Yoo Eun-Sun, Lee Kyung-Eun, Seo Jeong-Wan, Yoo Eun-Hee, Lee Mi-Ae, Im Seock-Ah, Mun Yeung-Chul, Lee Soon-Nam, Huh Jung-Won, Kim Mi-Jung, Jo Duck-Yeon, Ahn Jee-Young, Lee Sun-Mee, Chung Wha-Soon, Kim Jae-Hong, Seong Chu-Myong
Department of Pediatrics, College of Medicine, Ewha Women's University, Seoul, Korea.
Stem Cells. 2003;21(2):228-35. doi: 10.1634/stemcells.21-2-228.
Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.
造血作用依赖于造血干细胞与构成造血微环境的基质细胞之间的关联。脐带血（UCB）来源的内皮细胞在体外的发育尚未完全明确，且取得的成功非常有限。在本研究中，使用血小板生成素、fms样酪氨酸激酶3配体和粒细胞集落刺激因子，将UCB CD34(+)细胞在无基质液体培养系统中培养5周。到第4 - 5周时，我们发现形成了牢固黏附的成纤维细胞样细胞。这些细胞表现出内皮细胞的特征，表达血管性血友病因子、人血管细胞黏附分子-1、人细胞间黏附分子-1、人CD31、E-选择素和人巨噬细胞。此外，当将没有建立内皮单层的体外系统与有建立内皮单层的体外系统进行比较时，在培养过程中发现总核细胞、CD34(+)细胞以及集落形成单位（CFU）-粒细胞-巨噬细胞和CFU-粒细胞-红系-巨核细胞-巨噬细胞有更好的扩增。这种现象部分是由于在黏附单层上培养长达5周的CD34(+)细胞中凋亡分数显著降低。为了获取关于源自给定数量CD34细胞的内皮细胞数量的定量数据，我们使用泊松分布进行有限稀释分析：产生37%阴性培养物（对数标度）的测试细胞数量（线性标度）就是含有一个内皮细胞的细胞数量。通过这种方法，培养5周后从314个CD34(+)细胞中可能发现一个内皮细胞。这些结果表明，UCB CD34(+)细胞组分包含内皮细胞前体，可建立造血微环境，并通过下调凋亡对UCB扩增方案产生有益影响。这些观察结果可能为未来的细胞治疗或移植工程提供见解。
