The other discrimination technique was based on the assumption that CD11c+/high autofluorescent lung cells do not express MHCII and, therefore, are alveolar macrophages, whereas cDC are CD11c+MHCII+ (K. Vermaelen & Pauwels, 2004). This observation contrasts with the results obtained by us and others (Beaty, Rose, & Sung, 2007; Chapoval et al., 2009; Sung et al., 2006) (Figure 5). As it can be seen in Fig. 5, the levels of MHCII

**A** WT

Fig. 5. (continues on next page) Application of flow cytometry gating strategies used by Vermealen K and Pauwels R (Vermaelen and Pauwels 2004) and Beaty SR and associates (Sung, Fu et al. 2006; Beaty, Rose et al. 2007) for a detailed analysis of WT (panel A) and

VEGF tg (panel B) lung cDC (marked as myeloid, mDC) populations

expression on macrophages and cDC are the same.

We then further analyzed lung cDC using the defined above Abs to CD11c, MHCII, CD11b, DEC-205, and F4/80 cell surface markers in multicolour flow cytometry analysis using LSRII flow cytometer equipped with FACS Diva software. We identified lung cDC in WT mice as CD11c+MHCII+CD11b+F4/80lowDEC-205low cells (Figure 6). Although it should be noted here that using anti-DEC-205 Ab for IHC of lung tissues we were able to detect an equal low number of DEC-205+ (a lectin-type receptor, (Pollard & Lipscomb, 1990) cells between WT and VEGF tg mice without DOX-containing water exposure, and, therefore, in the absence of transgene expression (Chapoval et al., 2009; Lee et al., 2004). Lung VEGF tg cDC demonstrated the increased DEC-205 and MHCII and decreased F4/80 expression. Recently, mouse lung cDC were subdivided into two subpopulations, namely CD11b+cDC and CD103+ cDC (Beaty et al., 2007; Jakubzick, Tacke, et al., 2008; Sung et al., 2006).  Nevertheless, the use of this marker the helped to subdivide mouse lung tissue DC on three subclasses, pDC, CD11b+ cDC and CD103+ cDC (Beaty et al., 2007; Jakubzick, Helft, Kaplan, & Randolph, 2008; Lambrecht et

Lung cells were stained with CD11c-FITC, MHCII-PE, and CD11b-PerCP. The cells were first analyzed for forward and side scatters and gated out as shown in the blue box. Lung cDC in WT and VEGF tg mice were defined using CD11c-FITC marker and FL6 empty channel (Jakubzick, Helft, et al., 2008; Jakubzick, Tacke, et al., 2008; K. Vermaelen & Pauwels, 2004). CD11c/Fl6 dot plots of this gated population showed two CD11chigh WT cDC subpopulations (180 +585 cells for WT mouse lungs). Both of them were not autofluorescent in WT mice. These cells were then back-gated on the FCS and SSC dot plot. Dot plot distribution of selected cells is shown in red color. The relative and absolute numbers of the mainly macrophage-gate containing portion of WT lung cDC are equaled 2.25% / 585 cells. In contrast, the relative and absolute numbers of the portion of VEGF tg cDC were significantly higher than those found in WT mice and equaled 2.97% / 1961 cells. Population of tg lung cDC (2.25% of gated cells) showed low autofluorescence in empty

Single cell suspensions obtained from mouse lungs with omitting the enzymatic digestion step (Piggott et al., 2005) were stained with CD11c-FITC, MHCII-PE, and CD11b-PerCP. CD11chigh cells were gated out and analyzed for MHCII expression vs SSC. Two subpopulations of MHCII+ cells were observed in WT mouse lungs, namely SSClowMHCII+ (84.1%) cells and SSChighMHCII+ (14.4%) cells. The SSChighMHCII+ cells composed of autofluorescent lung macrophages and they are shown in red. Most of these cells are located on the border line of SSC and where gated out in further lung cDC analysis. Lung cDC are shown in green. Lower panel for WT mice shows that both populations of autofluorescent macrophages and cDC overlap on MHCII histogram. This also applies for VEGF tg lung macrophages and cDC (two lower panels). In VEGF tg mouse lungs the number of CD11c+ SSChighMHCII+ macrophages mounted to 54.6% of CD11c+ cells. Similarly to WT mice, these cells were located on the border line of SSC and where gated out from further cDC

These cells were not macrophages since macrophages were more autofluorescent and shifted more to up and right on FL6 empty channel. When macrophages were added to the dot plot, a significantly higher number of cells (8.68% / 12875 cells) in this selection box was observed. Therefore, there is a significant overlap between cDC and CD11chigh lung macrophages with a gating strategy based on CD11c marker and an empty channel

In human, lung cDC were characterized in the enzymatically digested tissues obtained at the lung surgery (Masten et al., 2006). cDC were defined as CD1c+CD11c+CD14- HLA-DR+ cells which comprised 2% of low autofluorescent mononuclear cells in a flow cytometry analysis. The expression of CD14 together with CD11c are critical important distinguishing markers for lung cDC and macrophages, as monocytes and macrophages were CD11c-CD14+ whereas CD11c+ cDC did not express CD14. Similarly to the mouse lung DC, human lung DC in a steady state condition are in immature state as defined by the absence of expression of differentiation markers CD83 and CD1a and limited expression of

al., 2000; Sung et al., 2006).

FL6.

analysis.

autofluorescence.

Fig. 6. Conventional DC in the lungs of WT and VEGF tg mice

**Conventional DC**

MHCII MHCII CD11b

100 0

0 0

80.9 19.1

0 0

Fig. 6. Conventional DC in the lungs of WT and VEGF tg mice

MHCII DEC-205 DEC-205 F4/80 cAb

66.7 33.3

100 0

0 0

99.3 0.66

0 0

0 0

99 1.01

0 0

CD11c

B

A

CD11c

1.2 ± 0.1

**WT**

2.7 ± 0.2

**VEGF tg\***

**WT**

**VEGF tg\***

is alpha E integrin which was detected on a subset of lung cDC and is also expressed on intraepithelial lymphocytes (Lehmann et al., 2002). Nevertheless, the use of this marker the helped to subdivide mouse lung tissue DC on three subclasses, pDC, CD11b+ cDC and CD103+ cDC (Beaty et al., 2007; Jakubzick, Helft, Kaplan, & Randolph, 2008; Lambrecht et al., 2000; Sung et al., 2006).

Lung cells were stained with CD11c-FITC, MHCII-PE, and CD11b-PerCP. The cells were first analyzed for forward and side scatters and gated out as shown in the blue box. Lung cDC in WT and VEGF tg mice were defined using CD11c-FITC marker and FL6 empty channel (Jakubzick, Helft, et al., 2008; Jakubzick, Tacke, et al., 2008; K. Vermaelen & Pauwels, 2004). CD11c/Fl6 dot plots of this gated population showed two CD11chigh WT cDC subpopulations (180 +585 cells for WT mouse lungs). Both of them were not autofluorescent in WT mice. These cells were then back-gated on the FCS and SSC dot plot. Dot plot distribution of selected cells is shown in red color. The relative and absolute numbers of the mainly macrophage-gate containing portion of WT lung cDC are equaled 2.25% / 585 cells. In contrast, the relative and absolute numbers of the portion of VEGF tg cDC were significantly higher than those found in WT mice and equaled 2.97% / 1961 cells. Population of tg lung cDC (2.25% of gated cells) showed low autofluorescence in empty FL6.

Single cell suspensions obtained from mouse lungs with omitting the enzymatic digestion step (Piggott et al., 2005) were stained with CD11c-FITC, MHCII-PE, and CD11b-PerCP. CD11chigh cells were gated out and analyzed for MHCII expression vs SSC. Two subpopulations of MHCII+ cells were observed in WT mouse lungs, namely SSClowMHCII+ (84.1%) cells and SSChighMHCII+ (14.4%) cells. The SSChighMHCII+ cells composed of autofluorescent lung macrophages and they are shown in red. Most of these cells are located on the border line of SSC and where gated out in further lung cDC analysis. Lung cDC are shown in green. Lower panel for WT mice shows that both populations of autofluorescent macrophages and cDC overlap on MHCII histogram. This also applies for VEGF tg lung macrophages and cDC (two lower panels). In VEGF tg mouse lungs the number of CD11c+ SSChighMHCII+ macrophages mounted to 54.6% of CD11c+ cells. Similarly to WT mice, these cells were located on the border line of SSC and where gated out from further cDC analysis.

These cells were not macrophages since macrophages were more autofluorescent and shifted more to up and right on FL6 empty channel. When macrophages were added to the dot plot, a significantly higher number of cells (8.68% / 12875 cells) in this selection box was observed. Therefore, there is a significant overlap between cDC and CD11chigh lung macrophages with a gating strategy based on CD11c marker and an empty channel autofluorescence.

In human, lung cDC were characterized in the enzymatically digested tissues obtained at the lung surgery (Masten et al., 2006). cDC were defined as CD1c+CD11c+CD14- HLA-DR+ cells which comprised 2% of low autofluorescent mononuclear cells in a flow cytometry analysis. The expression of CD14 together with CD11c are critical important distinguishing markers for lung cDC and macrophages, as monocytes and macrophages were CD11c-CD14+ whereas CD11c+ cDC did not express CD14. Similarly to the mouse lung DC, human lung DC in a steady state condition are in immature state as defined by the absence of expression of differentiation markers CD83 and CD1a and limited expression of

Flow Cytometry-Based Analysis and Sorting of Lung Dendritic Cells 249

13 samples analyzed. Unexpectedly, BDCA-2 expression was also seen in the cDC subpopulation, raising concerns about the pDC specificity of this marker for lung DC. Of note, BDCA-2 is not present in mice, and Siglec-H is used to define mouse pDC (Steinman,

Maraskovsky E and associates (Maraskovsky et al., 1996) were first to demonstrate that in vivo injections of Flt3L (FMS-related tyrosine kinase 3 ligand) into the mice will lead to a dramatic increase of DC subpopulations in different organs including the lungs. Five DC subpopulations were detected in spleens of injected mice based mainly on CD8α and CD11b

Populations (1-3) were also CD8α+, whereas DC in (4-5) were not expressing this lymphoid cell marker. Population (1) was highly enriched with immature granulocytes or myeloid cells as determined with Wright-Giemsa stain of sorted cell's cytospin. Last two DC subpopulations were highly enriched for veiled cells with dendritic processes. When compared for T cell stimulation activity, all 1-5 population were able to stimulate allogeneic T cells in MLR, however, DC in populations 1-2 were 30-times less efficient as compared to control freshly isolated DC. For distinguishing lung DC populations affected by Flt3L injections, the authors used DEC-205 as a selection marker considering a previously reported CD11c expression on a subset of lung macrophages. They noted a strong 8-15-times fold elevation in relative numbers of DEC-205+CD11b+ and DEC-205+CD11b- cells. A more recent publication by Masten BJ and associates (Masten, Olson, Kusewitt, & Lipscomb, 2004) focused on Flt3L injection's effect specifically on lung DC. Flt3L induced a 19-fold increase in the absolute numbers of CD11c+CD45R/B220-DC in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DC (CD11c+CD45R/B220-CD11b+ cells) and only a 3-fold increase of lymphoid DC (CD11c+CD45R/B220-CD11b- cell) from the lungs of Flt3L-treated mice over vehicletreated mice. The authors noted 4 subpopulations of lung DC under the study conditions, namely: (1) myeloid CD11b+ CD45R/B220- DC, (2) lymphoid CD11b- CD45R/B220- DC, (3) and (4) plasmacytoid DC expressing various levels of CD11c and CD45R/B220 markers. Therefore, the use of Flt3L may be efficient in augmenting vaccine responses against different lung infectious agents and promoting anti-tumor responses. However, as for the dampening of allergic lung responses, it might not be as effective considering the fact that myeloid lung DC which are mainly upregulated by Fl3L treatment, are necessary for allergic lung response generation and sustainability (Lambrecht et al., 2000; Lambrecht, Salomon,

A very detailed classification of DC based on organ and tissue distribution, marker's (including chemokine receptors) expression, migration, and function was recently presented in a comprehensive review article by Alvarez, Vollmann, and von Andrian (Alvarez,

**2.2.5 Other lung DC subtypes arising under different conditions**

2010).

expression, namely

1. CD11bbrightMHCII-GR1+CD11c-, 2. CD11bbrightCD11cdullMHCII+GR1+, 3. CD11bbrightCD11c+MHCII+GR1-, 4. CD11bdullCD11c+MHCIIbrightGR1-, and 5. CD11b-CD11c+MHCIIbrightGR1- cells.

Klatzmann, & Pauwels, 1998).

costimulatory molecules (Cochand et al., 1999). In the latter study, the loosely adherent on the Petri dishes cells were further separated into DC and autofluorescent macrophages with a Coulter EPICS V based on the presence or absence of autofluorescent inclusions with a coherent INNOVA 90 light source, using a 488-nm wavelength for excitation and a 588-nm filter for emission. The gates were set to remove cell debris and to select mature alveolar macrophages and nonphagocytic DC.

