Veillonella parvula promotes the proliferation of lung adenocarcinoma through the nucleotide oligomerization domain 2/cellular communication network factor 4/nuclear factor kappa B pathway.

Enrichment of Veillonella parvula in the lung microbiota is strongly associated with non-small cell lung cancer (NSCLC) and induces the progression of lung adenocarcinoma in vivo, but its actual role and mechanism remain unexplored. This study analyzed the correlation between NSCLC and V. parvula abundance based on 16 s rRNA sequencing results. The effects of V. parvula on the progression of lung adenocarcinoma were observed in vivo and in vitro using a C57 bl/6j mouse tumor-bearing model, a bacterial cell co-culture model, combined with transcriptome sequencing, and a TCGA database to explore and validate the growth promotion of lung adenocarcinoma by V. parvula and its molecular mechanism. 16 s rRNA sequencing revealed that V. parvula was significantly enriched in lung adenocarcinoma. In vivo, V. parvula promoted the growth of lung adenocarcinoma in mice by suppressing the infiltration of tumor-associated T lymphocytes and peripheral T lymphocytes. It showed a higher affinity for lung adenocarcinoma in vitro and promoted lung adenocarcinoma cell proliferation through adhesion or intracellular invasion. Further analysis of differential gene expression and KEGG enrichment by transcriptome sequencing revealed that V. parvula induced CCN4 expression and activated NOD-like receptor and NF-κB signaling pathway in lung adenocarcinoma cells. Further analysis clarified that V. parvula promoted activation of the NF-κB pathway via Nod2/CCN4 signaling, which promoted lung adenocarcinoma cell proliferation. Thus, V. parvula mediates activation of the Nod2/CCN4/NF-κB signaling pathway to promote non-small cell lung adenocarcinoma progression, thereby providing a potential target for diagnosing and treating lung adenocarcinoma.

Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells.

Previous studies transplanted human-induced pluripotent stem cells (hiPSCs)-derived mesenchymal stem cells (iMSCs) into thyroid cartilage defect of X-liked severe combined immunodeficiency (X-SCID) rats and confirmed transplanted cell survival and cartilage regeneration. Thus, this study aimed to investigate the contribution of iMSC transplantation to thyroid cartilage regeneration of nude rats. iMSCs were induced from hiPSCs via a neural crest cell lineage. Then, clumps formed from an iMSC/extracellular matrix complex were transplanted into thyroid cartilage defects in nude rats. The larynx was removed and histological and immunohistochemical analyses were performed 4 or 8 weeks after the transplantation. Human nuclear antigen (HNA)-positive cells were observed in 11 of 12 (91.7%) rats, which indicated that transplanted iMSCs survived in thyroid cartilage defects in nude rats. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells in 8 of 12 rats (66.7%), which indicated cartilage-like regeneration. Cartilage-like regeneration in nude rats in this study was comparable to the previous report on X-SCID rats (HNA-positive cells were observed in all 14 rats and cartilage-like regeneration was observed in 10 of 14 rats). This result suggests that nude rats could be an alternative to X-SCID rats in thyroid cartilage regeneration experiments using iMSCs, and this nude rat cartilage transplantation model may develop cartilage regeneration research concerning fewer problems such as infection due to immunosuppression.

Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair.

Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.

Anti-angiogenic therapy for advanced primary pulmonary lymphoepithelioma-like carcinoma: a retrospective multicenter study.

PURPOSE: Primary pulmonary lympho-epithelioma-like carcinoma (PPLELC) is a rare subtype of primary non-small cell lung cancer (NSCLC). Currently, there is still lack of research data on anti-angiogenic therapy of advanced PPLELC. The purpose of this study was to investigate the efficacy and safety of anti-angiogenic therapy combined with chemotherapy compared with traditional chemotherapy for these patients. METHODS: Advanced PPLELC patients admitted to six grade A hospitals from January 2013 to January 2021 were selected. The patients received anti-angiogenic therapy combined with chemotherapy (AT group) or chemotherapy (CT group) alone. RESULTS: A total of 65 patients were included in this study, including 31 patients in the AT group treated with anti-angiogenic therapy combined with chemotherapy and 34 patients in the CT group treated with chemotherapy alone. As of October 1, 2021, the median progression-free survival (PFS) in the AT group was 11.2 months [95% confidence interval (CI), 5.9-16.5]. The median PFS in the CT group was 7.0 months [95%CI, 5.1-8.9] [Hazard Ratio (HR), 0.49; 95%CI, 0.29-0.83; P = 0.008]. The 1-year PFS rates were 41.9% and 17.6%, respectively. The overall response rates (ORR) of two groups were 45.2% (95% CI, 0.27-0.64), 38.2% (95% CI, 0.21-0.56), (P = 0.571). The disease control rates (DCR) of two groups were 93.5% (95% CI, 0.84-1.03), 88.2% (95% CI, 0.77-1.00), (P = 0.756). CONCLUSION: Among patients with advanced PPLELC, the PFS of patients with anti-angiogenic therapy combined with chemotherapy is better than that of patients with chemotherapy alone. Anti-angiogenic therapy combined with chemotherapy is an optional treatment scheme.

