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.

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.

Cox2-mediated PGE2 production via p38/JNK-c-fos signaling inhibits cell apoptosis in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes.

Mesenchymal stem cells (MSCs), a class of adult stem cells, have attracted scientific and medical attention due to their self-renewing properties, multipotency, and trophic factor production. Although MSCs were originally studied on classical two-dimensional (2D) plastic plates, extensive scientific efforts have developed three-dimensional (3D) MSC culture systems, including MSCs spheroids and organoids that can mimic physical conditions. Moreover, we have recently developed 3D culture clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs) for novel bone regenerative cell therapy. Of note, even though it is widely accepted that cell detachment from the culture plate causes cell apoptosis, so called anoikis, these 3D MSCs constructs can be maintained in floating culture conditions. Currently, it is unclear why 3D floating-cultured MSCs constructs can escape from anoikis. To answer this question, the present study explored trophic factor production in 3D floating-cultured C-MSCs that play a cytoprotective role against anoikis and clarified the underlying molecular mechanism in vitro. Compared with cells cultured on 2D plastic plates, PGE2 production mediated by COX2 was significantly increased, and its inhibition drastically induced cell apoptosis in 3D floating-cultured C-MSCs. In the process of C-MSCs preparation, detachment of the cell sheet from culture plate activated the p38/JNK-c-Fos signaling pathway. Moreover, blockage of this signaling by chemical inhibitors abrogated COX2/PGE2 expressions and induced severe apoptosis. These results demonstrated that cell detachment facilitates cytoprotective COX2-mediated PGE2 synthesis via p38/JNK-c-Fos signaling, revealing a possible mechanism that allows resistance against anoikis in floating-cultured 3D MSCs constructs.

Archaeal community dynamics and detection of ammonia-oxidizing archaea during composting of cattle manure using culture-independent DNA analysis.

The composting process is carried out under aerobic conditions involving bacteria, archaea, and fungi. Little is known about the diversity of archaeal community in compost, although they may play an important role in methane production and ammonia oxidation. In the present study, archaeal community dynamics during cattle manure composting were analyzed using a clone library of the archaeal 16S rRNA gene. The results indicated that methane-producing archaea (methanogen) and ammonia-oxidizing archaea (AOA) may be the dominant microbes throughout the composting. The community consisted primarily of Methanocorpusculum-like and Methanosarcina-like sequences until day 2, while the number of Candidatus Nitrososphaera-like sequences increased from day 6 to day 30. Methanosarcina thermophila-like sequences were dominant from day 2, suggesting that M. thermophila-like species can adapt to increasing temperature or nutrient loss. A denaturant gradient gel electrophoresis analysis of the archaeal amoA genes revealed that the dominant amoA gene sequence with 99% homology to that of Candidatus Nitrososphaera gargensis was identical to those obtained from a different composting facility. These data suggested that AOA may play a role in ammonia oxidation in several composting practices. Our results provide fundamental information regarding archaeal community dynamics that will help in understanding the collective microbial community in compost.