Human vascular organoids with a mosaic AKT1 mutation recapitulate Proteus syndrome.

Vascular malformation, a key clinical phenotype of Proteus syndrome, lacks effective models for pathophysiological study and drug development due to limited patient sample access. To bridge this gap, we built a human vascular organoid model replicating Proteus syndrome's vasculature. Using CRISPR/Cas9 genome editing and gene overexpression, we created induced pluripotent stem cells (iPSCs) embodying the Proteus syndrome-specific AKTE17K point mutation for organoid generation. Our findings revealed that AKT overactivation in these organoids resulted in smaller sizes yet increased vascular connectivity, although with less stable connections. This could be due to the significant vasculogenesis induced by AKT overactivation. This phenomenon likely stems from boosted vasculogenesis triggered by AKT overactivation, leading to increased vascular sprouting. Additionally, a notable increase in dysfunctional PDGFRß+ mural cells, impaired in matrix secretion, was observed in these AKT-overactivated organoids. The application of AKT inhibitors (ARQ092, AZD5363, or GDC0068) reversed the vascular malformations; the inhibitors' effectiveness was directly linked to reduced connectivity in the organoids. In summary, our study introduces an innovative in vitro model combining organoid technology and gene editing to explore vascular pathophysiology in Proteus syndrome. This model not only simulates Proteus syndrome vasculature but also holds potential for mimicking vasculatures of other genetically driven diseases. It represents an advance in drug development for rare diseases, historically plagued by slow progress.

Lung-Mimetic Hydrofoam Sealant to Treat Pulmonary Air Leak.

Pulmonary air leak is the most common complication of lung surgery, contributing to post-operative morbidity in up to 60% of patients; yet, there is no reliable treatment. Available surgical sealants do not match the demanding deformation mechanics of lung tissue; and therefore, fail to seal air leak. To address this therapeutic gap, a sealant with structural and mechanical similarity to subpleural lung is designed, developed, and systematically evaluated. This "lung-mimetic" sealant is a hydrofoam material that has alveolar-like porous ultrastructure, lung-like viscoelastic properties (adhesive, compressive, tensile), and lung extracellular matrix-derived signals (matrikines) to support tissue repair. In biocompatibility testing, the lung-mimetic sealant shows minimal cytotoxicity and immunogenicity in vitro. Human primary monocytes exposed to sealant matrikines in vitro upregulate key genes (MARCO, PDGFB, VEGF) known to correlate with pleural wound healing and tissue repair in vivo. In rat and swine models of pulmonary air leak, this lung-mimetic sealant rapidly seals air leak and restores baseline lung mechanics. Altogether, these data indicate that the lung-mimetic sealant can effectively seal pulmonary air leak and promote a favorable cellular response in vitro.

Cellular plasticity of the bone marrow niche promotes hematopoietic stem cell regeneration.

Hematopoietic stem cells (HSCs) regenerate after myeloablation, a procedure that adversely disrupts the bone marrow and drives leptin receptor-expressing cells, a key niche component, to differentiate extensively into adipocytes. Regeneration of the bone marrow niche is associated with the resolution of adipocytes, but the mechanisms remain poorly understood. Using Plin1-creER knock-in mice, we followed the fate of adipocytes in the regenerating niche in vivo. We found that bone marrow adipocytes were highly dynamic and dedifferentiated to leptin receptor-expressing cells during regeneration after myeloablation. Bone marrow adipocytes could give rise to osteolineage cells after skeletal injury. The cellular fate of steady-state bone marrow adipocytes was also plastic. Deletion of adipose triglyceride lipase (Atgl) from bone marrow stromal cells, including adipocytes, obstructed adipocyte dedifferentiation and led to severely compromised regeneration of HSCs as well as impaired B lymphopoiesis after myeloablation, but not in the steady state. Thus, the regeneration of HSCs and their niche depends on the cellular plasticity of bone marrow adipocytes.

Modeling and countering the effects of cosmic radiation using bioengineered human tissues.

