Disease modeling using human pluripotent stem cells (hPSCs) has been crucial in drug discovery, and clinical applications. However, a challenge with working with hPSCs is that over time, as they're cultured in vitro, they can develop recurrent genomic abnormalities and copy number variations (CNVs) that may compromise research outcomes and therapeutic potentials. Some CNVs, such as the common 20q11.21 amplification, can confer selective advantages to cells but reduce their differentiation capacities. Therefore, monitoring and verifying the genomic integrity of hPSC cultures is an essential quality control measure in cell-based research.
In this article, we explore recent advancements in karyotyping methodologies, discussing their benefits and limitations, and highlighting their significance in maintaining genomic stability in hPSCs.
Karyotyping & Keeping Your Copy Numbers Correct
Cytogenetics is a branch of genetics focused on the study of chromosome structure and function within the cell nucleus. Karyotyping is a fundamental cytogenetic technique used to analyze the chromosomal composition of cells. It involves various methods—including staining/imaging, sequencing, microarray analysis, and polymerase chain reaction (PCR)-based applications—to detect chromosomal abnormalities.
Each karyotyping method offers unique advantages and has certain limitations. Understanding these can help researchers choose the most appropriate technique for their specific needs, ensuring accurate detection of genomic alterations that may impact their studies
The Risks of Genomic Abnormalities in Human Stem Cell Research
Human pluripotent stem cells have the remarkable ability to differentiate into any cell type in the human body, making them invaluable tools for:
- Disease Modeling: hPSCs can model a wide range of diseases at the cellular level, providing insights into disease mechanisms and progression.
- Drug Discovery and Toxicology: hPSC-derived cells serve as platforms for screening potential therapeutics and assessing drug toxicity.
- Regenerative Medicine: hPSCs hold promise for cell-based therapies aimed at repairing or replacing damaged tissues and organs.
However, genomic instability in hPSCs can lead to aberrant differentiation, altered cell behavior, and tumorigenicity, posing risks to both research validity and patient safety. Therefore, routine karyotyping is essential to monitor chromosomal integrity, detect abnormalities early, and ensure the reliability of experimental results and clinical applications.
The Many Ways to Karyotype
Recent technological advancements have introduced several new techniques for karyotypic analysis. Each method varies in terms of resolution, turnaround time (TAT), cost, and the types of chromosomal changes it can detect. Table 1 summarizes the estimated TAT and resolutions for different karyotyping approaches.
Table 1. Turnaround times and resolutions for different karyotyping methods.
* External services not available; TAT based on internal resources.
G-Banding
G-banding, or Giemsa banding, is a classic DNA staining technique used to visualize condensed chromosomes within a cell's nucleus. Named after Gustav Giemsa, who developed the staining method in 1902, G-banding remains a cornerstone in cytogenetics.
Principle:
- Giemsa Stain Binding: The Giemsa stain binds specifically to phosphate groups of DNA, attaching more readily to regions rich in adenine-thymine (A-T) base pairs. These regions appear as dark bands under a microscope, while guanine-cytosine (G-C) rich regions incorporate less stain and appear as light bands.
- Karyogram Creation: The resulting banding pattern creates a karyogram—a visual representation of chromosomes—that can identify chromosomal aberrations such as translocations, deletions, duplications, and inversions (Fig 1).
Figure 1. An example karyogram for observing chromosome numbers and abnormalities.
Advantages:
- Whole-Genome Overview: Provides a comprehensive visualization of all chromosomes in a single assay.
- Detection of Large Structural Abnormalities: Effective for identifying significant chromosomal changes (>5–10 Mb).
Limitations:
- Limited Resolution: Unable to detect smaller chromosomal alterations below 5–10 Mb.
- Time-Consuming: Requires culturing cells to metaphase, which can take several weeks.
- Subjectivity and Expertise Dependence: Interpretation relies heavily on the skill and experience of the cytogeneticist.
Next-Generation Sequencing (NGS)
Advancements in sequencing technologies have made Next-Generation Sequencing (NGS) a powerful option for karyotypic analysis of hPSCs, offering high-resolution detection of CNVs.
What is NGS?
NGS is a high-throughput process for determining DNA sequences. Platforms like Illumina use sequencing by synthesis (SBS), where DNA polymerase incorporates fluorescently labeled nucleotides into a DNA template. Each nucleotide is identified by its specific fluorescent label during DNA synthesis cycles. NGS can be targeted to hotspot regions known to harbor karyotypic abnormalities.
NGS Workflow:
- Library Preparation: Create short DNA or cDNA fragments with ligated adapters.
- Cluster Amplification: Load the library onto a flow cell for bridge amplification, forming clonal clusters.
- Sequencing: Detect single bases as they are incorporated using SBS technology.
- Alignment and Analysis: Align sequence reads to a reference genome using specialized software.
Figure 2. Example sequence alignment compared to the reference genome.
Advantages:
- High Resolution: Can detect genomic alterations at single-base resolution.
- Comprehensive Data: Provides detailed information on a wide range of genetic variations.
Limitations:
- Cost and Time Intensive: More expensive and time-consuming compared to other methods.
- Complex Data Analysis: Requires significant computational resources and bioinformatics expertise.
- Detection of Large Structural Variations: Short-read sequencing may struggle with identifying large structural rearrangements or repetitive regions.
