A GMP-compliant manufacturing method for Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

Efficient generation of induced pluripotent stem cell lines from healthy donors' peripheral blood mononuclear cells of different genders.

Peripheral blood mononuclear cell (PBMC) are recognized as a conveniently collected reprogramming resource. Several methods are available in academia to reprogram PBMC into induced pluripotent stem cells (iPSC). In this research, we reprogrammed PBMC of different genders by using non-integrative non-viral liposome electrotransfer containing the reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The three obtained iPSC cell lines were karyotypically normal and showed significant tritiated differentiation potential in vitro and in vivo. Our study provided an efficient procedure for reprogramming PBMC into iPSC and obtained three well-functioning iPSC, that may contribute to advance personalized cell therapy in the future.

Unveiling the functional heterogeneity of cytokine-primed human umbilical cord mesenchymal stem cells through single-cell RNA sequencing.

BACKGROUND: Mesenchymal stem cells (MSCs) hold immense promise for use in immunomodulation and regenerative medicine. However, their inherent heterogeneity makes it difficult to achieve optimal therapeutic outcomes for a specific clinical disease. Primed MSCs containing a certain cytokine can enhance their particular functions, thereby increasing their therapeutic potential for related diseases. Therefore, understanding the characteristic changes and underlying mechanisms of MSCs primed by various cytokines is highly important. RESULTS: In this study, we aimed to reveal the cellular heterogeneity, functional subpopulations, and molecular mechanisms of MSCs primed with IFN-γ, TNF-α, IL-4, IL-6, IL-15, and IL-17 using single-cell RNA sequencing (scRNA-seq). Our results demonstrated that cytokine priming minimized the heterogeneity of the MSC transcriptome, while the expression of MSC surface markers exhibited only slight changes. Notably, compared to IL-6, IL-15, and IL-17; IFN-γ, TNF-α, and IL-4 priming, which stimulated a significantly greater number of differentially expressed genes (DEGs). Functional analysis, which included Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, indicated that IFN-γ, TNF-α, and IL-4-primed hUC-MSCs are involved in interferon-mediated immune-related processes, leukocyte migration, chemotaxis potential, and extracellular matrix and cell adhesion, respectively. Moreover, an investigation of various biological function scores demonstrated that IFN-γ-primed hUC-MSCs exhibit strong immunomodulatory ability, TNF-α-primed hUC-MSCs exhibit high chemotaxis potential, and IL-4-primed hUC-MSCs express elevated amounts of collagen. Finally, we observed that cytokine priming alters the distribution of functional subpopulations of MSCs, and these subpopulations exhibit various potential biological functions. Taken together, our study revealed the distinct regulatory effects of cytokine priming on MSC heterogeneity, biological function, and functional subpopulations at the single-cell level. CONCLUSIONS: These findings contribute to a comprehensive understanding of the inflammatory priming of MSCs, paving the way for their precise treatment in clinical applications.

Circulating metabolites as potential biomarkers for the early detection and prognosis surveillance of gastrointestinal cancers.

BACKGROUND AND AIMS: Two of the most lethal gastrointestinal (GI) cancers, gastric cancer (GC) and colon cancer (CC), are ranked in the top five cancers that cause deaths worldwide. Most GI cancer deaths can be reduced by earlier detection and more appropriate medical treatment. Unlike the current "gold standard" techniques, non-invasive and highly sensitive screening tests are required for GI cancer diagnosis. Here, we explored the potential of metabolomics for GI cancer detection and the classification of tissue-of-origin, and even the prognosis management. METHODS: Plasma samples from 37 gastric cancer (GC), 17 colon cancer (CC), and 27 non-cancer (NC) patients were prepared for metabolomics and lipidomics analysis by three MS-based platforms. Univariate, multivariate, and clustering analyses were used for selecting significant metabolic features. ROC curve analysis was based on a series of different binary classifications as well as the true-positive rate (sensitivity) and the false-positive rate (1-specificity). RESULTS: GI cancers exhibited obvious metabolic perturbation compared with benign diseases. The differentiated metabolites of gastric cancer (GC) and colon cancer (CC) were targeted to same pathways but with different degrees of cellular metabolism reprogramming. The cancer-specific metabolites distinguished the malignant and benign, and classified the cancer types. We also applied this test to before- and after-surgery samples, wherein surgical resection significantly altered the blood-metabolic patterns. There were 15 metabolites significantly altered in GC and CC patients who underwent surgical treatment, and partly returned to normal conditions. CONCLUSION: Blood-based metabolomics analysis is an efficient strategy for GI cancer screening, especially for malignant and benign diagnoses. The cancer-specific metabolic patterns process the potential for classifying tissue-of-origin in multi-cancer screening. Besides, the circulating metabolites for prognosis management of GI cancer is a promising area of research.

