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Introduction: Extensive thermal injury remains a formidable clinical challenge,primarily due to the profound deficit of autologous donor skin,which necessitates prolonged hospitalization and escalates healthcare expenditures. While human embryonic stem cells (hESCs) offer a theoretically inexhaustible source for regenerative therapy,optimizing their differentiation and engraftment remains critical for clinical translation. Methods: We used a three-stage protocol to induce the differentiation of hESCs into keratinocytes (KCs). To optimize the delivery of hESC-derived keratinocytes (EKCs),human dermal fibroblasts (HFBs) were utilized to provide essential extracellular matrix (ECM) and microenvironmental support. The two cell types could self-assemble into 3D spheroids. After optimizing the size and cell proportion,these spheroids were subsequently transplanted onto full-thickness dorsal wounds in immunodeficient mice to evaluate their regenerative capacity. Results: hESC-derived keratinocytes exhibited the expression of stage-specific epidermal markers,confirming high differentiation efficiency. In vitro,EKCs demonstrate the capacity to form stratified epidermal structures. By self-assembling into spheres with dermal fibroblasts,the EKCs demonstrated successful engraftment and sustained survival in vivo. The transplantation of these 3D spheroids significantly accelerated wound closure and re-epithelialization compared with controls. Conclusions: This study establishes a robust cell therapy approach characterized by a short preparation cycle with high differentiation efficiency and high transplantation survival rate,offering a novel strategy for the treatment of extensive skin defects. View Publication

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation,development,and utilization. However,the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore,generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study,we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos,using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro,exhibiting high expression of key pluripotency markers,genomic stability,and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes. View Publication

Background: MSCs possess strong immunoregulatory properties and play a central role in maintaining immune homeostasis by limiting inflammatory responses. Their function is highly plastic and influenced by environmental cues,including viral signals. How SARS-CoV-2-derived antigens affect MSC immunoregulation remains incompletely understood. This study aimed to investigate the impact of SARS-CoV-2 peptides on MSC-mediated immune modulation of T-cells. Methods: MSCs were stimulated directly with SARS-CoV-2 spike protein S peptides or cocultured with SARS-CoV-2 peptide-activated T-cells. TLR4 surface expression and receptor downstream signaling were assessed to evaluate pathway activation. MSC immunoregulatory function was analyzed by measuring suppression of TNF-α and IFN-γ expression and induction of CD4+FOXP3+ regulatory T-cells. TLR4 inhibition and lipopolysaccharide (LPS) stimulation were used to examine pathway specificity and interaction. Results: SARS-CoV-2 peptides activated TLR4-associated signaling in MSCs,increasing TLR4 expression and NF-κB phosphorylation. Peptide-treated MSCs showed impaired suppression of pro-inflammatory cytokines and reduced induction of regulatory T-cells. TLR4 inhibition prevented these effects. LPS induced similar effects,while combining LPS and peptide stimulation partially restored physiological T-cell cytokine suppression. Conclusions: SARS-CoV-2 peptides modulate MSC immunoregulatory function on T-cells via TLR4-dependent mechanisms. View Publication

Resident cardiac macrophages,derived from primitive yolk sac precursors during embryogenesis,have increasingly been recognized for their distinct phenotype and functions in regulating homeostasis of the human heart. However,the profile of their extracellular vesicles (EVs) in cardiac signaling and regulation remains uncharted. Here,we employ differentiation of human pluripotent stem cell-derived primitive macrophages (Mac),harvesting their secreted EVs and performing in-depth characterization of associated microRNAs (miRNAs). Primitive macrophages secreted nanoscale EVs that expressed canonical EV markers,and miRNA sequencing highlighted a diverse and unique profile of miRNAs when compared to EVs sourced from other principal cardiac cell lineages and published data from monocyte-derived cells. In particular,we noted the abundance and enrichment of vascular-modulatory let-7 miRNAs and miR-126-3p. Functional screening of Mac-EVs in a 3D model of in vitro cardiac vasculogenesis confirmed enhanced early endothelial cell organization and branching. Establishing a reference for the human Mac-EV miRNome enables further hypothesis-driven mechanistic tests of Mac-EV miRNAs in mediating cardiac physiology and disease,opening the door to identification of therapeutic targets and modalities for cardiac repair. View Publication

