Dvorak A M, Mitsui H, Ishizaka T
Department of Pathology, Beth Israel Hospital, Boston, Massachusetts 02215.
J Leukoc Biol. 1993 Nov;54(5):465-85. doi: 10.1002/jlb.54.5.465.
The ability to culture human mast cells and, thus, to determine their ontogeny and possible relationships to other lineages has been facilitated by new studies using cocultures of cord blood cells with mouse fibroblasts and recombinant human or murine c-kit ligand-supplemented suspension cultures of cord blood cells. In this study, we examined c-kit ligand-supplemented cord blood cell suspension cultures designed so that the effects of growth factor source, individual cord sample, and culture time (3-17 weeks) on the developing mast cell lineage could be individually evaluated. We found that human mast cells, basophils, neutrophils, eosinophils, macrophages, megakaryocytes, and endothelial cells were present in these cultures. The numbers of mast cells and their granules increased with culture time; mature basophils, present in quantity in 3-week cultures, decreased in number and released granule contents with increased culture times. The mast cell lineage developed similarly, regardless of which factor preparation was added to cultures, but considerable variability existed among individual donors from whom cord bloods were obtained. Unlike the mature, crystal-containing mast cells that regularly developed in fibroblast cord blood cocultures (Furitsu et al. [1989] Proc. Natl. Acad. Sci. USA 86, 10039-10043), human mast cells failed to attain full maturity in the suspension cultures examined here, regardless of individual cord sample, added growth factor, or culture time. Furthermore, unlike cells of the basophil lineage in which granule content release was regularly observed, morphologic evidence of secretion from human mast cells was absent. Instead, these cells were actively undergoing granule building as determined by the increasing numbers of granules and filling of these containers over culture time. Crystal granules never developed, even at the maximum culture time of 17 weeks. We conclude that fibroblasts are necessary and sufficient for the differentiation and maturation of human mast cells in vitro from their agranular precursors in cord blood but that soluble c-kit ligand is not.
利用脐血细胞与小鼠成纤维细胞共培养以及补充重组人或鼠c-kit配体的脐血细胞悬浮培养的新研究，促进了培养人肥大细胞的能力，从而能够确定其个体发生以及与其他谱系的可能关系。在本研究中，我们检测了补充c-kit配体的脐血细胞悬浮培养，其设计目的是能够分别评估生长因子来源、个体脐血样本以及培养时间（3至17周）对发育中的肥大细胞谱系的影响。我们发现这些培养物中存在人肥大细胞、嗜碱性粒细胞、中性粒细胞、嗜酸性粒细胞、巨噬细胞、巨核细胞和内皮细胞。肥大细胞及其颗粒的数量随培养时间增加；3周培养物中大量存在的成熟嗜碱性粒细胞数量减少，且随着培养时间延长其颗粒内容物释放。无论向培养物中添加哪种因子制剂，肥大细胞谱系的发育都相似，但从其获取脐血的个体供体之间存在相当大的变异性。与在成纤维细胞-脐血共培养中正常发育的含有晶体的成熟肥大细胞不同（Furitsu等人[1989]美国国家科学院院刊86, 10039 - 10043），在此处检测的悬浮培养物中，人肥大细胞无论个体脐血样本、添加的生长因子或培养时间如何，都未能达到完全成熟。此外，与经常观察到颗粒内容物释放的嗜碱性粒细胞谱系细胞不同，人肥大细胞没有分泌的形态学证据。相反，通过颗粒数量的增加以及随着培养时间这些容器的填充情况确定，这些细胞正在积极进行颗粒构建。即使在最长17周的培养时间，晶体颗粒也从未形成。我们得出结论，成纤维细胞对于体外从脐血中的无颗粒前体分化和成熟人肥大细胞是必要且充分的，但可溶性c-kit配体并非如此。
