Establishment of normal, terminally differentiating mouse erythroid progenitors: molecular characterization by cDNA arrays

Abstract

Expression profiling with cDNA arrays is an excellent tool for molecular analysis of complex processes such as terminal erythroid differentiation. The shortcomings of the currently available erythroid in vitro differentiation models, however, severely impaired the usefulness of this approach to study erythropoiesis. Here, we describe a novel, murine erythroid cell system closely corresponding to in vivo erythroid progenitors. Mortal, long‐term proliferating erythroid progenitors of fetal liver or immortal strains of p53‐deficient erythroblasts were established in culture. Both cell types proliferated in serum‐free medium and were strictly dependent on physiologically relevant cytokines and hormones, stably retaining a diploid set of chromosomes. If exposed to physiological differentiation factors (erythropoietin plus insulin), cells synchronously recapitulated the normal in vivo differentiation program to mature terminally into enucleated erythrocytes and expressed stage‐specific erythroid transcription factors in the expected temporal order. Using cDNA arrays, we found a large number of genes differentially expressed at time points during differentiation. Already 6 h after differentiation induction, 17% of the expressed genes showed significant alterations in mRNA abundance, increasing to 53% (12% up‐regulated, 41% down‐regulated genes) by 48 h. Cluster analysis of mRNA expression kinetics during differentiation identified six distinct expression patterns. All genes on the array with a known function in erythropoiesis showed the expected variations in expression. The genes identified also allowed first insights into the sequence of events within the regulatory network responsible for erythroid maturation. In mortal wild‐type as well as immortal p53‐/‐ erythroblasts, changes in mRNA abundance of several well‐regulated gene products was verified at the protein level. Taken together, this novel hematopoietic cell system faithfully executes essential steps of normal erythropoiesis and allows us to dissect and characterize molecular mechanisms involved in erythropoiesis.

Global transcription analysis of immature avian erythrocytic progenitors: from self-renewal to differentiation

“…One analysed the variations in gene expression when those cells were induced to differentiate (Dolznig et al, 2001), whereas the second one focused on the cooperative signalling by Epo, SCF and Dex upon gene expression (Kolbus et al, 2003). It is important to underline that P53 À/À progenitors are more mature than T2EC cells and can be considered closer to the EpoR progenitors than the T2EC cells, as far as their level of differentiation is concerned (Bauer et al, 2001;Dazy et al, 2003).…”

Apoptotic mechanisms in the control of erythropoiesis

“…45 In a second cellular system, an immortalized murine cell line was used: these cells correspond to immature murine erythroid precursors isolated from p53 À/À mice that proliferate in medium containing SCF and Dexamethasone, while they differentiate in medium containing Epo and Insulin. 46 When shifted to a differentiating medium, these cells undergo terminal maturation in 72 h. Bcl-X L expression in these cells progressively increases, reaching maximal expression at terminal erythroid maturation. 46 In another study, immature murine erythroblasts, mainly corresponding to proerythroblasts, have been isolated from the spleen of mice infected 2 weeks earlier with an anemia-inducing strain of Friend virus and then induced in vitro to terminal erythroid maturation in the presence of Epo; alternatively, cells corresponding to CFU-E have been isolated after 1 week of culture in vitro of BFU-E in semisolid medium and then grown in vitro in the presence of Epo up to terminal maturation.…”

“…46 When shifted to a differentiating medium, these cells undergo terminal maturation in 72 h. Bcl-X L expression in these cells progressively increases, reaching maximal expression at terminal erythroid maturation. 46 In another study, immature murine erythroblasts, mainly corresponding to proerythroblasts, have been isolated from the spleen of mice infected 2 weeks earlier with an anemia-inducing strain of Friend virus and then induced in vitro to terminal erythroid maturation in the presence of Epo; alternatively, cells corresponding to CFU-E have been isolated after 1 week of culture in vitro of BFU-E in semisolid medium and then grown in vitro in the presence of Epo up to terminal maturation. In both these cell culture systems, Bcl-X L was expressed at very low levels in immature erythroblasts and its expression progressively and markedly increased during maturation up to terminal erythroblasts.…”

Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis

“…However, the GR and c-Kit are indispensable for stress erythropoiesis Broudy et al, 1996). Likewise, a potential function for Stat5 in stress hematopoiesis was recently identi®ed using genetically modi®ed mice, both in the myeloid and the erythroid lineage (Kieslinger et al, 2000;Deiner et al (2001) in preparation). Notably, STAT5-de®cient mice failed to develop certain types of leukemia (Levy and Gilliland, 2000).…”