Cosmic radiation is the most serious risk that will be encountered during the planned missions to the Moon and Mars. There is a compelling need to understand the effects, safety thresholds, and mechanisms of radiation damage in human tissues, in order to develop measures for radiation protection during extended space travel. As animal models fail to recapitulate the molecular changes in astronauts, engineered human tissues and "organs-on-chips" are valuable tools for studying effects of radiation in vitro. We have developed a bioengineered tissue platform for studying radiation damage in individualized settings. To demonstrate its utility, we determined the effects of radiation using engineered models of two human tissues known to be radiosensitive: engineered cardiac tissues (eCT, a target of chronic radiation damage) and engineered bone marrow (eBM, a target of acute radiation damage). We report the effects of high-dose neutrons, a proxy for simulated galactic cosmic rays, on the expression of key genes implicated in tissue responses to ionizing radiation, phenotypic and functional changes in both tissues, and proof-of-principle application of radioprotective agents. We further determined the extent of inflammatory, oxidative stress, and matrix remodeling gene expression changes, and found that these changes were associated with an early hypertrophic phenotype in eCT and myeloid skewing in eBM. We propose that individualized models of human tissues have potential to provide insights into the effects and mechanisms of radiation during deep-space missions and allow testing of radioprotective measures.

Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-ß signaling.

In tissue development and homeostasis, transforming growth factor (TGF)-ß signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-ß signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-ß signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-ß gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-ß signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.

Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

A multi-organ chip with matured tissue niches linked by vascular flow.

Engineered tissues can be used to model human pathophysiology and test the efficacy and safety of drugs. Yet, to model whole-body physiology and systemic diseases, engineered tissues with preserved phenotypes need to physiologically communicate. Here we report the development and applicability of a tissue-chip system in which matured human heart, liver, bone and skin tissue niches are linked by recirculating vascular flow to allow for the recapitulation of interdependent organ functions. Each tissue is cultured in its own optimized environment and is separated from the common vascular flow by a selectively permeable endothelial barrier. The interlinked tissues maintained their molecular, structural and functional phenotypes over 4 weeks of culture, recapitulated the pharmacokinetic and pharmacodynamic profiles of doxorubicin in humans, allowed for the identification of early miRNA biomarkers of cardiotoxicity, and increased the predictive values of clinically observed miRNA responses relative to tissues cultured in isolation and to fluidically interlinked tissues in the absence of endothelial barriers. Vascularly linked and phenotypically stable matured human tissues may facilitate the clinical applicability of tissue chips.

Engineering complexity in human tissue models of cancer.

Major progress in the understanding and treatment of cancer have tremendously improved our knowledge of this complex disease and improved the length and quality of patients' lives. Still, major challenges remain, in particular with respect to cancer metastasis which still escapes effective treatment and remains responsible for 90% of cancer related deaths. In recent years, the advances in cancer cell biology, oncology and tissue engineering converged into the engineered human tissue models of cancer that are increasingly recapitulating many aspects of cancer progression and response to drugs, in a patient-specific context. The complexity and biological fidelity of these models, as well as the specific questions they aim to investigate, vary in a very broad range. When selecting and designing these experimental models, the fundamental question is "how simple is complex enough" to accomplish a specific goal of cancer research. Here we review the state of the art in developing and using the human tissue models in cancer research and developmental drug screening. We describe the main classes of models providing different levels of biological fidelity and complexity, discuss their advantages and limitations, and propose a framework for designing an appropriate model for a given study. We close by outlining some of the current needs, opportunities and challenges in this rapidly evolving field.

Emerging Trajectories for Next Generation Tissue Engineers.

The field of tissue engineering has evolved from its early days of engineering tissue substitutes to current efforts at building human tissues for regenerative medicine and mechanistic studies of tissue disease, injury, and regeneration. Advances in bioengineering, material science, and stem cell biology have enabled major developments in the field. In this perspective, we reflect on the September 2021 virtual Next Generation Tissue Engineering symposium and trainee workshop, as well as our projections for the field over the next 15 years.