Induction of functional xeno-free MSCs from human iPSCs via a neural crest cell lineage.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that they secrete soluble factors into affected regions. We also found that the peroxidasin and IGF2 secreted by the XF-iMSCs partially contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine.

Alterations of lung microbiota in patients with non-small cell lung cancer.

The role of lung microbiota in non-small cell lung cancer remains unclear. We investigated the characteristics and functional roles of lung microbiota in non-small cell lung cancer. Bronchoalveolar lavage fluid samples were obtained from patients with non-small cell lung cancer (n = 46) and with benign lung disease (n = 29). The differences in composition and gene expression in the microbiota between the samples were analyzed using 16s rRNA sequencing. The oncogenic genus (Veillonella) was then evaluated in the progression of lung cancer in C57 BL/6 mice. Compared to benign lung disease, the lung microbiota in non-small cell lung cancer was significantly altered, both in terms of α- and ß-diversity. In terms of bacterial composition, the non-small cell lung cancer group was enriched with two Phyla (Firmicutes, Bacteroidetes) and three genera (Streptococcus, Prevotella, Veillonella). Prevotella and Veillonella were most strongly associated with non-small cell lung cancer, and Veillonella significantly promoted the progression of lung cancer in vivo. Moreover, metabolic prediction revealed that ribosomes, biosynthesis of secondary metabolites, and pyrimidine metabolism were among the enriched pathways that may be involved in the progression of non-small cell lung cancer. Overall, results suggest that the progression of non-small cell lung cancer is followed by significant changes in the composition and function of the lung microbiota. These differing genera may be potential diagnostic markers and therapeutic targets.

Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture.

Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.

In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells.

The laryngotracheal cartilage is a cardinal framework for the maintenance of the airway for breathing, which occasionally requires reconstruction. Because hyaline cartilage has a poor intrinsic regenerative ability, various regenerative approaches have been attempted to regenerate laryngotracheal cartilage. The use of autologous mesenchymal stem cells (MSCs) for cartilage regeneration has been widely investigated. However, long-term culture may limit proliferative capacity. Human-induced pluripotent stem cell-derived MSCs (iMSCs) can circumvent this problem due to their unlimited proliferative capacity. This study aimed to investigate the efficacy of iMSCs in the regeneration of thyroid cartilage in immunodeficient rats. Herein, we induced iMSCs through neural crest cell intermediates. For the relevance to prospective future clinical application, induction was conducted under xeno-free/serum-free conditions. Then, clumps fabricated from an iMSC/extracellular matrix complex (C-iMSC) were transplanted into thyroid cartilage defects in immunodeficient rats. Histological examinations revealed cartilage-like regenerated tissue and human nuclear antigen (HNA)-positive surviving transplanted cells in the regenerated lesion. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells. These results indicated that the transplanted C-iMSCs promoted thyroid cartilage regeneration and some of the iMSCs differentiated into chondrogenic lineage cells. Induced MSCs may be a promising candidate cell therapy for human laryngotracheal reconstruction.

Pro-angiogenic scaffold-free Bio three-dimensional conduit developed from human induced pluripotent stem cell-derived mesenchymal stem cells promotes peripheral nerve regeneration.

Although autologous nerve grafting is widely accepted as the gold standard treatment for segmental nerve defects, harvesting autologous nerves is highly invasive and leads to functional loss of the ablated part. In response, artificial nerve conduits made of artificial materials have been reported, but the efficacy of the nerve regeneration still needs improvement. The purpose of this study is to investigate the efficacy and mechanism of the Bio three-dimensional (3D) conduit composed of xeno-free human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). The 5-mm nerve gap of the sciatic nerve in immunodeficient rats was bridged with the Bio 3D conduit or silicone tube. Functional and histological recovery were assessed at 8 weeks after surgery. The regenerated nerve in the Bio 3D group was significantly superior to that in the silicone group based on morphology, kinematics, electrophysiology, and wet muscle weight. Gene expression analyses demonstrated neurotrophic and angiogenic factors. Macroscopic observation revealed neovascularization both inside and on the surface of the Bio 3D conduit. Upon their subcutaneous implantation, iMSCs could induce angiogenesis. The Bio 3D conduit fabricated from iMSCs are an effective strategy for nerve regeneration in animal model. This technology will be useful in future clinical situations.

