PI3 K/Akt/mTOR-mediated translational control regulates proliferation and differentiation of lineage-restricted RoSH stem cell lines

Background: We have previously derived highly similar lineage-restricted stem cell lines, RoSH and E-RoSH cell lines from mouse embryos and CD9hi SSEA-1- differentiated mouse embryonic stem cells, respectively. These cell lines are not pluripotent and differentiate readily into endothelial cells in vitro and in vivo. Results: We investigated the signaling pathway that maintains proliferation of these cells in an undifferentiated state, and demonstrate that PI3 K/Akt/mTOR, but not Raf/MEK/Erk, signaling in these cells was active during proliferation and was downregulated during endothelial differentiation. Inhibition of PI3 K/Akt/mTOR signaling, but not Raf/MEK/Erk, reduced proliferation and induced expression of endothelial specific proteins. During differentiation or inhibition of PI3 K/Akt/mTOR signaling, cyclinD2 transcript abundance in ribosome-enriched RNA but not in total RNA was reduced with a corresponding reduction in protein level. In contrast, transcript abundance of endothelial-specific genes e.g. Kdr, Tek and Pdgfrα in ribosome-enriched RNA fraction was not reduced and their protein levels were increased. Together these observations suggested that translational control mediated by PI3K/Akt/mTOR signaling was critical in regulating proliferation and endothelial differentiation of lineage-restricted RoSH-like stem cell lines. Conclusion: This study highlights translation regulation as a critical regulatory mechanism during proliferation and differentiation in stem cells

High Glucose Predisposes Gene Expression and ERK Phosphorylation to Apoptosis and Impaired Glucose-Stimulated Insulin Secretion via the Cytoskeleton

10.1371/journal.pone.0044988PLoS ONE79

Iodine Radiolabeled Mesenchymal Stem Cell (MSC)-Exosomes and Their CD73 Enzymatic Activities

IODINE RADIOLABELLED MESENCHYMAL STEM CELL (MSC)-EXOSOMES AND THEIR CD73 ENZYMATIC ACTIVITIES Chang-Tong Yang a,b*,Ruenn Chai Laic, Sai Kiang Limc, David Chee Eng Ng a,b a Department of Nuclear Medicine and Molecular Imaging, Radiological Sciences Division, Singapore General Hospital, Outram Road, 169608 Singapore; b Duke-NUS Medical School, 8 College Road, 169857 Singapore; cInstitute of Molecular & Cell Biology, 8A Biomedical Grove #05-16 Immunos, 138648, Singapore; *[email protected] Introduction MSC-derived exosomes have shown therapeutic potential in the areas of cardiovascular, orthopaedic, ophthalmologic, immune, dermatologic diseases and radiation sickness. Efficient radioisotope-labelling of exosomes remains as a challenging process. We demonstrate iodine-131 radiolabeled exosomes using both chloramine-T and Pierce Iodination methods, and characterized I-labelled exosomes via their CD73 enzymatic activities. Experimental methodology and results Two classic radio-iodination methods have been used to label exosomes due to several advantages: relatively long half-life of I-131 (half-life 8 days) and I-124 (half-life 4.2 days, 25.6% positron emission) could enable a desired tracking kinetics of exosomes in vitro and in vivo; radiolabeling of iodine to peptides and antibodies is a well-established chemistry; the unlabeled free iodine after radio-labelling can be easily removed to reach high radiochemical purity. By using chloramine-T, the radiolabeling yield of 131I-labeled-exosomes achieved ~30-40% with a radiochemical purity > 90% after running through PD10 column purification. Using Pierce Iodination, the radiolabeling yield drops to ~15-20%, radiochemical purity achieved >90% after the same purification process. The integrity of I-labeled-exosomes is important in the reproducibility and development of exosome clinical therapeutics. No radioactive iodine was labelled to exosome for characterization of their integrity. The results showed that chloramine T radiolabeling affected the structures of I-labeled-exosomes as the CD73 enzymatic activity of I-labeled exosomes was destroyed, the particle size became much larger and caused broader exosome size distribution. While with Pierce iodination the CD73 activity drops by 50 % when compared to that of the unlabeled exosomes, and the particles kept the same size. Conclusions Using chloramine T method showed that the CD73 enzymatic activity of I-labelled exosomes was destroyed, suggesting the labeling process damaged the structure of exosomes. By comparison, using Pierce Iodination method preserved the CD73 enzymatic activity, indicating that exosomes can be radiolabeled using Pierce Iodination for in vitro and in vivo tracking and pharmacokinetic studies

