Comparison of the Modulated Effects of Tretinoin and Calcitriol Treated Mesenchymal Stem Cell Supernatant on Macrophage Functions

Background: According to a number of studies, calcitriol and tretinoin are able to regulate differentiation as well as the growth and of mesenchymal stem cells (MSCs). Nevertheless, the relationship between the supernatant of macrophage and mesenchymal stem cells is still under investigation. In the present work, a comparison is made between the modulated impacts of calcitriol and tretinoin treated mesenchymal stem cell supernatant on macrophage functions. Materials and Methods: The isolation of mesenchymal stem cells was done using mouse bone marrow and the various concentrations of calcitriol (200 and 400 nM) and tretinoin (25, 50, and 100 nM) were used to pulse MSCs for 48 h. Macrophages were then applied to co-culture the supernatant of MSCs for 4 hr. Consequently, macrophages were assessed for respiratory burst. Results:  Based on the obtained results, supernatant of bone marrow‐derived MSCs pulsed with calcitriol and tretinoin can have the potential for decreasing the respiratory burst of macrophages considerably in comparison with the control group.  Conclusion: The anti‐inflammatory M2 macrophage polarization can be accelerated using calcitriol and tretinoin by mesenchymal stem cells

Investigating the Relation between miR-31 and RhoA Expressions in Breast Cancer Clinical Samples and Cell Lines: A Controversial Matter

Breast cancer is the most prevalent diagnosed cancer and the second cause of cancer death among women worldwide. There are different mechanisms that play crucial roles in the onset and progression of breast cancer including microRNAs. microRNAs are small noncoding RNAs that regulate gene expression by repressing translation post-transcriptionally. miR-31 is an integrin modulator implicated in different cellular processes such as apoptosis, cell cycle control, and DNA repair. According to the literature, RhoA is one of the genes regulated by miR-31. It has an important role in actin-myosin contraction and subsequently in cell motility and migration in metastasis cascade. Breast cancer cell lines, MCF-7 and MDA-MB-231, as well as normal breast cells, MCF-10A, were cultured. RNA extraction, cDNA synthesis, and SYBR Green I quantitative real-time PCR were used to investigate the expression of miR-31 and RhoA. In addition, 10 metastatic breast cancer clinical samples were analyzed to assess miR-31 and RhoA expression, and normal cells from the same patients were used as controls. Pearson’s correlation co-efficient was applied to find out any probable relation between miR-31 and RhoA expression. Gene expression analyses in MCF-7 cell line showed downregulation of miR-31 while RhoA was upregulated in the cell line (inverse correlation). miR-31 and RhoA were both upregulated in metastatic MDA-MB-231 cell line and downregulated in 90% of clinical samples. Pearson’s correlation co-efficient showed complete positive correlation between miR-31 and RhoA expression. The expression of miR-31 and RhoA is positively correlated, and it is declined in metastatic breast that cancer clinical samples save MDA-MB-231 cells. Unlike previous reports, we found that miR-31 is not the main silencer of RhoA expression. Therefore, more investigation on genes and miRNAs affecting metastasis process can elucidate new biomarkers and therapeutic targets for metastatic breast cancer.Highlights miR-31 is an important miRNA implicated in different cellular processes as well as cancer.The protein product of RhoA gene plays a role in actin-myosin contraction and cell motility in cancer metastasis.We approved bioinformatically and experimentally that RhoA is one of the genes regulated by miR-3

Over-expression of NOTCH1 as a biomarker for invasive breast ductal carcinoma

Breast cancer is the leading cause of cancer-related death in women worldwide. Invasive ductal carcinoma (IDC) is the most frequent invasive form of breast cancer followed by metastasis. There is no accepted marker for distinguishing this form from other less aggressive forms of breast cancer. Therefore, finding new markers especially molecularly detectable ones are noteworthy. It has been shown that NOTCH1 has been overexpressed in the patients with breast cancer, but no study has investigated the expression of NOTCH1 and its correlation with other molecular and hormonal markers of breast cancer so far. In the current study, 20 breast cancer tissues and 20 matched adjacent normal breast tissue from breast cancer patients were obtained and categorized in two groups: patients with IDC and patient with other types of breast cancer. Gene expression analysis using real-time PCR showed that the NOTCH1 gene was significantly overexpressed in patients with IDC. We also found a slight correlation between NOTCH1 overexpression and p53 accumulation in the cancerous cells confirmed by Immunohistochemistry (IHC). This results showed that it is possible to introduce NOTCH1 expression as a novel biomarker of IDC, alone or preferably accompanied by IHC of p53. We also can design new therapeutic agents targeting NOTCH1 expression for inhibition of metastasis in ductal breast carcinoma

