The effect of serum origin on cytokines induced killer cell expansion and function.

BACKGROUND: Cytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture. RESULTS: Cell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression. CONCLUSION: hPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.

Chromosome conformation capture-based DNA hybridization method for chromosomal translocation screening.

Background: DNA probes have been widely used as diagnostic tools for translocations. This study sought to design a screening tool using ssDNA probes and chromosome conformation capture (3C) library fragment hybridization. Method: The authors focused on developing a probe for the juxtaposed region of MYC and TRD. Fragments of the MYC gene with a thiol modification (MYC-Au NP probe) were functionalized by gold nanoparticles (Au NPs). Then TRD probes were immobilized on a nitrocellulose surface. Hybridization between DNA probes and 3C library fragments of SKW3 cells was determined by color intensity. Results: Optimal hybridization of the 3C library sample of the cell line to probes showed higher color intensity than human umbilical vein endothelial cells. Conclusion: Combining 3C-based techniques and DNA-DNA hybridization can identify rearrangements in cancer cells.

Evaluation of anticancer effects of frankincense on breast cancer stem-like cells.

BACKGROUND: Relapse and metastasis in breast cancer are linked to cancer stem cells (CSCs) resistant to anticancer therapies. The presence of cancer stem-like cells (CSLCs) and their ability to self-renew is determined by in vitro spheroid formation. AIMS: Many studies have found that frankincense has anticancer impacts, although these effects on breast CSLCs have never been evaluated. METHODS AND RESULTS: A population of heterogeneous breast tumor cells was extracted from the tumor mass after generating an animal model of triple-negative breast cancer (TNBC). Spheroid formation was used as an in vitro assay to determine the existence of CSLCs in these cells. MTT assay was used to determine frankincense's cytotoxic activity. An annexin V- propidium iodide (PI) staining and scratch test were used to assess the induction of apoptosis and antimetastatic effects of frankincense. The frankincense extract has significant cytotoxic and apoptotic effects on breast CSLCs. Although, the breast CSLCs are more resistant to these impacts than other breast cancer cells. CONCLUSION: Our study is the first report that indicates that frankincense extract has anticancer properties in breast CSLCs. Compared to many anticancer chemicals, which have limited potential to battle cancer stem cells, frankincense is an appropriate option to combat breast CSCs.

Expression of osteopontin-5 splice variant in the mouse primary and metastatic breast cancer cells.

OBJECTIVE: Osteopontin (OPN) is a well-known glycoprotein involved in numerous pathobiological processes, including cancer. Despite having five splice variants for osteopontin in mice, the main focus of most studies has been on total OPN (tOPN). There are some studies on other splice variants, but the expression of osteopontin-5 (OPN5) has not been addressed in mouse cancer cells. Therefore, this study sought to evaluate OPN5 expression in mouse breast cancer cells. RESULTS: The expression of OPN5 in primary and metastatic breast cancer cells of mice was confirmed in our study. These findings provided important insights regarding the OPN alternative splicing in mice for the first time. It is concluded that, like other OPN-SVs, OPN5 probably plays an essential role in tumor progression, which requires further investigation in different tumor models.

Xeno-free protocol for GMP-compliant manufacturing of human fetal pancreas-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have been suggested as an appropriate source for diabetes cell-based therapies. The high proliferation and differentiation capacity of fetal MSCs and the role of fetal pancreatic-derived MSCs (FPMSCs) in islet generation make them good candidates for diabetes treatment. To manufacture clinical-grade MSCs, animal-free culture protocols are preferred. The current study aimed to establish a xeno-free/GMP-compliant protocol for FPMSCs manufacturing. The focus was on the effects of fetal bovine serum (FBS) replacement with pooled human serum (HS). MATERIAL AND METHODS: FPMSCs were isolated and expanded from the pancreas of legally aborted fetuses with few modifications in our previously established protocol. The cells were expanded in two different culture media, including DMEM supplemented with 10% FBS or 10% pooled HS. A side-by-side comparison was made to evaluate the effect of each serum on proliferation rate, cell cycle, senescence, multi-lineage differentiation capacity, immunophenotype, and tumorigenesis of FPMSCs. RESULTS: Flow cytometry analysis and three-lineage differentiation ability demonstrated that fibroblast-like cells obtained from primary culture had MSCs' characteristics. The FPMSCs displayed similar morphology and CD markers expression in both sera. HS had a higher proliferative effect on FPMSCs than FBS. In FBS, the cells reached senescence earlier. In addition to normal karyotypes and anchorage-dependent growth, in vivo tumor formation was not seen. CONCLUSION: Our results demonstrated that HS was a better serum alternative than FBS for in vitro expansion of FPMSCs. Compared with FBS, HS increased FPMSCs' proliferation rate and decreased their senescence. In conclusion, HS can effectively replace FBS for clinical-grade FPMSCs manufacturing.

