Valatkaitė Elvina, Baušytė Raminta, Vitkevičienė Aida, Ramašauskaitė Diana, Navakauskienė Rūta
Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania.
Centre of Obstetrics and Gynaecology of the Institute of Clinical Medicine, Faculty of Medicine, Vilnius University, Vilnius, Lithuania.
Front Cell Dev Biol. 2021 Nov 19;9:765265. doi: 10.3389/fcell.2021.765265. eCollection 2021.
Human endometrium derived mesenchymal stem cells (hEndSCs) offer a great promise for regenerative medicine and reproductive system disorders treatment methods based on cell therapy due to their broad differentiation potential and highly efficient proliferation. In our study, we investigated the characteristics of hEndSCs that were isolated from two sources: endometrium and menstrual blood, which both contain endometrial origin stem cells. Changes in gene and protein expression levels during long-term cultivation and decidualization potential were examined in endometrial stem cells (EndSCs) and menstrual blood stem cells (MenSCs). The decidualization process was induced on early and late passages of hEndSCs using dibutyryl cyclic-AMP (db-cAMP) and medroxyprogesterone acetate (MPA) agents. We demonstrated that after long-term cultivation of hEndSCs the expression of typical mesenchymal stromal cell surface markers such as CD44, CD73, CD90, CD105 and perivascular marker CD146 remains at a similar level throughout long-term cultivation. Additionally, hematopoietic and endothelial markers CD34, CD45 were also tested, they were negative in all cases. Analyzed stem cells gene markers, such as OCT4, SOX2, NANOG, KLF4, showed similar expression in all passages of hEndSCs. RT-qPCR results demonstrated that the expression of cell cycle control associated genes - CDK2, CCNA2, CCNE2, p21, p53 and Rb, among all groups was very similar. Expression of genes associated with senescence (ATM, JUND, TOP2A, MYC) was maintained at a similar level throughout passaging. In addition, Western blot analysis was used to assess changes in proteins' levels associated to epigenetics (EZH2, SUZ12, H3K27me3) and cell cycle control (cyclinE1, p53) during long-term cultivation. The levels of proteins associated with epigenetic changes were fluctuated slightly depending on the patient. Also, we demonstrated that in all induced hEndSCs the expression of decidualization markers Prolactin (PRL), IGFBP1 and WNT4 was upregulated. In conclusion, we demonstrated successful decidualization of stem cells derived from two reproductive system resources: endometrium and menstrual blood by using db-cAMP and MPA regardless of the length of the stem cell passaging. According these findings, we suppose that endometrium derived stem cells and menstrual blood derived stem cells could have a potency not only for endometrium tissue regeneration, but could also become a successful therapy for reproductive system disorders, including infertility or recurrent pregnancy loss.
人子宫内膜间充质干细胞（hEndSCs）因其广泛的分化潜能和高效的增殖能力，在再生医学以及基于细胞治疗的生殖系统疾病治疗方法中具有巨大潜力。在我们的研究中，我们调查了从两个来源分离的hEndSCs的特性：子宫内膜和月经血，这两者都含有子宫内膜来源的干细胞。我们检测了子宫内膜干细胞（EndSCs）和月经血干细胞（MenSCs）在长期培养过程中基因和蛋白质表达水平的变化以及蜕膜化潜能。使用二丁酰环磷腺苷（db - cAMP）和醋酸甲羟孕酮（MPA）试剂诱导hEndSCs早期和晚期传代的蜕膜化过程。我们证明，hEndSCs长期培养后，典型的间充质基质细胞表面标志物如CD44、CD73、CD90、CD105和血管周围标志物CD146的表达在整个长期培养过程中保持在相似水平。此外，还检测了造血和内皮标志物CD34、CD45，在所有情况下它们均为阴性。分析的干细胞基因标志物，如OCT4、SOX2、NANOG、KLF4，在hEndSCs的所有传代中均表现出相似的表达。RT - qPCR结果表明，细胞周期调控相关基因 - CDK2、CCNA2、CCNE2、p21、p53和Rb在所有组中的表达非常相似。与衰老相关的基因（ATM、JUND、TOP2A、MYC）的表达在整个传代过程中保持在相似水平。此外，蛋白质印迹分析用于评估长期培养过程中与表观遗传学（EZH2、SUZ12、H3K27me3）和细胞周期调控（细胞周期蛋白E1、p53）相关的蛋白质水平变化。与表观遗传变化相关的蛋白质水平根据患者情况略有波动。此外，我们证明，在所有诱导的hEndSCs中，蜕膜化标志物催乳素（PRL）、胰岛素样生长因子结合蛋白1（IGFBP1）和WNT4的表达均上调。总之，我们证明了使用db - cAMP和MPA成功诱导了来自两种生殖系统资源：子宫内膜和月经血的干细胞的蜕膜化，且与干细胞传代次数无关。基于这些发现，我们推测子宫内膜来源的干细胞和月经血来源的干细胞不仅具有子宫内膜组织再生的潜能，还可能成为治疗生殖系统疾病（包括不孕症或复发性流产）的成功疗法。
