Comparison of osteogenic and chondrogenic differentiation ability of buccal fat pad derived mesenchymal stem cells and gingival derived cells

Statement of the Problem: One major goal of tissue engineering and regenerative medicine is to find an appropriate source of mesenchymal stem cells [MSCs] with higher differentiation ability

Purpose: In this experimental study, the osteogenic and chondrogenic differentiation ability of buccal fat pad derived MSCs [BFP-MSCs] with gingival derived cells [GDCs] were compared

Materials and Method: BFP-MSCs and GDCs were cultured enzymatically and expanded. The expanded cells were analyzed for membrane-associated markers, using flow cytometry. Then the ability of these cells to differentiate into osteocyte and chondrocyte was assessed morphologically and by mRNA expression of collagen I [COLL], BGLA and bone morphogenetic protein 2 [BMP2] using qRT-PCR

Results: Flow cytometry analysis showed that both BFP-MSCs and GDCs expressed the characteristic stem cell markers such as CD73, CD44, and CD90, whereas they did not express hematopoietic markers. Mineralized calcium deposition was observed apparently in BFP-MSCs cultured in osteogenic medium but GDCs showed fewer mineralized nodules. The mRNA expression levels of BGLA and BMP2 showed 7×105 and 733-fold more mRNA expression in BFP-MSCs treated with differentiation media compared to the control group. In chondrogenic differentiation, BFP-MSCs transformed from a spindle to a cuboidal shape while GDCs showed only a slight transformation. In addition, mRNA expression of COLL showed 282-fold higher expression in BFP-MSCs in comparison to the control group. Such significant difference in mRNA expression of BGLA, BMP2, and COLL was not observed in GDCs compared to their corresponding controls

Conclusion: Based on the present results, BFP yields a greater proportion of stem cells compared to gingiva. Therefore, this tissue can be introduced as an easily available source for the treatment of periodontal defects and other maxillofacial injuries

Comparative proteomic analysis of tumor mesenchymal-like stem cells derived from high grade versus low grade gliomas

Objective: Gliomas are the most common primary brain tumors, and have been ranked as the fourth leading cause of cancer death. Tumor mesenchymal-like stem cells [tMSCs] contribute to the aggressive behavior of glial tumors by providing a favorable microenvironment for the malignant cells. The aim of our study was to identify differential proteome of tMSCs derived from low vs. high grade glioma tumors

Materials and Methods: Patients with newly diagnosed low and high grade gliomas were included in this case control study. tMSCs were isolated from tumors using enzymatic digestion validated by flow cytometer analysis after sub-culturing. Differential proteomic analysis of tMSCs derived from low and high grade tumors was performed by two-dimensional gel electrophoresis and mass spectrometry. Protein spots with more than two fold differences and P values below 0.05 were considered as differentially expressed ones

Results: In tMSCs isolated from low and high grade gliomas, different isoforms of mesenchymal- related proteins vimentin and transgelin were differentially expressed. Overexpressed proteins in tMSCs isolated from low grade gliomas were mitochondrial manganese-containing superoxide dismutase [Mn-SOD], 40S ribosomal protein SA, and GTP-binding nuclear protein, while in tMSCs isolated from high grade gliomas, cathepsin B, endoplasmin, ezrin, peroxiredoxin1, and pyruvate kinase [PK] were found to be significantly overexpressed

Conclusion: For the first time, we analyzed the differential proteomic profiles of tMSCs isolated from glioma tumors with different grades. It was found that molecules related to mesenchymal cells [vimentin and transglin], in addition to those related to tumor aggressiveness with potential secretory behavior [e.g. cathepsin B] were among differentially expressed proteins

Adipose derived stem cells exert immunomodulatory effects on natural killer cells in breast cancer

Objective: Adipose derived stem cells [ASCs], as one of the important stromal cells in the tumor microenvironment, are determined with immunomodulatory effects. The principle aim of this study was to evaluate the immunosuppressive effects of ASCs on natural killer [NK] cells

Materials and Methods: In this experimental study, we assessed the expressions of indolamine 2, 3-dioxygenase [IDO1], IDO2 and human leukocyte antigen-G5 [HLA-G5] in ASCs isolated from breast cancer patients with different stages as well as normal individuals, using quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR]. Immunomodulatory effects of ASCs on the expression of CD16, CD56, CD69, NKG2D, NKp30, NKG2A and NKp44 was also assessed in peripheral blood lymphocytes [PBLs] by flow-cytometry

Results: Our result showed that IDO1, IDO2 and HLA-G5 had higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals [P>0.05]. mRNA expression of these molecules were higher in ASCs isolated from breast cancer patients with stage III tumors than those with stage II. The indirect culture of ASCs isolated from breast cancer patients and normal individuals with activated PBLs significantly reduced NKG2D+ and CD69+ NK cells [P<0.05]

Conclusion: Results of the present study suggest more evidences for the immunosuppression of ASCs on NK cells, providing conditions in favor of tumor immune evasion

Downregulation of MMP2 and Bcl-2 in adipose derived stem cells [ASCs] following transfection with IP-10 gene

