Genomic stability is one of the primary concerns for research scientists working on human pluripotent stem cells. Although these cells have amazing capabilities, they are prone to developing chromosomal abnormalities during their time in culture. This can also be the case after critical bottleneck events such as gene editing, basically every time the cells experience replicative stress imposed by in-vitro culture.
Recurrent genomic alterations, essentially copy number variations (CNVs), often affect the same genomic regions, such as 1q, 12p, 17q, 20q and the X chromosome. This provides the affected cells with a selective advantage which may favor proliferation, cell survival or reduce cell differentiation capacities.
Gain of 20q11.21 copy-number variant (CNV) is detected in more than 20% of human Pluripotent Stem Cells (hPSCs) cultured worldwide and represents 22.9% of the recurrent structural variants identified in hPSCs (Assou et al., 2020; Avery et al., 2013; Halliwell et al., 2020). This makes it the most common genomic abnormality in hPSCs. Unfortunately, it cannot always be detected by conventional methods.
Concurrently, as cell therapy evolves and an increasing number of studies reach the clinical stage, demand increases for more exhaustive analyses of copy number variations (CNVs) across the entire genome. With the advent of cutting-edge technologies such as next-generation sequencing (NGS), the ability to detect novel abnormalities has greatly improved. Many recurrent CNV regions previously described using these less sensitive techniques are now being re-evaluated and redefined with greater accuracy using NGS-based approaches. This improved sensitivity and resolution in CNV analysis is making a significant contribution to our understanding of genomic variation and its potential impact in various clinical and research settings.
Beyond CNV detection, it is also critical to look for molecular abnormalities and Single Nucleotide Variations in hPSCs to properly check your cells’ genomic stability. Single Nucleotide Variations (SNVs) are other genomic defects that need identifying, as they represent the most abundant type of genetic variations in human. Case in point, they caused the suspension of the first human trial using hPSCs at the RIKEN Institute in Japan when they were identified, alongside CNVs.
Several studies using WGS (Whole Genome Sequencing) have identified SNVs associated with cancer and other diseases that can compromise the proper development of cells in culture (Merkle et al. 2022).
In the case of human Pluripotent Stem Cells, the abnormalities that arise will persist following differentiation. Whether you differentiate your cells or not, this can invalidate your research, interfere with studies that use iPSC cells for disease modelling or drug screening or present substantial risks for patient health in clinical trials.
Finally, the other important factor to take into account when working on hPSCs is the speed at which those alterations can overcome the whole culture (Hastings et al., 2009). It can happen in 5 passages or less (Assou et al., 2020; McIntire et al., 2020; Pamies et al., 2017). It is therefore essential to perform tests routinely, and at early stages.
It is therefore essential for researchers to undertake regular in-process controls to identify recurrent (or frequent) CNVs (such as 20q gain) and to consider sequencing to detect SNV abnormalities (such as TP53 mutations). These combined methods will help scientists secure their hPSCs cultures.
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Based on our extensive experience working with hPSC research scientists, we recommend regular in- process testing at different sensitive stages in the workflow. Click on the graph for more details:
Pluripotent stem cells (PSCs). Cells capable of maintaining an undifferentiated state indefinitely and that give rise to all cells in the body.
Induced Pluripotent Stem Cells (iPSCs). A type of pluripotent stem cell derived from adult somatic cells and reprogrammed to an embryonic stem (ES) cell-like state by chemical or genetic reprogramming, commonly by inducing the expression of the transcription factors OCT4, SOX2, KLF4 and MYC (OSKM).
1981: Martin Evans of Cardiff University, UK, then at the University of Cambridge, is first to identify embryonic stem cells in mice.
1997: From the “Dolly the sheep” experiment, the first artificial animal clone, Iam Wilmut and his colleagues at the Roslin Institute, Edinburgh imagine that creating genetically matched tissue and organs in humans would be possible.
1998: James Thomson of the University of Wisconsin in Madison and John Gearhart of Johns Hopkins University in Baltimore isolate human embryonic stem cells and grow them in the lab.
2006: Shinya Yamanaka developed a revolutionary method for generating stem cells from existing cells of the body: inserting specific genes into the nuclei of adult cells, a process that results in the reversion of cells from an adult state to a pluripotent state.
2012: Nobel prize awarded to Shinya Yamanaka and John B. Gurdon for the discovery that mature cells could be reprogrammed.
This discovery marked a turning point in stem cell research, because it offered a way of obtaining human stem cells without the controversial use of human embryos.
Since then, iPSCs have demonstrated extraordinary applications in the fields of disease modelling, drug screening and regenerative medicine.
In order to follow recommended guidelines by the ISSCR, a suitable test needs to be selected. Although pertinent at certain key stages of the workflow, G-Banding, the traditionally used technique for genomic stability testing, is not suitable for regular in-process assessment. It would slow processes down and be cost-prohibitive.
With these constraints in mind, Stem Genomics has developed a rapid and cost-effective digital PCR- based solution called iCS-digital™ PSC dedicated to recurrent CNV detection.
This assay combines a fast turnaround with high resolution, enabling the precise detection of sub-karyotypic defects such as the 20q11.21 chromosomal abnormality.
For a more comprehensive approach looking at both CNVs and SNVs, the NGS-based Stem-Seq™ range includes the Stem-Seq™ Panel for SNV detection and the Stem-Seq™ Plus for SNV and CNV detection over the whole genome. This powerful technology will allow the detection of SNVs in 361 targeted locations and CNVs across the genome that may not have been previously considered or that could harbor unexpected variations. This comprehensive and unbiased view adds significant information on the genomic stability of your cells.
G-Banding: the traditional and best-known technique for controlling genomic stability in human stem cells. More on G-Banding.
iCS-digital™ PSC range: Available in 24 probes, 12 probes or 20q only, as a service or a kit, this unique test enables up to 92% detection of recurrent genomic abnormalities in hPSCs. More on iCS-digital™ PSC.
Duo iCS-Karyo: a powerful combination of traditional G-Banding karyotyping and iCS-digital™ PSC for comprehensive detection of PSC abnormalities. Available as a service only. More on Duo iCS-Karyo.
The Stem-Seq™ range: a high-resolution, highly-sensitive, targeted NGS panel specifically designed for SNV and Indel detection combined with a pangenomic view of CNVs. This comprehensive test comes with various levels of reports and interpretations about the significant identified variants. More on the Stem-Seq™ range.
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