Efficient generation of induced pluripotent stem cell lines from peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMCs) have been widely considered as a more convenient and almost unlimited reprogramming resource, while the reprogramming procedure and efficiency still need to be improved. We reprogrammed the PBMCs by using non-integrative non-viral vectors liposome electrotransfer containing the reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The iPSC lines exhibited a normal karyotype with their corresponding PBMCs and exhibited significant cellular pluripotency. Teratoma formation assay revealed that the iPSCs we generated could differentiate into three embryonic germ layers. Our study provides a more effective procedure for peripheral blood monocyte reprogramming to iPSC, and promotes its future application.

Interleukin-18-primed human umbilical cord-mesenchymal stem cells achieve superior therapeutic efficacy for severe viral pneumonia via enhancing T-cell immunosuppression.

Coronavirus disease 2019 (COVID-19) treatments are still urgently needed for critically and severely ill patients. Human umbilical cord-mesenchymal stem cells (hUC-MSCs) infusion has therapeutic benefits in COVID-19 patients; however, uncertain therapeutic efficacy has been reported in severe patients. In this study, we selected an appropriate cytokine, IL-18, based on the special cytokine expression profile in severe pneumonia of mice induced by H1N1virus to prime hUC-MSCs in vitro and improve the therapeutic effect of hUC-MSCs in vivo. In vitro, we demonstrated that IL-18-primed hUC-MSCs (IL18-hUCMSC) have higher proliferative ability than non-primed hUC-MSCs (hUCMSCcon). In addition, VCAM-1, MMP-1, TGF-ß1, and some chemokines (CCL2 and CXCL12 cytokines) are more highly expressed in IL18-hUCMSCs. We found that IL18-hUCMSC significantly enhanced the immunosuppressive effect on CD3+ T-cells. In vivo, we demonstrated that IL18-hUCMSC infusion could reduce the body weight loss caused by a viral infection and significantly improve the survival rate. Of note, IL18-hUCMSC can also significantly attenuate certain clinical symptoms, including reduced activity, ruffled fur, hunched backs, and lung injuries. Pathologically, IL18-hUCMSC transplantation significantly enhanced the inhibition of inflammation, viral load, fibrosis, and cell apoptosis in acute lung injuries. Notably, IL18-hUCMSC treatment has a superior inhibitory effect on T-cell exudation and proinflammatory cytokine secretion in bronchoalveolar lavage fluid (BALF). Altogether, IL-18 is a promising cytokine that can prime hUC-MSCs to improve the efficacy of precision therapy against viral-induced pneumonia, such as COVID-19.

Computationally rational design of metal-involving halogen bonds with π-covalency: Structures and bonding analysis.

Traditional π-covalent interactions have been proved in the non-metal halogen bond adducts formed by chloride and halogenated triphenylamine-based radical cations. In this study, we have rationally designed two metal-involving halogen bond adducts with π-covalency property, such as [L1-Pd···I-PTZ]+ (i.e., 1) and [L2-Pd···I-PTZ]+ (i.e., 2), in which the square-planar palladium complexes serve as halogen bond acceptor and 3,7-diiodo-10H-phenothiazine radical cation (i.e., [I-PTZ]•+ ) acts as halogen bond donor. Noncovalent interaction analysis and quantum theory of atoms in molecules analysis revealed that there are notable halogen bond interactions along the Pd···I direction without genuine chemical bond formed in both designed adducts. Energy decomposition analysis together with natural orbital for chemical valence calculations were performed to gain insight into their bonding nature, which demonstrated the presence of remarkable π-covalent interactions and σ-covalent interactions in both 1 and 2. We therefore proposed a new strategy for building the metal-involving halogen bonds with π-covalency property, which will help the further development of new types of halogen bonds.

A Comprehensive Mass Spectrometry-Based Workflow for Clinical Metabolomics Cohort Studies.