#### **2.2.4 Lung plasmacytoid DC**

Plasmacytoid DC are characterized as CD11cintermed/ B220+/GR1+ cells and CD123+CD11c-CD14-HLA-DR+ cells in mice and human, correspondingly (Chapoval et al., 2009; de Heer et al., 2004; Masten et al., 2006; Ritz et al., 2004). Plasmacytoid DC in the mouse lungs were first properly detected and functionally analyzed by Bart Lambrecht's group in 2004 (de Heer et al., 2004). This discrimination was based on CD11c and GR1 expression. The sorted cells based on this discrimination characteristic showed typical morphologies for different cell types such as eosinophils, lymphocytes, macrophages, neutrophils, and DC. pDC were targeted to the specific region based on CD11c and GR-1 expression, displayed a more immature phenotype than cDC and were MHCIIlow, B220+ and CD45RB+. Confocal microscopy targeted B220+Gr1+ pDC to the lung interstitium. The elimination of these cells with the in vivo use of anti-GR1 or 120G8 depleting Abs lead to the allergic eosinophilic response in the lungs to normally inert allergen applications (Asselin-Paturel, Brizard, Pin, Briere, & Trinchieri, 2003; de Heer et al., 2004).

Lung VEGF expression increases the number of cDC in tg mouse lungs. Mouse lung tissues were obtained on day 7 of DOX water administration and processed using the enzymatic digest method. Single cell suspensions were stained with corresponding Ab and analyzed by flow cytometry. (A-B) Autofluorescent macrophages (shown in black color in panel A) were removed from the DC analysis. An upregulation of MHCII, CD11b, DEC-205 but not F4/80 expression on tg lung cDC was detected. For histograms: solid line represents isotype control rat IgG2a staining whereas transparent line shows the level of DEC-205 expression. \*p<0.0025, WT vs tg lung mDC number (n=5/group).

We studied the effect of lung VEGF expression on local pDC number and activation employing VEGF tg mice (Chapoval et al., 2009). We have found that VEGF induced activation of lung pDC which were characterized as CD11cintB220+GR1+ cells and upregulated MHCII, CD40, CD80, and CD86 without a substantial modulation of ICOS-L expression.

Human lung plasmacytoid DC were characterized as CD123+CD11c+CD14+HLA-DR+ cells and comprised approximately 1.0% of the low autofluorescent mononuclear cells in flow cytometry analysis of cells from the lung tissues obtained upon surgery (Masten et al., 2006). A first discrimination of DC in this study was based on CD11c and CD14 expression. Morphological examination of cytospinned cells revealed cDC enriched CD11c+CD14- cell population and pDC-enriched CD11c-CD14- cell population. These populations were further analyzed for CD3, CD19, and CD56 expression to distinguish T, B and NK cells. The elimination of these cells subsets from the analysis demonstrated that approximately 7% of CD11c-CD14-human lung cell population were pDC. A lectin type receptor BDCA-2 (Demedts, Brusselle, Vermaelen, & Pauwels, 2005) expression was present only on 9 out of 13 samples analyzed. Unexpectedly, BDCA-2 expression was also seen in the cDC subpopulation, raising concerns about the pDC specificity of this marker for lung DC. Of note, BDCA-2 is not present in mice, and Siglec-H is used to define mouse pDC (Steinman, 2010).

#### **2.2.5 Other lung DC subtypes arising under different conditions**

Maraskovsky E and associates (Maraskovsky et al., 1996) were first to demonstrate that in vivo injections of Flt3L (FMS-related tyrosine kinase 3 ligand) into the mice will lead to a dramatic increase of DC subpopulations in different organs including the lungs. Five DC subpopulations were detected in spleens of injected mice based mainly on CD8α and CD11b expression, namely

1. CD11bbrightMHCII-GR1+CD11c-,

248 Flow Cytometry – Recent Perspectives

costimulatory molecules (Cochand et al., 1999). In the latter study, the loosely adherent on the Petri dishes cells were further separated into DC and autofluorescent macrophages with a Coulter EPICS V based on the presence or absence of autofluorescent inclusions with a coherent INNOVA 90 light source, using a 488-nm wavelength for excitation and a 588-nm filter for emission. The gates were set to remove cell debris and to select mature alveolar

Plasmacytoid DC are characterized as CD11cintermed/ B220+/GR1+ cells and CD123+CD11c-CD14-HLA-DR+ cells in mice and human, correspondingly (Chapoval et al., 2009; de Heer et al., 2004; Masten et al., 2006; Ritz et al., 2004). Plasmacytoid DC in the mouse lungs were first properly detected and functionally analyzed by Bart Lambrecht's group in 2004 (de Heer et al., 2004). This discrimination was based on CD11c and GR1 expression. The sorted cells based on this discrimination characteristic showed typical morphologies for different cell types such as eosinophils, lymphocytes, macrophages, neutrophils, and DC. pDC were targeted to the specific region based on CD11c and GR-1 expression, displayed a more immature phenotype than cDC and were MHCIIlow, B220+ and CD45RB+. Confocal microscopy targeted B220+Gr1+ pDC to the lung interstitium. The elimination of these cells with the in vivo use of anti-GR1 or 120G8 depleting Abs lead to the allergic eosinophilic response in the lungs to normally inert allergen applications (Asselin-Paturel, Brizard, Pin,

Lung VEGF expression increases the number of cDC in tg mouse lungs. Mouse lung tissues were obtained on day 7 of DOX water administration and processed using the enzymatic digest method. Single cell suspensions were stained with corresponding Ab and analyzed by flow cytometry. (A-B) Autofluorescent macrophages (shown in black color in panel A) were removed from the DC analysis. An upregulation of MHCII, CD11b, DEC-205 but not F4/80 expression on tg lung cDC was detected. For histograms: solid line represents isotype control rat IgG2a staining whereas transparent line shows the level of DEC-205 expression.

We studied the effect of lung VEGF expression on local pDC number and activation employing VEGF tg mice (Chapoval et al., 2009). We have found that VEGF induced activation of lung pDC which were characterized as CD11cintB220+GR1+ cells and upregulated MHCII, CD40, CD80, and CD86 without a substantial modulation of ICOS-L

Human lung plasmacytoid DC were characterized as CD123+CD11c+CD14+HLA-DR+ cells and comprised approximately 1.0% of the low autofluorescent mononuclear cells in flow cytometry analysis of cells from the lung tissues obtained upon surgery (Masten et al., 2006). A first discrimination of DC in this study was based on CD11c and CD14 expression. Morphological examination of cytospinned cells revealed cDC enriched CD11c+CD14- cell population and pDC-enriched CD11c-CD14- cell population. These populations were further analyzed for CD3, CD19, and CD56 expression to distinguish T, B and NK cells. The elimination of these cells subsets from the analysis demonstrated that approximately 7% of CD11c-CD14-human lung cell population were pDC. A lectin type receptor BDCA-2 (Demedts, Brusselle, Vermaelen, & Pauwels, 2005) expression was present only on 9 out of

macrophages and nonphagocytic DC.

Briere, & Trinchieri, 2003; de Heer et al., 2004).

\*p<0.0025, WT vs tg lung mDC number (n=5/group).

expression.

**2.2.4 Lung plasmacytoid DC**

Populations (1-3) were also CD8α+, whereas DC in (4-5) were not expressing this lymphoid cell marker. Population (1) was highly enriched with immature granulocytes or myeloid cells as determined with Wright-Giemsa stain of sorted cell's cytospin. Last two DC subpopulations were highly enriched for veiled cells with dendritic processes. When compared for T cell stimulation activity, all 1-5 population were able to stimulate allogeneic T cells in MLR, however, DC in populations 1-2 were 30-times less efficient as compared to control freshly isolated DC. For distinguishing lung DC populations affected by Flt3L injections, the authors used DEC-205 as a selection marker considering a previously reported CD11c expression on a subset of lung macrophages. They noted a strong 8-15-times fold elevation in relative numbers of DEC-205+CD11b+ and DEC-205+CD11b- cells. A more recent publication by Masten BJ and associates (Masten, Olson, Kusewitt, & Lipscomb, 2004) focused on Flt3L injection's effect specifically on lung DC. Flt3L induced a 19-fold increase in the absolute numbers of CD11c+CD45R/B220-DC in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DC (CD11c+CD45R/B220-CD11b+ cells) and only a 3-fold increase of lymphoid DC (CD11c+CD45R/B220-CD11b- cell) from the lungs of Flt3L-treated mice over vehicletreated mice. The authors noted 4 subpopulations of lung DC under the study conditions, namely: (1) myeloid CD11b+ CD45R/B220- DC, (2) lymphoid CD11b- CD45R/B220- DC, (3) and (4) plasmacytoid DC expressing various levels of CD11c and CD45R/B220 markers. Therefore, the use of Flt3L may be efficient in augmenting vaccine responses against different lung infectious agents and promoting anti-tumor responses. However, as for the dampening of allergic lung responses, it might not be as effective considering the fact that myeloid lung DC which are mainly upregulated by Fl3L treatment, are necessary for allergic lung response generation and sustainability (Lambrecht et al., 2000; Lambrecht, Salomon, Klatzmann, & Pauwels, 1998).

A very detailed classification of DC based on organ and tissue distribution, marker's (including chemokine receptors) expression, migration, and function was recently presented in a comprehensive review article by Alvarez, Vollmann, and von Andrian (Alvarez,

Flow Cytometry-Based Analysis and Sorting of Lung Dendritic Cells 251

BDCA1+ CD11chigh

CD207- CD14+ BDCA1+ CD11chigh ?

> CD207- CD11b+ CD11chigh

> CD207- CD11b+ CD11chigh

CADM1- CLEC9A-SIRPa+ CD11chigh RIG-I/MDA5/DAI+ NOD/NALPhigh ? CD4 T cell activation, humoral immunity, response to extracellular parasites

Fig. 7. A unifying model of human and mouse DC subsets (courtesy of Bernard Malissen). Human and mouse DC subsets were organized into five broad subsets irrespective of their primary location/tissue origin. These five subsets correspond to: (1) Langerhans cells (green), (2) DC-like cells (blue), (3) CD8a+ DC-like cells (violet), (4) pDC (brown), and (5) monocyte-derived inf-DC (orange). The authors suggest a general nomenclature for each DC subset (lower row, shaded colors) which is based on the unified phenotypic definition,

characteristic pattern recognition receptors and functional specialization

CD11b+ LC -like DC CD8a+-like DC pDC inf-DC

BDCA3+ CD11chigh

CD207- CD14- CD1a+ BDCA3+ CD11chigh ?

CD207+/- CD8a+ and/or CD103+ CD11b-/low CD11chigh

CD207+/- CD103+ CD11b-/low CD11chigh

CADM1+ CLEC9A+ SIRPa- CD11chigh TLR3high ? Cross-presentation, CD8 T cell activation, IL-12p70

?

BDCA2+ CD11c-

BDCA2+ CD11c-

SiglecH+ CD11clow

SiglecH+ CD11clow

None? TLR7/9high Innate defenses against viruses, IFN-a/ß

?

Ly6C+ MAC3+ CD11b+ CD11c+

Ly6C+ MAC3+ CD11b+ CD11c+

Awaiting human inf-DC identification TLR4/8high, NOD/NALPhigh ?

Classical phenotypic definition

Classical phenotypic definition

Classical phenotypic definition

**HUMAN & MOUSE**

Proposed unified phenotypic definition Typical PRRs Proposed functional specialization

**MOUSE**

Vollmann, & von Andrian, 2008) (Table 1 shows a fraction of a table presented in this comprehensive review). As for lung DC, the authors focused only on two myeloid DC subpopulations distinguished mainly by CD103 expression, lung interstitium CD11bloCD11c+CD103+ and conducting airway CD11bhiCD11c+Cd103- DC.

Table 1. Phenotype of dendritic cell subsets (modified from (Alvarez, Vollmann et al. 2008)

Finally, a unifying classification of human and mouse DC subsets was recently proposed by Guilliams M and associates (Guilliams et al.) which takes into account a functional specialization and specific marker expression (Figure 7). This classification is based on the expression of CD11b, CD103, and CD207. It highlights many unanswered questions in the DC research including the proper cell determination.

Vollmann, & von Andrian, 2008) (Table 1 shows a fraction of a table presented in this comprehensive review). As for lung DC, the authors focused only on two myeloid DC subpopulations distinguished mainly by CD103 expression, lung interstitium

CD45+Lin-c-Kit+Sca-1+

CD44+Lin-c-KitintCD11b-

CD44+Lin-c-KitintFlt3+

1+CD1a+CD11c+CD11b+CD24a+CD205+CD45loCD8a+/-

Langerin+MHCII+CD11cintCD11bloCD45hiCD8a+CD103+

Langerin-MHCII+CD11c+ CD24a-DEC205+

Monocyte subsets CD115+CD11b+Ly6C low/int/hi F4/80lowCD62L+/-

Langerhans cell Langerin+MHCII+Dectin-

CD103-

CD8a-CD4+DC CD8a-CD4+CD11b+MHCII+DCIR2+

subsets) CD11chiCD11b-CD205+CD103+

DC research including the proper cell determination.

CD8a+DC CD8a+MHCII+CD11c+CD4-CD205+SIRP-a+

CD8a-DC CD8a-MHCII+CD11c+CD11b+CD4-SIRP-a+DCIR2+

Plasmacytoid DC B220+CD11cloLy6C+MHCIIloCD4+/-CD8a+/-PDCA-1+

CD11bloCD11c+CD103- CD11bloCD11c+CD103+

CD11c+CD8a+CD11b-CD11c+CD8a+CD11b+ CD11c+CD8a-CD11b-

Table 1. Phenotype of dendritic cell subsets (modified from (Alvarez, Vollmann et al. 2008)

Finally, a unifying classification of human and mouse DC subsets was recently proposed by Guilliams M and associates (Guilliams et al.) which takes into account a functional specialization and specific marker expression (Figure 7). This classification is based on the expression of CD11b, CD103, and CD207. It highlights many unanswered questions in the

(human: CD123+BDCA-2+BDCA-4+)

CD11chiCD11b+CD205+CD103+ CD11cintCD11bintCD205-CD103- CD11cintCD11b+CD205-CD103-

CD11bloCD11c+CD103+ and conducting airway CD11bhiCD11c+Cd103- DC.

