Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran ; Faulty of Paramedics, Kermanshah University of Medical Science, Kermanshah, Iran.
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Cell J. 2015 Summer;17(2):201-10. doi: 10.22074/cellj.2016.3715. Epub 2015 Jul 11.
Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder.
In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+(CD34(+)) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test.
HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05).
FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture.
在过去十年中，使用脐带血（UCB）进行造血干细胞（HSCs）移植已经得到了改善。由于细胞数量有限，许多研究都集中在 HSCs 的体外扩增上。已经进行了大量的研究来引入最佳的细胞因子鸡尾酒以扩增 HSCs，其中大多数使用 Fms 相关酪氨酸激酶 3 配体（FLT3-L）作为关键成分。根据 FLT3-L 的生物学特性，在本研究中，我们假设 FLT3-L 仅在存在间充质干细胞（MSC）饲养细胞的情况下才能有效诱导 HSCs 扩增。
在这项实验研究中，分别从 UCB 和胎盘分离 HSCs 和 MSC。在存在和不存在 MSC 饲养细胞和细胞因子的情况下，将 HSCs 在不同的培养条件下培养。培养十天后，评估总核细胞计数（TNC）、分化簇 34+（CD34+）细胞计数、集落形成单位测定（CFU）、长期培养起始细胞（LTC-IC）、同源盒蛋白 B4（HoxB4）mRNA 和表面 CD49d 表达。通过 t 检验比较某些培养条件下的倍数增加。
在细胞因子和 MSC 饲养细胞的存在下，HSCs 得到了扩增。共培养条件下的扩增率是细胞因子培养的两倍（P<0.05）。FLT3-L 可以在共培养条件下将 HSCs 扩增到与存在干细胞因子（SCF）、血小板生成素（TPO）和无饲养细胞的 FLT3-L 相等的 20 倍水平。与细胞因子鸡尾酒条件相比，LTC-IC 和 CD49d 表达的提取菌落数量显著增加（P<0.05）。
FLT3-L 与 MSC 的共培养可以诱导 HSCs 的高产量扩增，并且可以替代无饲养细胞的 SCF、TPO 和 FLT3-L 的通用鸡尾酒。
