The anti-inflammatory effects of mesenchymal stem cells attenuate diffuse pulmonary hemorrhage.

There are few effective treatment options for diffuse pulmonary hemorrhage (DPH). We aimed to elucidate the therapeutic role and underlying mechanisms of mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) in DPH. Therapeutic effects of MSCs/MSC-EVs in pristane-induced DPH mice were evaluated via pulmonary function testing and histopathology. Transcriptome sequencing analyzed differentially expressed genes in control, DPH, and MSC groups. The proportion of macrophage polarization was evaluated in vivo and in vitro via fluorescence-activated cell sorting in control, DPH, MSC, MSC-EV inhalation, and MSC-EV intravenous groups. Intraperitoneal injection of pristane induced diffuse alveolar hemorrhage, early fibrosis, and inflammation in C57BL/6 mice. Monocytes were depleted in the peripheral blood in DPH mice and MSCs were recruited to the lungs, resulting in significantly attenuated diffuse alveolar hemorrhage and suppressed immunological response. This was more effective in the hyperacute hemorrhage phase than the early inflammatory phase. An MSC treatment-mediated anti-inflammatory effect was observed in DPH mice. Furthermore, MSC-EVs inhalation or tail-vein injection could effectively reduce DPH injury. MSCs could suppress macrophage M1 polarization in DPH in vivo and in vitro. MSCs displayed significant therapeutic effects in pristane-induced DPH, which may be a promising cell-free therapeutic approach.

Integration of 16S rRNA gene sequencing and LC/MS-based metabolomic analysis of early biomarkers of acute ischaemic stroke in Tibetan miniature pigs.

Acute ischaemic stroke (AIS) is a complex, systemic, pathological, and physiological process. Systemic inflammatory responses and disorders of the gut microbiome contribute to increased mortality and disability following AIS. We conducted 16S high-throughput sequencing and ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry-based non-targeted metabolomic analyses of the plasma from a Tibetan miniature pig middle cerebral artery occlusion (MCAO) model. A significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Actinobacteria were observed after the onset of AIS. Among the plasma metabolites, the levels of phospholipids and amino acids were considerably altered. Loading values and differential metabolite-bacterial group association analyses of the metabolome and microbiome indicated a correlation between the microbiome and metabolome of Tibetan miniature pigs after MCAO. Furthermore, significant changes were observed in the ABC transporter pathway and purine metabolism in the gut microbiome-plasma metabolome during the early stage of AIS. Kyoto Encyclopaedia of Genes and Genomes enrichment analysis showed that arginine, proline, and cyanoamino acid metabolism was upregulated while ABC transporter metabolism pathway and carbohydrate digestion and absorption were substantially downregulated. The results of this study suggest that AIS affects the gut microbiota and plasma metabolites in Tibetan miniature pigs and that faecal microbiota transplantation could be a potential therapeutic approach for AIS.

Human umbilical-cord-derived mesenchymal stem cells in combination with rapamycin reduce cartilage degradation via inhibition of the AKT/mTOR signaling pathway.

BACKGROUND AND AIMS: Mesenchymal stem cell (MSC) therapy is a promising strategy for treating osteoarthritis (OA). However, the inflammatory microenvironment, apoptosis of transplanted cells, and shear forces during direct injection limit the therapeutic efficacy. This study aimed to explore the role of rapamycin combined with human umbilical-cord-derived mesenchymal stem cells (hUMSCs) in OA rabbits in vivo. METHODS: OA rabbits received an intra-articular injection of a collagenase solution. Gross observations, X-ray examinations, and histological examinations were performed to detect cartilage degradation levels. The fluorescent membrane dye DiR was used to label hUMSCs. In the combination therapy group, rapamycin was injected into the rabbit knee joint one day post the intra-articular injection of hUMSCs. Bioinformatics and transcriptome profiling of the knee meniscus were used to evaluate the potential molecular mechanisms of the combination therapy. RESULTS: Our study shows that rapamycin combined with hUMSCs significantly ameliorated OA severity in vivo, enhancing matrix synthesis and promoting cartilage repair. The combination therapy was more efficient than rapamycin or hUMSC treatment alone. Moreover, bioinformatics and transcriptomic analyses revealed that combination therapy might enhance autophagy in chondrocytes, partially by inhibiting the mTOR pathway. CONCLUSIONS: Our study indicates that the combination therapy of rapamycin and hUMSCs may promote cartilage repair in OA rabbits through the mTOR pathway and offers a novel approach for OA therapy. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Our study provides new evidence to support the use of hUMSCs in combination with rapamycin as a potential candidate for OA treatment.

Multiparametric MRI Features Predict the SYP Gene Expression in Low-Grade Glioma Patients: A Machine Learning-Based Radiomics Analysis.

