The Stem Cell Dilemma: Reprogramming in-house versus off-the-shelf purchase of commercially produced iPSC-derived cell types
The case for using more physiologically-relevant cells for modelling disease pathophysiology, for understanding a drug’s mechanism of action, in toxicology/safety testing and in phenotypic drug screening assays is now well accepted.
Human induced pluripotent stem cells (iPSCs) are seen as a resource from which to differentiate unlimited quantities of almost any tissue cell, the resulting human iPSC-derived cells replacing cultured cell lines which may lack cellular function or primary cells where it may be impossible to obtain sufficient numbers from patients.
The stem cell workflow, which includes generating (reprogramming) iPSC and differentiating them into the desired cell type, is complex and to the inexperienced may represent a significant hurdle in the production of iPSC-derived cells. For this reason many prefer to outsource reprogramming or to bypass it altogether by purchasing commercial iPSC-derived cells.
A review of vendor updates on their iPSC offerings reveals useful coverage in terms of the range of different commercial iPSC-derived cell types now offered, and an increasing assortment of iPSC workflow tools and outsourced services.
New entrants wanting to access iPSC-derived cells now have a clear choice either to embark on reprogramming and differentiation in-house or gain immediate access by purchasing off-the-shelf iPSC-derived cell types or engage a third party to, say, create an isogenic cell line or disease model for them. What is clear is that iPSC-derived cells will feature to a greater extent in future disease models, toxicology/safety testing and phenotypic assays.
Alot of enthusiasm and optimism surrounds the use of stem cells, particularly induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC) in drug discovery applications. Cells-derived from human iPSC or ESC are thought to be more physiologically relevant and better suited for modelling disease pathophysiology, for understanding a drug’s mechanism of action, for toxicology/safety testing and for phenotypic drug screening assays.
Stem cells are unusual because they can survive and replicate indefinitely and have the capacity to differentiate into any cell type in the human body (pluripotency). There are two general types: 1) ESC derived from early stage embryos; and 2) iPSC generated from adult human cells (eg skin or blood cells). However, ESC-derived cells are generally less favoured as there are ethical concerns regarding the use of human embryos as a cell source and there is potential for immunogenicity in cell therapy applications.
The process of generating iPSC is referred to as reprogramming and involves the directed expression of pluripotency genes in adult cells. Reprogramming somatic cells to induced pluripotent stem cells is a critical and potentially time-consuming step in stem cell research. Once iPSC are generated they must be directed to differentiate into the tissue cells of interest, resulting in iPSCderived cells. This ability to differentiate into a desired cell type depends on the availability of an efficient protocol to achieve the differentiation, and protocols are not yet established for all cell types.
Although many vendors have made big efforts in providing tools and technologies such as reprogramming vectors, transfection kits, maintenance and differentiation media, immunocytochemistry and live staining kits, etc to aid the stem cell workflow, the scientific challenges of enabling iPSC generation and differentiation on a large scale sufficient to supply the cell demands of screening are significant. For this reason many prefer to outsource repro gramming or to bypass it altogether by purchasing commercial iPSC-derived cells from companies such as Cellular Dynamics International and Axiogenesis.
These companies have invested heavily in developing and optimising the reprogramming process of iPSC to enable the production of industrial quantities of differentiated tissue cells that can recapitulate relevant donor disease biology in the laboratory. The availability of unlimited quantities of cryopreserved stem cell aliquots is already significantly impacting drug discovery, with an expectation it will decrease the risk of late-stage attrition during drug development.
So far commercial producers of iPSC-derived cell types have focused mainly on cardiac, hepatic, neuronal and pancreatic cells. Cardiac differentiation from stem cells seems to have made the most progress with iPSC-derived human cardiomyocytes now used routinely for in vitro toxicity testing and were recently assigned by the FDA a pivotal role in the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative which aims to replace the current preclinical hERG assay.
In December 2015, HTStec undertook a market study on the use of stem cells and stem cell-derived cells (particularly iPSC), as well as their utilisation in different areas of drug discovery and development1. We now report some of the findings of that market today and review it together with vendor updates on commercial cells and tools used for reprogramming and the stem cell workflow.