As a comprehensive analysis of all metabolites in a biological system, metabolomics is being widely applied in various clinical/health areas for disease prediction, diagnosis, and prognosis. However, challenges remain in dealing with the metabolomic complexity, massive data, metabolite identification, intra- and inter-individual variation, and reproducibility, which largely limit its widespread implementation. This study provided a comprehensive workflow for clinical metabolomics, including sample collection and preparation, mass spectrometry (MS) data acquisition, and data processing and analysis. Sample collection from multiple clinical sites was strictly carried out with standardized operation procedures (SOP). During data acquisition, three types of quality control (QC) samples were set for respective MS platforms (GC-MS, LC-MS polar, and LC-MS lipid) to assess the MS performance, facilitate metabolite identification, and eliminate contamination. Compounds annotation and identification were implemented with commercial software and in-house-developed PAppLineTM and UlibMS library. The batch effects were removed using a deep learning model method (NormAE). Potential biomarkers identification was performed with tree-based modeling algorithms including random forest, AdaBoost, and XGBoost. The modeling performance was evaluated using the F1 score based on a 10-times repeated trial for each. Finally, a sub-cohort case study validated the reliability of the entire workflow.

Mechanistic study of the bismuth mediated fluorination of arylboronic esters and further rational design.

Density functional theory (DFT) calculations have been performed to gain insight into the catalytic mechanism of the bismuth redox catalyzed fluorination of arylboronic esters to deliver the widely used arylfluoride compounds (Science 2020, 367, 313-317). The study reveals that the whole catalysis can be characterized via three stages: (i) transmetallation generates the Bi(iii) intermediate 5, capitalizing on the use of KF as an activator. (ii) 5 then reacts with the electrophilic fluorination reagent 1-fluoro-2,6-dichloropyridinium 4via oxidative addition to give the Bi(v) intermediate IM4A. (iii) IM4A undergoes a reductive elimination step to yield aryl fluoride compounds and regenerates the bismuth catalyst for the next catalytic cycle. Each stage is kinetically and thermodynamically feasible. The transmetallation step, with a barrier of 25.4 kcal mol-1, is predicted to be the rate-determining step (RDS) during the whole catalytic cycle. Furthermore, based on a mechanistic study, new catalysts with the framework of tethered bis-anionic ligands were designed, which will help to improve current catalytic systems and develop new bismuth mediated fluorination of arylboronic esters.

Human Platelet Lysate Maintains Stemness of Umbilical Cord-Derived Mesenchymal Stromal Cells and Promote Lung Repair in Rat Bronchopulmonary Dysplasia.

Mesenchymal stromal cells (MSCs) show potential for treating preclinical models of newborn bronchopulmonary dysplasia (BPD), but studies of their therapeutic effectiveness have had mixed results, in part due to the use of different media supplements for MSCs expansion in vitro. The current study sought to identify an optimal culture supplement of umbilical cord-derived MSCs (UC-MSCs) for BPD therapy. In this study, we found that UC-MSCs cultured with human platelet lysate (hPL-UCMSCs) were maintained a small size from Passage 1 (P1) to P10, while UC-MSCs cultured with fetal bovine serum (FBS-UCMSCs) became wide and flat. Furthermore, hPL was associated with lower levels of senescence in UC-MSCs during in vitro expansion compared with FBS, as indicated by the results of ß-galactosidase staining and measures of senescence-related genes (CDKN2A, CDKN1A, and mTOR). In addition, hPL enhanced the proliferation and cell viability of the UC-MSCs and reduced their doubling time in vitro. Compared with FBS-UCMSCs, hPL-UCMSCs have a greater potential to differentiate into osteocytes and chondrocytes. Moreover, using hPL resulted in greater expression of Nestin and specific paracrine factors (VEGF, TGF-ß1, FGF2, IL-8, and IL-6) in UC-MSCs compared to using FBS. Critically, we also found that hPL-UCMSCs are more effective than FBS-UCMSCs for the treatment of BPD in a rat model, with hPL leading to improvements in survival rate, lung architecture and fibrosis, and lung capillary density. Finally, qPCR of rat lung mRNA demonstrated that hPL-UCMSCs had lower expression levels of inflammatory factors (TNF-α and IL-1ß) and a key chemokine (MCP-1) at postnatal day 10, and there was significant reduction of CD68+ macrophages in lung tissue after hPL-UCMSCs transplantation. Altogether, our findings suggest that hPL is an optimal culture supplement for UC-MSCs expansion in vitro, and that hPL-UCMSCs promote lung repair in rat BPD disease.

