Ashley L. Gard

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.

Drug development suffers from a lack of predictive and human-relevant in vitro models. Organ-on-chip (OOC) technology provides advanced culture capabilities to generate physiologically appropriate, human-based tissue in vitro, therefore providing a route to a predictive in vitro model. However, OOC technologies are often created at the expense of throughput, industry-standard form factors, and compatibility with state-of-the-art data collection tools. Here we present an OOC platform with advanced culture capabilities supporting a variety of human tissue models including liver, vascular, gastrointestinal, and kidney. The platform has 96 devices per industry standard plate and compatibility with contemporary high-throughput data collection tools. Specifically, we demonstrate programmable flow control over two physiologically relevant flow regimes: perfusion flow that enhances hepatic tissue function and high-shear stress flow that aligns endothelial monolayers. In addition, we integrate electrical sensors, demonstrating quantification of barrier function of primary gut colon tissue in real-time. We utilize optical access to the tissues to directly quantify renal active transport and oxygen consumption via integrated oxygen sensors. Finally, we leverage the compatibility and throughput of the platform to screen all 96 devices using high content screening (HCS) and evaluate gene expression using RNA sequencing (RNA-seq). By combining these capabilities in one platform, physiologically-relevant tissues can be generated and measured, accelerating optimization of an in vitro model, and ultimately increasing predictive accuracy of in vitro drug screening.

The Notch pathway plays a pivotal role in regulating cell fate decisions in many stem cell systems. However, the full repertoire of Notch target genes in vivo and the mechanisms through which this pathway activity is integrated with other signaling pathways are largely unknown. Here, we report a transgenic mouse in which the activation of the Notch pathway massively expands the neural stem cell (NSC) pool in a cell context-dependent manner. Using this in vivo system, we identify direct targets of RBPJ/N1ICD in cortical NSCs at a genome-wide level through combined ChIP-Seq and transcriptome analyses. Through a highly conservative analysis of these datasets, we identified 98 genes that are directly regulated by N1ICD/RPBJ in vivo. These include many transcription factors that are known to be critical for NSC self-renewal (Sox2, Pax6, Tlx, and Id4) and the transcriptional effectors of the Wnt, SHH, and Hippo pathways, TCF4, Gli2, Gli3, Yap1, and Tead2. Since little is known about the function of the Hippo-Yap pathway in NSCs, we analyzed Yap1 expression and function in NSCs. We show that Yap1 expression is restricted to the stem cell compartment in the developing forebrain and that its expression is sufficient to rescue Notch pathway inhibition in NSC self-renewal assays. Together, results of this study reveal a previously underappreciated complexity and breadth of Notch1 targets in vivo and show direct interaction between Notch and Hippo-Yap pathways in NSCs.