目的:使用脐带血(UCB)的造血干细胞(HSC)移植在过去十年中有所改善。由于细胞的局限性,一些研究集中在HSC的离体扩增。进行了许多研究以引入用于HSC扩增的最佳细胞因子混合物。大多数使用Fms相关酪氨酸激酶3配体(FLT3-L)作为关键组分。根据FLT3-L生物学,在这项研究中,我们研究了以下假设:FLT3-L仅在间充质干细胞(MSC)饲养细胞存在下有效诱导HSC扩增。
方法:在本实验研究中,从UCB和胎盘中分离出HSC和MSCs,分别。在存在和不存在MSC饲养细胞和细胞因子的情况下,在不同的培养条件下培养HSC。经过十天的培养,有核细胞总数(TNC),分化簇34+(CD34(+))细胞计数,菌落形成单位测定(CFU),长期培养起始细胞(LTC-IC),评价同源异型盒蛋白B4(HoxB4)mRNA和表面CD49d表达。通过t检验比较一些培养条件的倍数增加。
结果:HSC在细胞因子和MSC饲养物存在下扩增。共培养条件下的扩增速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以20倍的水平扩增HSC,血小板生成素(TPO)和FLT3-L无饲养细胞。与细胞因子混合物条件相比,从LTC-IC和CD49d表达中提取的菌落数量显著增加(P<0.05)。
结论:FLT3-L与MSCs共培养可诱导HSCs的高产量扩增,可替代SCF的通用混合物,无饲养培养中的TPO和FLT3-L。
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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).
CONCLUSIONS: 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.