Genomic stability control: arguments to justify the costs

Most academic laboratories have to operate within a tightly controlled budget. This implies setting priorities, and unfortunately sometimes cutting corners. As a research scientist working on human pluripotent stem cells, you may find for instance that funds are not always available to perform thorough quality controls on your cell lines. Most of you wish things could be different, particularly when you understand the importance of regular testing for the validity of your research work. It can also be challenging to find the right arguments to convince those who hold the purse strings.
This is why we have come up with 4 solid arguments for you to use when trying to pitch for more budget to support this most important step in your cell culture.

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1. Let’s be clear: not testing for genomic stability on hPSCs is a serious gamble

Human Pluripotent Stem Cells are subject to genomic instability during their time in culture. That’s a well-documented fact. You’ll find many studies on the topic and we have shortlisted a few that you can refer to (Andrews, 2021a) (Assou et al., 2020) (Avery et al., 2013).

Our field experience corroborates the facts in the studies: since the start of our company in 2018, we have determined that 28% of our clients’ cell lines presented recurrent abnormalities.

What does it mean to ignore those abnormalities?

Unlike somatic cells, genomic alterations in hPSCs can affect their behavior and compromise research work.

• Amongst the most frequently observed effects, signs of neoplastic progression have been noted, including reduced apoptosis, growth-factor independence and higher cloning efficiency (Baker et al., 2016a).

• They are also known to affect differentiation potential. For example, a culture-adapted H7 line displayed a reduced tendency for differentiation to endoderm (Fazeli et al., 2011). Similarly, variant cells with a gain of chromosome 20q11.1-11.2 showed differences in hematopoietic and neural differentiation propensity compared with their wild-type controls (Werbowetski-Ogilvie et al., 2009). Altered patterns of differentiation caused by accrued genetic changes may significantly affect the use of such cell lines in applications that require the production of differentiated derivatives. Commonly observed genetic changes in hPSCs are also frequently observed in embryonal carcinoma cells, the stem cells of malignant germ cell tumors termed teratocarcinomas (Harrison et al., 2007). Gain of chromosome 12p is even used as a diagnostic marker for testicular germ cell tumors. With hPSCs derivatives entering clinical trials, a possibility that genetic changes may confer malignant properties to hPSCs or their differentiated progeny is a cause of regulatory concern (Goldring et al., 2011).

Still want to take your chances?

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2. Testing with one G-Banding karyotype just for publishing is not enough

We often hear that testing for genomic stability happens just before submitting a piece of research for publication, usually by G-Banding analysis.

This won’t be robust enough for two main reasons:

1. Abnormalities in hPSCs in culture appear very early on and can become predominant in a culture within 5 passages (Andrews, 2021b; Assou et al., 2020; McIntire et al., 2020). In its 2011 study, the International Stem Cell Initiative observed that none of the abnormalities found in early passages disappeared over time, reflecting the selective advantage of the acquired abnormalities. Waiting for the end of your research work is therefore not a productive strategy as it ends up wasting resources and time. It is also a motivation killer for the team who has spent months working on a cell line…

2. G-Banding has a resolution of 5 Mb, which is not sufficient when it comes to identifying the most recurrent defects in hPSCs. Case in point: the gain of chromosome 20q.11.21. It is smaller than 5 Mb and therefore will fall beyond the limit of detection of G-Banding. It occurs in 20% of lines, and in many cases with no overt karyotyping changes (Amps et al. 2011). At Stem Genomics we take it very seriously, as we have detected it in 50% of the anomalies found by our platform. This is detected with a digital PCR specific hPSC assay, our iCS-digital™ PSC test, available as a service and as a kit, that presents a greater resolution than G-Banding at 200 bp.

We recommend regular testing throughout your workflow to pick up abnormalities early in the process and to ensure the stability of your cell line during its time in culture.

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Genomic stability control does not have to break the bank

3. Testing for genomic stability does not have to break the bank

Of course, a best-practice, comprehensive genomic stability testing workflow is not for every laboratory. For some, just performing one G-Banding during the whole time the cells are in culture is already stretching the budget to its limits. Our client denovoMATRIX recommends our iCS-digital™ PSC test as “an easy entry to quality control for anyone with a limited budget”. You will not have the exhaustive analysis that G-Banding can provide with structural and numerical variant analyses, but you will confidently detect the most recurrent abnormalities that can be found in hPSCs. At a fraction of the cost! This combined with the fast turn-around time enables our clients to test regularly.

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4. Setting up a genomic quality control does not have to be a hassle

Some tests are harder to set up than others. G-Banding requires stringent cell preparation that often fails to deliver the expected number of metaphases. SNP array requires a minimum number of samples before tests are run. WGS or NGS are just very expensive options and although extremely comprehensive, are not suited for regular in-process testing. A digital PCR-based assay such as iCS-digital™ PSC on the other hand offers a hassle-free solution.

