Lahm Harald, Doppler Stefanie, Dreßen Martina, Werner Astrid, Adamczyk Klaudia, Schrambke Dominic, Brade Thomas, Laugwitz Karl-Ludwig, Deutsch Marcus-André, Schiemann Matthias, Lange Rüdiger, Moretti Alessandra, Krane Markus
Department of Cardiovascular Surgery, Division of Experimental Surgery, German Heart Center Munich, Munich Heart Alliance.
Stem Cells. 2015 Feb;33(2):392-402. doi: 10.1002/stem.1872.
The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.
在许多物种中都已成功实现诱导多能干细胞（iPS细胞）的生成。然而，真正重编程的iPS细胞的鉴定仍然很费力，并且在大多数情况下，多能性标志物的检测需要固定细胞。在此，我们报告一种方法，即使用携带与金颗粒偶联的Cy3标记的正义寡核苷酸报告链的纳米颗粒。这些分子无需任何操作即可直接添加到培养的细胞中，过夜孵育后通过显微镜评估基因表达。为了同时检测不同物种中的基因表达，根据种间同源性选择探针序列。使用通用的靶标特异性探针，我们成功地证明了GAPDH管家基因在体细胞中的表达，以及多能性标志物NANOG和GDF3在小鼠、人类和猪源的胚胎干细胞和iPS细胞中的表达。可以通过荧光激活细胞分选纯化靶基因阳性细胞群体。在用慢病毒转导小鼠尾尖成纤维细胞后，Nanog特异性探针基于其Cy3荧光在发育过程中原位鉴定真正重编程的小鼠iPS细胞。Nanog特异性荧光的强度与单个克隆分化为所有三个胚层细胞的能力增强呈正相关。我们的方法提供了一种通用工具，可直接在任何所需来源的活细胞中检测细胞内基因表达，无需操作，从而保留靶细胞的遗传背景。此外，它代表了一种简单、可扩展的方法，用于高效筛选多能性，这在高通量细胞重编程期间以及多能干细胞基因组编辑后是非常需要的。
