Bring life to science with do-it-yourself, ready-made, and custom stem cell solutions
The International Society for Stem Cell Research (ISSCR) is the premier organization that connects scientists from around the world to advance the fields of stem cell research and regenerative medicine. The annual ISSCR conference was held in Boston, MA, on June 14–17, 2017, providing opportunities for attendees to share their research and expertise.
Adding to the body of expertise at ISSCR 2017, Takara Bio USA, Inc., offered attendees guidance, technologies, and services for advancing their stem cell studies. The new Cellartis iPSC gene editing systems are the latest addition to a broad portfolio that offers do-it-yourself, ready-made, and custom solutions. Additionally, we offer custom services for sourcing, banking, reprogramming, differentiation, and clinical hES cell line derivation so you can focus on your areas of expertise while we take care of the rest.
Check out the posters and Innovation Showcase talk we presented, as well as featured products and services, below.
Innovation Showcase Talk at ISSCR 2017
Footprint-free gene editing using CRISPR/Cas9 and single-cell cloning of edited human iPS cells
The combination of two powerful technologies (human induced pluripotent stem 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. Here we show a new workflow using 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, which results in a high number of edited and expandable hiPSC clones that maintain the hallmarks of pluripotency.
Download our poster presentations
By 2030, diabetes is predicted to be the seventh leading cause of death globally. Type I diabetes (T1D) is a chronic autoimmune variant of this disease characterized by pancreatic islet beta cell loss and dysfunction, which results in insufficient production of insulin and subsequent excess of blood glucose, leading to numerous complications. While advances in diabetes treatments, including the development of new classes of drugs, have made diabetes treatment more manageable, a gap in health outcomes between T1D patients and those without diabetes still remains. In addition to the development of new drugs, transplantation of pancreatic islets into patients, currently limited by donor availability, holds great promise for a diabetes cure. Stem cell-derived beta cells that faithfully recapitulate in vivo beta cell features have tremendous potential to advance diabetes treatments on both the drug discovery and regenerative medicine fronts. In vivo compounds known as incretins stimulate insulin production. Recent developments in therapeutics use incretin analogues, such as the GLP-1 analogue Exenatide, to induce insulin secretion. Finding new target receptors to treat diabetes requires in vitro models that secrete insulin in response to stimulation, like their in vivo counterparts. In particular, free-fatty acid receptors (FFARs) are under high scrutiny as likely candidates for new therapies.
One strong therapeutic target of interest today is FFAR1 (GPR40). We have recently developed a hiPSC-derived beta cell line (ChiPSC12) displaying beta cell markers like insulin, C-peptide, MAFA, and NKX6.1. Here, we present data from another hiPS cell line, ChiPSC22, which carries the HLA type A*02:01 that is strongly associated with the susceptibility to develop T1D. We present further characterization of these cell lines, showing expression data of GLP-1R and FFARs together with analysis of insulin secretion upon stimulation with incretins and GPR agonist, to demonstrate their suitability for drug development. In addition, these newly developed beta cell lines can be used in a format suitable for High-Throughput Screening (HTS), enabling a fast, reliable, and robust beta cell in vitro system for finding new diabetes therapies.
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. The ability to create hiPS cell lines from donors with disease specific-mutations and to edit mutations into specific backgrounds will enable discoveries with a new level of granularity. Despite progress in improving nuclease specificity and reducing off-target activity with 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, 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 that compromise their health and survival. Using the DEF-CS-500 culture system, we can culture hiPS cells in a monolayer with a very high rate of single-cell survival and clone expansion. We used 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, 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 efficiencies of up to 65% for the membrane protein CD81 in a hiPS cell population. We also achieved efficient, accurate knock-in using electroporation with long ssDNA donor fragments. We demonstrated that edited hiPS clones obtained with this workflow were still pluripotent and have a normal karyotype, even after further expansion. The data show this workflow using footprint-free editing via efficient delivery of Cas9/sgRNA RNP complexes and single-cell cloning of hiPS cells in modified media, results in a high number of edited and expandable hiPS clones that maintain the hallmarks of pluripotency.
