Analytical Methods and Testing for iPSC
Published on: July 03, 2024
Induced pluripotent stem cells (iPSC) are a type of cells like embryonic stem cells (ESCs), which can be generated by reprogramming of differentiated somatic cells. iPSCs possess self-renewal capabilities and pluripotency, potentially offering solutions for various diseases, including cancer, cardiovascular issues, metabolic disorders, immune system disease, and neurological disorders. However, in the development of these potential regenerative therapies, quality control of the final product is crucial for them to be considered as "medicines". This blog focuses on introducing methods for characterizing and analyzing iPSC final products.
The therapeutic application of iPSCs relies on their genetic stability and integrity, yet genetic instability is a common issue during long-term stem cell culture. In cell therapy, routine monitoring of genomic integrity and identifying severe mutations are crucial for product safety. Although not all genomic abnormalities are harmful, some variations may affect iPSC differentiation and potentially lead to tumor development.
Karyotyping is a well-established method for monitoring iPSC genetic integrity by detecting chromosomal abnormalities, ensuring that chromosome numbers and morphology are intact during reprogramming and differentiation process.
Pathological biology test primarily relies on histological/cytological morphological observation of biopsy tissues and DNA analysis to detect molecular and genetic changes in cells and tissues at the genetic level. However, iPSCs require more sophisticated techniques to decipher molecular clues, signal transduction, and accurate population detection. Therefore, a combination of various technologies is needed to provide a comprehensive snapshot of iPSC genomic abnormalities.
Alkaline phosphatase (AP) is expressed in most cell types, but its activity is significantly upregulated in pluripotent stem cells (PSCs), including undifferentiated ESCs, iPSCs, and embryonic germ cells (EGCs). This method is non-toxic to cells and can maintain cell vitality, making it suitable for early colony screening during reprogramming process and for detecting undifferentiated cells at later stages.
Alternative Methods (PluriTest, Teratoscore, ScoreCard)
In recent years, the field of iPSC testing has been continuously developing new alternative technologies to determine the developmental potential of human pluripotent cell lines, such as TeratoScore, PluriTest, and ScoreCard. These methods are often complemented by additional analyses like pluripotency assays (Affymetrix, Illumina) and RNA sequencing.
TeratoScore is an online platform that quantitatively evaluates the differentiation potential of human pluripotent stem cells in teratomas by analyzing gene expression patterns. The theoretical basis of this algorithm is that teratoma formation is one of the ‘gold standards’ for assessing the efficiency of hPSCs.
PluriTest is an online bioinformatics analysis platform used for verifying and characterizing the pluripotency potential of stem cell cultures and is a well-established method for assessing pluripotency. This platform compares the transcriptome of the test cell line with a large number of transcriptomes from pluripotent cell lines. Although the test results cannot directly assess the differentiation ability of the cell line being tested, they can exclude cells that significantly differ from undifferentiated stem cells. PluriTest is also suitable for rapid assessment of small number of cells in the early stage of PSC establishment.
ScoreCard is a qPCR-based method that accesses a cell line’s potential to differentiate into all three germ layers by measuring marker gene expression. This method provides a straightforward score for evaluating differentiation potential, particularly in conditions with or without trophoblasts.
Figure: PluriTest workflow. Start by uploading the microarray data of the cell line to the PluriTest website. The PluriTest algorithm and model evaluates the Pluripotency Scores and Novelty Scores of the uploaded samples and generates the results for reporting to the user. (data source: DOI: 10.3824/stembook.1.84.1)
Telomere analysis is crucial for assessing the differentiation potential of stem cells and iPSCs, as well as its importance in studies related to the quality of cell-derived products. The regulation, maintenance, and homeostasis of telomeres are vital for the long-term culture of iPSCs. Telomerase activation and telomere elongation during reprogramming, making telomere analysis a valuable tool for accessing the quality and differentiation potentials of stem cells.
Telomere analysis technology (TAT) measures the median telomere length in cell lines using high-throughput quantitative fluorescence in situ hybridization (Q-FISH ) technology.
Telomeric Repeat Amplification Protocol (Q-TRAP ) evaluates telomerase activity in whole-cell lysates from blood lymphocytes and other biological samples.
These two methods help access iPSC status and function and are useful for screening cell products with high differentiation potential. Studies have used telomere analysis to differentiate between freshly thawed iPSCs and those from later passages.
A recently reported novel microfluidic chip-based technique enables the quantification of human hematopoietic stem cells at very low quantity. This method, based on stem cell quantification cytometry, allows for ultra-sensitive capture, analysis, and counting of small quantities of hPSCs labeled with magnetic nanoparticles within low-cost, manufacturable microfluidic chips. Microfluidic technology has already been used to detect rare hPSCs in hPSC-derived cardiomyocytes and can be extended to detect other hPSC-derived products.
In addition to qPCR methods for detecting residual iPSCs, a sensitive ddPCR technique has been reported for detecting undifferentiated hiPSCs in cardiomyocytes. Using ddPCR (LIN28 probes and primers), researchers can detect extremely low levels of undifferentiated hiPSCs (<0.001%). These new technologies offer precise analysis and evaluation of iPSC product safety and characteristics.
Flow cytometry uses fluorescence-labeled antibodies to quantify cell surface antigen expression, facilitating cell type identification and purity assessment of stem cell products. PCR-based methods, including ddPCR, offer high sensitivity for detecting cell specific genes and product purity. While flow cytometry is efficient and cost-effective, PCR provides greater sensitivity, thus they can be used complementarily in clinical trials for risk assessment.
In iPSC projects, analytical identification is crucial, both during the preparation process and in the release testing phase of the final cell products. It is important to closely monitor cell phenotypes, secretion profiles, culture heterogeneity, and the presence of contaminant particles to ensure the safety and efficacy of the cellular therapeutics.
uBriGene Biosciences, a global CGT CDMO service provider, has established a robust iPSC generation platform, providing end-to-end services covering GMP master cell bank construction, GMP plasmid production, GMP production of gene editing components (Cas9/mRNA, dsDNA, gRNA, etc.) and iPSC cell banking service. Also, uBriGene has developed a comprehensive iPSC quality control system, includes tests for sterility, mycoplasma, endotoxins, differentiation factor residues, cell viability, and pluripotency, helping drug developers produce cell-based therapies that meet global biopharmaceutical standards and quality requirements.