Dendritic cell subset Phenotype

Precursor DC

(MDP)

subset)

subset)

Hematopoietic stem and progenitor cell (HSPC)

Macrophage DC precursor

Common dendritic projenitor (CDP)

Differentiated DC

Dermal DC (langerin+

Dermal DC (langerin-

Lung DC (2 subsets) Conducting airway DC Lung interstitium DC

Lamina propria DC (4

Peyer's patch DC (3

subsets)

Fig. 7. A unifying model of human and mouse DC subsets (courtesy of Bernard Malissen). Human and mouse DC subsets were organized into five broad subsets irrespective of their primary location/tissue origin. These five subsets correspond to: (1) Langerhans cells (green), (2) DC-like cells (blue), (3) CD8a+ DC-like cells (violet), (4) pDC (brown), and (5) monocyte-derived inf-DC (orange). The authors suggest a general nomenclature for each DC subset (lower row, shaded colors) which is based on the unified phenotypic definition, characteristic pattern recognition receptors and functional specialization

Flow Cytometry-Based Analysis and Sorting of Lung Dendritic Cells 253

expression induced lung cDC activation as they upregulate MHCII, CD40, CD80, CD86, and

To track the Ag uptake by lung DC in vivo, we applied 1 μg/50 μl/mouse of OVA-FITC i.n. to WT and VEGF tg mice one time (Chapoval et al., 2009). Lung tissue and local LN digests were analyzed by flow cytometry 6h and 24h after Ag application for FITC+ cells using CD11c/MHCII/CD11b markers. We observed an increase in FITC+ DC but not Mac-1+ cell number in VEGF tg lungs by flow cytometry. Therefore, intermediately mature cDC

Intratracheal instillations of OVA-FITC to mice with different deficiency can be used to assess the effect of such deficiency on lung DC migration. As an example, Vermaelen and associates (K. Y. Vermaelen et al., 2003) used OVA-FITC in MMP-9-/- mice to evaluate the effect of MMP-9 deficiency on lung DC migration. The study has shown that FITC+ cells gradually accumulate in the draining lymph nodes with a peak reaching at 24h of Ag application with no difference in lung DC migration between WT and MMP-9-/- mice.

Highly pure isolated populations of DC are needed to evaluate their exact biological properties and function. Despite the fact that most lung DC represent rare populations among other lung cells, the high level of DC purification could be reached with either immunomagnetic (f.e. MACS technology, Milenyi Biotech) cell separation or flow

Lung conventional DC were sorted using a triple marker combination (CD11c/MHCII/ CD11b) employing BD FACS Vantage or BD FACS Aria (both equipped with FACSDiva software), or Dako MoFlo (Summit software) high speed automated cell sorters (Chapoval et al., 2009) which all provide the state-of-the-art advances in the instrument set-up, cell sorting and integrated cell analysis. Autofluorescent macrophages and cell doublets are eliminated from further analysis by proper gating (Chapoval et al., 2009). The cDC sorting

Sorted lung DC then could be used in different in vitro assays to define the specific protein expression which could be further analyzed by flow cytometry (Beaty et al., 2007; Chapoval et al., 2009; K. Y. Vermaelen et al., 2001). The in vitro Ag uptake is an important technique to define the maturation status of DC and its functional activity. To study the in vitro Ag uptake, we subjected lung cells and sorted lung cDC to the in vitro cultures with or without increasing doses of OVA-FITC (Molecular Probes) ranging from 0.01 mg/ml to 1 mg/ml in RPMI (Life Technologies) in 24-well plates (Costar) for 30 min at 37ºC. After incubation, cells were extensively washed with RPMI medium and analyzed for Ag uptake by flow cytometry. VEGF tg mDC are significantly more efficient in Ag uptake with low doses of Ag used. For example, at 10 μg of Ag only 30.2 % of WT DC were FITC+ whereas for VEGF tg

obtained from the lung of VEGF tg mice were more efficient in Ag uptake.

 Lung DC sorting**

cytometry-based cell sorts.

**4.1 Methods of sorting**

strategy is shown in Figure 8.

**4.2 Analyses of sorted lung DC**

#### **3. Functional characteristics of lung DC**

As mentioned above, the crucial functions of DC are to capture antigens throughout the body, migrate to the local lymphoid organs to deliver Ag and stimulate or tolerize T cells. The in vitro and in vivo Ag sampling by DC can be easily and specifically measured by flow cytometry (Chapoval et al., 2009; Cleret et al., 2007; Lambrecht et al., 2000; K. Y. Vermaelen et al., 2001; K. Y. Vermaelen et al., 2003). When DC acquire Ag, they become mature, upregulate their adhesion and costimulatory molecule expression, and specifically regulate chemokine receptor expression what makes them attracted to the lymphoid tissue (McColl, 2002). Ag-stimulated peripheral organ/tissue DC which downregulated inflammatory chemokine expression and upregulated CCR7 migrate to the local lymphoid tissue. These cells are called activated DC (McColl, 2002).

#### **3.1 DC activation and maturation**

As mentioned above, DC populate the lung tissue in the immature state (K. Vermaelen & Pauwels, 2005). Immature DC display high endocytic abilities and low T cell stimulation activity (Figure 1). After antigen uptake, DC undergo a complex process called maturation when they upregulate adhesion and costimulatory molecule expression and cytokine production but shut down the antigen-capturing activity. The DC conversion from immature to mature state is accompanied by a marked cellular reorganization (Shin et al., 2006) which can be detected by flow cytometry. This includes the redistribution of MHC II molecules from late endosomal and lysosomal compartments to the plasma membrane (Trombetta & Mellman, 2005; Turley et al., 2000). In addition, as mentioned above, DC downregulate their Ag uptake ability. This downregulation of some forms of endocytosis slows the clearance of MHC II from the cell surface (Sallusto, Cella, Danieli, & Lanzavecchia, 1995; Wilson, El-Sukkari, & Villadangos, 2004). Shin J-S and associates have determined the regulation of surface MHC II (Shin et al., 2006) employing bone marrow-derived DC expressing WT MHCII or mutant MHCIIβ (where single lysine of β-chain cytoplasmic domain was replaced with arginine, K>R) in flow cytometry. They have demonstrated that the MHC II β-chain cytoplasmic tail is ubiquitinated in mouse immature DC. Although only partly required for the sequestration MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II was significantly downregulated with DC maturation, resulting in the cell surface accumulation of MHC II. Therefore, DC demonstrate a unique ability to regulate MHC II surface expression by selectively controlling MHC II ubiquitination.

#### **3.2 Costimulatory molecule expression and function**

In steady state conditions, lung cDC are immature and express CD11c, CD11b, low MHCII and low DEC-205 (Chapoval et al., 2009). The levels of costimulatory molecule expression on these cells vary from low to absent. To determine the effect of lung VEGF expression on local DC maturation state, we performed a flow cytometry analysis of lung digest single cell suspensions using the following marker-specific Abs for detection of costimulatory molecule expression on lung DC: anti-CD40-PE (3.23), anti-CD54-PE (3E2), anti-CD80-PE (16-10A1), anti-CD86-PE (GL1) PE-labeled, all from BD Biosciences. Anti-B7h/ICOS-L (HK5.3) Ab was obtained from eBioscience (Bhandari et al., 2006; Lee et al., 2004). PEconjugated rat IgG2a (R35-95) and rat IgG2b (R35-38) were used as isotype controls. VEGF expression induced lung cDC activation as they upregulate MHCII, CD40, CD80, CD86, and CD54 expression on their surface (Lee et al., 2004).

#### **3.3 In vivo antigen uptake**

252 Flow Cytometry – Recent Perspectives

As mentioned above, the crucial functions of DC are to capture antigens throughout the body, migrate to the local lymphoid organs to deliver Ag and stimulate or tolerize T cells. The in vitro and in vivo Ag sampling by DC can be easily and specifically measured by flow cytometry (Chapoval et al., 2009; Cleret et al., 2007; Lambrecht et al., 2000; K. Y. Vermaelen et al., 2001; K. Y. Vermaelen et al., 2003). When DC acquire Ag, they become mature, upregulate their adhesion and costimulatory molecule expression, and specifically regulate chemokine receptor expression what makes them attracted to the lymphoid tissue (McColl, 2002). Ag-stimulated peripheral organ/tissue DC which downregulated inflammatory chemokine expression and upregulated CCR7 migrate to the local lymphoid tissue. These

As mentioned above, DC populate the lung tissue in the immature state (K. Vermaelen & Pauwels, 2005). Immature DC display high endocytic abilities and low T cell stimulation activity (Figure 1). After antigen uptake, DC undergo a complex process called maturation when they upregulate adhesion and costimulatory molecule expression and cytokine production but shut down the antigen-capturing activity. The DC conversion from immature to mature state is accompanied by a marked cellular reorganization (Shin et al., 2006) which can be detected by flow cytometry. This includes the redistribution of MHC II molecules from late endosomal and lysosomal compartments to the plasma membrane (Trombetta & Mellman, 2005; Turley et al., 2000). In addition, as mentioned above, DC downregulate their Ag uptake ability. This downregulation of some forms of endocytosis slows the clearance of MHC II from the cell surface (Sallusto, Cella, Danieli, & Lanzavecchia, 1995; Wilson, El-Sukkari, & Villadangos, 2004). Shin J-S and associates have determined the regulation of surface MHC II (Shin et al., 2006) employing bone marrow-derived DC expressing WT MHCII or mutant MHCIIβ (where single lysine of β-chain cytoplasmic domain was replaced with arginine, K>R) in flow cytometry. They have demonstrated that the MHC II β-chain cytoplasmic tail is ubiquitinated in mouse immature DC. Although only partly required for the sequestration MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II was significantly downregulated with DC maturation, resulting in the cell surface accumulation of MHC II. Therefore, DC demonstrate a unique ability to regulate MHC II surface expression by

In steady state conditions, lung cDC are immature and express CD11c, CD11b, low MHCII and low DEC-205 (Chapoval et al., 2009). The levels of costimulatory molecule expression on these cells vary from low to absent. To determine the effect of lung VEGF expression on local DC maturation state, we performed a flow cytometry analysis of lung digest single cell suspensions using the following marker-specific Abs for detection of costimulatory molecule expression on lung DC: anti-CD40-PE (3.23), anti-CD54-PE (3E2), anti-CD80-PE (16-10A1), anti-CD86-PE (GL1) PE-labeled, all from BD Biosciences. Anti-B7h/ICOS-L (HK5.3) Ab was obtained from eBioscience (Bhandari et al., 2006; Lee et al., 2004). PEconjugated rat IgG2a (R35-95) and rat IgG2b (R35-38) were used as isotype controls. VEGF

**3. Functional characteristics of lung DC**

cells are called activated DC (McColl, 2002).

selectively controlling MHC II ubiquitination.

**3.2 Costimulatory molecule expression and function**

**3.1 DC activation and maturation**

To track the Ag uptake by lung DC in vivo, we applied 1 μg/50 μl/mouse of OVA-FITC i.n. to WT and VEGF tg mice one time (Chapoval et al., 2009). Lung tissue and local LN digests were analyzed by flow cytometry 6h and 24h after Ag application for FITC+ cells using CD11c/MHCII/CD11b markers. We observed an increase in FITC+ DC but not Mac-1+ cell number in VEGF tg lungs by flow cytometry. Therefore, intermediately mature cDC obtained from the lung of VEGF tg mice were more efficient in Ag uptake.

Intratracheal instillations of OVA-FITC to mice with different deficiency can be used to assess the effect of such deficiency on lung DC migration. As an example, Vermaelen and associates (K. Y. Vermaelen et al., 2003) used OVA-FITC in MMP-9-/- mice to evaluate the effect of MMP-9 deficiency on lung DC migration. The study has shown that FITC+ cells gradually accumulate in the draining lymph nodes with a peak reaching at 24h of Ag application with no difference in lung DC migration between WT and MMP-9-/- mice.

#### **4. Lung DC sorting**

Highly pure isolated populations of DC are needed to evaluate their exact biological properties and function. Despite the fact that most lung DC represent rare populations among other lung cells, the high level of DC purification could be reached with either immunomagnetic (f.e. MACS technology, Milenyi Biotech) cell separation or flow cytometry-based cell sorts.

#### **4.1 Methods of sorting**

Lung conventional DC were sorted using a triple marker combination (CD11c/MHCII/ CD11b) employing BD FACS Vantage or BD FACS Aria (both equipped with FACSDiva software), or Dako MoFlo (Summit software) high speed automated cell sorters (Chapoval et al., 2009) which all provide the state-of-the-art advances in the instrument set-up, cell sorting and integrated cell analysis. Autofluorescent macrophages and cell doublets are eliminated from further analysis by proper gating (Chapoval et al., 2009). The cDC sorting strategy is shown in Figure 8.

