Mingyao Meng

Global transcriptome-wide analysis of CIK cells identify distinct roles of IL-2 and IL-15 in acquisition of cytotoxic capacity against tumor

BackgroundCytokine-induced killer (CIK) cells are an emerging approach of cancer treatment. Our previous study have shown that CIK cells stimulated with combination of IL-2 and IL-15 displayed improved proliferation capacity and tumor cytotoxicity. However, the mechanisms of CIK cell proliferation and acquisition of cytolytic function against tumor induced by IL-2 and IL-15 have not been well elucidated yet.MethodsCIKIL-2 and CIKIL-15 were generated from peripheral blood mononuclear cells primed with IFN-γ, and stimulated with IL-2 and IL-15 in combination with OKT3 respectively. RNA-seq was performed to identify differentially expressed genes, and gene ontology and pathways based analysis were used to identify the distinct roles of IL-2 and IL-15 in CIK preparation.ResultsThe results indicated that CIKIL-15 showed improved cell proliferation capacity compared to CIKIL-2. However, CIKIL-2 has exhibited greater tumor cytotoxic effect than CIKIL-15. Employing deep sequencing, we sequenced mRNA transcripts in CIKIL-2 and CIKIL-15. A total of 374 differentially expressed genes (DEGs) were identified including 175 up-regulated genes in CIKIL-15 and 199 up-regulated genes in CIKIL-2. Among DEGs in CIKIL-15, Wnt signaling and cell adhesion were significant GO terms and pathways which related with their functions. In CIKIL-2, type I interferon signaling and cytokine-cytokine receptor interaction were significant GO terms and pathways. We found that the up-regulation of Wnt 4 and PDGFD may contribute to enhanced cell proliferation capacity of CIKIL-15, while inhibitory signal from interaction between CTLA4 and CD80 may be responsible for the weak proliferation capacity of CIKIL-2. Moreover, up-regulated expressions of CD40LG and IRF7 may make for improved tumor cytolytic function of CIKIL-2 through type I interferon signaling.ConclusionsThrough our findings, we have preliminarily elucidated the cells proliferation and acquisition of tumor cytotoxicity mechanism of CIKIL-15 and CIKIL-2. Better understanding of these mechanisms will help to generate novel CIK cells with greater proliferation potential and improved tumor cytolytic function.

The CIK cells stimulated with combination of IL-2 and IL-15 provide an improved cytotoxic capacity against human lung adenocarcinoma

Generation of cytokine-induced killer (CIK) cells is an emerging approach in adoptive donor lymphocyte infusion for patients with a wide range of tumors. However, our previous in vitro studies have shown that the killing efficacy of CIK cells against lung cancer was lower than other tumor cells, while the underlying mechanisms are not clear. We explored the feasibility to improve CIK cells mediated cytotoxicity against lung cancer. Interleukin (IL)-15 is a pleiotropic cytokine that stimulates cytolytic activity and cytokine secretion of NK cells, which may enhance the cytotoxic activity of CIK cells. In this study, we intended to stimulate the CIK cells by IL-2 in combination with IL-15 in cell expansion to achieve enhanced cytotoxicity against lung cancer cells. The different phenotypes of IL-2 or combination of IL-2 and IL-15 stimulated cytokine-induced killer cells were determined, and the improved cytotoxicity of IL-2 and IL-15 induced CIK cells against lung adenocarcinoma were evaluated both in vitro and in vivo. CIK cells stimulated with both IL-2 and IL-15 has shown greater proliferative potential than CIK cells treated with IL-2 alone. IL-15 induction also has driven the expansion of CD3+CD56+ subset and significantly enhanced cytotoxicity against tumor cells. Further analysis has demonstrated that CIKIL-2&IL-15 injected mice models have shown significant tumor regression and lower expression level of CyclinD1 in tumor tissue. This study has provided preclinical evidences that CIKIL-2&IL-15 with enhanced cytotoxicity may offer alternative treatment option for patients with lung cancer.

Anti-NSP4 antibody can block rotavirus-induced diarrhea in mice.

