Nakahata T, Ogawa M
J Cell Physiol. 1982 Jun;111(3):239-46. doi: 10.1002/jcp.1041110304.
We characterized murine hemopoietic colonies consisting of granulocytes, macrophages, megakaryocytes, and blast cells and yet lacking erythroid elements. Mouse marrow or spleen cells were cultured in methylcellulose media in the presence of 10% (v/v) pokeweek mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) and 2 units/ml erythropoietin for 8 days. Granulocyte-macrophage-megakaryocyte (GMM) colonies could be distinguished from granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies because the former lacked the typical appearance of bursts with red color. Analysis of Y-chromosomes in mixing experiments with male and female marrow cells confirmed the clonal nature of the GMM colonies. Differential counts of GMM colonies revealed varying, but significant, numbers of blast cells in all of the day-8 and day-12 colonies and in seven out of ten day-14 GMM colonies. In general, the percentages of blast cells were inversely related to the length of incubation in culture. Replating experiments confirmed the absence of late erythroid precursors such as CFU-E and normoblasts in all of the 50 day-8 GMM colonies. However, six out of the 50 GMM colonies contained early progenitors capable of erythroid expression, such as BFU-E, CFU-EM, CFU-GEM, and CFU-GEMM. In contrast, the three day-14 GMM colonies which did not reveal blast cells failed to produce secondary colonies. Thus, while the progenitors for the latter colonies are restricted to only granulocyte-macrophage-megakaryocyte differentiation, some of the apparent GMM colonies containing blast cells may have originated in early progenitors close to pluripotent stem cells. Detailed cytological analyses and replating experiments are necessary for characterization of true differentiation potentials of mixed colonies in culture.
我们对由粒细胞、巨噬细胞、巨核细胞和原始细胞组成但缺乏红系成分的小鼠造血集落进行了表征。将小鼠骨髓或脾细胞在含有10%（v/v）商陆丝裂原刺激的脾细胞条件培养基（PWM-SCM）和2单位/毫升促红细胞生成素的甲基纤维素培养基中培养8天。粒细胞-巨噬细胞-巨核细胞（GMM）集落可与粒细胞-红细胞-巨噬细胞-巨核细胞（GEMM）集落区分开来，因为前者缺乏带有红色的典型爆式集落外观。在雄性和雌性骨髓细胞混合实验中对Y染色体的分析证实了GMM集落的克隆性质。对GMM集落的差异计数显示，在所有第8天和第12天的集落以及十分之七的第14天GMM集落中，原始细胞数量各不相同但很显著。一般来说，原始细胞的百分比与培养中的孵育时间呈负相关。再接种实验证实，在所有50个第8天的GMM集落中均不存在晚期红系祖细胞，如CFU-E和正成红细胞。然而，50个GMM集落中有6个含有能够进行红系表达的早期祖细胞，如BFU-E、CFU-EM、CFU-GEM和CFU-GEMM。相比之下，未显示原始细胞的3个第14天GMM集落未能产生次级集落。因此，虽然后者集落的祖细胞仅限于粒细胞-巨噬细胞-巨核细胞分化，但一些含有原始细胞的明显GMM集落可能起源于接近多能干细胞的早期祖细胞。为了表征培养中混合集落的真正分化潜能，需要进行详细的细胞学分析和再接种实验。