#### **4.2 Analyses of sorted lung DC**

Sorted lung DC then could be used in different in vitro assays to define the specific protein expression which could be further analyzed by flow cytometry (Beaty et al., 2007; Chapoval et al., 2009; K. Y. Vermaelen et al., 2001). The in vitro Ag uptake is an important technique to define the maturation status of DC and its functional activity. To study the in vitro Ag uptake, we subjected lung cells and sorted lung cDC to the in vitro cultures with or without increasing doses of OVA-FITC (Molecular Probes) ranging from 0.01 mg/ml to 1 mg/ml in RPMI (Life Technologies) in 24-well plates (Costar) for 30 min at 37ºC. After incubation, cells were extensively washed with RPMI medium and analyzed for Ag uptake by flow cytometry. VEGF tg mDC are significantly more efficient in Ag uptake with low doses of Ag used. For example, at 10 μg of Ag only 30.2 % of WT DC were FITC+ whereas for VEGF tg

Flow Cytometry-Based Analysis and Sorting of Lung Dendritic Cells 255

In addition to the fluorescently-labeled Ag application directly to the airways to study the lung DC migration, lung sorted or bone marrow-derived labelled DC can be introduced to the airways to track their migration with a use of flow cytometry (Lambrecht et al., 2000). This approach helps to distinguish the lymph node resident DC and DC migrated there from the lung. Bone marrow CD11c+MHCII+CD86+HSA+CD44+ICAM-1+DC were pulsed with CFSE (carboxyfluoroscein succinimidyl ester) and 1x106 cells were instilled intratracheally into naive mice. DC were traced in the lungs and mediastinal lymph nodes at 12h, 36h, and 120h after application. The authors have demonstrated that CFSE+ DC could be detected in BALF, digested lung tissues, and draining lymph nodes by 12h after instillation. By 36h CFSE+DC disappear from the BALF and intensively accumulate in the draining lymph nodes. A majority of injected cells had disappeared by 120h. Another study using this approach has demonstrated that by 24h approximately 2% of draining lymph node cells were CFSE+DC migrated from the lungs (Legge & Braciale, 2003). These CFSE+ DC were more mature than local DC as they showed higher levels of costimulatory

molecule expression (CD40, CD80, and CD86) and upregulated MHCII expression.

Intratracheal adoptive transfer of CFSE-labeled exogenous DC has been used to study the effect of MMP-9 deficiency on DC trafficking from the lung to the mediastinal lymph nodes (K. Y. Vermaelen et al., 2003). Measuring the absolute numbers of FITC+ cells in the lymph nodes, they demonstrated no differences between MMP-9+ and MMP-9- DC reaching

In conclusion, we show and discuss in this chapter a high phenotypic and functional complexity of lung DC. None of DC subpopulation can be identified and isolated by cell sorting for further analysis based on one or two marker expression. Specific Abs at least to three-four molecules needed to be used simultaneously for a clear lung DC subpopulation differentiation. Multicolor flow cytometry measuring simultaneously 5 or more parameters has dramatically increased our ability in DC subtype biology characterization. However, the complexity of such analysis increases with the number of fluorochromes used. The use of appropriate gating strategy, inclusion of all necessary controls from the beginning of the experiment and all necessary isotype controls for the specific Abs used in the assay, proper instrument validation, correct compensation will highly contribute to obtaining relevant

Alvarez, D., Vollmann, E. H., & von Andrian, U. H. (2008). Mechanisms and consequences

of dendritic cell migration. *Immunity, 29*(3), 325-342.

**4.4 In vivo DC migration study using sorted labeled cells**

lymph nodes

**5. Summary**

biological data.

**7. References**

**6. Acknowledgment**

S.P.C. is supported by NIAID R21AI076736 grant.

DC this number increased to 73.9%. At high dose both WT and tg cDC are equally efficient in Ag uptake.

#### **4.3 Intracellular staining for cytokine and chemokine expression by DC**

Chemokine production by lung CD11c+ cells was studied using intracellular staining (Beaty et al., 2007). Lung DC were sorted using anti-CD11c-conjugated magnetic microbeads (Miltenyi Biotec). pDC were enriched from the flow-through CD11c- cells using antimPDCA-1 magnetic microbeads and further sorted as PDCA-1+B220+I-Aint low scattered cells. CD11c+ cell fraction was further separated into subpopulations (CD103+ DC and CD11bhiDC) by cell sorting on Vantage SE sorter with FACSDiva sofrware (BD Biosciences). The authors demonstrated that lung DC even without stimulation have accumulated detectable amounts of many chemokines and cytokines. Readily detectable were MIP-2, IP-10, MIP-1a, MIP-1b, RANTES, CXCL16, C10, TARC, and MDC for CD11bhi DC. Lung CD103+ DC expressed MIP-1a, CXCL16, TARC, and MDC but at much less levels. Therefore, the lung CD11bhi DC and CD103+ DC differentially express chemokines in naive mice. This difference further deepens with corresponding cell activation.

Fig. 8. Conventional lung DC acquisition and morphology. Single lung cell suspensions obtained from WT and VEGF tg mice (n=4-5 mice per experiment) were prepared with an omitting the enzymatic digestions step (Chapoval et al., 2009; Piggott et al., 2005). Cell were stained with anti-CD11c, -CD11b, and –MHCII Abs and analyzed using either FACSDiva or Summit software on cell sorters. Assigned cell populations were sorted and analyzed morphologically by Diff-Quick stain of cytospin slides prepared with 100 ml of sorted cells. The sorted cDC selected for further analysis represent CD11c+MHCIIlow population in WT mice and CD11c+MHCIIhigh population of cells in VEGF tg mice. The cells in gate 4 (CD11cintermed/highMHCIIhigh) displayed a granulocyte-like morphology

#### **4.4 In vivo DC migration study using sorted labeled cells**

In addition to the fluorescently-labeled Ag application directly to the airways to study the lung DC migration, lung sorted or bone marrow-derived labelled DC can be introduced to the airways to track their migration with a use of flow cytometry (Lambrecht et al., 2000). This approach helps to distinguish the lymph node resident DC and DC migrated there from the lung. Bone marrow CD11c+MHCII+CD86+HSA+CD44+ICAM-1+DC were pulsed with CFSE (carboxyfluoroscein succinimidyl ester) and 1x106 cells were instilled intratracheally into naive mice. DC were traced in the lungs and mediastinal lymph nodes at 12h, 36h, and 120h after application. The authors have demonstrated that CFSE+ DC could be detected in BALF, digested lung tissues, and draining lymph nodes by 12h after instillation. By 36h CFSE+DC disappear from the BALF and intensively accumulate in the draining lymph nodes. A majority of injected cells had disappeared by 120h. Another study using this approach has demonstrated that by 24h approximately 2% of draining lymph node cells were CFSE+DC migrated from the lungs (Legge & Braciale, 2003). These CFSE+ DC were more mature than local DC as they showed higher levels of costimulatory molecule expression (CD40, CD80, and CD86) and upregulated MHCII expression.

Intratracheal adoptive transfer of CFSE-labeled exogenous DC has been used to study the effect of MMP-9 deficiency on DC trafficking from the lung to the mediastinal lymph nodes (K. Y. Vermaelen et al., 2003). Measuring the absolute numbers of FITC+ cells in the lymph nodes, they demonstrated no differences between MMP-9+ and MMP-9- DC reaching lymph nodes

#### **5. Summary**

254 Flow Cytometry – Recent Perspectives

DC this number increased to 73.9%. At high dose both WT and tg cDC are equally efficient

Chemokine production by lung CD11c+ cells was studied using intracellular staining (Beaty et al., 2007). Lung DC were sorted using anti-CD11c-conjugated magnetic microbeads (Miltenyi Biotec). pDC were enriched from the flow-through CD11c- cells using antimPDCA-1 magnetic microbeads and further sorted as PDCA-1+B220+I-Aint low scattered cells. CD11c+ cell fraction was further separated into subpopulations (CD103+ DC and CD11bhiDC) by cell sorting on Vantage SE sorter with FACSDiva sofrware (BD Biosciences). The authors demonstrated that lung DC even without stimulation have accumulated detectable amounts of many chemokines and cytokines. Readily detectable were MIP-2, IP-10, MIP-1a, MIP-1b, RANTES, CXCL16, C10, TARC, and MDC for CD11bhi DC. Lung CD103+ DC expressed MIP-1a, CXCL16, TARC, and MDC but at much less levels. Therefore, the lung CD11bhi DC and CD103+ DC differentially express chemokines in naive

**4.3 Intracellular staining for cytokine and chemokine expression by DC**

mice. This difference further deepens with corresponding cell activation.

Fig. 8. Conventional lung DC acquisition and morphology. Single lung cell suspensions obtained from WT and VEGF tg mice (n=4-5 mice per experiment) were prepared with an omitting the enzymatic digestions step (Chapoval et al., 2009; Piggott et al., 2005). Cell were stained with anti-CD11c, -CD11b, and –MHCII Abs and analyzed using either FACSDiva or Summit software on cell sorters. Assigned cell populations were sorted and analyzed morphologically by Diff-Quick stain of cytospin slides prepared with 100 ml of sorted cells. The sorted cDC selected for further analysis represent CD11c+MHCIIlow population in WT mice and CD11c+MHCIIhigh population of cells in VEGF tg mice. The cells in gate 4

(CD11cintermed/highMHCIIhigh) displayed a granulocyte-like morphology

in Ag uptake.

In conclusion, we show and discuss in this chapter a high phenotypic and functional complexity of lung DC. None of DC subpopulation can be identified and isolated by cell sorting for further analysis based on one or two marker expression. Specific Abs at least to three-four molecules needed to be used simultaneously for a clear lung DC subpopulation differentiation. Multicolor flow cytometry measuring simultaneously 5 or more parameters has dramatically increased our ability in DC subtype biology characterization. However, the complexity of such analysis increases with the number of fluorochromes used. The use of appropriate gating strategy, inclusion of all necessary controls from the beginning of the experiment and all necessary isotype controls for the specific Abs used in the assay, proper instrument validation, correct compensation will highly contribute to obtaining relevant biological data.

#### **6. Acknowledgment**

S.P.C. is supported by NIAID R21AI076736 grant.

#### **7. References**

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Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function.

Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar

Mellman, I. (2000). Transport of peptide-MHC class II complexes in developing

Drexhage, H. A. (1994). Dendritic cells and their precursors isolated from human bronchoalveolar lavage: immunocytologic and functional properties. *Am J Respir*

pulmonary dendritic cell and macrophage populations by flow cytometry:

migratory dendritic cells rapidly transport antigen from the airways to the thoracic

(2003). Matrix metalloproteinase-9-mediated dendritic cell recruitment into the airways is a critical step in a mouse model of asthma. *J Immunol, 171*(2), 1016-

present self antigens in their immature state in vivo and regulate antigen presentation by controlling the rates of MHC class II synthesis and endocytosis.

Masten, B. J., Olson, G. K., Kusewitt, D. F., & Lipscomb, M. F. (2004). Flt3 ligand

Masten, B. J., Olson, G. K., Tarleton, C. A., Rund, C., Schuyler, M., Mehran, R., . . . Lipscomb,

McColl, S. R. (2002). Chemokines and dendritic cells: a crucial alliance. *Immunol Cell Biol,*

Metlay, J. P., Witmer-Pack, M. D., Agger, R., Crowley, M. T., Lawless, D., & Steinman,

Niu, N., Le Goff, M. K., Li, F., Rahman, M., Homer, R. J., & Cohn, L. (2007). A novel pathway

Piggott, D. A., Eisenbarth, S. C., Xu, L., Constant, S. L., Huleatt, J. W., Herrick, C. A., &

Pollard, A. M., & Lipscomb, M. F. (1990). Characterization of murine lung dendritic cells:

Ray, P., Tang, W., Wang, P., Homer, R., Kuhn, C., Flavell, R. A., Elias, J. A. (1997). Regulated

dependent and -independent phenotypes. *J Clin Invest*, 100(10), 2501–2511. Ritz, S. A., Cundall, M. J., Gajewska, B. U., Swirski, F. K., Wiley, R. E., Alvarez, D., . . .

Sallusto, F., Cella, M., Danieli, C., & Lanzavecchia, A. (1995). Dendritic cells use

Sertl, K., Takemura, T., Tschachler, E., Ferrans, V. J., Kaliner, M. A., & Shevach, E. M. (1986).

Shin, J. S., Ebersold, M., Pypaert, M., Delamarre, L., Hartley, A., & Mellman, I. (2006).

Solovjov, D. A., Pluskota, E., & Plow, E. F. (2005). Distinct roles for the alpha and

Spencer, S. C., & Fabre, J. W. (1990). Characterization of the tissue macrophage and the

the lungs of mice. *J Immunol, 172*(7), 4077-4083.

intranasal antigen. *J Clin Invest, 115*(2), 459-467.

antigen in vivo. *Clin Exp Immunol, 138*(2), 213-220.

ubiquitination. *Nature, 444*(7115), 115-118.

tissue of rat heart. *J Exp Med, 171*(6), 1841-1851.

cytokines and bacterial products. *J Exp Med, 182*(2), 389-400.

parenchyma, and visceral pleura. *J Exp Med, 163*(2), 436-451.

human lung. *J Immunol, 177*(11), 7784-7793.

*80*(5), 489-496.