The potential of the immune system to decrease cancer progression is widely recognized and has led to the development of innovative anti-cancer immunotherapies. Here,we studied human macrophages derived from genetically engineered iPSCs (iMac) with angiotensin-converting enzyme (ACE) expression regulatable by a doxycycline (dox)-inducible promoter as a novel anti-cancer immunotherapy. Increased ACE expression in iMac (cells now termed ACE-iMac) augments polarization towards an M1 macrophage phenotype characterized by increased production of proinflammatory cytokines,reactive oxygen species,nitric oxide,and an RNA profile indicating an aggressive immune response. ACE-iMac kills tumor cells in vitro significantly better than iMac. In vivo,studies using tumor xenografts for melanoma,breast cancer,and head and neck squamous cell carcinoma (HNSCC) showed a highly significant 3.4- to 7.2-fold reduction in solid tumor size following ACE-expressing ACE-iMac immunotherapy as compared to results with iMac. To further investigate the impact of ACE on human anti-tumor responses,we developed a humanized BLT-NSG mouse model with a fully functional adaptive immune system. Here,ACE-iMac treatment significantly reduced the growth of human melanoma xenografts by enhancing the activation of human T cells and NK cells. In conclusion,enhancing ACE expression in human-derived macrophages (ACE-iMac) greatly amplifies their anti-cancer phenotype,offering a compelling new therapeutic strategy with the potential to improve clinical outcomes for cancer patients. View Publication

Cryptococcus gattii is an emerging fungal pathogen that is acquired through the respiratory tract and causes invasive infections in both immunocompromised and otherwise healthy people. Many of these apparently immunocompetent patients are subsequently found to have autoantibodies against the pleiotropic cytokine GM-CSF. In this study,we investigated the potential role of GM-CSF (or CSF2) in the host response to C. gattii using a murine model of infection. Interestingly,infected Csf2-/- mice were found to have significantly improved survival and decreased lung fungal burden compared to wild type (WT) mice. We determined that during C. gattii infection,GM-CSF promotes the differentiation of monocytes into alveolar and interstitial macrophages. When these macrophages are ablated in CCR2-DTR+ mice,there is a corresponding improvement in survival with decreased lung fungal burden,thus phenocopying Csf2-/- mice. WT bone-marrow derived macrophages challenged with C. gattii and interstitial and alveolar macrophages from infected WT mice are unable to undergo M1 polarization,suggesting that monocyte-derived macrophages (moMacs) are rendered permissive for fungal proliferation. Therefore,GM-CSF and moMacs mediate immune responses that are harmful to the host. We further found that GM-CSF and moMacs preferentially promote the influx of eosinophils over neutrophils into the infected lung which is associated with substantial inflammatory lung pathology. Ablation of neutrophils using Mrp8cretg iDTR+ mice significantly increased C. gattii burden in the lungs,indicating that GM-CSF and moMacs block the entry of these beneficial,fungal-clearing granulocytes during infection. Altogether,our results show that GM-CSF plays a key role in impeding host anti-fungal responses to C. gattii by coordinating monocyte-derived macrophages and granulocyte activity and crosstalk. Author summaryCryptococcus gattii is an environmental fungus that can cause severe lung and brain infections after inhalation through the respiratory tract. C. gattii causes disease in patients with known immune deficits but also in individuals that are apparently healthy. Studies on otherwise healthy people who become infected with C. gattii suggest that they may have a previously unrecognized problem involving granulocyte macrophage-colony stimulating factor (GM-CSF),a cytokine,or messenger protein,that is an important part of the immune system. Here,we investigate the role of GM-CSF in the immune response to C. gattii using a mouse model of infection. We find that C. gattii increases GM-CSF in the lungs,leading to the influx of immune cells,including monocyte-derived macrophages and eosinophils,while inhibiting the entry of neutrophils. The macrophages and eosinophils allow the fungus to proliferate and cause inflammatory damage to the lungs,which is ultimately fatal. The absence of neutrophils also contributes to fungal growth,as these immune cells would otherwise be able to help kill the fungus. Our study provides new insight into how GM-CSF regulates immunity to C. gattii and has important implications as to the mechanisms that govern susceptibility to this infection. View Publication

