Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives, including thyroid, thymus, lungs, liver, and pancreas, has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage, three lineage models with two, three, and four populations and three growth models are constructed. Additionally, three models for error distribution are defined, resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration, model selection, and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184, a finding corroborated by existing literature. Additionally, space-limited growth models, such as logistic and Gompertz growth, outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model, it is predicted that the optimal differentiation period is between 1.9 and 2.4 days, plating populations closer to 300 000 cells per well result in the highest yield efficiency, and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols, providing insights into developmental biology.

In this work, non-invasive high-spatial resolution three-dimensional (3D) X-ray micro-computed tomography (μCT) of healthy mouse lung vasculature is performed. Methodologies are presented for filtering, segmenting, and skeletonizing the collected 3D images. Novel methods for the removal of spurious branch artefacts from the skeletonized 3D image are introduced, and these novel methods involve a combination of distance transform gradients, diameter-length ratios, and the fast marching method (FMM). These new techniques of spurious branch removal result in the consistent removal of spurious branches without compromising the connectivity of the pulmonary circuit. Analysis of the filtered, skeletonized, and segmented 3D images is performed using a newly developed Vessel Network Extraction algorithm to fully characterize the morphology of the mouse pulmonary circuit. The removal of spurious branches from the skeletonized image results in an accurate representation of the pulmonary circuit with significantly less variability in vessel diameter and vessel length in each generation. The branching morphology of a full pulmonary circuit is characterized by the mean diameter per generation and number of vessels per generation. The methods presented in this paper lead to a significant improvement in the characterization of 3D vasculature imaging, allow for automatic separation of arteries and veins, and for the characterization of generations containing capillaries and intrapulmonary arteriovenous anastomoses (IPAVA).

A microrobotic method for fabricating multilayered poly(lactic acid) (PLA) biological scaffolds using micropipettes for tissue engineering applications is presented. Biological scaffolds are fabricated over several different substrates by drawing and solidification of a viscous liquid polymer solution pumped continuously through a glass micropipette. The proposed method produces highly aligned, multilayered, crisscrossed fiber scaffolds with user specified pore sizes and diameters in the range from 1 to 10 micrometer. Attachment, proliferation and differentiation of C2C12 mouse pluripotential cells seeded on individual, parallel, and intersecting fibers is successfully demonstrated. The proposed robotic methodology consistently provides parameterized biological scaffolds to aid studies in tissue engineering and to develop novel MEMS, filtration and controlled drug delivery devices