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interstitial dendritic cell as distinct leukocytes normally resident in the connective

**13**

*Iran*

Arash Zaminy

**Stem Cell Characterization**

*Shahid Beheshti University of Medical Sciences, Tehran,*

One of the main problems in stem cell studies is how researchers can identify and

Identification and characterization of stem cells is a difficult and often evolving procedure. Stem cells not only must exhibit the appropriate markers, but also a healthy and robust stem cell population must also lack specific markers. In addition to the difficulty of this area of stem cell biology markers, profiles change based on the species, site of origin and maturity (totipotent vs. multipotent) of a given population. Furthermore, stem cell populations may consist of several specific phenotypes which are often indicators of the population′s general health. Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. It is a widely used method for characterizing and

This chapter tries to explain what stem cells are, as well as to summarize current knowledge

All life forms initiate with a stem cell, which is defined as a cell that has the dual capacity to self-renew and to produce progenitors and different types of specialized cells in the organism. Scientists mostly work with two kinds of stem cells from animals and humans:

At the beginning of human life, one fertilized egg cell – the zygote – divides into two and two becomes four (Carlson, 1996). Within 5 to 7 days, some 40 cells are produced which build up the inner cell mass encircled by an outer cell layer subsequently forming the placenta. At this phase, each of these cells in the inner cell mass has the potential to give rise to all tissue types and organs – that is, these cells are pluripotent. Finally, the cells forming the inner cell mass will give rise to the some 1013 cells that constitute a human body, organized in 200 differentiated cell types (Sadler, 2002). Many somatic, tissue-specific or adult stem cells are produced during the foetal period. These stem cells have a more limited ability than the pluripotent embryonic stem cells (ESCs) and they are multipotent – that is, they have the potential to give rise only to a limited number of cell lineages. These adult

on stem cell characterization and usage of stem cells markers.

embryonic stem cells and non-embryonic "somatic" or "adult" stem cells.

**1. Introduction**

characterize a stem cell.

separating individual cells.

**2. Stem cells**

*Department of Anatomy & Cell Biology,*

Zeng, X., Wert, S.E., Federici, R., Peters, K.G. & Whitsett, J.A. (1998). VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. *Dev Dyn*, 211, 215−227.

### **Stem Cell Characterization**

#### Arash Zaminy

*Department of Anatomy & Cell Biology, Shahid Beheshti University of Medical Sciences, Tehran, Iran*

#### **1. Introduction**

260 Flow Cytometry – Recent Perspectives

Zeng, X., Wert, S.E., Federici, R., Peters, K.G. & Whitsett, J.A. (1998). VEGF enhances

215−227.

pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. *Dev Dyn*, 211,

One of the main problems in stem cell studies is how researchers can identify and characterize a stem cell.

Identification and characterization of stem cells is a difficult and often evolving procedure. Stem cells not only must exhibit the appropriate markers, but also a healthy and robust stem cell population must also lack specific markers. In addition to the difficulty of this area of stem cell biology markers, profiles change based on the species, site of origin and maturity (totipotent vs. multipotent) of a given population. Furthermore, stem cell populations may consist of several specific phenotypes which are often indicators of the population′s general health. Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. It is a widely used method for characterizing and separating individual cells.

This chapter tries to explain what stem cells are, as well as to summarize current knowledge on stem cell characterization and usage of stem cells markers.

#### **2. Stem cells**

All life forms initiate with a stem cell, which is defined as a cell that has the dual capacity to self-renew and to produce progenitors and different types of specialized cells in the organism. Scientists mostly work with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells.

At the beginning of human life, one fertilized egg cell – the zygote – divides into two and two becomes four (Carlson, 1996). Within 5 to 7 days, some 40 cells are produced which build up the inner cell mass encircled by an outer cell layer subsequently forming the placenta. At this phase, each of these cells in the inner cell mass has the potential to give rise to all tissue types and organs – that is, these cells are pluripotent. Finally, the cells forming the inner cell mass will give rise to the some 1013 cells that constitute a human body, organized in 200 differentiated cell types (Sadler, 2002). Many somatic, tissue-specific or adult stem cells are produced during the foetal period. These stem cells have a more limited ability than the pluripotent embryonic stem cells (ESCs) and they are multipotent – that is, they have the potential to give rise only to a limited number of cell lineages. These adult

Stem Cell Characterization 263

beam. Now, multiple fluorescent labels are available with emitted light that differ in colour

Researchers exploit the combination of the chemical properties of fluorescence and unique receptor patterns on cell surfaces to identify specific numbers of stem cells. One approach for using markers is a technique known as fluorescence-activated cell sorting (FACS) (Bonner et al., 1972; Herzenberg, 2000; Julius, 1972). Researchers frequently use a FACS instrument to sort out the rare stem cells from the millions of other cells. By this method, a suspension of tagged cells (i.e. fluorescently-labelled antibodies are bound to the cell surface markers) is sent under pressure through a very fine nozzle. Upon exiting the nozzle cells pass through a light source, usually a laser, and then through an electric field. Operators apply a series of criteria. If the cell stream meets the criteria, they become negatively or positively charged. When cells are passing among an electric field, the charge difference permits the desired cells to be separated from other cells. The researchers now have a population of cells that have all of the same marker characteristics and with these cells they

A second method uses stem cell markers and fluorescent antibodies to visually assess cells as they exist in tissues. Often researchers want to assess how stem cells appear in tissues and in doing so they use a microscope to evaluate them rather than the FACS instrument. In this case, a thin slice of tissue is prepared and the stem cell markers are tagged by the antibodies that have the fluorescent tag attached. The fluorescent tags are then activated either by special light energy or a chemical reaction. The stem cells will emit a fluorescent light that

An embryonic stem cell (ESC) is described by its origin. It is obtained from the blastocyst stage of the embryo. Embryonic stem cells are unique cell populations with the capability of both self-renewal and differentiation, and thus ESCs can give rise to any adult cell type. Pluripotent embryonic stem (ES) cells, like embryonal carcinoma cells, were rst used as a tool to examine thoroughly early differentiation. However, the properties of ESCs identify them as being highly appropriate for making specic cell lineages in vitro. The ability of embryonic stem cells to almost limitless self-renewal and differentiation capacity has opened up the panorama of widespread applications in biomedical research and

ESCs are harvested from the inner cell mass of the pre-implantation blastocyst and have been derived from rodents (Martin, 1981; Evans & Kaufman, 1981; Doetschman et al., 1988; Graves & Moreadith, 1993), primates (Thomson et al., 1995) and humans (Thomson et al.,

Some approaches have been applied to characterize ESCs, but the most widely used approach is analysis of cell surface antigens by flow cytometry and evaluation of gene expression profile by RT-PCR or microarrays. Many cell surface antigens used to identify hESCs were first detected with antibodies prepared against pre-implantation mouse embryos and/or against mouse or human embryonal carcinoma cells (Pera et al., 2000).

and intensity.

can conduct their research.

**4. Embryonic stem cells**

regenerative medicine.

1998; Reubinoff et al., 2000).

**4.1 Embryonic stem cell markers**

can easily be seen under the microscope.

stem cells keep on in the related organs to varying degrees over the whole of a person's lifetime.

Stem cells are well-known from other cell types because of two important characteristics. First, they are unspecialized cells and the have ability to renew themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special abilities (Thrasher, 1966; Merok & Sherley, 2001). In some organs, such as the gut and bone marrow, stem cells divide regularly to repair and restore exhausted or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

#### **3. Stem cells markers**

In recent years researchers have revealed a broad range of stem cells that have unique capabilities to self-renew, grow indefinitely and differentiate or develop into multiple kinds of cells and tissues. Researchers now know that many different types of stem cells exist, but they are all found in very small populations in the human body, in some cases one stem cell in 100,000 cells in circulating blood. In addition, when scientists study these cells under a microscope, they are similar to other cells in the tissue where they are found. So, like the search for a needle in a haystack, how do scientists recognize these uncommon types of cells found in many different cells and tissues? The answer is rather simple, thanks to stem cell "markers."

What are stem cell markers? The surface of every cell in the body has specialized proteins called receptors that have the capability of selectively binding or adhering to other "signalling" molecules. There are many different types of receptors that differ in their composition and affinity for the signalling molecules. Generally, cells use these receptors and the molecules that bind to them as a way of communicating with other cells, and to perform their correct functions in the body. These cell surface receptors are commonly used as cell markers. Each cell type, for example a liver cell, has a certain combination of receptors on their surface that makes them distinguishable from other types of cells. Scientists have taken advantage of the biological exclusivity of stem cell receptors and the chemical properties of certain compounds to label or mark cells. Researchers owe much of the past success in finding and characterizing stem cells to the use of markers.

Stem cell markers are given shorthand names based on the molecules that attach to the stem cell surface receptors. For example, a cell that has the receptor stem cell antigen -1 on its surface is known as Sca-1. In many cases, a mixture of multiple markers is used to identify a particular stem cell type. So now, researchers often identify stem cells in shorthand by a combination of marker names reflecting the presence (+) or absence (-) of them. For example, a special type of haematopoietic stem cell from blood and bone marrow is described as (CD34-/low, c-Kit+, Sca-1+) (Jackson et al., 2001).

Researchers employ antibody molecules that selectively bind with the receptors on the surface of the cell as a way to identify stem cells. In former years a method was developed to attach to the antibody molecule another molecule (or tag) that has the ability to fluoresce or emit light energy when triggered by an energy source such as an ultraviolet light or laser

stem cells keep on in the related organs to varying degrees over the whole of a person's

Stem cells are well-known from other cell types because of two important characteristics. First, they are unspecialized cells and the have ability to renew themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special abilities (Thrasher, 1966; Merok & Sherley, 2001). In some organs, such as the gut and bone marrow, stem cells divide regularly to repair and restore exhausted or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide

In recent years researchers have revealed a broad range of stem cells that have unique capabilities to self-renew, grow indefinitely and differentiate or develop into multiple kinds of cells and tissues. Researchers now know that many different types of stem cells exist, but they are all found in very small populations in the human body, in some cases one stem cell in 100,000 cells in circulating blood. In addition, when scientists study these cells under a microscope, they are similar to other cells in the tissue where they are found. So, like the search for a needle in a haystack, how do scientists recognize these uncommon types of cells found in many different cells and tissues? The answer is rather simple, thanks to stem cell

What are stem cell markers? The surface of every cell in the body has specialized proteins called receptors that have the capability of selectively binding or adhering to other "signalling" molecules. There are many different types of receptors that differ in their composition and affinity for the signalling molecules. Generally, cells use these receptors and the molecules that bind to them as a way of communicating with other cells, and to perform their correct functions in the body. These cell surface receptors are commonly used as cell markers. Each cell type, for example a liver cell, has a certain combination of receptors on their surface that makes them distinguishable from other types of cells. Scientists have taken advantage of the biological exclusivity of stem cell receptors and the chemical properties of certain compounds to label or mark cells. Researchers owe much of

Stem cell markers are given shorthand names based on the molecules that attach to the stem cell surface receptors. For example, a cell that has the receptor stem cell antigen -1 on its surface is known as Sca-1. In many cases, a mixture of multiple markers is used to identify a particular stem cell type. So now, researchers often identify stem cells in shorthand by a combination of marker names reflecting the presence (+) or absence (-) of them. For example, a special type of haematopoietic stem cell from blood and bone marrow is

Researchers employ antibody molecules that selectively bind with the receptors on the surface of the cell as a way to identify stem cells. In former years a method was developed to attach to the antibody molecule another molecule (or tag) that has the ability to fluoresce or emit light energy when triggered by an energy source such as an ultraviolet light or laser

the past success in finding and characterizing stem cells to the use of markers.

described as (CD34-/low, c-Kit+, Sca-1+) (Jackson et al., 2001).

lifetime.

"markers."

under special conditions.

**3. Stem cells markers**

beam. Now, multiple fluorescent labels are available with emitted light that differ in colour and intensity.

Researchers exploit the combination of the chemical properties of fluorescence and unique receptor patterns on cell surfaces to identify specific numbers of stem cells. One approach for using markers is a technique known as fluorescence-activated cell sorting (FACS) (Bonner et al., 1972; Herzenberg, 2000; Julius, 1972). Researchers frequently use a FACS instrument to sort out the rare stem cells from the millions of other cells. By this method, a suspension of tagged cells (i.e. fluorescently-labelled antibodies are bound to the cell surface markers) is sent under pressure through a very fine nozzle. Upon exiting the nozzle cells pass through a light source, usually a laser, and then through an electric field. Operators apply a series of criteria. If the cell stream meets the criteria, they become negatively or positively charged. When cells are passing among an electric field, the charge difference permits the desired cells to be separated from other cells. The researchers now have a population of cells that have all of the same marker characteristics and with these cells they can conduct their research.

A second method uses stem cell markers and fluorescent antibodies to visually assess cells as they exist in tissues. Often researchers want to assess how stem cells appear in tissues and in doing so they use a microscope to evaluate them rather than the FACS instrument. In this case, a thin slice of tissue is prepared and the stem cell markers are tagged by the antibodies that have the fluorescent tag attached. The fluorescent tags are then activated either by special light energy or a chemical reaction. The stem cells will emit a fluorescent light that can easily be seen under the microscope.

#### **4. Embryonic stem cells**

An embryonic stem cell (ESC) is described by its origin. It is obtained from the blastocyst stage of the embryo. Embryonic stem cells are unique cell populations with the capability of both self-renewal and differentiation, and thus ESCs can give rise to any adult cell type. Pluripotent embryonic stem (ES) cells, like embryonal carcinoma cells, were rst used as a tool to examine thoroughly early differentiation. However, the properties of ESCs identify them as being highly appropriate for making specic cell lineages in vitro. The ability of embryonic stem cells to almost limitless self-renewal and differentiation capacity has opened up the panorama of widespread applications in biomedical research and regenerative medicine.

ESCs are harvested from the inner cell mass of the pre-implantation blastocyst and have been derived from rodents (Martin, 1981; Evans & Kaufman, 1981; Doetschman et al., 1988; Graves & Moreadith, 1993), primates (Thomson et al., 1995) and humans (Thomson et al., 1998; Reubinoff et al., 2000).

#### **4.1 Embryonic stem cell markers**

Some approaches have been applied to characterize ESCs, but the most widely used approach is analysis of cell surface antigens by flow cytometry and evaluation of gene expression profile by RT-PCR or microarrays. Many cell surface antigens used to identify hESCs were first detected with antibodies prepared against pre-implantation mouse embryos and/or against mouse or human embryonal carcinoma cells (Pera et al., 2000).

