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We used high-resolution optical mapping (~50 µm) to investigate potential arrhythmia mechanisms following transplantation of engineered cardiac tissue. We induced myocardial infarction in 6 immunosuppressed pigs and implanted cardiac spheroids into the border zone. One week later, 600-µm-thick cardiac slices containing implanted spheroids were harvested and electrical propagation was imaged. Histology showed low connexin-43 expression, scar, and misaligned muscle fibers at the graft-host interface. We observed propagation from host-to-graft in 10 slices from 3 pigs. Host-graft electrical bridges were spaced by millimeters. Propagation was ~4-fold slower in the graft than host. One graft beat spontaneously, but activation did not propagate from graft-to-host in this, or any other slice. We did not observe reentry, but slow in-graft conduction and sparse electrical bridges provided opportunity for reentry induction. These data reveal potential for reentrant or focal arrhythmias 1 week post-implant, which may resolve with maturation of the graft and the graft-host interface.

Renal failure due to drug nephrotoxicity or disease is frequently observed in patients. The development of in vitro models able to recapitulate kidney biology offers new possibilities to study drug toxicity or model diseases. Induced pluripotent stem cell–derived kidney organoids already show promise, but several drawbacks must be overcome to maintain them in culture, among which is the presence of non-renal cell populations such as cartilage. We modified the culture protocol and maintained kidney organoids in medium containing FGF9 for 1 additional week compared to the control protocol (Takasato). In comparison to the control, the FGF9-treated kidney organoids had reduced cartilage at day 7 + 25 and diminished chondrocyte marker expression. Importantly, the renal structures assessed by immunofluorescence were unaffected by the FGF9 treatment. This reduction of cartilage produces a higher quality kidney organoid that can be maintained longer in culture to improve their maturation for further in vivo work. Subject terms: Pluripotent stem cells, Stem-cell differentiation, Kidney

Spinal cord injury (SCI) remains a significant clinical challenge and poses a dramatic threat to the life quality of patients due to limited neural regeneration and detrimental post-injury alternations in tissue microenvironment. We developed a therapeutic approach by transplanting spinal neural progenitor cells (spNPGs), derived from human induced pluripotent stem cell (iPSC)-generated neuromesodermal progenitors, into a contusive SCI model in NOD-SCID mice. Single-cell RNA sequencing mapped the in vitro differentiation of iPSC-spNPGs, confirming their specification into spinal neuronal lineages. Single-nucleus transcriptomics at 1 week post-transplantation showed that the grafted cells differentiated in vivo into motor neurons and two interneuron subtypes (V2 and dI4). Additionally, spNPGs integrated into host neural circuits, enhancing synaptic connectivity, while simultaneously modulating the injury microenvironment by shifting microglia and astrocyte polarization toward anti-inflammatory and neuroprotective phenotypes. This dual mechanism promoted axonal regrowth, remyelination, and significant sensorimotor recovery, as evidenced by improved locomotor scores. Our findings highlight the therapeutic potential of human iPSC-spNPGs in reconstructing neural networks and mitigating secondary damage, providing compelling preclinical evidence for advancing stem cell-based SCI therapies. Subject terms: Stem-cell differentiation, Spinal cord injury

Nanoplastics (NPs) are emerging environmental pollutants that pose growing concerns due to their potential health risks. However, the effects of inhaled NP exposure during pregnancy on fetal brain development remain poorly understood. In this study, we investigated the impact of maternal exposure to polypropylene nanoplastics (PP-NPs) on fetal brain development and neurobehavioral outcomes in a mouse model and further explored its mechanism in human cerebral organoids. Maternal exposure to PP-NPs significantly impaired neuronal differentiation and proliferation in the fetal cortex. Neurobehavioral assessments revealed significant deficits in offspring following maternal exposure, including impaired spatial memory, reduced motor coordination, and heightened anxiety-like behavior. Furthermore, human brain organoids exposed to PP-NPs exhibited reduced growth and neuronal differentiation, with significant downregulation of key neuronal markers such as TUJ1, MAP2, and PAX6. Transcriptomic analysis identified alterations in gene expression, particularly in neuroactive ligand-receptor interaction pathway. Molecular docking and fluorescence co-localization analysis further suggested CYSLTR1 and PTH1R as key molecular targets of PP-NPs. These findings provide novel insights into the toxicological effects of NPs on the developing brain and emphasize the need for preventive measures to protect fetal neurodevelopment during pregnancy. The online version contains supplementary material available at 10.1186/s12951-025-03561-1.

CACNA1A encodes the pore-forming α 1A subunit of the Ca V 2.1 calcium channel, whose altered function is associated with various neurological disorders, including forms of ataxia, epilepsy, and migraine. In this study, we generated isogenic iPSC-derived neural cultures carrying CACNA1A loss-of-function mutations differently affecting Ca V 2.1 splice isoforms. Morphological, molecular, and functional analyses revealed an essential role of CACNA1A in neurodevelopmental processes. We found that different CACNA1A loss-of-function mutations produce distinct neurodevelopmental deficits. The F1491S mutation, which is located in a constitutive domain of the channel and therefore causes a complete loss-of-function, impaired neural induction at very early stages, as demonstrated by changes in single-cell transcriptomic signatures of neural progenitors, and by defective polarization of neurons. By contrast, cells carrying the Y1854X mutation, which selectively impacts the synaptically-expressed Ca V 2.1[EFa] isoform, behaved normally in terms of neural induction but showed altered neuronal network composition and lack of synchronized activity. Our findings reveal previously unrecognized roles of CACNA1A in the mechanisms underlying neural induction and neural network dynamics and highlight the differential contribution of the divergent variants Ca V 2.1[EFa] and Ca V 2.1[EFb] in the development of human neuronal cells. The online version contains supplementary material available at 10.1007/s00018-025-05740-7.

