No abstract

We have developed a time-lapse camera system to follow the replication history and the fate of hematopoietic stem cells (HSC) at a single-cell level. Combined with single-cell culture, we correlated the early replication behavior with colony development after 14 days. The membrane dye PKH26 was used to monitor cell division. In addition to multiple, synchronous, and symmetric divisions, single-sorted CD34+/CD38− cells derived from fetal liver (FLV) also gave rise to a daughter cell that remained quiescent for up to 8 days, whereas the other daughter cell proliferated exponentially. Upon separation and replating as single cells onto medium containing a cytokine cocktail, 60.6% ± 9.8% of the initially quiescent cells (PKH26 bright) gave rise again to colonies and 15.8% ± 7.8% to blast colonies that could be replated. We have then determined the effects of various regulatory molecules on symmetry of initial cell divisions. After single-cell sorting, the CD34+/CD38− cells derived from FLV were exposed to flt3-ligand, thrombopoietin, stem cell factor (SCF), or medium containing a cytokine cocktail (with SCF, interleukin-3, interleukin-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin). Whereas mitotic rate, colony efficiency, and asymmetric divisions could be altered using various regulatory molecules, the asymmetric division index, defined as the number of asymmetric divisions versus the number of dividing cells, was not altered significantly. This observation suggests that, although lineage commitment and cell proliferation can be skewed by extrinsic signaling, symmetry of early divisions is probably under the control of intrinsic factors.

A primitive human hematopoietic myeloid progenitor cell line, KG1a, characterized by high expression of the CD34 surface antigen has been observed to extend long, thin pseudopodia. Once extended, these pseudopods may take on one of two newly described morphologies, tenupodia or magnupodia. Tenupodia are very thin and form in linear segments. They adhere to the substrate, can bifurcate multiple times, and often appear to connect the membranes of cells more than 300 μm apart. Magnupodia are much thicker and have been observed to extend more than 330 μm away from the cell. Magnupods are flexible and can exhibit rapid dynamic motion, extending or retracting in a few seconds. During retraction, the extended material often pools into a bulb located on the pod. Both morphologies can adhere to substrates coated with fibronectin, collagen IV, and laminin as well as plastic. The CD34 and CD44 antigens are also present on the surface of these podia. Primary human CD34+ cells from fetal liver, umbilical cord blood, adult bone marrow, and mobilized peripheral blood extend these podia as well. The morphology that these pseudopods exhibit suggest that they may play both sensory and mechanical roles during cell migration and homing after bone marrow transplantation.

Background:The phototoxic effects of the well-known fluorescent membrane dyes PKH2 and PKH26 have been unknown, although their use in cell tracking experiments has increased dramatically. To eliminate the phototoxicityinduced alteration in cell function and morphology, it is essential to examine the suspicious phototoxicity of these dyes. Methods: Chemical and phototoxic effects of PKH dyes on the human hematopoietic KG1a cell line were examined. To minimize phototoxicity in long-term cell tracking experiments lasting up to 18 h with a fluorescence microscope system, time-lapse monitoring with different time intervals and exposure times was introduced. Results: There were no significant effects of the two PKH dyes on cell viability and growth when using dye concen-trations up to 5 µM. However, when stained cells were exposed to excitation light, cell viability decreased dramatically, showing the phototoxicity of the PKH dyes. More than 60% of cells stained with 5 µM PKH26 died after 5 min of continuous light exposure. The phototoxic effect was more extensive in cells stained with higher concentrations of the dyes. Conclusions: We present guidelines for the optimal use of these dyes by using a defined hardware configuration.