Stem Cell Characterization 265

A haematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate a variety of specialized cells and can mobilize out of the bone marrow into circulating blood. Since HSCs look and behave in culture like ordinary white blood cells, it has been a challenge to identify them by morphology (size and shape). Even now, scientists must rely on cell surface proteins, which generally serve as markers of white blood cells.

HSCs have an identity problem. First, the ones with long-term replicating ability are rare. Second, there are multiple types of stem cells. Third, the stem cells look like many other blood or bone marrow cells. So how do researchers find the desired cell populations? The

A variety of markers has been found to help distinguish and separate HSCs. Early marker efforts focused on cell size, density and recognition by lectins (carbohydrate-binding proteins derived largely from plants) (Bauman et al., 1988), but more recent attempts have focused mostly on cell surface protein markers, as defined by monoclonal antibodies. For mouse HSCs, these markers contain panels of 8 to 14 different monoclonal antibodies that recognize cell surface proteins present on differentiated haematopoietic lineages, such as the red blood cell and macrophage lineages (thus, these markers are collectively referred to as Lin) (Spangrude et al., 1988; Uchid & Weissman, 1992) as well as the proteins Sca-1 (Spangrude et al., 1988; Uchid & Weissman, 1992), CD27 (Weissman et al., 2000), CD34 (Osawa et al., 1996), CD38 (Randall et al., 1996), CD43 (Moore et al., 1994), CD90.1 (Thy-1.1) (Spangrude et al., 1988; Uchid & Weissman, 1992), CD117 (c-Kit) (Ikuta & Weissman, 1992), AA4.1 (Jordan et al., 1996) and MHC class I (Bauman et al., 1988), and CD150 (Kiel et al.,

Human HSCs have been described with respect to staining for CD34 (Civin et al., 1984), CD38 (Kiel et al., 2005), CD43 (Moore et al., 1994), CD45RO (Lansdorp et al., 1990), CD45RA (Lansdorp et al., 1990), CD59 (Hill et al., 1996), CD90 (Bauman et al., 1988), CD109 (Sutherland et al., 1996), CD117 (Gunji et al., 1993), CD133 (Miraglia et al., 1997; Yin et al., 1997), CD166 (Uchida et al., 1997), HLA DR (human) (Srour et al., 1992; Tsukamoto et al., 1995) and lacking expression of lineage (Lin) markers (Baum et al., 1992). It is important to note that lineage markers are cell surface antigens that can be used for immunophenotyping cells of a particular developmental lineage. Cells that do not express these marker antigens,

While none of these markers recognize functional stem cell activity, combinations (typically with 3 to 5 different markers, see examples below) led to the purification of nearhomogenous populations of HSCs. The ability to obtain pure preparations of HSCs, albeit in limited numbers, has greatly facilitated the functional and biochemical characterization of these important cells. However, now there has been limited impact of these discoveries on clinical practice, as highly purified HSCs have only rarely been used to treat patients. The irrefutable advantages of using purified cells (e.g.*,* the absence of contaminating tumour cells in autologous transplantations) have been offset by practical difficulties and increased

HSC assays, when combined with the ability to purify HSCs, have provided increasingly detailed insight into the cells and the early steps involved in the differentiation process.

or express them at very low levels, are said to be lineage marker negative [lin(-)].

most common approach is through markers that emerge on the surface of cells.

**5.1 Haematopoietic stem cell markers**

2005).

purification costs.

Although the functions of those antigens in the continuance of undifferentiated human embryonal carcinoma cells are not necessarily clear, they may represent helpful markers for the recognition of pluripotent stem cells. These antigens include the globo-series glycolipid antigens, stage-specific embryonic antigen-3 (SSEA-3) and -4 (SSEA-4), keratan sulphate antigens TRA-1-60, TRA-1-81, GCTM2 and GCTM343, a set of various protein antigens comprising the two liver alkaline phosphatase antigens TRA-2-54 and TRA-2-49, Thy1, CD9, HLA class 1 antigens, Oct3/4, Nanog and the absence of hESC negative markers, such as SSEA-1 (Pera et al., 2000; Carpenter et al., 2003; Chambers et al., 2003; Draper et al., 2004; Heins et al., 2004; Nichols et al., 1998).

Some cell surface biomarkers are also listed in Table 1. The International Stem Cell Initiative (ISCI) established by the International Stem Cell Forum (http://www.stemcellforum.org.uk) carried out a comparative study of a large and different set of hESC lines derived from and maintained in different laboratories worldwide (Adewumi et al., 2007). Fifty-nine independent hESC lines derived from 17 laboratories in 11 countries were investigated for the expression of 17 cell surface antigens and 93 genes, which have been chosen as potential markers of undifferentiated stem cells or their differentiated derivatives (Adewumi et al., 2007). All of the independent hESC lines displayed a common expression profile for a specific set of marker antigens, despite the fact that they had different genetic backgrounds and were produced by different techniques in each laboratory. All examined cell lines expressed a comparable spectrum of cell surface marker antigens characteristic of hESCs, suggesting that there is a common set of markers that can be used to monitor, in general, the presence of pluripotent stem cells. SSEA-3 and SSEA-4 were expressed in all hESCs tests, indicating that these molecules are valuable operational markers of this cell type; however, a study revealed that they are not necessary for the pluripotency of hESCs (Brimble et al., 2007).

Table 1. Marker expression and growth properties of mouse and primate pluripotent cells

#### **5. Haematopoietic stem cells**

Blood cells are responsible for continuous preservation and immune protection of every cell type of the body. This persistent and brutal work requires blood cells, along with skin cells, to have the greatest power of self-renewal of any adult tissue. The stem cells that form blood and immune cells are known as haematopoietic stem cells (HSCs). HSCs are among the best characterized adult stem cells and the only stem cells being regularly used in clinics.

Although the functions of those antigens in the continuance of undifferentiated human embryonal carcinoma cells are not necessarily clear, they may represent helpful markers for the recognition of pluripotent stem cells. These antigens include the globo-series glycolipid antigens, stage-specific embryonic antigen-3 (SSEA-3) and -4 (SSEA-4), keratan sulphate antigens TRA-1-60, TRA-1-81, GCTM2 and GCTM343, a set of various protein antigens comprising the two liver alkaline phosphatase antigens TRA-2-54 and TRA-2-49, Thy1, CD9, HLA class 1 antigens, Oct3/4, Nanog and the absence of hESC negative markers, such as SSEA-1 (Pera et al., 2000; Carpenter et al., 2003; Chambers et al., 2003; Draper et al., 2004;

Some cell surface biomarkers are also listed in Table 1. The International Stem Cell Initiative (ISCI) established by the International Stem Cell Forum (http://www.stemcellforum.org.uk) carried out a comparative study of a large and different set of hESC lines derived from and maintained in different laboratories worldwide (Adewumi et al., 2007). Fifty-nine independent hESC lines derived from 17 laboratories in 11 countries were investigated for the expression of 17 cell surface antigens and 93 genes, which have been chosen as potential markers of undifferentiated stem cells or their differentiated derivatives (Adewumi et al., 2007). All of the independent hESC lines displayed a common expression profile for a specific set of marker antigens, despite the fact that they had different genetic backgrounds and were produced by different techniques in each laboratory. All examined cell lines expressed a comparable spectrum of cell surface marker antigens characteristic of hESCs, suggesting that there is a common set of markers that can be used to monitor, in general, the presence of pluripotent stem cells. SSEA-3 and SSEA-4 were expressed in all hESCs tests, indicating that these molecules are valuable operational markers of this cell type; however, a study revealed that they are not necessary for the pluripotency of hESCs (Brimble et al.,

> SSEA-1 + - SSEA-3 - + SSEA-4 - + TRA-1-60 - + TRA-1-81 - + GCTM-2 - + Alkaline phosphatase + + Oct-4 + + GDF-3 + ?

Table 1. Marker expression and growth properties of mouse and primate pluripotent cells

Blood cells are responsible for continuous preservation and immune protection of every cell type of the body. This persistent and brutal work requires blood cells, along with skin cells, to have the greatest power of self-renewal of any adult tissue. The stem cells that form blood and immune cells are known as haematopoietic stem cells (HSCs). HSCs are among the best

characterized adult stem cells and the only stem cells being regularly used in clinics.

**5. Haematopoietic stem cells**

2007).

A haematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate a variety of specialized cells and can mobilize out of the bone marrow into circulating blood. Since HSCs look and behave in culture like ordinary white blood cells, it has been a challenge to identify them by morphology (size and shape). Even now, scientists must rely on cell surface proteins, which generally serve as markers of white blood cells.

#### **5.1 Haematopoietic stem cell markers**

HSCs have an identity problem. First, the ones with long-term replicating ability are rare. Second, there are multiple types of stem cells. Third, the stem cells look like many other blood or bone marrow cells. So how do researchers find the desired cell populations? The most common approach is through markers that emerge on the surface of cells.

A variety of markers has been found to help distinguish and separate HSCs. Early marker efforts focused on cell size, density and recognition by lectins (carbohydrate-binding proteins derived largely from plants) (Bauman et al., 1988), but more recent attempts have focused mostly on cell surface protein markers, as defined by monoclonal antibodies. For mouse HSCs, these markers contain panels of 8 to 14 different monoclonal antibodies that recognize cell surface proteins present on differentiated haematopoietic lineages, such as the red blood cell and macrophage lineages (thus, these markers are collectively referred to as Lin) (Spangrude et al., 1988; Uchid & Weissman, 1992) as well as the proteins Sca-1 (Spangrude et al., 1988; Uchid & Weissman, 1992), CD27 (Weissman et al., 2000), CD34 (Osawa et al., 1996), CD38 (Randall et al., 1996), CD43 (Moore et al., 1994), CD90.1 (Thy-1.1) (Spangrude et al., 1988; Uchid & Weissman, 1992), CD117 (c-Kit) (Ikuta & Weissman, 1992), AA4.1 (Jordan et al., 1996) and MHC class I (Bauman et al., 1988), and CD150 (Kiel et al., 2005).

 It is important to note that lineage markers are cell surface antigens that can be used for immunophenotyping cells of a particular developmental lineage. Cells that do not express these marker antigens, or express them at very low levels, are said to be lineage marker negative [lin(-)].

While none of these markers recognize functional stem cell activity, combinations (typically with 3 to 5 different markers, see examples below) led to the purification of nearhomogenous populations of HSCs. The ability to obtain pure preparations of HSCs, albeit in limited numbers, has greatly facilitated the functional and biochemical characterization of these important cells. However, now there has been limited impact of these discoveries on clinical practice, as highly purified HSCs have only rarely been used to treat patients. The irrefutable advantages of using purified cells (e.g.*,* the absence of contaminating tumour cells in autologous transplantations) have been offset by practical difficulties and increased purification costs.

HSC assays, when combined with the ability to purify HSCs, have provided increasingly detailed insight into the cells and the early steps involved in the differentiation process.

Stem Cell Characterization 267

The SP fraction expresses an ABC transporter, Bcrp-1(ABCG2), on the cell surface and this transporter contributes to efflux of the Hoechst dye from the cells, leading to low levels of staining (Zhou et al., 2001). Interestingly, the bone marrow and peripheral immune system in ABCG2 transporter knockouts animals, is normal, suggesting that the capability to efflux Hoechst 33342 is characteristic of stem cells, but not essential for function (Uchida et al., 2002). Similar SP subpopulations have been observed in primates and humans (Kim et al., 2002; Allen et al., 2002). The SP phenotype, therefore, has become a significant marker for stem cell activity in the identication of these cells and in their physical isolation by

Stem cells from adult tissues are an interesting source for cell therapy, gene therapy and tissue engineering. These cells normally have limited lineage potential in comparison to embryonic stem cells and this can be advantageous from the viewpoint of controlling cell growth and differentiation in certain therapeutic applications (Barrilleaux et al., 2006; Barry

In 1961, bone marrow was shown to have haematopoietic progenitor cells (Till & McCulloch, 1961). In the early 1970s, many investigators confirmed that bone marrow also had cells with fibroblastic morphology that could differentiate into bone, cartilage, fat and muscle (Prockop, 1997). These cells have been variously designated as marrow stromal cells or mesenchymal stem cells, and abbreviated as ''MSCs.'' It has been demonstrated that individual cells from the bone marrow stromal population possessed multilineage potential (Pittenger et al., 1999). Since the recognition of MSCs in bone marrow, cells with the same multilineage potential have been isolated from other tissues, including trabecular bone (Noth et al., 2002; Sottile et al., 2002) , adipose tissue (Lee et al., 2004; Zuk et al., 2001) and umbilical cord (Secco, 2008). The presence of MSCs in adipose tissue has generated special interest because harvesting fat tissue is generally less invasive to the donor than harvesting bone marrow and larger quantities may be available. Adipose-derived MSCs are also called adipose-derived stem cells (ADSCs) and adipose-derived adult stromal or stem cells (ADAS cells). But in addition to ADSCs, umbilical cord is also another interesting source for MSCs

Cell surface proteins may characterize particular cell types or lineages. In some cases, the role of a specific surface protein and its role in the biology of the cell type is known. However, often the function of the protein has not been determined, but the protein has been shown to be related to a certain type of cell and can serve as a marker.  Surface marker antigens can be used to distinguish the cells in a specific preparation and monitor their differentiation. Surface markers that are exclusively positive for a different cell type, for example, the haematopoietic surface markers CD45 and CD34, can be used to search for

uorescence-activated cell sorting.

