PluriCon 2024
Postscript
We are thrilled to share a wrap up of the iPSC-targeted discussions and insights from PluriCon 2024, Pluristyx’s inaugural iPSC Innovation Symposium held in Seattle on September 10, 2024.
Translating Today’s Discoveries into Tomorrow’s Therapies: Observations and experiences in the complexities of moving academic iPSC research into the clinic.
Designing a Better Cell Therapy – Revealing cutting edge approaches to iPSC differentiation and scalability,
Testing for Success – Identifying strategies to identify iPSC genetic instability.
Culture Consistency – Understanding variability in iPSC cell culture to ensure developmental success.
Regulatory Challenges and Opportunities – Navigating the global regulatory environment of highly complex iPSC-based therapies.
From Lab to Clinic – A case study Advancing Parkinson’s Disease Therapy.
The Last Mile – Identifying the critical role cryopreservation plays in iPSC genetic stability and commercialization.
The Last Mile: Cryopreservation Challenges for iPSC Products
Dr. Brian Hawkins
Chief Technology Officer, Pluristyx
Cryopreservation is an often overlooked, but absolutely critical, component of development that can make or break the success of a promising iPSC-derived therapy, from research through large-scale clinical manufacturing.
Key insights from Dr. Hawkins’ talk included:
- Learn from the past – Cryopreservation, especially when performed improperly, clearly impacts iPSC genetic stability. Dr. Hawkins utilized real-world examples along with ideas on how to leverage yesterday’s experiences in CAR T-cell therapies for Tomorrow’s iPSC breakthroughs.
- Cryopreservation is more than just freezing – The science behind cell cryopreservation is complex and a continuum. What happens both BEFORE and AFTER the freezing event are the key to preventing cell damage and improving post-thaw function.
- Customization is key – Groups should not rely simply on historical protocols, but rather identify all the critical process parameters, collect the relevant data, and then implement proper controls to enable consistent cryopreservation outcomes. This optimization should occur early in development to ensure iPSCs remain genetically stable and can be transformed into breakthrough living medicines.
- Always consider the clinic – The goal of any cell therapy research should be the patient. Proper consideration should be given to cryopreservation that can easily transition to the clinic with minimal impact on cryopreservation efficacy.
- Go Big – Proper consideration should be given to cryopreservation processes that can follow iPSC development from laboratory to industrial scale. Procrastinating scale up until the last minute is a recipe for disaster for complex and expensive iPSC therapies.
Dr. Hawkins' presentation was a timely reminder that cryopreservation is essential for iPSC therapies and should be treated as a continuous process, ensuring scalability and effectiveness of Tomorrow’s Cell Therapies, Today®.
Safeguarding Genomic Stability in iPSC Line Development
Dr. Raluca Marcu
Director of Cell Line Development, Pluristyx
Dr. Marcu, Director of Cell Line Development at Pluristyx, discussed the challenges and solutions involved in maintaining genomic stability during the development of unmodified and engineered pluripotent stem cell lines. Her talk focused on identifying sources of genomic instability, improving testing methods, and optimizing workflows to create stable, genetically robust iPSC lines suitable for clinical applications.
Key insights from Dr. Marcu’s talk included:
- Sources of Genomic Instability in iPSCs – Genomic instability can arise from mutations that pre-exist in the donor material or are acquired during iPSC derivation and expansion, particularly at population bottlenecks that introduce stress on the genome (reprogramming, gene editing, cloning). Mutations presented in the form of DNA sequence changes and/or genome structural changes can result in altered gene expression and impact biological functions of iPSC.
- The Importance of Comprehensive Testing – Standard genomic stability tests like G-band karyotyping have limitations in specificity, resolution, and sensitivity. Dr. Marcu presented a case where G-band initially missed genomic instability in iPSC Seed Banks, leading to failures in later-stage Master Cell Banks. Recent ISSCR and FDA guidelines stress the need for detailed genomic characterization to detect mutations and mosaicism in iPSCs. Testing methods should be chosen based on detection capability, resolution, sensitivity, and timing of characterization. Advanced methods like digital PCR and targeted NGS now allow earlier issue detection, preventing costly downstream failures.
- Pluristyx’ Optimized Workflow – Pluristyx has developed an optimized iPSC derivation workflow to reduce culture-induced stress and enhance genomic stability. This is achieved by minimizing passage numbers, eliminating bottlenecks, standardizing culture protocols and integrating enhanced in-process and bank testing. Utilizing efficient mRNA-based reprogramming and bulk iPSC passaging, this workflow includes gene editing of polyclonal pools to maintain genetic and epigenetic diversity, with cloning deferred until later stages. A stress test on iPSC Seed Banks is conducted to evaluate the long-term genomic stability of iPSCs under anticipated usage conditions.
