Stem cell, gene editing, and protein purification expertise for the screening and automation community
The Society for Laboratory Automation and Screening (SLAS) promotes the synthesis of new technologies, partnerships, and ideas to improve laboratory workflows. The SLAS conference, held on February 4–8, 2017 in Washington, D.C., is an essential hub for a community of experts who are forging ahead to apply state-of-the-art technologies to find solutions for significant biological challenges in an interdisciplinary manner.
Adding to the body of expertise at SLAS2017, Takara Bio USA, Inc. offered attendees expert guidance, technologies, and services for advancing their stem cell, gene editing, and protein purification research. Our products, such as Cellartis hiPS beta cells, provide researchers with novel screening tools that bring unparalleled consistency to drug screening and automation efforts.
Check out the posters and exhibitor tutorial we presented, below.
Exhibitor tutorial at SLAS2017
Efficient, footprint-free gene editing of iPS cells using CRISPR/Cas9 for disease modeling and drug screening applications
The combination of two powerful technologies (human induced pluripotent stem [hiPS] cells and precise, footprint-free editing using CRISPR/Cas9) allows for a new level of sophistication in development of disease models, which should allow for rapid advancement in therapeutic technologies. The ability to create hiPS cell lines from different donors and to determine the effects of specific mutations created via gene editing within the donor-specific genetic background will enable discoveries with a new level of granularity. Despite progress in improving designer nuclease specificity and reducing off-target activity with the introduction of precise tools like CRISPR/Cas9, a major challenge for successful gene editing in hiPSCs is the lack of culture systems that allow researchers to isolate single hiPSCs with the desired mutations and to generate stable, healthy, clonal lines from edited cells.
Traditionally, hiPS cells are grown and passaged as colonies. In order to obtain single cells for cloning purposes, the colonies must first be dissociated into a single-cell suspension, which often results in cell death or premature differentiation. Furthermore, gene editing protocols often subject stem cells to harsh conditions (e.g., electroporation) that compromise their health and survival. In contrast to traditional methods, the Cellartis DEF-CS 500 Culture System allows culturing of hiPS cells in a monolayer and permits single-cell isolation. The specific compositions of the DEF-CS culture medium and coating reagent allow for a very high rate of single-cell survival and clone expansion. We applied this culture system to develop a complete workflow, starting with CRISPR/Cas9-mediated editing, using Cas9/sgRNA ribonucleoprotein (RNP) complexes delivered into hiPS cells via either electroporation or cell-derived nanoparticles called gesicles, and followed by successful single-cell cloning of edited hiPS cells. We chose non-DNA-based delivery methods to guarantee footprint-free editing of the hiPS cells. We achieved endogenous gene knockout (KO) efficiencies of up to 65% for the membrane protein CD81 in a hiPS cell population. Individual, edited hiPS cells were seeded into a 96-well plate either by limiting dilution or fluorescence activated cell sorting. Notably, the use of limiting dilution resulted in a very high recovery rate of single hiPS cells that went on to form clonal colonies. We also demonstrated that edited hiPS clones obtained with the described workflow were still pluripotent, even after further expansion.
The data show this workflow—footprint-free editing via efficient delivery of Cas9/sgRNA RNP complexes and single-cell cloning of hiPS cells using a modified DEF-CS culture protocol—results in a high number of edited and expandable hiPS clones that maintain the hallmarks of pluripotency.
Download our poster presentations
SLAS2017 track: Assay development and screening
By 2030, diabetes is predicted to be the seventh leading cause of death globally. Human induced pluripotent stem (iPS) cell-derived beta cells have tremendous potential to advance the treatment of diabetes. Insulin-secreting beta cells could serve as a renewable, in vitro model system for toxicity testing, vaccine development, and drug discovery. Drug discovery requires large numbers of beta cells with consistent characteristics from batch to batch, and until recently, cell production methods have been unable to deliver. To address this need, we have developed a standardized four-step differentiation protocol, mimicking embryonic development, which can generate industrial-scale quantities of insulin-producing beta cells for in vitro applications. Now commercially available, Cellartis hiPS beta cells allow vastly easier access to human beta cells and reduced variability compared to primary islet beta cells, and can be used as a predictive cellular assay for screening compounds that regulate insulin secretion.
Novel systems for rapid purification of recombinant proteins and antibodies: Capturem high-capacity membranes >>
Recombinant protein production is of immense importance in nearly all research settings, including academic research institutions, biopharmaceutical organizations, and enzyme and agricultural industries. Protein fusion tags are widely used in order to improve yields and enable purification and characterization of protein structure and function. Here, we describe a novel, nylon-membrane-based IMAC system that has protein binding capacity comparable to, or better than, resins at 75 mg or more per cm3 of membrane, due to the chemically enhanced surface area of the pores. However, unlike traditional resin-based systems, the entire purification process—from loading the lysate to eluting pure protein—can be completed at room temperature in less than five minutes.