Cryogel-based Injectable 3D Microcarrier Co-culture for Support of Hematopoietic Progenitor Niches.

Although hematopoietic stem cell (HSC) transplantation can restore functional hematopoiesis upon immune or chemotherapy-induced bone marrow failure, complications often arise during recovery, leading to up to 25% transplant-related mortality in treated patients. In hematopoietic homeostasis and regeneration, HSCs in the bone marrow give rise to the entirety of cellular blood components. One of the challenges in studying hematopoiesis is the ability to successfully mimic the relationship between the stroma and hematopoietic stem and progenitor cells (HSPCs). This study and the described protocols propose an advantageous method for culturing and assessing stromal hematopoietic support in three dimensions, representing a simplified in vitro model of the bone marrow niche that can be transplanted in vivo by injection. By co-culturing OP9 bone marrow-derived stromal cells (BMSCs) and cKit+ Sca-1+ Lin- (KLS+ ) HSPCs on collagen-coated carboxymethylcellulose scaffolds for 2 weeks in the absence of cytokines, we established a methodology for in vivo subcutaneous transplantation. With this model we were able to detect early signs of extramedullary hematopoiesis. This work can be useful for studying various stromal cell populations in co-culture, as well as simple transfer by injection of these scaffolds in vivo for heterotopic regeneration of the marrow microenvironment. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of HSPCs from mice Basic Protocol 2: Co-seeding of HSPCs and BMSCs on collagen-coated CCMs Basic Protocol 3: Maintenance, real-time imaging, and analysis of co-seeded scaffolds Basic Protocol 4: End-point analysis of co-seeded scaffolds using flow cytometry and CFU assays Basic Protocol 5: Transplantation of scaffolds by subcutaneous injection Support Protocol: Preparation of custom scaffold drying device.

Harnessing organs-on-a-chip to model tissue regeneration.

Tissue engineering has markedly matured since its early beginnings in the 1980s. In addition to the original goal to regenerate damaged organs, the field has started to explore modeling of human physiology "in a dish." Induced pluripotent stem cell (iPSC) technologies now enable studies of organ regeneration and disease modeling in a patient-specific context. We discuss the potential of "organ-on-a-chip" systems to study regenerative therapies with focus on three distinct organ systems: cardiac, respiratory, and hematopoietic. We propose that the combinatorial studies of human tissues at these two scales would help realize the translational potential of tissue engineering.

Engineered models of tumor metastasis with immune cell contributions.

Most cancer deaths are due to tumor metastasis rather than the primary tumor. Metastasis is a highly complex and dynamic process that requires orchestration of signaling between the tumor, its local environment, distant tissue sites, and immune system. Animal models of cancer metastasis provide the necessary systemic environment but lack control over factors that regulate cancer progression and often do not recapitulate the properties of human cancers. Bioengineered "organs-on-a-chip" that incorporate the primary tumor, metastatic tissue targets, and microfluidic perfusion are now emerging as quantitative human models of tumor metastasis. The ability of these systems to model tumor metastasis in individualized, patient-specific settings makes them uniquely suitable for studies of cancer biology and developmental testing of new treatments. In this review, we focus on human multi-organ platforms that incorporate circulating and tissue-resident immune cells in studies of tumor metastasis.

MarrowQuant Across Aging and Aplasia: A Digital Pathology Workflow for Quantification of Bone Marrow Compartments in Histological Sections.

The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-µCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in, MarrowQuant, which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner. MarrowQuant discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature, AdipoQuant, fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with MarrowQuant lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of in vivo recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies MarrowQuant successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.

Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety.

