Potential of 5-azacytidine induction decidual stromal cells from maternal human term placenta towards cardiomyocyte-like cells in serum-free medium.

Decidual stromal cells (DSCs) from maternal term placenta represent a potential source of cells for the treatment of cardiovascular and graft-versus-host diseases. However, it is not clear whether DSCs could be induced towards cardiomyocyte-like differentiation. We chose the placentas which should bred male new-baby. We isolated DSCs from placenta by tissue adherence. The morphology, immunophenotype, and multi-lineage potential were analyzed. Karyotype analysis (G-band) was performed to determine the source and karyotype stability of DSCs. DSCs were induced by 5-azacytidine. Expression of Myf5, α-cardiac actin, Cardiac troponin T (cTnT) and GAPDH was assessed by PCR, and cTnT expression was also analyzed by immunofluorescence. Karyotype analyses indicated that cells were derived from the maternal matrix. After induction with 5-azacytidine, DSCs expressed the cardiac-specific markers Myf5, myogenin and cTnT, indicating differentiation towards cardiomyocyte-like cells.

Monitoring the biology stability of human umbilical cord-derived mesenchymal stem cells during long-term culture in serum-free medium.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology, immunophenotype and multi-lineage potential were analyzed at passages 3, 5, 10, 15, 20, and 25 (P3, P5, P10, P15, P20, and P25, respectively). The cell cycle distribution, apoptosis, and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3, P5, P10, P15, P20, and P25. From P3 to P25, the three defining biological properties of hUC-MSCs [adherence to plastic, specific surface antigen expression, multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased, compared with the cells at P3 (P < 0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183 %), compared to those at P3 (P < 0.01). Within subculture generations 3-20 (P3-P20), the differences between the cell apoptotic rates were not statistically significant (P > 0.05). There were no detectable chromosome eliminations, displacements, or chromosomal imbalances, as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN, 2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations.

The Exosomal lncRNA KLF3-AS1 From Ischemic Cardiomyocytes Mediates IGF-1 Secretion by MSCs to Rescue Myocardial Ischemia-Reperfusion Injury.

The purpose of the study was to explore the mechanism by which myocardial ischemia-reperfusion (I/R) injury-induced exosomes modulate mesenchymal stem cells (MSCs) to regulate myocardial injury. In this study, we established an I/R injury model in vivo and a hypoxia-reoxygenation (H/R) model in vitro. Then, exosomes isolated from H/R-exposed H9c2 cells were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot analysis. CCK-8 assays and flow cytometry were performed to assess cell injury. ELISA was applied to determine the level of insulin-like growth factor 1 (IGF-1). Echocardiography was used to assess cardiac function in vivo. HE staining and TUNEL assays were conducted to analyze myocardial injury in vivo. In the present study, H/R-exposed H9c2 cells induced IGF-1 secretion from MSCs to inhibit cell myocardial injury. Moreover, exosomes derived from H/R-exposed H9c2 cells were introduced to MSCs to increase IGF-1 levels. The lncRNA KLF3-AS1 was dramatically upregulated in exosomes derived from H/R-treated H9c2 cells. Functional experiments showed that the exosomal lncRNA KLF3-AS1 promoted IGF-1 secretion from MSCs and increased H9c2 cell viability. In addition, miR-23c contains potential binding sites for both KLF3-AS1 and STAT5B, and miR-23c directly bound to the 3'-UTRs of KLF3-AS1 and STAT5B. Furthermore, the lncRNA KLF3-AS1 promoted IGF-1 secretion from MSCs and rescued myocardial cell injury in vivo and in vitro by upregulating STAT5B expression. The lncRNA KLF3-AS1 may serve as a new direction for the treatment of myocardial I/R injury.

Comparison of the Effects of Different Cryoprotectants on Stem Cells from Umbilical Cord Blood.

