It remains a significant challenge to reactivate the cell cycle activity of adult mammalian cardiomyocytes (CMs). This study created a hypo-immunogenic human induced pluripotent stem cell (hiPSC) line using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing to knockout β2-microglobulin in hiPSCs (B2MKOhiPSCs) for manufacturing nanovesicles (B2MKOhiPSC-NVs). Approximately 9500 B2MKOhiPSC-NVs were produced from a single B2MKOhiPSC. Proteomic analyses indicated that, compared to B2MKOhiPSCs, the cargos of B2MKOhiPSC-NVs were enriched in spindle and chromosomal proteins, as well as proteins that regulate the cell cycle and scavenge reactive oxygen species (ROS). When administrated to hiPSCs derived CMs (hiPSC-CMs), B2MKOhiPSC-NVs reduced lactate dehydrogenase leakage and apoptosis in hypoxia-cultured hiPSC-CMs through activating the AKT pathway, protected hiPSC-CMs from H2O2-induced damage by ROS scavengers in the NV cargo, increased hiPSC-CM proliferation via the YAP pathway, and were hypoimmunogenic when co-cultured with human CD8+ T cells or delivered to mice. Furthermore, when B2MKOhiPSC-NVs or 0.9 % NaCl were intramyocardially injected into mice after cardiac ischemia/reperfusion injury, cardiac function and infarct size, assessed 4 weeks later, were significantly improved in the B2MKOhiPSC-NV group, with increased mouse CM survival and cell cycle activity. Thus, the proteins in the B2MKOhiPSC-NV cargos convergently activated the AKT pathway, scavenged ROS to protect CMs, and upregulated YAP signaling to induce CM cell cycle activity. Thus, B2MKOhiPSC-NVs hold great potential for cardiac protection and regeneration.
© 2025 The Authors.

Autologous T cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily due to their individualized manufacturing requirements. To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select HLA Class I and II alleles ( HLA-A , HLA-B , and the class II transactivator ( CIITA )), while retaining HLA-C to protect from NK cell rejection. Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus (AAV) template allowed for high localized insertion rate while simultaneously removing the endogenous TCR and preventing GvHD. Using a lipid nanoparticle (LNP)-based delivery system of the editing components enabled a robust cell engineering process, achieving high editing rates and cell expansion. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene editing approach generated cells with minimal chromosomal aberrations. The Allo strategy has also been applied to induced pluripotent stem cells (iPSCs), suggesting potential applications in regenerative medicine applications.

Extended pluripotent stem cells (EPSCs) possess a high differentiation capacity, potentially as a superior seed resource for generating cardiomyocytes. Here, we present a protocol for generating feeder-free EPSCs (ffEPSCs), cardiomyocytes, and engineered heart tissues (EHTs). We describe steps for converting human embryonic stem cells or induced pluripotent stem cells (ESCs/iPSCs) into ffEPSCs, followed by their long-term maintenance, cryopreservation, seed preservation, and differentiation into cardiomyocytes. We then detail procedures for constructing and culturing three-dimensional EHTs followed by their contraction force measurement and optical mapping. For complete details on the use and execution of the protocol, please refer to Zheng et al.1 and Li et al.2.
Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.

RNA-binding proteins (RBPs) are essential in cardiac development. However, a large of them have not been characterized during the process.
We applied the human embryonic stem cells (hESCs) differentiated into cardiomyocytes model and constructed SAMD4A-knockdown/overexpression hESCs to investigate the role of SAMD4A in cardiomyocyte lineage specification.
SAMD4A, an RBP, exhibits increased expression during early heart development. Suppression of SAMD4A inhibits the proliferation of hESCs, impedes cardiac mesoderm differentiation, and impairs the function of hESC-derived cardiomyocytes. Correspondingly, forced expression of SAMD4A enhances proliferation and promotes cardiomyogenesis. Mechanistically, SAMD4A specifically binds to FGF2 via a specific CNGG/CNGGN motif, stabilizing its mRNA and enhancing translation, thereby upregulating FGF2 expression, which subsequently modulates the AKT signaling pathway and regulates cardiomyocyte lineage differentiation. Additionally, supplementation of FGF2 can rescue the proliferation defect of hESCs in the absence of SAMD4A.
Our study demonstrates that SAMD4A orchestrates cardiomyocyte lineage commitment through the post-transcriptional regulation of FGF2 and modulation of AKT signaling. These findings not only underscore the essential role of SAMD4A in cardiac organogenesis, but also provide critical insights into the molecular mechanisms underlying heart development, thereby informing potential therapeutic strategies for congenital heart disease.
© 2025. The Author(s).

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have potential applications in regenerative medicine. The quality by design (QbD) approach enables the efficiency and quality assurance in the manufacturing of hiPSC-derived products. It requires a molecular understanding of hiPSC differentiation throughout the differentiation process; however, information on cardiac differentiation remains limited. Proteins associated with the early stages of cardiac differentiation would be useful in the cardiomyocyte quality assessment. Here, we performed quantitative proteomics of hiPSC intermediate cells in the early phase of cardiac differentiation to better understand their molecular characteristics. Proteomic profiles suggested that day 5-7 cells were in the morphogenetic stage of cardiac differentiation. Trophoblast glycoprotein (TPBG) was the most up-regulated protein in the morphogenetic stage; it was previously shown to be up-regulated during differentiation into neural stem cells. Proteomics of TPBG-knockdown cells revealed that TPBG is involved in cell proliferation and is related to the cardiomyocyte yield, suggesting that it could be used as a marker in QbD development. Our approach helps us understand the molecular basis of hiPSC differentiation and could be a powerful tool in QbD-based manufacturing.
© 2024 The Authors. Published by American Chemical Society.