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Ensuring Reproducibility in Research Using Human Pluripotent Stem Cell Models

Ensuring Reproducibility in Research Using Human Pluripotent Stem Cell Models

Ensuring Reproducibility in Research Using Human Pluripotent Stem Cell Models

Insights from the Network

June 2024

Provided by STEMCELL Technologies

Human induced pluripotent stem cells (iPSCs) are a transformative tool for a wide range of biomedical research applications, including disease modeling, drug discovery, and regenerative medicine. Reproducible research with human iPSCs depends on thoroughly characterized and quality-controlled iPSC lines and cell banks. Fortunately, the research community and various organizations have come together to create tools and best practices to ensure scientific reproducibility and maximize the potential of iPSCs. Read on to understand some common challenges for iPSC users, the importance of standardizing iPSC data reporting and quality control measures, and ways to address the challenges for the best chance of success for your research goals.

Standardizing iPSC Data Reporting and Quality Control Measures

Many iPSC lines are available, but not all of them meet the necessary quality standards. In 2018, it was estimated that over 10,000 iPSC lines had been reported in the literature, based on an analysis of more than 3,500 iPSC research papers.¹ This figure, combined with the finding that approximately one-third of the lines submitted to the WiCell Stem Cell bank for distribution were found to fail minimum quality criteria,² raises concerns about the lack of consensus on iPSC quality attributes and reproducibility between research groups. To obtain reproducible and reliable results with iPSCs, standard criteria for stem cell characterization, quality control, and maintenance are crucial. Without standardization, interpreting basic scientific observations, extrapolating clinically meaningful conclusions, reproducibility, and comparison to other studies is difficult. To address these factors, the International Society for Stem Cell Research (ISSCR) released the Standards for Human Stem Cell Use in Research, a document that outlines a set of recommendations that establish the minimum characterization and reporting criteria for working with human stem cells.

To plan and conduct your human pluripotent stem cell (hPSC)-based research following the ISSCR’s Standards for Human Stem Cell Use in Research, it is crucial to understand the factors listed below:

1. Technical Processes

High-quality iPSC culture is a technically demanding process that requires skilled personnel to continuously monitor cell morphology, perform feeds, and passage cultures to maintain culture quality. Cells must remain undifferentiated, and any regions of spontaneous differentiation should promptly be removed. Extensive characterization measures should continuously be performed, including after the generation of master and working cell banks. These labor-intensive processes involve generating large numbers of vials of cryopreserved iPSCs, followed by in-depth characterization procedures to ensure consistency between and within batches. Any variation must be identified and addressed to ensure the reproducibility of experiments.

2. Contamination

According to a 2019 study by WiCell,² over one-third of assumed good-quality iPSC lines failed routine testing, with 4% found to be non-sterile. Separately, surveys of cell culture laboratories and cell banks substantiate that on average, 15 – 35% of all cell cultures may be contaminated with mycoplasma.³,⁴ Contamination has a detrimental impact on iPSC cultures, leading to increased rates of apoptosis, changes in cell morphology, and decreased viability and proliferation rates. Furthermore, microbial contamination results in changes in gene expression, such as the downregulation of undifferentiated cell markers, and the upregulation of genes involved in the stress response and immune activation. As a result, stringent microbiological control measures and aseptic techniques are required to minimize the risk of iPSC culture contamination.

3. Genetic Stability

Human iPSC culture can acquire recurrent genetic and epigenetic variants due to potential selective events, such as replating during passaging or cloning. These recurrent variants often involve gains of chromosomes 12, 17, 20, and X, similar variants to those found in human cancers. Karyotypic abnormalities such as these can affect cell behavior and compromise the utility of iPSCs in disease modeling, drug screening, and cell therapy applications. Other aberrations may include large chromosomal abnormalities, sub-chromosomal and copy number variants (CNVs), and nucleotide point mutations. Unfortunately, the identification of each type of abnormality requires a different detection method. Mutations in tumor suppressor genes TP53⁵ and BCOR⁶ have also been found to accumulate as iPSCs are passaged, limiting their clinical potential. Regular genetic analysis of long-term iPSC cultures and a robust banking strategy are crucial for identifying and addressing any mutations that may arise.

