3 burning questions about stem cell sequencing

We are launching a (quarterly) e-newsletter focused on the hot topic of QC for stem cell scientists. Read about key opinion leaders’ views looking at the various challenges they face from different angles. Let’s get the conversation going!

In this edition, 3 questions answered by our R&D Director, Dr. Reda ZENAGUI, and Founder and Scientific Advisor, Dr. Said ASSOU.

Should we choose a targeted or a wide-genome approach?

Dr. ZENAGUI: Choosing between a targeted approach and a whole-genome approach depends on several factors, including the specific goals of your study.

In theory, the ideal scientific approach could lean towards a wide-genome approach, to achieve the most exhaustive screening possible. The issue there is that science doesn’t have all the answers yet to explain the impact of certain identified variants. This leaves scientists working in the field hesitant when confronted with the analysis results. What should they do with their cell lines when a high number of non-defined variants comes up? Are they truly detrimental to the cell line and ultimately to the therapeutic product?

This is why we believe a targeted approach is best suited to work aimed at therapeutic applications. Chromosomal abnormalities such as the Copy Number Variation (CNV) 20q.11.21 and punctual mutations like the Single-Nucleotide Variant (SNV) TP53  have been well researched, and even though some are still not sufficiently documented (particularly in the case of SNVs), we can already confidently identify some abnormalities that should not be ignored.

The specificity of targeted approaches allows us to accurately assess the potential impact of variants in these regions of focus, making the data more manageable and the results more clinically relevant.

The specificity of targeted approaches allows us to accurately assess the potential impact of variants in these regions of focus, making the data more manageable and the results more clinically relevant.

Additionally, the interpretation of observed abnormalities in the targeted approach is more straightforward because we have many references to classify these variants and make informed decisions about our cell culture.

Dr. ASSOU: The choice between these approaches depends on the specific goals, resources, and the stage of development. Both techniques are interesting for scientists. Whole-genome assays are often used at the very final stage of a workflow. They provide a holistic view of the genome, enabling the detection of off-target effects, genetic stability, and unexpected mutations and offer additional safety before injection to the patient. The challenge is still the interpretation of the vast amount of data they generate. This is where the targeted approach has merits. It is generally less expensive than a whole-genome assay, with a faster turnaround time. It can either be used in-process or at key stages of a workflow (cell acquisition, bank characterization, end of workflow, etc.). With a targeted approach, the challenge is to find the right panel of key genes and critical markers designed for your cell type, of course!

Therefore, for a robust quality control, a combination of both approaches should ideally be used at different stages to ensure both comprehensive initial characterization and efficient ongoing monitoring.

A robust quality control will be a combination of both approaches ideally used at different stages to ensure both comprehensive initial characterization and efficient ongoing monitoring.

What should the depth of sequencing be?

Dr. ZENAGUI: Genomic abnormalities can occur at a very early stage (as soon as 5 passages) and can rapidly take over a pluripotent stem cell culture. Variants can also be very small in size (<5-10 Mb), and go undetected by conventional karyotyping methods. To detect the presence of these small variants as early as possible in the process, low mosaicism makes sense. Therefore, deep screening of genomic defects should be the answer to make your research more robust.

In my experience, deep sequencing can provide valuable insights into mosaicism within a cell culture by offering high-resolution data for both single nucleotide variants (SNVs) and copy number variants (CNVs). This increased depth allows for the detection of low-frequency variants and sub-clonal populations that may be missed with lower coverage, thereby giving a more comprehensive understanding of genetic heterogeneity and the potential impact on cell behavior and experimental outcomes. However, findings of variants with very low allele frequencies (around 1%) should be interpreted with caution, as the clinical and biological significance of such low-frequency variants is still not fully understood. It is a broad topic that we can discuss at length.

Findings of variants with very low allele frequencies (around 1%) should be interpreted with caution, as the clinical and biological significance of such low-frequency variants is still not fully understood.

To provide you with the best possible advice, we recommend that you surround yourself with professionals with a proven track record in understanding the true impact of the identified variants for your cell type.

Dr. ASSOU:  To detect low-frequency variants or in applications requiring very high sensitivity (e.g. detecting mosaicism or sub-clonal populations), 60x or higher coverage might be necessary. For targeted panels, coverage requirements can vary depending on the panel design and the genes of interest. 500-1000x coverage is often targeted to ensure accurate detection of low-frequency variants. The sequencing depth should be optimized based on the specific needs of your quality control process, balancing between sensitivity, specificity, and cost.

