2% fetal calf serum (FCS), 10 mM HEPES, 1% penicillin/streptomycin and add 5 μg per ml Hoechst 33342. The verapamil control should additionally contain 50 μM verapamil. Incubate the tubes for 90 min at 37°C in the dark and shake every 10 min. Add 2 μg propidium iodide (PI) and store the tubes on ice until measurement. Adjustments could be necessary for the concentration of Hoechst 33342 in samples and controls or verapamil in the controls. Depending on the type of tumor and the type and content of ABC transporter proteins, the use of a different inhibitor could be necessary. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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However, variations of the Hoechst protocol exist and these variations have to be considered in data interpretation (111). Commonly, the final Hoechst 33342 dye concentration in the reaction tube is 5 μg ml−1 but some investigators work with lower e.g., 2.5 μg ml−1 (121) or even higher concentrations e.g., 6.2 μg ml−1 (122) or 10 μg ml−1 (123). This may lead to different results and give rise to scepticism and uncertainty about the general validity of this technique (124). Ibrahim et al. investigated the correlation of Hoechst dye concentration and incubation time (125) and found the dye uptake kinetics to be critical for isolating and characterizing stem cells. Figure 2 demonstrates the effects of different Hoechst 33342 concentrations (1, 2.5, 5, and 10 μg ml−1) on the identification of the SP phenotype in the Ewing sarcoma cell line STA-ET-1. Apart from a lower overall fluorescence, which needs an adjustment of the instrument's gain, the number of cells detectable in the SP increases by decreasing the dye concentration.

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Figure 2. Determination of SP in STA-ET-1 with different Hoechst 33342 concentrations. The Hoechst 33342 concentration has a huge impact on the number of cells in the SP. The verapamil control sample with the commonly used Hoechst 33342 concentration of 5 μg ml−1 reveals 0.15% SP cells, while the corresponding sample without verapamil reveals 0.52%. A Hoechst dye concentration of 10 μg ml−1 shows 0.10% of SP cells, 2.5 μg ml−1 a proportion of 1.43%, and 1 μg ml−1 a proportion of 5.66%. Typical verapamil control and SP histograms are shown below. Cells in the gate R2 represent the SP population. It should be noted, that a very high dye concentration can compromise even those transporters that are not verapamil sensitive. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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A critical point in SP analysis is the way in which ABC transporter inhibitors like verapamil are used in the control samples. The inhibitor concentration ranges from 50 to 100 μM (111, 126) in different studies and the incubation times are variable. Some groups use 30 min (122) or 60 min (35), while others incubate for 90 min (127) or 80 min with a further 10 min of preincubation without Hoechst dye and inhibitor (13). Additionally, the specificity of inhibitors differs. While verapamil is described to block the ABCB1 and ABCC1 transporter proteins most effectively, probenecid specifically inhibits ABCC1. Hoechst efflux can also be performed by the less verapamil/probenecid-sensitive ABCG2 transporter, which can be blocked by fumitremorgin C (118). Reserpine has been shown to be a pan-ABC transporter inhibitor (128). Therefore, there are a lot of differences in the used protocols which may have consequences on the comparability and significance of results.

Some technical requirements are of high relevance in flow cytometric SP analysis. A powerful and expensive 375 nm UV-laser is essential to obtain clear Hoechst 33342 fluorescence signals. A standard 488 nm blue laser is necessary for Rh123. For the measurement of DyeCycle Violet, a violet laser diode is needed, which conveniently is relatively inexpensive. Hoechst signals should be slivered by a 610 nm dichroic mirror into a blue channel equipped with a 455 nm or 475 nm band pass optical filter and a red channel with a 665-nm long pass filter (113). The results should be displayed in a dotplot-histogram with Hoechst red on the x-axis and Hoechst blue on the y-axis. The exact adjustment of the optical paths is critical for the detection of the relatively weak signals of the SP.

