ANDROS Day Surgery, Reproductive Medicine Unit, Palermo, Italy.
Eur J Obstet Gynecol Reprod Biol. 2013 Oct;170(2):381-6. doi: 10.1016/j.ejogrb.2013.07.032. Epub 2013 Aug 6.
To compare multipotent mesenchymal stem cells (MSCs) obtained from chorionic villi (CV), amniotic fluid (AF) and placenta, with regard to their phenotype and gene expression, in order to understand if MSCs derived from different extra-embryonic tissues, at different stages of human ontological development, present distinct stemness characteristics.
MSCs obtained from 30 samples of CV, 30 of AF and 10 placentas (obtained from elective caesarean sections) were compared. MSCs at second confluence cultures were characterized by immunophenotypic analysis with flow cytometry using FACS CANTO II. The expression of the genes Oct-4 (Octamer-binding transcription factor 4, also known as POU5F1), Sox-2 (SRY box-containing factor 2), Nanog, Rex-1 (Zfp-42) and Pax-6 (Paired Box Protein-6), was analyzed. Real-time quantitative PCR was performed by ABI Prism 7700, after RNA isolation and retro-transcription in cDNA. Statistical analysis was performed using non-parametric test Kruskal-Wallis (XLSTAT 2011) and confirmed by REST software, to estimate fold changes between samples. Each gene was defined differentially expressed if p-value was <0.05.
Cells from all samples were negative for haematopoietic antigens CD45, CD34, CD117 and CD33 and positive for the typical MSCs antigens CD13, CD73 and CD90. Nevertheless, MSCs from AF and placentas showed different fluorescence intensity, reflecting the heterogeneity of these tissues. The gene expression of OCT-4, SOX-2, NANOG was not significantly different among the three groups. In AF, REX-1 and PAX-6 showed a higher expression in comparison to CV.
MSCs of different extra-embryonic tissues showed no differences in immunophenotype when collected from second confluence cultures. The expression of OCT-4, NANOG and SOX-2 was not significantly different, demonstrating that all fetal sources are suitable for obtaining MSCs. These results open new possibilities for the clinical use of MSCs derived from easily accessible sources, in order to develop new protocols for clinical and experimental research.
比较来源于绒毛膜（CV）、羊水（AF）和胎盘的多能间充质干细胞（MSCs），以了解在不同的胚胎外组织、在人类胚胎发生的不同阶段获得的 MSCs 是否具有不同的干性特征。
对 30 例 CV、30 例 AF 和 10 例胎盘（剖宫产时获得）样本中获得的 MSCs 进行比较。使用流式细胞仪（FACS CANTO II）对第二代汇合培养物中的 MSCs 进行免疫表型分析。分析 Oct-4（八聚体结合转录因子 4，也称为 POU5F1）、Sox-2（SRY 盒包含因子 2）、Nanog、Rex-1（Zfp-42）和 Pax-6（配对盒蛋白-6）的基因表达。通过 ABI Prism 7700 进行实时定量 PCR，在 RNA 分离和 cDNA 反转录后进行。使用非参数检验 Kruskal-Wallis（XLSTAT 2011）进行统计分析，并使用 REST 软件进行确认，以估计样品之间的倍数变化。如果 p 值<0.05，则定义每个基因的差异表达。
所有样本的细胞均为造血抗原 CD45、CD34、CD117 和 CD33 阴性，而典型的 MSCs 抗原 CD13、CD73 和 CD90 阳性。然而，AF 和胎盘的 MSCs 显示出不同的荧光强度，反映了这些组织的异质性。三组之间 OCT-4、SOX-2、NANOG 的基因表达没有显著差异。在 AF 中，REX-1 和 PAX-6 的表达与 CV 相比更高。
从第二代汇合培养物中收集的不同胚胎外组织的 MSCs 在免疫表型上没有差异。OCT-4、NANOG 和 SOX-2 的表达没有显著差异，表明所有胎儿来源都适合获得 MSCs。这些结果为从易于获得的来源获得的 MSCs 的临床应用开辟了新的可能性，以便为临床和实验研究开发新的方案。
