Stem Cell Pluripotency Assays
Pluripotent stem cells (PSCs) are characterized by their innate ability to self-renew and capacity to differentiate into any cell type. PSCs comprise two main categories, induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). Unlike ESCs, iPSCs avoid many ethical concerns and have rapidly become a gold-standard, scalable model system for regenerative medicine, disease modeling, and cell-based therapies.
Unlocking the full potential of these valuable cell types depends on precisely optimized culture conditions that preserve their genetic stability, pluripotency, health, and longevity. However, stem cell culture is technical, costly and time intensive and most methods for quality control or characterization are qualitative or limited to destructive end-point analyses.
As translational, physiologically relevant stem cell models become more widely used, there is a growing need for non-perturbing solutions that enable quantitative real-time assessments throughout culture. Kinetic morphological changes accompanying spontaneous loss of pluripotency can be captured at all stages with live-cell imaging.
The Incucyte® Live-Cell Analysis System in combination with the Incucyte® AI Stem Cell QC Analysis Software module offers a label-free, real-time tool for monitoring iPSC pluripotency. This approach enhances quality control, enables data-driven decisions, and due to its non-invasive manner, enables valuable stem cells to be preserved for downstream applications.
Buy Now - Incucyte® AI stem cell QC analysis software module
Solutions for Stem Cell QC Assays
Incucyte® AI Stem Cell QC Analysis Software Module
The Incucyte®AI Stem Cell QC Analysis Software Module is an all-in-one label-free analysis tool that segments feeder-free pluripotent stem cell colonies in HD phase-contrast images and classifies them as pluripotent or non-pluripotent. It allows users to perform analyses in real-time without the need for fluorescent labels or destructive protocols, generating accurate and objective data with minimal user input, reduced time requirements and no perturbance to precious stem cell samples.
The Incucyte® AI Stem Cell QC Analysis Software Module is available to purchase for Incucyte® S-series and CX3 systems and requires software version 2026B or higher.
Incucyte® AI Stem Cell QC Analysis Software Workflow
Figure 1. Phase-contrast images are captured at specific intervals and processed using purpose-built algorithms to segment and classify stem cell colonies. Colony segmentation and Pluripotent/Non-Pluripotent classification masks are displayed and provide robust quantification of colony growth and pluripotency over time.
| Related Products | Item No. | Buy Now |
| Incucyte® AI Stem Cell QC Analysis Software | BA-04909 | Buy Now |
Key Advantages of Stem Cell QC Assays
Perform AI-driven, label-free segmentation and classification of feeder-free pluripotent stem cell colonies over time with integrated software
[Automatically classify colonies as pluripotent or non-pluripotent using pre-trained models, reducing user bias and improving reproducibility.
Eliminate fluorescent labels and destructive end-point assays to generate accurate QC metrics with minimal user input and reduced analysis time.
Extract detailed colony-level growth and morphology metrics or combine with fluorescence readouts for additional biological insights.
Example Data for Stem Cell QC Assays
Identify and Segment Colonies Label-Free
Perform AI-driven, label-free segmentation and classification of feeder-free pluripotent stem cell colonies over time with integrated software.
Figure 2. Kinetically monitor loss of pluripotency label-free. hiPSCs were seeded into a VTN coated Incucyte® Imagelock 96-well plate in optimized medium supplemented with rock inhibitor. Once compact colonies had formed, they were maintained in either optimized medium or switched to non-optimized medium and monitored label-free every 15 minutes using the Incucyte® Live-Cell Analysis System and AI Stem Cell QC Analysis. Phase videos show rapid spontaneous loss of pluripotency in non-optimized conditions with AI-driven classification mask outlines shown for pluripotent colonies (blue) and non-pluripotent colonies (magenta).
Objectively Classify Colony Pluripotency
Automatically classify colonies as pluripotent or non-pluripotent using pre-trained models, reducing user bias and improving reproducibility.
