iPSCs are mature adult cells, such as skin or blood cells, that have been reprogrammed back into a stem‑cell‑like state, allowing them to develop into almost any cell type. Since iPSCs can be generated in large numbers, tailored to individual patients, and genetically modified, they are a promising foundation for cell therapies.
But before iPSC‑derived cells can be used safely in patients, the starting iPSC material itself must undergo rigorous quality control. Quality control (QC) assays are standardised tests used to confirm that cells have the correct identity, remain genetically stable, and behave as expected before they are used as a foundation for downstream differentiation and manufacturing.
Inconsistent or poorly validated QC methods at this early stage can introduce variability that carries through the entire production process, undermining reliability and increasing risks in clinical translation.
“iPSC‑based therapies will only reach their full potential if we can trust the quality of the cells, regardless of where and how they are produced,” says Professor Anna Falk, Director of the LU‑ATMP Centre and head of the Neural Stem Cells research group at Lund University’s Lund Stem Cell Center.
Fragmented workflows across the world
Today, even the most basic quality control assays are often performed using different protocols, reagents, markers, and analysis strategies. This makes it difficult to compare results between laboratories or ensure consistent standards across regions.
“To make iPSC‑based therapies a truly global option, we need quality controls that are not only robust, but also feasible and reproducible across countries and laboratories,” says Alice Hägg, first author of the study and doctoral student in the Neural Stem Cells research group. “At the moment, many quality control workflows are fragmented and region‑specific. That lack of standardisation makes them expensive and difficult to implement beyond a small number of well‑resourced centres.”
To address this, the first large-scale international effort was launched to systematically evaluate how reproducible two fundamental QC assays are when performed in different laboratories: Flow cytometry–based assessment of the undifferentiated (pluripotent) cell state, and qPCR‑based analysis of genomic integrity.
An unprecedented international benchmarking effort
The study was conducted within the framework of the Global Alliance of iPSC Therapies (GAiT), with coordination support from the Scottish National Blood Transfusion Service and in collaboration with the CiRA Foundation, National Institute for Biological Standards and Control, and Stem Cell Technologies.
More than 80 researchers from 23 institutes across 12 countries and five continents participated by responding to an open call. Two quality assessment rounds were carried out in 2019 and 2023, followed by a validation round in 2024.
In the first round, all participating laboratories received identical cell samples but analysed them using their own in‑house flow cytometry protocols. Although the approaches used were similar, the results showed a lot of variation.
“We were surprised by how large the differences were,” says Alice Hägg. “Even among experienced labs, the flow cytometry results were all over the place. This underscores how small local protocol differences can translate into major reproducibility gaps.”
In contrast, the genomic integrity assay, performed using a standardised kit and protocol, showed much higher consistency across sites, highlighting the impact of harmonised workflows.
Standardisation reveals which markers are truly reliable
In the second assessment round, the researchers introduced fully standardized flow cytometry protocols, and analysis criteria, including predefined go/no‑go thresholds. This significantly reduced inter‑laboratory variability and enabled systematic comparison of pluripotency markers.
Using this approach, the team identified a small set of markers that consistently performed well across laboratories. Interestingly, one marker, SSEA‑5, which is rarely used in routine workflows, proved to be one of the most robust markers. While one of the most used markers in the field, SSEA‑4, showed poor performance as a discriminator of undifferentiated cells, as it remained expressed even during early differentiation.
“This was an important and somewhat unexpected finding, which we followed up in our validation round,” says Alice Hägg. “Many labs rely on SSEA‑4 for iPSC quality control, but our data show that it can mask partially differentiated cells.”
Toward data‑driven global standards
Beyond identifying individual markers, the study provides a roadmap for how international quality control benchmarking studies can be designed and implemented. The authors emphasize the importance of data‑driven decision‑making, clear analysis thresholds, and cross‑site validation.
“This work shifts the focus from expert opinion to empirical evidence,” says Alice Hägg. “It shows that we can define standards based on large‑scale data rather than tradition or convenience.”
Anna Falk adds that harmonised QC protocols could also accelerate equitable global access to iPSC‑based therapies: “Using validated, standardised quality controls, reduces the need to re‑validate methods in every new setting. That saves time, reduces costs, and ultimately benefits patients.”
Although no further assessment rounds are currently planned, the team strongly encourages the field to expand this type of benchmarking to additional QC assays and to engage regulatory bodies and international organizations in the next steps.
“Our study shows that this kind of global collaboration is possible,” says Alice Hägg.
