Acquisition of CD34 Correlates with Increased Hematopoietic and Self Renewal Activity of CD34−CD133+ Cord Blood Cells.

CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.