小鼠與豬羊水幹細胞之細胞治療及命運之研究

Abstract

羊水來源幹細胞為胎兒脫落之細胞且已被證實具有多分化潛能。本試驗利用本研究室產製之攜帶紅色螢光蛋白轉基因豬與攜帶綠色螢光蛋白轉基因小鼠來建立羊水來源幹細胞，以做為後續之追蹤。經本試驗結果得知小鼠羊水來源幹細胞可表現幹細胞之標誌例如CD29, CD44, Sca-1，但不表現造血、淋巴等免疫細胞或是內皮細胞之標誌例如CD31, CD45, CD34, CD166, CD11b, CD117, CD133, CD86, CD105。豬羊水幹細胞可表現幹細胞之標誌例如CD44, CD90，但不表現內皮細胞、巨噬細胞、淋巴球細胞等標誌例如CD31,CD4a。此類羊水來源幹細胞於體外可分化成多種特異性之細胞，例如硬骨細胞(ARS 染鈣離子沉澱)，軟骨細胞(Toluidine blue染glycosaminoglycan)，脂肪細胞(Oil Red O 染neutral lipid vacuoles)，心肌細胞 (myosin heavy chain 與cardiac troponin I抗體結合)等，分化後之羊水來源幹細胞仍然表現螢光蛋白，此結果顯示未來細胞移植可長期追蹤其細胞命運。迄今，尚無研究探討羊水來源幹細胞於體內之命運，因此吾人以注射方式移植小鼠或豬之羊水來源前驅細胞於12.5-13.5天懷孕母鼠子宮內探討此細胞之遷移。結果顯示羊水來源幹細胞具有遷移至腸子，肝臟，腎臟等參與至小鼠三個胚層細胞之能力且發現植入的細胞會與接受者小鼠的細胞進行細胞融合之現象 (藉由組織染色切片與流氏細胞儀方式測得知以同種異體移植為例，帶有綠色螢光之小鼠羊水幹細胞可與全身攜帶紅色螢光蛋白的小鼠細胞做融合而呈現黃色之現象;以異種移植為例，帶有紅色螢光之豬羊水幹細胞可與全身攜帶綠色螢光蛋白之小鼠做融合而呈現黃色之現象)，經由病理組織判讀顯示，同種異體或是異種細胞移植的各個器官均無發現癌化或是病變的現象。此發現未來可於先天性疾病胎兒治療提供重要的臨床參考資訊。

Amniotic fluid-derived stem cells (AFSCs) are multipotent and shed from the fetus into the amniotic fluid. We established AFSCs from Ds-red transgenic pig and EGFP transgenic mouse which were produced in our lab for tracing and tracking purpose. The result indicated that mouse derived AFSCs can express stem cells related markers such as CD29, CD44, Sca-1, whereas do not express hematopoietic、lymphocyte、immune related or endothelial cells markers such as CD31, CD45, CD34, CD166, CD11b, CD117, CD133, CD86, CD105. Pig derived AFSCs can express stem cells related markers such as CD44, CD90, yet do not express neutrophile, macrophages, endothelial cells or lymphocyte related markers such as CD31,CD4a. AFSCs can give rise to different lineages cells such as ostoblast (ARS for calcium deposition)、chondrocyte (Toluidine blue staining for glycosaminoglycan)、adipocyte (Oil Red O staining for neutral lipid vacuoles)、spontaneously beating cardiomyocyte like cells (myosin heavy chain and cardiac troponin I antibody conjugation). After differentiation of AFSCs, the cells still remain expressing fluorescent protein. The outcome demonstrated here implies that after transplantation of the cells into the receipient, we can trace the cell fate and cell destiny. Hence, AFSCs are capable of expressing stem cell like markers and differentiating into specific cell type in the experiments in vitro. However, no study has fully investigated the potentiality and destiny of these cells in in vivo experiments. Mouse fetuses (on day 12.5-13.5 of pregnancy) were transplanted in utero with mouse or pig amniotic fluid-derived stem cells. Our results demonstrated that transplantability of AFSCs into intestine, liver and kidney ect these three germ layers were observed. By means of histological section and flow cytometry methods, allogenic transplantation for example, EGFP harboring mouse AFSCs hold the potentiality to fuse with the mouse recipient Ds-red harboring cells and eventually present in yellow color. In terms of xenotransplantation, Ds-red harboring pig AFSCs are capable of fusing with the mouse recipient EGFP harboring cells and finially present in yellow color. The pathological section results reveal that after either allotransplantion or xenotransplantaion, all organs were not found tumor formation risk. The findings may justify a clinical trial of in utero transplantation during gestation for patients who have inherited genetic disorders.