Human induced pluripotent stem cell (hiPSC) models inherit genetic and phenotypic variation from their donors that can influence differentiation, function, and disease phenotypes, with implications for model validity and generalizability. While diversity is widely discussed in biomedical research and extensively theorized in fields like genomics, comparable guidance remains limited in hiPSC research. Effectively, diversity-related decisions are often made implicitly without clear criteria for when inclusion is methodologically warranted. We address this by (1) analyzing how diversity is defined and operationalized in (pre)clinical research, (2) extending genomics frameworks to outline stem cell-specific recommendations for describing and reporting diversity, and (3) introducing a decision framework based on four criteria (experimental purpose, biological plausibility, platform readiness, and statistical power) to determine when and how diversity should be incorporated. The overall goal is to establish a shared basis for transparent and reproducible diversity-related design and reporting.

Gene syntax-the order and arrangement of genes and their regulatory elements-shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus perturbs the transcription of adjacent genes, but the molecular basis of this effect remains poorly understood. In this work, we show that supercoiling-mediated feedback arises from transcription and regulates expression of adjacent genes in a syntax-specific manner. Using a suite of assays, we measured syntax- and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter, we built and improved compact gene circuits, tuning the expression mean, noise, and stoichiometry across delivery methods and cell types. Integrating supercoiling mediated feedback into models of gene regulation will expand our understanding of native and synthetic systems.

Cardiotoxicity is a significant challenge in cancer therapies, particularly with doxorubicin, a widely used anthracycline. More predictive in vitro models are needed to understand doxorubicin-induced cardiac damage and patient-specific responses. Here, human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs), cardiac fibroblasts (hPSC-cFBs), and endothelial cells (hPSC-ECs) were cultured in mono- or multi-cell-type formats and repeatedly treated with doxorubicin to mimic cumulative clinical exposure. A machine learning-based tool enabled continuous quantification of the early toxicity marker caspase-3/7 and accurately identified hPSC-CMs within mixed cultures. Notably, hPSC-ECs were more sensitive to doxorubicin than hPSC-CMs or hPSC-cFBs, with nitric oxide signaling contributing to the elevated cardiomyocyte toxicity observed in co-culture. These results question the conventional in vitro focus on cardiomyocytes regarding drug-induced cardiac damage, highlighting the interplay among different cardiac cell types in mediating the toxic effects of doxorubicin. Furthermore, the work demonstrates the potential of AI-based tools to provide scalable strategies for assessing drug-induced cardiotoxicity.

Background: Tumor organoid and tumor-on-chip (ToC) platforms replicate aspects of the anatomical and physiological states of tumors. They, therefore, serve as models for investigating tumor microenvironments, metastasis, and immune interactions, especially for precision drug testing. To map the changing research diversity and focus in this field, we performed a quality-controlled text analysis of categorized academic publications and clinical studies. Methods: Previously, we collected metadata of academic publications on organoids or organ-on-chip platforms from PubMed, Web of Science, Scopus, EMBASE, and bioRxiv, published between January 2011 and June 2023. Here, we selected documents from this metadata corpus that were computationally determined as relevant to tumor research and analyzed them using an in-house text analysis algorithm. Additionally, we collected and analyzed metadata from ClinicalTrials.gov of clinical studies related to tumor organoids or ToC as of March 2023. Results and Discussion: From 3551 academic publications and 139 clinical trials, we identified 55 and 24 tumor classes modeled as tumor organoids and ToC models, respectively. The research was particularly active in neural and hepatic/pancreatic tumor organoids, as well as gastrointestinal, neural, and reproductive ToC models. Comparative analysis with cancer statistics showed that lung, lymphatic, and cervical tumors were under-represented in tumor organoid research. Our findings also illustrate varied research topics, including tumor physiology, therapeutic approaches, immune cell involvement, and analytical techniques. Mapping the research geographically highlighted the focus on colorectal cancer research in the Netherlands, though overall the specific research focus of countries did not reflect regional cancer prevalence. These insights not only map the current research landscape but also indicate potential new directions in tumor model research.