Our clients like it for the following 3 main reasons:

1. The convenience of sample preparation with 3 sampling options:

• Genomic DNA sent at room temperature
• Cells in culture medium sent at room temperature
• Cell pellets sent on dry ice

2. Fast and clearly interpreted results

Once the test is performed, the results are available as a straightforward graphical interpretation that gives confidence in the outcome of the sample analyzed. This will be done and dusted within 3 days maximum of reception of the samples! According to our long-lasting client Ncardia, “the results are obtained very quickly; the reports are clearly interpreted and there is a smooth line of communication between us and Stem Genomics”.

3. No minimum number of samples

To top it off, we don’t need you to send a minimum number of samples before we start the work. We can start with just 1 sample!

 

Ready to try it out? Let’s talk!

 

Amps, K., Andrews, P. W., Anyfantis, G., Armstrong, L., Avery, S., Baharvand, H., Baker, J., Baker, D., Munoz, M. B., Beil, S., Benvenisty, N., Ben-Yosef, D., Biancotti, J. C., Bosman, A., Brena, R. M., Brison, D., Caisander, G., Camarasa, M. v., Chen, J., … Zhou, Q. (2011). Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage. Nature Biotechnology, 29(12), 1132–1144. https://doi.org/10.1038/nbt.2051
Andrews, P. W. (2021a). Human pluripotent stem cells: Genetic instability or stability? In Regenerative Medicine (Vol. 16, Issue 2, pp. 113–115). Future Medicine Ltd. https://doi.org/10.2217/rme-2021-0013
Andrews, P. W. (2021b). Human pluripotent stem cells: Genetic instability or stability? In Regenerative Medicine (Vol. 16, Issue 2, pp. 113–115). Future Medicine Ltd. https://doi.org/10.2217/rme-2021-0013
Assou, S., Girault, N., Plinet, M., Bouckenheimer, J., Sansac, C., Combe, M., Mianné, J., Bourguignon, C., Fieldes, M., Ahmed, E., Commes, T., Boureux, A., Lemaître, J. M., & de Vos, J. (2020). Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR. Stem Cell Reports, 14(1), 1–8. https://doi.org/10.1016/j.stemcr.2019.12.004
Avery, S., Hirst, A. J., Baker, D., Lim, C. Y., Alagaratnam, S., Skotheim, R. I., Lothe, R. A., Pera, M. F., Colman, A., Robson, P., Andrews, P. W., & Knowles, B. B. (2013). BCL-XL mediates the strong selective advantage of a 20q11.21 amplification commonly found in human embryonic stem cell cultures. Stem Cell Reports, 1(5), 379–386. https://doi.org/10.1016/j.stemcr.2013.10.005
Baker, D., Hirst, A. J., Gokhale, P. J., Juarez, M. A., Williams, S., Wheeler, M., Bean, K., Allison, T. F., Moore, H. D., Andrews, P. W., & Barbaric, I. (2016a). Stem Cell Reports Resource Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells. https://doi.org/10.1016/j.stemcr.2016.10.003
Baker, D., Hirst, A. J., Gokhale, P. J., Juarez, M. A., Williams, S., Wheeler, M., Bean, K., Allison, T. F., Moore, H. D., Andrews, P. W., & Barbaric, I. (2016b). Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells. Stem Cell Reports, 7(5), 998–1012. https://doi.org/10.1016/j.stemcr.2016.10.003
Fazeli, A., Liew, C. G., Matin, M. M., Elliott, S., Jeanmeure, L. F. C., Wright, P. C., Moore, H., & Andrews, P. W. (2011). Altered patterns of differentiation in karyotypically abnormal human embryonic stem cells. International Journal of Developmental Biology, 55(2), 175–180. https://doi.org/10.1387/ijdb.103177af
Goldring, C. E. P., Duffy, P. A., Benvenisty, N., Andrews, P. W., Ben-David, U., Eakins, R., French, N., Hanley, N. A., Kelly, L., Kitteringham, N. R., Kurth, J., Ladenheim, D., Laverty, H., McBlane, J., Narayanan, G., Patel, S., Reinhardt, J., Rossi, A., Sharpe, M., & Park, B. K. (2011). Assessing the Safety of Stem Cell Therapeutics. Cell Stem Cell, 8(6), 618–628. https://doi.org/10.1016/J.STEM.2011.05.012
Harrison, N. J., Baker, D., & Andrews, P. W. (2007). Culture adaptation of embryonic stem cells echoes germ cell malignancy. International Journal of Andrology, 30(4), 275–281. https://doi.org/10.1111/j.1365-2605.2007.00762.x
McIntire, E., Taapken, S., Leonhard, K., & Larson, A. L. (2020). Genomic Stability Testing of Pluripotent Stem Cells. Current Protocols in Stem Cell Biology, 52(1). https://doi.org/10.1002/cpsc.107
Werbowetski-Ogilvie, T. E., Bossé, M., Stewart, M., Schnerch, A., Ramos-Mejia, V., Rouleau, A., Wynder, T., Smith, M. J., Dingwall, S., Carter, T., Williams, C., Harris, C., Dolling, J., Wynder, C., Boreham, D., & Bhatia, M. (2009). Characterization of human embryonic stem cells with features of neoplastic progression. Nature Biotechnology, 27(1), 91–97. https://doi.org/10.1038/nbt.1516

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