Background: Rotavirus infection is the most common cause of infectious diarrhea and gastroenteritis among children worldwide. The viral proteins (VP), especially VP4- and VP7-induced neutralizing antibodies, were considered to be critical in protective immunity to rotavirus disease. However, whether the antibody to rotavirus nonstructural protein 4 (NSP4) protects against rotavirus-induced diarrhea directly is not completely clear, especially for the protective time course. Materials and Methods: To obtain direct evidence, 12-day-old ICR mice were treated with NSP4 and entire rotavirus to induce diarrhea. Results: Both NSP4 and rotavirus-treated mice developed diarrhea, which was accompanied by histological changes in the small intestine compared to age-matched control mice. Anti-NSP4 antibody demonstrated protection against both entire rotavirus-induced diarrhea and NSP4-induced diarrhea. The histological changes in the small intestinal were reversible. These data show that early intervention with anti-NSP4 antibody can prevent rotavirus-induced diarrhea in mice; late intervention with anti-NSP4 antibody could halt diarrhea progression in mice. Conclusions: Our findings demonstrate for the first time that administration of anti-NSP4 antibody is effective both prior to and during the time course of rotavirus infection. These observations extend our knowledge of rotavirus infection and its therapeutic options.

Comparative transcriptome analysis of atrial septal defect identifies dysregulated genes during heart septum morphogenesis.

Congenital heart disease (CHD) is one of most common birth defects, causing fetal loss and death in newborn all over the world. Atrial and ventricular septal defects were the most common CHD subtypes in most districts. During the past decades, several genes were identified to control atrial septum formation, and mutations of these genes can cause cardiac septation defects. However, the pathogenic mechanism of ASD on transcriptional levels has not been well elucidated yet. Herein, we performed comparative transcriptome analysis between normal and atrial septal defect (ASD) patients by Illumina RNA sequencing (RNA-seq). Advanced bioinformatic analyses were employed to identify dysregulated genes in ASD. The results indicated that cardiac specific transcriptional factors (GATA4 and NKX2-5), extracellular signal molecules (VEGFA and BMP10) and cardiac sarcomeric proteins (MYL2, MYL3, MYH7, TNNT1 and TNNT3) were downregulated in ASD which may affect heart atrial septum formation, cardiomyocyte proliferation and cardiac muscle development. Importantly, cell cycle was dominant pathway among downregulated genes, and decreased expression of the proteins included in cell cycle may disturb cardiomyocyte growth and differentiation during atrial septum formation. Our study provided evidences of understanding pathogenic mechanism of ASD and resource for validation of CHD genomic studies.

Stemness gene expression profile analysis in human umbilical cord mesenchymal stem cells.

Umbilical cord mesenchymal stem cells (UC-MSCs) have several advantages for clinical therapy: the material is easily obtainable, the donation procedure is painless and there is low risk of viral contamination. UC-MSCs play important roles in tissue regeneration, tissue damage repair, autoimmune disease and graft-versus-host disease. In this study, we investigated the normal mRNA expression profile of UC-MSCs, and analyzed the candidate proteins responsible for the signaling pathway that may affect the differentiation characteristics of UC-MSCs. UC-MSCs were isolated by mincing UC samples into fragments and placing them in growth medium in a six-well plate. The immunophenotype characteristics and multilineage differentiation potential of the UC-MSCs were measured by flow cytometry and immunohistochemical assays. In addition, the pathway-focused gene expression profile of UC-MSCs was compared with those of normal or tumorous cells by realtime quantitative polymerase chain reaction. We successfully isolated and cultured UC-MSCs and analyzed the appropriate surface markers and their capacity for osteogenic, adipogenic and neural differentiation. In total, 168 genes focusing on signal pathways were examined. We found that the expression levels of some genes were much higher or lower than those of control cells, either normal or tumorous. UC-MSCs exhibit a unique mRNA expression profile of pathway-focused genes, especially some stemness genes, which warrants further investigation.