Organ-on-chips (OoCs) are controlled microfluidic platforms that replicate specific organ-level functionalities and pathological processes using cultured cells. OoCs promise to enhance drug discovery, reduce dependence on animal models, and enable personalized treatments. However, OoCs also introduce ethical challenges. This article provides a mapping review of the philosophical and ethical issues associated with developing and using OoCs. Given the limited literature on OoC ethics, the review takes a comparative analytical approach, drawing on organoid, digital twin, and precision medicine literature. Ethical issues are categorized across three consecutive phases-research, clinical testing, and implementation. Nine key themes are identified: privacy and confidentiality, informed consent, evidence, ontology and moral status, animal experimentation, evidence standards, patient care, intellectual property and commercialization, and distributive justice. Overall, the review highlights several key challenges that require further normative inquiry and hold significance for both research and policy. These include underrepresented groups in OoC research, complexities and limitations related to different consent models, the need for clear criteria to determine evidence standards for replacing animal models, accountability in the standardization of OoC research, and sustainability.

Advancing the use of human stem cell-based models on preclinical and regulatory testing fields requires the performance of rigorous and reproducible research. Quality standards and reporting best practices should be promoted to ensure the reliability and translatability of stem cell models and results. Strategies to increase awareness and implementation of best practices and standards will require training initiatives and collaboration across relevant stakeholders. Overall, improving the quality and reproducibility of stem cell-based models and methods through best practices and standards will accelerate their adoption in industrial and regulatory contexts and ultimately drive the development of effective therapies and safer chemicals.

Current cardiac cell models for drug screening often face a trade-off between cellular maturity and throughput. 3D human-induced pluripotent stem cell (hiPSC)-based heart models typically exhibit adult-like features, but their use often requires large cell numbers or complex equipment. In this study, we developed cost-effective methods to scale the production of stem cell-derived cardiac microtissues (cMTs) containing three cardiac cell types and assess calcium transients and action potential metrics for high-throughput screening (HTS). Automating the procedure revealed reproducible drug responsiveness and predictive accuracy in a reference compound screen. Furthermore, an arrhythmic phenotype was reliably triggered in cMTs containing cardiomyocytes with an RYR2 mutation. Screening a library of more than 2000 compounds demonstrated the suitability of the assay for identifying potential antiarrhythmic agents. Our findings underscore the scalability of cMTs and their utility in disease modeling and HTS. The advanced technology readiness level of cMTs supports their regulatory uptake and acceptance within the pharmaceutical industry.

A subset of coronavirus disease 2019 (COVID-19) patients develops severe symptoms, characterized by acute lung injury, endothelial dysfunction and microthrombosis. Viral infection and immune cell activation contribute to this phenotype. It is known that systemic inflammation, evidenced by circulating inflammatory factors in patient plasma, is also likely to be involved in the pathophysiology of severe COVID-19. Here, we evaluate whether systemic inflammatory factors can induce endothelial dysfunction and subsequent thromboinflammation. We use a microfluidic Vessel-on-Chip model lined by human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs), stimulate it with plasma from hospitalized COVID-19 patients and perfuse it with human whole blood. COVID-19 plasma exhibited elevated levels of inflammatory cytokines compared to plasma from healthy controls. Incubation of hiPSC-ECs with COVID-19 plasma showed an activated endothelial phenotype, characterized by upregulation of inflammatory markers and transcriptomic patterns of host defense against viral infection. Treatment with COVID-19 plasma induced increased platelet aggregation in the Vessel-on-Chip, which was associated partially with formation of neutrophil extracellular traps (NETosis). Our study demonstrates that factors in the plasma play a causative role in thromboinflammation in the context of COVID-19. The presented Vessel-on-Chip can enable future studies on diagnosis, prevention and treatment of severe COVID-19.

