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.
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
Human pluripotent stem cells have the remarkable ability to differentiate into any cell type in the human body, making them invaluable tools for:
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.
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, 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.
Figure 1. An example karyogram for observing chromosome numbers and abnormalities.
Advancements in sequencing technologies have made Next-Generation Sequencing (NGS) a powerful option for karyotypic analysis of hPSCs, offering high-resolution detection of CNVs.
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.
Figure 2. Example sequence alignment compared to the reference genome.
Array-based karyotyping offers whole-genome coverage for detecting chromosomal copy number changes with higher resolution than traditional methods.
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.
Figure 3. Whole-genome view from a KaryoStat™ karyotyping report.
Digital droplet PCR (ddPCR) is an advanced PCR technique that allows precise quantification of nucleic acids by partitioning the sample into thousands of droplets.
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.
Figure 4. Example ddPCR report showing copy number assessment for 24 common hotspots.
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
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.
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.
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.