Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with...

Flow cytometry has been used in recent years to study antigenic and physical changes accompanying hematopoietic cell differentiation. Such studies have provided the basis for rapid and objective assays that are potential alternatives to the colony assays currently in widespread use. In this report, erythropoiesis was examined in growth factor-supplemented perfused cultures of bone marrow mononuclear cells (BMMNC) using flow cytometric analysis of the transferrin receptor (CD71), glycophorin antigen expression occurred with time, Initially, fewer than 10% of the cells were (CD71++, but by day 8, 19-34% of the cells were CD71++gly A-. This was followed by the appearance of gly A on 10-60% of the CD71++ cells. After day 10, CD71 expression on many gly A+ cells decreased so that a population of CD71-gly A+ cells (11-54 accumulated by day 14. Each phenotype was sorted for morphologic identification and colony assay analysis. CD71++gly A- cells were 85% blasts, one-half of which were erythroblasts, and were significantly enriched for burst-forming units-erythroid (BFU-E). The time-varying number of CD71++gly A- cells in these cultures was found to correlate with the number of BF.78-0.97). Three-color analysis was next used to examine CD33 expression on BFU-E, and in fresh BM, most were found to be CD33-. During culture, however, the number of BFU-E recovered from CD33+ populations first increased and then decreased. Therefore, CD33 was not particularly useful for identifying BFU-E. In contrast, CFU-GM were mostly found to be in the CD71+CD33+ population throughout the culture period. When erythropoietin (Epo) was not added to these cultures, the percentage of gly A+ cells was reduced from 33 to 3.3%. Further, the omission of Epo caused an 80% decrease in the number of BFU-E and a corresponding 94% decrease in the number of CD71++gly A- cells, thereby maintaining the relationship between CD71++gly A- cells and BFU-E. Therefore, flow cytometric analysis was found to be useful in assessing erythroid development, and this approach may be used to develop flow cytometric assays for other populations of interest in hematopoietic cell cultures.