FGF2 promotes the expansion of parietal mesothelial progenitor pools and inhibits BMP4-mediated smooth muscle cell differentiation.

Mesothelial cells, in the outermost layer of internal organs, are essential for both organ development and homeostasis. Although the parietal mesothelial cell is the primary origin of mesothelioma that may highjack developmental signaling, the signaling pathways that orchestrate developing parietal mesothelial progenitor cell (MPC) behaviors, such as MPC pool expansion, maturation, and differentiation, are poorly understood. To address it, we established a robust protocol for culturing WT1+ MPCs isolated from developing pig and mouse parietal thorax. Quantitative qPCR and immunostaining analyses revealed that BMP4 facilitated MPC differentiation into smooth muscle cells (SMCs). In contrast, FGF2 significantly promoted MPC progenitor pool expansion but blocked the SMC differentiation. BMP4 and FGF2 counterbalanced these effects, but FGF2 had the dominant impact in the long-term culture. A Wnt activator, CHIR99021, was pivotal in MPC maturation to CALB2+ mesothelial cells, while BMP4 or FGF2 was limited. Our results demonstrated central pathways critical for mesothelial cell behaviors.

The perspective for next-generation lung replacement therapies: functional whole lung generation by blastocyst complementation.

PURPOSE OF REVIEW: Blastocyst complementation represents a promising frontier in next-generation lung replacement therapies. This review aims to elucidate the future prospects of lung blastocyst complementation within clinical settings, summarizing the latest studies on generating functional lungs through this technique. It also explores and discusses host animal selection relevant to interspecific chimera formation, a challenge integral to creating functional human lungs via blastocyst complementation. RECENT FINDINGS: Various gene mutations have been utilized to create vacant lung niches, enhancing the efficacy of donor cell contribution to the complemented lungs in rodent models. By controlling the lineage to induce gene mutations, chimerism in both the lung epithelium and mesenchyme has been improved. Interspecific blastocyst complementation underscores the complexity of developmental programs across species, with several genes identified that enhance chimera formation between humans and other mammals. SUMMARY: While functional lungs have been generated via intraspecies blastocyst complementation, the generation of functional interspecific lungs remains unrealized. Addressing the challenges of controlling the host lung niche and selecting host animals relevant to interspecific barriers between donor human and host cells is critical to enabling the generation of functional humanized or entire human lungs in large animals.

Ephrin Forward Signaling Controls Interspecies Cell Competition in Pluripotent Stem Cells.

In the animal kingdom, evolutionarily conserved mechanisms known as cell competition eliminate unfit cells during development. Interestingly, cell competition also leads to apoptosis of donor cells upon direct contact with host cells from a different species during interspecies chimera formation. The mechanisms underlying how host animal cells recognize and transmit cell death signals to adjacent xenogeneic human cells remain incompletely understood. In this study, we developed an interspecies cell contact reporter system to dissect the mechanisms underlying competitive interactions between mouse and human pluripotent stem cells (PSCs). Through single-cell RNA-seq analyses, we discovered that Ephrin A ligands in mouse cells play a crucial role in signaling cell death to adjacent human cells that express EPHA receptors during interspecies PSC co-culture. We also demonstrated that blocking the Ephrin A-EPHA receptor interaction pharmacologically, and inhibiting Ephrin forward signaling genetically in the mouse cells, enhances the survival of human PSCs and promotes chimera formation both in vitro and in vivo . Our findings elucidate key mechanisms of interspecies PSC competition during early embryogenesis and open new avenues for generating humanized tissues or organs in animals, potentially revolutionizing regenerative medicine.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation.

Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.

Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

An immunostimulatory glycolipid that blocks SARS-CoV-2, RSV, and influenza infections in vivo.

Prophylactic vaccines for SARS-CoV-2 have lowered the incidence of severe COVID-19, but emergence of viral variants that are antigenically distinct from the vaccine strains are of concern and additional, broadly acting preventive approaches are desirable. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon-[Formula: see text]. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics long before vaccines or drugs are developed.

A local translation program regulates centriole amplification in the airway epithelium.