4. Variability and Standardization

Reproducibility is a critical aspect of any scientific research program, but the high variability among human iPSC lines makes it challenging to obtain consistent results and compare data from different studies. Line-to-line variability in iPSCs arises from genetic and epigenetic differences between individuals, reprogramming processes, and culture conditions. Passage-to-passage variability can result from the use of varying passaging techniques, culture conditions, and genetic drift. Non-standardized experimental protocols and the absence of clear reporting of experimental details are also contributing factors. To address these issues, several initiatives have been developed, including the ISSCR Standards Initiative and hPSCreg®, which aim to provide standardized reporting guidelines and a centralized bank for registered, traceable, and validated cell lines.

5. Cost

Human iPSC research can be a costly endeavor. The long-term culture of iPSCs demands that specialized and highly trained personnel perform experiments and maintain the cells. Experiments need to be repeated, and the optimization of experimental procedures, such as differentiation protocols, can quickly increase labor costs. iPSCs require specialized culture conditions, including the use of defined media supplemented with highly qualified growth factors. Even small variations in reagent quality can affect cell viability and functionality, so commercially manufactured and qualified media systems can be of great benefit. Variability in cell line quality necessitates rigorous quality control checks, which can dramatically increase the cost of research, especially if performed frequently. Other factors include equipment costs and regulatory costs, such as obtaining ethical approvals for the donation of patient tissue for reprogramming. Learn more about cell quality here.

Highly Characterized Tools to Standardize and Speed Up Your iPSC-Based Research

Reproducible research using iPSCs can be simplified by using thoroughly characterized and quality-controlled cell lines. Whether you’re looking to have high-quality iPSCs in long-term maintenance or to get to your downstream applications quickly, STEMCELL Technologies has you covered. Start your research from high-quality iPSC sources, including our recently launched iPSC line for basic research and commercial use, SCTi003-A, derived from peripheral blood mononuclear cells (PBMCs) of a healthy female donor. For maintenance-free iPSCs, choose the innovative single-use, iPSCdirect™ which has been derived from its parent line, SCTi003-A.

SCTi003-A: An Extensively Characterized, High-Quality iPSC Control Line

Whether you’re looking to source a commercially available iPSC line for long-term culture or larger-scale expansion, SCTi003-A provides researchers with the option to choose, with the bonus of having been manufactured under extensive quality control standards. SCTi003-A is a healthy control iPSC line and can be used as such, for example, in comparative studies with diseased cell lines, to validate processes and/or protocols, gene editing, and more. Our iPSC quality assessments and release criteria have been developed based on recommendations and guidance from the International Stem Cell Banking Initiative,⁷ the Global Alliance for iPSC Therapy,⁸ and the consensus workshop hosted in June 2022 by the ISSCR Standards Initiative.

Meet regulatory requirements for academic or commercial purposes with ethically sourced iPSCs that have been collected using Institutional Review Board (IRB) protocols. Donations are performed in the United States in compliance with applicable federal, state, and local laws, regulations, and guidance. Additionally, donors in our healthy pool are pre-screened using a health questionnaire aimed at excluding any donors with diseases, blood disorders, or other health concerns. SCTi003-A has been certified by hPSCreg®, a global registry for PSC lines that aims to provide the community with a central and searchable hub and act as a trustworthy data source by verifying ethical and biological conformity based on community standards. The hPSCreg® cell line certificate is essential for certain funding agencies (e.g. using a hPSC line in research funded by the European Union). To learn more about SCTi003-A on the hPSCreg® website, click here.

Table 1. iPSC Line SCTi003-A Is Derived from a Healthy Female Donor

Note 1: Only iPSC vials from the commercial cell bank are made available for commercial sale.