Mesenchymal Stem Cells [MSCs] are recently introduced as novel immunological gene carriers for treatment of cancer. It is believed that balance between the expression of angiogenic and anti-angiogenic factors, such as SDF-1 and IP-10, may regulate neovascularization within the tumor. In this study, we compared the expression of important tumor promoting mediators in IP-10-transfected Adipose Derived Stem Cells [ASCs] to those transfected with SDF-1. ASCs were isolated from adipose tissue of a normal subject undergoing cosmetic mamoplasty surgery using collagenase. ASCs were transfected with IP-10 or SDF-1 propagated plasmids by electroporation method and Lipofectamin 2000. Expressions of SDF-1, CXCR4, IP-10, Bcl-2, MMP2, IL-10, IGF-1, and VEGF were detected in transfected ASCs using quantitative Real-Time Polymerase Chain Reaction [qRT-PCR]. Results showed that the expressions of SDF-1, CXCR4, Bcl-2, MMP2, IL-10, IGF-1, and VEGF were upregulated in SDF-1-transfected ASCs. In contrast, Bcl-2 and MMP2 transcripts showed 45×103 and 10 fold lower expression in ASCs transfected with IP-10 compared to non-transfected cells. Anti-angiogenic chemokines such as IP-10 may modulate tumor promoting properties of ASCs and would be introduced as novel candidates for tumor immunotherapy; however, further studies are needed to be conducted

IL-23/IL-27 ratio in peripheral blood of patients with breast cancer

Background: Interleukin [IL]-23 and IL-27 are two IL-12-related cytokines which their function may dramatically influence the inflammatory response to tumor development. IL-12 and IL-27 seem to have antagonistic roles with IL-23 in tumor site. In this study, IL-23 and IL-27 mRNA expressions were analyzed in peripheral blood of patients with breast cancer and healthy volunteers using quantitative real-time PCR

Methods: Peripheral blood samples were collected from 50 women with breast cancer and 50 healthy ones. The total RNA was extracted from peripheral blood after lysis with ammonium chloride and TRizol reagent and the cDNA was synthesized. The expression of IL-23 and IL-27 gene transcripts was determined with real-time polymerase chain reaction [qRT-PCR] using Syber Green PCR Master Mix

Results: It is found that IL-23 and IL-27 transcripts had significantly higher expression in peripheral blood of patients compared with the healthy controls. The ratio of IL-23 transcript expression to IL-27 was 3.4 fold lower in the studied patients compared with the normal individuals

Conclusion: It is concluded that the over expression of IL-23 and IL-27 gene transcript in peripheral blood of breast cancer patients may be an immune response against tumor development and the inflammatory response plays a critical role in tumor development via up regulating the corresponding cytokines. However, the IL-23/IL-27 ratio may play an important role in cytokine-based immunotherapy against cancer. Further research should be carried out to assess these cytokines in a larger sample size

[Apoptosis of gingival connective tissue cells in diabetic individuals with chronic periodontitis]

Apoptosis or programmed cell death plays an important role in the pathogenesis of many diseases. Previous studies suggest that apoptosis is involved in the pathogenesis of periodontal disease. On the other hand, it is also suggested that diabetes mellitus enhances apoptosis of connective tissue cells. Thus, we measured expression of proteins which are relevant to apoptosis in the gingival tissue of diabetic patients with chronic periodontitis in comparison to non diabetic individuals. 25 patients with diabetes and chronic periodontitis and 16 non diabetic controls with chronic periodontitis were included in this study. 4 weeks after scaling and root planning and oral hygiene instructions, periodontal surgery was done and gingival tissues obtained during surgery, were sent to lab to investigate expression of Fas, P53, Bcl-2 and Survivin using real-time PCR technique. Data were analyzed using Mann-Whitney and Chi-squared. Pro-apoptotic proteins [Fas, P53] were significantly [P<0.05] higher in gingival tissues of diabetics [9.5x10[-6], 2.4x10[-6], respectively] in comparison to non diabetics [9.4x10[-7], 5.6x10[-7]], whereas the difference in expression of anti-apoptotic proteins [Bcl-2, Survivin] between 2 groups was not significant [9.7x10[-8], 3.5x10[-7] in comparison to 1.4x10[-7], 3.1x10[-7], respectively] [P=0.91, P=0.29 respectively]. Apoptosis was increased in gingival connective tissue of diabetic patients with chronic periodontitis in comparison with non diabetic ones. Therefore, intervention in expression or function of pro-apoptotic proteins [Fas, P53] could be a new goal in the treatment of periodontal disease of diabetic patients.

Mesenchymal stem cells do not suppress lymphoblastic leukemic cell line proliferation

Several studies have demonstrated the immunosuppresive effects of mesenchymal stem cells [MSCs] in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this regard. To investigate if adipose derived MSCs could inhibit Jurkat lymphoblastic leukemia T cell proliferation during coculture. Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial characterization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells [PBMCs] were labeled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increasing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, initial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant [50% vol/vol] had no significant effect on Jurkat cell proliferation [p>0.6]. The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of different sources are needed to fully characterize the immunological properties of MSCs be-fore planning clinical applications