**6. Mesenchymal stem cells**

& Murphy, 2004; Haynesworth et al., 1998).

and has recently gained some attention.

**6.1 Mesenchymal stem cell markers**

Several marker combinations have been developed that describe murine HSCs, including [CD117high, CD90.1low, Linneg/low, Sca-1pos] (Morrison & Weissman, 1994), [CD90.1low, Linneg, Sca-1posRhodamine123low] (Kim et al., 1998), [CD34neg/low, CD117pos, Sca-1pos, Linneg] (Osawa et al., 1996), [CD150 pos, CD48neg, CD244neg] (Kiel et al., 2005) and side-population; cells using Hoechst-dye (Goodell et al., 1996). Each of these combinations allows purification of HSCs to near-homogeneity. Similar strategies have been widened to purify human HSCs, employing markers such as CD34, CD38, Lin, CD90, CD133 and fluorescent substrates for the enzyme, aldehyde dehydrogenase. The use of highly purified human HSCs has been mostly experimental and clinical use normally employs enrichment for one marker, usually CD34. CD34 enrichment yields a population of cells enriched for HSC and blood progenitor cells, but still contains many other cell types. However, limited trials in which highly FACSpurified CD34pos CD90pos HSCs were used as a source of reconstituting cells have demonstrated that rapid reconstitution of the blood system can reliably be obtained using only HSCs (Negrin et al., 2000; Vose et al., 2001).

None of the HSC markers currently used are directly linked to crucial HSC function, and consequently, even within species, markers can differ depending on genetic alleles (Spangrude & Brooks, 1992), mouse strains (Spangrude & Brooks, 1993), developmental stages (Morrison et al., 1995) and cell activation stages (Randall & Weissman, 1997; Sato et al., 1999). In spite of this, there is an obvious connection with HSC markers between divergent species such as humans and mice. However, unless the current efforts at defining the complete HSC gene expression patterns will yield usable markers that are linked to essential functions for maintaining the stemness of the cells (Ramalho et al., 2002; Ivanova et al., 2002), functional analysis will remain necessary to identify HSCs clearly (Domen et al., 1999).

Table 2. Proposed cell surface markers of undifferentiated haematopoietic stem cells

#### **5.2 Side population**

When in adult mouse haematopoietic tissue, unpuried bone marrow cells are labelled with the membrane-permeate DNA binding dye Hoechst 33342, a very small fraction of cells extrudes this dye via a membrane pump (Goodell et al., 1996, 1997, 2001). Analysis of these cells on a ow cytometer equipped with an ultraviolet (UV) laser source allows finding of these cells; when Hoechst-labelled cells are analysed simultaneously through blue and red emission lters, the SP forms a dim tail extending from the normal G1 cell populations. These cells can reconstitute the bone marrow of lethally irradiated mice at an ED50 (Effective Dose 50) of fewer than 100 cells, indicating that they are highly enriched for totipotent stem cells. The SP cell subpopulation is also enriched for cells expressing the murine stem cell markers Sca-1 and c-kit, further suggesting that they contain very early haematopoietic progenitors (Goodell et al., 1996).

Several marker combinations have been developed that describe murine HSCs, including [CD117high, CD90.1low, Linneg/low, Sca-1pos] (Morrison & Weissman, 1994), [CD90.1low, Linneg, Sca-1posRhodamine123low] (Kim et al., 1998), [CD34neg/low, CD117pos, Sca-1pos, Linneg] (Osawa et al., 1996), [CD150 pos, CD48neg, CD244neg] (Kiel et al., 2005) and side-population; cells using Hoechst-dye (Goodell et al., 1996). Each of these combinations allows purification of HSCs to near-homogeneity. Similar strategies have been widened to purify human HSCs, employing markers such as CD34, CD38, Lin, CD90, CD133 and fluorescent substrates for the enzyme, aldehyde dehydrogenase. The use of highly purified human HSCs has been mostly experimental and clinical use normally employs enrichment for one marker, usually CD34. CD34 enrichment yields a population of cells enriched for HSC and blood progenitor cells, but still contains many other cell types. However, limited trials in which highly FACSpurified CD34pos CD90pos HSCs were used as a source of reconstituting cells have demonstrated that rapid reconstitution of the blood system can reliably be obtained using

None of the HSC markers currently used are directly linked to crucial HSC function, and consequently, even within species, markers can differ depending on genetic alleles (Spangrude & Brooks, 1992), mouse strains (Spangrude & Brooks, 1993), developmental stages (Morrison et al., 1995) and cell activation stages (Randall & Weissman, 1997; Sato et al., 1999). In spite of this, there is an obvious connection with HSC markers between divergent species such as humans and mice. However, unless the current efforts at defining the complete HSC gene expression patterns will yield usable markers that are linked to essential functions for maintaining the stemness of the cells (Ramalho et al., 2002; Ivanova et al., 2002), functional analysis will remain necessary to identify HSCs clearly (Domen et al.,

Mouse Human CD34low/- CD 34+ SCA-1+ CD59+ Thy1+/low Thy1+ CD38+ CD38low/- C-kit+ C-kit -/low Table 2. Proposed cell surface markers of undifferentiated haematopoietic stem cells

When in adult mouse haematopoietic tissue, unpuried bone marrow cells are labelled with the membrane-permeate DNA binding dye Hoechst 33342, a very small fraction of cells extrudes this dye via a membrane pump (Goodell et al., 1996, 1997, 2001). Analysis of these cells on a ow cytometer equipped with an ultraviolet (UV) laser source allows finding of these cells; when Hoechst-labelled cells are analysed simultaneously through blue and red emission lters, the SP forms a dim tail extending from the normal G1 cell populations. These cells can reconstitute the bone marrow of lethally irradiated mice at an ED50 (Effective Dose 50) of fewer than 100 cells, indicating that they are highly enriched for totipotent stem cells. The SP cell subpopulation is also enriched for cells expressing the murine stem cell markers Sca-1 and c-kit, further suggesting that they contain very early haematopoietic

only HSCs (Negrin et al., 2000; Vose et al., 2001).

1999).

**5.2 Side population**

progenitors (Goodell et al., 1996).

The SP fraction expresses an ABC transporter, Bcrp-1(ABCG2), on the cell surface and this transporter contributes to efflux of the Hoechst dye from the cells, leading to low levels of staining (Zhou et al., 2001). Interestingly, the bone marrow and peripheral immune system in ABCG2 transporter knockouts animals, is normal, suggesting that the capability to efflux Hoechst 33342 is characteristic of stem cells, but not essential for function (Uchida et al., 2002). Similar SP subpopulations have been observed in primates and humans (Kim et al., 2002; Allen et al., 2002). The SP phenotype, therefore, has become a significant marker for stem cell activity in the identication of these cells and in their physical isolation by uorescence-activated cell sorting.

#### **6. Mesenchymal stem cells**

Stem cells from adult tissues are an interesting source for cell therapy, gene therapy and tissue engineering. These cells normally have limited lineage potential in comparison to embryonic stem cells and this can be advantageous from the viewpoint of controlling cell growth and differentiation in certain therapeutic applications (Barrilleaux et al., 2006; Barry & Murphy, 2004; Haynesworth et al., 1998).

In 1961, bone marrow was shown to have haematopoietic progenitor cells (Till & McCulloch, 1961). In the early 1970s, many investigators confirmed that bone marrow also had cells with fibroblastic morphology that could differentiate into bone, cartilage, fat and muscle (Prockop, 1997). These cells have been variously designated as marrow stromal cells or mesenchymal stem cells, and abbreviated as ''MSCs.'' It has been demonstrated that individual cells from the bone marrow stromal population possessed multilineage potential (Pittenger et al., 1999). Since the recognition of MSCs in bone marrow, cells with the same multilineage potential have been isolated from other tissues, including trabecular bone (Noth et al., 2002; Sottile et al., 2002) , adipose tissue (Lee et al., 2004; Zuk et al., 2001) and umbilical cord (Secco, 2008). The presence of MSCs in adipose tissue has generated special interest because harvesting fat tissue is generally less invasive to the donor than harvesting bone marrow and larger quantities may be available. Adipose-derived MSCs are also called adipose-derived stem cells (ADSCs) and adipose-derived adult stromal or stem cells (ADAS cells). But in addition to ADSCs, umbilical cord is also another interesting source for MSCs and has recently gained some attention.

#### **6.1 Mesenchymal stem cell markers**

Cell surface proteins may characterize particular cell types or lineages. In some cases, the role of a specific surface protein and its role in the biology of the cell type is known. However, often the function of the protein has not been determined, but the protein has been shown to be related to a certain type of cell and can serve as a marker.  Surface marker antigens can be used to distinguish the cells in a specific preparation and monitor their differentiation. Surface markers that are exclusively positive for a different cell type, for example, the haematopoietic surface markers CD45 and CD34, can be used to search for

Stem Cell Characterization 269

more lineage restricted, progenitors; accordingly making the retrospective studies of NSC biology skewed to an unknown degree. Adding to this is the increasing evidence that implies clear functional differences between neural stem and progenitor cells (Galli et al., 2003; Cai & Rao, 2002). Consequently, there is a critical need to use strategies to identify and isolate pure populations of NSCs and other type cells with the aim of resolving their shared or unique biological properties with respect to cell-fate determination and lineage

NSCs are immunoreactive for a range of neural precursor/radial glia markers such as Nestin, Vimentin, RC2, 3CB2, Sox-2 and brain lipid-binding protein (BLBP). However, subtle differences exist between mouse and human NSCs. For example, hNS cells display moderate levels of glial fibrillary acidic protein (GFAP) expression unlike mouse NSCs (Conti et al., 2005), reflecting the differences between the species in vivo (Malatesta et al.,

So, as mentioned above, the cells which are gathered from neural tissue are heterogeneous and identifying cells is required. Therefore, some markers that are used in studies are listed

Neural stem cells GFAP, Nestin, Prominin, SOX-2

Immature neurons beta Tubulin,DCX,PSA-NCAM

Germ cells are specific cells that transfer the genetic information of an individual to the next generation. Making functional germ cells is vital for continuation of the germ line of the species. Spermatogenesis, the process of male germ cell production, takes place in the seminiferous tubules of the postnatal testis and is an extremely productive system in the body. In the mammalian testis, more than 20 million sperms per gram of tissue are created daily (Amann, 1986). The high productivity relies on spermatogonial stem cells (SSCs). Similar to other kinds of stem cells in adult tissues, SSCs are self-renewing and produce daughter cells that assign to differentiate throughout the life of the male (Meistrich & van Beek, 1993). In addition, in mammals, SSCs are unique among stem cells in the adult body, because they are the only cells that undergo self-renewal and transmit genes to subsequent generations. Furthermore, SSCs provide an excellent model to study stem cell biology due to the availability of a functional assay that clearly identifies the stem cell (Weissman et al.,

Spermatogonial stem cells derive from primordial germ cells (PGCs), which in turn originate from epiblast cells (embryonal ectoderm) (Lawson KA et al., 1992). Soon after the development of the PGCs, they migrate from the base of the allantois, along the hindgut, finally reaching the genital ridges. The PGCs increase in number during migration, when

Mature neurons NeuN, MAP-2, NF, BLBP

Proliferating cells Ki-67,BrdU, PCNA

Radial glia GLAST, RC2

Oligdendrocytes O4, MBP, RIP

Oligodendrocyte precursors NG2

progression.

2000; Rakic, 2003).

Table 4. Neural stem cells markers

**8. Spermatogonial stem cells**

below:

2001).

contamination of MSC preparations with other cell types. Surface markers have also been used for positive and negative immunoselection of MSC cell populations (Buhring et al., 2007; Simmons & Torok-Storb, 1991).

The expressed genes that appear on the hMSC surface include receptors for growth factors, matrix molecules and other cells, and point out how the hMSC will interact with its environment. The flow cytometry analysis also indicates the homogeneity of the hMSC population or whether it is a mixture of different cell types. A wide-ranging, yet incomplete, list of the surface molecules on hMSCs is provided in Table 3.

Table 3. Mesenchymal stem cells markers

#### **7. Neural stem cells**

Neurogenesis is defined as the procedure of generating new neurons from neural stem cells (NSCs),which consists of the proliferation and fate determination of NSCs, migration and survival of young neurons, and maturation and integration of recently matured neurons (Ming & Song, 2005).

NSCs are defined as undifferentiated cells that developmentally originate from the neuroectodermal layer during early embryogenesis. After neural tube closing, these undifferentiated precursor cells and their immediate progeny compose the neuroepithelial layer that surrounds the lateral, third and fourth ventricles in the midbrain and forebrain, and the central canal in the spinal cord. They are the main source of cells that later form all major structures of the brain and spinal cord (Maric & Barker, 2004).

NSCs have recently attracted a great deal of attention because of their inherent ability to generate all major classes of cells of the nervous system. NSCs have therefore been supposed as a useful resource for potentially repairing and restoring the physiological functions to damaged, diseased or aging neural tissues (Gang, 2000; Anderson, 2001; Temple, 2001; Vaccarino et al., 2001; Vescovi et al., 2001; Weissman et al., 2001).

However, with the accelerated interest in and growth of the NSC field, there has been growing uncertainty around the understanding of what cell phenotype actually makes up a neural stem cell. NSCs in their undifferentiated shape are characterized by a unique bipolar morphology that can help identify them from the heterogeneity associated with early culture. Derivation from human foetal material gives rise to an apparently mixed population of NSCs, exhibiting both classic bipolar NSC morphology and other cell morphologies.