Current work carried out with stem cells
The majority (66%) of survey respondents’ stem cell work carried out today (2015) involves induced pluripotent stem cells (iPSC) or iPSCderived cells. This was followed by human adult stem cells (11%), and then human embryonic stem cells (ESC) (10%), other (8%) and animal ESC or iPSC (5%) (Figure 1).
Survey respondents undertaking different tasks with iPSC or iPSC-derived cells are presented in Figure 2. In order of greatest use by survey respondents, these were: 27% undertaking phenotypic screening assays in-house using iPSCderived cells; 26% undertaking toxicology/safety testing in-house using iPSC-derived cells; 23% undertaking in-house stem cell differentiation; 23% undertaking disease modelling in-house using iPSC-derived cells; 23% whose work involves generation of iPSC or iPSC-derived cells; and 23% undertaking disease modelling in-house using iPSC-derived cells.
Main obstacles limiting stem cell work
Survey respondents rated cost of commercially available cells as the main obstacle limiting work with stem cell-derived cells. This was followed by batch to batch variation of cells; difficulty to direct differentiation to desired cell phenotypes; and then issues of assay sensitivity, robustness and reproducibility. Rated least limiting were ethical issues and intellectual property (Figure 3).
Main focus of in-house differentiation
The cell lineages into which survey respondents were most attempting to differentiate stem cells were cardiac muscle cells (56% attempting), brain/neuronal/glial cells (54% attempting) and hepatocytes (28%). All other cell lineages had less than 20% attempting differentiation (Figure 4).
Main barriers to sourcing stem cells commercially
Survey respondents rated the cost of commercial cells as the main barrier to sourcing their iPSC or iPSC-derived cell needs commercially. This was followed by cell types needed are not commercially available; issues with the general quality of commercially available cells; and then issues with reproducible quality of commercially available cells. Rated least limiting were potential legal issues (Figure 5).
Survey respondents rated level of characterisation/ validation of cell type as the factors that most influence expected purchasing on iPSC and iPSCderived cells. This was closely followed by price of commercial cells; availability of specific iPSC type(s) from desired species/phenotype; and then internal data demonstrating value of stem cells. Rated least important was availability of a certain cell format (eg plated cells) (Figure 6).
Main focus of commercially purchased cells
The commercially available iPSC-derived cell types of most interest to survey respondents today were cardiomyocytes (mixed population, 45% wanting). This was closely followed by cardiomyocytes (ventricular enriched, 43% wanting); and then CNS neurons (32% wanting); cardiac fibroblasts (25% wanting) and dopaminergic neurons (25% wanting). Least interest was for skeletal myoblasts and hematopoietic progenitor cells (Figure 7).
Toxicology/safety testing with stem cells
The percentage of current toxicology/safety testing done by survey respondents using different cell types today (2015) was: 41% stem cells, 39% cell lines and 20% primary cells (Figure 8).
The breakdown of different iPSC-derived cell types used in toxicology/safety testing assays today (2015) by survey respondents was split as follows: cardiomyocytes 60%; neural cells 23%; hepatocytes 9%; and all other iPSC types 8% (Figure 9).
Survey respondents rated calcium transients/ voltage sensitive dye assays as the assay technology they were most interested in applying to toxicology/ safety testing using iPSC-derived cells. This was followed by multichannel electrode arrays (MEA); and then high content analysis (HCA) and automated patch clamp. Least interest was expressed for vector motion analysis (Figure 10).
Phenotypic screening assays with stem cells
The current (2015) breakdown of phenotypic screening assays reported by all respondents between different cell types was: 55% stem cells, 24% cell lines and 21% primary cells (Figure 11).
Other application areas for iPSC
Interest in other application areas of iPSC or iPSCderived cells was greatest for investigation of stem cells in regenerative medicine/tissue engineering/ cellular therapies and molecular pathway analysis (both with 67% interested). This was followed by reporter assays (47% interested); investigation of stem cells in precision medicine (45% interested); general research into stem cell biology and generation of stem cells (40% interested); and then stem cell biobanking (25% interested) (Figure 12).
Latest developments in iPSC
The following vendor snapshots provide additional details and describe some of the latest developments in commercial iPSC and the tools used for reprogramming and the stem cell workflow.
