In vitro apoptotic cell death during erythroid differentiation

被引:25
作者
Zamai, L
Burattini, S
Luchetti, F
Canonico, B
Ferri, P
Melloni, E
Gonelli, A
Guidotti, L
Papa, S
Falcieri, E
机构
[1] Univ Urbino, Ist Sci Morfol, I-61029 Urbino, Italy
[2] Univ Urbino, Carlo BO, Ctr Citometria & Citomorfol, I-61029 Urbino, Italy
[3] Univ Ferrara, Sez Anat Umana, Dipartimento Morfol & Embriol, I-44100 Ferrara, Italy
[4] Univ Bologna, Ist Istol & Embriol Gen, I-40126 Bologna, Italy
[5] IOR, Unit Bologna, CNR, ITOI, Bologna, Italy
关键词
annexin-V; apoptosis; DNA fragmentation; erythropoiesis; flow cytometry;
D O I
10.1023/B:APPT.0000018805.63663.a5
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages ( day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.
引用
收藏
页码:235 / 246
页数:12
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