DETERMINATION OF ALDH1 ACTIVITY

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

Nineteen isoforms of ALDH exist in humans and most of them are able to catalyze the oxidation of different aldehydes (129). The ALDH1 isoform catalyzes the conversion of retinol to retinoic acid in normal and malignant stem cells. The reaction is important for the normal development and homeostasis of different organs like liver, prostate, and kidney. Furthermore, it acts as a morphogen during embryogenesis (130, 131). ALDH1 is highly expressed in stem cells of the hematopoietic system and the intestine. It is assumed that ALDH activity is necessary for hematopoietic stem cell differentiation on environmental cues (37). High ALDH activity has been shown in liver cells or in the bronchial epithelium of smokers caused by carcinogenic aldehydes in cigarette smoke (132). Ginestier et al. stated that ALDH1 is a marker for stem/progenitor cells of the normal breast (133). It is also active in stem cells of several tumors like breast cancer or lung cancer (37, 134). Its activity might be crucial for both stem cell longevity and the resistance of CSCs to chemotherapy. ALDH1 is able to mediate resistance against chemotherapeutic drugs that act through toxic aldehyde intermediates, e.g., cisplatin or cyclophosphamide (135).

Cells with ALDH1 activity can be detected using Bodipy™-aminoacetaldehyde (BAAA), a visible light excitable fluorochrome which diffuses freely across the plasma membrane of intact and viable cells. Intracellular ALDH converts BAAA into the fluorescent product Bodipy™-aminoacetate (BAA). BAA becomes trapped inside the cells since it is negatively charged and exclusion through ABC transporter proteins is blocked by inhibitors added to the assay system (136). The cellular fluorescence is detected with the green fluorescence channel (527/30 nm band pass filter) of a standard flow cytometer equipped with a 488-nm laser and compared with cells treated with the ALDH inhibitor diethylamino-benzaldehyde (DEAB). This detection system was initially designed by Storms et al. in 1999 (136) and is now commercially available (STEMCELL Technologies, Grenoble, France). The staining procedure and measurement are straightforward and the system works with cultured cells as well as with cells isolated from solid tumors. As a single marker, ALDH1 activity has limitations since its activity can be altered by chemotherapeutic treatment (94). However, ALDH1 activity can be combined with other more stable CSC-specific detection methods like cell surface marker expression analysis and/or SP analysis, which is described in the following section.

COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

If physiological stem cell features like SP and ALDH or cell surface markers are measured exclusively, they have their limitations to identify CSCs unequivocally, especially when tumor tissue is analyzed (137). For example, SP analysis alone was not sufficient to define a CSC phenotype in glioblastoma multiforme (138) and this was also the case for CD44+/CD24- cells in breast cancer (139). Thus, it might be more advantageous to use several markers and properties in combination. It is possible to combine SP or ALDH1 analysis with cell surface markers and to combine SP and ALDH1 analysis as well. SP analysis is usually performed with Hoechst 33342 and propidium iodide, both of which can be excited by a 375 nm UV-laser. The emissions are captured at 455 and 665 nm (113). The ALDH1 substrate BAAA is excited at 488 nm and emission is captured at 527 nm. If SP and ALDH1 are combined, the 488-nm laser also excites propidium iodide and raises signals at 610 nm. These signals can be used for a life-gate, to visualize only living propidium iodide-negative cells in the 527 nm BAAA channel. However, the number of phenotype markers which can be used in parallel to SP and ALDH1 analysis depends on the equipment of the used flow cytometer and the availability of appropriate antibody conjugates (Table 2).