ATCC (www.atcc.org/ipsc) continues to lead the iPSC field by offering iPSCs derived from bone marrow CD34+ hematopoietic stem cells with gender and ethnic diversity. This offering includes iPSCs of African American, Asian, Caucasian and Hispanic ethnic backgrounds. The cells in this collection may be employed as a complete suite for high-throughput screening in toxicological experiments, disease modelling, or pharmacological target validation. Or, you can use an individual iPSC line from this diverse offering for testing cosmetic and/or other health products for a targeted market.
In addition, ATCC has iPSCs derived from cystic fibrosis, Parkinson’s disease and Down syndrome patients for disease-specific studies. ATCC has also responded to the movement away from feeder celland serum-dependent culture by creating a feederfree and serum-free iPSC culture system. This culture system relies on plating the iPSCs on a biological matrix and culturing the cells in a specialised, defined, serum- and xeno-free stem cell medium. Further, the iPSC culture system has been highly optimised to ensure that the iPSCs retain their undifferentiated state after many passages, and is validated for large-scale production of iPSCs.
Finally, recent advances in gene editing have opened opportunities for introducing gene knockouts, gene knock-ins, mutations and mutation corrections into cells. This has enabled iPSCs to be gene-edited by zinc finger nuclease, TALENs, or CRISPR/Cas9 to create models to study disease biology, identify novel drug targets, and further the development of therapies for any genetic disease under study in the laboratory (Figure 13).
Axiogenesis (www.axiogenesis.com) is a leading provider of unlimited volumes of pure, tissue-specific cell types derived from human iPSC and is an expert on the application of these cells on commercial assay platforms. Axiogenesis, founded in 2000, has been providing first-in-class stem cell-derived cells since 2005. Axiogenesis’ focus lies on the development and validation of functional assays using neuronal and cardiac cells.
Axiogenesis has a global distribution network and 40 employees including a team of 10 customer service staff and six scientists providing technical assistance. Axiogenesis is a preferred partner to pharmaceutical companies and CROs as they can lend unbiased expert advice in setting up, validating and troubleshooting assays. Cardiac products encompass iPSC-derived Cor.4U® cardiomyocytes and CorV.4U® ventricular cardiomyocytes used in cardiac safety and HTS applications as well as isogenic FibroCor.4U® electro-competent cardiac fibroblasts used in tissue modelling.
Neuronal products include Dopa.4U™ dopaminergic neurons and CNS.4U™ central nervous system neurons used for neurodegenerative disease modelling and neurotox applications, as well as isogenic Astro.4U™ astrocytes for neuronal co-culture. The Peri.4U™ peripheral neurons are validated for structural and functional neurotoxicity assays and used for novel neurotoxin screening.
New developments include smooth muscle cells, skeletal muscle cells, sensory neurons and atrial cardiomyocytes. A strong IP position through key licences, including the Yamanaka iPSC technology and the Geron stem cell patents, as well as its own patent portfolio enable Axiogenesis to offer FTO in the use of iPSC-derived cells including disease models such as cardiac hypertrophy (also hypertrophic cardiomyopathy, HCM) (Figure 14).
Axol Bioscience (www.axolbio.com) specialises in the use of stem cell technology to manufacture disease- relevant human cell-based assay systems for the drug discovery industry. It offers a growing range of primary and iPSC-derived cells, as well as complementary media and supplements, for robust cell growth in vitro. The iPSC-derived neural cells offer a valuable tool for high-throughput screening and disease modelling.
Currently these cells include highly validated, electrically active neural stem cells, cortical neurons, astrocytes and dopaminergic neurons from healthy and patient donors including Huntington’s, Alzheimer’s and Parkinson’s diseases. Axol also offers spontaneously beating iPSC-derived cardiomyocytes that are ideal for use in cardiotoxicity testing, drug screening and validation, electrophysiological applications, metabolic and mitochondrial studies. All cells are commercially available in large batches, offering researchers a continuous source from a single donor for consistent and reproducible results across multiple assays.
Axol also provides a complete suite of cell sourcing, reprogramming, differentiation and gene editing services. Using CRISPR/Cas9 technology to correct mutations in cells derived from patients as well as introduce disease- relevant mutations into cells derived from healthy donors, Axol creates isogenic and reporter cell lines for disease modelling and drug discovery.
In addition, Axol also offers a range of primary cells including fibroblasts and peripheral blood mononuclear cells (PBMCs) from both healthy and diseased backgrounds including autoimmune conditions, neurodegenerative disorders, leukaemias and other cancers. These can be reprogrammed into iPSCs using Axol’s integration-free system under fully-defined conditions and differentiated to generate in vitro models for screening novel compounds and drug combinations (Figure 15).
The limitations of conventional cell-based assays used for drug discovery are well-documented: immortalised cell lines lack native cellular function, primary cells are available in limited quantities, and cell lines may not be representative of normal physiology.
Cellular Dynamics International (CDI) (www.cellulardynamics.com) develops and manufactures biologically relevant human cells derived from iPSCs. Because CDI’s differentiated cell products are highly pure, highly reproducible, strongly predictive and available in industrial quantity, they have demonstrated the ability to overcome these problems to enable disease modelling, screening and toxicity applications. CDI’s iPSC-derived product portfolio consists of iCell® and donor-specific MyCell® human cells.
iCell products are inventoried, terminally differentiated cells including cardiomyocytes, hepatocytes and neurons. MyCell products are human cells derived from researcher-provided or biobanked samples that can serve as models of disease or genetic variation among populations. CDI’s recent access to newly available biobanks of high quality iPSCs from hundreds of donors further enhances the MyCell product offering. Providing a limitless supply of relevant human cell types, CDI cells are ideal for phenotypic screening efforts. The intrinsic, biologically relevant behaviours of iPSC-derived differentiated cells facilitate the building of new drug discovery models.
For toxicity testing applications, the ready availability of consistent iPSC-derived cells (including iCell Hepatocytes and iCell Cardiomyocytes) is especially valuable for revealing unwanted toxic effects earlier in the drug development process. All of these factors combined indicate that the use of CDI’s iPSC-derived differentiated cells for drug discovery purposes will increase the likelihood of discovering more efficacious and safer drugs (Figure 16).
The use of iPSC in drug discovery applications is hindered by the difficulty of directing their differentiation into the desired cell types and by high batchto- batch variability.
The Cellartis® brand, offered by Clontech Laboratories, a Takara Bio Company, (www.clontech.com/stem-cell-partnership) provides off-the-shelf solutions to these challenges, enabling researchers to direct the differentiation of their own iPS cell lines. With more than 14 years of experience with human iPS cell differentiation, its team has developed robust protocols for efficient differentiation into hepatocytes or pancreatic beta cells.
The Cellartis iPS Cell to Hepatocyte Differentiation System is a complete solution for dif ferentiating disease- or patient-specific iPS cells into hepatocytes; it combines a standardised protocol with optimised media, supplements and coating reagents. Within three weeks, hepatocytes express high levels of drug-metabolising enzymes and transporters, showing stable functionality for at least 11 days.
This novel system allows differentiation of multiple iPS cell lines from virtually any genet ic background, providing an accurate reflection of human metabolic diversity while delivering reproducible results. Pluripotent cells can also be differentiated into beta cells, highly desirable in drug discovery due to their potential to treat diabetes and metabolic disorders.
Cellartis iPS-derived beta cells express mature betacell markers including insulin, and demonstrate Glucose-Stimulated Insuli n Secretion (GSIS) after cryopreservation, indicating their suitability for in vitro assays (Figure 17). By providing highly functional iPS-derived cell models including hepatocytes and beta cells with reduced variability compared to primary cells, Cellartis products are helping scientists understand disease biology and find better drug candidates, speeding up the drug discovery process.
Corning (www.co rning.com/lifesciences) brings together an industry-leading portfolio of cell culture surfaces, vessels, media and high throughput screening platforms that enables stem cell scientists to unlock the potential of their research in new drug discovery and disease treatment. As the leading product in its portfolio, Corning® Matrigel® Matrix has proven through the years to be a critical and robust research reagent for pluripotent stem cell expansion and differentiation. Matrigel matrix is also a key reagent in building biologically relevant organoid models of human disease.
As stem cell applications move from research into the therapeutic and drug discovery arena, Corning continues to develop new surfaces and technologies that improve the efficiency and productivity of stem cell expansion. Recent additions to its portfolio include pre-coated cultureware with animalfree peptides and recombinant proteins that replace the use and function of biological extracellular matrices. Corning® PureCoat™ ECM Mimetic and PureCoat rLaminin 521 cultureware are ready-to-use vessels that address many of the technical, quality and handling concerns experienced by stem cell scientists as they scale-up pluripotent cells for clinical use.
Coupling surfaces with its closed system options, Corning’s complete solution allows researchers to go from researchscale cell culture to cGMP cell processing quickly and efficiently. Finally, for researchers using pluripotent stem cells in drug discovery and screening applications, the Corning® Spheroid Microplates offer a truly enabling class of product that combines embryoid body formation and downstream analysis in a single microplate with no transfer steps.
Available in both 96- and 384-well formats, the spheroid microplate is a powerful tool for large-scale, high throughput screening in 3D cell format. As the market leader in cell culture, Corning is committed to providing its users with a full workflow solution enabling stem cell research. (Figure 18).
MilliporeSigma (www.emdmillipore.com/simplicon) offers workflow solutions for all areas of stem cell biology including cellular reprogramming. Various methods utilising viruses, DNA, RNA, miRNA and protein have been developed to generate integrationfree iPSCs. Disadvantages to existing methods include low reprogramming efficiency (ie DNA and protein), a requirement for negative selection and recloning steps to remove persistent traces of the virus (ie Sendai virus) or for daily transfections of four individual in vitro-generated RNAs over a 14- day period (ie mRNA-based).
MilliporeSigma’s Simplicon™ RNA Reprogramming technology is a safe and efficient method to generate integrationfree, virus-free human iPS cells using a single transfection step of self-replicating RNA. The technology is based upon a positive strand, single-stranded RNA species derived from non-infectious (nonpackaging), self-replicating Venezuelan equine encephalitis (VEE) virus.
The Simplicon RNA replicon is a synthetic in vitro transcribed RNA expressing all four reprogramming factors (OKS-iG; Oct4, Klf4, Sox2 and Glis1) in a single polycistronic transcript that is able to self-replicate for a limited number of cell divisions, offering a more controlled method of cellular reprogramming (Figure 19).
MTI-GlobalStem (www.mti-globalstem.com) is utilising its expertise in gene delivery systems and stem cell biology to develop and supply scientists with novel, high-quality tools and reagents used to advance stem cell and drug discovery research. MTI-GlobalStem recently introduced its PluriQ™ G9 Reprogramming and Maintenance Media, two defined, xeno-free cell culture systems for creating iPSC from somatic cells and expanding and maintaining undifferentiated cells in culture long-term, respectively.
These G9 cell culture systems support a highly flexible experimental set-up, where stem cells currently grown in other feeder-free media can be directly passaged into PluriQ™ G9 medium after harvesting. The G9 media systems were optimised for and have been validated with MTIGlobalStem’s mRNA-based transfection reagents, mRNA-In® and mRNA-In® Stem, for researchers interested in a highly efficient, safe mRNA-based iPS reprogramming method without the worry of genomic integration or to efficiently direct the differentiation of the pluripotent stem cells into terminally differentiated cell types.
The process of reprogramming cells requires cell culture media that has consistent high quality from one lot to the next. Each lot of cell culture medium from MTIGlobalStem is rigorously tested under its PluriQ™ standards for quality, purity and the effective maintenance of pluripotent stem cells over multiple passages to ensure culture health, morphology and lack of unwanted differentiation (Figure 20).
Pluriomics (www.pluriomics.com) is the only company working at the interphase of human cardiac cell products, novel assay development and screening services. Pluriomics developed a novel proprietary method for differentiation of fully functional human iPSC-derived cardiomyocytes using a 100% chemically defined, serum-free medium that enhances their maturation and function.
Pluriomics has focused its effort on the production and industrialisation of high-quality, fully functional Pluricyte® Cardiomyocytes which are now available for customers. Pluriomics also commercialises Pluricyte® Maturation Medium and has just launched a Pluricyte® Cardiomyocytes differentiation kit to generate high-quality contracting cardiomyocytes in adherent monolayer formats. Pluricyte® Cardiomyocytes have a high purity with a predominant ventricular phenotype.
Pluriomics recently demonstrated that its cells also present the unique advantages to exhibit a relatively high level of maturity, when compared to other human stem cell-derived cardiomyocytes, which is indicated by improved contractility force, increased upstroke velocities, lower resting membrane potentials and improved sarcomeric organisation. Moreover, Pluricyte® Cardiomyocytes cells are fully functional and have well-pronounced depolarisation and repolarisation peaks, enabling easy detection of field potential durations in multielectrode array (MEA) assays.
The company also leverages Pluricyte® Cardiomyocytes advantages for the development and realisation of electrophysiology-, biochemistry- and contraction-based assays for predictive safety pharmacology, toxicology testing and efficacy screening. Pluriomics works closely with its partners to develop customised assays and offers these in a fee-for-service testing model (Figure 21).
ReproCELL (www.reprocell.com), the first stem cell company founded in Japan (2003), and its group companies, BioServe (US), Stemgent (US), Reinnervate (UK) and Biopta (UK), serve as a single source solution for reagents, services and training across the entire regenerative medicine workflow – patient to patient. More specifically, ReproCELL supplies reagents to support researchers in the derivation, maintenance and differentiation of iPS cell lines, eg the ReproNaive™ cell culture medium for conversion of primed state iPSC to naive state cells.
Uniquely, ReproCELL is able to procure primary patient samples from both normal and a diverse array of disease backgrounds. Subsequent application of Stemgent’s industry-leading RNA reprogramming technology enables researchers to generate clinically compliant and integration-free iPS cell lines from both human blood and fibroblasts in their own labs or through Stemgent’s service portal.
Additionally, ReproCELL group companies supply a variety of reagents such as small molecules, growth factors and culture media to support the differentiation of pluripotent stem cell lines. For researchers looking for readily-available differentiated cell types, ReproCELL offers off-the-shelf iPS-derived hepatocytes (ReproHepato), neurons (ReproNeuro) and cardiomyocytes (ReproCardio), ready for use in endpoint assays. Notably, by applying Reinnervate 3D cell culture technology to these differentiated cell types, ReproCELL is able to enhance their performance.
Researchers enter the stem cell research space from diverse backgrounds and experience. As such ReproCELL is able to support more experienced stem cell researchers with individual stem cell reagents and kits while supporting those less experienced through a combination of services (primary cell line establishment, RNA reprogramming and differentiation), training and off-the-shelf differentiated cells (Figure 22).
Thermo Fisher Scientific (www.thermofisher.com/ stemcells) focuses on developing tools that allow you to simplify your stem cell workflow and provide you with more control – allowing for faster, more efficient systems. It offers tools ranging from manipulation of PSCs using novel approaches for reprogramming, long-term culture and propagation, and characterisation of these cells through to gene editing and differentiation of the final desired cell type.
The unique aspect of these products is that they can be valuable in applications ranging from basic research to disease modelling to cell therapy research. Recently launched in to the Gibco stem cell media portfolio, Essential 8 Flex medium offers a paradigm change to traditional PSC culture approaches by offering the only commercially available media kit to go two consecutive days without feeding cells.
In addition, this optimised variation of the original Essential 8 Medium consistently maintains pluripotency by stabilising heat sensitive components such as FGF 2 and offers flexibility across multiple substrates and dissociation reagents. Also recently added to the Gibco stem cell media portfolio are simple and efficient induction and differentiation medium such as the launch of the PSC Dopaminergic Neuron Differentiation Kit, which enables differentiation of PSCs to dopaminergic neurons with increased flexibility, speed and scalability, all while retaining proper biological relevance. This kit uniquely allows creation of workable banks of precursor cells and helps significantly increase resultant DA neuron purity (Figure 23).
Table 1 summarises the latest iPSC product offerings, supporting tools and services reported in the vendor updates. Offerings reported fall into three broad categories: 1) commercial cells; 2) stem cell workflow tools; and 3) services.
1. Commercial cells: These are pure, tissue-specific cell types derived from human iPSC available in unlimited quantities as ‘off-the shelf’ terminally differentiated cell aliquots. The majority of commercial cells purchased today are either cardiomyocytes, neurons or hepatocytes, although many other iPSC-derived cell types are becoming available, eg smooth muscle cells, skeletal muscle cells and pancreatic beta cells to name a few.
