iPSC Characterization and Downstream Applications Using Advanced Flow Cytometry

Advanced Flow Cytometry for iPSC Characterization and Downstream Applications

Since the discovery of induced Pluripotent Stem Cells (iPSCs), stem cell biology has rapidly expanded and iPSCs form the basis of many new areas of research. Major benefits of the use of iPSCs are the variety of cell types that can be differentiated from them and their capacity for infinite expansion. This flexibility provides many opportunities for the development of specific, physiologically relevant cell and tissue models (in 2D and 3D) for pharmacological testing, cancer research, organoid modelling and neurodevelopmental biology, reducing the need for animal models. In addition, iPSCs are increasingly used in translational applications, targeting eventual use in the clinic via autologous cell therapies and for individualized medicine approaches. 

Limitations are inherent in any system, however, and iPSCs are high-maintenance, expensive and require constant monitoring to ensure they maintain pluripotency, viability and homogeneity. Long term culture of iPSCs can result in genotypic and phenotypic heterogeneity, even in a cell line derived from a single source cell. Therefore, it is vital that methods for monitoring, detecting and reducing heterogeneity in iPSC lines are developed. The increasing use of stem cells in clinical and research settings calls for fast, robust and cost-effective solutions for the growth, characterization and maintenance of this valuable biological resource. Traditional methods for monitoring iPSC characteristics during culture - such as traditional flow cytometry - often:

  • Demand labor intensive and time-consuming techniques, requiring multiple steps including fixation, staining and washing
  • Require large sample volumes, using more precious cells and reducing the remaining sample for downstream expansion, characterization and differentiation
  • Use low-throughput instrumentation, increasing workflow time and reducing capacity for intra- and inter- experiment replication 
  • Necessitate in-depth manual manipulation and analysis of raw data and require compensation optimization 

In this application page, we describe methods for the assessment, monitoring, differentiation and scale up of iPSCs in a combined workflow approach. This is achieved using real-time imaging on the Incucyte® Live-Cell Analysis system for easy morphological analysis and rapid expression profiling using minimal sample volumes (10 µL) on the iQue® Advanced Flow Cytometry platform. Characterizing precious iPSC lines has never been easier!


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Workflow

Assay Workflow

Figure 1. Schematic highlighting the combined iQue® and Incucyte® workflow for iPSC cell line selection and differentiation monitoring

Figure 2. iPSC characterization workflow

Key Advantages

  • Maximize Productivity - Characterize pluripotency and viability in multiple iPSC lines as part of a high-throughput, multiplexed assay
  • Flexible Assay Format - Measure the effects of growth conditions on marker expression and morphology in 2D- and 3D-cultured iPSCs
  • Gain More Biological Insights - Analyze changing phenotypic characteristics of iPSCs undergoing directed differentiation for pharmacological outputs with enhanced biological relevance

Maximize productivity – High-throughput, multiplexed assay to characterize pluripotency and viability in multiple iPSC lines.

Figure 3. Characterize pluripotency and viability in multiple iPSC lines using a high-throughput, multiplexed assay.

Three iPSC lines were labelled using fluorophore conjugated anti-SSEA-1 (marker of differentiated cells), anti-SSEA-4 and anti-TRA-1-60 (two pluripotency markers) antibodies and a viability dye (iQue® Cell Membrane Integrity Dye (B/Red). THP-1 cells were also labelled as a differentiated cell control and NCCIT cells as a control cell line known to express stem cell pluripotency markers. Marker expression and viability were measured using the iQue® Advanced Flow Cytometer. (A) Table listing cell lines, and characteristics. (B) Marker expression as a percentage of live cells for each cell type. Pluripotent refers to SSEA-1 negative, SSEA-4 and TRA-1-60 double positive phenotype. (C) Heat map showing % viability for each cell type (n=4). (D) Incucyte® images of colonies of each iPSC cell line taken 48 hours after seeding illustrating tightly packed colony morphology.

Flexible assay format - Assess the effects of growth conditions on marker expression and morphology in 2D- and 3D-cultured iPSCs.

Figure 4. Evaluate optimal growth conditions to retain pluripotency in 2D- and 3D-cultured iPSCs.

iPSCs were grown in 2D (A) and 3D (B) in optimized (mTESR Plus 2D and regular passaging 3D) and non-optimized (RPMI 2D and no passaging 3D) culture conditions to induce ‘differentiation’ for 4 days in 2D and 18 days in 3D (± SEM, n=4). ‘Pluripotent’ are a population of SSEA-1 negative cells that are positive for both SSEA-4 and TRA-1-60, representing pluripotent cells. Dot plots showing the raw data collected by the iQue® system at 4 days in 2D and 18 days in 3D comparing the optimized and non-optimized iPSCs, note the shift in SSEA-1 expression in the non-optimized iPSCs and the subsequent losses in pluripotency marker expression (n=4). iPSCs grown in optimized and non-optimized growth conditions display distinct morphological differences both in 2D and 3D linked to differentiation which can be imaged and analyzed effectively using the Incucyte® (representative images taken at 10x magnification, scale bar indicates 400 μm).

Gain more biological insights – Examine changing phenotypic characteristics of iPSCs undergoing directed differentiation for pharmacological output.

Figure 5. Characterize iPSC differentiation through marker expression and morphological analysis.

Expression of markers in the hepatocyte differentiation pathway, definitive endoderm (CD184) and mature hepatocytes (CD99), were measured on the iQue®. AU565 cells were used as a differentiated cell control. HepG2s were included as a liver cell line. (A) Table describing cell line characteristics. (B) Percentage expression analysis shows marker expression of control cells from a single timepoint (left), and changes in hepatocyte marker expression of iPSCs during differentiation into hepatocytes (right). (C) Contour plots show CD184 expression over time and Incucyte® images from representative time points show morphological and spatial changes in cells during differentiation compared to iPSCs grown conventionally in mTESR.

Ordering Information

Product

Size

Catalog Number

Platform: Compatible with iQue® 3/iQue® Screener Plus – VBR and BR Configurations

iQue® Cell Membrane Integrity Dye (V/Blue)

5 x 384
20 x 384
50 x 384 

97057
97058
97059

iQue® Cell Membrane Integrity Dye (R/Red)

5 x 384
20 x 384
50 x 384 

90350
90351
90352

iQue® Cell Membrane Integrity Dye (B/Green)

5 x 384
20 x 384
50 x 384 

90342
90343
90344

iQue® Cell Membrane Integrity Dye (B/Red)

5 x 384
20 x 384
50 x 384 

90346
90347
90348

iQue® Cell Proliferation and Encoding Dye (R/Red) 

5 x 384
20 x 384
50 x 384 

90358
90359
90360

iQue® Cell Proliferation and Encoding Dye (B/Green)

5 x 384
20 x 384
50 x 384 

90354
90355
90356

iQue® Cell Proliferation and Encoding Dye V/Blue (Tag-it Violet™)

5 x 384
20 x 384
50 x 384 

97054
97055
97056


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Resources

Literature and Documentation

Application Note

iPSC Characterization and Optimization

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Application Note

iPSC Pluripotency and Differentiation

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Technical Note

Intracellular Staining

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