Three‐dimensional analysis of CD34 sialomucin distribution on cord blood and bone marrow

Abstract

Summary. Determining the cellular distribution of key adhesion molecules may aid in understanding haematopoietic progenitor/stem cell (HPSC) homing to bone marrow (BM). CD34, a well-characterized marker for blast-like HPSC, is widely used for the isolation and enumeration of HPSC. Functional studies have yet to identify a ligand for CD34. However, growing evidence suggests that CD34 may aid the regulation of HPSC differentiation and modulate the expression of other HPSC adhesion molecules necessary for homing. This study aimed to determine CD34 distribution on umbilical cord blood (CB) and BM. CD34-selected cells were adhered to positively charged gold slides at room temperature, before indirect fluorescent antibody labelling with fluorescein isothiocyanate. Fluorescent distribution was determined by 1-lm interval, confocal laser scanning microscope Z-sections. Initial analysis showed CD34 distributed within peripheral halos and dense pocket regions. The development of three-dimensional imaging software enabled spatial visualization of CD34 distribution on CB and BM in association with differential interference contrast cell image. This showed that CD34 was distributed within peripheral halos, with magnupodia-associated 'meridian-shaped crescents', extending from points of cell-slide adhesion towards the top of the cell. True CD34 distribution, not previously discernible by confocal laser scanning microscopy alone, suggests a possible role in cell adhesion/homing revealed by three-dimensional imaging.

Three‐dimensional distribution patterns of CD34 antigen on nonactivated cord blood cells

“…On all cell surfaces the antigen patches are spread out flat, thus covering the cell surface membrane at varying degrees. Interestingly, two recent microscopical studies (3, 4) and electron microscopic analysis (11) on contact‐activated cells observed protruding, dense CD34 pockets; these were not found in our studies, which focus on patch distribution patterns of not contact‐activated progenitor cells.…”

“…3E) show low variations (<0.55 μm); this suggests that all patches are localized at similar distance from the center of the cell, and that the membrane approximates an “ideal” sphere. Therefore extrusions as observed previously in activated cells (3, 4) are absent in suspended cells.…”

“…Little is known about CD34 antigen distribution on the cell membrane; it is therefore tempting to study its distribution patterns and find out whether different types of such patterns exist. Two recent studies have employed confocal microscopy: in one study, circulating CD34 + cells were studied after adherence to glass slides (3), whereas, in another, bone marrow cells were fixed on gold support (4). In both studies cells were activated, which goes along with actin polymerization and enhanced adhesiveness (5).…”

Thrombopoietin, flt3‐ligand and c‐kit‐ligand modulate HOX gene expression in expanding cord blood CD133+ cells

“…CD133 +/– cells were obtained from MNC after immunomagnetic separation using the CD133 mini‐magnetic activated cell sorting (MACS) selection kit (Miltenyi Biotec, Bergish Gladbach, Germany) following the manufacturer's instructions as previously reported (Forraz et al . 2002a; Whiting et al . 2003; McGuckin et al .…”

Human hematopoietic microenvironments

“…HSPCs were classified according to cellular morphology, as well as to CD34-and CD38-immunofluorescence intensity and pattern (Fig 2 ). A cell profile was classified as an HSC/MPP when it fulfilled the following criteria: i) a representative nuclear profile; ii) intermediately to intensively positive for CD34 and negative for CD38; iii) high nucleus/cytoplasm ratio; iv) sparse CD34 fluorescence that distributes in dense fluorescent pockets and, depending on how the cell profile was sectioned, as a perinuclear partial halo (Fig 2) [37,38]. A cell profile was classified as a progenitor when it fulfilled the following criteria: i) a representative nuclear profile; ii)…”