Kusadasi N, Koevoet J L, van Soest P L, Ploemacher R E
Institute of Hematology, Erasmus University Rotterdam, The Netherlands.
Leukemia. 2001 Sep;15(9):1347-58. doi: 10.1038/sj.leu.2402204.
Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.
目前在1至2周内对造血干细胞/祖细胞（HSPC）进行体外数字扩增的技术，不足以保证其移植后在重建时间上有显著缩短。为了更严格地测试影响HSPC扩增的参数，我们对CD34 +选择的脐带血（UCB）HSPC进行了长达10周的体外培养，并研究了基质支持和添加细胞因子的影响。细胞因子组合包括FL + TPO、FL + TPO加SCF和/或IL6，或SCF + IL6。为了鉴定未培养和培养材料中的HSPC，我们通过表型分析确定了集落形成细胞（CFC）、鹅卵石区域形成细胞（CAFC）的数量、NOD/SCID重建造血能力（SRA）以及CD34 +亚群。对于无基质培养，CD34 +和CD34 + CD38 -细胞数量的最高扩增倍数分别为1197和30937，对于有基质支持的培养分别为4066和117235。一般来说，在含FL + TPO的组中，直到第5周CFC生成每周增加，扩增倍数为28至195倍，此后每周CFC产量稳定。基质支持在第6周最大程度地增强了CAFC的扩增，在FL + TPO + IL6刺激下扩增了11至89倍。在无基质和有基质支持的培养2周时，至少用FL + TPO刺激的培养物使CD34 + UCB细胞多谱系植入亚致死照射的NOD/SCID小鼠骨髓的能力估计扩增了10至14倍，而在第5周及以后，所有组的估计SRA降至低水平或无法检测到的水平。我们的结果表明，基质和FL + TPO，以及牛血清白蛋白的加入，通过体外测定法极大地增加了HSPC的长期生成，并且对于CD34 + CD38 - CXCR4 +细胞的长期扩增是必不可少的。然而，用于量化HSPC的不同替代方法（CD34 + CD38 -、CFC、第6周的CAFC和SRA）随着培养时间的增加显示出越来越大的不一致性，特别是表型分析和CFC生成在10周培养中极大地高估了CAFC和SRA的扩增。
