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Stem Cells 2003;21:162-170 www.StemCells.com
© 2003 AlphaMed Press

Colocalization Analysis of Sialomucins CD34 and CD164

Colin P. McGuckina,b, Nicolas Forraza,b,c, Marc-Olivier Baradeza,d, Carlos Lojo-Riala,b, David Wertheimd, Karen Whitinga,c, Suzanne M. Watte,f, Ruth Pettengella,c

a King-George Laboratory, St. George’s Hospital Medical School and Kingston University, London, United Kingdom;
b School of Life Sciences, Kingston University, Kingston Upon Thames, United Kingdom;
c Department of Haematology, St. George’s Hospital Medical School, London, United Kingdom;
d School of Computer and Information Systems, Kingston University, Kingston Upon Thames, United Kingdom;
e National Blood Service, Stem Cell Laboratory, The John Radcliffe Hospital, Oxford, United Kingdom;
f Nuffield Department of Clinical and Laboratory Sciences, University of Oxford, Oxford, United Kingdom

Key Words. CD34 • CD164 • Stem cells • Progenitor cells • Confocal microscopy

Correspondence: Colin P. McGuckin, Ph.D., School of Life Sciences, Faculty of Science, Kingston University, Penrhyn Road, Kingston Upon Thames, Surrey KT1 2EE, United Kingdom. Telephone: 44-797-126-6764; Fax: 44-208-547-7562; e-mail: c.mcguckin{at}kingston.ac.uk.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell-to-cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34+ cells to adhere to bone marrow stroma. In this study, we employed high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface co-localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three-dimensional computer-generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo-like pattern and second in antigen-dense pockets. Three-dimensional computer analyses further revealed that this pocketing corresponded to dense crest-like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Identification of hemopoietic stem and progenitor cell (HSPC) populations relies primarily upon flow-cytometric strategies. Although in vitro cell culture and in vivo-related protocols are increasingly used to check the functional elements of harvested cellular populations, little is known of the specific antigen distribution status of the cells before transplantation.

Regulation of HSPC communication and the process of homing and adhesion to bone marrow (BM) stroma are believed to involve glycoprotein cell adhesion molecules (CAMs). These molecules, which include integrins, selectins, immunoglobulins, the CD44 "homing" receptor family, and the sialomucins, may also regulate HSPC cycle status as well as proliferation potential [1-3]. Little is known about the distribution of adhesion molecules on HSPCs, but their analysis may aid understanding of the relocation process of HSPCs to BM stroma during homing. Further, since it is known that more than one such molecule is involved in this process, a better comprehension of how these antigens interact would be useful to characterize which cells have a higher potential to engraft.

Sialomucins are transmembrane glycoproteins ranging from 50-3,000 kD that share limited homology to one another at the amino acid and nucleotide levels [2, 4]. Nevertheless, they bear numerous O-glycosylations, which infer multiple kinds of cell-cell or cell-extracellular matrix interactions. At least six members of the sialomucin family have been reported to be expressed on primitive HSPCs: CD43, CD45RA, CD162 (PSGL-1), PCLP-1, CD34, and CD164. CD34 and CD164 sialomucin thread-like structures might be related to their suggested role in both cellular adhesion and the proliferation status of early HSPCs. A linked role/interaction for these two molecules has indeed been implied with respect to HSPC growth and cytoadhesion to the BM stroma [5, 6].

CD34 is well characterized as a marker for HSPCs during early hemopoiesis, and is frequently used as a surrogate marker for hemopoietic cell harvest protocols [7-9]. Although CD34 involvement in HSPC cytoadhesion has been established through ligation and signal transduction assays [10–13], the intrinsic mechanisms linking the CD34 molecule to HSPC cellular adhesion and homing remain to be elucidated.

We and others have reported that CD164 (as the 103B2/9E10 epitope) is expressed on a very primitive HSPC subset [1, 6, 14]. Binding of CD164 class II epitope suggested that CD164 may functionally facilitate CD34+ cell adhesion to BM stroma and inhibit CD34+ HSPC proliferation [5]. Such results suggest a possible functional interaction between CD34 and CD164. Despite this, CD164 expression is not limited to early hemopoietic cells, but rather is more heterogeneously expressed than CD34 [6, 15]. Furthermore, although CD34 protrudes from the membrane as a single entity, the CD164 molecule is essentially a homodimeric molecule [5, 15–17]. Whether this structural difference is related to function has yet to be fully discovered. Additionally, we have already reported the flow-cytometric antigen expression patterns of CD164 and CD34 in comparison with other antigens of the HSPC subset including AC133, CD117, CD164, Thy-1, and CD38 [14, 18–20]. In that work, we demonstrated that although CD164 shows a wide expression profile among HSPC subsets, higher levels are found in cells, which are translating, and in early presentation of the CD34 glycoprotein on the HSPC membrane [6, 14].

