de Oliveira Pinheiro Alessandra, Lara Valéria M, Souza Aline F, Casals Juliana B, Bressan Fabiana F, Fantinato Neto Paulo, Oliveira Vanessa C, Martins Daniele S, Ambrosio Carlos E
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil.
Private Veterinary Practice, Pirassununga, São Paulo, Brazil.
Stem Cells Cloning. 2020 May 7;13:43-55. doi: 10.2147/SCCAA.S237686. eCollection 2020.
Amniotic membrane stem cells have a high capacity of proliferation, cell expansion, and plasticity, as well as immunomodulatory properties that contribute to maternal-fetal tolerance. Owing to the lack of research on human amniotic membrane at different gestational stages, the canine model is considered ideal because of its genetic and physiological similarities. We aimed to characterize the canine amniotic membrane (CAM) cell lineage in different gestational stages and evaluate the expression of immunomodulatory genes.
Twenty CAMs from early (20-30 days) (n=7), mid- (31-45 days) (n=7), and late gestation (46-63 days) (n=6) stages were studied. The cell features were assessed by cell viability tests, growth curve, colony-forming units, in vitro differentiation, cell labeling for different immunophenotypes, and pluripotent potential markers. The cells were subjected to RT-PCR and qPCR analysis to determine the expression of , and genes.
CAM cells exhibited a fibroblastoid morphology and adherence to plastic with an average cell viability of 78.5%. The growth curve indicated a growth peak in the second passage and we obtained an average of 138.2 colonies. Osteogenic, chondrogenic, and adipogenic lineages were confirmed by in vitro differentiation assays. Cellular immunophenotyping experiments confirmed the presence of positive mesenchymal markers (CD90 and CD105) and the low or negative expression of hematopoietic markers (CD45 and CD34). Qualitative analysis of the immunomodulatory functions indicated the expression of the , and genes. When stimulated by interferon-gamma, CAM cells exhibited higher IDO levels throughout gestation.
The CAMs from different gestational stages presented features consistent with mesenchymal stem cell lineage; better results were observed during the late gestation stage. Therefore, the gestational stage is a key factor that may influence the functionality of therapies when using fetal membrane tissues from different periods of pregnancy.
羊膜干细胞具有高增殖能力、细胞扩增能力和可塑性，以及有助于母胎耐受的免疫调节特性。由于缺乏对不同妊娠阶段人羊膜的研究，犬模型因其遗传和生理相似性而被认为是理想的。我们旨在表征不同妊娠阶段犬羊膜（CAM）细胞谱系，并评估免疫调节基因的表达。
研究了来自妊娠早期（20 - 30天）（n = 7）、中期（31 - 45天）（n = 7）和晚期（46 - 63天）（n = 6）阶段的20个CAM。通过细胞活力测试、生长曲线、集落形成单位、体外分化、不同免疫表型的细胞标记以及多能性潜能标志物来评估细胞特征。对细胞进行逆转录聚合酶链反应（RT-PCR）和定量聚合酶链反应（qPCR）分析，以确定 、 和 基因的表达。
CAM细胞呈现成纤维细胞样形态并贴壁生长，平均细胞活力为78.5%。生长曲线表明在第二代出现生长高峰，我们平均获得138.2个集落。通过体外分化试验证实了成骨、成软骨和成脂谱系。细胞免疫表型实验证实了间充质阳性标志物（CD90和CD105）的存在以及造血标志物（CD45和CD34）的低表达或阴性表达。免疫调节功能的定性分析表明了 、 和 基因的表达。当受到干扰素-γ刺激时，整个妊娠期CAM细胞表现出更高的吲哚胺2,3-双加氧酶（IDO）水平。
来自不同妊娠阶段的CAM呈现出与间充质干细胞谱系一致的特征；在妊娠晚期阶段观察到更好的结果。因此，妊娠阶段是使用不同孕期胎膜组织进行治疗时可能影响治疗功能的关键因素。
