Department of Hematology, Singapore General Hospital, Outram Road, Singapore.
J Hematol Oncol. 2008 Oct 23;1:19. doi: 10.1186/1756-8722-1-19.
Excessive maturation of hematopoietic cells leads to a reduction of long-term proliferative capability during cord blood (CB) expansion. In this study, we report the effects of anit-CD52 (Alemtuzumab, Campath) on both short- and long-term ex vivo expansion of CB hematopoietic stem cells (HSC) by evaluating the potential role of Alemtuzumab in preserving the repopulating capability in CB HSC and nonlymphoid progenitors.
Ex vivo expansion experiments were carried out using freshly purified CB CD34(+)cells in StemSpantrade mark SFEM medium in the presence of stem cell factor, Flt3-Ligand and thrombopoietin at 50 ng/ml. Alemtuzumab (10 microg/ml) was used to deplete CD52(+) cells during the cultures. Flow cytometry was used to monitor CB HSC and their differentiation. Colony forming unit (CFU) assays and long term culture-initiating cell (LTC-IC) assays were performed on cells obtained from day 0 (before culture) and day 14 after cultures. Secondary cultures was performed using CD34(+) cells isolated at 35 days from primary cultures and further cultured in StemSpantrade mark SFEM medium for another 14 days to confirm the long term effect of alemtuzumab in liquid cultures.
Compared to cytokines alone, addition of alemtuzumab resulted in a significant increase in total nucleated cells, absolute CD34(+) cells, myeloid and megakaryocytic progenitors, multi-lineage and myeloid CFU and LTC-IC.
The results from current study suggested that the use of alemtuzumab for ex vivo expansion of CBHSC maybe advantageous. Our findings may improve current technologies for CBHSC expansion and increase the availability of CB units for transplantation. However, in vivo studies using animal models are likely needed in further studies to test the hematopoietic effects using such expanded CB products.
造血细胞的过度成熟会导致脐血（CB）扩增过程中长期增殖能力的降低。在这项研究中，我们报告了抗 CD52（阿仑单抗，坎帕斯）对 CB 造血干细胞（HSC）短期和长期体外扩增的影响，评估了阿仑单抗在保留 CB HSC 和非淋巴祖细胞的重建能力方面的潜在作用。
使用新鲜纯化的 CB CD34+细胞在 StemSpan 商标 SFEM 培养基中进行体外扩增实验，在培养基中添加 50ng/ml 的干细胞因子、Flt3 配体和血小板生成素。在培养过程中使用阿仑单抗（10μg/ml）来去除 CD52+细胞。流式细胞术用于监测 CB HSC 及其分化。在细胞获得后第 0 天（培养前）和第 14 天进行集落形成单位（CFU）检测和长期培养起始细胞（LTC-IC）检测。在初级培养的第 35 天，使用 CD34+细胞进行二次培养，并在 StemSpan 商标 SFEM 培养基中进一步培养 14 天，以确认阿仑单抗在液体培养中的长期作用。
与单独使用细胞因子相比，添加阿仑单抗可显著增加总核细胞、绝对 CD34+细胞、髓系和巨核细胞祖细胞、多系和髓系 CFU 和 LTC-IC。
目前的研究结果表明，阿仑单抗用于 CBHSC 的体外扩增可能是有利的。我们的发现可能会改进目前用于 CBHSC 扩增的技术，并增加 CB 单位用于移植的可用性。然而，在进一步的研究中，可能需要使用动物模型进行体内研究，以测试使用此类扩增的 CB 产品的造血效果。