Myocardial infarction (MI) causes the loss of millions of cardiomyocytes, and current treatments do not address this root issue. New therapies focus on stimulating cardiomyocyte division in the adult heart, inspired by the regenerative capacities of lower vertebrates and neonatal mice. This review explores strategies for heart regeneration, offers insights into cardiomyocyte proliferation, evaluates in vivo models, and discusses integrating in vitro human cardiac models to advance cardiac regeneration research.

Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, TP53 is knocked out in hPSCs using CRISPR-Cas9 and compared them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency, and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. The results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, can impact their behavior and safety in a clinical setting.

Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is a vascular disorder caused by mutations in ACVRL1. We generated human induced pluripotent stem cell (hiPSC) lines from two HHT2 patients with a heterozygous 1 bp deletion in exon 7 of ACVRL1 (c.1042delG) by reprogramming skin fibroblasts. Gene-corrected isogenic hiPSCs were created using CRISPR-Cas9. All lines displayed normal karyotypes, expressed markers of the undifferentiated state, differentiated into all germ layers in vitro, and showed no off-target effects. These patient-derived, genetically matched hiPSC pairs provide a robust platform for modeling HHT2 in vitro and investigating molecular mechanisms of pathogenesis.

Engineered heart tissues (EHTs) have shown great potential in recapitulating tissue organization, functions, and cell-cell interactions of the human heart in vitro. Currently, multiple EHT platforms are used by both industry and academia for different applications, such as drug discovery, disease modelling, and fundamental research. The tissues' contractile force, one of the main hallmarks of tissue function and maturation level of cardiomyocytes, can be read out from EHT platforms by optically tracking the movement of elastic pillars induced by the contractile tissues. However, existing optical tracking algorithms which focus on calculating the contractile force are customized and platform-specific, often not available to the broad research community, and thus hamper head-to-head comparison of the model output. Therefore, there is the need for robust, standardized and platform-independent software for tissues' force assessment. To meet this need, we developed ForceTracker: a standalone and computationally efficient software for analyzing contractile properties of tissues in different EHT platforms. The software uses a shape-detection algorithm to single out and track the movement of pillars' tips for the most common shapes of EHT platforms. In this way, we can obtain information about tissues' contractile performance. ForceTracker is coded in Python and uses a multi-threading approach for time-efficient analysis of large data sets in multiple formats. The software efficiency to analyze circular and rectangular pillar shapes is successfully tested by analyzing different format videos from two EHT platforms, developed by different research groups. We demonstrate robust and reproducible performance of the software in the analysis of tissues over time and in various conditions. ForceTracker's detection and tracking shows low sensitivity to common incidental defects, such as alteration of tissue shape or air bubbles. Detection accuracy is determined via comparison with manual measurements using the software ImageJ. We developed ForceTracker as a tool for standardized analysis of contractile performance in EHT platforms to facilitate research on disease modeling and drug discovery in academia and industry.

Tissue fibrosis, with the excessive accumulation of extracellular matrix, leads to organ dysfunction. The ovary shows signs of fibrosis from an early age, creating a permissive environment for ovarian cancer. A robust culture-platform to study human ovarian fibrosis would enable screens for antifibrotic drugs to prevent or even reverse this process. Based on previous results showing that androgen therapy can induce ovarian fibrosis, we characterized the fibrotic state of ovaries from transmasculine donors of reproductive age. Anti-inflammatory and antioxidant drugs, such as Pirfenidone, Metformin, and Mitoquinone, could reduce and revert the excess collagen content of the ovarian cortical tissue during culture. We demonstrated that Metformin exerts an antioxidant role and prevents a glycolytic metabolic shift in non-immune ovarian stromal cells in the human ovary, while promoting early folliculogenesis during culture. These results may contribute to develop strategies to manage pro-tumorigenic fibrotic ovarian stroma in advanced age and metabolic disorders.

