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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. 

The past decade has witnessed significant advances in cancer immunotherapy, particularly through the adoptive transfer of engineered T cells in treating advanced leukemias and lymphomas. Despite these excitements, challenges remain with scale, cost, and ensuring quality control of engineered immune cells, including chimeric antigen receptor T, natural killer cells, and macrophages. The advent of human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, has transformed immunotherapy by providing a scalable, off-the-shelf source of any desired immune cells for basic research, translational studies, and clinical interventions. The tractability of hPSCs for gene editing could also generate homogenous, universal cellular products with custom functionality for individual or combinatory therapeutic applications. This review will explore various immune cell types whose directed differentiation from hPSCs has been achieved and recently adapted for translational immunotherapy and feature forward-looking bioengineering techniques shaping the future of the stem cell field. 

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. 

Heart diseases are leading cause of death around the world. Given their unique capacity to self-renew and differentiate into all types of somatic cells, human pluripotent stem cells (hPSCs) hold great promise for heart disease modeling and cardiotoxic drug screening. hPSC-derived cardiac organoids are emerging biomimetic models for studying heart development and cardiovascular diseases, but it remains challenging to make mature organoids with a native-like structure in vitro . In this study, temporal modulation of Wnt signaling pathway co-differentiated hPSCs into beating cardiomyocytes and cardiac endothelial-like cells in 3D organoids, resulting in cardiac endothelial-bounded chamber formation. These chambered cardiac organoids exhibited more mature membrane potential compared to cardiac organoids composed of only cardiomyocytes. Furthermore, a better response to toxic drugs was observed in chamber-contained cardiac organoids. In summary, spatiotemporal signaling pathway modulation may lead to more mature cardiac organoids for studying cardiovascular development and diseases. 

Abstract Spatiotemporally controlled presentation of morphogens and elaborate modulation of signaling pathways elicit pattern formation during development. Though this process is critical for proper organogenesis, unraveling the mechanisms of developmental biology have been restricted by challenges associated with studying human embryos. Human pluripotent stem cells (hPSCs) have been used to model development in vitro, however difficulties in precise spatiotemporal control of the cellular microenvironment have limited the utility of this model in exploring mechanisms of pattern formation. Here, a simple and versatile method is presented to spatially pattern hPSC differentiation in 2‐dimensional culture via localized morphogen adsorption on substrates. Morphogens including bone morphogenetic protein 4 (BMP4), activin A, and WNT3a are patterned to induce localized mesendoderm, endoderm, cardiomyocyte (CM), and epicardial cell (EpiC) differentiation from hPSCs and hPSC‐derived progenitors. Patterned CM and EpiC co‐differentiation allows investigation of intercellular interactions in a spatially controlled manner and demonstrate improved alignment of CMs in proximity to EpiCs. This approach provides a platform for the controlled and systematic study of early pattern formation. Moreover, this study provides a facile approach to generate 2D patterned hPSC‐derived tissue structures for modeling disease and drug interactions. 

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC‐derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC‐derived ECs with hPSC‐derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC‐derived CMs.