Traditional drug screening models are often unable to faithfully recapitulate human physiology in health and disease, motivating the development of microfluidic organs-on-a-chip (OOC) platforms that can mimic many aspects of human physiology and in the process alleviate many of the discrepancies between preclinical studies and clinical trials outcomes. Linsitinib, a novel anti-cancer drug, showed promising results in pre-clinical models of Ewing Sarcoma (ES), where it suppressed tumor growth. However, a Phase II clinical trial in several European centers with patients showed relapsed and/or refractory ES. We report an integrated, open setting, imaging and sampling accessible, polysulfone-based platform, featuring minimal hydrophobic compound binding. Two bioengineered human tissues - bone ES tumor and heart muscle - were cultured either in isolation or in the integrated platform and subjected to a clinically used linsitinib dosage. The measured anti-tumor efficacy and cardiotoxicity were compared with the results observed in the clinical trial. Only the engineered tumor tissues, and not monolayers, recapitulated the bone microenvironment pathways targeted by linsitinib, and the clinically-relevant differences in drug responses between non-metastatic and metastatic ES tumors. The responses of non-metastatic ES tumor tissues and heart muscle to linsitinib were much closer to those observed in the clinical trial for tissues cultured in an integrated setting than for tissues cultured in isolation. Drug treatment of isolated tissues resulted in significant decreases in tumor viability and cardiac function. Meanwhile, drug treatment in an integrated setting showed poor tumor response and less cardiotoxicity, which matched the results of the clinical trial. Overall, the integration of engineered human tumor and cardiac tissues in the integrated platform improved the predictive accuracy for both the direct and off-target effects of linsitinib. The proposed approach could be readily extended to other drugs and tissue systems.

Oxygen-Sensing Biomaterial Construct for Clinical Monitoring of Wound Healing.

OBJECTIVE: Oxygen is essential to wound healing; therefore, accurate monitoring can guide clinical decisions. Clinical wound assessment is often subjective, and tools to monitor wound oxygen are typically expensive, indirect, and highly variable. This study demonstrates the utility of a novel, low-cost oxygen-sensing thin film for serial assessment of wound oxygenation. DESIGN: Dual-layer films were fabricated with boron oxygen-sensing nanoparticles (BNPs) impregnated into a chitosan-polycaprolactone layer for direct wound bed contact with a relatively oxygen impermeable calcium alginate surface layer. The BNPs are a dual-emissive difluoroboron ß-diketonate dye incorporated into poly(lactic acid) nanoparticles. Under UV excitation, the BNPs emit fluorescence based on concentration and oxygen-sensitive phosphorescence. The fluorescence/phosphorescence ratio is directly proportional to oxygen concentration. METHODS: A series of in vitro oxygen challenges and in vivo murine and porcine wound healing models were used to validate the utility of the film in sensing wound oxygenation. MAIN RESULTS: In vitro testing demonstrated the oxygen-sensing capability of the BNP film and its ability to shield ambient oxygen to isolate wound oxygen. In vivo testing demonstrated the ability of the film to accurately monitor relative oxygen changes in a murine wound over time, measuring a 22% fluorescence/phosphorescence increase during acute healing. CONCLUSIONS: This study presents a low-cost, noninvasive, direct, and serial oxygen mapping technology to detect spatial differences in wound oxygenation. Clinical use of the films has the potential to monitor wound healing trajectories and guide wound care decisions.

From arteries to capillaries: approaches to engineering human vasculature.

From micro-scaled capillaries to millimeter-sized arteries and veins, human vasculature spans multiple scales and cell types. The convergence of bioengineering, materials science, and stem cell biology has enabled tissue engineers to recreate the structure and function of different hierarchical levels of the vascular tree. Engineering large-scale vessels has been pursued over the past thirty years to replace or bypass damaged arteries, arterioles, and venules, and their routine application in the clinic may become a reality in the near future. Strategies to engineer meso- and microvasculature have been extensively explored to generate models to study vascular biology, drug transport, and disease progression, as well as for vascularizing engineered tissues for regenerative medicine. However, bioengineering of large-scale tissues and whole organs for transplantation, have failed to result in clinical translation due to the lack of proper integrated vasculature for effective oxygen and nutrient delivery. The development of strategies to generate multi-scale vascular networks and their direct anastomosis to host vasculature would greatly benefit this formidable goal. In this review, we discuss design considerations and technologies for engineering millimeter-, meso-, and micro-scale vessels. We further provide examples of recent state-of-the-art strategies to engineer multi-scale vasculature. Finally, we identify key challenges limiting the translation of vascularized tissues and offer our perspective on future directions for exploration.

Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis.

Modeling the interaction between the supportive stroma and the hematopoietic stem and progenitor cells (HSPC) is of high interest in the regeneration of the bone marrow niche in blood disorders. In this work, we present an injectable co-culture system to study this interaction in a coherent in vitro culture and in vivo transplantation model. We assemble a 3D hematopoietic niche in vitro by co-culture of supportive OP9 mesenchymal cells and HSPCs in porous, chemically defined collagen-coated carboxymethylcellulose microscaffolds (CCMs). Flow cytometry and hematopoietic colony forming assays demonstrate the stromal supportive capacity for in vitro hematopoiesis in the absence of exogenous cytokines. After in vitro culture, we recover a paste-like living injectable niche biomaterial from CCM co-cultures by controlled, partial dehydration. Cell viability and the association between stroma and HSPCs are maintained in this process. After subcutaneous injection of this living artificial niche in vivo, we find maintenance of stromal and hematopoietic populations over 12 weeks in immunodeficient mice. Indeed, vascularization is enhanced in the presence of HSPCs. Our approach provides a minimalistic, scalable, biomimetic in vitro model of hematopoiesis in a microcarrier format that preserves the HSPC progenitor function, while being injectable in vivo without disrupting the cell-cell interactions established in vitro.

Design of a student-led organizational partnership to host an annual statewide Science Olympiad K-12 outreach tournament.

Since fall 2015, the University of Virginia's (UVA) Engineering Student Council (ESC) has partnered with the nonprofit Virginia Science Olympiad (VASO) organization to host a Science Olympiad (SciOly) state tournament in Charlottesville, Virginia, each spring. This annual tournament brings over 2,000 middle and high school students, teachers, and parents to the UVA campus, and teams of 15-17 people from roughly 90 schools across Virginia participate in 46 different events (23 middle school, Division B; 23 high school, Division C) relating to the science, technology, engineering, and mathematics (STEM) fields throughout the day-long competition. The national SciOly organization sets the events and rules to comply with national education standards, and the VASO board coordinates the teams and tournaments within the state. By collaborating with VASO, UVA ESC was able to plan a large-scale SciOly tournament at UVA in approximately 10 mo with the support of the UVA School of Engineering and Applied Science. Since this event was planned and executed solely by undergraduates in cooperation with the nonprofit organization, there were institutional hurdles that were overcome through the months of planning. The Virginia SciOly state tournament has continued to be held at UVA with the support and cooperation of the UVA ESC and VASO, and bringing this tournament to UVA has allowed for increased excitement for participating K-12 students and a mitigated burden to the VASO organizers in planning the state competition. This paper aims to provide a resource for other universities to support STEM activities in K-12 outreach organizations, like SciOly, in the future.

Design and implementation of a student-taught course on research in regenerative medicine.

In the Undergraduate School of Engineering and Applied Sciences (SEAS) at the University of Virginia (UVa), there are few opportunities for undergraduate students to teach, let alone develop, an introductory course for their major. As two undergraduate engineering students (D. N. Tavakol and C. J. Broshkevitch), we were among the first students to take advantage of a new initiative at UVa SEAS to offer student-led courses. As part of this new program, we designed a 1000-level, 1-credit, pass-fail course entitled Introduction to Research in Regenerative Medicine. During a student's first year at the University, opportunities to build research skills and gain exposure to topics within the field of the biomedical sciences are relatively rare, so, to fill this gap, we focused our course on teaching primarily freshman undergraduate students how to synthesize and contextualize scientific literature, covering both basic science and clinical applications. At the end of the course, students self-reported increased confidence in reading and discussing scientific papers and review articles. The critical impact of this course lies not only in an early introduction to the popularized field of regenerative medicine, but also encouragement for younger students to participate in research early on and to appreciate the value of interdisciplinary interactions. The teaching model can be extended for implementation of student-taught introductory courses across diverse undergraduate major tracks at an institution.