Table 2. Overview of important dyes used in cancer stem cell analysis
DyeExcitationEmission PeakDetectionUseCrosstalk
Hoechst 33342375 nm475 nmDetected in FL4 (BP 455/50 nm)Used to identify cells in SP by staining the DNACrosstalk with FL5 (LP 665 nm), necessary to identify SP cells
Propidium Iodide (PI)375 and 488 nm617 nmDetected in FL3 (BP 610/20 nm)Used to identify dead cells by staining the DNAHeavy crosstalk with FL2 (BP 575/26 nm), PE and PI have low compatibility
BAAA488 nm545 nmDetected in FL1 (BP 527/30 nm)Used to identify ALDH1 enzyme activityCrosstalk with FL2 (BP 575/26 nm)and FL3 (BP 610/20 nm), compensation necessary
Phycoerythrin (PE)488 nm575 nmDetected in FL2 (BP 575/26 nm)Used for CD-classificationCrosstalk with FL1 (BP 527/30 nm)and FL3 (BP 610/20 nm), compensation necessary
Allophycocyanin (APC)633 nm660 nmDetected in FL5 (BP 675/20 nm)Used for CD-classificationThe use together with SP analysis depends on the equipment of the flow cytometer

Another important point which has not yet been discussed is the sequence of the different protocols. Some staining procedures are not compatible. The ALDH1 assay buffer contains channel inhibitors (e.g., verapamil) which prevent the active efflux of BAAA. Unfortunately, the same substances act as inhibitors in the SP assay. Thus, protocols have to take the correct order into account (140). Pierre-Louis et al. have described this in detail (141), proposing staining the SP cells first and then performing the ALDH1 staining. Further analysis of additional phenotype markers should be performed in a final step (Fig. 3).

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Figure 3. Proposed sequence of the different staining protocols in case of a combined measurement. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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They found that the coexpression of SP and ALDH markers refines the LinCD34+CD38 hematopoietic compartment and identifies an SP/ALDHBr cell subset enriched in quiescent primitive hematopoietic stem cells (141). Costaining of the stem cell markers SP and CD133+ was performed to identify CSCs in the DAOY medulloblastoma cell line. Interestingly, CD133+ cells were present in both the SP and non-SP fraction. The proportion of CD133+ cells was almost fourfold greater in the non-SP cells than in the SP population. The authors stated that this may explain the similar tumor sphere formation efficiencies they found in the CD133+ and CD133 fractions (111).

SORTING OF CSCs

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

All of the staining techniques described above are also suitable for the isolation of CSCs by flow cytometry (66, 142, 143). Two main sorting principles exist, which are designated as electrostatic sorting (144) and mechanical sorting (145). The electrostatic deflection of target cells is realized in the droplet sorter. A vibrating nozzle in the focus of the flow stream generates small cell-containing droplets. By passing a laser beam, the type of event is identified and the droplets containing the events of interest become charged electrostatically. The droplets are then separated by their electrostatic charges in a high voltage field and collected in separate tubes, 96-well plates or slides (144, 146, 147). However, the jet-in-air cell presentation provides less optical sensitivity than cuvette systems and may negatively influence the identification of extremely rare target cells (119). A further disadvantage is the generation of aerosols, which raises safety concerns regarding the sorting of samples containing toxic substances (e.g., fluorescent dyes or fixative solutions). Special biosafety guidelines are required to address this and contamination of the sample with pathogens such as bacteria (e.g. Mycobacterium tuberculosis) or viruses (e.g., hepatitis B, C, D, virus, HIV) could represent a potential health risk to the operator (148). The sorting process itself generates high pressures and the cell-containing droplets will impact with a high speed in the collection tubes. This might damage the rare sorted cells and could have a negative influence on cell survival (149, 150). However, the high sorting speed of up to 100,000 events per second and the ability to sort different populations in parallel make this technique well suited for the sorting of rare events like stem cells.

Compared to the electrostatic sorting procedures, the mechanical sorting systems are fully isolated, thus eliminating the risk of aerosol generation. Two systems have been developed to date. The original system (FACS-Calibur, Becton Dickinson, NJ) works with a catcher tube located in the upper portion of the flow cuvette. If the analyzing unit recognizes a cell as a sorting target, the mechanical unit moves into the flow stream, collects the cell, and directs it into a separate tube. If the instrument settings are adjusted properly, this unit sorts the target cells with high purity. However, the speed of sorting is low and only one population per run can be collected (151), both of which are disadvantages for collecting rare cells.