CD4+ T helper (TH)-17 cells play a pivotal role in mucosal immune defense and are implicated in autoimmune diseases and cancer. Although Th17 cell plasticity is well-studied in mice,the factors driving their transition between pro-inflammatory and immunomodulatory states in humans remain less understood. Our study explored the transcriptional and epigenetic landscapes of single-cell cultures of human memory TH17 cells,focusing on clones that produce either immunomodulatory IL-10 or pro-inflammatory IFNγ and IL-22. We found that IL-10+ TH17 cells exhibit a T cell exhaustion-like profile with increased CTLA-4 expression and reduced IL-2 levels,while Ikaros zinc finger (IkZF) transcription factors,Aiolos and Eos,are differentially expressed in IL-10+ and IL-22+ TH17 cells,respectively. While exogenous IL-2 promotes IL-10 production in TH17 cells,lenalidomide induces IL-2 and promotes inflammatory TH17 cells,shifting TH17 cells towards a pro-inflammatory phenotype by reducing IL-10 and increasing IL-22 and IFNγ levels. Conversely,upregulation of Eos enhanced pro-inflammatory cytokine production. These findings highlight the crucial role of IkZF transcription factors in regulating human TH17 cell functions. Moreover,single-cell RNA sequencing of PBMCs from lenalidomide-treated patients confirmed an enrichment of inflammatory signatures,including interferon and IL-2/STAT5 pathways in TH17 cells. The ability to modulate this axis through targeted interventions,such as lenalidomide-induced Aiolos degradation or enforced Eos expression,presents new therapeutic opportunities for managing TH17 cell states in cancer and autoimmune diseases. View Publication

Objective and designChronic rhinosinusitis (CRS) is a heterogeneous inflammatory disease of the paranasal sinuses,which is divided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRSwNP is typically caused by type 2 inflammation,which is characterized by elevated IL-4 and IL-13 levels,impairment of the epithelial barrier,and tissue remodeling. While the involvement of immune cells is well known,it remains unclear to what extent structural cells intrinsically maintain disease-specific functional programs. The aim of this study was to determine whether epithelial cells and fibroblasts derived from CRSwNP and CRSsNP differ in their barrier properties,inflammatory reactivity,and type 2-associated functional characteristics.MethodsAir–liquid interface (ALI) epithelial cultures and primary fibroblast cultures were generated from CRSwNP and CRSsNP tissue. Epithelial barrier integrity was assessed by transepithelial electrical resistance (TEER),and inflammatory responses to TLR stimulation were analyzed by qRT-PCR. Fibroblast migration was evaluated using scratch assays. Cellular responses to IL-4/IL-13 with or without Dupilumab were quantified by qRT-PCR.ResultsCRSwNP-derived epithelial cells exhibited delayed tight junction formation and impaired differentiation compared to CRSsNP cells. Poly(I:C) stimulation induced stronger expression of Th2-associated cytokines in CRSwNP cultures. CRSwNP fibroblasts showed reduced migratory capacity and a heightened induction of Th2 cytokines and extracellular matrix genes following IL-4/IL-13 stimulation relative to CRSsNP fibroblasts.ConclusionEpithelial cells and fibroblasts derived from CRSwNP retain disease-associated type 2 characteristics in vitro,indicating persistent disease-aligned programmed functional alterations of the polyp microenvironment. In contrast,CRSsNP-derived cells lacked comparable enhanced type 2 responsiveness. These findings support CRSwNP as a distinct,self-sustaining inflammatory endotype and underscore the value of patient-derived models for investigating disease mechanisms and targeted therapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12950-026-00497-7. View Publication

Neural cells in the adult human central nervous system (CNS) display extensive transcriptional heterogeneity. How different layers of epigenetic regulation underpin this heterogeneity is poorly understood. Here we profile,at the single-nuclei epigenomic level,distinct regions of the adult human CNS,for chromatin accessibility and simultaneously for the histone modifications H3K27me3 and H3K27ac. We unveil a putative SOX10 enhancer and primed chromatin signatures at HOX loci in spinal-cord-derived human oligodendroglia (OLG) and astrocytes,but not microglia. These signatures in adult OLG were reminiscent of developmental profiles but were decoupled from robust gene expression. Moreover,using high-resolution Micro-C,we show that induced pluripotent stem-cell-derived human OLGs exhibit a HOX chromatin architecture compatible with the primed chromatin in adult OLGs,bearing a strong resemblance not only to OLG developmental architecture but also to high-grade pontine gliomas. Thus,epigenetic memory from developmental states in adult OLG not only enables them to promptly transcribe Hox family genes during regeneration but also makes them susceptible to gliomagenesis. Single-nucleus epigenomic maps of the adult human brain and spinal cord reveal that adult oligodendroglia retain developmental chromatin patterns,suggesting a molecular memory that may shape repair processes and cancer vulnerability. View Publication