Amniotic fluid (AF) is a rich source of mesenchymal stromal cells (MSCs) that have the ability to differentiate into multiple lineages rendering them a promising and powerful tool for regenerative medicine. However, information regarding the differences among AFMSCs derived from different gestational stages is limited. In the present study, AFMSCs derived from 125 pregnant rats at four embryonic day (E) stages (E12, E15, E18, and E21) were isolated and cultured. The primary E15 cells were the smallest in size and the easiest to culture and usually grew in a spherical shape that resembled the growth morphology of embryonic stem cells (ESCs). Once adhered, the E12 and E15 AFMSCs grew faster and could be passaged more than 60 times while still maintaining a continuous proliferative state; however, AFMSCs derived from E18 and E21 could normally be maintained for only 10 passages. To identify the possible reasons for this difference, RT-qPCR was used to examine several genes associated with self-renewal ability and cell origin. The Sox2 expression levels indicated that AFMSCs from E12 and E15 possessed stronger self-renewal capability. The K19, Col2A1, FGF5, AFP, and SPC expression levels indicated there were mixed-population cells co-existing in the AFMSC culture. In conclusion, E15 cells were easier to culture than E12 cells, could be passaged more often, and had a higher Sox2 expression than E18 or E21 cells. The E15-derived AFMSCs had higher viability and proliferative capacity than cells from the later stages. Therefore, AF cells from the early stages could be a good choice for exploring potential treatments involving AFMSCs.

羊水 (AF) 是间充质基质细胞 (MSC) 的丰富来源，具有分化成多个谱系的能力，使它们成为再生医学的有前途和强大的工具。然而，关于来自不同妊娠阶段的 AFMSCs 之间差异的信息是有限的。在本研究中，分离和培养了 125 只处于四个胚胎日 (E) 阶段 (E12、E15、E18 和 E21) 的怀孕大鼠的 AFMSC。原代 E15 细胞体积最小，最容易培养，通常呈球形，类似于胚胎干细胞 (ESC) 的生长形态。一旦粘附，E12和E15 AFMSCs生长更快，可传代60多次，同时仍保持持续增殖状态；然而，源自 E18 和 E21 的 AFMSCs 通常只能维持 10 次传代。为了确定这种差异的可能原因，RT-qPCR 用于检查与自我更新能力和细胞起源相关的几个基因。Sox2 表达水平表明来自 E12 和 E15 的 AFMSCs 具有更强的自我更新能力。K19、Col2A1、FGF5、AFP 和 SPC 的表达水平表明在 AFMSC 培养物中存在混合群细胞。总之，E15 细胞比 E12 细胞更容易培养，可以更频繁地传代，并且比 E18 或 E21 细胞具有更高的 Sox2 表达。E15 衍生的 AFMSCs 比后期细胞具有更高的活力和增殖能力。因此，早期的 AF 细胞可能是探索涉及 AFMSC 的潜在治疗方法的不错选择。