Hepatocytes derived from human pluripotent stem cells (hPSC) have the potential to serve as a predictive human in vitro model systems for disease modeling and drug development studies, provided that they possess relevant hepatocyte functions. The liver performs over 500 functions and many in vitro models lack the ability to faithfully recapitulate these functions. In addition, existing differentiation protocols have not been robust enough for use with multiple hPSC lines, further hampering the use of hPSC-derived hepatocytes. Therefore, we have developed a robust differentiation protocol which recapitulates in vivo liver development and allows derivation of hepatocytes from multiple hPSC lines. Of 25 different hPSC lines tested, all lines were efficiently differentiated into highly homogenous hepatocyte cultures that exhibit important adult hepatocyte features, such as substantial CYP activities, low expression of fetal genes, and high expression of adult genes. More importantly, hepatocytes derived from multiple hPSC lines show diverse CYP activity profiles, thus reflecting the inter-individual variation present in the population. To allow generation of panels of cryopreserved hepatocytes from multiple hPSC lines, we have also developed a cryopreservation method for hPSC-derived hepatocytes. Like their fresh counterparts, the cryopreserved hPSC-derived hepatocytes have substantial CYP activities in the same range as in human primary hepatocytes. Importantly, a novel maintenance medium significantly improves hepatocyte functions, such as albumin and urea secretion, gluconeogenesis, glycogen storage, LDL-uptake, and lipid storage. We show the utility of the cryopreserved hPSC-derived hepatocytes for chronic toxicity studies. These functions are maintained for up to two weeks, nearly an order of magnitude greater than primary hepatocytes which rapidly lose their functionality within days in conventional 2D cultures. Taken together, our robust differentiation protocol together with the improved maintenance medium allow the reliable generation of mature hepatocytes from multiple hPSC lines. This can provide an inexhaustible source of human hepatocytes for use in in vitro disease modeling, drug discovery, metabolism, and chronic toxicity studies.
Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling as well as promising candidates for cell therapy applications. Here we present the development of a defined, feeder-free medium, without human- or animal-derived components. hiPSCs 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. Using this complete, clinical-grade culture medium, eight different hiPSC lines that were expanded as a 2D monolayer (2D culture) maintain high expression of pluripotent stem cell markers and lack any expression of differentiation markers over the 12–20 passages tested. In addition, no karyotype abnormalities were reported for any of the tested cell lines. In order to generate clinically relevant quantities of hiPSCs—109 and beyond—it is essential to develop efficient, yet robust 3D suspension cultures maintaining the same stability as 2D monolayer cultures. Previous reports in the literature of suspension cultures have typically described a reduced growth rate compared to monolayer cultures with a final cell concentration of 1–2 million cells per milliliter. We demonstrate that our culture system supports large-scale, 3D, non-adherent expansion of hiPSCs in suspension culture in a perfusion bioreactor. Furthermore, by optimizing perfusion rates and dissolved oxygen levels, we were able to expand hiPSCs by 1,100-fold within three passages over 11 days to a final concentration of five million cells per milliliter using our 3D suspension culture system. In summary, our clinical-grade culture system allows for efficient, robust, and scalable production of hiPSCs, thus facilitating the use of hiPSCs for research and large-scale 3D suspension clinical applications.
Featured Products and Services at ISSCR 2017
Guiding gene editing in stem cells
The CRISPR/Cas9 system is leading the way as an easy, robust editing mechanism 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.
Our new Cellartis iPSC gene editing kits provide a complete solution for creating edited clonal human iPSC lines via electroporation- or gesicle-based delivery of Cas9-sgRNA complexes. Already using an optimized editing protocol? Our single-cell cloning kit is ideal for promoting development of healthy clones from your edited hiPSCs.
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.
Beta cells were 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.
Cellartis Human Pluripotent Stem Cell Services
Reprogrammed somatic cells from patients are powerful precursors for the cellular models used to study diseases and find treatments. Making an effective disease model requires finding the right starting material from a qualified individual and achieving successful reprogramming.
With Cellartis Human Pluripotent Stem Cell Services, our team will perform tailored clinical grade hES derivation, sourcing, reprogramming, directed differentiation and/or banking services that deliver pure hiPSCs for your disease model.
Create >90% pure hiPS cell-derived hepatocytes with diverse genetic backgrounds
The Cellartis iPS Cell to Hepatocyte Differentiation System provides a complete solution for generating functional, hiPS cell-derived hepatocytes within three weeks. As an alternative to using primary hepatocytes for toxicology studies, these hepatocytes exhibit sufficient expression levels of drug-metabolizing enzymes and transporters and demonstrate stable functionality over time in culture. Derived from your own patient- or disease-specific hiPS cell lines, these cells can provide an accurate reflection of the metabolic diversity observed in the human population.