SLAS2017 track: Cellular technologies
The combination of two powerful technologies (human induced pluripotent stem [hiPS] cells and precise, footprint-free editing using CRISPR/Cas9) allows for a new level of sophistication in cell biology research and disease model development. The ability to create hiPS cell lines from different donors and to determine the effects of specific mutations created via gene editing within the donor-specific genetic background will enable discoveries with a new level of granularity.
However, while the introduction of CRISPR/Cas9 technology has made gene editing easier to achieve (even in hiPS cells), obtaining single-cell clones of edited hiPS cells has been a major bottleneck. Traditionally, hiPS cells are grown and passaged as colonies. In order to obtain single cells for cloning purposes, the colonies must first be dissociated into a single-cell suspension, which often results in cell death or premature differentiation. In contrast to traditional methods, the Cellartis DEF-CS 500 Culture System allows culturing of hiPS cells in a monolayer and permits single-cell isolation.
The data show this workflow—footprint-free editing via efficient delivery of Cas9/sgRNA RNP complexes and single-cell cloning of hiPS cells using a modified DEF-CS culture protocol—results in a high number of edited and expandable hiPSC clones that maintain the hallmarks of pluripotency.
Here we present the development of a defined, feeder-free medium, without human- or animal-derived components. Human pluripotent stem cells (hPSCs) that are cultured in this medium for an extended period of time express expected stem cell markers, remain diploid, and can differentiate into cell types from the three germ layers. Our clinical-grade culture system allows for efficient, robust, and scalable production of hPSCs, thus facilitating the use of hPSCs for research and large-scale 3D suspension clinical applications.
Featured products and services at SLAS2017
Pluripotent stem cell culture system for gene editing and single-cell cloning
Gene editing protocols often subject stem cells to harsh conditions that compromise their survival (e.g., electroporation), a problem that is compounded by the innate challenges of single-cell culture for mutant isolation and characterization.
Overcoming these challenges, the Cellartis DEF-CS 500 Culture System sustains a feeder-free hiPS cell monolayer culture while performing the gene-editing technique of choice and ensures survival and proliferation once single cells are plated and expanded into clonal lines. The highly reproducible nature of the system, coupled with its ability to ensure an efficient and predictable growth rate, makes the DEF-CS culture system ideal for the expansion and scale-up of a homogeneous population of edited iPS cells.
Guiding gene editing in stem cells
No matter which gene-editing protocol you choose (transgene delivery via electroporation, virus, or cell-derived nanovesicles called gesicles), we have the tools to enable successful gene engineering in stem cells. While the Cellartis DEF-CS 500 Culture System provides the foundation for hiPS cell survival and the formation of clonal edited lines, our Guide-it tools support the overall editing workflow.
The CRISPR/Cas9 system is leading the way as an easy, robust editing mechanism in stem cells. Guide-it tools improve every step of your CRISPR/Cas9 workflow, from sgRNA production and delivery to indel identification.
Beta cells for modeling diabetes and metabolic disorders
The Cellartis hiPS Beta Cells (from ChiPSC12) Kit is a complete kit for investigating beta-cell function, modeling diabetes and pancreatic disorders, and screening compounds that regulate insulin expression and secretion. The kit contains beta cells frozen in a single-cell suspension along with media, supplements, and coating matrix.
The kit contains beta cells generated in vitro from the human iPS cell line ChiPSC12 using a standardized protocol that mimics embryonic development. The cells express insulin, C-peptide, MAFA, NKX6.1, PDX1, and UCN3 mRNA and protein, and they also secrete insulin and C-peptide in response to incretin stimulation. This makes them ideal for obtaining consistent results from drug screening studies.
A revolutionary solution for recombinant protein and antibody purification
Recombinant protein production is an important part of many studies in nearly all research settings, from academic institutions to biopharmaceutical and agricultural industries. Having the right tools to produce and purify tagged proteins or antibodies can make a big difference in the ability to efficiently obtain meaningful data and make the most of every experiment. While conventional methods require a lot of time and effort, Capturem His-Tagged Purification Kits mark the beginning of a protein purification revolution. By uniting speed, ease of use, and high capacity in one powerful system, protein and antibody work can move forward as never before.
Cellartis human pluripotent stem cell services
With Cellartis services, you can focus on your areas of expertise and leave the challenges to us. Since our scientists have over 15 years’ experience with human pluripotent stem cell culture and differentiation, you can be confident that we will deliver custom services to expand and enhance the stem cell capabilities and success of your research team.
Custom services include clinical-grade hES cell line derivation, sourcing, reprogramming, banking, and directed differentiation. Expect a close, worry-free collaboration with straightforward and prompt communication about timelines and deliverables.