Down-Regulation of c-Kit Receptor on Hematopoietic Cells By Stem Cell Factor (SCF; c-Kit-ligand): No Impact on Transplantation

Presence of the c-Kit tyrosine kinase receptor is a hallmark of the mouse hematopoietic stem cells (HSCs) and progenitors routinely used for their identification and separation. c-Kit is activated after binding of its ligand, the stem cell factor (SCF; c-Kit-ligand). c-Kit receptors with bound SCF form dimers that are rapidly internalized and degraded. This activates the c-Kit signaling pathways supporting cell survival, proliferation or quiescence and self-renewal. Although there is a consensus that c-Kit signaling is important for functioning of HSCs, published results are partly controversial.

We have defined HSCs and progenitors as Lineage^- Sca-1⁺c-Kit⁺ cells (LSK cells) and characterized them further by means of the CD150 and CD48 markers. We used anti-c-Kit antibody minus (FMO; Fluorescence Minus One) samples to distinguish between c-Kit⁺ and c-Kit^- bone marrow cells and analyzed the distribution of c-Kit on the immature hematopoietic cells carrying different phenotypes. Further, we exposed bone marrow cells to a wide range of concentration of a recombinant mouse SCF in vitro and measured a change in c-Kit presence and distribution on these different cell types. Also, SCF was injected to mice in vivo and their bone marrow was similarly analyzed for a change in c-Kit expression. Bone marrow cells exposed to SCF concentrations that deeply down-regulated c-Kit receptors were transplanted to recipient mice, and their transplantation efficiency was compared to that of normal bone marrow.

c-Kit was unevenly but characteristically distributed on different types of LSK CD150/CD48 cells, showing the highest and the most homogeneous density on cells with the LSK CD150⁺CD48^- phenotype. Exposure of bone marrow cells to SCF in ranges of concentrations from 0.3-2000 ng/ml induced progressive down-regulation of c-Kit. However, the cells mostly remained c-Kit^+(low). The response to SCF was the most prominent in a range of SCF concentrations between 1-100 ng/ml. Cells with the phenotype LSK CD150⁺CD48⁺were relative low-responders. In vivo administration of SCF to mice in doses exceeding 300 ng/mouse, either intraperitoneally or intravenously, had similar effect on c-Kit expression by bone marrow cells as their incubation with SCF in vitro.

Next we investigated correlation of the intensity of c-Kit receptor expression on bone marrow cells with their repopulating capacity after transplantation. A significantly decreased c-Kit expression on transplanted cells, induced by exposure of the cells to SCF, did not decrease contribution of the cells to chimeric hematopoiesis in competitive transplantation assays. Formation of spleen colonies was also not affected in the CFU-S assay.

Experiments which measured the effect of SCF administered to normal mice in vivo demonstrated an effect that lasted for less than 12 hours. c-Kit turnover on hematopoietic cells is thus rapid, and this fact may explain why down-regulation of c-Kit, on otherwise normal bone marrow cells, does not affect their capacity to be transplanted.

In conclusion, c-Kit receptor density on hematopoietic cells does not appear to be a critical factor for the homing of transplanted hematopoietic stem and progenitor cells into the blood-forming tissues and their engraftment into specific niches. Also their performance in establishing productive hematopoiesis is not altered by the SCF-induced down-regulation of the c-Kit receptor density in time of their transplantation.