Chang, Yun

Abstract Glioblastoma (GBM) is the most common malignant tumor of the CNS, accounting for 80% of brain tumors. Recent advances in gene editing and immunology have led to the development of chimeric antigen receptors (CARs), including CARs incorporating chlorotoxin (CLTX) receptor domains for GBM targeting. While CAR-engineered neutrophils have shown promise, their systemic administration can induce severe off-target effects. Extracellular vesicles (EVs), secreted by these cells, retain properties of their parent cells—including CAR expression and tumor-lytic activity—yet their potential has not been explored in CAR Neutrophils. To address this, CAR-CLTX DNA was inserted into the AAV1 safe harbor locus of induced pluripotent stem cells, which were subsequently differentiated into neutrophils. Conditioned media were collected during differentiation and processed using extraPEG-based EV isolation. EVs were quantified via nanoparticle tracking analysis, electron microscopy, and western blotting. Proteomic and microRNA (miRNA) profiling of EV cargo was conducted at days 12 and 21 of differentiation. Cytotoxic effects of EVs were assessed in U87MG and LN229 glioblastoma cells in 2D culture, 3D organoids, and in vivo models. EVs were secreted in high quantities within the 100–200 nm size range, expressed canonical exosomal markers, and exhibited characteristic morphology. Proteomic analysis identified neutrophil-associated proteins involved in migration, chemotaxis, and degranulation. miRNA sequencing revealed high levels of tumor-suppressive miRNAs (e.g., miR-182) implicated in apoptotic signaling, T cell receptor pathways, cytokine-cytokine receptor interactions, Jak-STAT, Hedgehog, and NK cell-mediated cytotoxicity. EV uptake by glioma cells was demonstrated in vitro. Cytotoxic effects of CAR Neutrophil EVs were observed across glioblastoma cell lines, organoids, and in vivo. These findings demonstrate that CAR Neutrophil-derived EVs carry anti-tumor protein and miRNA cargo, are efficiently taken up by GBM cells, and mediate glioma cell cytotoxicity. This supports the feasibility of correlating EV cargo composition with function in GBM treatment.

Background: Cellular transplantation has emerged as promising approach for treating cardiac diseases. However, a poor engraftment rate limits our understanding on how transplanted cardiomyocytes contribute to cardiac function in the recipient’s heart. Methods: The CRISPR/Cas9 technique was employed for stable and constitutive gene expression in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). Myocardial infarction was induced in adult immunodeficient mice, followed by intramyocardial injection of hiPSC-CMs expressing either CCND2/channelrhodopsin 2 (hiPSC-CCND2OE/ChR2OECMs) or CCND2/luciferase (hiPSC-CCND2OE/LuciOECMs). Six months later, hemodynamics and intramural electrocardiogram were recorded upon blue light illuminations in anesthetized, open-chest mice. Results: Blue light resets automaticity of spontaneously beating hiPSC-CCND2OE/ChR2OECMs in culture, but not that of hiPSC-CCND2OE/LuciOECMs. Response to blue light was also observed in mice carrying large (>106 cells) intracardiac grafts of hiPSC-CCND2OE/ChR2OECM but not in mice carrying hiPSC-CCND2OE/LuciOECMs. The former exhibited single premature ventricular contractions upon light illumination or ventricular quadrigeminy upon second-long illuminations. At the onset of premature ventricular contractions, maximal systolic ventricular pressure decreased while ventricular volume rose concomitantly. Light-induced changes reversed upon resumption of sinus rhythm. Conclusions: We established an in vivo model for optogenetic-based modulation of the excitability of donor cardiomyocytes in a functional, reversible, and localized manner. This approach holds unique value for studying electromechanical coupling and molecular interactions between donor cardiomyocytes and recipient hearts in live animals.

AbstractProper cell‐cycle progression is essential for the self‐renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination‐based cell‐cycle indicator (FUCCI) has allowed the dual‐color visualization of the G1 and S/G2/M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage‐specific FUCCI reporters have not yet been developed to analyze complex tissue‐specific cell‐cycle progression during hPSC differentiation. Desiring a robust tool for spatiotemporal reporting of cell‐cycle events in hPSCs, we employed the CRISPR/Cas9 genome editing tool and successfully knocked the FUCCI reporter into the AAVS1 safe harbor locus of hPSCs for stable and constitutive FUCCI expression, exhibiting reliable cell‐cycle‐dependent fluorescence in both hPSCs and their differentiated progeny. We also established a cardiac‐specific TNNT2‐FUCCI reporter for lineage‐specific cell‐cycle monitoring of cardiomyocyte differentiation from hPSCs. This powerful and modular FUCCI system should provide numerous opportunities for studying human cell‐cycle activity, and enable the identification and investigation of novel regulators for adult tissue regeneration.