Programmed Cell Death Ligand 1 Is Enriched in Mammary Stem Cells and Promotes Mammary Development and Regeneration.

Programmed cell death ligand 1 (PD-L1) is widely expressed in a variety of human tumors, and inhibition of the PD-L1/PD-1 pathway represents one of the most promising therapy for many types of cancer. However, the physiological function of PD-L1 in tissue development is still unclear, although PD-L1 mRNA is abundant in many tissues. To address this puzzle, we investigated the function of PD-L1 in mammary gland development. Interestingly, we found that PD-L1 is enriched in protein C receptor (Procr)-expressing mammary stem cells (MaSCs), and PD-L1-expressing mammary basal cells (PD-L1+ basal cells) exhibit robust mammary regeneration capacity in transplantation assay. The lineage tracing experiment showed that PD-L1+ cells can differentiate into all lineages of mammary epithelium cells, suggesting that PD-L1+ basal cells have the activities of MaSCs. Furthermore, PD-L1 deficiency significantly impairs mammary development and reduces mammary regeneration capacity of mammary basal cells, suggesting that PD-L1 is not only enriched in MaSCs but also improves activities of MaSCs. In summary, these results demonstrated that PD-L1 is enriched in MaSCs and promotes mammary gland development and regeneration. Mechanistically, our data indicated that PD-L1 expression is induced by continuous activation of Wnt/ß-catenin signaling. In conclusion, these results demonstrated that PD-L1 is a marker of MaSCs, and PD-L1 is essential for mammary development. Our study provides novel insight into the physiological functions of PD-L1 in tissue development.

Human umbilical cord mesenchymal stromal cells attenuate pulmonary fibrosis via regulatory T cell through interaction with macrophage.

BACKGROUND: Pulmonary fibrosis (PF) is a growing clinical problem with limited therapeutic options. Human umbilical cord mesenchymal stromal cell (hucMSC) therapy is being investigated in clinical trials for the treatment of PF patients. However, little is known about the underlying molecular and cellular mechanisms of hucMSC therapy on PF. In this study, the molecular and cellular behavior of hucMSC was investigated in a bleomycin-induced mouse PF model. METHODS: The effect of hucMSCs on mouse lung regeneration was determined by detecting Ki67 expression and EdU incorporation in alveolar type 2 (AT2) and lung fibroblast cells. hucMSCs were transfected to express the membrane localized GFP before transplant into the mouse lung. The cellular behavior of hucMSCs in mouse lung was tracked by GFP staining. Single cell RNA sequencing was performed to investigate the effects of hucMSCs on gene expression profiles of macrophages after bleomycin treatment. RESULTS: hucMSCs could alleviate collagen accumulation in lung and decrease the mortality of mouse induced by bleomycin. hucMSC transplantation promoted AT2 cell proliferation and inhibited lung fibroblast cell proliferation. By using single cell RNA sequencing, a subcluster of interferon-sensitive macrophages (IFNSMs) were identified after hucMSC infusion. These IFNSMs elevate the secretion of CXCL9 and CXCL10 following hucMSC infusion and recruit more Treg cells to the injured lung. CONCLUSIONS: Our study establishes a link between hucMSCs, macrophage, Treg, and PF. It provides new insights into how hucMSCs interact with macrophage during the repair process of bleomycin-induced PF and play its immunoregulation function.

Efficacy and safety of oral Panax notoginseng saponins for unstable angina patients: A meta-analysis and systematic review.

BACKGROUND: Panax notoginseng saponins (PNS) is one of the most important active ingredients in Panax notoginseng, which plays an important role against cardiovascular diseases in Traditional Chinese Medicine (TCM). METHODS: This review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement. We searched the following databases from their inception to February 2017: CENTRAL, MEDLINE, EMBASE Database, WHO ICTRP, CNKI, WANFANG, VIP and SinoMed. All the randomized controlled trials (RCTs) based on PNS in patients with unstable angina (UA) which meet the standard were included. RESULT: Seventeen studies were included in this systemic review. The included studies indicated that PNS has promising therapeutic effects on reduction of the primary end point [RR 0.05 (95% CI -0.07, -0.02); P < 0.001], electrocardiography (ECG) [RR 0.32 (95% CI 0.23, 0.46); P < 0.001], the frequency and duration of angina attacks [MD -1.88 (95% CI -2.03, -1.72); P < 0.001], and dosage of nitroglycerin [MD -1.13 (95% CI -1.70, -0.56); P < 0.001] of UA patients. Adverse events were described 9 included RCTs. CONCLUSION: Oral PNS could reduce the end point, and improve the ECG, the frequency and duration of angina pectoris, dosage of nitroglycerin and lipids in UA patients. And the results indicated oral PNS is safe up to now. However, we need more multi-centre, large-sample, high-quality RCTs to provide high-quality evidence.

