Rapid differentiation of a rare subset of adult human Lin(-)CD34(-)CD38(-)cells stimulated by multiple growth factors in vitro

Recently, several reports of lineage-negative (lin(-)) CD34(-) cells with i n vivo hematopoietic activity have focused interest on the properties and g rowth factor response characteristics of these cells. We have now identifie d a combination of 5 growth factors that are necessary and sufficient to st imulate a marked mitogenic and differentiation response by a subset of huma n lin(-)CD34(-)CD38(-) cells present in normal adult human marrow and granu locyte colony-stimulating factor (G-CSF)-mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin(-)CD34(-)CD38(- ) cells from these 2 sources formed colonies directly in semisolid medium o r generated such cells after 6 weeks in long-term culture. Nevertheless, ap proximately 1% of the same lin(-)CD34(-)CD38(-) cells were able to prolifer ate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper-IL-6 to produce a net 28- +/- 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% +/- 2% were CD34(+) and 2.5 % +/- 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multili neage colony-forming cells (CFC) (13 +/- 7 CFC per lin(-)CD34(-)CD38(-) pre -CFC). In contrast to lin(-)CD34(+)CD38(-) cells, this response of lin(-)CD 34(-)CD38(-) cells required exposure to all of the 5 growth factors used. U p to 1.7 x 10(5) lin(-)CD34(-) adult marrow cells failed to engraft subleth ally irradiated NOD/SCID-beta(2)M(-/-) mice. These studies demonstrate uniq ue properties of a rare subset of lin(-)CD34(-)CD38(-) cells present in bot h adult human marrow and mobilized blood samples that allow their rapid pro liferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults. (C) 1 999 by The American Society of Hematology.