Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells

Abstract

We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3), or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the alpha and beta subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and alpha- and beta-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (> or = 17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the alpha subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The beta subunits of the IL-3 and GM-CSF receptor, the alpha subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, alpha- and beta-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the alpha subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor, in particular, is activated both in erythroid (alpha- and beta-globin positive and in myeloid (alpha- and beta-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis.

Constitutive and inducible expression of megakaryocyte‐specific genes in Friend erythroleukaemia cells

“…A semiquantitative RT-PCR assay has been developed to analyse mRNA level modifications under different culture conditions; a detailed description of the theoretical and technical aspects of the procedure is reported elsewhere (Ashihara et al, 1997). Briefly, in preliminary experiments the RT-PCR reaction was optimized for each couple of primers so that the assumption could be made that, within the linear portion of the curve, the amplification kinetics were dependent on the amount of input cDNA.…”

Increased expression of the distal, but not of the proximal,Gata1 transcripts during differentiation of primary erythroid cells

“…The majority of the amplifications were in the linear phase between 20 and 30 cycles with the exception of the amplifications for ␣and ␤-globin cDNA, which were linear between 12 and 18 cycles. We considered as a measure of the mRNA for a certain gene the amount of its cDNA product (as a ratio of the product for actin) obtained at either 14 cycles (␣-and ␤-globin) or 30 cycles (all the other mRNAs; see also Ashihara et al, 1997;Moroni et al, 1997). S1 nuclease protection analysis of the levels of proximal and distal Gata1 transcripts in day 1 and day 3 PHZ cells The BamHI-Pst I fragment of the murine distal Gata1 cDNA (corresponding to nucleotides 1-1188; Ito et al, 1993) was purified on low-melting agarose gel and digested with EcoRI to obtain two fragments: a 371-bp fragment, corresponding to the distal-specific I Gata1 exon, the common II exon, and the first 37 bp of the III exon; and an 817-bp fragment, corresponding to the region from the III exon to the first part of VI exon (see Fig.…”

“…Gata1 mRNA is detectable not only in erythroid cells but also, although at lower levels, in megakaryocytes (Martin et al, 1990;Romeo et al, 1990), in mast cells (Crotta et al, 1980;Martin et al, 1990), and in immature granulocytes (Crotta et al, 1990;Ashihara et al, 1997). Gata1 mRNA has also been detected in the Sertoli cells of prepuberal mouse testis (Ito et al, 1993).…”

Role of GATA‐1 in Normal and Neoplastic Hemopoiesis

“…The first indication that the activation of GATA‐1 expression is regulated in a lineage‐specific fashion was the observation that, although GATA‐1 is expressed in erythroid, megakaryocytic, mast, and eosinophilic cells, its level of expression differs greatly among the various cell types 2,16. Furthermore, purified murine stem cells, which do not express detectable levels of GATA‐1,17,18 activate its expression within 24 h in culture (i.e., before they are induced to cycle) when exposed to both erythroid‐ and myeloid‐specific growth factor combinations 19. However, whereas myeloid growth factors activate the expression from only the promoter proximal to the first AUG, a consistent level of expression from an alternative promoter located 8 kb upstream from the AUG (called the distal or testis promoter because it was originally cloned from Sertoli cells of the testis9) is observed only in cells that are exposed to erythroid‐specific growth factors 19.…”

“…GATA‐2 is expressed in the hemopoietic stem cells and, together with GATA‐1 , in multipotent progenitor cells 17,18. The expression ratios between GATA‐2 and GATA‐1 change as the progenitor cells progress along the differentiation pathway: GATA‐2 is expressed at high levels in early cells, and GATA‐1 expression increases as differentiation advances 17,18. The mechanism regulating the GATA‐2:GATA‐1 expression ratio has been proposed to be a possible mitotic clock at the progenitor cell level 80.…”