Using magnetic resonance relaxometry to evaluate the safety and quality of induced pluripotent stem cell-derived spinal cord progenitor cells.

Background: The emergence of induced pluripotent stem cells (iPSCs) offers a promising approach for replacing damaged neurons and glial cells, particularly in spinal cord injuries (SCI). Despite its merits, iPSC differentiation into spinal cord progenitor cells (SCPCs) is variable, necessitating reliable assessment of differentiation and validation of cell quality and safety. Phenotyping is often performed via label-based methods including immunofluorescent staining or flow cytometry analysis. These approaches are often expensive, laborious, time-consuming, destructive, and severely limits their use in large scale cell therapy manufacturing settings. On the other hand, cellular biophysical properties have demonstrated a strong correlation to cell state, quality and functionality and can be measured with ingenious label-free technologies in a rapid and non-destructive manner.
Method: In this study, we report the use of Magnetic Resonance Relaxometry (MRR), a rapid and label-free method that indicates iron levels based on its readout (T ₂ ). Briefly, we differentiated human iPSCs into SCPCs and compared key iPSC and SCPC cellular markers to their intracellular iron content (Fe ³⁺ ) at different stages of the differentiation process.
Results: With MRR, we found that intracellular iron of iPSCs and SCPCs were distinctively different allowing us to accurately reflect varying levels of residual undifferentiated iPSCs (i.e., OCT4 ⁺ cells) in any given population of SCPCs. MRR was also able to predict Day 10 SCPC OCT4 levels from Day 1 undifferentiated iPSC T ₂ values and identified poorly differentiated SCPCs with lower T ₂ , indicative of lower neural progenitor (SOX1) and stem cell (Nestin) marker expression levels. Lastly, MRR was able to provide predictive indications for the extent of differentiation to Day 28 spinal cord motor neurons (ISL-1/SMI-32) based on the T ₂ values of Day 10 SCPCs.
Conclusion: MRR measurements of iPSCs and SCPCs has clearly indicated its capabilities to identify and quantify key phenotypes of iPSCs and SCPCs for end-point validation of safety and quality parameters. Thus, our technology provides a rapid label-free method to determine critical quality attributes in iPSC-derived progenies and is ideally suited as a quality control tool in cell therapy manufacturing.
Competing Interests: Declarations. Ethics approval and consent to participate: This study utilized deidentified iPSC lines and does not involve animals, clinical experiments, or primary patient-derived human tissues. Derivation of the CLEC23 iPSC line had received prior IRB approval (document no.: IRB-2020-05-026, date of approval: 02/06/2020, name of institution: NTU). BJ-iPSCs were derived from the BJ (ATCC CRL-2522) cell line obtained from ATCC ( https://www.atcc.org/products/crl-2522 ) and does not require approvals for derivation as it does not involve the use of patient-derived material. NTU-IRB waived the need for ethical approvals regarding this study in accordance with relevant guidelines and regulations following the Singapore’s Human Tissue Framework under the Human Biomedical Research Act. In accordance to Singapore’s Human Tissue Framework, any form of human biological material which has been substantially manipulated and de-identified are no longer considered human material, and, therefore, ethical approvals or consent to participate does not apply. Consent for publication: Not applicable. Competing interests: J.H., S.Y.C., D.N.R, J.T. filed an intellectual property with a patent that is co-owned by SMART Singapore-MIT Alliance for Research and Technology and NTU. The other authors declared no potential conflicts of interest.
(© 2024. The Author(s).)