Zlatska A V, Rodnichenko A E, Gubar O S, Zubov D O, Novikova S N, Vasyliev R G
Biotechnology Laboratory ilaya.regeneration, Medical Company ilaya, Kyiv 03115, Ukraine.
Institute of Molecular Biology & Genetics of NAS of Ukraine, Kyiv 03680, Ukraine.
Exp Oncol. 2017 Sep;39(3):197-202.
We aimed to study biological properties of human endometrial stromal cells in vitro.
The endometrium samples (n = 5) were obtained by biopsy at the first phase of the menstrual cycle from women with endometrial hypoplasia. In all cases, a voluntary written informed consent was obtained from the patients. Endometrial fragments were dissociated by enzymatic treatment. The cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mМ L-glutamine and 1 ng/ml FGF-2 in a multi-gas incubator at 5% CO and 5% O. At P3 the cells were subjected to immunophenotyping, multilineage differentiation, karyotype stability and colony forming efficiency. The cell secretome was assessed by BioRad Multiplex immunoassay kit.
Primary population of endometrial cells was heterogeneous and contained cells with fibroblast-like and epithelial-like morphology, but at P3 the majority of cell population had fibroblast-like morphology. The cells possessed typical for MSCs phenotype CD90CD105CD73CD34CD45HLA-DR. The cells also expressed CD140a, CD140b, CD146, and CD166 antigents; and were negative for CD106, CD184, CD271, and CD325. Cell doubling time was 29.6 ± 1.3 h. The cells were capable of directed osteogenic, adipogenic and chondrogenic differentiation. The cells showed 35.7% colony forming efficiency and a tendency to 3D spheroid formation. The GTG-banding assay confirmed the stability of eMSC karyotype during long-term culturing (up to P8). After 48 h incubation period in serum-free medium eMSC secreted anti-inflammatory IL-1ra, as well as IL-6, IL-8 and IFNγ, angiogenic factors VEGF, GM-CSF and FGF-2, chemokines IP-10 and MCP-1.
Thus, cultured endometrial stromal cells meet minimal ISCT criteria for MSC. Proliferative potential, karyotype stability, multilineage plasticity and secretome profile make eMSC an attractive object for the regenerative medicine use.
我们旨在研究人子宫内膜基质细胞的体外生物学特性。
从子宫内膜发育不全的女性月经周期第一阶段通过活检获取子宫内膜样本（n = 5）。所有病例均获得患者自愿签署的知情同意书。通过酶处理使子宫内膜碎片解离。细胞在补充有10%胎牛血清、2 mM L-谷氨酰胺和1 ng/ml FGF-2的DMEM/F12培养基中，于多气培养箱中在5% CO₂和5% O₂条件下培养。在P3代时，对细胞进行免疫表型分析、多向分化、核型稳定性和集落形成效率检测。细胞分泌组通过BioRad多重免疫分析试剂盒进行评估。
子宫内膜细胞的原代群体具有异质性，包含具有成纤维细胞样和上皮样形态的细胞，但在P3代时，大多数细胞群体具有成纤维细胞样形态。这些细胞具有间充质干细胞典型的表型CD90、CD105、CD73、CD34、CD45、HLA-DR阴性。细胞还表达CD140a、CD140b、CD146和CD166抗原；而CD106、CD184、CD271和CD325为阴性。细胞倍增时间为29.6 ± 1.3小时。这些细胞能够定向进行成骨、成脂和成软骨分化。细胞显示出35.7%的集落形成效率以及形成三维球体的倾向。GTG显带分析证实了子宫内膜基质细胞在长期培养（直至P8代）过程中核型的稳定性。在无血清培养基中孵育48小时后，子宫内膜基质细胞分泌抗炎因子IL-1ra，以及IL-6、IL-8和IFNγ、血管生成因子VEGF、GM-CSF和FGF-2、趋化因子IP-10和MCP-1。
因此，培养的子宫内膜基质细胞符合间充质干细胞的最低国际细胞治疗协会标准。增殖潜能、核型稳定性、多向可塑性和分泌组特征使子宫内膜基质细胞成为再生医学应用中一个有吸引力的对象。
