Human Pluripotent Stem Cell-Derived Alveolar Organoid with Macrophages.

Alveolar organoids (AOs), derived from human pluripotent stem cells (hPSCs) exhibit lung-specific functions. Therefore, the application of AOs in pulmonary disease modeling is a promising tool for understanding disease pathogenesis. However, the lack of immune cells in organoids limits the use of human AOs as models of inflammatory diseases. In this study, we generated AOs containing a functional macrophage derived from hPSCs based on human fetal lung development using biomimetic strategies. We optimized culture conditions to maintain the iMACs (induced hPSC-derived macrophages) AOs for up to 14 days. In lipopolysaccharide (LPS)-induced inflammatory conditions, IL-1ß, MCP-1 and TNF-α levels were significantly increased in iMAC-AOs, which were not detected in AOs. In addition, chemotactic factor IL-8, which is produced by mononuclear phagocytic cells, was induced by LPS treatment in iMACs-AOs. iMACs-AOs can be used to understand pulmonary infectious diseases and is a useful tool in identifying the mechanism of action of therapeutic drugs in humans. Our study highlights the importance of immune cell presentation in AOs for modeling inflammatory pulmonary diseases.

Injectable and Pathogen-Mimicking Hydrogels for Enhanced Protective Immunity against Emerging and Highly Pathogenic Influenza Virus.

Seasonal emerging infectious diseases such as influenza A impose substantial risk and need new translational strategies to achieve active immunomodulation. Here, a novel injectable pathogen-mimicking hydrogel (iPMH) that can enhance both cellular and humoral immune responses is suggested. By the help of poly(γ-glutamic acid) that has abundant carboxylate groups and dispersion helper function, hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPLA) molecules and viral antigens (PR8, W150) can be successfully combined as pathogen-mimicking adjuvants. Polyelectrolyte complex between the poly(γ-glutamic acid)-based adjuvants and collagens generate in situ gel-forming hydrogel at physiological temperature. When the iPMH are immunized, they act as a pathogen-mimicking (MPLA, H1N1, H5N1) immune priming center and a depot for continuous stimulation of immune system, resulting in the induction of high levels (8.5 times higher) of antigen-specific IgG titers in the sera of mice and the increased number of IFN-γ-producing cells (7.3 times higher) compared with those in the groups immunized with antigen plus clinically used aluminum gels. Following the intranasal infection of the mouse adapted virus (emerging infectious 2009 H1N1 and highly pathogenic 2006 H5N1) at 50 times the 50% lethal dose, the mice immunized with viral antigens plus iPMH exhibit 100% protective immunity against lethal virus challenge.

An Electrostatically Self-Assembled Ternary Nanocomplex as a Non-Viral Vector for the Delivery of Plasmid DNA into Human Adipose-Derived Stem Cells.

In this study, we developed electrostatically self-assembled ternary nanocomplexes as a safe and effective non-viral vector for the delivery of plasmid DNA (pDNA) into human adipose-derived stem cells (hASCs). Although polyethylenimine (PEI) polymers initially showed excellent performance as gene delivery carriers, their broad use has been limited by cytotoxicity resulting from their strong positive charge. To reduce the cytotoxicity, we utilized anionic hyaluronic acid (HA) as a corona layer material for pDNA/PEI binary nanocomplexes. HA was also introduced to increase the targeting efficiency of pDNA/PEI nanocomplexes because HA has can bind CD44 that is highly expressed on the surface of hASCs. We confirmed that the addition of HA changed the surface charge of pDNA/PEI nanocomplexes from positive to negative. The pDNA/PEI/HA ternary nanocomplexes showed high transfection efficiency and low cytotoxicity compared with commercially available products. When hASCs were pretreated with HA to passivate CD44, the transfection efficiency of pDNA/PEI/HA nanocomplexes was significantly reduced. These results suggest that HA that can act as a targeting ligand to CD44 contributed to the improved transfection of pDNA into hASCs. Our novel pDNA/PEI/HA nanocomplexes may be used as an effective non-viral pDNA delivery system for hASCs.

Evaluation of the novel near-infrared fluorescence tracers pullulan polymer nanogel and indocyanine green/γ-glutamic acid complex for sentinel lymph node navigation surgery in large animal models.