Figure 3. Label-free, non-perturbing readouts are comparable to standard fluorescence methods. hiPSCs expressing a pluripotency reporter (OCT-4 GFP) were cultured in optimized medium until compact colonies formed and were then either maintained in the optimized medium or switched into non-optimized medium. Pluripotency was temporally assessed label-free using AI Stem Cell QC. Representative phase and fluorescence videos are shown; Pluripotent (blue outline) and Non-Pluripotent (magenta outline) classification masks indicate accurate label-free identification of pluripotency loss. Quantification revealed a decrease in the percentage of pluripotent colonies (label-free) with a temporal profile comparable to the loss of OCT-4 expression (fluorescence).
Streamline QC with Faster, Non-Destructive Workflows
Eliminate fluorescent labels and destructive end-point assays to generate accurate QC metrics with minimal user input and reduced analysis time.
Figure 4. Assess stem cell colony growth and pluripotency without disturbing cultures. hiPSCs were cultured in 6-well plates using single-cell dissociation or aggregate passaging methods. Growth and pluripotency were monitored label-free throughout the culture using AI Stem Cell QC. Representative phase images are shown with classification mask outlines for Pluripotent (blue outline) and Non-Pluripotent (magenta outline) colonies. Quantification shows how stem cell culture can be streamlined for different passage methods, using real-time monitoring of colony growth to determine the optimal timing of passaging and assess pluripotency to ensure robust QC.
Figure 5. Examine colony-level morphological features. hiPSCs were seeded into 96-well plates pre-coated with different matrices commonly used for iPSC culture. Colony morphology and pluripotency was assessed label-free using AI Stem Cell QC, with comparisons being made at similar confluencies (~40%). Representative phase images are shown with colony segmentation (yellow outline). All conditions showed high levels of pluripotency (>98%, data not shown). Colony level quantification of average object area, eccentricity, and texture revealed variances in morphological features for the different matrices (n = 25 images).
Perform Reproducible Phenotypic Profiling
Extract detailed colony-level growth and morphology metrics or combine with fluorescent readouts for additional biological insights
Figure 6. Multiplexed fluorescence readouts for enhanced biological insights. hiPSCs stably expressing Incucyte® Nuclight Green were seeded into 96-well plates. Once compact colonies had formed, cultures were either maintained in optimized medium or switched to a range of non-optimized medium (25 – 100%) in the presence of SSEA-4 Alexa Fluor 555, a marker of pluripotency and monitored using AI Stem Cell QC at 4x. Representative phase and Pluripotent (blue outline) or Non-pluripotent (magenta outline) classification masks are shown. Label-free quantification of pluripotency indicated a comparable morphological change across all non-optimized conditions compared to optimized control. Additional fluorescence readouts revealed a non-optimized percentage-dependent effect on nuclear and SSEA-4 intensity.
Stem Cell QC Assays Technical Resources
Featured Resources
Stem Cell QC Assays FAQ
The Colony Segmentation Mask can be improved by adjusting the Colony Splitting sensitivity and using Parameter refinement through Cleanup options (e.g., hole fill) and Filters (e.g., area or eccentricity).
No, the AI-driven models are pre-trained on a variety of feeder-free pluripotent stem cell conditions, this allows you to simply perform the analysis with minimal user input.
Incucyte® AI Stem Cell QC is enabled for most acquisition types (Standard, Adherent Cell-by-Cell, AI Scan or Imagelock) at either 4x or 10x objectives.
No, Incucyte® AI Stem Cell QC was trained and validated on feeder-free human induced pluripotent stem cell (hiPSCs) conditions. The analysis is expected to be applicable to non-human or embryonic pluripotent stem cells, provided that their morphology is comparable to that of hiPSCs (e.g., compact colonies with well-defined edges and high nucleus-to-cytoplasm ratio). The analysis is not recommended for feeder-dependent cultures or multipotent stem cells (e.g., MSCs).