Which genomic assay when working
on human Pluripotent Stem Cells (hPSCs)

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.