Each tissue and organ in the body has its own type of vasculature. Here, we demonstrate that organotypic vasculature for the heart can be recreated in a three-dimensional cardiac microtissue (MT) model composed of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs). ECs in cardiac MTs upregulated expression of markers enriched in human intramyocardial ECs, including CD36, CLDN5, APLNR, NOTCH4, IGFBP3, and ARHGAP18. We further show that the local microenvironment largely dictates the organ-specific identity of hiPSC-derived ECs: we compared ECs derived from cardiac and paraxial mesoderm and found that, regardless of origin, they acquired similar identities upon integration into cardiac MTs. Overall, the results indicated that while the initial gene profile of ECs was dictated by developmental origin, this could be modified by the local tissue environment. This developmental "plasticity" in ECs has implications for multiple pathological and disease states.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) alone often lack the maturity and intricacy required to act as a reliable cardiac disease model. 3D cardiac models have been developed to bridge the limitations of a 2D system. Previously, we have shown that a tri-cellular combination of cardiomyocytes, endothelial cells, and fibroblasts in 3D cardiac microtissues (MTs) enhances maturation. Here, we aim to generate cardiac MTs in a scalable method suitable for high-throughput drug screening and drug response prediction. Furthermore, using a MT model of catecholaminergic polymorphic ventricular tachycardia (CPVT1) and a calcium sensitive fluorescent dye we can assess, and subsequently rescue, cardiac arrhythmias. Differentiation protocols for all 3 cell types utilized a low-cost, small molecule based protocol in 2D. MTs were then generated and maintained in a 384-well plate using a Tecan Fluent® automated workflow, followed by end-point calcium analysis and acute drug treatment using the FDSS/μCELL screening system. Initial assay development confirmed reproducible generation of cardiac MTs over staggered days. Calcium assays were performed several weeks after MT generation, showing arrhythmias could be detected in CPVT1 MTs at all timepoints. The end-point calcium assay further confirmed acute rescue with flecainide, halting arrhythmias in ~97 % of arrhythmogenic CPVT1 MTs at each timepoint. Overall our results show that the generation of MTs can be streamlined using robotics, reliably producing MTs of similar quality to manual seeding. Using a 3D model of CPVT1 we are also able to observe an arrhythmogenic phenotype, apply acute drug treatment and monitor rescue in a high-throughput manner. End-point analysis of cardiac MTs can be performed as early as 14 days after generation, reducing the requirement for costly long-term culture. In summary, this study confirms the potential of using 3D cardiac MTs for cardiac disease modeling and high-throughput drug screening.

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influence transgene silencing, demonstrating the impact these design features have on reporter gene expression and forward programming of hiPSCs into neurons, motor neurons, and endothelial cells. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of different transcription factors within the same cell. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

Generation of functional vasculature within organoids is considered important for their development and maturation. However, direct differentiation of endothelial cells (ECs) in vasculature often still requires transplantation into host animals. This review discusses recent strategies for generating preorganoids, that include co-differentiation of ECs using growth factors or (an inducible transcription factor) ETV2, controlled assembly of tissue organoids with hPSC-derived ECs or Blood Vessel Organoids (BVOs), and 3D bioprinting. Additionally, the potential and key challenges of organ-on-chip technology for creating perfusable and functional vascular networks in organoids are explored, highlighting their implications for advancing research and improving experimental models of human tissue and disease.

Stem cell research is the product of cumulative, integrated effort between and within laboratories and disciplines. The many collaborative steps that lead to that special “Eureka moment”, when something that has been a puzzle perhaps for years suddenly become clear, is among the greatest pleasures of a scientific career. In this essay, the serendipitous pathway from first acquaintance with pluripotent stem cells to advanced cardiovascular models that emerged from studying development and disease will be described. Perhaps inspiration for later generations of stem cell researchers simply to follow whatever they find interesting.