Electron microscopy demonstration of CD34 antigen on progenitor pseudopodia

Current theory predicts stem cells are CD34-negative, despite CD34 use for therapeutic progenitor purification. Expressed on early haemopoietic cells, it is implicated in cellular adhesion, and marrow stromal cell homing. There are 2 CD34 forms, one intracellularly truncated, hypothesised for adhesion alone, with the extended variant capable of signal transduction. Often assumed as evenly expressed over the cell surface, our transmission electron microscopy (TEM) CD34 tests do not agree. Immunomagnetically selected CD34+ cells (MACS, Miltenyi Biotec) from human umbilical cord blood, and KG1a (CD34+) & HL60 (CD34-) leukaemia cell lines were anti-CD34 antibody-labelled prior to secondary isotype-matched gold antibody (between 30 to 120 mins 4°C). Blocking controls used unspecific mouse IgG1 primary antibody. Cells were fixed in Karnovskyʹs, postfixed in osmium tetroxide, embedded in “Epon” and sections were uranyl acetate counterstained before observing by TEM. HL60 cells lacked CD34. KG1a & Cord Blood distributed antigen that was not even over the surface, but specific to small pseudopodia. Smooth membrane presented no gold-labelled antigen, unless 2 adjacent cells had interacted. Time of incubation, even at 4°C was critical. Extended KG1a incubation caused CD34 gold-label to internalise. 60 mins incubation of selected CD34+ cord blood showed extensive magnetic iron bead- & gold-labelled CD34 antigen capping. Antigen internalisation, complete with iron beads was also seen. This suggests CD34 antigens not only possess adhesive properties, but that cells can detect adhesion and are stimulated to respond. This suggests clinically selected CD34+ populations may be artificially stimulated before therapeutic use.