Flow Cytometric Measurement of Reactive Oxygen Species to Assess the Effects of Preconditioning Total Body Irradiation on NOG Mice.

BACKGROUND: Preclinical development of new drugs for cancer immunotherapy requires preconditioning total body irradiation (TBI) of mice to be humanized via hematopoietic stem cell transplantation. To assess the effect of preconditioning TBI, we detected the reactive oxygen species (ROS), Annexin V, propidium iodide (PI) level in bone marrow samples by flow cytometer. METHODS: We divided all NOG mice between irradiated (n = 20) and control groups (n = 10) for two time points. Irradiated mice were exposed to 3.5 Gy of radiation. After sacrificing BM samples were collected, the flow cytometric percentage of ROS, Annexin V, and PI markers were investigated on days 2 and 14 after exposure. RESULTS: At the first time point, the level of ROS was higher in the irradiated group than in the control group, and this difference was statistically significant (P < 0.05). Also, at the second time point, the mean differences of all markers in the irradiated group were significantly compared to the control group (P < 0.05). CONCLUSION: Thus, in NOG mice, the measurement of ROS level is helpful to the assessment of preconditioning TBI.

Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy.

BACKGROUND: The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. RESULTS: T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96-98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. CONCLUSIONS: Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

Comparison of insulin secretion by transduced adipose-derived and endometrial-derived stem cells in 2D and 3D cultures on fibrin scaffold.

Type 1 diabetes is a metabolic disorder caused by the loss or dysfunction of ß-cells in the pancreas. Organ shortage is a critical concern of diabetic patients in need of beta islet transplantation. Tissue engineered islets are promising alternatives to traditional organ transplantation. Recent progress in stem cell biology and gene cloning techniques has raised hopes for the generation of insulin producing cells (IPCs) without the need of immunosuppression. The purpose of this study was to produce IPCs using human adipose-derived stem cells (hADSCs) and human endometrial-derived stem cells (hEnSCs) and also to compare the level of insulin secretion by these cells in 2D and 3D culture systems on fibrin scaffolding. Stem cells differentiation was carried out through transduction with an insulin over expression lentiviral vector. Real-time PCR and immunocytochemistry confirmed the successful transduction of both cell types. Both cell types showed comparable insulin secretion by ELISA.3D culture resulted in higher amounts of insulin secretion of the two cell types versus 2D as control. This study showed that insulin gene delivery to the stem cells could be an efficient method for producing IPCs and fibrin encapsulation enhances the functionality of these cells.

Hypoxia-Preconditioned Wharton's Jelly-Derived Mesenchymal Stem Cells Mitigate Stress-Induced Apoptosis and Ameliorate Human Islet Survival and Function in Direct Contact Coculture System.

Protection of isolated pancreatic islets against hypoxic and oxidative damage-induced apoptosis is essential during a pretransplantation culture period. A beneficial approach to maintain viable and functional islets is the coculture period with mesenchymal stem cells (MSCs). Hypoxia preconditioning of MSCs (Hpc-MSCs) for a short time stimulates the expression and secretion of antiapoptotic, antioxidant, and prosurvival factors. The aim of the present study was to evaluate the survival and function of human islets cocultured with Hpc-MSCs. Wharton's jelly-derived MSCs were subjected to hypoxia (5% O2: Hpc) or normoxia (20% O2: Nc) for 24 hours and then cocultured with isolated human islets in direct and indirect systems. Assays of viability and apoptosis, along with the production of reactive oxygen species (ROS), hypoxia-inducible factor 1-alpha (HIF-1α), apoptotic pathway markers, and vascular endothelial growth factor (VEGF) in the islets, were performed. Insulin and C-peptide secretions as islet function were also evaluated. Hpc-MSCs and Nc-MSCs significantly reduced the ROS production and HIF-1α protein aggregation, as well as downregulation of proapoptotic proteins and upregulation of antiapoptotic marker along with increment of VEGF secretion in the cocultured islet. However, the Hpc-MSCs groups were better than Nc-MSCs cocultured islets. Hpc-MSCs in both direct and indirect coculture systems improved the islet survival, while promotion of function was only significant in the direct cocultured cells. Hpc potentiated the cytoprotective and insulinotropic effects of MSCs on human islets through reducing stressful markers, inhibiting apoptosis pathway, enhancing prosurvival factors, and promoting insulin secretion, especially in direct coculture system, suggesting the effective strategy to ameliorate the islet quality for better transplantation outcomes.

