背景:稳定可靠的体外和体内原代细胞模型对于研究骨髓增生异常肿瘤(MDS)的病理机制和确定新的治疗策略是必要的。MDS衍生的造血干细胞和祖细胞(HSPC)依赖于骨髓(BM)衍生的间充质基质细胞(MSC)的支持。因此,MCSs的分离和扩增是成功建立本病模型的关键.对于从人类BM分离的健康MSC的临床使用,脐带血或脂肪组织,一些研究表明,与在胎牛血清(FBS)存在下培养的MSC相比,无异种(XF)培养条件导致优异的生长动力学。在本研究中,我们调查,用XF培养基替换含有FBS的市售MSC扩增培养基是否有利于扩增通常难以培养的MDS患者的BM来源的MSC。
方法:将从MDS患者的BM分离的MSC培养并在含有FBS或XF补充剂的MSC扩增培养基中扩增。随后,培养基对生长动力学的影响,形态学,免疫表型,克隆潜能,分化能力,评估了在免疫缺陷小鼠模型中的基因表达谱和移植能力。
结果:与含有FBS的培养基相比,在用XF培养基培养MDSSCs期间观察到显著更高的细胞数目和克隆形成潜能的增加。用XF扩增后,差异基因表达显示与MSC干性相关的转录本增加。此外,MSCs的免疫表型及其向成骨细胞分化的能力,脂肪细胞或成软骨细胞保持稳定。用XF培养基扩增的MSC与用FBS扩增的MSC类似地支持在体内产生MDS异种移植物。
结论:我们的数据表明,使用XF培养基,在体外和体内实验模型中,可以获得更高细胞数量的MDSMSC,具有整体改善的特征。
Robust and reliable in vitro and in vivo models of primary cells are necessary to study the pathomechanisms of Myelodysplastic Neoplasms (MDS) and identify novel therapeutic strategies. MDS-derived hematopoietic stem and progenitor cells (HSPCs) are reliant on the support of bone marrow (BM) derived mesenchymal stroma cells (MSCs). Therefore, isolation and expansion of MCSs are essential for successfully modeling this disease. For the clinical use of healthy MSCs isolated from human BM, umbilical cord blood or adipose tissue, several studies showed that xeno-free (XF) culture conditions resulted in superior growth kinetics compared to MSCs cultured in the presence of fetal bovine serum (FBS). In this present study, we investigate, whether the replacement of a commercially available MSC expansion medium containing FBS with a XF medium is beneficial for the expansion of MSCs derived from BM of MDS patients which are often difficult to cultivate.
MSCs isolated from BM of MDS patients were cultured and expanded in MSC expansion medium with FBS or XF supplement. Subsequently, the impact of culture media on growth kinetics, morphology, immunophenotype, clonogenic potential, differentiation capacity, gene expression profiles and ability to engraft in immunodeficient mouse models was evaluated.
Significant higher cell numbers with an increase in clonogenic potential were observed during culture of MDS MSCs with XF medium compared to medium containing FBS. Differential gene expression showed an increase in transcripts associated with MSC stemness after expansion with XF. Furthermore, immunophenotypes of the MSCs and their ability to differentiate into osteoblasts, adipocytes or chondroblasts remained stable. MSCs expanded with XF media were similarly supportive for creating MDS xenografts in vivo as MSCs expanded with FBS.
Our data indicate that with XF media, higher cell numbers of MDS MSCs can be obtained with overall improved characteristics in in vitro and in vivo experimental models.