While extremely interesting, it is also uncomfortable to think about the genetic stability of cultured induced pluripotent stem cells (iPSCs). Our team members Fabian Zanella and Raluca Marcu discussed this topic in a recent publication, and we are expanding some of those insights and highlighting how the approaches and technologies available at Pluristyx can help.
There’s an abundance of evidence demonstrating that as cells are continuously propagated, it’s not a matter of if, but rather a matter of when genetic abnormalities will develop. Thus, this topic has generated nervousness within initiatives to bring PSC-derived cell therapies to the clinic. Our general understanding is that those genetic changes occur stochastically. However, selective pressures of cell culture can favor genetic alterations that confer growth advantages to the affected cells, allowing those affected cells to take over a culture within a few expansion cycles. In the context of cell therapy, if altered cells are placed in a niche that favors their expansion, an uncontrolled and potentially malignant outgrowth could arise, leading to risk for the patients receiving that therapy.
Thankfully, much work has been carried out to understand, minimize, and mitigate the occurrence of those genetic occurrences, as well as work towards the elimination of potentially dangerous cells that could put patients in jeopardy. At Pluristyx, we are very attuned to this topic and here are some of our views:
- Beginning with the starting material, donors must be carefully selected. Age, gender and blood type may be consequential for the cell therapy under development. The bodily location where donor cells are collected may also play a role in downstream genetic integrity. There is evidence that skin fibroblasts more exposed to UV radiation tend to have higher propensity to present TP53 mutations. Thus, Pluristyx favors the collection of skin fibroblasts from less sun-exposed areas, and adoption of alternative sources such as mesenchymal stem cells, which due to their internal location are more protected from UV radiation. Once the donor material is established, robust screening of donor cells, including NGS-based probing for genetic abnormalities, is key to establish confidence on the genomic integrity of that starting material.
- Once an appropriate donor material is selected, cell reprogramming and initial banking must be conducted under conditions that minimize cell stress to enable a healthy and stable starting population of pluripotent stem cells. Plursityx has developed robust cell expansion protocols that ensure cell health and can be efficiently scaled up to generate a sufficient number of iPSCs for downstream product development. That includes leveraging appropriate small molecules that facilitate cell survival in conditions of anoikis, such as inhibitors of the Rho-associated, coiled-coil containing protein kinase (ROCK), a popular strategy in human pluripotent stem cell culture. This approach may reduce the selective pressure towards cells that developed genetic alterations to enable their survival under those conditions. On the other hand, it has been proposed that supplementation of cell culture medium with nucleosides to meet the high DNA synthesis demands of a rapidly-dividing culture can mitigate the occurrence and propagation of DNA alterations. Pluristyx has devised careful strategies with stage-appropriate genetic integrity checkpoints during cell expansion and after banking to be able to detect eventual genetic abnormalities as soon as possible in our processes, building confidence towards the final cells delivered to our partners and clients. As a first layer of analysis, our strategy encompasses an usage test, where banked cells are thawed and cultivated for 4 passages, and re-tested to verify the preservation of genetic integrity at the later passage point. Examples of those are analysis of copy-number variations (CNVs) across the genome and probing for the amplification of specific genes observed to enable selective advantage to growing PSCs, such as BCL2L1.
- Finally, if DNA alterations are detected and cannot be circumvented, or if they are developed after delivery of the cell therapy, a final layer of control, harnessing molecular safety switches can be used. Technologies like the cell cycle-linked FailSafe™ mechanism available at Pluristyx involve an inducible cell suicide switch, which can be efficiently activated with the pro-drug Ganciclovir, leading to the removal of target cells.