Laponite Lights Calcium Flickers by Reprogramming Lysosomes to Steer DC Migration for An Effective Antiviral CD8+ T-Cell Response.

Immunotherapy using dendritic cell (DC)-based vaccination is an established approach for treating cancer and infectious diseases; however, its efficacy is limited. Therefore, targeting the restricted migratory capacity of the DCs may enhance their therapeutic efficacy. In this study, the effect of laponite (Lap) on DCs, which can be internalized into lysosomes and induce cytoskeletal reorganization via the lysosomal reprogramming-calcium flicker axis, is evaluated, and it is found that Lap dramatically improves the in vivo homing ability of these DCs to lymphoid tissues. In addition, Lap improves antigen cross-presentation by DCs and increases DC-T-cell synapse formation, resulting in enhanced antigen-specific CD8+ T-cell activation. Furthermore, a Lap-modified cocktail (Lap@cytokine cocktail [C-C]) is constructed based on the gold standard, C-C, as an adjuvant for DC vaccines. Lap@C-C-adjuvanted DCs initiated a robust cytotoxic T-cell immune response against hepatitis B infection, resulting in > 99.6% clearance of viral DNA and successful hepatitis B surface antigen seroconversion. These findings highlight the potential value of Lap as a DC vaccine adjuvant that can regulate DC homing, and provide a basis for the development of effective DC vaccines.

Establishment of a Novel Mouse Hepatocellular Carcinoma Model for Dynamic Monitoring of Tumor Development by Bioluminescence Imaging.

In this study, a novel mouse model of hepatocellular carcinoma (HCC) was established by simultaneously knocking out Pten and p53 suppressor genes and overexpressing c-Met and △90-ß-catenin proto-oncogenes in the livers of mice via hydrodynamic injection (HDI). The mutations were introduced using the CRISPR/Cas9 and Sleeping Beauty transposon systems. In this way, a primary liver cancer model was established within six weeks. In addition, macrophages expressing arginase-1(Arg1) promoter coupled with firefly luciferase were engineered for bioluminescence imaging (BLI) of the tumor microenvironment. This novel, rapidly-generated model of primary hepatocellular carcinoma can be monitored noninvasively, which can facilitate not only applications of the model, but also the development of new drugs and treatment strategies of HCC.

Leaf Coloration in Acer palmatum Is Associated with a Positive Regulator ApMYB1 with Potential for Breeding Color-Leafed Plants.

Anthocyanin biosynthesis and accumulation is closely associated with tissue/organ coloring in plants. To gain insight into the physiological and molecular mechanisms of leaf coloring in Acer palmatum, a deciduous tree during autumnal senescence, we first investigated concentration dynamics of pigments (i.e., chlorophyll, carotenoid and anthocyanin) in leaves with differential coloring. It was found that compared to green leaves (GN), anthocyanins were accumulated actively in semi-red (SR) and total-red (TR) leaves, accompanied with chlorophyll and carotenoid degradation. Then transcriptional profiling on GN and SR leaves identified thousands of transcripts with differential expression in SR compared to GN leaves. An annotation search showed that the entire flavonoid/anthocyanin biosynthesis pathway from the production of naringenin chalcone to modification of flavonoid backbone was extensively activated at the transcriptional level in SR leaves. Phylogenetic analysis of putative MYB proteins identified ApMYB1 as a putative regulator promoting anthocyanin biosynthesis. Expression of ApMYB1 in leaves was induced by exogenous hormones including abscisic acid. Stable overexpression of ApMYB1 in tobacco resulted in leaves with higher accumulation of anthocyanins. Collectively, our results identified ApMYB1 as a positive regulator associated with leaf coloring in Acer palmatum during autumnal senescence, which may be regarded a potential target for breeding color-leafed plants.

Large-Sized Graphene Oxide Nanosheets Increase DC-T-Cell Synaptic Contact and the Efficacy of DC Vaccines against SARS-CoV-2.

Dendritic cell (DC) vaccines are used for cancer and infectious diseases, albeit with limited efficacy. Modulating the formation of DC-T-cell synapses may greatly increase their efficacy. The effects of graphene oxide (GO) nanosheets on DCs and DC-T-cell synapse formation are evaluated. In particular, size-dependent interactions are observed between GO nanosheets and DCs. GOs with diameters of >1 µm (L-GOs) demonstrate strong adherence to the DC surface, inducing cytoskeletal reorganization via the RhoA-ROCK-MLC pathway, while relatively small GOs (≈500 nm) are predominantly internalized by DCs. Furthermore, L-GO treatment enhances DC-T-cell synapse formation via cytoskeleton-dependent membrane positioning of integrin ICAM-1. L-GO acts as a "nanozipper," facilitating the aggregation of DC-T-cell clusters to produce a stable microenvironment for T cell activation. Importantly, L-GO-adjuvanted DCs promote robust cytotoxic T cell immune responses against SARS-CoV-2 spike 1, leading to >99.7% viral RNA clearance in mice infected with a clinically isolated SARS-CoV-2 strain. These findings highlight the potential value of nanomaterials as DC vaccine adjuvants for modulating DC-T-cell synapse formation and provide a basis for the development of effective COVID-19 vaccines.

