Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.
Lahm H, Doppler S, Dreßen M, Werner A, Adamczyk K, Schrambke D, Brade T, Laugwitz KL, Deutsch MA, Schiemann M, Lange R, Moretti A, Krane M.
Department of Cardiovascular Surgery, Division of Experimental Surgery, German Heart Center Munich, Munich Heart Alliance.
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.
KEYWORDS: Embryonic stem cells; Fluorescent nanoparticles; Induced pluripotent stem cells; Live staining; Pluripotency
SmartFlareTM Probes – a novel approach to detect pluripotency gene expression in live cells.
It is now almost a decade ago that murine fibroblasts have successfully been reprogrammed to a pluripotent state, generating so-called induced pluripotent stem cells (iPS cells) after retroviral transduction with a cocktail of appropriate transcription factors (Takahashi and Yamanaka, 2006). Quite rapidly, this technique has effectively been transferred to cells of other species including humans. Within two weeks after viral transduction the first iPS colonies emerge and are visible under the microscope. An experienced researcher might identify those colonies which are truly reprogrammed by visual inspection at a high frequency. Nevertheless, this “gut feeling” has to be substantiated by confirming the pluripotency status of these colonies with molecular methods, a procedure which is laborious and also time-consuming.
A few membrane-bound iPS specific markers exist, e.g. TRA1-81, which can be detected directly on growing cells (Figure 1) though the interaction with the antibody might nevertheless bother the cell. Secondly, after fixation of the cells it is possible to validate the expression of endogenous pluripotency factors, those which have been used for reprogramming (Figure 2) or other independent factors (e.g. NANOG, REX1). Finally, the expression of the pluripotency factors can be evaluated by qRT-PCR after cell lysis (Figure 3). The latter two approaches require replicates as the treated cells are lost for further cell culture.
In our work we describe an approach with gene-specific nanoparticles (so-called SmartFlareTM probes) which allow the detection of gene expression of endogenously expressed genes directly in live cells. In addition, this method does not require any manipulation of the target cell (Lahm et al., 2015) and can be applied to almost every cell type since the nanoparticles are engulfed by endocytosis. After application of the nanoparticles and overnight incubation a gene-specific fluorescence in the target cell can be observed visually under a fluorescence microscope. We have used nanoparticles specific for NANOG and GDF3. An example of a NANOG staining of a human iPS colony is shown in Figure 4. Another advantage is that these SmartFlareTM treated cells can be further cultured and evaluated in downstream experiments. Cy3 positive cells can also be identified by flow cytometry and sorted. Finally, we have tested whether an application of NANOG-specific nanoparticles interferes with gene- and protein-expression but we did not see any significant differences.
Therefore, these nanoparticles might be a powerful tool in detecting truly reprogrammed iPS cells in situ. Disease modeling and drug-screening are research areas with a high demand for human iPS cells. Pluripotency gene-specific nanoparticles might be helpful in high throughput screening and shorten the time to identify truly reprogrammed iPS colonies. Finally, the application of gene-specific nanoparticles may also be a powerful tool in identifying rare cell populations in many research areas, e.g. cancer research.
Lahm H, Doppler S, Dreßen M, Adamczyk K, Deutsch M-A, Ulrich H, Schiemann M, Lange R, Krane M. Detection of intracellular gene expression in live cells of murine, human, and porcine origin using fluorescence-labeled nanoparticles. J. Vis. Exp., in press, (2015)
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126: 663-676 (2006).