Application of Chromosome Conformation Capture Method for Detection MYC/TRD Chromosomal Translocation in Leukemia Cell Line.

Background: Chromosomal breakpoints are the most common cause of hereditary diseases and cancers. Today, many standard clinical methods such as cytogenetic and PCR based techniques are used which have limitation regarding detection resolution. Chromosome conformation capture is a method for detecting gene proximity and chromosomal rearrangements. Materials and Methods: In this study, SKW3 cell line was used for detecting t(8;14)(q24;q11) using a 3C-based technique. SKW3 cell line was used for 3C library preparation. For Inverse PCR, two regions were selected in upstream and downstream of the viewpoint locus on chromosome 8-MYC gene based on EcoRI restriction sites. The captured sequence with intra-chromosomal interaction between chr8-c-MYC and chr14-TRD was selected for the translocation PCR primer design. Results: The DNA fragment captured in 3C PCR showed a specific TRD sequence translocated downstream of the MYC gene. Translocation PCR demonstrated the existence of (8; 14) (q24; q11) MYC /TRD in both library and genomic DNA. Conclusion: This result demonstrated 3C- based method could be used as a useful low-cost easy operating technique in chromosomal rearrangements detection. In this study, the integration of whole genome library monitoring and PCR method was used as a high- through put method in chromosomal breakpoints detection.

Significant reduction of apoptosis induced via hypoxia and oxidative stress in isolated human islet by resveratrol.

BACKGROUND AND AIMS: Successful islet transplantation as a promising treatment of diabetes type 1 is threatened with the loss of islets during the pre-transplant culture due to hypoxia and oxidative stress-induced apoptosis. Therefore, optimization of culture in order to preserve the islets is a critical point. In this study, we investigated the effect of resveratrol, as a cytoprotective agent, on the cultured human islets. METHODS AND RESULTS: Isolated islets were treated with different concentrations of resveratrol for 24 and 72 h. Islets' viability, apoptosis, apoptosis markers, and insulin and C-peptide secretion, along with the production of reactive oxygen species (ROS), hypoxia inducible factor 1 alpha (HIF-1α), and its target genes in the islets were investigated. Our findings showed that the islets were exposed to hypoxia and oxidative stress after isolation and during culture. This insult induced apoptosis and decreased viability during 72 h. The presence of resveratrol significantly attenuated HIF-1α and ROS production, reduced apoptosis, promoted the VEGF secretion, and increased the insulin and C-peptide secretion. In this regard, resveratrol improved the islet's survival and function in the culture period. CONCLUSIONS: Using resveratrol can attenuate the stressful condition for the islets in the pre-transplant culture and subsequently ameliorate their viability and functionality that lead to successful outcome after clinical transplantation.

Effects of human placenta-derived mesenchymal stem cells with NK4 gene expression on glioblastoma multiforme cell lines.

Poor prognosis and low survival are commonly seen in patients with glioblastoma multiforme (GBM). Due to the specific nature of solid tumors such as GBM, delivery of therapeutic agents to the tumor sites is difficult. So, one of the major challenges in the treatment of these tumors is a selection of appropriate method for drug delivery. Mesenchymal stem cells (MSCs) have a unique characteristic in migration toward the tumor tissue. In this regard, the present study examined the antitumor effects of manipulating human placenta-derived mesenchymal stem cells (PDMSCs) with NK4 expression (PDMSC-NK4) on GBM cells. After separation and characterization of PDMSCs, these cells were transduced with NK4 which was known as the antagonist of hepatocyte growth factor (HGF). The results indicated that engineered PDMSCs preferably migrate into GBM cells by transwell coculture system. In addition, the proliferation of the GBM cells significantly reduced after coculture with these cells. In fact, manipulated PDMSCs inhibited growth of tumor cells by induction of apoptosis. Our findings suggested that besides having antitumor effects, PDMSCs can also be applied as an ideal cellular vehicle to target the glioblastoma multiforme.