Determining the optimal protocol for preparing an acellular scaffold of tissue engineered small-diameter blood vessels

Although detergent-based decellularization protocols have been widely used to obtain a natural extracellular matrix (ECM) scaffold in tissue engineering, some key challenges still exist. To achieve an optimum natural decellularized scaffold for the construction of tissue-engineered small-diameter blood vessels (TEBV), porcine carotid arteries (PCAs) were decellularized by combining sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) and Triton X-100 (Triton) in different concentrations. Tissue samples were processed and their histological, biochemical and biomechanical characteristics were investigated. Results showed that only two methods 0.5% (SDS + SDC) and 1% (SDS + SDC) could completely remove of the cellular contents and preserve the native ECM architecture. Furthermore, 1% (SDS + SDC) based methods acquire preferable porosity and suitable mechanical strength. Residual Triton in the ECM scaffold holds intensive cytotoxity. In conclusion, 1%(SDS + SDC) based method can obtain a superior PCAs scaffold for the construction of TEBV.

Compared to the amniotic membrane, Wharton’s jelly may be a more suitable source of mesenchymal stem cells for cardiovascular tissue engineering and clinical regeneration

BackgroundThe success of developing cardiovascular tissue engineering (CTE) grafts greatly needs a readily available cell substitute for endothelial and interstitial cells. Perinatal annexes have been proposed as a valuable source of mesenchymal stem cells (MSCs) for tissue engineering and regenerative medicine. The objective of the present study is to evaluate the potential of human Wharton’s jelly MSCs (WJ-MSCs) and amniotic membrane MSCs (AM-MSCs) as a seeding cell in CTE and cardiovascular regenerative medicine.MethodsWJ-MSCs/AM-MSCs were isolated and characterized in vitro according to their morphology, proliferation, self-renewal, phenotype, and multipotency. More importantly, the characteristics of hemocompatibility, extracellular matrix deposition, and gene expression and viability of both MSCs were investigated.ResultsFibroblast-like human WJ-MSCs and AM-MSCs were successfully isolated and positively expressed the characteristic markers CD73, CD90, and CD105 but were negative for CD34, CD45, and HLA-DR. Both MSCs shared trilineage differentiation toward the adipogenic, osteogenic, and chondrogenic lineages. The proliferative and self-renewal capacity of WJ-MSCs was significantly higher than that of AM-MSCs (P < 0.001). WJ-MSCs provided comparable properties of antiplatelet adhesion and did not activate the coagulation cascade to endothelial cells. However, aggregated platelets were visualized on the surface of AM-MSCs-derived cell sheets and the intrinsic pathway was activated. Furthermore, WJ-MSCs have superior properties of collagen deposition and higher viability than AM-MSCs during cell sheet formation.ConclusionsThis study highlights that WJ-MSCs could act as a functional substitute of endothelial and interstitial cells, which could serve as an appealing and practical single-cell source for CTE and regenerative therapy.

MicroRNA profiling of CD3 + CD56 + cytokine-induced killer cells

Studies have proven that IL-2 and IL-15 showed contrasting roles during CIK cells preparation. By employing microarray, we analyzed miRNA expression profiles of PBMC, CIKIL-2 and CIKIL-15. Advanced bioinformatic analyses were performed to explore the key miRNAs which may regulate cell proliferation and anti-tumor activity of CIK. We identified 261 differentially expressed miRNAs (DEMs) between PBMC and CIKIL-2, and 249 DEMs between PBMC and CIKIL-15. MiR-143-3p/miR-145-5p was miRNA cluster which may positively regulate cell proliferation. In contrast, miR-340-5p/miR-340-3p cluster may negatively regulate cell proliferation via induction apoptosis, which may cause decreased cell proliferation capacity of CIKIL-2. MiRNA-target interaction analysis indicated that 10 co-downregulated miRNAs may synergistically turn on the expression of a pool of tumor cytotoxic genes in CIK cells. The DEMs between CIKIL-2 and CIKIL-15 may contribute to enhanced tumor cytotoxic capacity of CIKIL-2. Importantly, we found that repressed miR-193a-5p may regulate the expressions of inhibitory receptor KLRD1. The results of the validation assay have shown that KLRD1 were upregulated in CIK cells. Our findings have provided new insights into mechanisms of CIK cells production and tumor cytotoxic function, and shed light on their safety for clinical trial.