Gunji Y, Nakamura M, Osawa H, Nagayoshi K, Nakauchi H, Miura Y, Yanagisawa M, Suda T
Department of Pediatrics and Medicine, Jichi Medical School, Tochigi-ken, Japan.
Blood. 1993 Dec 1;82(11):3283-9.
To clarify the phenotypes of various classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with CD34 and a newly developed mouse antihuman c-kit proto-oncogene product (KIT) monoclonal antibody (MoAb). We characterized three cell fractions in CD34+ cells that express KITlow and KIThigh cells in addition to KIT- cells. A clonogenic assay showed that most granulocyte-macrophage colony-forming cells (GM-CFC) were present in CD34+KIThigh populations, whereas erythroid burst-forming cells (BFU-E) were detected mainly in the CD34+KITlow population. CD34(+)-KIT- fraction contained a small number of BFU-E. Morphologic analysis showed that blast-like cells were more enriched in the CD34+KITlow fraction. KITlow cells contained CD34+CD38- cells that were considered to be very primitive progenitor cells, as determined by a replating assay. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell functions by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At week 2, more CFC recovered from the culture in the fraction initiated with a CD34+KIThigh population. However, more LTC-IC were present during weeks 5 to 9 in the CD34+KITlow population. These results indicate that primitive progenitors are more enriched in the KITlow population and that the KIThigh population contains many GM-committed progenitor cells. We also showed that anti-KIT MoAb inhibited the ability of CD34+ cells to generate CFC on the stromal layer in the LTC system. This suppressive effect was more evident in the generation of BFU-E by CD34+KITlow cells. Moreover, we confirmed that CD34+KIThigh cells emerged from CD34+KITlow cells during coculture with allogeneic stromal cells or from liquid culture in the presence of stem cell factor (SCF), interleukin-6, and erythropoietin. These results emphasize the pivotal role of the KIT and SCF interaction in hematopoiesis and indicate that KITlow cells are more primitive than KIThigh cells.
为了阐明各类人类造血祖细胞的表型，我们采用了一种多色染色方案，并结合CD34以及一种新开发的小鼠抗人c-kit原癌基因产物（KIT）单克隆抗体（MoAb）。我们对CD34+细胞中的三个细胞亚群进行了表征，除了KIT-细胞外，还包括表达KITlow和KIThigh的细胞。克隆形成试验表明，大多数粒细胞-巨噬细胞集落形成细胞（GM-CFC）存在于CD34+KIThigh群体中，而红系爆式集落形成细胞（BFU-E）主要在CD34+KITlow群体中被检测到。CD34(+)-KIT-亚群含有少量BFU-E。形态学分析表明，原始样细胞在CD34+KITlow亚群中更为富集。通过再接种试验确定，KITlow细胞含有被认为是非常原始的祖细胞的CD34+CD38-细胞。为了阐明这两个亚群之间的生物学差异，我们通过评估基质细胞上的长期培养起始细胞（LTC-IC）来检测更原始的祖细胞功能。在第2周时，从以CD34+KIThigh群体起始的培养物中回收的集落形成细胞（CFC）更多。然而，在第5至9周期间，CD34+KITlow群体中存在更多的LTC-IC。这些结果表明，原始祖细胞在KITlow群体中更为富集，并且KIThigh群体包含许多已向粒细胞-巨噬细胞系定向分化的祖细胞。我们还表明，抗KIT MoAb抑制了CD34+细胞在LTC系统的基质层上产生CFC的能力。这种抑制作用在CD34+KITlow细胞产生BFU-E的过程中更为明显。此外，我们证实CD34+KIThigh细胞在与异基因基质细胞共培养期间或在存在干细胞因子（SCF）、白细胞介素-6和促红细胞生成素的液体培养中从CD34+KITlow细胞分化而来。这些结果强调了KIT与SCF相互作用在造血过程中的关键作用，并表明KITlow细胞比KIThigh细胞更原始。
