A multiparametric approach using the Attune NxT Flow Cytometer

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The ability to direct human pluripotent stem cells (hPSCs) towards differentiated cell phenotypes offers tremendous potential for personalized and regenerative medicine [1,2]. The identification of key transcriptional regulators of pluripotency—as well as chemically defined media and cell culture conditions that drive PSCs towards distinct cell fates—have enabled researchers to derive a multitude of differentiated cell types with a high degree of control and precision [3]. One of the hallmarks of the transition from pluripotency towards terminal differentiation is the orchestrated nuclear expression of various transcription factors that act as regulators of cell-fate determination. In the case of hPSC-derived cardiomyocytes, the down-regulation and eventual loss of pluripotency markers is followed by the sequential expression of other factors that act to restrict cell-fate potential [4].

Quantifying the dynamic expression patterns of transcription factors that underlie cardiomyocyte differentiation often relies on detection of mRNA transcripts via qRT-PCR in a heterogeneous cell population. While this approach is highly sensitive and can be performed using small amounts of input material, it does not provide information about transcription factor expression in individual cells. An alternative approach is to use specific antibodies for the detection and quantification of transcription factor expression at the single-cell level (Figure 1) using either high-content imaging and analysis or multiparameter flow cytometry. Here we describe a flow cytometric method for the simultaneous quantification of Oct4, a canonical marker of pluripotency, and Nkx2.5, a marker of cardiac fate, in hPSCs that have been induced to differentiate towards cardiomyocytes.

Figure 1. Workflow for cardiomyocyte differentiation. H9 human pluripotent stem cells (hPSCs) were differentiated into cardiomyocytes over a 10-day period with the use of the Gibco™ PSC Cardiomyocyte Differentiation Kit (Cat. No. A2921201), which is a complete, ready-to-use, xeno-free system. Each day during the differentiation process, cells were detached from plates using Gibco™ TrypLE™ Express Enzyme solution (Cat. No.12605010) and combined into a single cell suspension. Cell counts and viability measurements were made using the Invitrogen™ Countess™ II Automated Cell Counter (Cat. No. AMQAX1000). A total of 1 × 106 cells from each time point were prepared and stained with an Invitrogen™ Alexa Fluor™ 488 anti-Oct4 antibody and an anti-Nkx2.5 antibody that was detected using Invitrogen™ Alexa Fluor™ 647 donkey anti–rabbit IgG secondary antibody. Cells were analyzed on the Invitrogen™ Attune™ NxT Flow Cytometer at a flow rate of 200 μL/min with stop criteria set on 10,000 total events using a forward-scatter threshold.

Advantages of the Attune NxT Flow Cytometer for stem cell research

Stem cells and cardiomyocytes represent traditionally challenging samples for flow cytometric testing due to their size, fragility, and scarcity. The Invitrogen™ Attune™ NxT Flow Cytometer is ideally suited for these samples because the acoustic-assisted hydrodynamic focusing technology and advanced fluidics are designed to minimize clogging and effectively handle a broad range of cell types with no loss in data quality. With its short acquisition times, the Attune NxT Flow Cytometer enables the detection of rare events without excess sample manipulation in a wide range of samples, including those with large cells that tend to clump as well as those with very low cell concentrations (e.g., due to high dilution or very small sample size).

Flow cytometric analysis of cardiomyocyte differentiation

In this experiment, H9 hPSC differentiation was monitored through the differential expression levels of the key nuclear differentiation markers, Oct4 and Nkx2.5, via flow cytometry (Figures 1 and 2). Two-parameter plots of the staining profiles of the singlet cells show that initially nearly all cells were positive for the Oct4 transcription factor and negative for the Nkx2.5 transcription factor, which is consistent with a pluripotent state (Figure 2B). Over time, cells gradually began to show reduced levels of Oct4 and increased levels of the cardiac marker Nkx2.5 (Figures 2C–2J). Prior to differentiation, 97% of all cells were Oct4-positive and Nkx2.5-negative; after day 3, the frequency of Oct4-positive cells began to decline—consistent with a loss of pluripotency and transition to a terminally differentiated cardiomyocyte phenotype—and by day 9, more than half of the cells were expressing Nkx2.5 (Figure 3).

Figure 2. Two-parameter plots representing staining profiles for Oct4 and Nkx2.5 in H9 hPSCs during cardiomyocyte differentiation. A dual-parameter plot of forward scatter height vs. forward scatter width was used to identify singlet cells (A), and a gate was drawn around the singlet-cell population. Using this gate, a dual-parameter plot of Oct4 Alexa Fluor™ 488 fluorescence vs. Nkx2.5 Alexa Fluor™ 647 fluorescence was created, and a quadrant gate was used to identify the cell population as it differentiated through time into Oct4-positive events (green), Nkx2.5-positive events (red), and dual-negative events (blue).

Summary of cell populations over the 10-day cardiomyocyte differentiation

Figure 3. Summary of cell populations over the 10-day cardiomyocyte differentiation. The percentages of Oct4+ (green) and Nkx2.5+ (red) expression over 10 days in culture were determined by placing quadrant gates on the Oct4 vs. Nkx2.5 dual-parameter plots of singlet cells, shown in Figure 2. Prior to differentiation, 97% of all cells expressed the Oct4+ Nkx2.5 phenotype, consistent with a pluripotent state. With induction, expression of Oct4 declines, consistent with a loss of pluripotency, and a transition to a terminally differentiated cardiomyocyte phenotype is seen as expression of Nkx2.5 increases.

Bring Attune NxT technology to your multiparametric experiments

The Attune NxT Flow Cytometer enables the single-cell quantification of cells expressing markers of pluripotency (Oct4) and cardiomyocyte specification (Nkx2.5) in H9 hPSCs as they differentiate into cardiomyocytes. This flow cytometry assay, which uses specific antibodies against the two transcription factors, produced results consistent with published data using qRT-PCR quantification of Oct4 and Nkx2.5 mRNA transcripts [4,5]. With sample throughput rates over 10 times faster than those of other cytometers, the Attune NxT Flow Cytometer allows users to process samples more quickly without loss in data quality, enabling the detection of rare events even in the case of challenging samples.

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