Debili N, Coulombel L, Croisille L, Katz A, Guichard J, Breton-Gorius J, Vainchenker W
INSERM U362, Institut Gustave Roussy, Villejuif, France.
Blood. 1996 Aug 15;88(4):1284-96.
The aim of the present study was to determine if the human erythroid (E) and megakaryocytic (MK) lineages were closely linked to the existence of a bipotent burst-forming unit (BFU) E/MK progenitor. In methylcellulose cultures, BFU-E/MK colonies were observed at day 12 and closely resembled mature BFU-E with the exception that the erythroid component was surrounded by MK. These colonies were quite different from the colony forming unit (CFU)-GEMM-derived colonies, which were composed of a larger number of erythroblasts and which developed later in culture. The existence of these bilineage colonies composed of 100 to 1,000 erythroblasts intermingled with a few MK and without granulocytic cells was confirmed by the plasma clot technique and immunoalkaline phosphatase labeling of the MK. To investigate if this bipotent progenitor belonged to the compartment of primitive progenitors, CD34+ marrow cells were subfractionated according to expression of the CD38 antigen. The bipotent BFU-E/MK progenitor as well as a large fraction of MK progenitors were found in the CD34+ CD38+/- or in the CD34+ CD38- cell fractions. Growth of this bipotent BFU-E/MK progenitor required the combination of stem cell factor (SCF), Interleukin-3 (IL-3), and Epo in serum free conditions. Addition of IL-6 had only a marginal effect, whereas megakaryocyte growth and development factor (MGDF) was not an absolute requirement, but slightly increased the plating efficiency of CFU-MK and of BFU-E/MK progenitors when combined with SCF, IL-3, and Epo. In contrast, when these cultures were performed in the presence of 30% fetal calf serum, no BFU-E/MK colonies were observed irrespective of the combination of growth factors used, including the presence of MGDF; however, inclusion of the MS-5 cell line restored the growth of this bipotent progenitor. In contrast, in cultures performed in the presence of human normal or aplastic plasma, MS-5 had only a slight effect on the cloning efficiency but improved MK cytoplasmic maturation and MK size, suggesting that the main effect of MS-5 is to diminish the inhibitory effect of the fetal calf serum on the MK differentiation. The clonal origin of bipotent BFU-E/MK colonies was demonstrated in liquid culture of single CD34+ CD38low cells by immunophenotyping individual clones. At day 12, 30% of the clones contained erythroblasts (glycophorin A+) and some MK (CD41+) without granulocytes (G) or macrophages (M) (CD14+ and CD15+). At day 20, clones containing erythroblasts and MK were rare (5%). In contrast multilineage clones could be frequently detected at this time without passage from BFU-E/MK clones at day 12 to GEMM at day 20. These results suggest that a bipotent BFU-E/MK progenitor may be a nonrandom step in the hierarchical development of stem cells.
本研究的目的是确定人类红系（E）和巨核系（MK）谱系是否与双能爆式形成单位（BFU）E/MK祖细胞的存在密切相关。在甲基纤维素培养中，第12天观察到BFU-E/MK集落，其与成熟BFU-E非常相似，不同之处在于红系成分被MK包围。这些集落与集落形成单位（CFU）-GEMM衍生的集落有很大不同，后者由大量成红细胞组成，且在培养后期才出现。通过血浆凝块技术和MK的免疫碱性磷酸酶标记证实了这些由100至1000个成红细胞与少量MK混合且无粒细胞的双谱系集落的存在。为了研究这种双能祖细胞是否属于原始祖细胞区室，根据CD38抗原的表达对CD34+骨髓细胞进行了亚分类。在CD34+ CD38+/-或CD34+ CD38-细胞亚群中发现了双能BFU-E/MK祖细胞以及大部分MK祖细胞。在无血清条件下，这种双能BFU-E/MK祖细胞的生长需要干细胞因子（SCF）、白细胞介素-3（IL-3）和促红细胞生成素（Epo）的联合作用。添加IL-6仅有轻微作用，而巨核细胞生长和发育因子（MGDF）并非绝对必需，但与SCF、IL-3和Epo联合使用时可略微提高CFU-MK和BFU-E/MK祖细胞的接种效率。相反，当在30%胎牛血清存在下进行这些培养时，无论使用何种生长因子组合，包括MGDF存在与否，均未观察到BFU-E/MK集落；然而，加入MS-5细胞系可恢复这种双能祖细胞的生长。相比之下，在人正常或再生障碍性血浆存在下进行培养时，MS-5对克隆效率仅有轻微影响，但可改善MK的细胞质成熟和MK大小，这表明MS-5的主要作用是减轻胎牛血清对MK分化的抑制作用。通过对单个CD34+ CD38low细胞进行液体培养并对单个克隆进行免疫表型分析证实了双能BFU-E/MK集落的克隆起源。在第12天，30%的克隆含有成红细胞（血型糖蛋白A+）和一些MK（CD41+），无粒细胞（G）或巨噬细胞（M）（CD14+和CD15+）。在第20天，含有成红细胞和MK的克隆很少见（5%）。相反此时可频繁检测到多谱系克隆，且不存在从第12天的BFU-E/MK克隆到第20天的GEMM克隆的传代情况。这些结果表明，双能BFU-E/MK祖细胞可能是干细胞分级发育中的一个非随机步骤。