Purpose. Cryoprotectants (CPA) for stem cells from umbilical cord blood (UCB) have been widely developed based on empirical evidence, but there is no consensus on a standard protocol of preservation of the UCB cells. Methods. In this study, UCB from 115 donors was collected. Each unit of UCB was divided into four equal parts and frozen in different kinds of cryoprotectant as follows: group A, 10% ethylene glycol and 2.0% dimethyl sulfoxide (DMSO) (v/v); group B, 10% DMSO and 2.0% dextran-40; group C, 2.5% DMSO (v/v) + 30 mmol/L trehalose; and group D, without CPA. Results. CD34(+), cell viability, colony forming units (CFUs), and cell apoptosis of pre- and postcryopreservation using three cryoprotectants were analyzed. After thawing, significant differences in CD34(+) count, CFUs, cell apoptosis, and cell viability were observed among the four groups (P < 0.05). Conclusion. The low concentration of DMSO with the addition of trehalose might improve the cryopreservation outcome.

Mesenchymal Stem Cells and Mononuclear Cells From Cord Blood: Cotransplantation Provides a Better Effect in Treating Myocardial Infarction.

The aim of this study was to evaluate the effect of cotransplanting mononuclear cells from cord blood (CB-MNCs) and mesenchymal stem cells (MSCs) as treatment for myocardial infarction (MI). Transplanting CD34+ cells or MSCs separately has been shown effective in treating MI, but the effect of cotransplanting CB-MNCs and MSCs is not clear. In this study, MSCs were separated by their adherence to the tissue culture. The morphology, immunophenotype, and multilineage potential of MSCs were analyzed. CB-MNCs were separated in lymphocyte separation medium 1.077. CD34+ cell count and viability were analyzed by flow cytometry. Infarcted male Sprague-Dawley rats in a specific-pathogen-free grade were divided into four treatment groups randomly: group I, saline; group II, CB-MNCs; group III, MSCs; and group IV, CB-MNCs plus MSCs. The saline, and CB-MNCs and/or MSCs were injected intramyocardially in infarcted rats. Their cardiac function was evaluated by echocardiography. The myocardial capillary density was analyzed by immunohistochemistry. Both cell types induced an improvement in the left ventricular cardiac function and increased tissue cell proliferation in myocardial tissue and neoangiogenesis. However, CB-MNCs plus MSCs were more effective in reducing the infarct size and preventing ventricular remodeling. Scar tissue was reduced significantly in the CB-MNCs plus MSCs group. MSCs facilitate engraftment of CD34+ cells and immunomodulation after allogeneic CD34+ cell transplantation. Cotransplanting MSCs and CB-MNCs might be more effective than transplanting MSCs or CB-MNCs separately for treating MI. This study contributes knowledge toward effective treatment strategies for MI.

Comparison of biological characteristics of mesenchymal stem cells derived from maternal-origin placenta and Wharton's jelly.

INTRODUCTION: Although mesenchymal stem cells (MSCs) from different sources share many similar characteristics, they also exhibit individual properties. In this study, we compared MSCs derived from Wharton's jelly in the umbilical cord with those derived from the decidual basalis in the maternal part of the placenta to better understand the similarities and differences between these two cell types. METHOD: The morphology, immunophenotype (as assessed using flow cytometry), and multi-lineage differentiation potential were analyzed. Karyotype analysis was carried out to determine the origin of the MSCs. Growth kinetics were evaluated using analysis of the population doubling time and cell cycle. Immunosuppressive function was analyzed using mixed lymphocyte culture. RESULTS: MSCs from Wharton's jelly and the decidua basalis exhibited similar morphology, immunophenotype, and differentiation potential to osteogenesis and adipogenesis. The percentage of MSCs in the G0/G1 phase was higher in the case of Wharton's jelly than in the case of the decidua basalis (P < 0.05). Decidual MSCs displayed more remarkable immunosuppressive effects on phytohemagglutinin-stimulated T-cell proliferation (P < 0.05). CONCLUSION: MSCs from both sources had similar basic biological properties, but decidual MSCs had slower proliferation and stronger immunosuppressive function.

