Ex Vivo Organ Perfusion Systems for Disease Modeling and Therapeutic Applications in Small Animal Models

Digital light processing (DLP)-based bioprinting technology has been exploited for a wide range of applications in tissue repair, tissue regeneration, disease modeling and drug screening. In article number e270, Li and co-workers comprehensively summarize works about DLP-based bioprinting strategy to deliver biomedical materials and/or specific cells to create sophisticated structures for various tissue modeling and regeneration.

Simple SummaryHeart disease is a leading cause of death for both humans and dogs. Inherited heart diseases, including dilated cardiomyopathy (DCM), account for a proportion of these cases. Human and canine patients with DCM suffer from an enlarged heart that can no longer pump efficiently, resulting in heart failure. This causes symptoms or clinical signs like difficulty breathing, irregular heartbeat, and eventually death. The symptoms or clinical signs of this disease vary in age of onset at the beginning of symptoms, sex predisposition, and overall disease progression. Despite the many similarities in DCM in both species, only a few candidate genes so far have been linked to this disease in dogs versus tens of genes identified in human DCM. Additionally, the use of induced pluripotent stem cells, or engineered stem cells, has been widely used in the study of human genetic heart disease but has not yet been fully adapted to study heart disease in dogs. This review describes the current knowledge on the genetics and subtypes of naturally occurring DCM in dogs, and how advances in research might benefit the dog but also the human patient. Additionally, a novel method using canine engineered stem cells to uncover unknown contributions of mistakes in DNA to the progression of DCM will be introduced along with its applications for human DCM disease modeling and treatment.Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment.

Purpose of Review Regenerative medicine is transforming modern healthcare by offering personalized approaches to the treatment of diseases and injuries. With rapid market growth and significant breakthroughs in cell and biomaterial technologies, the field is poised to reshape the future of medicine. This short review highlights emerging trends, and the critical roles of human induced pluripotent stem cells (hiPSCs) and 3D biomanufacturing in advancing regenerative solutions and challenges. Recent Findings Recent studies have shown that the potentials of hiPSCs have been significantly limited by current monolayer (2D) or 3D suspension technologies. hiPSCs require physiologically relevant 3D microenvironment to support their migration and signaling and interact with the 3D environment as well as between hiPSCs themselves, regulating and sustaining their proliferation and pluripotency, thereby enabling long-term maintenance, expansion, and differentiation. Summary hiPSCs play a significant role in regenerative medicine, including applications in drug development, disease modeling, and tissue regeneration. Synthetic peptide hydrogels (PepGel), such as PGmatrix, have demonstrated the ability to enable physiologically relevant 3D biomanufacturing of hiPSCs by supporting the maintenance and growth of hiPSCs in a biologically natural manner while preserving their pluripotent integrity for high performance somatic cells and organoids production. Advanced automation and AI aided processing have been aligned to traditional 2D culture and 3D suspension systems, which need to be adapted into physiologically relevant biomanufacturing of 3D hiPSC and their derived products.

Bioprinting, an advanced three-dimensional (3D) printing technology that uses living cells and biomaterials as bioinks, has emerged as a transformative tool in regenerative medicine and tissue engineering. This article presents a detailed exploration of bioprinting's potential to revolutionize medical treatments, with a focus on its applications in tissue regeneration, organ printing, and disease modeling. We discuss the key technologies, challenges, and future directions in this rapidly evolving field, emphasizing its profound impact on healthcare and the development of personalized medicine.

Recent advances in applications of nanoparticles and decellularized ECM for organoid engineering

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.

Background The advent of human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) provided exciting tools for cardiovascular physiological studies, disease modeling and drug testing applications. Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single-cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. Objective To establish a novel platform to assess the active and passive mechanical properties of single-cell hPSC-CMs at different loading conditions and to demonstrate the potential of this approach for drug testing and disease modeling applications. Methods and results To allow morphological maturation, hPSC-CMs were treated with Tri-iodo-thyronine hormone, dexamethasone and Insulin-like growth factor-1. The hPSC-CM were then lifted and attached to a highly sensitive optical-force transducer and a piezoelectric length controller and electrically-stimulated. The attached hPSC-CM remained intact and contractile allowing evaluation of their passive and active mechanical properties. Utilizing this technique, single-cell hPSC-CMs exhibited positive length-tension (Frank-Starling) relationships, and appropriate inotropic, klinotropic, and lusitropic changes in response to treatment with isoproterenol. The unique potential of the approach for drug testing and disease modeling was exemplified by treating the cells with doxorubicin (a potential cardiotoxic anti-cancer agent) and omecamtiv mecarbil (a positive ionotropic drug currently in stage 3 clinical trial). The results of these studies recapitulated the drugs' known actions to suppress (doxorubicin) and augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility. Finally, novel insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties. Conclusion A novel method that allows direct active and passive force measurements from single hPSC-CMs at different loading conditions for the first time was established and validated. Our results highlight the potential implications of this novel approach for pharmacological studies and disease modeling studies. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): European Research Council

Advances in Cerebral Organoid Systems and their Application in Disease Modeling

The ABCs of autologous blood collection for ex vivo organ preservation

Hepatic organoids as a platform for liver disease modeling and the development of novel therapies.

