Vaccination with human amniotic epithelial cells confer effective protection in a murine model of Colon adenocarcinoma.

As a prophylactic cancer vaccine, human amniotic membrane epithelial cells (hAECs) conferred effective protection in a murine model of colon cancer. The immunized mice mounted strong cross-protective CTL and antibody responses. Tumor burden was significantly reduced in tumor-bearing mice after immunization with hAECs. Placental cancer immunotherapy could be a promising approach for primary prevention of cancer. In spite of being the star of therapeutic strategies for cancer treatment, the results of immunotherapeutic approaches are still far from expectations. In this regard, primary prevention of cancer using prophylactic cancer vaccines has gained considerable attention. The immunologic similarities between cancer development and placentation have helped researchers to unravel molecular mechanisms responsible for carcinogenesis and to take advantage of stem cells from reproductive organs to elicit robust anti-cancer immune responses. Here, we showed that vaccination of mice with human amniotic membrane epithelial cells (hAECs) conferred effective protection against colon cancer and led to expansion of systemic and splenic cytotoxic T cell population and induction of cross-protective cytotoxic responses against tumor cells. Vaccinated mice mounted tumor-specific Th1 responses and produced cross-reactive antibodies against cell surface markers of cancer cells. Tumor burden was also significantly reduced in tumor-bearing mice immunized with hAECs. Our findings pave the way for potential future application of hAECs as an effective prophylactic cancer vaccine.

Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone-marrow-derived mesenchymal stem cells to functional hepatocyte-like cells.

OBJECTIVES: The aim of this study was to find out substitution effect of fetal bovine serum (FBS) with human platelet releasate (HPR) as a major growth factor source during expansion and differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) into hepatocytes. METHODS: Propagation and differentiation potential of hBMSCs into hepatocyte-like cells in a medium fortified with HPR instead of FBS were investigated with morphological, cytochemical and molecular experiments. RESULTS: Multiplex analysis showed that HPR was more efficient than FBS in supporting hBMSC outgrowth. The proliferation rate of MSC in presence of HPR (derived from 10(9) platelets/ml) was about threefold greater than that of FBS (P < 0.001). Despite the differences in MTT value, hBMSCs-driven HPR or FBS did not differ in terms of gross morphology, immunophenotype and osteogenic differentiation potential. Hepatic differentiation of hBMSCs was successfully performed in the media enriched with HPR. Immunoreactivity of cells with monoclonal antibodies against for albumin and alpha-fetoprotein (AFP) was even more positive in hepatocytes differentiated in presence of HPR as compared to that of FBS. The gene expression of albumin, AFP and cytokeratin-18 at mRNA levels in differentiated cells attest to supporting role of HPR in hepatic differentiation media. These findings were further confirmed with greater urea production (approximately twofold) in the culture media of cells differentiated under HPR compared to that in FBS (P < 0.001). CONCLUSION: Human platelet releasate is an efficient and safe substitute for FBS in culture media used for expansion and differentiation of hBMSCs to hepatocyte.

Functional hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel 3-dimensional biocompatible nanofibrous scaffold.

AIM: To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold. METHODS: After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers. RESULTS: Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and alfa-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (p<0.05). CONCLUSION: Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.

Modified protocol for improvement of differentiation potential of menstrual blood-derived stem cells into adipogenic lineage.

OBJECTIVES: To characterize potency of menstrual blood-derived stem cells (MenSCs) for future cell therapies, we examined differentiation potential of MenSCs into adipocytes. MATERIALS AND METHODS: Differentiation potential of MenSCs in comparison to bone marrow stem cells (BMSCs) was assessed in conventional culture medium. Differentiation potential of MenSCs into adipocytes was improved using different combinations of growth factors and hormones. RESULTS: First, we demonstrated that MenSCs preserve their appearance and karyotypic stability during passages. Although these cells express mesenchymal stem cells markers, they cannot simply be classified as mesenchymal stem cells due to expression of embryonic stem cells marker, OCT-4. Oil red O staining showed that differentiated MenSCs in conventional medium with/without retinoic acid (protocols 1 and 2) did not attain adipocyte characteristics, whereas differentiated BMSCs in conventional medium accumulated oil vacuoles typically. Nevertheless, real-time RT-PCR results showed that LPL gene expression was up-regulated in both protocols 1 and 2, whereas LEPR was up-regulated only in protocol 2 (fortified with retinoic acid). Surprisingly, protocol 3 (including rosiglitazone) had odd influence on mRNA expression of all genes (LEPR, LPL and PPAR-γ). Oil red O staining confirmed fat-producing ability of MenSCs under protocol 3. CONCLUSIONS: Presented data suggest an efficient differentiation protocol for in vitro production of MenSC-derived adipocytes. These cells are suggested to be an apt alternative to BMSCs for future stem cell therapy of soft tissue injuries.

Role of Wnt signaling on proliferation of menstrual blood derived stem cells.

AIM: Menstrual blood derived stem cells (MenSCs) are unique stem cells that have been isolated and identified recently. The special traits of MenSCs can be related to the cell signaling pathways. In this study, in order to find out the role of Wnt signaling on MenSCs proliferation, we evaluated ß-catenin expression as a key participant in Wnt signaling pathway in response to Lithium chloride (LiCl). METHODS: MenSCs were isolated from healthy women by combining gradient density centrifugation with plastic adherence. After characterization of the isolated cells, cell proliferation of MenSCs in presence of 10-15 mM LiCl was evaluated by MTT assay. ß-catenin expression of the treated cells was examined using immunofluorescence technique. RESULTS: Flow cytometric analysis revealed that both mesenchymal and embryonic stem cell markers are expressed on menstrual blood stem cells. MTT value decreased depending on the LiCl concentration. The proliferation of MenSCs cultivated in culture media containing 15mM LiCl was approximately two fold less than those grown without LiCl (p<0.01). Moreover, nuclear accumulation of ß-catenin protein in cells treated by LiCl was greater than cells without LiCl. CONCLUSION: The MenSCs are stem cell populations with high proliferation ability and unique immunophenotyping properties. Our results demonstrated that Wnt signaling pathway regulates MenSCs proliferation via trans-localization of activated-ß-catenin protein.