iPSC Characterization and Downstream Applications Using High-Throughput Screening (HTS) by Cytometry
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:
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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Figure 1. Schematic highlighting the combined iQue® and Incucyte® workflow for iPSC cell line selection and differentiation monitoring
Figure 2. iPSC characterization workflow
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.
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).
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.
Size
Catalog Number
Platform: Compatible with iQue® 3/iQue® Screener Plus – VBR and BR Configurations
5 x 38420 x 38450 x 384
970579705897059
iQue® Cell Membrane Integrity Dye (R/Red)
903509035190352
iQue® Cell Membrane Integrity Dye (B/Green)
903429034390344
iQue® Cell Membrane Integrity Dye (B/Red)
903469034790348
iQue® Cell Proliferation and Encoding Dye (R/Red)
903589035990360
iQue® Cell Proliferation and Encoding Dye (B/Green)
903549035590356
iQue® Cell Proliferation and Encoding Dye V/Blue (Tag-it Violet™)
970549705597056
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Utilizing High-Throughput Screening (HTS) by Cytometry Based Techniques, in iPSC Culture, Characterization, and Optimization.
Outlines methods for single cell dissociation of ACM, such as spheroids and organoids for surface marker analysis using flow cytometry.
A Guide to HTS Cytometry Assays and Workflows
Learn about an accelerated and streamlined workflow approach for the culture, monitoring and characterization of iPSCs.
Perform comprehensive, multiplexed analysis of cells, beads and secreted proteins all from the same cells at the same timepoint.
The iQue® High-Throughput Screening (HTS) by Cytometry Platform allows researchers to acquire and analyze high content, multiplexed assays.
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