Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal.

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.

Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation.

Embryonic stem (ES) cells hold great promise with respect to their potential to be differentiated into desired cell types. Of interest are organs derived from the definitive endoderm, such as the pancreas and liver, and animal studies have revealed an essential role for Nodal in development of the definitive endoderm. Activin A is a related TGFß member that acts through many of the same downstream signaling effectors as Nodal and is thought to mimic Nodal activity. Detailed characterization of ES cell-derived endodermal cell types by gene expression analysis in vitro and functional analysis in vivo reveal that, despite their similarity in gene expression, Nodal and Activin-derived endodermal cells exhibit a distinct difference in functional competence following transplantation into the developing mouse embryo. Pdx1-expressing cells arising from the respective endoderm populations exhibit extended differences in their competence to mature into insulin/c-peptide-expressing cells in vivo. Our findings underscore the importance of functional cell-type evaluation during stepwise differentiation of stem cells.

Protein kinase A signalling via CREB controls myogenesis induced by Wnt proteins.

Select members of the Wnt family of secreted glycoproteins have been implicated in inducing the myogenic determinant genes Pax3, MyoD and Myf5 during mammalian embryogenesis, but the mechanism of induction has not been defined. We describe an unexpected role for protein kinase A (PKA) signalling via CREB in this induction. Using a combination of in vitro explant assays, mutant analysis and gene delivery into mouse embryos cultured ex vivo, we demonstrate that adenylyl cyclase signalling via PKA and its target transcription factor CREB are required for Wnt-directed myogenic gene expression. Wnt proteins can also stimulate CREB-mediated transcription, providing evidence for a Wnt signalling pathway involving PKA and CREB. Our findings raise the possibility that PKA/CREB signalling may also contribute to other Wnt-regulated processes in embryonic patterning, stem cell renewal and cancer.

Optimizing drug discovery by Investigative Toxicology: Current and future trends.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing.

Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFß as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

Reprogramming within hours following nuclear transfer into mouse but not human zygotes.

Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation.

Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.

An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.

Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.

Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc.

Derivation of human embryonic stem cells by immunosurgery.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass.

Prospective isolation and global gene expression analysis of definitive and visceral endoderm.

In spite of the therapeutic importance of endoderm derivatives such as the pancreas, liver, lung, and intestine, there are few molecular markers specific for early endoderm. In order to identify endoderm-specific genes as well as to define transcriptional differences between definitive and visceral endoderm, we performed microarray analysis on E8.25 definitive and visceral endoderm. We have developed an early endoderm gene expression signature, and clarified the transcriptional similarities and differences between definitive and visceral endoderm. Additionally, we have developed methods for flow cytometric isolation of definitive and visceral endoderm. These results shed light on the mechanism of endoderm formation and should facilitate investigation of endoderm formation from embryonic stem cells.

Targeting gene expression in the mouse somite: adenovirus-mediated gene delivery and whole embryo culture.

We report here a novel approach to direct gene expression in the mouse somite based on the combined application of adenovirus-mediated gene delivery and whole embryo ex vivo culture. As proof of principle, we show functional analysis of somites microinjected with an engineered virus expressing an activated form of Smoothened, the signaling receptor for Sonic Hedgehog (SHH). As adenovirus can infect many embryonic tissues in the mouse, this method may provide an effective alternative to conventional transgenesis for targeted spatial and temporal gene expression.

Microarray analysis of somitogenesis reveals novel targets of different WNT signaling pathways in the somitic mesoderm.

WNT signaling plays a major role in patterning the dermomyotome of the somitic mesoderm. However, knowledge of downstream target genes and their regulation is limited. To identify new genes involved in the development and early patterning of the somite, we performed a comparison of gene expression by microarray between the presomitic mesoderm and the 5 most recently formed somites of the mouse at embryonic day 9.5. We identified 207 genes upregulated and 120 genes downregulated in somite formation. Expression analysis and functional categorization of these genes demonstrate this to be a diverse pool that provides a valuable resource for studying somite development. Thus far, we have found three genes expressed in the dermomyotome of the early somite. Consistent with their expression patterns, these genes are transcriptional targets of WNT signals, but display differential activation by different WNTs. We further demonstrate that 1 of these genes, Troy, is a direct target of canonical WNT signaling, while the other 2 genes, Selp and Arl4, are not. Thus, our microarray study using microdissected tissues not only provides global information on gene expression during somite development, it also provides novel targets to study the inductive signaling pathways that direct somite patterning.