BACKGROUND: This study aimed to examine tracers designed to overcome the disadvantages of indocyanine green (ICG), which disperses quickly to multiple lymph nodes, using a near-infrared (NIR) imaging system in animal models. METHODS: Diluted ICG, ICG/poly-γ-glutamic acid (PGA) complex, and IRDye900-conjugated pullulan-cholesterol nanoprobe "near-infrared polynagogel" (NIR-PNG) were injected into the stomachs of dogs and pigs, and the patterns of dispersion were observed using an NIR imaging system. To compare retention times, fluorescence signals were evaluated in the stomach and small bowel of animals 1 week after injection. RESULTS: A diluted concentration (~0.1 mg/ml) of ICG was optimal for NIR imaging compared with the conventional concentration (5 mg/ml) for visual inspection. When injected into the stomach, the signals of ICG and ICG/PGA complex were relatively large at the injection site, and signals were detected at multiple sentinel nodes and lymph nodes beyond them. The NIR-PNG signal intensity was relatively small at the injection site and limited to only one sentinel node with no additional node. When evaluated 1 week after injection, only the NIR-PNG signal was detected in the canine stomach, and the signal intensity at the lymph nodes of the porcine small bowel was the highest with NIR-PNG, followed by ICG/PGA complex and finally ICG. CONCLUSION: NIR-PNG showed the best characteristics of less dispersion and longer retention in the sentinel nodes, and ICG/PGA complex remained longer than diluted ICG. These tracers could potentially be used as optimal tracers for sentinel node navigation surgery in gastric cancer.

In vivo imaging of islet transplantation using PLGA nanoparticles containing iron oxide and indocyanine green.

PURPOSE: We determined whether poly(lactic-co-glycolic acid) nanoparticles would be a useful reagent for the successful monitoring of isolated islets by magnetic resonance imaging and optical imaging systems, without clinically relevant toxicity in vitro or in vivo. METHODS: We used iron oxide for MR imaging and a cyanide dye approved by the Food and Drug Administration (indocyanine green) for optical imaging and estimated the in vivo detection of transplanted pancreatic islets. RESULTS: The poly(lactic-co-glycolic acid) nanoparticles were associated with the islets in vitro and were successfully detected by 4.7 T (MR) and optical imaging, without other toxic effects. When labeled islets were transplanted under the mouse kidney capsule, in vivo T2/ T2*-weighted scans with 4.7 T MR detected as few as 300 labeled islets by 4 weeks. Optical in vivo imaging revealed indocyanine green fluorescence by 2 and 4 days after transplantation of islets containing 250 and 500 µg/mL poly(lactic-co-glycolic acid) nanoparticles, respectively. These results were further supported by the immunohistochemical results for insulin and iron in the recipient mouse kidney and pancreas. CONCLUSIONS: Taken together, these data indicate that poly(lactic-co-glycolic acid) nanoparticles may be used to label transplanted islets and may be imaged with in vivo MR and optical imaging systems.

Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

Highly secreted tryptophanyl tRNA synthetase 1 as a potential theranostic target for hypercytokinemic severe sepsis.

Despite intensive clinical and scientific efforts, the mortality rate of sepsis remains high due to the lack of precise biomarkers for patient stratification and therapeutic guidance. Secreted human tryptophanyl-tRNA synthetase 1 (WARS1), an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4 against infection, activates the genes that signify the hyperinflammatory sepsis phenotype. High plasma WARS1 levels stratified the early death of critically ill patients with sepsis, along with elevated levels of cytokines, chemokines, and lactate, as well as increased numbers of absolute neutrophils and monocytes, and higher Sequential Organ Failure Assessment (SOFA) scores. These symptoms were recapitulated in severely ill septic mice with hypercytokinemia. Further, injection of WARS1 into mildly septic mice worsened morbidity and mortality. We created an anti-human WARS1-neutralizing antibody that suppresses proinflammatory cytokine expression in marmosets with endotoxemia. Administration of this antibody into severe septic mice attenuated cytokine storm, organ failure, and early mortality. With antibiotics, the antibody almost completely prevented fatalities. These data imply that blood-circulating WARS1-guided anti-WARS1 therapy may provide a novel theranostic strategy for life-threatening systemic hyperinflammatory sepsis.

Polymer nanomicelles for efficient mucus delivery and antigen-specific high mucosal immunity.

Micelles for mucosal immunity: A mucosal vaccine system based on γ-PGA nanomicelles and viral antigens was synthesized. The intranasal administration of the vaccine system induces a high immune response both in the humoral and cellular immunity (see picture).

A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances cancer immunotherapy and reduces immune cell exhaustion.

Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.

Self-fluorescence of chemically crosslinked MRI nanoprobes to enable multimodal imaging of therapeutic cells.

Old chemistry for novel materials: Self-fluorescent high-relaxivity T(2)-weighted magnetic resonance imaging (MRI) contrast agents are produced. They are a novel type of MR/optical dual-modality in vivo imaging nanoprobe using glutaraldehyde crosslinking chemistry, and they are used to label and monitor therapeutic cells both in vitro and in vivo.

Bioderived polyelectrolyte nanogels for robust antigen loading and vaccine adjuvant effects.

