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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

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

Multi-kilobase knock-ins (KIs) are a necessary,yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI,but the insert size is limited by the viral packaging capacity. Here,we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs,we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets),where relevant genomic regions are enriched via hybridization,achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles,imprecision in the insert and donor concatenation,genotypes of non-KI alleles,and more importantly,uniquely identifies and localizes random integration of the full or partial donor(s). Additionally,the multi-rAAV donor approach is successfully applied to cell lines,including lines intolerant of plasmid DNA,whereas LOCK-seq reliably and efficiently screens for KI clones. Together,the two approaches significantly improve the creation and precision of knock-in models. 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