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Abstract Lipid droplets (LDs) are dynamic organelles that serve as metabolic hubs and emerging regulators of immune cell fate. Although LDs have been implicated in immune regulation, how LD metabolism is remodeled during neutrophil differentiation and how stage‐specific LD dynamics shape mature neutrophil function remain poorly defined. Here, we profiled LD dynamics during neutrophil development and evaluated their role in innate immune function. Using stimulated Raman scattering (SRS) microscopy, we performed label‐free, quantitative mapping of LD accumulation in two complementary differentiation systems: murine Hoxb8 myeloid progenitors and human pluripotent stem cell (hPSC)‐derived neutrophils. To define metabolic requirements, we pharmacologically modulated LD biosynthesis and catabolism throughout neutrophil differentiation. Perturbing LD metabolism did not impair differentiation efficiency or lineage commitment, but significantly altered the functional output of mature neutrophils. In the murine system, inhibition of adipose triglyceride lipase (ATGL)‐mediated LD breakdown enhanced reactive oxygen species (ROS) production and increased anti‐tumor cytotoxicity against GL261 glioma cells. In the hPSC model, ATGL inhibition during the myeloid progenitor‐to‐neutrophil transition selectively increased intracellular LD accumulation without compromising neutrophil yield or purity. This metabolic rewiring also elevated ROS production in hPSC‐derived neutrophils, although cytotoxic enhancement against U87MG glioblastoma cells was less pronounced than in the murine system. Collectively, these findings define a stage‐specific LD metabolic landscape during neutrophil development and highlight targeted LD modulation as a potential strategy to enhance the functional potency of therapeutic neutrophils. 

ABSTRACT Organoids are considered a novel modeling platform for studying human biology and advancing health research. With the ability to demonstrate complex 3D structure and multicellular interactions, organoids have advanced studies in all major organs as a reliable model. In this study, we generated an advanced cardiovascular organoid by using a genome‐edited human pluripotent stem cell line with inducibleSOX17expression, enabling controlled endothelial specification, adjustable cell‐type composition, and human heart‐like morphology. Our organoids recapitulated the cardiotoxic phenotypes of FDA‐approved chemotherapeutic doxorubicin, manifesting as decreased cell viability and diminished contractile activity. Cryoinjury‐induced myocardial infarction in our organoids led to reduced beating, viability, and α‐actinin expression, along with increased fibroblast formation, which were mitigated by Captopril. Lastly, isoproterenol treatment increased peak Ca²⁺transient amplitude and shortened APD₅₀in our organoids, consistent with previously reported β‐adrenergic responses. In summary, we established a protocol for generating in vitro 3D cardiovascular organoids with controllable cellular composition and heart‐like structures, providing a robust and easy‐to‐produce platform for future studies of human heart disease. 

Cardiac fibrosis is a pathological hallmark of almost all forms of heart disease, characterized by excessive deposition of extracellular matrix (ECM) proteins by activated fibroblasts, leading to cardiomyocyte hypertrophy, arrhythmias, and heart failure. Current treatments, predominantly pharmacological, target signaling pathways involved in fibroblast activation but often come with side effects such as cardiac toxicities. There is a critical need for therapies that specifically target activated cardiac fibroblasts to mitigate these adverse effects. Recent advances have shown that chimeric antigen receptor (CAR)-T cells targeting fibroblast activation protein (FAP), expressed by activated fibroblasts, can significantly reduce fibrosis and improve cardiac function in mouse models. However, CAR-T cell therapies face challenges such as the requirement for large quantities of healthy primary immune cells, lengthy process, and the high cost of personalized treatments. To address these issues, we propose an innovative strategy using off-the-shelf CAR-neutrophils derived from human pluripotent stem cells (hPSCs). We hypothesize that hPSC-derived CAR-neutrophils engineered to target FAP will effectively reduce cardiac fibrosis and improve cardiac function post-injury due to their potent cytotoxic effects and ability to infiltrate infarct regions. To test this hypothesis, anti-FAP CAR hPSCs were generated by CRISPR/Cas9 genome editing and differentiated into neutrophils. The differentiated anti-FAP CAR hPSC-neutrophils exhibited molecular characteristics comparable to unmodified hPSC-neutrophils. We also established an in vitro cardiac fibrosis model utilizing a previously reported protocol for the generation of hPSC-derived epicardial fibroblasts. Importantly, our anti-FAP hPSC-neutrophils exhibited significant cytotoxicity against activated epicardial fibroblasts, while unmodified hPSC-neutrophils showed no/minimal killing efficiency. This study suggests a proof-of-concept therapeutic approach against cardiac fibrosis utilizing FAP-targeting CAR-neutrophils. This strategy can potentially be adapted to treat fibrosis in other organs, thereby having a broad and significant impact on the treatment of various fibrotic diseases, ultimately contributing to longer, healthier human lives. 

Single-cell RNA sequencing (scRNA-seq) has transformed biomedical research by enabling transcriptomic analysis at single-cell resolution. Yet, existing computational approaches remain primarily data-driven and lack the ability to integrate research context, limiting their interpretability and impact on hypothesis generation or experimental planning. We present scChat, a large language model (LLM)–powered co-pilot for contextualized scRNA-seq analysis. Unlike conventional pipelines restricted to tasks such as cell type annotation or enrichment analysis, scChat has an interactive, reasoning-based framework. It combines quantitative algorithms with retrieval-augmented generation and a multi-agent architecture to support hypothesis validation, mechanistic interpretation, and next-step experimental design. Through showcase and benchmarking studies, we demonstrate that scChat not only achieves high accuracy in cell type annotation but also provides biologically grounded explanations and contextual insights. 

Background: Glioblastoma (GBM) is an aggressive brain tumor giving a poor prognosis with the current treatment options. The advent of chimeric antigen receptor (CAR) T-cell therapy revolutionized the field of immunotherapy and has provided a new set of therapeutic options for refractory blood cancers. In an effort to apply this therapeutic approach to solid tumors, various immune cell types and CAR constructs are being studied. Notably, macrophages have recently emerged as potential candidates for targeting solid tumors, attributed to their inherent tumorinfiltrating capacity and abundant presence in the tumor microenvironment. Materials and methods: In this study, we developed a chemically defined differentiation protocol to generate macrophages from human pluripotent stem cells (hPSCs). A GBM-specific CAR was genetically incorporated into hPSCs to generate CAR hPSC-derived macrophages. Results: The CAR hPSC-derived macrophages exhibited potent anticancer activity against GBM cells in vitro. Conclusion: Our findings demonstrate the feasibility of generating functional CAR-macrophages from hPSCs for adoptive immunotherapy, thereby opening new avenues for the treatment of solid tumors, particularly GBM. 

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.