The ε4 isoform of apolipoprotein E (ApoE) is the most significant genetic risk factor for Alzheimer’s disease. Glial cells are the main source of ApoE in the brain, and in microglia, the ε4 isoform of ApoE has been shown to impair mitochondrial metabolism and the uptake of lipids and Aβ42. However, whether the ε4 isoform alters autophagy or lysosomal activity in microglia in basal and inflammatory conditions is unknown. Altogether, microglia-like cells (iMGs) from eight APOE 3/3 and six APOE 4/4 human induced pluripotent stem cell (iPSC) lines were used in this study. The responses of iMGs to Aβ42, LPS and IFNγ were studied by metabolomics, proteomics, and functional assays. Here, we demonstrate that iMGs with the APOE 4/4 genotype exhibit reduced basal pinocytosis levels compared to APOE 3/3 iMGs. Inflammatory stimulation with a combination of LPS and IFNγ or Aβ42 induced PI3K/AKT/mTORC signaling pathway, increased pinocytosis, and blocked autophagic flux, leading to the accumulation of sequestosome 1 (p62) in both APOE 4/4 and APOE 3/3 iMGs. Exposure to Aβ42 furthermore caused lysosomal membrane permeabilization, which was significantly stronger in APOE 4/4 iMGs and positively correlated with the secretion of the proinflammatory chemokine IL-8. Metabolomics analysis indicated a dysregulation in amino acid metabolism, primarily L-glutamine, in APOE 4/4 iMGs. Overall, our results suggest that inflammation-induced metabolic reprogramming places lysosomes under substantial stress. Lysosomal stress is more detrimental in APOE 4/4 microglia, which exhibit endo-lysosomal defects. The online version contains supplementary material available at 10.1186/s12974-025-03470-y.

The development of immunocompetent skin models marks a significant advancement in in vitro methods for detecting skin sensitizers while adhering to the 3R principles, which aim to reduce, refine, and replace animal testing. This study introduces for the first time an advanced immunocompetent skin model constructed entirely from induced pluripotent stem cell (iPSC)-derived cell types, including fibroblasts (iPSC-FB), keratinocytes (iPSC-KC), and fully integrated dendritic cells (iPSC-DC). To evaluate the skin model’s capacity, the model was treated topically with a range of well-characterized skin sensitizers varying in potency. The results indicate that the iPSC-derived immunocompetent skin model successfully replicates the physiological responses of human skin, offering a robust and reliable alternative to animal models for skin sensitization testing, allowing detection of extreme and even weak sensitizers. By addressing critical aspects of immune activation and cytokine signaling, this model provides an ethical, comprehensive tool for regulatory toxicology and dermatological research.

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless, dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS), taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated, the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition, transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together, our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology, Innate immunity, Neural stem cells

Understanding the interaction between genetic and epigenetic variation remains a challenge due to confounding environmental factors. We propose that human induced Pluripotent Stem Cells (iPSCs) are an excellent model to study the relationship between genetic and epigenetic variation while controlling for environmental factors. In this study, we have created a comprehensive resource of high-quality genomic, epigenomic, and transcriptomic data from iPSC lines and three iPSC-derived cell types (neural stem cell (NSC), motor neuron, monocyte) from three healthy donors. We find that epigenetic variation is most strongly associated with genetic variation at the iPSC stage, and that relationship weakens as epigenetic variation increases in differentiated cells. Additionally, cell type is a stronger source of epigenetic variation than genetic variation. Further, we elucidate a utility of studying epigenetic variation in iPSCs and their derivatives for identifying important loci for GWAS studies and the cell types in which they may be acting. Subject terms: Epigenomics, Genomics, Transcriptomics

Pericytes play a crucial role in controlling inflammation and vascular functions in the central nervous system, which are disrupted in Parkinson’s disease (PD). Still, there is a lack of studies on the impact of pericytes on neurodegenerative diseases, and their involvement in the pathology of PD is unclear. Our objective was to investigate the molecular and functional differences between healthy pericytes and pericytes with the LRRK2 G2019S mutation, which is one of the most common mutations associated with PD. Our study employed pericyte-like cells obtained from induced pluripotent stem cells produced from PD patients with the LRRK2 G2019S mutation as well as from healthy individuals. We examined the gene expression profiles of the cells and analyzed how the alterations reflect on their functionality. We have shown differences in the expression of genes related to inflammation and angiogenesis. Furthermore, we observe modified migration speed in PD pericyte-like cells as well as enhanced secretion of inflammatory mediators, such as soluble VCAM-1 and MCP-1, in these pericyte-like cells following exposure to proinflammatory stimuli. In summary, our findings support the notion that pericytes play a role in the inflammatory and vascular changes observed in PD. Further investigation of pericytes could provide valuable insight into understanding the pathogenesis of PD. The online version contains supplementary material available at 10.1186/s12987-024-00592-y.

The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of “off-the-shelf” cancer immunotherapies. However, the differentiation of NK cells from iPSCs remains poorly understood, particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. To investigate this question, we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term, clinically compatible feeder-free protocol corresponding to primitive hematopoiesis, and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Our work demonstrated that both protocols are capable of producing functional iNK cells. However, the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile, which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell–based adoptive immunotherapies.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives, particularly extracellular vesicles (EVs), for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs, alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction, indicating a potential mechanism for their immunomodulatory effects. Furthermore, iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally, iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably, priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics, given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3.