An easy but robust strategy for the synthesis of bioderived polyelectrolyte nanogels for protein antigen loading and vaccine adjuvant systems that can improve both humoral (Th2) and cellular immunity (Th1) is presented. The synthesized polyelectrolyte nanogels promote the uptake of antigens into antigen-presenting cells and strongly induce ovalbumin-specific INF-γ producing cells, cytotoxic T cell activity, and antibody production.

pH-stimuli-responsive near-infrared optical imaging nanoprobe based on poly(γ-glutamic acid)/poly(ß-amino ester) nanoparticles.

pH-stimuli-responsive near-infrared optical imaging nanoprobes are designed and synthesized in this study in a facile one-step synthesis process based on the use of the biocompatible and biodegradable polymer poly(γ-glutamic acid) (γ-PGA)/poly(ß-amino ester) (PBAE). PBAE has good transfection efficiency and promotes degradation properties under acidic conditions. This pH-responsive degradability can be used for the effective release of encapsulating materials after cellular uptake. As an optical imaging probe, indocyanine green (ICG) is an FDA-approved near-infrared fluorescent dye with a quenching property at a high concentration. In this regard, we focus here on the rapid degradation of PBAE in an acidic environment, in which the nanoparticles are disassembled. This allows the ICG dyes to show enhanced fluorescence signals after being releasing from the particles. We demonstrated this principle in cellular uptake experiments. We expect that the developed pH-stimuli-responsive smart nanoprobes can be applied in intracellular delivery signaling applications.

A first-in-human study of KMRC011, a potential treatment for acute radiation syndrome, to explore tolerability, pharmacokinetics, and pharmacodynamics.

KMRC011 is a novel Toll-like receptor 5 agonist under development as a treatment for acute radiation syndrome (ARS). The aim of this first-in-human study was to investigate the tolerability, pharmacokinetics, and pharmacodynamics of a single intramuscular dose of KMRC011 in healthy subjects. A randomized, single-blind, placebo-controlled, single dose-escalation study was conducted with the starting dose of 5 µg. Eight (4 only for 5 µg cohort) subjects per cohort were randomly assigned to KMRC011 or placebo in a 3:1 ratio. Dose-limiting toxicity (DLT) was assessed throughout the study. Serum concentrations of KMRC011, granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6) were measured up to 48 h postdose. Based on safety review, the dose of KMRC011 escalated up to 20 µg, and consequently, a total of 4 dose levels (5, 10, 15, and 20 µg) were explored. The most common adverse event was injection site reaction, showing no dose-related trend. Three DLTs (2 cases of hepatic enzyme increased and 1 of pyrexia) were observed; 1 in the 15 µg cohort and 2 in the 20 µg cohort. A developed method could not detect any KMRC011 in serum. KMRC011 15 µg and 20 µg showed significant increases of G-CSF, IL-6, and absolute neutrophil counts, compared with the placebo. A single intramuscular administration of KMRC011 ranging from 5 to 15 µg was tolerated in healthy subjects. Doses of KMRC011 equal to or greater than 15 µg exerted TLR5 agonist-like activities by increasing serum G-CSF and IL-6. It suggests that KMRC011 has the potential for a treatment for ARS.

Hypoxia Pretreatment Promotes Chondrocyte Differentiation of Human Adipose-Derived Stem Cells via Vascular Endothelial Growth Factor.

BACKGROUND: Human adipose tissue-derived stem cells (ADSCs) are attractive multipotent stem cell sources with therapeutic potential in various fields requiring repair and regeneration, such as acute and chronically damaged tissues. ADSC is suitable for cell-based therapy, but its use has been hampered due to poor survival after administration. Potential therapeutic use of ADSC requires mass production of cells through in vitro expansion. Many studies have consistently observed the tendency of senescence by mesenchymal stem cell (MSC) proliferation upon expansion. Hypoxia has been reported to improve stem cell proliferation and survival. METHODS: We investigated the effects of hypoxia pretreatment on ADCS proliferation, migration capacity, differentiation potential and cytokine production. We also analyzed the effects of vascular endothelial growth factor (VEGF) on osteogenic and chondrogenic differentiation of ADSCs by hypoxia pretreatment. RESULTS: Hypoxia pretreatment increased the proliferation of ADSCs by increasing VEGF levels. Interestingly, hypoxia pretreatment significantly increased chondrogenic differentiation but decreased osteogenic differentiation compared to normoxia. The osteogenic differentiation of ADSC was decreased by the addition of VEGF but increased by the depletion of VEGF. We have shown that hypoxia pretreatment increases the chondrogenic differentiation of ADSCs while reducing osteogenic differentiation in a VEGF-dependent manner. CONCLUSION: These results show that hypoxia pretreatment can provide useful information for studies that require selective inhibition of osteogenic differentiation, such as cartilage regeneration.

