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Discriminating between specific cell types derived from embryonic stem cells using fluorescent probes Evolving Science For The Future | Articles

Written by Patricia Viard, PhD

One major challenge in stem cell research is to identify and sort the various subpopulations of cells co-existing within heterogeneous cultures. Indeed, stem cells (SCs), obtained either directly from the inner cell mass of preimplantation embryos (ESCs) [1;2] or by reprogramming somatic cells (induced Pluripotent SCs/iPSCs) [3;4;5], exist in two main pluripotency states, either primed or naive, with a range of intermediate states[6;7]. In addition to their greater self-renewal abilities, naive SCs have a greater capacity to differentiate into cell-types of interest when receiving appropriate cues [7;8]. Then again, there is a strong variability within the cells regarding the efficiency and timing of the differentiation process. Only a fraction of SCs reach the desired phenotype and can be used for cell-based therapy or pharmaceutical studies[9] . Furthermore, SCs which fail to differentiate are potentially teratogenic and accurate cell selection is primordial for the safety of clinical applications [10]. Hence, reliable techniques are needed to isolate naive SCs and, after in vitro differentiation, SC-derived cells of interest.

Seminal studies in SC selection were performed using transgenic animal models expressing fluorescent reporter genes [3;7;11]. However, there are inherent biases associated with the use of transgenes. Their expression may interfere with the normal function of the cell by competing with endogenous proteins, preventing interactions with partners or regulators. In addition, random insertion of transgenes into the genomic DNA may alter endogenous gene regulation and/or integrity. Alternatively, immunodetection of cell surface markers has been used to achieve cell type selection in live conditions [12;13;14]. However, immunolabeling is not readily reversible. Hence, none of these techniques are suitable for human therapy. Thus, there is a growing interest for non-invasive techniques to detect endogenous cell markers of either pluripotency or differentiation. A range of fluorescent probes have been developed to select either pluripotent SCs or differentiated cells. Remarkable examples are described below.

Isolation of stem cells with distinct pluripotency states

In order to evaluate the pluripotency of SCs, a range of fluorescent small molecules were chemically engineered using a Diversity Oriented Fluorescence Library Approach (DOFLA) [15]. One such probe, CDy 1 (Compound of Designation yellow 1) is a rosamine-based dye which selectively labels mouse ESCs and iPSCs [16;17]. Remarkably, the orange/red fluorescence signal of CDy1 (excitation and emission peaks respectively at 535 nm and 570 nm) could be detected prior to the expression of the transcription factor Oct4-GFP in mouse iPSCs [17]. This result indicated that CDy1 could be used to select early pluripotent stages preceding the expression of this canonical pluripotency marker. Remarkably, CDy1 has also been used to target and eliminate undifferentiated SCs [18], in order to prevent the risk of teratoma formation associated with stem cell therapy [19].

Discriminating between specific cell types derived from embryonic stem cells using fluorescent probes

Another red fluorescent probe, rhodamine Kyoto Probe-1 (KP-1) was shown to specifically stain human iPSCs [20]. In differentiated cells, KP-1 is actively exported out of the cells via ATP Binding Cassette (ABC) transporters, hence the lack of staining. Expression of these transporters is repressed in human iPSCs, but also in cancer cells. Accordingly, KP¬-1 also labeled HeLa cells, a cervical cancer cell line [20]. Hence, KP-1 is a marker of pluripotency but is not specific for stem cells.

In a later study, Cho and collaborators showed that another compound, CDy9, specifically labels naive (versus primed) mouse ESCs [21;22]. CDy9 penetrates into cells via Slc (Solute carrier) 13a5, a membrane transporter which is highly expressed in naive mouse ESCs [22]. CDy9 was derived from a synthetic orange/red BODIPY intermediate. Its fluorescence properties only slightly differ from CDy1, with maximum excitation and emission peaks at 563 nm and 578 nm, respectively. After a 1-hour incubation with 1 μM CDy9, CDy9-positive cells were sorted using fluorescence-activated cell sorting (FACS) [22]. This strategy allowed for specific isolation of naive pluripotent cells, as confirmed by the absence of immunolabeling for the stage-specific embryonic antigen-4 (SSEA-4), a classic surface marker of the primed state.

Selection of differentiated cells derived from SCs

One well described example is the detection of hepatocyte-like cells using indocyanine green (ICG). ICG is a non-toxic fluorescent organic anion which is selectively taken up by hepatocytes, and commonly used for clinical evaluation of liver function [23;24]. In a study from Yoshie and collaborators [25], ESCs from the mouse R1 cell line were differentiated into hepatocyte-like cells. After incubation with 5 μg/ml ICG for 30 minutes, the differentiating cells were analysed with a FISHMAN-R flow cytometer (On-Chip technologies) equipped with a 785-nm excitation laser and 815-850 nm bandpass filter, in order to detect ICG-positive cells. The authors showed that mouse ESC-derived hepatocyte-like cells selectively took up ICG. The hepatocyte phenotype was confirmed by periodic acid-Schiff staining and by real-time PCR quantification of hepatocytes specific gene transcripts. Using this flow cytometry strategy, the authors also showed that ICG specifically stained rat primary hepatocytes and human hepatocellular carcinoma cells (HepG2). In contrast, non-hepatocyte cell lines, such as rat pancreatic adenocarcinoma (AR42J), human breast adenocarcinoma (MCF7) and embryonic kidney (HEK293T) cells, remained essentially unlabeled under these experimental conditions. Of note, the fluorescence properties of ICG are altered at higher concentrations. It is therefore recommended to use low concentrations and short incubation times with ICG to avoid accumulation of ICG in hepatocytes, as well as non-specific labeling.

Other fluorescent probes and perspectives

Discriminating between specific cell types derived from embryonic stem cells

Beside ICG, a few other fluorescent probes have proven useful in discriminating specific cell types derived from stem cells. Amongst these, the BODIPY derivative compound of designation red 3 (CDr3) specifically recognized both mouse and human neural stem cells [26]. CDr3 binds to fatty acid binding protein 7 (FABP7), which is highly expressed in the cytoplasm of neural stem cells. Other non-genetic modifying approaches, while remaining invasive, are worthy of consideration. For instance, the so-called molecular beacons (MB), become fluorescent when binding to cell type-specific mRNAs [27], such as the transcript encoding Irx4 (Iroquois homeobox protein 4), a transcription factor specific for ventricular cardiomyocytes [28]. Nanoparticles carrying fluorescent (Cy3-labeled) oligonucleotides have also been reported to detect pluripotency markers [29].

Nonetheless, fluorophores specifically designed to identify cells of interest are still too few and mostly validated on animal models. With the increasing variety of cell types differentiated from human stem cells, there is a growing need for novel tools to specifically detect human cell-specific endogenous markers with non-invasive techniques. Sorting methods shall involve short incubation times, maintain sterile conditions throughout and preserve the integrity of the cells. These requirements are essential to safeguard the potential of SC-derived cells for regenerative medicine.

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