Biogenesis of organelles requires targeting of a subset of proteins to specific subcellular domains by signal peptides or mechanisms controlling mRNA localization and local translation. How local distribution and translation of specific mRNAs for organelle biogenesis is achieved remains elusive and likely to be dependent on the cellular context. Here we identify Trinucleotide repeat containing-6a (Tnrc6a), a component of the miRNA pathway, distinctively localized to apical granules of differentiating airway multiciliated cells (MCCs) adjacent to centrioles. In spite of being enriched in TNRC6A and the miRNA-binding protein AGO2, they lack enzymes for mRNA degradation. Instead, we found these apical granules enriched in components of the mRNA translation machinery and newly synthesized proteins suggesting that they are specific hubs for target mRNA localization and local translation in MCCs. Consistent with this, Tnrc6a loss of function prevented formation of these granules and led to a broad reduction, rather than stabilization of miRNA targets. These included downregulation of key genes involved in ciliogenesis and was associated with defective multicilia formation both in vivo and in primary airway epithelial cultures. Similar analysis of Tnrc6a disruption in yolk sac showed stabilization of miRNA targets, highlighting the potential diversity of these mechanisms across organs.

Construction of a bioactive copper-based metal organic framework-embedded dual-crosslinked alginate hydrogel for antimicrobial applications.

Metal-organic frameworks (MOFs) containing bioactive metals have the potential to exhibit antimicrobial activity by releasing metal ions or ligands through the cleavage of metal-ligand bonds. Recently, copper-based MOFs (Cu-MOFs) with sustained release capability, porosity, and structural flexibility have shown promising antimicrobial properties. However, for clinical use, the controlled release of Cu2+ over an extended time period is crucial to prevent toxicity. In this study, we developed an alginate-based antimicrobial scaffold and encapsulated MOFs within a dual-crosslinked alginate polymer network. We synthesized Cu-MOFs containing glutarate (Glu) and 4,4'-azopyridine (AZPY) (Cu(AZPY)-MOF) and encapsulated them in an alginate-based hydrogel through a combination of visible light-induced photo and calcium ion-induced chemical crosslinking processes. We confirmed Cu(AZPY)-MOF synthesis using scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, and thermogravimetric analysis. This antimicrobial hydrogel demonstrated excellent antibacterial and antifungal properties against two bacterial strains (MRSA and S. mutans, with >99.9 % antibacterial rate) and one fungal strain (C. albicans, with >78.7 % antifungal rate) as well as negligible cytotoxicity towards mouse embryonic fibroblasts, making it a promising candidate for various tissue engineering applications in biomedical fields.

Towards human organ generation using interspecies blastocyst complementation: Challenges and perspectives for therapy.

Millions of people suffer from end-stage refractory diseases. The ideal treatment option for terminally ill patients is organ transplantation. However, donor organs are in absolute shortage, and sadly, most patients die while waiting for a donor organ. To date, no technology has achieved long-term sustainable patient-derived organ generation. In this regard, emerging technologies of chimeric human organ production via blastocyst complementation (BC) holds great promise. To take human organ generation via BC and transplantation to the next step, we reviewed current emerging organ generation technologies and the associated efficiency of chimera formation in human cells from the standpoint of developmental biology.

Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication.

Salivary glands act as virus reservoirs in various infectious diseases and have been reported to be targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanisms underlying infection and replication in salivary glands are still enigmatic due to the lack of proper in vitro models. Here, we show that human induced salivary glands (hiSGs) generated from human induced pluripotent stem cells can be infected with SARS-CoV-2. The hiSGs exhibit properties similar to those of embryonic salivary glands and are a valuable tool for the functional analysis of genes during development. Orthotopically transplanted hiSGs can be engrafted at a recipient site in mice and show a mature phenotype. In addition, we confirm SARS-CoV-2 infection and replication in hiSGs. SARS-CoV-2 derived from saliva in asymptomatic individuals may participate in the spread of the virus. hiSGs may be a promising model for investigating the role of salivary glands as a virus reservoir.

Prominin 1 and Notch regulate ciliary length and dynamics in multiciliated cells of the airway epithelium.

Differences in ciliary morphology and dynamics among multiciliated cells of the respiratory tract contribute to efficient mucociliary clearance. Nevertheless, little is known about how these phenotypic differences are established. We show that Prominin 1 (Prom1), a transmembrane protein widely used as stem cell marker, is crucial to this process. During airway differentiation, Prom1 becomes restricted to multiciliated cells, where it is expressed at distinct levels along the proximal-distal axis of the airways. Prom1 is induced by Notch in multiciliated cells, and Notch inactivation abolishes this gradient of expression. Prom1 was not required for multicilia formation, but when inactivated resulted in longer cilia that beat at a lower frequency. Disruption of Notch resulted in opposite effects and suggested that Notch fine-tunes Prom1 levels to regulate the multiciliated cell phenotype and generate diversity among these cells. This mechanism could contribute to the innate defense of the lung and help prevent pulmonary disease.