In order to understand this possible linked functional duality, we have extended our study using high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface colocalization patterns on BM and umbilical cord blood (CB), and to compare coexpression using a three-dimensional representation of antigen distribution.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CB and BM Collection and Mononuclear Cell (MNC) Isolation
Umbilical cord blood specimens (n = 11) were collected from full-term deliveries scheduled for elective cesarean sections. BM samples (n = 5) were obtained from informed hematologically normal volunteers. CB and BM specimens were collected with appropriate ethical consent. Specimens were treated as reported previously [14, 21]. Briefly, samples were diluted one in four in phosphate-buffered saline (PBS) supplemented with a citrate-based anticoagulant (0.6% ACD-A; Baxter; Maurepas, France; http://www.baxter.com) and bovine serum albumin (0.5% fraction V; Sigma Aldrich, Poole, United Kingdom; http://www.sigmaaldrich.com) at pH 7.4 and referenced (ACD-A buffer). Diluted CB was carefully overlaid in a 1:4 ratio onto a research-grade Ficoll-Hypaque solution (d:1.077 g/cm3; Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com) prior to centrifugation (400 g, 30 minutes, 22°C). The MNC layer was collected, washed twice in ACD-A buffer, pelleted (400 g, 10 minutes) before being resuspended in ACD-A buffer, and cell aliquots were taken for cell viability/enumeration using trypan blue (Sigma Aldrich).

HSPC Immunophenotyping for Flow Cytometry Analysis
For each sample, CB or BM MNC aliquots were incubated in human gammaglobulins (20 minutes, 4°C, 2% in PBS; Sigma Aldrich) to block nonspecific Fc receptors before incubation with mouse anti-human CD164 class II epitope (IgG3, 103B2/9E10, 30 minutes, 4°C). Cells were then washed twice in staining buffer (400 g, 10 minutes, 4°C) prior to secondary labeling with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse F(ab')2 fragments (IgG3 specific, 30 minutes, 4°C; Southern Biotech; Birmingham, AL; http://www.southernbiotech.com). After two washes, cells were directly labeled with monoclonal mouse anti-human antibodies (30 minutes, 4°C): anti-AC133 phycoerythrin (PE) conjugated (IgG1, AC133/2; Miltenyi Biotec; Bergish Gladbach Germany; http://www.miltenyibiotec.com) and anti-CD34 peridinin chlorophyll A-protein (IgG1, HPCA-II; Pharmingen; San Diego, CA; http//www.bdbiosciences.com/pharmingen). Cells were then washed twice in staining buffer (400 g, 10 minutes, 4°C) prior to fixation in paraformaldehyde (1%, BDH Laboratory Supply; Poole, Dorset, United Kingdom; http://www.bdh.com). Relevant matched isotype-negative antibody controls were used to determine background-labeling levels. Fluorescent events were acquired on a FACScan flow cytometer (Becton Dickinson; San Diego, CA; http://www.bd.com) with CELLQuest software prior to analysis with WinMDI software. Results are expressed as mean percentage expression ± standard error.

Laser-Scanning Confocal Microscopy Imaging of CD34 and CD164 Antigens on CB and BM HSPCs
Cord blood or BM MNCs were adhered at room temperature on gold positive slides (BDH) before incubation at 4°C with human gammaglobulins (2%; Sigma Aldrich) to block Fc receptors. Adhered cells were then indirectly labeled using primary mouse anti-human CD164 (IgG3, 103B2/9E10 labeling class II epitope reported to be ubiquitously distributed on most primitive HSPC subsets [6]) and/or CD34 (IgG1, HPCA-II labeling class III epitope reported to be expressed on more primitive HSPC subsets than class I/II counterparts [8, 22]) antibodies (Becton Dickinson). Cells were also labeled against isotype-matched monoclonal antibody controls: mouse IgG1 pure and mouse IgG3 pure, respectively (Becton Dickinson). Secondary labels used were FITC-conjugated goat anti-mouse IgG3-specific, F(ab')2 fragments (Southern Biotech) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-mouse IgG F(ab')2 fragments (DAKO; Glostrup, Denmark; http://www.dako.dk) against IgG1 primary antibodies. All labeled cells were fixed in a 3.5% paraformaldehyde solution (BDH) and stored at 4°C prior to images acquisition with a Zeiss LSM.440 laser-scanning confocal microscope.

