Characterization of human induced pluripotent stems cells: Current approaches, challenges, and future solutions

Abstract

Human induced pluripotent stem cells (iPSC) have demonstrated massive potentials for use in regenerative and personalized medicine due to their ability to expand in culture and differentiate into specialized cells with therapeutic benefits. However, in order to industrialize iPSC-derived therapies, it is necessary to address the existing challenges surrounding the analytics implemented in the manufacturing process to evaluate and monitor cell expansion, differentiation, and quality of the final products. Here, we review some of the key analytical methods used as part of identity, potency, or safety for in-process or final product release testing and highlighted the challenges and potential solutions for consideration in the Chemistry, Manufacturing and Controls (CMC) strategy for iPSC-based therapies. Some of the challenges associated with characterization and testing of iPSC-based products are related to the choice of analytical technology (to ensure fit-for-purpose), assay reliability and robustness. Automation of analytical methods may be required to reduce hands on time, and improve reliability of the methods through reducing assay variability. Indeed, we have shown that automation of analytical methods is feasible (evaluated using an ELISA based assay) and would result in more precise measurements (demonstrated by lower co-efficient of Variation and standard deviation), less hands-on time, and swift compared to a manually run assay. Therefore, in order to support commercialization of iPSC-based therapies we suggest a well-designed testing strategy to be established in the development phase while incorporating robust, reproducible, reliable, and potentially automated analytics in the manufacturing process.

Keywords: Analytical methods; Assay robustness; Automation; Cell therapies; iPSCs.

Graphical abstract

Fig. 1

Common Release and Characterization Assays: depicts the assays used for (A) sterility and safety includes sterility, mycoplasma, endotoxin, reprogramming clearance and karyotype testing. (B) identity and purity includes flow cytometry and alkaline phosphatase testing. (C) safety use includes telomere analysis, HLA characterization and congenital disease markers. (D) content release testing of iPSC banks includes cell count and viability assay.

Fig. 2

(A) Co-efficient of Variation (%CV) represented in Box plot as interquartile range is lower for the Automation (Tecan) compared with manual load. (B) Absorbance reading is comparable between the Automation and Manual runs while the standard deviation in the manual run is higher compared to automation runs. Statistical method t-test was used to calculate the p value (*p values - Standard 1: 0.418, Standard 2: 0.599, Standard 3: 0.105, Standard 4: 0.017, Standard 5: 0.818, Standard 6: 0.035).

Fig. 3

(A) Illustration of iPSC cell bank generation from CD34+ cells and differentiation into cardiomyocytes, T-cells and NK-cells. (B) Proposed Release assays from the generated iPSC bank includes identity and purity by flow cytometry assay, CCV using NC-200 and ViCell, Reprogramming clearance using ddPCR and qPCR, Master Cell Bank Viral Testing using FPERT and QPERT. (C) Proposed Characterization assays includes conventional Karyotyping, Histology, ALP Staining, Alternative methods such as Pluritest and scorecard analysis, Telomere analysis as a potential test and novel assays devoted to iPSC manufacturing.