Note 2: Commercial cell bank iPSCs have been characterized for all assessment criteria at varying stages of the manufacturing process.

Demographic, health, and genetic characteristics of the SCTi003-A donor were compiled based on self-reported information and whole-exome sequencing. Sex was determined by karyotype. Ancestry was calculated by EthSEQ analysis from whole-exome sequencing data. HLA haplotype was determined by next-generation sequencing, sequence-base typing, and sequence-specific oligonucleotide probes as needed to obtain the required resolution. Other genetic variants were determined from whole-exome sequencing using ClinVar analysis. Blood type (ABO/Rh blood group) was determined by next-generation sequencing. Height, weight, and BMI were calculated at the donation facility.

SCTi003-A has been optimized for compatibility with the TeSR™ family of feeder-free culture media for maintenance and with STEMdiff™ for standardized, high-quality maintenance and robust differentiation. To date, the compatibility of SCTi003-A has been tested with over 20 off-the-shelf STEMdiff™ differentiation protocols, including differentiation to neural progenitor cells (Figure 1), microglia, forebrain neurons, midbrain neurons, astrocytes, cerebral organoids, atrial and ventricular cardiomyocytes, hematopoietic progenitor cells, endothelial cells, intestinal organoids, and many more.

Figure 1. SCTi003-A Human Pluripotent Stem Cells Can Efficiently Differentiate into Neural Progenitor Cells

Expression was quantified for (A) PAX6, (B) SOX1, and (C) class III β-tubulin (TUJ1). (D) NPCs displayed the expected small, teardrop-shaped morphology. (E) Marker expression was quantified for NPC markers and mature neuronal markers. Error bars represent standard deviation (n = 2 biological replicates).

iPSCdirect™: Fast-Track Your PSC Experiments

Skip the challenges involved with maintaining human iPSCs in long-term culture and increase the efficiency and productivity of your stem cell research with iPSCdirect™, a high-density, single-use source of singularized iPSCs immediately ready to use upon thaw (Figure 2). Save time and reduce variability by starting with the same source of highly characterized iPSCs for every experiment. Highly consistent and robust, iPSCdirect™ avoids the cost and effort of developing and characterizing master and working cell banks. Derived from the healthy control human iPSC line, SCTi003-A, iPSCdirect™ has undergone rigorous quality control procedures and demonstrates high levels of consistency and performance. iPSCdirect™ has been optimized for compatibility with mTeSR™ Plus and CloneR™2 for seeding and STEMdiff™ for differentiation (Figure 2).

Schematic for a Generalized Monolayer Protocol to Thaw and Culture iPSCdirect™ for Downstream Assays

Figure 2. Schematic for a Generalized Monolayer Protocol to Thaw and Culture iPSCdirect™ for Downstream Assays

iPSCdirect™ is thawed and plated into mTeSR™ Plus supplemented with CloneR™2 and incubated overnight according to product instructions. After 24 hours, cells are ready for STEMdiff™ or customized monolayer workflows.

iPSCdirect™ is a research-use-only (RUO) product, approved for both academic and commercial use. The cells are also karyotypically stable, demonstrate trilineage differentiation potential, and express undifferentiated cell markers. An immediate source of human iPSCs, iPSCdirect™ is suitable for numerous applications in the field of stem cell research, including basic research to study the mechanisms of stem cell self-renewal, differentiation, and maintenance. After thawing and incubation overnight, iPSCdirect™ is ready for differentiation into cell types such as ventricular cardiomyocytes, hepatocyte-like cells, and neural progenitor cells within 24 hours after seeding (Figure 4). The ability to bypass time-consuming and continual iPSC maintenance using iPSCdirect™ enhances the drug discovery potential of human iPSCs to screen potential drug candidates for safety and efficacy, identify lead compounds, and optimize drug dosing and delivery strategies.

