Stem cells have long been controversial in research but remain of particular interest for biological exploration due to their flexibility and longevity. While debate still surrounds embryonic stem cells, other cell types are providing a wealth of possibility for research into health and disease, as well as further expansion into possible clinical applications.
Given that induced pluripotent stem cells (iPSCs) are derived from skin or blood cells by reprogramming them to an embryonic-like pluripotent state, less controversy surrounds these cells, easing development of new applications and transforming how stem cell research is approached. Investigations involving iPSCs in basic biology, drug-cell interactions and screening, and organoid generation have been accelerated by novel technologies aimed specifically to improve iPSC generation, growth, modification, and monitoring.
The relevance of iPSCs
iPSCs retain the genome of the donor, can regenerate indefinitely, and can be differentiated into virtually any cell type of interest using a range of published protocols. Susan L. Solomon, CEO and co-founder of The New York Stem Cell Foundation (NYSCF) Research Institute, describes these patient-specific, disease-relevant cells as incredibly powerful models in which we can study disease mechanisms using functional assays, genetic contributors using genome editing, and even patient-specific therapies using in vitro drug testing.
Access to tissue and cells from healthy and diseased donors can be limited, resulting in substantial barriers to applied research and treatment exploration. "Induced pluripotent stem cells give researchers unprecedented access to limitless quantities of cells (and tissues), made from a simple blood donation from donors with particular diseases,” offers Seimi Satake, chairman and CEO of FUJIFILM Cellular Dynamics Inc. (FCDI). The result has enabled researchers to not only generate unique and previously unattainable disease models but also generate these models in a human background, decreasing the time and cost of drug discovery while increasing the efficacy and safety of new therapeutics. Companies like FCDI and institutes like NYSCF are working to build publicly accessible banks of iPSCs derived from large diseased donor cohorts. By providing the research community with a broad array of human-based models, these banks will accelerate both basic and applied research efforts and ultimately lead to new knowledge and medicines.
“Human stem cells represent a relevant population of cells that can be manipulated and differentiated to model a wide range of diseases that are uniquely human in nature such as Alzheimer’s disease. In fact, species differences likely represent a contributing factor to drug efficacy discrepancies when moving from preclinical mouse studies to clinical trials in humans,” explains Ashley Watson, associate product manager for pluripotent stem cells at STEMCELL Technologies. It has been estimated in research by Niall Shanks and colleagues at Wichita State University that animal models of disease can only correctly predict toxicity in approximately 70% of cases, resulting in time and effort wasted on ineffective or unsafe drugs.
iPSCs provide a more biologically relevant system for human biology than alternative models, recapitulating what is occurring in the body and modeling more closely what is happening in a disease state. Radhika Dixit, applications scientist at Harvard Bioscience, notes that iPSCs such as cardiomyocytes are transforming research by enabling novel approaches for generating disease-specific or lineage-specific cell lines and organoids that have opened the door to studying all aspects of a disease in a model system and associating data to donor phenotypes.
Expanding research
Human pluripotent stem cells are unique in their capacity for indefinite self-renewal and the capability to differentiate into multiple downstream cell types. Differentiation combined with recent advances in genome-editing techniques has paved the way for more effective disease modeling due to the ease in which iPSCs can be genetically modified. Watson expounds, “Disease-causing mutations can now be introduced or corrected in iPSC lines to create or rescue disease-associated phenotypes, and this can be studied in relevant human cell types through directed differentiation. The ability to perform efficacy or toxicity assays on models with the exact mutation or disease-causing phenotype of a patient can greatly inform the discovery of treatments for disease.” Furthermore, the ability to differentiate patient-specific iPSCs into hard to access primary tissues with technologies like the STEMdiff™ kits for iPSC differentiation to a range of lineages provides relevant cell sources, enabling more and better drug discovery research for diseases that affect those tissues.
Generation of iPSCs can be accomplished through several different approaches, including virus-mediated modifications, electroporation, and transfection. “Electroporation has been up and coming in recent years due to its ability to adapt to a variety of approaches regarding iPSC studies and deliver more complex molecules. Genetically modifying iPSCs using CRISPR to knock-in a gene, for example, requires delivery of a complex mixture of protein, guideRNA complexes, and donor DNA comprised of larger molecules. Electroporation can be the most efficient and sometimes the only viable option for successful generation of these modifications,” suggests Michelle Ng, global product manager at Harvard Bioscience.
Specific to electroporation, the use of more technologically advanced electrodes that can be used for cells and for organoids allows various molecules including CRISPR complexes, large DNA molecules, or nanoparticles to enter cells while maintaining their three-dimensional structure. And with CRISPR extending the iPSC toolkit, much more can be accomplished using disease-specific iPSCs with knock-in or knock-out modifications, easing the transition into the generation and use of tissue-specific organoids.