Dr. Marcu concluded by emphasizing the need for rigorous, multi-step testing and process optimization to generate genetically stable iPSC lines, which are critical for clinical success in cell therapy development.
An integrated paradigm for robust expansion, cryopreservation and recovery of iPSCs
Dr. Fabian Zanella
Process & Analytical Development Director, Pluristyx
Dr. Zanella’s talk focuses on the importance of harmonization of iPSC culture methods across different labs and industries. However, due to the multitudes of reagents and growth methods, there is significant fragmentation in iPSC growth protocols. This issue is further complicated by needing to find alternatives for components that are not fully GMP compliant.
He noted that while achieving full standardization may be difficult, developing specific paradigms for certain applications could improve reproducibility and efficiency in iPSC research and clinical production.
Dr. Zanella shared his work on optimizing iPSC expansion, cryopreservation, and recovery, ensuring robust growth and preserving genetic integrity and pluripotency. His team developed a controlled, cost-effective paradigm to minimize variability and stressors, resulting in a reproducible culture protocol tailored for Pluristyx’s PluriBank™ RUO Failsafe® iPSC lines.
Experiment Design
To establish a growth paradigm using reagents that support iPSC pluripotency, expansion, cryopreservation and recovery, while having a clear path towards clinical applications, two commercially available iPSC lines were tested under two conditions: daily media changes and a "skip-day" paradigm (media changes every other day). Over several passages, viability and growth metrics were tracked. At the final passage, cells were cryopreserved and later thawed to assess recovery, viability, pluripotency, and maintenance of genetic integrity.
A Controlled and reproducible iPSC Growth Paradigm with a path to the clinic.
Both paradigms achieved success criteria, including ≥8-fold growth in 4 days, 18–24h doubling time, and ≥95% viability at harvest. Post-cryopreservation viability exceeded 85%, with preserved pluripotency and maintenance of genetic integrity. Furthermore, the skip-day method matched daily feeding, offering a less labor-intensive alternative.
The final result is a tailored culture paradigm that included:
- Laminin-521 as cell culture substrate
- HiDef-B8 Medium (a modified version of Essential 8) as PSC medium
- PBS-EDTA as cell dissociation reagent for clump passaging
- CEPT Cocktail a blend of pro-survival molecules to aid in recovery
- PluriFreeze 10 a cryopreservation reagent developed specifically for PSCs
iPSC-Based Therapies and Beyond
Dr. Jeanne Loring
Co-founder of Aspen Neuroscience
Dr. Loring highlighted the development of Aspen Neurosciences’ autologous dopamine neuron replacement therapy for Parkinson’s disease, which has reached Phase 1/2 clinical trials.
- Securing Funding: Starting with grassroots efforts, she built the foundation for millions in support from CIRM, NIH, and venture capitalists. Dr. Loring emphasized the importance of crafting a compelling narrative to secure funding and the long-term dedication needed to move therapies from research to clinical application.
- Freedom to Operate: She stressed the necessity of doing freedom to operate analysis and securing the right IP licenses, such as those for reprogramming factors.
- Ensuring Safety: Since stressors such as cell division or gene editing can lead to loss of genomic integrity in the iPSCs and compromised safety for the patient, Dr. Loring stressed the importance of frequent genomic testing to ensure that therapeutic cells remain safe. Her team employed whole genome sequencing, SNP genotyping, and other bioinformatics assays such as PluriTest and NeuriTest to ensure the safety, maturity and authenticity of the transplanted cells. She also encourages the continued development of new analysis tools such as optical genome mapping, to meet the growing need in the field for higher resolution genomic characterization tools.
Stem cells in space
Outside of therapeutic applications of iPSCs, Dr. Loring’s team is studying the effect of microgravity on brain cells related to Parkinson’s and multiple sclerosis by sending iPSC-derived neural organoids into space. Remarkably, the cells were found to have accelerated maturation in space, prompting further research into why microgravity accelerates cell development. You can read more about the findings here.
Rhino Conservation
Dr. Loring’s team has also been participating in the conservation of the northern white rhino, a functionally extinct species. Her team successfully reprogrammed frozen fibroblasts from the remaining rhinos into iPSCs, with the ultimate goal of using these cells to help restore the species.
Stem cells hold the key to healing.
And longevity.
A bright future lies ahead.
-haiku from ChatGPT
Contact us for copies of slides from these talks
(where available)