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A G-banded karyotype is a widely used genetic assay for detecting chromosomal abnormalities, offering researchers critical insights into the genetic stability of their samples. This method involves visualizing chromosomes using a specific staining technique called Giemsa-Trypsin-Wright (GTW), which produces characteristic light and dark banding patterns unique to each chromosome. These patterns enable the detection of significant structural changes, including aneuploidies (abnormal chromosome numbers), translocations, inversions, and other alterations larger than 5 Mb.

Chromosomal abnormalities are frequently observed in hPSCs maintained in long-term culture, particularly at higher passage numbers. The most recurrent changes include gains in regions of chromosomes 1, 12, 17, 20, and X, as well as losses in parts of chromosomes 10 and 18. These genetic alterations can confer altered phenotypes to hPSCs, such as enhanced growth properties and modified differentiation capacities. Detecting these changes early is essential for maintaining the biological relevance, reproducibility, and reliability of research outcomes, as highlighted by the ISSCR Standards for Human Stem Cell Use in Research (2023).

At ���ϳԹ�, our G-banded karyotyping service is designated for research use only (RUO) and adheres to the ISCN (2024) guidelines to ensure consistent and accurate reporting. While highly effective at identifying large-scale structural abnormalities larger than 5 Mb, G-banded karyotyping may not detect submicroscopic changes, such as microdeletions, which often require complementary techniques like fluorescence in situ hybridization (FISH) for detection. For higher-resolution analyses, FISH is recommended to capture these smaller but significant genetic changes.

Our karyotype analysis report provides a comprehensive overview of chromosomal findings for your sample, following ISCN (2024) guidelines.

Here’s what the report includes:

• Summary of Findings: A clear overview of any detected chromosomal abnormalities, with descriptions and interpretations.
• Karyogram Image: A high-quality image of the chromosomes, visually highlighting any observed abnormalities.
• Sample and Analysis Details: Key information such as sample ID, passage number, and dates related to sample receipt and report generation.
• Chromosome Count and Quality Control Data: The number of cells analyzed and the band resolution, which indicates the quality of the analysis.

This report aligns with the ISSCR Standards for Human Stem Cell Use in Research (2023), which emphasize the importance of monitoring genetic stability in RUO stem cell samples to help maintain consistency and reliability in research outcomes. For more information, refer to a sample karyotype report or contact our Product and Scientific Support Team at techsupport@stemcell.com.

The 20q11.21 (BCL2L1) FISH test is a high-resolution assay specifically designed to detect genetic changes in the 20q11.21 region of hPSCs. This region is frequently gained as a copy number variant (CNV) in hPSCs, with duplications ranging in size from 0.55 Mb to 4.6 Mb. Due to their relatively small size and potential low-level mosaicism, these changes can be challenging to detect using G-banded karyotyping. As duplications of 20q11.21 are among the most common genetic alterations observed in hPSCs, reliable detection methods like FISH are essential for maintaining genetically stable cultures.

The assay utilizes FISH probes to target the BCL2L1 gene locus at 20q11.21 and a control region at 20p11.21. A minimum of 200 interphase cells are analyzed, with signal patterns evaluated against established thresholds to identify abnormal CNVs. This high-resolution approach allows for the detection of genetic changes that might be missed in standard karyotyping, including smaller aberrations and low-level mosaicism, making it a critical tool for ensuring the integrity of hPSC cultures.

The 20q11.21 (BCL2L1) FISH test adheres to the ISSCR Standards for Human Stem Cell Use in Research (2023), which emphasize the importance of high-resolution assays like FISH to detect small but recurrent genetic changes, particularly in preclinical research or after significant changes in cell behavior. This test is designated for research use only (RUO) and provides a robust solution for genetic monitoring in hPSC cultures.

For further details, please contact our Product and Scientific Support Team at techsupport@stemcell.com.

Our 20q11.21 (BCL2L1) FISH analysis report provides a detailed summary of genetic findings in the 20q11.21 region, following ISCN (2024) guidelines. Here’s a breakdown of common components:

• Summary of Findings: An overview of any detected abnormalities in the 20q11.21 region, with interpretations explaining the potential implications.
• Signal Pattern Analysis: A breakdown of observed fluorescent signals across a minimum of 200 cells, indicating the proportion of cells with typical or abnormal signal patterns (e.g. increased or decreased signals at the BCL2L1 locus).
• Sample and Analysis Details: Information such as sample ID, passage number, and dates for sample receipt and report generation, ensuring traceability and accuracy.

