Sawada K, Krantz S B, Dai C H, Sato N, Ieko M, Sakurama S, Yasukouchi T, Nakagawa S
Second Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo, Japan.
J Cell Physiol. 1991 Oct;149(1):1-8. doi: 10.1002/jcp.1041490102.
The course of the differentiation and proliferation of the human erythroid burst-forming units (BFU-E) to colony-forming units (CFU-E) was directly investigated using a combination of highly purified BFU-E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU-E with a purity of 45-79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU-E required not only rEP but also rIL-3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 +/- 20-fold above the starting cells, while erythroid progenitors increased 156 +/- 74-fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rEP combined with or without rIL-3 showed the following: 1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. 2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rIL-3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU-E. These studies provide a quantitative assessment of the loss of IL-3 dependency by BFU-E and indicate that the size of the generated erythroid colonies and their IL-3 requirement correlate with the erythroid differentiated state.
利用高度纯化的红系爆式集落形成单位（BFU-E）、液体培养系统以及以下克隆分析方法，直接研究了人类BFU-E向集落形成单位（CFU-E）分化和增殖的过程。将纯度为45%-79%的高度纯化的人类血液BFU-E在含有重组人促红细胞生成素（rEP）和重组人白细胞介素-3（rIL-3）的液体培养基中培养，以生成更多分化的红系祖细胞。每天收集培养的细胞，以研究其形态、细胞数量的增加以及克隆性。90%的纯化BFU-E克隆发育不仅需要rEP，还需要rIL-3。液体培养7天时，总细胞数比起始细胞增加了237±20倍，而红系祖细胞增加了156±74倍。随着液体培养孵育时间的增加，细胞形态持续分化。用rEP联合或不联合rIL-3进行再接种实验，结果如下：1）作为红系集落一部分的成红细胞数量随着红系祖细胞的分化和增殖而减少。2）随着液体培养孵育时间的增加，红系祖细胞对rIL-3的依赖性逐渐丧失，液体培养3天后观察到完全丧失依赖性。此时，85%的红系祖细胞形成了超过100个成红细胞的集落，相当于成熟的BFU-E。这些研究对BFU-E丧失IL-3依赖性进行了定量评估，并表明所生成的红系集落的大小及其对IL-3的需求与红系分化状态相关。
