Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture

Recently, culture conditions that stimulate the proliferation of primitive hematopoietic cells defined by various phenotypic and functional endpoints in vitro have been identified. However, evidence that they support a high p robability of self-renewal leading to a large net expansion in vitro of tra nsplantable cells with lympho-myeloid repopulating ability has been more di fficult to obtain. The present study was designed to investigate whether th e low overall expansion of human repopulating hematopoietic cells seen in v itro reflects a selective unresponsiveness of these rare cells to the growt h factors currently used to stimulate them or, alternatively, whether they do proliferate in vitro but lose engrafting potential, For this, we used a high-resolution procedure for tracking and reisolating cells as a function of their proliferation history based on the loss of cellular fluorescence a fter staining with (5- and 6-) carboxyfluorescein diacetate succinimidyl es ter. The results show that the vast majority of long-term culture-initiatin g cells and in vivo lympho-myeloid competitive repopulating units present i n 5-day suspension cultures initiated with CD34(+) human cord blood and fet al liver cells are the progeny of cells that have divided at least once in response to stimulation by interleukin-3, interleukin-6, granulocyte colony -stimulating factor, Steel factor, and Flt3-ligand. Thus, most human repopu lating cells from these two sources are stimulated to undergo multiple divi sions under currently used short-term suspension culture conditions and a p roportion of these retain engraftment potential. (C) 1999 by The American S ociety of Hematology.