iPSC Characterization and Downstream Applications Using High-Throughput Screening (HTS) by Cytometry

High-Throughput Screening (HTS) by 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. The workflow includes both the Incucyte® Live-Cell Analysis System and the iQue® HTS Platform, which together achieve both real-time morphology monitoring, and rapid profiling expression. Characterizing precious iPSC lines has never been easier!

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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® High-Throughput Screening 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® HTS Platform 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


Literature and Documentation


First Edition High-Throughput Screening Cytometry Handbook

A Guide to HTS Cytometry Assays and Workflows

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Solutions for Culturing, Maintaining and Characterizing iPSCs
Application Note

Solutions for Culturing, Maintaining and Characterizing iPSCs

Learn about an accelerated and streamlined workflow approach for the culture, monitoring and characterization of iPSCs.

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characterization and optimization of induced pluripotent stem cell culture using advanced flow cytometry and live-cell analysis
Application Note

iPSC Characterization and Optimization

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utilizing advanced flow cytometry and live-cell imaging to evaluate iPSC pluripotency During Cell Line Selection
Application Note

iPSC Pluripotency and Differentiation

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Technical Note

Intracellular Staining

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iQue®️ Reagent Kits brochure cover

Complete Product Listing of iQue® Reagents

Perform comprehensive, multiplexed analysis of cells, beads and secreted proteins all from the same cells at the same timepoint.

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iQue®️ Advanced Flow Cytometry Platform brochure cover

iQue® 3 - Truly Fast. Simply Effortless

The iQue® High-Throughput Screening (HTS) by Cytometry Platform allows researchers to acquire and analyze high content, multiplexed assays.

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