Epigenetic mechanisms in coronary artery disease: The current state and prospects.

Coronary artery disease (CAD) is the leading cause of morbidity and mortality. CAD has both genetic and environmental causes. In the past two decades, the understanding of epigenetics has advanced swiftly and vigorously. It has been demonstrated that epigenetic modifications are associated with the onset and progression of CAD. This review aims to improve the understanding of the epigenetic mechanisms closely related to CAD and to provide a novel perspective on the onset and development of CAD. Epigenetic changes include DNA methylation, histone modification, microRNA and lncRNA, which are interrelated with critical genes and influence the expression of those genes. In addition, miRNA plays a diverse role in the pathological process of CAD. Numerous studies have found that some cardiac-specific miRNAs have potential as certain diagnostic biomarkers and treatment targets for CAD. In this review, the aberrant epigenetic mechanisms that contribute to CAD will be discussed. We will also provide novel insight into the epigenetic mechanisms that target CAD.

The role of DNA methylation in coronary artery disease.

Epigenetic studies have identified DNA methylation in coronary artery disease (CAD). How the critical genes interact at the cellular level to cause CAD is still unknown. The discovery of DNA methylation inspired researchers to explore relationships in genomic coding and disease phenotype. In the past two decades, there have been many findings regarding the relationship between DNA methylation and CAD development, and the DNA methylation of critical genes have been found to be significantly changed during CAD, including DNA methylation at homocysteine, Alu and long Interspersed Element 1 (LINE-1) repetitive elements. Here, we provide a brief overview of the biology and mechanisms of DNA methylation and its roles in CAD. We also discuss recent findings regarding DNA methylation of homocysteine, Alu and LINE-1 and some genes on CAD in vitro and in vivo. Finally, we provide some perspectives on DNA methylation in CAD.

Panax notoginseng Saponins for Treating Coronary Artery Disease: A Functional and Mechanistic Overview.

Coronary artery disease (CAD) is a major public health problem and the chief cause of morbidity and mortality worldwide. Panax notoginseng, a valuable herb in traditional Chinese medicine (TCM) with obvious efficacy and favorable safety, shows a great promise as a novel option for CAD and is increasingly recognized clinically. Firstly, this review introduced recent clinical trials on treatment with PNS either alone or in combination with conventional drugs as novel treatment strategies. Then we discussed the mechanisms of P. notoginseng and Panax notoginseng saponins (PNS), which can regulate signaling pathways associated with inflammation, lipid metabolism, the coagulation system, apoptosis, angiogenesis, atherosclerosis, and myocardial ischaemia.

Modern researches on Blood Stasis syndrome 1989-2015: A bibliometric analysis.

BACKGROUND: Blood Stasis syndrome (BSS) is one of the major syndromes in Traditional East Asia medicine (TEAM). Modern research of BSS began in the late1980s. METHODS: We searched in PubMed for BSS-related articles published between 1989 and 2015. The publication information, study contents, and bibliometric indicators were documented and analyzed. RESULTS: Most of the BSS-related studies were conducted by Chinese researchers in China. The number of publications on BSS-related increased rapidly in recent years, so did the quality of them. The disease diversity of BSS-related studies increased along with the number of publications. Yet the academic influence of them remained on average low; further, the diagnostic criteria of BSS were not uniform. CONCLUSIONS: BSS-related studies have increased in quantity and quality, but there are limitations to them which require improvement in research productivity and academic influence.

The dynamic changes of DNA methylation in primordial germ cell differentiation.

The discovery of DNA methylation has provided a new perspective on how DNA may be dynamically regulated in the mammalian genome. DNA methylation is a dynamic process with a demethylation and de novo methylation from primordial germ cell to differentiation. DNA methylation and demethylation have been proposed to play important roles in somatic cell reprogramming. Some essential components were discussed, such as hydroxymethylation which has recently been confirmed as a modification of developmental importance.