Mechanosignaling YAP/TAZ-TEAD Axis Regulates the Immunomodulatory Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have gained significant attention in cell therapies due to their multipotency and immunomodulatory capacities. The transcriptional co-activators YAP/TAZ, central to the mechanotransduction system in MSCs, dominantly direct MSCs lineage commitment. However, their role in immunomodulation remains elusive. Accordingly, this present study aimed to investigate the role of mechanotransducer YAP/TAZ and their binding target transcriptional factor, TEAD, in the immunomodulatory capacities of human bone marrow-derived MSCs. Reducing YAP/TAZ activity by altering the matrix stiffness, disrupting the F-actin integrity with chemical inhibitors, or using siRNAs increased the expression of immunomodulatory genes, such as TSG-6 and IDO, upon TNF-α stimulation. Similarly, transfection of TEAD siRNA also increased the immunomodulatory capacities in MSCs. RNA-seq analysis and inhibition assays demonstrated that the immunomodulatory capacities caused by YAP/TAZ-TEAD axis disruption were due to the NF-κB signaling pathway activation. Then, we also evaluated the in vivo anti-inflammatory efficacy of MSCs in a dextran sulfate sodium (DSS)-induced mice colitis model. The administration of human MSCs transfected with TEAD siRNA, which exhibited enhanced immunomodulatory properties in vitro, significantly ameliorated inflammatory bowel disease symptoms, such as body weight loss and acute colon inflammation, in the DSS-induced mice colitis model. Our findings underscore the mechanosignaling YAP/TAZ-TEAD axis as a regulator of MSCs immunomodulation. Targeting these signaling pathways could herald promising MSCs-based therapies for immune disorders.

A Cartilaginous Construct with Bone Collar Exerts Bone-Regenerative Property Via Rapid Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be implanted into tissue defects with no artificial scaffolds. In addition, the cellular properties and characteristics of the ECM in C-MSCs can be regulated in vitro. Most bone formation in the developmental and healing process is due to endochondral ossification, which occurs after bone collar formation surrounding cartilage derived from MSCs. Thus, to develop a rapid and reliable bone-regenerative cell therapy, the present study aimed to generate cartilaginous tissue covered with a mineralized bone collar-like structure from human C-MSCs by combining chondrogenic and osteogenic induction. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free (XF) growth medium. Confluent cells that formed cellular sheets were detached from the culture plate using a micropipette tip. The floating cellular sheet contracted to round clumps of cells (C-MSCs). C-MSCs were maintained in XF-chondro-inductive medium (CIM) and XF-osteo-inductive medium (OIM). The biological and bone-regenerative properties of the generated cellular constructs were assessed in vitro and in vivo. C-MSCs cultured in CIM/OIM formed cartilaginous tissue covered with a mineralized matrix layer, whereas CIM treatment alone induced cartilage with no mineralization. Transplantation of the cartilaginous tissue covered with a mineralized matrix induced more rapid bone reconstruction via endochondral ossification in the severe combined immunodeficiency mouse calvarial defect model than that of cartilage generated using only CIM. These results highlight the potential of C-MSC culture in combination with CIM/OIM to generate cartilage covered with a bone collar-like structure, which can be applied for novel bone-regenerative cell therapy.

Clumps of mesenchymal stem cells/extracellular matrix complexes directly reconstruct the functional periodontal tissue in a rat periodontal defect model.

Periodontitis is an inflammatory disease characterized by tooth-supporting periodontal tissue destruction, including the cementum, periodontal ligament, and alveolar bone. To regenerate the damaged periodontal tissue, mesenchymal stem cells (MSCs) have attracted much scientific and medical attention. Recently, we generated clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs), which consist of cells and self-produced ECM. C-MSCs can be transplanted into lesion areas without artificial scaffold to induce tissue regeneration. To develop reliable scaffold-free periodontal tissue regenerative cell therapy by C-MSCs, this study investigated the periodontal tissue regenerative capacity of C-MSCs and the behavior of the transplanted cells. Rat bone marrow-derived MSCs were isolated from rat femur. Confluent cells were scratched using a micropipette tip and then torn off. The sheet was rolled to make a three-dimensional round clump of cells, C-MSCs. Then, ten C-MSCs were grafted into a rat periodontal fenestration defect model. To trace the grafted cells in the defect, PKH26-labeled cells were also employed. Micro-CT and histological analyses demonstrated that transplantation of C-MSCs induced successful periodontal tissue regeneration in the rat periodontal defect model. Interestingly, the majority of the cells in the reconstructed tissue, including cementum, periodontal ligaments, and alveolar bone, were PKH26 positive donor cells, suggesting the direct tissue formation by MSCs. This study demonstrates a promising scaffold-free MSCs transplantation strategy for periodontal disease using C-MSCs and offers the significance of multipotency of MSCs to induce successful periodontal tissue regeneration.

Xenotransplantation of cryopreserved human clumps of mesenchymal stem cells/extracellular matrix complexes pretreated with IFN-γ induces rat calvarial bone regeneration.