Protective effect of nobiletin on isolated human islets survival and function against hypoxia and oxidative stress-induced apoptosis.

Islets transplantation, as a treatment of type 1 diabetes, faces challenges, including the loss of islets in the process of isolation and pre-transplantation due to cellular stresses-induced apoptosis. Accordingly, the optimization of culture plays a decisive role in the transplantation success. In this study, we evaluated the effect of nobiletin on the cultured human islets. Isolated human islets were treated by different concentrations of nobiletin and cultured for 24 and 72 hours. Then, the islets viability, apoptosis, insulin and C-peptide secretion, and apoptosis markers were evaluated. Also, the production of reactive oxygen species (ROS), hypoxia inducible factor 1 alpha (HIF-1α), and its target genes in the islets were examined. Our findings showed that the islets were encountered with hypoxia and oxidative stress after isolation and during culture. These insults induced apoptosis and reduced viability during culture period. Moreover, the secretion of insulin and C-peptide decreased. Nobiletin treatments significantly improved the islets survival through reduction of HIF-1α and ROS production and suppression of apoptosis, along with increased islets function. Islet protective effect of nobiletin might be related to its anti-oxidant, anti-apoptotic and insulinotropic properties. Hence, in order to achieve viable and functional islets for clinical transplantation, the application of nobiletin during pre-transplantation period is useful.

Apoptotic Resistance of Metastatic Tumor Cells in Triple Negative Breast Cancer: Roles of Death Receptor-5.

Background: Metastasis is a major cause of death from cancer in triple-negative breast cancer (TNBC). Apoptosis evasion is a critical feature of metastatic tumor cells. Chemopreventive and apoptotic potential of curcumin has been shown in breast cancer. However, the precise mechanism of these effects against metastatic tumor cells has not been clearly addressed yet. Methods: 4T1 cell line was used for induction of metastatic animal model of breast cancer. Primary and metastatic tumor cells were extracted from subcutaneous tumor and lung of cancerous mice, respectively. MTT assay was used to determine the effect of curcumin on viability of tumor cells. Quantitative real-time polymerase chain reaction was performed to analyze the effect of curcumin on death receptor-5 (DR-5) gene expression. Results: Our data revealed that, compared with primary tumor cells, metastatic tumor cells were more resistance to apoptosis effects of curcumin. The DR-5 gene expression was up-regulated in both primary and metastatic tumor cells after curcumin treatment, but this up-regulation was significantly higher in primary tumor cells compared with metastatic cells. Conclusion: These findings provided important insights regarding the molecular mechanism of apoptosis resistance of metastatic tumor cells and can be used for designing a targeted therapeutic strategies in combat with metastatic TNBC.

Effect of tempol on the passive avoidance and novel object recognition task in diabetic rats.

Diabetes mellitus (DM) has several effects, including cognitive impairment. Oxidative stress is associated with complications from diabetes. It seems that antioxidants can reduce some complications of the diabetes induced by oxidative stress. The objective of this study was to evaluate the effect of synthetic antioxidant, tempol on the passive avoidance (PA) memory and novel object recognition (NOR) tests in the diabetic rats. Forty male Wistar rats randomly divided into the control, diabetic, diabetic receiving tempol and healthy receiving tempol groups. Diabetes was induced by injection of streptozotocin (STZ) (60 mg/kg, i.p.). Then, the rats received saline or tempol (30 mg/kg) orally by gavages for 60 days. After that, they were assessed using the PA memory and NOR tests. The results of NOR test showed that the discrimination index (DI) in the healthy receiving tempol group and diabetic control group was significantly lower than control group. Also the amount of this index in diabetic receiving tempol group was significantly higher than diabetic group. The results of PA test indicated that the number of trials to acquisition in the diabetic rats is significantly more than control and diabetic tempol treated groups. Also, the time spent in the dark compartment (TDC) in the control and diabetic receiving tempol groups was less than diabetic group. TDC in the healthy receiving tempol group was more than control group. It can be concluded that although use of tempol is restricted as a cognitive enhancer in non-diabetic subjects but long-term administration of synthetic antioxidant, tempol, is able to dramatically improve diabetes-induced learning and memory deficit in both PA and NOR tests.