Multiplexed imaging of therapeutic cells with multispectrally encoded magnetofluorescent nanocomposite emulsions.

Here, we describe the fabrication of multispectrally encoded nanoprobes, perfluorocarbon (PFC)/quantum dots (QDs) nanocomposite emulsions, which could provide both multispectral MR and multicolor optical imaging modalities. Our strategy exploited the combination of the multispectral MR properties of four different PFC materials and the multicolor emission properties of three different colored CdSe/ZnS QDs. The PFC/QDs nanocomposite emulsions were fabricated by exchanging hydrophobic ligands coated onto CdSe/ZnS QDs using 1H,1H,2H,2H-perfluorooctanethiol, which renders the QDs to be dispersible in the PFC liquids. To provide biocompatibility, the PFC liquids containing QDs were emulsified into aqueous solutions with the aid of phospholipids. The distinct (19)F-based MR images of PFC/QDs nanocomposite emulsions were obtained by selective excitation of the nanocomposite emulsions with magnetic resonance frequency of each PFC, while a specific fluorescence image of them could be selected using appropriate optical filters. The uptake of PFC/QDs nanocomposite emulsions was high in phagocytic cells such as macrophages (90.55%) and dendritic cells (85.34%), while it was low in nonphagocytic T cells (33%). We have also shown that the nanocomposite emulsions were successfully applied to differentially visualize immunotherapeutic cells (macrophages, dendritic cells, and T cells) in vivo. The PFC/QDs nanocomposite emulsions are expected to be a promising multimodality nanoprobe for the multiplexed detection and imaging of therapeutic cells both in vitro and in vivo.

Noninvasive imaging of dendritic cell migration into lymph nodes using near-infrared fluorescent semiconductor nanocrystals.

Effective tracking of immunotherapeutic cells is essential for monitoring the migration of injected cells to the target tissue. Here we report the use of near-infrared (NIR) -emitting fluorescent semiconductor nanocrystals, called quantum dots (QDs), for noninvasive in vivo tracking of dendritic cell (DC) migration into lymph nodes. The effect of QDs on DC viability and maturation was systematically investigated using MTT assays and FACS analysis. We found that the labeling of DCs with QDs had no effect on DC phenotype or maturation potential. Cytokine and migration assays revealed that there were no significant changes in either cytokine production or chemokine-dependent migration of QD-labeled DCs relative to unlabeled cells; in both labeled and unlabeled cells, cytokine production and migratory capacity was increased by stimulation with lipopolysaccharide. Furthermore, QDs did not influence allogenic naive T cell activation or uptake of exogenous antigens. Notably, we also demonstrated that it was possible to track QD-labeled DCs injected into the footpad into popliteal and inguinal lymph nodes using NIR fluorescence. Taken together, our protocols establish the potential of noninvasive in vivo imaging of NIR-emitting QDs for tracking immunotherapeutic cells.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy.

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN-gamma) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Lyophilizable and Multifaceted Toll-like Receptor 7/8 Agonist-Loaded Nanoemulsion for the Reprogramming of Tumor Microenvironments and Enhanced Cancer Immunotherapy.

The low therapeutic efficacy of current cancer immunotherapy is related to nonimmunogenic and immunosuppressive tumor microenvironments (TMEs). To overcome these limitations, both the immune priming of antitumoral lymphocytes and the reprogramming of immunosuppressive factors in TMEs are essential. Here, we suggest a nanoemulsion (NE)-based immunotherapeutic platform that can not only modulate tumor-induced suppression but also induce an effective cell-mediated immune response for T cell proliferation. Multifunctional NEs can be fabricated by integrating the efficacy of NEs as delivery systems and the multifaceted immunomodulation characteristics (i.e., immunostimulation and reprogramming of immunosuppression) of small molecule-based Toll-like receptor 7/8 agonists. Local in situ vaccination of melanoma and cervical tumor models with tumor antigens (protein and peptide) adjuvanted with NE loaded with TLR7/8 agonists [NE (TLR7/8a)] induced the recruitment and activation of innate immune cells, infiltration of lymphocytes, and polarization of tumor-associated M2 macrophages, which resulted in inhibition of tumor growth and prolonged survival in both primary and rechallenged tumor models. Antibody-depletion experiments also suggested that macrophages, type I IFN (IFN-α and IFN-ß), CD8+ T cells, and NK1.1+ cells contributed to the antitumor effect of NE (TLR7/8a). The combination of antitumoral lymphocytes and reprogramming of immunosuppressive TMEs induced by NE (TLR7/8a) treatment evoked a synergistic antitumor immune response with immune checkpoint blockade therapy (anti-PD-1 and anti-PD-L1).

The anaphase promoting complex promotes NHEJ repair through stabilizing Ku80 at DNA damage sites.

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APCCdh1 and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.