#### **7.1 Neural stem cell markers**

The major research limitation is that the cellular preparations used as a source of NSCs are themselves naturally heterogeneous and consisting of both NSCs and self-renewing, but

contamination of MSC preparations with other cell types. Surface markers have also been used for positive and negative immunoselection of MSC cell populations (Buhring et al.,

The expressed genes that appear on the hMSC surface include receptors for growth factors, matrix molecules and other cells, and point out how the hMSC will interact with its environment. The flow cytometry analysis also indicates the homogeneity of the hMSC population or whether it is a mixture of different cell types. A wide-ranging, yet incomplete,

Positive CD13, CD29, CD44, CD49b(Integrin alpha 2,5), CD54(ICAM1), cd71(Transferrin Rec), CD73(SH-3), CD105(Endoglin.SH-2), CD106(VCAM), CD166(ALCAM)

Neurogenesis is defined as the procedure of generating new neurons from neural stem cells (NSCs),which consists of the proliferation and fate determination of NSCs, migration and survival of young neurons, and maturation and integration of recently matured neurons

NSCs are defined as undifferentiated cells that developmentally originate from the neuroectodermal layer during early embryogenesis. After neural tube closing, these undifferentiated precursor cells and their immediate progeny compose the neuroepithelial layer that surrounds the lateral, third and fourth ventricles in the midbrain and forebrain, and the central canal in the spinal cord. They are the main source of cells that later form all

NSCs have recently attracted a great deal of attention because of their inherent ability to generate all major classes of cells of the nervous system. NSCs have therefore been supposed as a useful resource for potentially repairing and restoring the physiological functions to damaged, diseased or aging neural tissues (Gang, 2000; Anderson, 2001; Temple, 2001;

However, with the accelerated interest in and growth of the NSC field, there has been growing uncertainty around the understanding of what cell phenotype actually makes up a neural stem cell. NSCs in their undifferentiated shape are characterized by a unique bipolar morphology that can help identify them from the heterogeneity associated with early culture. Derivation from human foetal material gives rise to an apparently mixed population of NSCs, exhibiting both classic bipolar NSC morphology and other cell morphologies.

The major research limitation is that the cellular preparations used as a source of NSCs are themselves naturally heterogeneous and consisting of both NSCs and self-renewing, but

D18 (Integrin beta 2), CD31 (PECAM), CD49d (Intergrin alpha 4), CD50 (ICAM3),

Negative CD3, CD4, CD6, CD9, CD10, CD11a, b, CD14, CD15, CD34, CD45,

CD62E (E-Selectin), CD117(c-kit), CD133

major structures of the brain and spinal cord (Maric & Barker, 2004).

Vaccarino et al., 2001; Vescovi et al., 2001; Weissman et al., 2001).

2007; Simmons & Torok-Storb, 1991).

Table 3. Mesenchymal stem cells markers

Surface antigens

**7. Neural stem cells**

(Ming & Song, 2005).

**7.1 Neural stem cell markers**

list of the surface molecules on hMSCs is provided in Table 3.

more lineage restricted, progenitors; accordingly making the retrospective studies of NSC biology skewed to an unknown degree. Adding to this is the increasing evidence that implies clear functional differences between neural stem and progenitor cells (Galli et al., 2003; Cai & Rao, 2002). Consequently, there is a critical need to use strategies to identify and isolate pure populations of NSCs and other type cells with the aim of resolving their shared or unique biological properties with respect to cell-fate determination and lineage progression.

NSCs are immunoreactive for a range of neural precursor/radial glia markers such as Nestin, Vimentin, RC2, 3CB2, Sox-2 and brain lipid-binding protein (BLBP). However, subtle differences exist between mouse and human NSCs. For example, hNS cells display moderate levels of glial fibrillary acidic protein (GFAP) expression unlike mouse NSCs (Conti et al., 2005), reflecting the differences between the species in vivo (Malatesta et al., 2000; Rakic, 2003).

So, as mentioned above, the cells which are gathered from neural tissue are heterogeneous and identifying cells is required. Therefore, some markers that are used in studies are listed below:

Table 4. Neural stem cells markers

#### **8. Spermatogonial stem cells**

Germ cells are specific cells that transfer the genetic information of an individual to the next generation. Making functional germ cells is vital for continuation of the germ line of the species. Spermatogenesis, the process of male germ cell production, takes place in the seminiferous tubules of the postnatal testis and is an extremely productive system in the body. In the mammalian testis, more than 20 million sperms per gram of tissue are created daily (Amann, 1986). The high productivity relies on spermatogonial stem cells (SSCs). Similar to other kinds of stem cells in adult tissues, SSCs are self-renewing and produce daughter cells that assign to differentiate throughout the life of the male (Meistrich & van Beek, 1993). In addition, in mammals, SSCs are unique among stem cells in the adult body, because they are the only cells that undergo self-renewal and transmit genes to subsequent generations. Furthermore, SSCs provide an excellent model to study stem cell biology due to the availability of a functional assay that clearly identifies the stem cell (Weissman et al., 2001).

Spermatogonial stem cells derive from primordial germ cells (PGCs), which in turn originate from epiblast cells (embryonal ectoderm) (Lawson KA et al., 1992). Soon after the development of the PGCs, they migrate from the base of the allantois, along the hindgut, finally reaching the genital ridges. The PGCs increase in number during migration, when

Stem Cell Characterization 271

however, for this to be possible more research needs to be performed to determine the

2005) FC, MACS, IHC, ISH, WM

SOX3 (Raverot et al., 2005) KO, IHC; c-RET (Meng et al., 2000) IHC, MACS

WM EE2 (Koshimizu et al., 1995) WB , IHC

GCNA1 (Enders & May., 1994) FC, WB, IHC

TAF4B (Falender et al., 2005) FC, KO, IHC

CD9 (Kanatsu-Shinohara et al., 2004) FC, IHC, MACS

OCT4 (Pesce et al., 1998) FC, ISH, IHC, WM TG; NGN3 (Yoshida et al., 2004) ISH, TG, WM, ISH; NOTCH1 (Von Schonfeldt et al., 2004) RT–PCR, IHC,

A(s) and GFRalpha-1(Von Schonfeldt et al., 2004, Hofmann et al,

A(s), A (pr) and A(al) PLZF (Buaas et al., 2004) (Costaya et al., 2004) FC, ISH,

IHC, WM, Mu;

Spermatogonia EP-CAM (Anderson et al., 1999) FC, IHC, MACS

Premeiotic germ cells STRA8 (Oulad Abdelghani et al., 1996) RT–PCR, ISH, IHC,

A(s), A-single; A (pr), pair of spermatogonia; A (al), A-aligned spermatogonia; FC, ow cytometry (including FACS); Mu, mutant mouse; TG, transgenic mouse; KO, Knockout mouse; IHC, immunohistochemistry; WM, whole mount immunostaining; WB, Western blot; ISH, in situ hybridization; RT–PCR, reverse transcriptase– PCR; MACS, magnetic-activated cell sorting

The skin is the body's strong outer cover that maintains the inside of the body being moist and protects the body from outside assaults by physical, environmental and biological factors. Skin and its associated hair follicles and glandular structures, sebaceous and sweat glands, are made by a stratified epithelium where the position of the cell within the tissue relates to its state of differentiation. The terminally differentiated stratum corneum, hairs and oil-filled sebocytes have a limited lifespan and are constantly shed from the body throughout the adult life. This continual shedding requires that the epithelium is replenished and restored by a stem cell population during normal maintenance of the skin and also in response to injury (Fuchs & Horsley, 2008; Watt et al., 2006). By definition, adult stem cells (ASCs) have the ability to both self-renew and make differentiated progeny (Lajitha, 1979). In healthy skin, epidermal stem cells divide uncommonly, but upon skin

There has been important progress in the recognizing of epidermal stem cells (ESCs) since the 1970s, when the idea of interfollicular epidermis was firstly suggested; later, much work was focused on the specific region of the hair follicle outer root sheath, mainly the bulge

A spermatogonia RBM (Jarvis et al., 2005) RT–PCR, IHC

Table 6. Overview of markers used to identify spermatogonial stem cells

injury, stem cells quickly divide to repair the lesion.

optimal procedures and combinations of markers.

Cells on basal membrane and

Spermatogonia, spermatocytes

and round spermatids

Premeiotic spermatogonia and postmeiotic spermatid

**9. Epidermal stem cells**

interstitium

these cells have reached the genital ridges; their number increases to about 10,000 per gonad (Tam and Snow, 1981). PGCs are single cells that under certain culture conditions can make colonies of cells which morphologically are similar to undifferentiated embryonic stem cells (ESCs) (Resnick JL et al., 1992).When they have arrived in the genital ridges, the PGCs are surrounded by the differentiating Sertoli cells, so seminiferous cords are formed.

The germ cells present within the seminiferous cords are different morphologically from PGCs and are called gonocytes (Clermont and Perey, 1957; Sapsford CS et al., 1962; Huckins & Clermont, 1968) or various subsequent types of pro-spermatogonia (Hilscher B et al;1974). Shortly after birth, the gonocytes restart proliferation to give rise to adult types of spermatogonia (Sapsford CS et al., 1962; Huckins & Clermont, 1968; Vergouwen RPFA et al., 1991; Novi & Saba, 1968; Bellye AR et al., 1977). This happening indicates the start of spermatogenesis.

#### **8.1 Spermatogonial stem cell markers**

Since the establishment of the transplantation technique, several new markers and characteristics of spermatogonial stem cells have been identified that can be used to isolate a population from the testis that is enriched for spermatogonial stem cells - Tables 5 and 6.

Table 5. Overview of markers that have been successfully used to isolate spermatogonial stem cell populations from the testis by either positive or negative selection

#### **8.2 Testicular side population**

So far, four groups have separated a side population of testicular cells; meanwhile, different results were drawn as to whether these were spermatogonial stem cells. The first group reported the existence of a testicular side population. Amazingly, they did not find this population to be capable of colonizing a recipient testis after transplantation and concluded that it did not contain spermatogonial stem cells (Kubota et al., 2003).

Then, two other groups found the testicular side population to be enriched for spermatogonial stem cells (Falciatori et al., 2004; Lassalle et al., 2004). A fourth group then explained that testicular side population cells contain Leydig cell progenitors (Lo et al., 2004; de Rooij.,2004) and later failed to find spermatogonial stem cells in this population (Lo et al., 2005).

The controversial results can probably be explained by the strictness of the FACS gating and the different procedures used to separate the side population. It may be possible to isolate a very pure population of spermatogonial stem cells from the testis using the side population technique, alone or in combination with membrane markers (van Bragt et al., 2005),

these cells have reached the genital ridges; their number increases to about 10,000 per gonad (Tam and Snow, 1981). PGCs are single cells that under certain culture conditions can make colonies of cells which morphologically are similar to undifferentiated embryonic stem cells (ESCs) (Resnick JL et al., 1992).When they have arrived in the genital ridges, the PGCs are

The germ cells present within the seminiferous cords are different morphologically from PGCs and are called gonocytes (Clermont and Perey, 1957; Sapsford CS et al., 1962; Huckins & Clermont, 1968) or various subsequent types of pro-spermatogonia (Hilscher B et al;1974). Shortly after birth, the gonocytes restart proliferation to give rise to adult types of spermatogonia (Sapsford CS et al., 1962; Huckins & Clermont, 1968; Vergouwen RPFA et al., 1991; Novi & Saba, 1968; Bellye AR et al., 1977). This happening indicates the start of

Since the establishment of the transplantation technique, several new markers and characteristics of spermatogonial stem cells have been identified that can be used to isolate a population from the testis that is enriched for spermatogonial stem cells - Tables 5 and 6.

2003)

stem cell populations from the testis by either positive or negative selection

that it did not contain spermatogonial stem cells (Kubota et al., 2003).

Table 5. Overview of markers that have been successfully used to isolate spermatogonial

So far, four groups have separated a side population of testicular cells; meanwhile, different results were drawn as to whether these were spermatogonial stem cells. The first group reported the existence of a testicular side population. Amazingly, they did not find this population to be capable of colonizing a recipient testis after transplantation and concluded

Then, two other groups found the testicular side population to be enriched for spermatogonial stem cells (Falciatori et al., 2004; Lassalle et al., 2004). A fourth group then explained that testicular side population cells contain Leydig cell progenitors (Lo et al., 2004; de Rooij.,2004) and later failed to find spermatogonial stem cells in this population (Lo et al.,

The controversial results can probably be explained by the strictness of the FACS gating and the different procedures used to separate the side population. It may be possible to isolate a very pure population of spermatogonial stem cells from the testis using the side population technique, alone or in combination with membrane markers (van Bragt et al., 2005),

CD9 (Kanatsu-Shinohara M et al., 2004), integrin alpha 6 (Shinohara T et al., 1999), integrin beta 1 (Shinohara T et al., 1999), THY-1, CD24 (Kubota H et al., 2003)

c-kit, MHC1, Ly6A(Sca-1), CD34 (Kubota H et al.,

surrounded by the differentiating Sertoli cells, so seminiferous cords are formed.

spermatogenesis.

**8.1 Spermatogonial stem cell markers**

Markers for positive selection of spermatogonial stem cells

Markers for negative selection of spermatogonial stem cells

**8.2 Testicular side population**

2005).

A(s), A-single; A (pr), pair of spermatogonia; A (al), A-aligned spermatogonia; FC, ow cytometry (including FACS); Mu, mutant mouse; TG, transgenic mouse; KO, Knockout mouse; IHC, immunohistochemistry; WM, whole mount immunostaining; WB, Western blot; ISH, in situ hybridization; RT–PCR, reverse transcriptase– PCR; MACS, magnetic-activated cell sorting

Table 6. Overview of markers used to identify spermatogonial stem cells