Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany.
Stem Cell Rev Rep. 2013 Oct;9(5):555-68. doi: 10.1007/s12015-012-9416-1.
Pluripotent cells hold great promise for cell replacement therapies in regenerative medicine. All known protocols for directed in vitro differentiation of pluripotent cells did not yield pure populations complicating the characterization of the derived cells. In addition, the risk of tumor formation due to residual undifferentiated cells is a serious unresolved problem. In the present study the tissue-specific mouse Pdx1 promoter was used to control the expression of the reporter gene GFP2 in mouse ES cells in order to purify them via FACS during in vitro differentiation. The background fluorescence of transduced ES cells hampered the purification of Pdx1-positive cells due to a contaminating population of partially undifferentiated cells. MicroRNAs (mir) are important regulators of gene expression and were used to enhance promoter specificity during differentiation towards pancreatic progenitor cells. The mouse mmu-mir-294 was found to be mainly expressed during pluripotency, whereas the expression of the mir-302 cluster was increased during early differentiation. Integration of a microRNA target site for the mmu-mir-294 into the lentiviral vector reduced the background fluorescence specifically during pluripotency and permitted re-occurrence of GFP2 expression upon differentiation. A combination of the microRNA target site with the Pdx1 promoter fragment allowed the purification of pancreatic progenitors from differentiated ES cells. This population reflected an early pancreatic progenitor population without other contaminating cell lineages. In conclusion, microRNA target sites are efficient regulatory elements to control transgene expression and to enhance tissue specificity as presented in this study facilitating the sorting and purification of Pdx1-positive pancreatic progenitor cells.
多能细胞在再生医学的细胞替代疗法中具有巨大的应用潜力。所有已知的诱导多能细胞体外定向分化的方案都未能获得纯细胞群体，这使得衍生细胞的特征复杂化。此外，由于残留未分化细胞导致肿瘤形成的风险是一个严重的未解决问题。在本研究中，使用组织特异性的小鼠 Pdx1 启动子来控制报告基因 GFP2 在小鼠胚胎干细胞中的表达，以便在体外分化过程中通过 FACS 对其进行纯化。转导的 ES 细胞的背景荧光会阻碍 Pdx1 阳性细胞的纯化，因为存在部分未分化细胞的污染群体。微小 RNA（mir）是基因表达的重要调节因子，并在向胰腺祖细胞分化过程中用于增强启动子特异性。发现小鼠 mmu-mir-294 主要在多能性期间表达，而 mir-302 簇的表达在早期分化过程中增加。将 mmu-mir-294 的微小 RNA 靶位点整合到慢病毒载体中可以特异性地降低背景荧光，并且在分化时允许 GFP2 表达重新出现。微小 RNA 靶位点与 Pdx1 启动子片段的组合允许从分化的 ES 细胞中纯化胰腺祖细胞。该群体反映了一种早期的胰腺祖细胞群体，没有其他污染的细胞谱系。总之，微小 RNA 靶位点是控制转基因表达和增强组织特异性的有效调节元件，如本研究所示，可促进 Pdx1 阳性胰腺祖细胞的分选和纯化。