Dimethyl Sulfoxide-Free Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells Based on Zwitterionic Betaine and Electroporation.

The effective cryopreservation of mesenchymal stem cells (MSCs) is indispensable to the operation of basic research and clinical transplantation. The prevalent protocols for MSC cryopreservation utilize dimethyl sulfoxide (DMSO), which is easily permeable and able to protect MSCs from cryo-injuries, as a primary cryoprotectant (CPA). However, its intrinsic toxicity and adverse effects on cell function remain the bottleneck of MSC cryopreservation. In this work, we cryopreserved human umbilical cord mesenchymal stem cells (UCMSCs) using zwitterionic betaine combined with electroporation without any addition of DMSO. Betaine was characterized by excellent compatibility and cryoprotective properties to depress the freezing point of pure water and balance the cellular osmotic stress. Electroporation was introduced to achieve intracellular delivery of betaine, intending to further provide comprehensive cryoprotection on UCMSCs. Compared with DMSO cryopreservation, UCMSCs recovered from the protocol we developed maintained the normal viability and functions and reduced the level of reactive oxygen species (ROS) that are harmful to cell metabolism. Moreover, the in vivo distribution of thawed UCMSCs was consistent with that of fresh cells monitored by a bioluminescence imaging (BLI) system. This work opens a new window of opportunity for DMSO-free MSC cryopreservation using zwitterionic compounds like betaine combined with electroporation.

Large-sized graphene oxide synergistically enhances parenchymal hepatocyte IL-6 expression monitored by dynamic imaging.

Graphene oxides (GOs) have received significant attention as emerging biomedical materials due to their special properties. The application of GOs in biological systems has raised considerable concern about their hepatotoxicity, however their biological effects on parenchymal hepatocytes remain unclear, despite the fact that GOs have shown size-dependent interactions with immunocytes in the liver. Herein we chose pleiotropic cytokine IL-6 as the model parameter to investigate inflammation responses upon exposure to GOs. An early and sensitive reporter mouse model was constructed, allowing non-invasive and longitudinal imaging of parenchymal hepatocyte IL-6 expressions. GOs of various lateral dimensions were assessed by using the reporter mice. The results demonstrated that large-sized GOs (L-GO) induced much stronger IL-6 activation. A detailed analysis uncovered that L-GO induced ROS production and TLR-4 activation promoted macrophage polarization and secretion of pro-inflammatory cytokines IL-1ß and TNF-α, activated via> the NF-κB signaling pathway, which in turn initiated the expression of IL-6 in hepatocytes. These in-depth investigations are expected to help modulate the inflammatory responses involved in hepatotoxicity and provide extended information to design sub-hepatic distribution and cell subset targeting by controlling the nanoparticle sizes.

Bone Marrow-Derived Mesenchymal Stem Cells Promoted Cutaneous Wound Healing by Regulating Keratinocyte Migration via ß2-Adrenergic Receptor Signaling.

Mesenchymal stem cells (MSCs) play an important role in cutaneous wound healing; however, the functional mechanisms involved in the healing process are poorly understood. A series of studies indicate that keratinocytes that migrate into the wound bed rely on an epithelial-mesenchymal transition (EMT)-like process to initiate re-epithelialization. We therefore examined whether bone marrow-derived MSCs (BMSCs) could affect biological behavior and induce EMT-like characteristics in the human epidermal keratinocytes (HEKs) and in the immortalized human keratinocyte cell line HaCaT cells, and we investigated the signaling pathways of BMSC-mediated phenotypic changes. By assessing the expression of EMT-related markers including E-cadherin, α-SMA, and Snail family transcription factors by ß2-adrenergic receptor (ß2-AR) blockage using ICI-118,551, a ß2-AR selective antagonist, or ß2-AR small interfering RNA (siRNA), we showed an involvement of ß2-AR signaling in the induction of EMT-like alterations in human keratinocytes in vitro. ß2-AR signaling also affected collective and individual cell migration in human keratinocyte cell lines, which was attenuated by administration of ICI-118,551. Treating the cells with BMSC-conditioned media (BMSC-CM) not only recapitulated the effect of isoproterenol (ISO) on cell migration but also induced the expression of ß2-AR and a panel of proteins associated with mesenchymal phenotype in HEKs and HaCaT cells. Similarly, a blockade of the ß2-AR by either ICI-118,551 or ß2-AR siRNAs reversed both responses of the epidermal keratinocyte cell lines relative to BMSC-CM exposure. These results were further verified in our vivo findings and indicated that the exogenous application of MSCs promoted cutaneous wound healing and endowed the keratinocytes surrounding the wound area with an increased migratory phenotype through activation of ß2-AR signaling. Our findings suggest a biochemical mechanism underlying the function of MSCs in wound re-epithelization, which provides a reliable theoretical basis for the wide application of MSCs in the treatment of chronic wounds.

Targeting ectodysplasin promotor by CRISPR/dCas9-effector effectively induces the reprogramming of human bone marrow-derived mesenchymal stem cells into sweat gland-like cells.

BACKGROUND: Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs). METHODS: Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model. RESULTS: After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly. CONCLUSIONS: These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns.