STRINGENTLY PURIFIED HUMAN HEMATOPOIETIC PROGENITORS STEM-CELLS - ANALYSIS OF CELLULAR MOLECULAR MECHANISMS UNDERLYING EARLY HEMATOPOIESIS

Analysis of the cellular/molecular basis of the early steps of hematop oietic proliferation and differentiation is hindered by the rarity of hematopoietic progenitors and stem cells (HP/HSC). The intensive effor ts devoted to the development of purification methods for early HP and HSC, although initially largely unsuccessful, have recently provided a high level of HP/HSC yield and/or recovery. The methodology develope d by our group, recently improved, provides not only virtually complet e purification, but also abundant recovery of early HP/HSC such as col ony forming units granulocyte/erythroid/macrophage/megakaryocyte (CFU- GEMM), burst forming units erythroid (BFU-E), CFU granulocyte/macropha ge (CFU-GM)/CFU blast cells (CFU-B), and long-term culture initiating cells (LTC-IC) from adult peripheral and cord blood (CB). We have also developed a serum-free liquid suspension culture for unilineage eryth roid (E), granulocytic (G) or monocytic (M) differentiation of stringe ntly purified HP/HSC. These culture systems allow sequential collectio n and cellular/molecular analysis of discrete populations of hematopoi etic cells at a homogenous stage of differentiation specifically along a unilineage pathway. These experimental tools have been utilized to investigate cellular/molecular mechanisms underlying early hematopoies is. The transcription factor (TF) GATA-1 is considered to be the ''mas ter'' gene of erythropoiesis. In highly purified HP/HSC undergoing E o r GM differentiation, GATA-1 expression is characterized initially by proliferation-dependent activation and at later stages by sustained ex pression in the E pathway and suppression in the GM pathway. Hypotheti cally, similar on/off switches of lineage-restricted TF may underlie t he binary fate decisions of early HP differentiation. The expression a nd modulation of hematopoietic growth factor receptors (HGFR) in early hematopoiesis have been extensively analyzed. The results suggest a m odel of transactivation cascade for HGFR such as interleukin 6 recepto r (IL-6R), IL-3R, GM colony stimulating factor receptor (GM-CSFR), and erythropoietin receptor (EpR), whereby each HGF upmodulates the R(s) for distal-acting HGF(s). Finally, we have investigated the effect of HGF on reactivation of hemoglobin F (HbF) in clonogenic or liquid susp ension serum-free culture of purified adult HP. The results suggest th at c-kit ligand (KL) plays a key role in the reactivation of HbF synth esis in adult life, and IL-3/GM-CSF potentiate this effect at low KL l evel. The KL-induced HbF reactivation is seemingly related to an enhan ced proliferation of early E progenitors in their differentiation path way.