Human umbilical cord-derived mesenchymal stem cells do not undergo malignant transformation during long-term culturing in serum-free medium.

BACKGROUND: Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are in the foreground as a preferable application for treating diseases. However, the safety of hUC-MSCs after long-term culturing in vitro in serum-free medium remains unclear. METHODS: hUC-MSCs were separated by adherent tissue culture. hUC-MSCs were cultured in serum-free MesenCult-XF medium and FBS-bases DMEM complete medium. At the 1st, 3rd, 5th, 8th, 10th, and 15th passage, the differentiation of MSCs into osteogenic, chondrogenic, and adipogenic cells was detected, and MTT, surface antigens were measured. Tumorigenicity was analyzed at the 15th passage. Conventional karyotyping was performed at passage 0, 8, and 15. The telomerase activity of hUC-MSCs at passage 1-15 was analyzed. RESULTS: Flow cytometry analysis showed that very high expression was detected for CD105, CD73, and CD90 and very low expression for CD45, CD34, CD14, CD79a, and HLA-DR. MSCs could differentiate into osteocytes, chondrocytes, and adipocytes in vitro. There was no obvious chromosome elimination, displacement, or chromosomal imbalance as determined from the guidelines of the International System for Human Cytogenetic Nomenclature. Telomerase activity was down-regulated significantly when the culture time was prolonged. Further, no tumors formed in rats injected with hUC-MSCs (P15) cultured in serum-free and in serum-containing conditions. CONCLUSION: Our data showed that hUC-MSCs met the International Society for Cellular Therapy standards for conditions of long-term in vitro culturing at P15. Since hUC-MSCs can be safely expanded in vitro and are not susceptible to malignant transformation in serum-free medium, these cells are suitable for cell therapy.

Polypeptide N-acetylgalactosaminyltransferase 2 regulates cellular metastasis-associated behavior in gastric cancer.

Aberrant glycosylation of cell surface glycoprotein due to specific alterations of glycosyltransferase activity is usually associated with invasion and metastasis of cancer, particularly of gastric carcinomas. Polypeptide N-acetylgalactosaminyltransferase 2 (ppGalNAc-T2), which catalyzes initiation of mucin-type O-glycosylation, is also involved in tumor migration and invasion. However, a comprehensive understanding of how ppGalNAc-T2 correlates with the metastasic potential of human gastric cancer is not currently available. In the present study, ppGalNAc-T2 was detected in a variety of human poorly differentiated tumor cells, and expression appeared to be higher in SGC7901 gastric cancer cells. In addition, we investigated the potential effects of ppGalNAc-T2 on growth and metastasis-associated behavior in SGC7901 cells after stable transfection with ppGalNAc-T2 sense and antisense vectors. We found that cell proliferation, adhesion and invasion were decreased in ppGalNAc-T2 overexpressed cells but increased in ppGalNAc-T2 downregulated cells. Therefore, we attempted to clarify the mechanisms underlying the anti-metastatic activities of ppGalNAc-T2. Further investigation indicated that overexpression of ppGalNAc-T2 is involved in the inhibition of matrix metalloproteinase (MMP)-2 expression at both the protein and mRNA levels, which may be associated with ppGalNAc-T2 suppressing the expression of transforming growth factor (TGF)-ß1. However, it did not exhibit any apparent correlation with MMP-14 expression levels. Our data show the effect of ppGalNAc-T2 on proliferation, adhesion or invasion of SGC7901 gastric cancer cells, suggesting that ppGalNAc-T2 may exert anti-proliferative and anti-metastatic activity through the decrease of MMP-2 and TGF-ß1. These results indicate that ppGalNAc­T2 may be used as a novel therapeutic target for human gastric cancer treatment.