Folic acid supplementation normalizes the endothelial progenitor cell transcriptome of patients with type 1 diabetes: a case-control pilot study

Background: Endothelial progenitor cells play an important role in vascular wall repair. Patients with type 1 diabetes have reduced levels of endothelial progenitor cells of which their functional capacity is impaired. Reduced nitric oxide bioavailability and increased oxidative stress play a role in endothelial progenitor cell dysfunction in these patients. Folic acid, a B-vitamin with anti-oxidant properties, may be able to improve endothelial progenitor cell function. In this study, we investigated the gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes compared to endothelial progenitor cells from healthy subjects. Furthermore, we studied the effect of folic acid on gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes. Methods: We used microarray analysis to investigate the gene expression profiles of endothelial progenitor cells from type 1 diabetes patients before (n = 11) and after a four week period of folic acid supplementation (n = 10) compared to the gene expression profiles of endothelial progenitor cells from healthy subjects (n = 11). The probability of genes being differentially expressed among the classes was computed using a random-variance t-test. A multivariate permutation test was used to identify genes that were differentially expressed among the two classes. Functional classification of differentially expressed genes was performed using the biological process ontology in the Gene Ontology database. Results: Type 1 diabetes significantly modulated the expression of 1591 genes compared to healthy controls. These genes were found to be involved in processes regulating development, cell communication, cell adhesion and localization. After folic acid treatment, endothelial progenitor cell gene expression profiles from diabetic patients were similar to those from healthy controls. Genes that were normalized by folic acid played a prominent role in development, such as the transcription factors ID1 and MAFF. Few oxidative-stress related genes were affected by folic acid. Conclusion: Folic acid normalizes endothelial progenitor cell gene expression profiles of patients with type 1 diabetes. Signaling pathways modulated by folic acid may be potential therapeutic targets to improve endothelial progenitor cell function

EV-Associated MMP9 in High-Grade Serous Ovarian Cancer Is Preferentially Localized to Annexin V-Binding EVs

High-grade serous ovarian cancer (HGSOC) is the most aggressive type of ovarian cancer and is responsible for most deaths caused by gynecological cancers. Numerous candidate biomarkers were identified for this disease in the last decades, but most were not sensitive or specific enough for clinical applications. Hence, new biomarkers for HGSOC are urgently required. This study aimed to identify new markers by isolating different extracellular vesicle (EV) types from the ascites of ovarian cancer patients according to their affinities for lipid-binding proteins and analyzing their protein cargo. This approach circumvents the low signal-to-noise ratio when using biological fluids for biomarker discovery and the issue of contamination by large non-EV complexes. We isolated and analyzed three distinct EV populations from the ascites of patients with ovarian cancer or cirrhosis and observed that Annexin V-binding EVs have higher levels of matrix metalloproteinase 9 in malignant compared to portal-hypertensive ascites. As this protein was not detected in other EV populations, this study validates our approach of using different EV types for optimal biomarker discovery. Furthermore, MMP9 in Annexin V-binding EVs could be a HGSOC biomarker with enhanced specificity, because its identification requires detection of two distinct components, that is, lipid and protein

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs

10.1186/1479-5876-9-47Journal of Translational Medicine9

Perinatal derivatives: How to best validate their immunomodulatory functions

Perinatal tissues, mainly the placenta and umbilical cord, contain a variety of different somatic stem and progenitor cell types, including those of the hematopoietic system, multipotent mesenchymal stromal cells (MSCs), epithelial cells and amnion epithelial cells. Several of these perinatal derivatives (PnDs), as well as their secreted products, have been reported to exert immunomodulatory therapeutic and regenerative functions in a variety of pre-clinical disease models. Following experience with MSCs and their extracellular vesicle (EV) products, successful clinical translation of PnDs will require robust functional assays that are predictive for the relevant therapeutic potency. Using the examples of T cell and monocyte/macrophage assays, we here discuss several assay relevant parameters for assessing the immunomodulatory activities of PnDs. Furthermore, we highlight the need to correlate the in vitro assay results with preclinical or clinical outcomes in order to ensure valid predictions about the in vivo potency of therapeutic PnD cells/products in individual disease settings

Entomologic and Virologic Investigation of Chikungunya, Singapore

Data from longitudinal analyses can be useful in the design and implementation of control strategies

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9(hi), SSEA-1(−) Cells in Embryonic Stem Cell-Derived Embryoid Bodies

BACKGROUND: Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2) = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi), SSEA-1(−) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(−) cells that constituted 1%–10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 0.95) and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs