1. Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran ; 2. Iran Blood Transfusion Organization Research Center, Tehran, Iran.
Cell J. 2011 Fall;13(3):173-8. Epub 2011 Sep 23.
Despite of many benefits, umbilical cord blood (UCB) hematopoietic stem cell (HSC) transplantation is associated with low number of stem cells and slow engraftment; in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte (MK) progenitor cells can shorten this period. In this study, we evaluated the cytokine conditions for maximum expansion and MK differentiation of CD133(+) HSCs.
In this experimental study, The CD133(+) cells were separated from three cord blood samples by magnetic activated cell sorting (MACS) method, expanded in different cytokine combinations for a week and differentiated in thrombopoietin (TPO) for the second week. Differentiation was followed by the flow cytometry detection of CD41 and CD61 surface markers. Colony forming unit (CFU) assay and DNA analysis were done for colonogenic capacity and ploidy assay.
CD133(+) cells showed maximum expansion in the stem span medium with stem cell factor (SCF) + FMS-like tyrosine kinase 3-ligand (Flt3-L) + TPO but the maximum differentiation was seen when CD133(+) cells were expanded in stem span medium with SCF + Interleukin 3 (IL-3) + TPO for the first and in TPO for the second week. Colony Forming Unit-MK (CFU-MK) was formed in three sizes of colonies in the mega-cult medium. In the DNA analysis; 25.2 ± 6.7% of the cells had more than 2n DNA mass.
Distinct differences in the MK progenitor cell count were observed when the cells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in the second week. Such strategy could be applied for optimization of CD133(+) cells expansion followed by MK differentiation.
尽管脐带血（UCB）造血干细胞（HSC）移植有许多益处，但与干细胞数量少和植入缓慢有关; 特别是血小板。因此，扩大 HSC 并共输注巨核细胞（MK）祖细胞可以缩短这段时间。在这项研究中，我们评估了最大程度扩增和 CD133(+) HSC 分化为 MK 的细胞因子条件。
在这项实验研究中，通过磁激活细胞分选（MACS）方法从三个脐带血样本中分离出 CD133(+)细胞，在不同细胞因子组合中扩增一周，然后在血小板生成素（TPO）中分化两周。分化后通过流式细胞术检测 CD41 和 CD61 表面标志物。进行集落形成单位（CFU）测定和 DNA 分析，以评估集落形成能力和倍性分析。
CD133(+)细胞在含有干细胞因子（SCF）+ FMS 样酪氨酸激酶 3 配体（Flt3-L）+ TPO 的干细胞扩增培养基中显示出最大扩增，但当 CD133(+)细胞在含有 SCF+白细胞介素 3（IL-3）+ TPO 的干细胞扩增培养基中扩增第一周和第二周 TPO 时，观察到最大分化。在 mega-cult 培养基中形成了三种大小的 CFU-MK 集落。在 DNA 分析中; 25.2±6.7%的细胞具有超过 2n 的 DNA 质量。
当细胞在含有 TPO、SCF、IL-3 的干细胞扩增培养基中培养，然后在第二周再使用 TPO 时，观察到 MK 祖细胞计数存在明显差异。这种策略可用于优化 CD133(+)细胞的扩增，然后再分化为 MK。