Array-Based Karyotyping: KaryoStat™ and KaryoStat™ Plus
Array-based karyotyping offers whole-genome coverage for detecting chromosomal copy number changes with higher resolution than traditional methods.
What is Array-Based Karyotyping?
This method uses microarrays printed with thousands of oligonucleotide probes to detect unbalanced chromosomal abnormalities. It addresses limitations of G-banding by providing finer resolution but cannot detect balanced abnormalities (e.g., reciprocal translocations) that do not affect copy number.
Workflow:
- DNA Labeling: Extract and fluorescently label DNA from samples.
- Hybridization: Mix and hybridize labeled DNA to the microarray.
- Visualization: Use a fluorescence scanner to detect signals and compare them to a reference genome.
Figure 3. Whole-genome view from a KaryoStat™ karyotyping report.
Advantages:
- Improved Resolution: Can detect CNVs as small as 1–2 Mb, better than G-banding.
- Genome-Wide Assessment: Simultaneously analyzes the entire genome for copy number changes.
Limitations:
- Cannot Detect Balanced Rearrangements: Unable to identify structural changes without copy number differences, such as inversions or balanced translocations.
- Detection Limitations: May miss smaller CNVs below the resolution threshold.
- Dependence on Probe Density: Sensitivity and coverage depend on the number and distribution of probes.
Digital Droplet PCR (ddPCR)
Digital droplet PCR (ddPCR) is an advanced PCR technique that allows precise quantification of nucleic acids by partitioning the sample into thousands of droplets.
What is ddPCR?
ddPCR refines conventional PCR by dividing the sample into approximately 20,000 droplets, each acting as an individual PCR reaction. This massive partitioning enables highly sensitive and absolute quantification of target DNA sequences.
Workflow:
- Sample Preparation: Combine DNA with primers, probes, and ddPCR supermix.
- Droplet Generation: Create uniform PCR-ready droplets using a droplet generator.
- PCR Amplification: Amplify DNA within each droplet using a thermal cycler.
- Droplet Reading: Analyze droplets individually with a two-color detection system for multiplexing.
- Analysis: Positive droplets containing at least one copy of the target exhibit increased fluorescence over negative droplets. The fraction of positive droplets is then fitted to a Poisson distribution to determine the absolute initial copy number of the target DNA molecule in the input reaction mixture in units of copies/µl. Manufacture-specific ddPCR software allows you to visualize the data in a variety of ways to determine the concentration in copies/µl.
Figure 4. Example ddPCR report showing copy number assessment for 24 common hotspots.
Advantages:
- High Sensitivity and Precision: Capable of detecting small CNVs and low-level mosaicism.
- Targeted Analysis: Focuses on specific genomic regions known to be problematic.
Limitations:
- Limited Genomic Coverage: Not suitable for whole-genome analysis; requires prior knowledge of target regions.
- Technical Expertise Required: Demands specialized equipment and training.
- Potential for Errors: More prone to errors if not performed meticulously.
EditCo Karyotyping Case Study
Detecting the Previously Undetectable: Why Resolution Matters
Each karyotyping method has its strengths and weaknesses. At EditCo, we routinely used Thermo Fisher's array-based karyotyping services for our iPSC lines. Recently, Thermo Fisher upgraded their platform from KaryoStat™ to KaryoStat+™, improving detection resolution from >2 Mb to >1 Mb.
With this enhanced resolution, we detected the common 20q11.21 amplification in some edited clones and our parental cell bank—an abnormality often undetected by the previous less sensitive method (Fig 5).
Figure 5. Normal Karyostat results for PGP1 parental cell lines
Understanding the 20q11.21 Amplification
- Prevalence: Detected in over 20% of cultured hPSCs worldwide (Assou et al., 2020; Baker et al., 2016; Avery et al., 2013).
- Significance: Represents 22.9% of recurrent structural variants in hPSCs (Assou et al., 2020).
- Selective Advantage: Cells with this amplification have a growth advantage and can overtake cultures within a few passages (Assou et al., 2018).
- Detection Challenges: Often below detection limits of conventional G-banding and some microarray methods.
To confirm these findings, we employed ddPCR using the iCS-digital™ PSC 24 Probes Kit from Stem Genomics. The ddPCR results corroborated the array-based detection of the 20q11.21 amplification.
Figure 6. KaryoStat™ Plus results showing the 20q11.21 amplification.
Comparing Even Earlier Passage Cells with ddPCR
Interestingly, when we tested a low-passage vial of the same cell line using both methods, the array-based karyotyping showed a normal karyotype (Fig. 7), while ddPCR detected the 20q11.21 amplification at low levels (Fig. 8). This suggests that the abnormality was present below the detection threshold of the array-based method but detectable with the higher sensitivity of ddPCR.
Figure 7. Array-based karyotyping of low-passage cells showing normal karyotype.
Figure 8. ddPCR revealing low-level 20q11.21 amplification in the same sample at a lower passage.
The Future of Karyotyping
As karyotyping technologies continue to advance, we anticipate the discovery of additional chromosomal abnormalities in hPSCs used worldwide. Enhanced precision in karyotyping methods will provide deeper insights into genomic integrity, which is crucial for both research and clinical applications.
Maintaining genomic stability in hPSCs is essential for their reliability in disease modeling and therapeutic use. By leveraging advanced karyotyping techniques, researchers can better monitor and ensure the quality of their cell cultures, ultimately accelerating progress in regenerative medicine and personalized therapies.