Coculture of adipose-derived mesenchymal stem cells/macrophages on decellularized placental sponge promotes differentiation into the osteogenic lineage

BACKGROUND: Several factors like three-dimensional microstructure, growth factors, cytokines, cell-cell communication, and coculture with functional cells can affect the stem cells behavior and differentiation. The purpose of this study was to investigate the potential of decellularized placental sponge as adipose-derived mesenchymal stem cells (AD-MSCs) and macrophage coculture systems, and guiding the osteogenic differentiation of stem cells. METHODS: The decellularized placental sponge (DPS) was fabricated, and its mechanical characteristics were evaluated using degradation assay, swelling rate, pore size determination. Its structure was also investigated using hematoxylin and eosin staining and scanning electron microscopy. Mouse peritoneal macrophages and AD-MSCs were isolated and characterized. The differentiation potential of AD-MSCs co-cultured with macrophages was evaluated by RT-qPCR of osteogenic genes on the surface of DPS. The in vivo biocompatibility of DPS was determined by subcutaneous implantation of scaffold and histological evaluations of the implanted site. RESULTS: The DPS had 67% porosity with an average pore size of 238 μm. The in vitro degradation assay showed around 25% weight loss during 30 days in PBS. The swelling rate was around 50% during 72 hours. The coculture of AD-MSCs/macrophages on the DPS showed a significant upregulation of four differentiation osteogenic lineage genes in AD-MSCs on days 14 and 21 and a significantly higher mineralization rate than the groups without DPS. Subcutaneous implantation of DPS showed in vivo biocompatibility of scaffold during 28 days follow up. CONCLUSIONS: Our findings suggest the decellularized placental sponge as an excellent bone substitute providing a naturally derived matrix substrate with biostructure close to the natural bone that guided differentiation of stem cells toward bone cells and a promising coculture substrate for crosstalk of macrophage and mesenchymal stem cells in vitro

Decellularized placental sponge: a platform for coculture of mesenchymal stem cells/macrophages to assess an M2 phenotype and osteogenic differentiation in vitro and in vivo

Effective communication between immune and bone-forming cells is crucial for the successful healing of bone defects. This study aimed to assess the potential of a decellularized placental sponge (DPS) as a coculture system for inducing M1/M2 polarization in macrophages and promoting osteogenic differentiation in adipose-derived mesenchymal stem cells (AD-MSCs), both in vitro and in vivo. We prepared the DPS and conducted a comprehensive characterization of its biomechanical properties, antibacterial activity, and biocompatibility. In vitro, we examined the influence of the DPS on the polarization of macrophages cocultured with AD-MSCs through nitric oxide assays, cytokine assays, phagocytosis tests, and real-time polymerase chain reaction (PCR). For in vivo assessment, we utilized micro-CT imaging, histological evaluations, and real-time PCR to determine the impact of the DPS seeded with Whartonâ s jelly mesenchymal stem cells (WJ-MSCs) on bone regeneration in a calvarial bone defect model. The coculture of AD-MSCs and macrophages on the DPS led to increased production of IL-10, upregulation of CD206, Arg1, and YM1 gene expression, and enhanced phagocytic capacity for apoptotic thymocytes. Concurrently, it reduced the secretion of TNF-α and nitric oxide (NO), downregulated the expression of CD86, NOS2, and IRF5 genes, and decreased macrophage phagocytosis of yeast. These results indicated polarization of macrophages toward an M2-like phenotype. In vivo, the presence of the DPS resulted in enhanced bone formation at the defect site. Immunostaining demonstrated that both the DPS and DPS + WJ-MSC constructs induced macrophage polarization toward an M2 phenotype, as compared to the control defect. In conclusion, this immunomodulatory effect, coupled with its biocompatibility and biomechanical properties resembling natural bone, positions the DPS as an attractive candidate for further exploration in the field of bone tissue engineering and regenerative medicine.This study was supported financially by a grant from the Cellular and Molecular Research Center, Iran University of Medical Sciences under grant number (18932)