This report offers a high-resolution view of genetic stability in hPSC cultures, aligning with the ISSCR Standards for Human Stem Cell Use in Research (2023). For more information, refer to a sample FISH analysis report or contact our Product and Scientific Support Team at techsupport@stemcell.com.

At ���ϳԹ�, we follow stringent quality control measures in both karyotyping and FISH analysis to ensure the accuracy and reliability of results.

For karyotyping, we use ISCN (2024) guidelines and analyze a minimum of 20 metaphase spreads, assessing band resolution and quality to detect chromosomal abnormalities. This analysis ensures that large-scale chromosomal changes are detected with high fidelity.

For FISH analysis, our quality control includes evaluating a minimum of 200 interphase cells to verify signal patterns accurately. Each cell's signal pattern is reviewed against established thresholds to determine any amplification, deletion, or aneuploidy in the target region (e.g. 20q11.21 for BCL2L1). All thresholds and controls are set based on data from large control populations to account for natural background noise and technical variability.

These measures ensure that our reports provide consistent and accurate genetic information, are aligned with best practices for stem cell research, and support the rigorous monitoring recommended by the ISSCR Standards for Human Stem Cell Use in Research (2023).

Genetic abnormalities are a recognized issue in hPSC cultures, with studies indicating that up to 30 - 35% of cultures analyzed by G-banding harbor a genetic abnormality. These abnormalities often arise due to selective pressures that favor variant cells with genetic changes, allowing them to outcompete wild-type cells over time in culture. Commonly observed abnormalities include gains in chromosomal regions such as 1q, 12p, and 20q11.21, which harbor genes associated with increased proliferation or resistance to apoptosis.^1-5

These culture-acquired chromosomal changes can significantly affect research consistency and reproducibility. To address this, the ISSCR Standards for Human Stem Cell Use in Research (2023) recommend routine genetic monitoring at key stages of hPSC culture, including karyotyping for broad chromosomal changes and FISH for detecting specific, smaller aberrations. Adhering to these guidelines helps maintain genetic stability and aligns with ISSCR Recommendations 3.1.1 and 3.2.2.

According to the ISSCR Standards for Human Stem Cell Use in Research (2023), karyotyping at several critical stages is recommended to ensure genetic integrity throughout your experiments:

• Before starting experiments: When establishing a master or working cell bank, initial karyotyping provides a baseline of the cells' genetic status (ISSCR Recommendation 3.2.1).
• Approximately every 10 passages during experiments: Regular karyotyping around every 10 passages is advisable to detect culture-acquired abnormalities early. Gains in chromosomes 12, 17, and 20, among other changes, can confer growth advantages, potentially altering the cell population within 5 - 10 passages (ISSCR Recommendation 3.2.2).
• After major culture bottlenecks: Events like cloning or genetic modifications may increase the risk of clonal expansion of abnormal cells. Karyotyping after these procedures helps detect any resulting genetic shifts (ISSCR Recommendation 3.2.3).
• At the end of experiments or if significant changes in cell traits are observed: If unexpected changes in growth or differentiation are noted, karyotyping can determine whether these shifts result from chromosomal abnormalities or undetected copy number variants (ISSCR Recommendation 3.2.2).

Following these steps helps maintain consistency and reliability in research outcomes by rigorously monitoring the genetic stability of stem cell cultures. For additional guidance on karyotyping schedules or genetic stability assessments, please contact our Product and Scientific Support Team at techsupport@stemcell.com.

Mosaicism refers to the presence of two or more genetically distinct cell populations within a single culture, often resulting from acquired genetic changes in a subset of cells. This can occur in hPSC cultures over time and may influence experimental outcomes if left undetected. Monitoring for mosaicism is essential to ensure the genetic stability and reproducibility of your cultures.

The sensitivity of karyotyping and FISH for detecting mosaicism differs:

Karyotyping typically detects mosaicism at levels exceeding 10 - 20% of the cell population. Because this method examines a limited number of metaphase spreads (typically 20), smaller abnormal cell populations may not be consistently detected.