Image Processing
Acquired images were first processed with Adobe Photoshop (version 5) or Paintshop Pro 5 (Jasc Software Inc.; Eden Prarie, MN; http://www.jasc.com) for differential interference contrast (DIC) and fluorescence overlays (CD34-TRITC represented in red; CD164-FITC represented in green) to give an overall impression of the staining patterns. Software systems were developed using MATLAB (MathWorks Inc.; Natick, MA; http://www.mathworks.com) to allow further processing to obtain three-dimensional contour-slice representation of CD34 and CD164 antigen distributions using a series of one µm (depth) spaced cell fluorescent slices (Z axis). Confocal microscope images used for the three-dimensional contour-slice representation were reduced in size from 1,024 x 1,024 pixels to 256 x 256 pixels using Paintshop Pro 5.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD34+CD164+ Cells Represent a Discrete HSPC Population Enriched for AC133+ Cells
Flow cytometry analysis characterized CD34+ cells as representing 1.10% ± 0.16% (n = 11) and 2.57% ± 1.22% (n = 5) of CB and BM MNCs, respectively. CD164+ cells represented 19.49% ± 5.1% and 22.92% ± 7.28% of CB and BM MNCs, respectively. CD34 and CD164 sialomucins were coexpressed on a discrete CD34+CD164+ cell subset (0.58% ± 0.06% and 0.81% ± 0.27% of MNCs in CB and BM, respectively). In addition 62.28% ± 8.25% in CB and 52.53% ± 5.87% in BM of coexpressing CD34+CD164+ cells also expressed AC133.

DIC Microscopy of BM and CB MNCs Identified Blasts with Membrane Activity Upon Adhesion
Differential interference contrast imaging, at a range of magnifications, allowed visualization of adhering MNCs (Fig. 1Go). A primary morphological analysis identified adhering MNCs at various stages of differentiation. At high magnification (x630x8), for BM and CB, several cells showed typical blast-progenitor morphology characterized by a large nucleus and a thin, discrete cytoplasm. Interestingly, these adhering BM blast cells consistently produced plasma membrane protrusions.



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  		Figure 1. DIC image of hemopoietic cells acquired by laser-scanning confocal microscope. A) shows a low magnification picture of CB MNCs (x630). B) reveals hemopoietic progenitor cells from CB at a medium magnification (x630x6). C) and D) are DIC images of hemopoietic progenitor cells from CB and BM, respectively, at high magnification (x630x8). Both BM and CB progenitor cells had typical "blast" morphology with a discrete cytoplasm and a large nucleus. Interestingly, BM progenitor cells revealed increased membrane activity with multiple protrusions, reflecting their physiological interacting status in the marrow when compared to normally circulating CB progenitor cells. Membrane contours represented in yellow (computer generated). White scale bar is equivalent to 10 µm.

 
CD34 and CD164 Sialomucins Distribute Along Low-Intensity Peripheral Halos and Concentrate to Antigen-Dense Pockets on BM and CB Progenitor Cell Membranes
Cells positive for only one antigen fluorescence, CD34 or CD164, were subsequently analyzed. In both CB and BM, antigen-fluorescence expression overlaid to cell DIC images revealed that CD34 (represented in red) was distributed evenly around the cell membrane following a low-intensity peripheral halo in conjunction with multiple high-density pockets (Fig. 2Go). Interestingly, on cells positive for CD164 only, CD164 antigen distribution patterns (in green) were similar to CD34, with a fluorescent halo and more dense fluorescent pockets (Fig. 3Go).



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  		Figure 2. CD34+ hemopoietic progenitor cells analyzed by laser-scanning confocal microscopy. Overlay of DIC image with CD34 antigen fluorescence (indirect stain TRITC fluorochrome, represented in red). In BM and CB, CD34 sialomucin expression followed a peripheral halo-like pattern with occasional dense pockets of antigen. x630x6 magnification. White scale bar is equivalent to 10 µm.