Induced pluripotent stem cell-derived mesenchymal stem cells prolong hind limb survival in a rat vascularized composite allotransplantation model.

BACKGROUND: The reduction of systemic immunosuppressive agents is essential for the expansion of vascularized composite allotransplantation (VCA) in a clinical setting. The purpose of this study is to compare human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) with four other types of mesenchymal stem cells (human bone marrow-derived MSCs [BMMSCs], human adipose-derived MSCs [ADMSCs], rat BMMSCs, and rat ADMSCs) in vitro, and to investigate the in vivo immunomodulatory effect of iMSCs in a rat VCA model. MATERIALS AND METHODS: One Brown Norway (BN) rat, 2 Lewis (LEW) rats, and 1 Wistar rat were used in the mixed lymphocyte reaction (MLR), and 9 BN rats and 3 LEW rats (for donors), and 24 LEW rats (for recipients) were used in the VCA model. The abovementioned five types of MSCs were imaged to examine their morphology and were also tested for suppressor function using a MLR. The 24 recipient LEW rats were divided randomly into four groups, and subjected to orthotopic hind limb transplantation. The three control groups were the Iso group, in which transplantation was performed on from three to six LEW rats without immunosuppressive treatment (n = 6); the FK group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus alone (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) (n = 6); and the UT group, in which transplantation was performed from BN rats to LEW rats without any immunosuppressive treatment (n = 6). The experimental group was the iMSC group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) and injected with iMSCs (2 × 106 cells, day 7, intravenously) (n = 6). Hind limb survival was assessed by daily inspection of gross appearance until 50 days postoperatively. Histology of the skin and muscle biopsy were investigated on day 14 postoperatively. A time series of the plasma cytokine level (before transplantation, and at 10, 14, and 17 days after transplantation) was also analyzed. RESULTS: The size of adherent and trypsinized iMSCs was 67.5 ± 8.7 and 9.5 ± 1.1 µm, respectively, which was the smallest among the five types of MSCs (p < .01). The absorbance in MLR was significantly smaller with rat ADMSCs (p = .0001), human iMSCs (p = .0006), rat BMMSCs (p = .0014), human ADMSCs (p = .0039), and human BMMSCs (p = .1191) compared to without MSCs. In vivo, iMSC treatment prolonged hind limb survival up to 12.7 days in macroscopic appearance, which is significantly longer than that of the FK group (p < .01). Histology of the skin and muscle biopsy revealed that mononuclear cell infiltration was significantly reduced by iMSC injection (p < .01). iMSC treatment also affected proinflammatory cytokines (interferon-gamma (IFNγ) and tumor necrosis factor α (TNFα)) and the anti-inflammatory cytokine (interleukin-10 (IL-10)) of the recipient plasma. The IFNγ levels at Δ14 and the TNFα levels at Δ14 and Δ17 of the iMSC group were significantly lower than those of the FK group (p = .0226, .0004, and .004, respectively). The IL-10 levels at Δ10 and Δ14 of the iMSC group were significantly higher than those of the FK group (p = .0013 and .0374, respectively). CONCLUSIONS: iMSCs induce T cell hyporesponsiveness to prolong hind limb survival in a rat VCA model. This immunomodulatory property against acute rejection could provide one of the promising strategies capable of enabling the toxicities of immunosuppressants to be avoided in clinical settings.

An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.

Generation and Applications of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are adult stem cells with fibroblast-like morphology and isolated from the bone marrow via plastic adhesion. Their multipotency and immunoregulatory properties make MSCs possible therapeutic agents, and an increasing number of publications and clinical trials have highlighted their potential in regenerative medicine. However, the finite proliferative capacity of MSCs limits their scalability and global dissemination as a standardized therapeutic product. Furthermore, adult tissue provenance could constrain accessibility, impinge on cellular potency, and incur greater exposure to disease-causing pathogens based on the donor. These issues could be circumvented by the derivation of MSCs from pluripotent stem cells. In this paper, we review methods that induce and characterize MSCs derived from induced pluripotent stem cells (iPSCs) and introduce MSC applications to disease modeling, pathogenic mechanisms, and drug discovery. We also discuss the potential applications of MSCs in regenerative medicine including cell-based therapies and issues that should be overcome before iPSC-derived MSC therapy will be applied in the clinic.

Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1, a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling, we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo, the underlying mechanisms have yet to be revealed. To this end, we developed a high-throughput screening (HTS) system using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds, we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models, an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC-based HO model mouse, revealed critical roles for mTOR signaling in vivo. Moreover, we identified ENPP2, an enzyme that generates lysophosphatidic acid, as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis.

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.

Facilitated hyperpolarization signaling in vascular smooth muscle-overexpressing TRIC-A channels.

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.