Most of the iPSC-derived cells available from Axiogenesis, Axol Bioscience, Clontech, CDI, Pluriomics and ReproCELL can been described as representing ‘typical’ human biology. Although in the case of cardiomyocytes there are now quite a large number of different offerings available for purchase, most are based on mixed cell populations, with varying degrees of ventricular and atrial enrichment and levels of cell maturity.
End-users need to be mindful that not all commercially available iPSC-derived cardiomyocytes are the same or can be expected to elicit identical responses to drug or reference compounds in the various cardiac disorders or diseases modelled. Some vendors (ATCC, Axol, CDI and ReproCELL) describe iPSC-derived offerings that are representative of specific genders, age or ethnic backgrounds or are derived from healthy patients as well as those with specific disease backgrounds.
These are expected to have greater relevancy for drug discovery applications as they serve as more pertinent models of disease conditions and take into account genetic variation among populations.
2. Stem cell workflow tools: These tools aid the competent researcher to reprogramme, culture, differentiate, expand and characterise iPSC for themselves. Included in this category are a number of new iPSC culture systems that are either feederfree, serum-free or xeno-free, eg ReproCELL’s ReproNaive™ medium promotes the conversion of primed state iPSC to naive state cells; Thermo Fisher Scientific Gibco Essential 8 Flex medium that is optimised for maintaining pluripotency of stem cells; and MTI-GlobalStem PluriQ™ G9 Reprogramming Media for creating iPSC from somatic cells.
Details are also given of a reprogramming technology from MilliporeSigma that generates integration free, virus-free human iPS cells using a single transfection step of self-replicating synthetic RNA. Also available are off-the-shelf solutions that enable researchers to direct the differentiation of their own iPS cell lines, eg Cellartis iPS Cell to Hepatocyte Differentiation System and Pluriomics Pluricyte® Cardiomyocytes differentiation kit. Thermo Fisher Scientific also offers gene editing tools.
Those based on CRISPR/Cas9 are generating a lot of interest for their ability to correct/ repair mutations in iPSC lines derived from patients as well as the ability to introduce diseaserelevant mutations in human iPSC derived from matched healthy donors.
3. Services: These offer those less skilled in stem cells or those lacking internal resources or capacity the ability to commission the derivation of a specific iPSC line, the custom development of a disease model or assay, screening against an iPSC-derived cellular system, or gene editing services. Of note here is Axiogenesis’ specific expertise in the development and validation of functional assays using neuronal and cardiac cells and its partnerships with pharmaceutical companies and CROs to lend unbiased expert advice in setting up, validating and troubleshooting iPSC-derived assays.
Axol Bioscience also provides a complete suite of services including cell sourcing, reprogramming, differentiation and gene editing. Using gene editing Axol can create isogenic and reporter cell lines for disease modelling and drug discovery. Pluriomics also works closely with its partners to develop customised cardiomyocytes assays and offers screening against these in a fee-for-service testing model.
In conclusion, there is now useful coverage in terms of the range of commercial iPSC-derived cells types offered, together with an increasing assortment of iPSC workflow tools and outsourced services. It is mainly down to individual labs (ie their budget, skill set, capacity, priorities, etc) how they choose the square the stem cell dilemma either by embarking on reprogramming and differentiation in-house or gaining immediate access by purchasing off-the-shelf iPSC-derived cell types or by engaging a third party to, say, create an isogenic cell line and disease model for them. What is clear is that iPSC-derived cells will feature to a greater extent in future toxicology/safety testing and phenotypic drug screening assays. DDW
Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation; assay methodologies and reagent offerings) to drug discovery and the life sciences. Since its formation 14 years ago, HTStec has published 122 market reports on enabling technologies and Dr Comley has authored 56 review articles in Drug Discovery World. Please contact firstname.lastname@example.org for more information about HTStec reports.
1 Induced Pluripotent Stem Cells In Research & Drug Discovery Trends 2015. Published by HTStec Limited, Godalming, UK, December 2015.
2 Ribeiro, MC et al (2015). Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro e Correlation between contraction force and electrophysiology.
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