The derivation of induced pluripotent stem cells (iPSCs) from mammalian somatic cells by ectopic expression of defined factors has rekindled scientific enthusiasm for studying flexibility in the differentiated states of mammalian somatic cells. Perhaps more importantly, it has also provided a practical technology that enables unprecedented access to human tissues with diverse genotypes and phenotypes via the generation of patient-specific pluripotent stem cells. Already, iPSCs have produced applications in disease modeling, toxicity testing, drug development, and clinical therapies. While we are only beginning to understand the molecular and epigenetic determinants that enable the successful conversion of somatic cells to iPSCs, the technological advancement in methods of iPSC derivation and their applications has been rapid. Nevertheless, many issues need to be resolved before the potential of iPSCs can be fully realized. In this chapter, we will discuss recent progress and current challenges for iPSC applications.

Extensive research has focused on the vasculature, aiming to understand its structural characteristics, functions, interactions with surrounding tissues, and the mechanisms underlying vascular-related pathologies. However, advancing our understanding of vascular biology requires more complex and physiologically relevant models that integrate physical, chemical, and biological factors. Traditional in vitro dish models cannot replicate three-dimensional (3D) architecture, multi-cell-type interactions, and extracellular environments. In vivo animal models, while more complex, present ethical concerns, high costs, and limited relevance to human physiology. As a result, increasing attention is being directed toward in vitro models, specifically vascular microphysiological systems (MPS) based on organ-on-a-chip (OoC) technologies. This review highlights the relevance and potency of vascular MPS, which leverage microfluidic channels and 3D structures to mimic the physiological environment, incorporate diverse cellular and acellular components, and support complex biological processes. Vascular MPS are already enabling deep investigation into vascular responses to physiological cues, interactions with healthy and pathological tissues, and applications in disease modeling and drug development.

Microphysiological systems (MPS) provide a highly controlled environment for the development and testing of human-induced pluripotent stem cell-based cardiac microtissues, with promising applications in disease modeling and drug development. Through optical measurements in such systems, we can quantify mechanical features such as motion and velocity during contraction. While these are useful for evaluating relative changes in muscle twitch, it remains challenging to quantify and characterize the actual active tension driving the contraction. Here, we aimed to quantify the active tension over time and space by solving an inverse problem in cardiac mechanics expressed by partial differential equations (PDEs). We formulated this as a PDE-constrained optimization problem based on a mechanical model defined for two-dimensional representations of the microtissues. Our optimization predicts active tension generated by the tissue as well as the fiber direction angle distribution. We used synthetic as well as experimental data to investigate the performance of our inversion protocol. Next, we employed the procedure to evaluate active tension changes in drug escalation studies of the inotropes omecamtiv mecarbil and Bay K8644. For both drug compounds, we observed a comparable increase in displacement, strain, and model-predicted active strain values upon higher drug doses. The estimated active tension was observed to be highest in the middle part of the tissue, and the fiber direction was mostly aligned with the longitudinal direction of the tissue. The computational framework presented here allows for spatiotemporal estimation of active tension in cardiac microtissues based on optical measurements. In the future, such methodologies might develop into valuable tools in drug development protocols.

Chapter 30 - Induced Pluripotent Stem Cells

Adipose-derived stem cells (ADSCs) have promising applications in tissue regeneration. Currently, there are only a few ADSC products that have been approved for clinical use. The clinical application of ADSCs still faces many challenges. Here, we review emerging strategies to improve the therapeutic efficacy of ADSCs in tissue regeneration. First, a great quantity of cells is often needed for the stem cell therapies, which requires the advanced cell expansion technologies. In addition cell-derived products are also required for the development of ‘cell-free’ therapies to overcome the drawbacks of cell-based therapies. Second, it is necessary to strengthen the regenerative functions of ADSCs, including viability, differentiation and paracrine ability, for the tissue repair and regeneration required for different physiological and pathophysiological conditions. Third, poor delivery efficiency also restricts the therapeutic effect of ADSCs. Effective methods to improve cell delivery include alleviating harsh microenvironments, enhancing targeting ability and prolonging cell retention. Moreover, we also point out some critical issues about the sources, effectiveness and safety of ADSCs. With these advanced strategies to improve the therapeutic efficacy of ADSCs, ADSC-based treatment holds great promise for clinical applications in tissue regeneration.