Decellularization of porcine kidney with submicellar concentrations of SDS results in the retention of ECM proteins required for the adhesion and maintenance of human adult renal epithelial cells.

When decellularizing kidneys, it is important to maintain the integrity of the acellular extracellular matrix (ECM), including associated adhesion proteins and growth factors that allow recellularized cells to adhere and migrate according to ECM specificity. Kidney decellularization requires the ionic detergent sodium dodecyl sulfate (SDS); however, this results in a loss of ECM proteins important for cell adherence, migration, and growth, particularly glycosaminoglycan (GAG)-associated proteins. Here, we demonstrate that using submicellar concentrations of SDS results in a greater retention of structural proteins, GAGs, growth factors, and cytokines. When porcine kidney ECM scaffolds were recellularized using human adult primary renal epithelial cells (RECs), the ECM promoted cell survival and the uniform distribution of cells throughout the ECM. Cells maintained the expression of mature renal epithelial markers but did not organize on the ECM, indicating that mature cells are unable to migrate to specific locations on ECM scaffolds.

Rapid target validation in a Cas9-inducible hiPSC derived kidney model.

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Aligned nanofiber scaffolds improve functionality of cardiomyocytes differentiated from human induced pluripotent stem cell-derived cardiac progenitor cells.

Cardiac progenitor cells (CPCs), capable of differentiating into multiple cardiac cell types including cardiomyocytes (CMs), endothelial cells, and smooth muscle cells, are promising candidates for cardiac repair/regeneration. In vitro model systems where cells are grown in a more in vivo-like environment, such as 3D cultures, have been shown to be more predictive than 2D culture for studying cell biology and disease pathophysiology. In this report, we focused on using Wnt inhibitors to study the differentiation of human iPSC-CPCs under 2D or 3D culture conditions by measuring marker protein and gene expression as well as intracellular Ca2+ oscillation. Our results show that the 3D culture with aligned nanofiber scaffolds, mimicing the architecture of the extracellular matrix of the heart, improve the differentiation of iPSC-CPCs to functional cardiomyocytes induced by Wnt inhibition, as shown with increased number of cardiac Troponin T (cTnT)-positive cells and synchronized intracellular Ca2+ oscillation. In addition, we studied if 3D nanofiber culture can be used as an in vitro model for compound screening by testing a number of other differentiation factors including a ALK5 inhibitor and inhibitors of BMP signaling. This work highlights the importance of using a more relevant in vitro model and measuring not only the expression of marker proteins but also the functional readout in a screen in order to identify the best compounds and to investigate the resulting biology.

A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell-derived kidney model for drug discovery.

Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

Phenotypic Screen for Cardiac Regeneration Identifies Molecules with Differential Activity in Human Epicardium-Derived Cells versus Cardiac Fibroblasts.

Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.

Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-ß Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-ß type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed, demonstrating the value of this biologically relevant and reproducible technology. In addition, this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells.

Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes.

Human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) hold great potential as an unlimited cell source for toxicity testing in drug discovery research. However, little is known about mechanisms of compound toxicity in hiPSC-Hep. In this study, modified mRNA was used to reprogram foreskin fibroblasts into hiPSC that were differentiated into hiPSC-Hep. The hiPSC-Hep expressed characteristic hepatic proteins and exhibited cytochrome P450 (CYP) enzyme activities. Next, the hiPSC-Hep, primary cryopreserved human hepatocytes (cryo-hHep) and the hepatic cell lines HepaRG and Huh7 were treated with staurosporine and acetaminophen, and the toxic responses were compared. In addition, the expression of genes regulating and executing apoptosis was analyzed in the different cell types. Staurosporine, an inducer of apoptosis, decreased ATP levels and activated caspases 3 and 7 in all cell types, but to less extent in Huh7. Furthermore, a hierarchical clustering and a principal component analysis (PCA) of the expression of apoptosis-associated genes separated cryo-hHep from the other cell types, while an enrichment analysis of apoptotic pathways identified hiPSC-Hep as more similar to cryo-hHep than the hepatic cell lines. Finally, acetaminophen induced apoptosis in hiPSC-Hep, HepaRG and Huh7, while the compound initiated a direct necrotic response in cryo-hHep. Our results indicate that for studying compounds initiating apoptosis directly hiPSC-Hep may be a good alternative to cryo-hHep. Furthermore, for compounds with more complex mechanisms of toxicity involving metabolic activation, such as acetaminophen, our data suggest that the cause of cell death depends on a balance between factors controlling death signals and the drug-metabolizing capacity.

Improved expression of recombinant human factor IX by co-expression of GGCX, VKOR and furin.

Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94% yield.

Expression and characterization of recombinant ecarin.

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K (m) value of 0.4 µM prethrombin-2 was determined but only a rough estimate could be made of the K (m) for prothrombin of 0.9 µM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.