探討小鼠羊水幹細胞之體內及體外分化潛能

Abstract

羊水幹細胞 (amniotic fluid stem cell, AFSCs) 可以透過胎兒產前分析之羊膜穿刺樣本分離取得，是目前最容易取得較早期的體幹細胞種類。其中包含了許多不同的幹細胞種類，因此可以被廣泛運用於治療或緩解多種疾病模式。除此之外， AFSCs 被發現有一定比例的細胞會表現多能性幹細胞標誌，因此 AFSCs 中包含了分化潛能較為原始的細胞，甚至可能含有多能性幹細胞 (pluripotent stem cell)。本研究的目的在於探討 AFSCs是否可以利用抗體篩選的方式，取得分化潛能較為原始的細胞，並確認該細胞群是否具有參與胚胎分化之能力。 結果顯示，小鼠 AFSCs (mAFSCs) 會表現如骨髓間葉幹細胞之貼附性細胞特性，且能進行中胚層之硬骨細胞及脂肪細胞分化。除此之外，mAFSCs 亦會表現多能性特異標誌 Oct-4。本試驗使用磁珠抗體分離來篩選細胞，並選用已知的多能性幹細胞表面抗原CD117（c-kit），取得其中分化潛能較原始的幹細胞。將篩選後與未篩選之 mAFSCs 注射入野生型小鼠囊胚腔中，並使用表現綠螢光蛋白質之胚幹細胞作為本試驗之正控制組。結果顯示，注射綠螢光胚幹細胞之組別，經胚移置後產下綠色螢光嵌合體 (chimera) 5 隻，以確認胚胎操作技術之熟練度；注射未經篩選之 mAFSCs 組別，一天後觀察與囊胚內細胞群有少數細胞聚集 (aggregation) 現象，且胚移置後將 E8.5、E10.5 及 E12.5 之胎兒取出觀察，並無綠色螢光嵌合，最終產下 56 隻子代，成體皆無綠色螢光嵌合；而注射c-kit+ mAFSCs 之組別，一天後觀察有較高比例的細胞與內細胞群有聚集現象，且胚移置後將相同天數之胎兒取出觀察，胎兒本身並無綠色螢光嵌合，但於 E10.5 觀察到綠色螢光胚外組織嵌合體，最終產下21 隻子代，成體亦無綠色螢光嵌合。此結果顯示，未經篩選之 mAFSCs 與內細胞群差異過大，無法與內細胞群聚集，因此沒有參與胚胎分化之能力；而 c-kit+ mAFSCs 與內細胞群性質較為相似，因此部分細胞可以與內細胞群聚集，但並沒有能力參與胚胎本體分化，只能參與部分胚外組織分化。 綜觀上述而言，本試驗有助於了解mAFSCs之分化潛能，並得知以 CD117 篩選 mAFSCs 能使其更接近內細胞群之特性，且有部分細胞能夠參與胚外組織之分化，對於未來幹細胞及再生醫學工程提供基礎研究。

Amniotic fluid stem cells (AFSCs) can be isolated from amniocentesis samples from prenatal analysis of the fetus. AFSCs population contains many different stem cell types and can therefore be widely used to treat or alleviate multiple disease models. In addition, AFSCs were found that a certain proportion of cells express pluripotent stem cell markers. Therefore, AFSCs contain cells with primitive differentiation potential and may even contain pluripotent stem cells. The purpose of this study was to investigate whether AFSCs can use antibody sorting to obtain cells with primitive differentiation potential and to confirm whether it has the ability to participate in embryo differentiation. The results show that mouse AFSCs (mAFSCs) exhibit adherent cell characteristics of mesenchymal stem cells, and is capable of differentiating to osteocytes and adipocytes. In addition, mAFSCs also exhibit the pluripotent stem cell marker, Oct-4. In this experiment, we chose stem cell surface antigen CD117 (c-kit) microbeads to obtain stem cells with primitive differentiation potential. The sorted and unsorted mAFSCs were injected into the wild-type blastocysts respectively, and GFP ESCs were used as the positive control in this experiment. The results showed that 5 GFP chimeras were born after embryo transfer in GFP ESCs control group that confirmed the embryo manipulation technology. As the unsorted mAFSCs group, there is no aggregation between mAFSCs and the inner cell mass (ICM) of host embryos. The fetus of E8.5, E10.5, E12.5 and 56 adult offspring showed no GFP chimeric; while the group of c-kit+ mAFSCs, some cells aggregated with ICM one day after injection. All of the fetus taken out after embryo transfer showed no GFP chimeric. However, we detected GFP expression in E10.5. So far, we haven’t obtain germline transmission ability in those adult GFP chimeras. This result shows that c-kit+ mAFSCs may have similar characteristic with the ICM. The c-kit+ mAFSCs show ability to contribute into extraembryonic tissue but not be able to participate into embryo development. Taken together, this experiment is helpful to understand the differentiation potential of mAFSCs, and CD117 sorting can make its characteristic closer to the ICM. In addition, some of them participate in the development of extraembryonic tissue. This result provides basic research for stem cell and regenerative medicine projects in the future.