THE EXPRESSION OF CYTOKINE RECEPTORS BY PURIFIED HEMATOPOIETIC STEM-CELLS

Sorted fractions from mouse bone marrow containing highly purified hem opoietic stem and progenitor cells were studied for the expression of growth factor receptors. With the use of rhodamine 123 WGA+, 15-1.1-, low density cells were separated into quiescent pluripotent stem cells and committed progenitor cells. RNA was extracted and cDNA was prepar ed by reverse transcription. Using primers specific for growth factor receptors, the cDNA of each sorted fraction was amplified by polymeras e chain reaction (PCR). The quiescent rhodamine 123 dull stem cell fra ction was found to express the interleukin 3 (IL-3) receptor beta unit and c-kit, but not the granulocyte-macrophage colony stimulating fact or (GM-CSF) receptor beta unit nor flk-2. The rhodamine 123 bright fra ction with activated stem cells and mostly committed progenitor cells similarly expressed the IL-3-Rbeta, and c-kit. However, this fraction also expressed flk-2 and GM-CSF-Rbeta. Since the expression of c-kit i n the stem cell fraction does not correspond with the poor response to the kit-ligand stem cell factor (SCF) by these cells, we further anal yzed the fractions with respect to their binding of biotinylated SCF. The SCF-binding cells were found to be all rhodamine 123 bright. This indicates that the expression of c-kit is not sufficient to yield a fu nctional receptor for SCF; c-kit probably needs a partner molecule to form a functional high-affinity binding site for SCF. Similar to the b eta unit of the GM-CSF receptor, this partner is then not expressed in the stem cell fraction. Its expression would be upregulated upon diff erentiation and commitment. It may be speculated that this partner mol ecule of c-kit is flk-2. Alternatively, it may be speculated that the stem cells contain an isoform splice product of kit that has a reduced capability for dimerization.