However, in my opinion, the field still lacks experience in that particular topic. The definite answer to this question will lie with scientists working in the field, sharing their findings with the rest of the community. Technologies are developing quickly to enable deeper sequencing, but we are still debating the sensitivity or mosaicism to ensure maximum safety from cell therapy. The challenge is to strike the right balance: we should aim for safety, but not to the point where scientists have their hands tied.

The challenge is to strike the right balance: we should aim for safety, but not to the point where scientists have their hands tied.

Regulatory bodies will have a role to play, but the way I see it, expertise and recommendations will come from the field, and from experts who do not have conflicts of interests.

After finding the SNVs or CNVs, what other orthogonal validations do you suggest for follow up?

Dr. ZENAGUI: I would provide a different answer depending on the identified variants. CNVs are well documented, and this helps in drawing rapid and robust conclusions based on scientific insights. I would add that if your detection method is sufficiently sensitive (less than 5 Mb), a second validation method might not be required.

With the exception of TP53 and BCOR, SNVs are less researched and can leave scientists wondering: should I discard the cell lines with SNVs that are not TP53 or BCOR? Or should I keep monitoring their progression to see how the frequency evolves? When in doubt, we recommend close monitoring. If the abnormal clone takes over, the cell culture is going to be impacted. We can observe unexpected behaviors (cell population developing too quickly, for example) or see the cells struggling to differentiate as expected. But sometimes the frequency remains stable and abnormalities can even disappear over time. This is often the case with MSCs (mesenchymal stromal cells).

In summary, when it comes to SNVs, a second validation method can be useful. It does not have to be molecular: a phenotypic observation of the cell line can serve as validation (e.g. morphologic observation).

CNVs are well documented, and this helps in drawing rapid and robust conclusions based on scientific insights... when it comes to SNVs, a second validation method can be useful.

At the end of the day, you don’t have to focus solely on which complementary technique to add for confirmation, as different methods can yield varying results. Instead, you should consider which result is the most accurate and trustworthy. The key is to utilize a test where the analysis is complete and interpreted beyond the bioinformatics stage. This comprehensive interpretation will provide you with robust information, allowing you to make confident decisions about the fate of your cell culture. 

Dr. ASSOU: Regarding genomic stability, digital PCR offers highly sensitive and absolute quantification of DNA molecules, making it suitable for detecting CNVs, rare SNVs and quantifying their frequency in a sample.  Sequencing individual cells can help confirm whether identified variants are present across the entire cell population or confined to subpopulations. Beyond genomic stability checks, I would strongly recommend testing for residual pluripotency genes before injecting a cell therapy product. Quite a few papers have been published on this aspect of QC (Fujikawa et al. 2005, Blum & Benvenisty 2008, Sui et al. 2013, Lee et al. 2009), as it may lead to tumor formation or to the growth of immature tissues. The choice of method will depend on the specific variant type, the available resources, and the context of your research or clinical application.

Several methods can be used depending on the specific variant type, the available resources, and the context of your research or clinical application.

Contributors:

Dr. Reda ZENAGUI

With a PhD in Molecular Genetics from the University of Montpellier, Dr. Reda Zenagui was a key contributor at the Montpellier Hospital for over a decade. He specializes in cutting-edge next-generation sequencing (NGS) techniques and bioinformatics analysis for targeted genetic studies. His expertise in variant interpretation ensures accurate patient diagnosis. In his current role as R&D Director at Stem Genomics, he continues his contribution to the genomic field by identifying molecular and chromosomal abnormalities, drawing on his extensive experience in testing the genomic integrity of stem cells.

Dr. Said ASSOU

Dr Said Assou is a specialist in stem cell genomic integrity. He works at the Institute for Regenerative Medicine and Biotherapy (IRMB), and he is the research manager of the group “Genomic instability of pluripotent stem cells”. For years, his work has been focused on the design and development of diagnostic tools to translate basic research findings into clinical applications for regenerative medicine, and particularly tools for the analysis of induced pluripotent stem cells in culture. His research has led to numerous scientific publications and several patents.