FISH is more sensitive and can identify mosaicism at levels as low as 5 - 10% of the cell population, depending on the number of interphase nuclei analyzed. By examining hundreds of cells, FISH can detect smaller subpopulations with specific chromosomal abnormalities, such as changes at the 20q11.21 region, which is commonly altered in hPSC cultures.

Following the ISSCR standards for routine genetic monitoring, using a combination of both karyotyping and FISH can provide a more comprehensive assessment of mosaicism, especially when targeting specific regions. This dual approach is beneficial in capturing both large- and small-scale chromosomal changes, offering a fuller picture of your culture’s genetic stability.

For more details on detecting mosaicism in your samples, or if you have specific questions about assay sensitivity, please contact our Product and Scientific Support Team at techsupport@stemcell.com.

In karyotyping, abnormalities are classified as clonal or non-clonal to help determine whether a genetic change is stable within a cell culture. Clonal abnormalities are defined as changes that appear in two or more cells, indicating that the abnormality is present in a population of cells that share the same genetic change. This suggests the change is a consistent feature of that cell population, likely due to selective growth advantages or other factors leading to its expansion over time. If clonal abnormalities, i.e. mosaicism, are detected, the proportion of abnormal to normal cells is specified in the report. For example, a result might read “46,XX[18]/47,XX,+12[2],” indicating that 18 cells were normal while 2 cells displayed trisomy 12, a mosaic pattern of abnormality.

Non-clonal abnormalities, by contrast, are those observed in only a single cell. These are typically regarded as isolated occurrences, which may result from technical artifacts during sample preparation rather than a stable or proliferative feature of the culture. While non-clonal abnormalities are included in reports to provide a complete record of all findings, they are usually not considered representative of the genetic profile of the culture as a whole.

Clonal and non-clonal distinctions are reported according to ISCN (2024) guidelines, providing clarity on the stability and significance of each observed abnormality in the sample.

Yes, ���ϳԹ�’s karyotype and FISH services align with the ISSCR Standards for Human Stem Cell Use in Research (2023), which emphasize the necessity of routine genetic monitoring for hPSC cultures. The ISSCR standards underscore that culture-acquired genetic changes can affect critical properties of hPSCs, such as growth rate, differentiation potential, functionality, and tumorigenicity, ultimately impacting the reproducibility and reliability of research data (ISSCR Recommendation 3.1.1).

Our G-banded karyotype service is a well-established method for detecting large-scale chromosomal abnormalities, such as aneuploidies and structural changes larger than 5 Mb. In addition, our 20q11.21 (BCL2L1) FISH assay targets specific sub-chromosomal changes that frequently occur in hPSC cultures but may be undetectable by karyotyping alone. This high-resolution FISH analysis provides added sensitivity for commonly acquired abnormalities in the 20q11.21 region, helping researchers monitor genetic stability in line with ISSCR guidance, which suggests refined assays for frequently observed variants (ISSCR Recommendation 3.2.1).

Because no single assay can detect all possible genetic changes, the ISSCR recommends using complementary methods with different detection limits to ensure a thorough assessment of genetic stability. Regularly reviewing and updating testing methods as best practices evolve is also encouraged (ISSCR Appendix 5). By combining karyotyping and FISH, you gain a comprehensive view of both large and subtle chromosomal changes in your hPSC cultures, which enhances the monitoring strategy across the experimental timeline (ISSCR Recommendation 3.2.2).

In addition to karyotyping and FISH analysis services, ���ϳԹ� offers an hPSC Genetic Analysis Kit for detecting common karyotypic abnormalities in your own lab.

Each method offers distinct advantages and is suited for specific applications in the genetic monitoring of hPSC cultures.

We may be able to accommodate requests for FISH services targeting custom genomic regions; however, these are assessed on a case-by-case basis. Custom FISH projects involve sourcing specific probes, and costs and turnaround times can vary depending on probe availability and the complexity of the request.

If you are interested in exploring a custom FISH assay, please contact us at iPSCservices@stemcell.com with the details of your project. Our team will review your requirements, evaluate feasibility, and provide guidance on the next steps.