Figure 3. iPSCdirect™ Can Be Seeded to Reach a Range of Confluencies After 24 Hours

To reach the desired confluency for downstream experiments, thaw and plate iPSCdirect™ into mTeSR™ Plus with CloneR™2. These representative examples of (A) low confluency, (B) medium confluency, and (C) high confluency were cultured after thaw on Corning® Matrigel® hESC-Qualified Matrix and imaged at a magnification of 4X.

iPSCdirect™ SCTi003-A Human Pluripotent Stem Cells Can Successfully Differentiate into Hepatocytes

Figure 4. iPSCdirect™ SCTi003-A Human Pluripotent Stem Cells Can Successfully Differentiate into Hepatocytes

iPSCdirect™ SCTi003-A cells were plated according to the iPSCdirect™ Product Information Sheet, in preparation for Day 1 of the STEMdiff™ Hepatocyte Kit (Catalog #100-0520) protocol outlined in the STEMdiff™ Hepatocyte Kit Product Information Sheet. (A) Monolayers of hepatocyte-like cells with characteristic polygonal morphology and binucleation are apparent by Day 21 of differentiation. (B) Marker expression was quantified by flow cytometry for hepatic markers albumin (ALB) and alpha1-antitrypsin (A1AT). Error bars represent standard deviation (n = 3 biological replicates).

Table 2 details the distinctions between SCTi003-A from STEMCELL’s iPSC Repository and iPSCdirect™. In summary, SCTi003-A is designed for long-term maintenance and the generation of cell banks, whereas iPSCdirect™ is single-use only, but with identical donor and cell line properties. Downstream assays, such as differentiation, can be initiated from iPSCdirect™ cells 24 hours after thawing (Figure 4). Whether you need high-quality iPSCs in long-term maintenance or to get to your downstream applications quickly, STEMCELL Technologies has you covered.

Table 2. Differences Between iPSCdirect™ and SCTi003-A

Table 2. Differences Between iPSCdirect™ and SCTi003-A

*iPSCdirect™ is available in a 10pk format, applicable for use as a master cell bank.

Despite both commercial products being derived from the same donor tissue and cell line, iPSCdirect™ and SCTi003-A have a number of key differences including format, licensing, and usage restrictions.

For more information about STEMCELL’s iPSC lines, refer to our Frequently Asked Questions on iPSCs. For any other queries, click here to contact STEMCELL’s iPSC Team or email us at iPSCrequests@stemcell.com.

References

  1. Guhr A et al. (2018) Recent Trends in Research with Human Pluripotent Stem Cells: Impact of Research and Use of Cell Lines in Experimental Research and Clinical Trials. Stem cell reports 11(2): 485–96
  2. Felkner D et al. (2019) “Human pluripotent stem cell quality: A scientific wake‐up call.” Poster presented at the ISSCR Annual Meeting, Los Angeles, California
  3. Drexler HG et al. (2002) Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39(2): 75-90
  4. Uphoff C et al. (2005). Detection of Mycoplasma Contaminations. Humana Press 290
  5. Merkle F et al. (2017) Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature 545, 229–233
  6. Rouhani FJ et al. (2022) Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells. Nat Genet 54, 1406–1416
  7. The International Stem Cell Banking Initiative. (2009) Consensus Guidance for Banking and Supply of Human Embryonic Stem Cell Lines for Research Purposes. Stem Cell Rev and Rep 5, 301–314
  8. Sullivan S et al. (2018) Quality control guidelines for clinical-grade human induced pluripotent stem cell lines Regenerative Medicine 13(7): 859-866
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STEMCELL Technologies provides high-quality cell culture media, cell separation technologies, instruments, accessory products, and educational resources to scientists around the world working on stem cell, immunology, cancer, regenerative medicine, and cellular therapy research. At STEMCELL, we are dedicated to improving lives through advanced knowledge and scientific discovery, through our commitment to fostering diversity and inclusion in STEM and the life sciences industry, and through our investments in sustainability, community, and social responsibility. Rooted in scientific expertise and rigor, we are a company of Scientists Helping Scientists, and we care deeply about making the world a better place.

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