Biology subjects

Stevens, K. R.

Publications and source records attributed to Stevens, K. R..

6 recordsLinked to original sources

bioRxiv2024

LiverMap pipeline for 3D imaging of human liver reveals volumetric spatial dysregulation of cirrhotic vasculobiliary architecture

The liver contains an intricate microstructure that is critical for liver function. Architectural disruption of this spatial structure is pathologic. Unfortunately, 2D histopathology - the gold standard for pathological understanding of many liver diseases - can misrepresent or leave gaps in our understanding of complex 3D structural features. Here, we utilized immunostaining, tissue clearing, microscopy, and computational software to create 3D multilobular reconstructions of both non-fibrotic and cirrhotic human liver tissue. We found that spatial architecture in human cirrhotic liver samples with varying etiologies had sinusoid zonation dysregulation, reduction in glutamine synthetase-expressing pericentral hepatocytes, regression of central vein networks, disruption of hepatic arterial networks, and fragmentation of biliary networks, which together suggest a pro-portalization/decentralization phenotype in cirrhotic tissue. Further implementation of 3D pathological analyses may provide a deeper understanding of cirrhotic pathobiology and inspire novel treatments for liver disease.

bioengineering↗

bioRxiv2024

Grayscale 4D Biomaterial Customization at High Resolution and Scale

Hydrogel biomaterials have proven indispensable for three-dimensional (3D) cell culture but have fallen short in replicating the innate physiochemical complexity of real tissue. Though traditional photolithography affords localized material manipulation, robust methods that govern when, where, and to what extent such phototailoring occurs throughout materials would be profoundly enabling towards fabricating more-realistic 3D tissue constructs. Here, we introduce "grayscale image z-stack-guided multiphoton optical-lithography" (GIZMO) as a generalizable and intuitive strategy to rapidly photomodulate materials in full 3D non-binary patterns at submicron resolutions spanning large volumes (>mm3). Highlighting its versatility, we employ GIZMO to variably photopattern biomolecule release from, protein immobilization to, and degradation within hydrogels based on biologically derived or synthetic grayscale image stacks with unprecedented complexity. We anticipate that GIZMO will enable new opportunities to probe and manipulate cell fates, as well as to engineer complex functional tissue.

bioengineering↗

bioRxiv2023

Highly Parallel Tissue Grafting for Combinatorial In Vivo Screening

Material- and cell-based technologies such as engineered tissues hold great promise as human therapies. Yet, the development of many of these technologies becomes stalled at the stage of pre-clinical animal studies due to the tedious and low-throughput nature of in vivo implantation experiments. We introduce a plug and play in vivo screening array platform called Highly Parallel Tissue Grafting (HPTG). HPTG enables parallelized in vivo screening of 43 three-dimensional microtissues within a single 3D printed device. Using HPTG, we screen microtissue formations with varying cellular and material components and identify formulations that support vascular self-assembly, integration and tissue function. Our studies highlight the importance of combinatorial studies that vary cellular and material formulation variables concomitantly, by revealing that inclusion of stromal cells can "rescue" vascular self-assembly in manner that is material-dependent. HPTG provides a route for accelerating pre-clinical progress for diverse medical applications including tissue therapy, cancer biomedicine, and regenerative medicine.

bioengineering↗

bioRxiv2022

Temporal dynamics of metabolic acquisition in grafted engineered human liver tissue

Liver disease affects millions globally and end-stage liver failure is only cured by organ transplant. Unfortunately, there is a growing shortage of donor organs and disparities in equitable access to transplants among different populations. Less than 10% of global transplantation needs are currently met, highlighting the demand for alternative therapies. Engineered liver tissue grafts that supplement organ function could address these demands. While engineered liver tissues built from human hepatocytes, endothelial cells, and fibroblasts encased in hydrogel have been successfully engrafted in rodent models previously, the extent to which these tissues express human liver metabolic genes and proteins remains unknown. Here, we built engineered human liver tissues and characterized their engraftment, expansion, and metabolic phenotype at sequential stages post-implantation by RNA sequencing, histology, and host serology. Expression of metabolic genes was observed at weeks 1-2, followed by cellular organization into hepatic cords by weeks 4-9.5. Furthermore, grafted engineered tissues exhibited progressive spatially restricted expression of critical functional proteins known to be zonated in the native human liver. To our knowledge, this is the first report of engineered human liver tissue zonation after implantation in vivo, which could have important translational implications for this field.

bioengineering↗

bioRxiv2020

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

Recent single cell analyses have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single cell behavior.

cell biology↗

bioRxiv2019

An omentum-inspired 3D PEG hydrogel for identifying ECM drivers of drug resistant ovarian cancer

Ovarian cancer (OvCa) is a challenging disease to treat due to poor screening techniques and late diagnosis. There is an urgent need for additional therapy options, as patients recur in 70% of cases. The limited availability of clinical treatment options could be a result of poor predictions in early stage drug screens on standard tissue culture polystyrene (TCPS). TCPS does not capture the mechanical and biochemical cues that cells experience in vivo, which can impact how cells will respond to a drug. Therefore, an in vitro model that captures some of the microenvironment features that the cells experience in vivo could provide better insights into drug response. In this study, we formed 3D multicellular tumor spheroids (MCTS) in microwells, and encapsulated them in 3D omentum-inspired hydrogels. SKOV-3 MCTS were resistant to Paclitaxel in our 3D hydrogels compared to a monolayer on TCPS. Toward clinical application, we tested cells from patients (ovarian carcinoma ascites spheroids (OCAS)), and drug responses predicted by using the 3D omentum-inspired hydrogels correlated with the reported pathology reports of those same patients. Additionally, we observed the presence of collagen production around the encapsulated SKOV-3 MCTS, but not on TCPS. Our results demonstrated that our 3D omentum-inspired hydrogel is an improved in vitro drug testing platform to study OvCa drug response for patient-derived cells, and helped us identify collagen 3 as a potential driver of Paclitaxel resistance.

bioengineering↗