A modern mechanical sorting system works with a Y-shaped cuvette (waste and sorting arm) where two-thirds of the flow stream passes the waste arm and one-third the sort arm. All unsorted cells naturally run through the waste arm and if the analyzing system recognizes a cell of interest, a piezo crystal generates a pressure pulse in the fluidic stream and deflects the cell into the sorting arm (145). Because of the permanent fluidic stream in the sorting arm, cells become collected in a relatively large volume and have to be concentrated for further analysis. The sorting rate is in the order of 1,000 cells per second and one population per run can be sorted with high purity. The sorted cells are in a good condition and are usually viable; however, the low sorting rate and the large collection volume limit its use for the collection of very rare cells.

All flow cytometer-based sorting systems require an excellent alignment of both the detection unit and the sorting unit. The aim of the CSC sorting process is to isolate rare cells from a large heterogeneous cell population. The few sorted cells are collected in polypropylene tubes commonly supplemented with PBS. Electrostatic interactions between the isolated cells and the surface of the collection tube might lead to a high or complete loss of cells. This is particularly true for droplet sorters, where substantial electrostatic charges are involved. A similar phenomenon has been described for micro particles in a protein-free environment (152). An effective measure for the prevention of electrostatic adhesion is the coating of the collection tubes with fetal calf serum or other proteins like bovine serum albumin (153).

FUTURE DIRECTIONS IN CSC ANALYSIS

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited

Cancer is initiated by genetic alterations, including mutations in tumor-suppressor genes and oncogenes as well as chromosomal abnormalities. Furthermore, altered regulatory elements like epigenetic alterations or a modified microRNA expression could be responsible for cancer development (154). However, within the tumor bulk, the CSCs reveal a unique gene expression profile consisting of stem cell-specific genes like Sox-2 (155), Oct-4 (156), musashi (157), notch (158), nestin (159), and nanog (160). These predominantly intracellular markers would be useable for a specific labeling of CSCs if suitable molecular probes were available which are capable of entering living cells.

Rhee and Bao published a promising new method based on using a molecular beacon to visualize Oct-4 mRNA in live mouse embryonic carcinoma stem cells (161). Molecular beacons are short single-stranded nucleotide sequences with a fluorophore at the 5′-end and a matching quencher dye at the 3′-end. In the absence of target binding, it develops a loop structure where the fluorophore comes into close contact to the quencher, which erases the emitted fluorescence (162). In the presence of a complementary mRNA sequence, the beacon hybridizes to it by opening its loop sequence. The quencher becomes separated from the fluorophore and fluorescence is emitted upon appropriate excitation (Fig. 4). Suitable combinations of quenchers and fluorophores are available over a wide spectrum from UV to the near infrared. The transfer of beacons into living cells can be achieved by a peptide modification causing a spontaneous uptake (162), by a reversible permeabilization of cells with streptolysin O (163) or by electroporation. Transfection agents like Lipofectamine™ or DharmaFect™ can also be employed. If different beacons are used in parallel, this technique allows the measurement of a gene expression signature profile on a single cell level. Therefore, in the near future, this new technology is expected to greatly expand the repertoire of relevant stem cell markers aiding the identification and sorting of CSCs. However, the construction of molecular beacons and the transfection procedure require further development and testing, thus limiting the applicability of this innovative technique at the present time.

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Figure 4. Mode of functioning of a molecular beacon. In the absence of target binding, the beacon develops a loop structure where the fluorophore comes into close contact to the quencher, which erases the emitted fluorescence. In the presence of a complementary mRNA sequence, the beacon hybridizes to it by opening its loop sequence and emits fluorescence. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Literature Cited

  1. Top of page
  2. Abstract
  3. CELL SURFACE MARKER ANALYSIS
  4. SIDE POPULATION ANALYSIS
  5. DETERMINATION OF ALDH1 ACTIVITY
  6. COMBINATION OF SP, ALDH1, AND CELL SURFACE MARKERS
  7. SORTING OF CSCs
  8. FUTURE DIRECTIONS IN CSC ANALYSIS
  9. Literature Cited