GROWTH-FACTOR RECEPTOR EXPRESSION DURING IN-VITRO DIFFERENTIATION OF PARTIALLY PURIFIED POPULATIONS CONTAINING MURINE STEM-CELLS

We have investigated, by semiquantitative RT-PCR, the kinetics of acti vation of hematopoietic receptors and differentiation markers in parti ally purified murine hematopoietic stem cells (HSC) induced to differe ntiate in serum-free culture with combinations of growth factor (CF). The combinations of CF used sustained either multilineage [stem cell f actor (SCF) + interleukin 3 (IL-3)], or erythroid [SCF + IL-3 + erythr opoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulatin g factor (G-CSF)] differentiation. The GF receptor genes investigated were the alpha and beta subunits of the IL-3 and granulocyte-macrophag e colony-stimulating factor (GM-CSF) receptor, the erythropoietin rece ptor, the C-CSF receptor, and c-Fms, the receptor for macrophage colon y-stimulating factor (M-CSF). The expression of Gata1 and alpha- and b eta-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, wh ich include partitioning of lineage-negative bone marrow cells with th e mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (greater than or equal to 17% of which reconstitute long-term hematopoiesis in recipie nt mice) and into Rho-bright (which are as capable as Rho-dull of mult ilineage differentiation but do not permanently reconstitute the host) . The following pattern of expression was observed: the a subunit of t he IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over lime in c ulture. The beta subunits of the IL-3 and CM-CSF receptor, the or subu nit of the CM-CSF receptor, the Epo and C-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their e xpression increased in cells cultured both in erythroid and in myeloid CF combinations. Gata1 was expressed maximally in Rho-bright cells bu t was below the level of detection in Rho-dull cells. Rho-dull cells e xpressed Gata1 when cultured both in erythroid and in myeloid GF combi nations. In contrast, alpha- and beta-globin, which also were not expr essed in the purified cells, were induced only in cells stimulated wit h Epo. These results indicate that the genes for all the CF receptors investigated (with the exception of the a subunit of the IL-3 receptor ) are expressed at low levels, ii any, in purified Rho-bright or Rho-d ull cells, but are expressed in their progeny cultured either in eryth roid or myeloid GF combinations. The expression of the Epo receptor,in particular, is activated both in erythroid (alpha- and beta-globin po sitive) and in myeloid (alpha- and beta-globin negative) cells. Theref ore, activation of the expression of the Epo receptor gene and activat ion of the erythroid differentiation program are two independent event s in normal hematopoiesis. (C) 1997 Wiley-Liss, Inc.