PURPOSE: Synaptophysin (SYP) gene expression levels correlate with the survival rate of glioma patients. This study aimed to explore the feasibility of applying a multiparametric magnetic resonance imaging (MRI) radiomics model composed of a convolutional neural network to predict the SYP gene expression in patients with glioma. METHOD: Using the TCGA database, we examined 614 patients diagnosed with glioma. First, the relationship between the SYP gene expression level and outcome of survival rate was investigated using partial correlation analysis. Then, 7266 patches were extracted from each of the 108 low-grade glioma patients who had available multiparametric MRI scans, which included preoperative T1-weighted images (T1WI), T2-weighted images (T2WI), and contrast-enhanced T1WI images in the TCIA database. Finally, a radiomics features-based model was built using a convolutional neural network (ConvNet), which can perform autonomous learning classification using a ROC curve, accuracy, recall rate, sensitivity, and specificity as evaluation indicators. RESULTS: The expression level of SYP decreased with the increase in the tumor grade. With regard to grade II, grade III, and general patients, those with higher SYP expression levels had better survival rates. However, the SYP expression level did not show any significant association with the outcome in Level IV patients. CONCLUSION: Our multiparametric MRI radiomics model constructed using ConvNet showed good performance in predicting the SYP gene expression level and prognosis in low-grade glioma patients.

TERT Mutation Is Accompanied by Neutrophil Infiltration and Contributes to Poor Survival in Isocitrate Dehydrogenase Wild-Type Glioma.

Mutation of the telomerase reverse transcriptase (TERT) promoter has been demonstrated as an unfavorable prognostic marker in patients with isocitrate dehydrogenase wild-type (IDHwt) glioma. This study aimed to investigate the immune role of TERT promoter mutation status which could improve prognostic prediction in IDHwt. TERT mutation status, IDH mutation, and 1p-19q codeletion status data were obtained from 614 glioma cases from the Cancer Genome Atlas, and 325 cases from the Chinese Glioma Genome Atlas. The same information was obtained from 49 clinical glioma tissues. TERT mutation is preferentially present in glioblastoma and IDH-wt gliomas and is associated with poor prognosis. Moreover, TERT mutation was associated with infiltration of neutrophils and expression of neutrophil chemokines. which might partially contribute to the poor outcome in IDH-wt glioma. Furthermore, patients with IDH-wt glioma did not harbor increased peripheral neutrophils, implying that the infiltrated neutrophil in the tumor environment might due to cytokine chemotaxis. In this study, we hereby propose that TERT mutation might be a molecular driver of the dysfunctional immune microenvironment in IDH-wt glioma. TERT mutation may be a potential immune therapeutic target for optimizing treatment combinations and patient selection for glioma immunotherapy.

Genetically blocking HPD via CRISPR-Cas9 protects against lethal liver injury in a pig model of tyrosinemia type I.

Hereditary tyrosinemia type I (HT1) results from the loss of fumarylacetoacetate hydrolase (FAH) activity and can lead to lethal liver injury (LLI). Therapeutic options for HT1 remain limited. The FAH -/- pig, a well-characterized animal model of HT1, represents a promising candidate for testing novel therapeutic approaches to treat this condition. Here, we report an improved single-step method to establish a biallelic (FAH -/- ) mutant porcine model using CRISPR-Cas9 and cytoplasmic microinjection. We also tested the feasibility of rescuing HT1 pigs through inactivating the 4-hydroxyphenylpyruvic acid dioxygenase (HPD) gene, which functions upstream of the pathogenic pathway, rather than by directly correcting the disease-causing gene as occurs with traditional gene therapy. Direct intracytoplasmic delivery of CRISPR-Cas9 targeting HPD before intrauterine death reprogrammed the tyrosine metabolism pathway and protected pigs against FAH deficiency-induced LLI. Characterization of the F1 generation revealed consistent liver-protective features that were germline transmissible. Furthermore, HPD ablation ameliorated oxidative stress and inflammatory responses and restored the gene profile relating to liver metabolism homeostasis. Collectively, this study not only provided a novel large animal model for exploring the pathogenesis of HT1, but also demonstrated that CRISPR-Cas9-mediated HPD ablation alleviated LLI in HT1 pigs and represents a potential therapeutic option for the treatment of HT1.

Lung adenocarcinoma organoids harboring EGFR 19Del and L643V double mutations respond to osimertinib and gefitinib: A case report.

INTRODUCTION: It has been well reported that non-small-cell lung cancer (NSCLC) patients with single epithelial growth factor receptor (EGFR) activating mutation have high objective response rate when treated with EGFR-TKIs. However, due to rarity of cases, the response of patients with EGFR double or multiple mutations is not yet well understood. Patient-derived organoid technology has become to a powerful tool in cancer personalized medicine. PATIENT CONCERNS: A 60-year-old nonsmoking female was admitted to hospital for lung cancer after Chest CT. DIAGNOSES: The patient had no obvious clinical symptoms. Postoperative pathology confirmed a stage I of NSCLC. An EGFR double mutation 19Del/L643V was detected in the sequence of patient's cancer specimen. INTERVENTIONS: The patient was in good condition after surgical resection, with no sign of lung cancer recurrence. The patient has not yet started on targeted medicine. OUTCOMES: A lung cancer organoid culture was established from the cancer tissue of the patient, which recapitulated the morphological and molecular characteristics of cancer tissue. The drug sensitivity test showed that the cancer organoids that retained original mutations were sensitive to anticancer agents osimertinib and gefitinib, while resistant to erlotinib and icotinib. CONCLUSION: The uncommon EGFR double mutation exhibits distinctive sensitivities towards different target drugs of EGFR-TKIs. Our findings provide a better understanding of EGFR-TKIs' effects on patient-derived cancer organoids harboring uncommon EGFR double mutation(s).

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway.

BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

BACKGROUND: Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs. METHODS: Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493-2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation. RESULTS: PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice. CONCLUSIONS: In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don't display completely hairless phenotype as expected.