Injectable hyaluronic acid hydrogel encapsulated with Si-based NiO nanoflower by visible light cross-linking: Its antibacterial applications.

Bacterial infections have become a severe threat to human health and antibiotics have been developed to treat them. However, extensive use of antibiotics has led to multidrug-resistant bacteria and reduction of their therapeutic effects. An efficient solution may be localized application of antibiotics using a drug delivery system. For clinical application, they need to be biodegradable and should offer a prolonged antibacterial effect. In this study, a new injectable and visible-light-crosslinked hyaluronic acid (HA) hydrogel loaded with silicon (Si)-based nickel oxide (NiO) nanoflowers (Si@NiO) as an antibacterial scaffold was developed. Si@NiO nanoflowers were synthesized using chemical bath deposition before encapsulating them in the HA hydrogel under a mild visible-light-crosslinking conditions to generate a Si@NiO-hydrogel. Si@NiO synthesis was confirmed using scanning electron microscopy, transmission electron microscopy, and powder X-ray diffraction. As-prepared Si@NiO-hydrogel exhibited enhanced mechanical properties compared to a control bare hydrogel sample. Moreover, Si@NiO-hydrogel exhibits excellent antibacterial properties against three bacterial strains (P. aeruginosa, K. pneumoniae, and methicillin-resistant Staphylococcus aureus (>99.9% bactericidal rate)) and negligible cytotoxicity toward mouse embryonic fibroblasts. Therefore, Si@NiO-hydrogel has the potential for use in tissue engineering and biomedical applications owing to its injectability, visible-light crosslink ability, degradability, biosafety, and superior antibacterial property.

E2F4's cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6.

Multiciliated cells play critical roles in the airway, reproductive organs, and brain. Generation of multiple cilia requires both activation of a specialized transcriptional program and subsequent massive amplification of centrioles within the cytoplasm. The E2F4 transcription factor is required for both roles and consequently for multiciliogenesis. Here we establish that E2F4 associates with two distinct components of the centriole replication machinery, Deup1 and SAS6, targeting nonhomologous domains in these proteins. We map Deup1 and SAS6 binding to E2F4's N-terminus and show that this domain is sufficient to mediate E2F4's cytoplasmic role in multiciliogenesis. This sequence is highly conserved across the E2F family, but the ability to bind Deup1 and SAS6 is specific to E2F4 and E2F5, consistent with their shared roles in multiciliogenesis. By generating E2F4/E2F1 chimeras, we identify a six-residue motif that is critical for Deup1 and SAS6 binding. We propose that the ability of E2F4 and E2F5 to recruit Deup1 and/or SAS6, and enable centriole replication, contributes to their cytoplasmic roles in multiciliogenesis.

Generation of functional lungs via conditional blastocyst complementation using pluripotent stem cells.

Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.

Jagged and Delta-like ligands control distinct events during airway progenitor cell differentiation.

Notch signaling regulates cell fate selection during development in multiple organs including the lung. Previous studies on the role of Notch in the lung focused mostly on Notch pathway core components or receptor-specific functions. It is unclear, however, how Jagged or Delta-like ligands collectively or individually (Jag1, Jag2, Dll1, Dll4) influence differentiation of airway epithelial progenitors. Using mouse genetic models we show major differences in Jag and Dll in regulation and establishment of cell fate. Jag ligands had a major impact in balancing distinct cell populations in conducting airways, but had no role in the establishment of domains and cellular abundance in the neuroendocrine (NE) microenvironment. Surprisingly, Dll ligands were crucial in restricting cell fate and size of NE bodies and showed an overlapping role with Jag in differentiation of NE-associated secretory (club) cells. These mechanisms may potentially play a role in human conditions that result in aberrant NE differentiation, including NE hyperplasias and cancer.

Wnt/Fgf crosstalk is required for the specification of basal cells in the mouse trachea.

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of ß-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.

Author Correction: Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis.

This corrects the article DOI: 10.1038/ncomms15857.