Center of Haematology and Hemotherapy, University of Campinas, Campinas, São Paulo Brazil.
Stem Cells Dev. 2010 Mar;19(3):413-22. doi: 10.1089/scd.2009.0098.
Umbilical cord blood (UCB), an ideal source for transplantable hematopoietic stem cells (HSC), is readily available and is rich in progenitor cells. Identification of conditions favoring UCB-HSC ex vivo expansion and of repopulating potential remains a major challenge in hematology. CD133+ cells constitute an earlier, less-differentiated HSC group with a potentially higher engraftment capacity. The presence of SCF, Flt3-L, and TPO are essential for CD133+ and/or CD34+ cells ex vivo expansion; however, IL-3 and IL-6 influence has not yet been clearly established. We investigated this influence on CD133+ cells from UCB ex vivo expansion and the effect of these cytokines upon cell phenotype. Immediately after isolation an 85% of CD133+ cell purity was obtained, diminishing after 4 and 8 days of ex vivo expansion. CD133+ fold-increase was higher using IMDM with SCF, Flt3-L, and TPO (BM)+IL-3 or BM+IL-3+IL-6 on day 8 (13.83- and 17.47-fold increase, respectively). BM+IL-6 presented no significant difference from BM alone. We demonstrated that 5.1% of the CD133+ cells expressed IL-6 receptor (IL-6R) after isolation. After 4 and 8 days in culture, the percentage of CD133+ cells that expressed IL-6R was as follows: BM alone (9.8% and 22.02%, respectively); BM+IL-3 (8.33% and 16.74%); BM+IL-6 (9.2% and 17.67%); and BM+IL-3+IL-6 (12.5% and 61.20%). Cell cycle analysis revealed quiescent cells after isolation, 95.5% CD133+ cells in the G0/G1 phase. Regardless of culture period or cytokine incubation, CD133+ cell cycle altered to 70% of CD133+ in the G0/G1 phase. Colony-forming unit (CFU) doubled in BM+IL-3+IL-6 after 8 days of incubation compared with BM group. SOX-2 and NANOG-relative gene expression was detected on day 0 after isolation. BM+IL-6 prevented the decrease in NANOG and SOX-2 gene expression level compared to BM+IL-3 or BM+IL-3+IL-6 incubated cells. Our results indicated that UCB-isolated CD133+ cells were better ex vivo expanded in the presence of SCF, Flt3-L, TPO, IL-3+IL-6. IL-3 probably promotes higher CD133+ cell expansion and IL-6 maintains immature phenotype.
脐带血（UCB）是一种理想的可移植造血干细胞（HSC）来源，其来源丰富，且富含祖细胞。鉴定有利于 UCB-HSC 体外扩增和重编程潜力的条件仍然是血液学中的主要挑战。CD133+细胞构成了一个更早、分化程度更低的 HSC 群体，具有更高的植入能力。SCF、Flt3-L 和 TPO 的存在对于 CD133+和/或 CD34+细胞的体外扩增是必不可少的；然而，IL-3 和 IL-6 的影响尚未得到明确证实。我们研究了这些细胞因子对 UCB 来源的 CD133+细胞的体外扩增的影响，以及这些细胞因子对细胞表型的影响。在体外扩增的第 4 天和第 8 天，CD133+细胞的 fold-increase 更高，使用含 SCF、Flt3-L 和 TPO 的 IMDM（BM）+IL-3 或 BM+IL-3+IL-6（分别为 13.83-和 17.47 倍）。BM+IL-6 与 BM 相比没有显著差异。我们证明，分离后 5.1%的 CD133+细胞表达 IL-6 受体（IL-6R）。在培养 4 天和 8 天后，表达 IL-6R 的 CD133+细胞的百分比如下：BM 单独（分别为 9.8%和 22.02%）；BM+IL-3（8.33%和 16.74%）；BM+IL-6（9.2%和 17.67%）；以及 BM+IL-3+IL-6（12.5%和 61.20%）。细胞周期分析显示，分离后细胞处于静止状态，95.5%的 CD133+细胞处于 G0/G1 期。无论培养时间或细胞因子孵育，CD133+细胞周期都改变为 G0/G1 期的 70%。与 BM 组相比，BM+IL-3+IL-6 孵育 8 天后集落形成单位（CFU）增加了一倍。在分离后的第 0 天检测到 SOX-2 和 NANOG-相对基因表达。与 BM+IL-3 或 BM+IL-3+IL-6 孵育的细胞相比，BM+IL-6 可防止 NANOG 和 SOX-2 基因表达水平的下降。我们的结果表明，在 SCF、Flt3-L、TPO、IL-3+IL-6 的存在下，UCB 分离的 CD133+细胞在体外扩增更好。IL-3 可能促进更高的 CD133+细胞扩增，而 IL-6 维持未成熟表型。