For example, recent research from the Salk Institute for Biological Studies described the development of a glioma cancer model in human cerebral organoids by electroporation of CRISPR/Cas9 components that allowed direct observation of tumor initiation. Results showed that transformed cells rapidly become invasive and destroy surrounding organoid structures. Studies like this demonstrate that organoids can allow visualization of both proliferation and invasive behaviors, offering a relevant platform to explore the natural progression of cancer.
NYSCF uses iPSCs to study many disease processes, including multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, diabetes, cancer, and more. These also include organoid models of neurodegenerative diseases, where it is necessary to co-culture several different cell types implicated in these diseases to better understand their functional interplay. “Our research programs focus on building tools and protocols to accelerate work across the stem cell field, modeling disease to investigate root causes and new therapeutic targets and bringing better treatments to the clinic as fast as we can. iPS cells are the tool that makes all of this work possible,” Solomon says.
Improving reproducibility
“While stem cells harbor great potential, several challenges remain, including reproducibility issues, immaturity of iPSC-derived cell types, and a lack of quality standards in the field to authenticate iPSCs,” offers Watson. Reproducibility of experiments can be marred by the variability between cell lines or sample sources. While this can be addressed through use of gene-editing technologies to derive isogenic lines for control of genetic background differences, it only works when modeling disorders with a defined genetic component. In addition, other culture variabilities such as karyotypic abnormalities can be acquired during long-term culture of iPSCs, prompting improvements in possible suboptimal culture conditions. Watson suggests using robust culture systems that contain pre-screened quality components for consistency and routine monitoring of culture quality. STEMCELL Technologies offers quality control products including the TeSR™ family of feeder-free media to ensure consistency and experimental reproducibility and a qPCR-based hPSC Genetic Analysis Kit for rapid detection of the most commonly reported karyotypic abnormalities in hPSC culture.
Dixit believes that whether working with plasmids, RNA, proteins, or other molecules, optimized delivery tools like electroporation can offer better control over an experiment, maintain efficiency, and ensure reproducibility. New advances in instrumentation now permit researchers to save all data on the electroporation unit itself, enabling researchers to review and repeat previous experiment parameters for increased reproducibility and experimental success. Dixit also emphasizes that the ability to monitor the system with oscilloscopes to confirm exact measurements further assists in ensuring consistency throughout an experiment.
To tackle the important issue of reproducibility in iPSC reprogramming, NYSCF built their Global Stem Cell Array, a robotic system capable of fully automated, high-throughput, standardized derivation and quality control of iPSCs. They have demonstrated that the automation and standardization of this process significantly improves the quality and stability of the resulting iPSC lines, and reduces line-to-line variation compared to manual protocols. Using this platform, NYSCF has generated thousands of lines for the scientific community.
Introducing quality control measures to iPSC processes from an experimental design perspective ensures reproducibility across an organization. “Improving reproducibility is an important issue with iPSC-derived cells. Currently, FCDI is working on two initiatives that will improve consistency in quality processes. The first is to implement a cGMP-compliant quality management system that will support internal therapeutics programs, as well as support any CDMO business objective. The second initiative is to centralize the quality function at FCDI, which will leverage quality resources and experiences within FCDI's quality function,” comments Satake. Managing compliance expectations into QbD processes commonly used in the pharmaceutical industry promotes rigorous design of experiments that model how critical process parameters can influence the critical quality attributes of a product, resulting in robust control of product performance. Satake offers that while QbD is a pharmaceutical practice, concepts could be applied at FCDI, resulting in predictable product performance across any laboratory or organization.
Looking forward
Satake believes that the introduction of new instruments and assays in iPSC research that yield big data will be increasingly more valuable with the development of better bioinformatics tools. In addition, FCDI is developing several key platform technologies such as imaging for the manufacturing of stem cells. Fujifilm artificial intelligence technology enables the monitoring of various QC metrics during the manufacturing process.
While iPSCs offer a useful technology for health and disease state research, the lack of standardized methods to characterize, authenticate, and bank stem cell lines is still a challenge. Several initiatives exist including the International Stem Cell Initiative, International Stem Cell Banking Initiative, the National Academies of Sciences, Engineering, and Medicine’s Forum on Regenerative Medicine, and the Global Alliance for iPSC Therapies (GAiT) that aim to support standardized practice across the field.
As a founding member of GAiT, NYSCF is a strong proponent of standardization, offering a Certificate of Analysis with each iPSC line they distribute to verify that it meets their stringent quality control standards. Watson adds that STEMCELL Technologies has a strong focus on cell quality and aims to provide high-quality culture systems, reagents, and tools to give scientists confidence in their data and support innovative research, including efforts to work collaboratively with both basic research- and clinically-focused groups to help define standards in the field.
With so many new tools on the horizon that are supporting and accelerating iPSC research and applications, collaborations could help take research to the next level suggests Dixit. Furthermore, with iPSC-generated organoids enhancing our knowledge of cell-cell interactions, taking iPSCs to the clinic might become a not so distant reality. “The potential applications for iPSCs in disease research, drug discovery, and regenerative medicine are almost limitless,” adds Solomon.