Introduction: Three-dimensional (3D) clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes, composed with cells and self-produced intact ECM, can be grafted into defect areas without artificial scaffold to induce successful bone regeneration. Moreover, C-MSCs pretreated with IFN-γ (C-MSCsγ) increased the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) expression and thereby inhibited T cell activity. Xenotransplantation of human C-MSCsγ suppressed host T cell immune rejection and induced bone regeneration in mice. Besides, we have also reported that C-MSCs retain the 3D structure and bone regenerative property even after cryopreservation. To develop the "off-the-shelf" cell preparation for bone regenerative therapy that is promptly provided when needed, we investigated whether C-MSCsγ can retain the immunosuppressive and osteogenic properties after cryopreservation. Methods: Confluent human MSCs that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The round cell clumps were incubated with a growth medium for 3 days, and then C-MSCs were obtained. To generate C-MSCsγ, after 2 days' culture, C-MSCs were stimulated with 50 ng/ml of IFN-γ. Both C-MSCs and C-MSCsγ were cryopreserved for 2 days and then thawed to obtain Cryo-C-MSCs and Cryo-C-MSCsγ, respectively. The biological properties of those cell clumps were assessed in vitro. In addition, to test whether human Cryo-C-MSCsγ attenuates immune rejection to induce bone regeneration, a xenograft study using a rat calvarial defect was performed. Results: Both IFN-γ pretreatment and cryopreservation process did not affect the 3D structure and cell viability in all human cell clumps. Interestingly, Cryo-C-MSCsγ showed significantly increased IDO mRNA expression equivalent to C-MSCsγ. More importantly, xenotransplantation of human C-MSCsγ and Cryo-C-MSCsγ induced rat calvarial bone regeneration by suppressing rat T cells infiltration and the grafted human cells reduction in the grafted area. Finally, there were no human donor cells in the newly formed bone, implying that the bone reconstruction by C-MSCsγ and Cryo-C-MSCsγ can be due to indirect host osteogenesis. Conclusion: These findings implied that Cryo-C-MSCsγ can be a promising bone regenerative allograft therapy that can be certainly and promptly supplied on demand.

Stepwise Evolution of a Klebsiella pneumoniae Clone within a Host Leading to Increased Multidrug Resistance.

Five blaCTX-M-14-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.

Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes Generated with Xeno-Free Chondro-Inductive Medium Induce Bone Regeneration via Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be transplanted into tissue defect site with no artificial scaffold. Importantly, most bone formation in the developing process or fracture healing proceeds via endochondral ossification. Accordingly, this present study investigated whether C-MSCs generated with chondro-inductive medium (CIM) can induce successful bone regeneration and assessed its healing process. Human bone marrow-derived MSCs were cultured with xeno-free/serum-free (XF) growth medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The cell clumps, i.e., C-MSCs, were maintained in XF-CIM. C-MSCs generated with XF-CIM showed enlarged round cells, cartilage matrix, and hypertrophic chondrocytes genes elevation in vitro. Transplantation of C-MSCs generated with XF-CIM induced successful bone regeneration in the SCID mouse calvaria defect model. Immunofluorescence staining for human-specific vimentin demonstrated that donor human and host mouse cells cooperatively contributed the bone formation. Besides, the replacement of the cartilage matrix into bone was observed in the early period. These findings suggested that cartilaginous C-MSCs generated with XF-CIM can induce bone regeneration via endochondral ossification.

Molecular analysis of virulence factors of hypermucoviscous Klebsiella pneumoniae in a diabetes patient with multifocal intramuscular and musculoskeletal abscesses.

Unusual community-acquired invasive Klebsiella pneumoniae infection has been reported worldwide, particularly in Asia. Recently, several virulence-associated genes of the isolates have been investigated. We report a case of multifocal intramuscular and musculoskeletal abscesses caused by K. pneumoniae in a 61-year-old male diabetes patient. A string test of the K. pneumoniae isolate, which was recovered from abscesses obtained by surgical debridement and drainage, was positive. We used whole-genome sequencing to analyze the virulence-associated gene profile of the isolate. The isolate belonged to the K2 genotype with sequence type 375. The isolate harbored rmpA and rmpA2, which induce serum resistance (hypermucoviscosity). The isolate also carried siderophores, i.e., aerobactin and salmochelin, which are associated with enhanced bacterial growth. The isolate did not harbor K1-unique virulence factors, such as colibactin, microcin, and yersiniabactin. Our K2 strain harbored a combination of virulence plasmid-associated genes-rmpA/A2 and siderophores (aerobactin and salmochelin). Hence, we advocate that essential molecular virulence factors of isolates that cannot be identified by a string test and capsular serotyping alone may exist.

"Pulmonary Actinomycosis attributable to Actinomyces meyeri presenting as cardiac tamponade: a case report".

BACKGROUND: Recently, it is reported that Actinomyces meyeri is the most common species causing actinomycosis. However, to our knowledge, there was no report about pulmonary actinomycosis attributable to A. meyeri presenting as cardiac tamponade. CASE PRESENTATION: Hereby we describe a case of pulmonary actinomycosis attributable to A. meyeri presenting as cardiac tamponade. At first, the patient was diagnosed with bacterial pericarditis with lung abscess in the left lower lung lobe and underwent pericardial drainage. Three days after the removal of the drainage tube, atrial fibrillation followed by cardiac arrest with asystole occurred and immediate cardiopulmonary resuscitation restored his circulation. Thereafter, he underwent pneumocentesis of the lung abscess and the culture grew A. meyeri. He was successfully treated with penicillin G. CONCLUSION: This is the first case of pulmonary actinomycosis attributable to A. meyeri presenting as cardiac tamponade. We believe that an increased awareness of the disease is necessary to expedite diagnosis therefore minimizing morbidity and mortality.