 


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  		Figure 3. CD164+ hemopoietic progenitor cells analyzed by laser-scanning confocal microscopy. Overlay of DIC image with CD164 antigen fluorescence (indirect stain via FITC fluorochrome represented in green). In BM and CB, CD164 sialomucin was distributed along peripheral halo with sporadic denser clusters of antigen. x630x6 magnification. White scale bar is equivalent to 10 µm.

 
Nevertheless, when dually expressed on BM or CB progenitor cells, CD34 and CD164 sialomucins showed a high degree of colocalization (represented in yellow) on the cell membrane (Fig. 4Go). When coexpressed, their distribution patterns (low-intensity halo and dense pockets) were consistent with those observed on cells expressing only one sialomucin. It was also noted that CD164 and CD34 pocket colocalization often correlated with short membrane protrusions observable on the DIC image. Specificity of the stain was confirmed by small regions on the membrane being either CD164 or CD34 positive only. Several controls were also performed to assess antigen specificity when staining. On positively stained slides, levels of cells expressing CD164 and CD34 were largely in line with our immunophenotyping data acquired by flow cytometry on CB and BM cell populations [14]. However, the lower numbers of cells available on a confocal slide precludes a direct comparison.



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  		Figure 4. CD34 and CD164 coexpression analysis on BM and CB MNCs by laser-scanning confocal microscopy. Overlay of CD164 fluorescence (indirect stain via FITC fluorochrome, represented in green), CD34 fluorescence (indirect stain via TRITC fluorochrome, represented in red) and CD164/CD34 colocalization, which appears in yellow with DIC image. The top image (CB1) shows a low-magnification image (x630) with several single CD164+ cells and one cell dually positive for CD34 and CD164. (CB2) shows the latter cell at higher magnification (x630x8). (BM) also shows a BM progenitor cell dually positive for CD34 and CD164 (x630x8 magnification). For both BM and CB, CD34 and CD164 showed high levels of colocalization on progenitor cells. Interestingly, dense pockets of antigen dual expression correlated with short membrane protrusions visible on DIC image. White scale bar is equivalent to 10 µm.

 
Three-Dimensional Fluorescence Analysis Characterizes Sialomucin Distribution to Adhesion-Dependent Crests on CB and BM Progenitor Cells
Using laser-scanning confocal microscope images and the software developed using MATLAB, 1-µm antigen fluorescence "Z" scans were stacked to give a three-dimensional contour representation of sialomucin distribution on both CB and BM progenitor cells.

CD34 and CD164 antigen-dense fluorescent pockets (also observable in two dimensions) reflected, in a three-dimensional representation, sialomucin distribution along crests. Figure 5Go shows a representative example of a cell positive for CD164 only. Sialomucin distribution appeared to be related to cell adhesion to the microscope slide, as each crest appeared to originate from the adhesion contact point on the slide growing toward the opposite pole of the cell. Further computer-aided analysis defined variable levels of colocalization of the two sialomucins (Fig. 6Go). On cells analyzed here, it was found that CD34 appears at a higher concentration than CD164.



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  		Figure 5. Examples of CD164 sialomucin distribution in a three-dimensional representation using MATLAB software. A) represents an overlay of CD164 fluorescence (indirect stain via FITC fluorochrome, represented in green) with differential interference contrast of a CB progenitor cell positive for CD164 only. This cell shows typical CD164 antigen distribution along a peripheral halo, with occasional denser antigen pockets arrowed 1, 2, and 3, respectively (x630x8 magnification; white scale bar is equivalent to 10 µm). B) and C) show three-dimensional representation of CD164 distribution in two different orientations. This provides a finer analysis by relating the two-dimensional dense-antigen pockets to three-dimensional meridian-shaped crescents growing from the cell-adhering contact point on the slide toward the top of the cell.

 



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  		Figure 6. Representative example of CD34 and CD164 sialomucin colocalization on a BM progenitor cell. This two-dimensional representation was performed by image processing of CD34 and CD164 fluorescence slices. Slices are numbered from the cell’s contact point with the slide (S1) to the top of the cell (S10). The representation allows outcrops from the cell membrane to be highlighted for antigen expression. Interestingly, CD164 was found to be rarely expressed on its own on the cell membrane, whereas pockets bearing only the CD34 antigen were more numerous towards the top of the cell. Furthermore, this representation emphasizes the high degree of CD164 and CD34 colocalization, when dually expressed, throughout the progenitor cell membrane. When coexpressed on progenitor cells, CD164 and CD34 membrane distribution patterns were consistent in both BM (n = 4) and CB (n = 11) and are here highlighted by the color map.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of adhesion molecules in hemopoiesis is a complex Pandora’s Box that has yet to be completely understood. Despite this, some adhesion molecules such as CD34 have reached prominent status, not for their actual role in vivo, but for the indirect HSPC-harvesting potential they offer in the transplantation arena. However, what transplantation biologists rarely consider is that using these antigens in the harvest procedure may in fact affect the cells, changing their previous in vivo role. A better understanding of CAM distribution and functional interactions in relation to the BM is therefore essential to optimize transplantation technologies. We have investigated here two sialomucin adhesion molecules, CD34 and CD164, both considered to be important in transplantation and engraftment.

Laser-scanning confocal microscopy DIC images revealed clues to the environmental surroundings of CB and BM HSPCs. On CB DIC images, the membrane was often more spherical than the corresponding BM cell, which in contrast appeared to have a higher proportion of membrane extensions. The BM microenvironment is one that occurs with a variety of structures, adhesion molecules, and even cytokine receptors holding developing HSPCs in a protected three-dimensional mold allowing "stromal cell-mediated hemopoiesis" to regulate development [23]. The extensions seen from the BM cell membranes are likely to relate to this and are an obvious difference to the circulating CB cells, which have yet to home to the marrow.

In our previous studies, we reported that CD164 (103B2/9E10 class II epitope) was more widely expressed on HSPC populations than the CD34 class III epitope, and indeed CD164+CD34– cells appeared in our confocal-based analysis [6, 14]. However, not all the CD34+ (class III epitope) cells coexpressed the cell surface CD164 103B2/9E10 epitope. This lends support for a role of glycosyl transferases in epitope expression, as the CD34 class II epitope is also less extensively expressed than the CD34 class III epitope [22]. Therefore, although there may be a role for interaction between these two antigens, it must be considered that the in vivo functional roles of CD34 and CD164 may not be mutually exclusive, and could in fact be wider.

Imaging of CD34 and CD164 expression identified halo-like peripheral expression patterns with occasional denser antigen clusters correlating with cell membrane activity. Such striking similarities in antigen distribution on HSPC membrane may relate to the intrinsic structural nature of sialomucin, as CD34 and CD164 are heavily O-glycosylated proteins. This typical glycosylation pattern results in extended thread-like structures protruding above the glycocalyx [15]. Such a configuration would provide an optimal interface for interaction with multiple terminal sugar moieties on opposite cells [15, 24].

Our sialomucin-distribution imaging study is consistent with published functional studies indicating a related role for CD34 and CD164 in homing, cell cycle inhibition, and adhesion to BM stroma [5]. Engagement of the 103B2/9E10 epitope of CD164 on CD34+ HSPCs with monoclonal antibodies suggested that CD164 might cooperate with CD34 in facilitating marrow stromal adhesion and/or regulating CD34+ HSPC growth and differentiation. The study here supports this, since all cells dually expressing the two CD34/CD164 antigens also revealed consistent colocalization patterns, particularly so where the cell membranes attached to the slides. However, an equally important finding is that CD164 surface expression on these dually expressing cells is not as high as CD34. This finding was further highlighted through the appearance on our computer three-dimensional antigen distribution representations of condensed crest-like areas radiating from the point of adherence to the microscope slide toward the top of the cell. In our other studies, where we have "pre-fixed" the cells before staining (data not shown), these antigen-dense crests are not so apparent. Whether sialomucin crest activity is really adhesion motivated will require more analysis, but such a hypothesis would correlate with possible interaction of CD164 and CD34 in homing, cell cycle inhibition, and adhesion to BM stroma. If CD164 is indeed believed to cooperate with CD34 [5, 6, 15], then perhaps cell membrane activation by physical adhesion or by circulating growth factors may recruit both CD164 and CD34 to perform their functions, as suggested by their condensation to these crest-like structures.

Whether the cells are in fact "adherence-aware" following contact with the confocal slide has to be considered. Whether CD34 and CD164 are directly involved in this process cannot be ruled out, but was not investigated here. Despite this, in our other electron microscopy investigations, HSPCs magnetically selected via the CD34 antigen displayed extensive membrane movement and activity that other nonmagnetic selected cells did not display [25, and work in preparation]. However, the immunomagnetic cell separation protocol applied to the electron microscopy work used the mouse anti-human QBEND10 antibody, labeling the class II CD34 molecule epitope, which has been reported to induce actin polymerization in hemopoietic cells and to strongly enhance cytoadhesiveness [10, 13]. This may indicate that clinically selected CD34+ cells may be artificially stimulated prior to transplant, the long-term implication of which is not known. It is worth noticing that in our present study, we used the HPCA-2 monoclonal antibody labeling the class III CD34 molecule epitope for unfractionated MNCs. This was used to prevent artificial activation/adhesion of the cells, but also because CD34 class III monoclonal antibody was reported to label a broader range of CD34+ cells than the class I and II counterparts [22].

We have avoided immunomagnetic selection here to initially study sialomucin localization patterns on HSPCs at new harvest. Immunophenotyping and flow cytometry characterized CD34+CD164+ cells as a very discrete cell population that required thorough and time-consuming confocal slide examination, revealing a consistent distribution profile for CD34 and CD164. In addition, as CD34 and CD164 colocalization often correlated with membrane activity at the adhesion point, the association between CD34 and CD164 membrane distribution and the HSPC activation status may be of importance. Further studies in our laboratory are focusing on comparing sialomucin localization profiles on cell populations enriched for quiescent HSPCs, or cytokine-stimulated proliferating HSPCs. This work has already indicated that the profile changes upon migratory cytokine stimulation (work in progress).

We would also speculate that the HSPC maturation level might also influence CD34 and CD164 membrane distribution. CD34+ cells still encompass a heterogeneous HSPC population [26]. It would therefore be interesting to further characterize CD34 and CD164 confocal localization patterns by comparing their distribution on the more immature CD34+CD38– HSPCs with more committed CD34+CD38+ cells. Our existing fluorescence-activated cell sorting data in this area (submitted work) has indicated that CD34+CD38– HSPCs express higher levels of AC133 and CD164 than the more mature CD34+CD38+ group. We would speculate that the CD34+CD164+ cell subset is relatively immature, as it was highly enriched with the AC133+ cells previously reported to have high proliferation potential and increased long-term culture initiating cell frequency [27, 28]. Our ongoing work investigates negatively selected populations to focus on the immature populations in comparison to standard immunomagnetically selected CD34+ and AC133+ populations.

Other studies support our theory on sialomucin coclustering upon adhesion. While studying common tyrosine phosphorylation signaling pathways for CD34 and CD43 (another sialomucin), Tada et al. [13] observed the formation of distinct CD34 and CD43 antigen clusters associated with F-actin reorganization upon cytoadhesion. CD43 clustering to cellular uropodia was also previously reported in activated T-lymphocyte and chemotactic neutrophils [29, 30]. They hypothesized that CD34 and CD43 clustering may be involved in HSPC polarization, which may be essential for adequate cell motility and cytoadhesion to the BM microenvironment. More recently, Krause et al. [9] observed that CD34 was upregulated in donor cells shortly after transplantation and prior to successful engraftment in the BM of a murine model. Taken together, these data highlight the importance of sialomucin colocalization along dense crests in adhering CB and BM HSPCs. CD34 and CD164 (and possibly CD43) may recruit one another along these dense crests to adequately polarize the progenitor cell prior to firmly anchoring itself to BM niches in vivo. A possible role for CD164 may, therefore, be to control and regulate CD34 condensation along the clustered crests.

In future experiments we will investigate further whether CD34 recruits CD164 into these antigen-dense crest-like areas. A dual linked function for these two sialomucins has implications for the current transplantation arena in which CD34 positive cells are widely employed.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are grateful to St. George’s Hospital Delivery Suite staff for their help in collecting umbilical cord blood samples, to the Haematology Department staff, St. George’s Hospital Medical School for provision of bone marrow samples, and to Professor Gordon-Smith for his advice and support. The authors would like to thank Mr. Ray Moss for his advice and assistance with the confocal microscope. We are very grateful to the Nuffield Foundation for awarding a vacation scholarship to Mr. Lojo-Rial (ref. # NUFURB00), and to the Wellcome Trust for a vacation scholarship awarded to Mr. Baradez (ref. # VS/00/kin/002/CH/TH/LC). We also thank the States of Jersey, Department of Education for supporting Mrs. Whiting. We acknowledge the contribution from the National Blood Service and The Leukaemia Research Fund (UK) to S.M. Watt.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on June 24, 2002; accepted for publication on September 11, 2002.




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