Clonal isolation of endothelial colony-forming cells from early gestation chorionic villi of human placenta for fetal tissue regeneration

BACKGROUND Endothelial colony-forming cells(ECFCs)have been implicated in the process of vascularization,which includes vasculogenesis and angiogenesis.Vasculogenesis is a de novo formation of blood vessels,and is an essential physiological process that occurs during embryonic development and tissue regeneration.Angiogenesis is the growth of new capillaries from pre-existing blood vessels,which is observed both prenatally and postnatally.The placenta is an organ composed of a variety of fetal-derived cells,including ECFCs,and therefore has significant potential as a source of fetal ECFCs for tissue engineering.AIM To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi(CV-ECFCs)of the placenta,and assess their potential for tissue engineering.METHODS The early gestation chorionic villus tissue was dissociated by enzyme digestion.Cells expressing CD31 were selected using magnetic-activated cell sorting,and plated in endothelial-specific growth medium.After 2-3 wks in culture,colonies displaying cobblestone-like morphology were manually picked using cloning cylinders.We characterized CV-ECFCs by flow cytometry,immunophenotyping,tube formation assay,and Dil-Ac-LDL uptake assay.Viral transduction of CVECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector,and transduction efficiency was tested by fluorescent microscopy and flow cytometry.Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved,small intestinal submucosa extracellular matrix scaffold.RESULTS After four passages in 6-8 wks of culture,we obtained a total number of 1.8×107 CV-ECFCs using 100 mg of early gestational chorionic villus tissue.Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31,CD144,CD146,CD105,CD309,only partially expressed CD34,and did not express CD45 and CD90.CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation,similar to cord blood-derived ECFCs(CB-ECFCs).CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%.Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold.CONCLUSION In summary,we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs.A substantial number of CV-ECFCs can be obtained within a short time frame,representing a promising novel source of ECFCs for fetal treatments.

Effects of Tremella Polysaccharide on Immune Function of Physically-Stressed Mice

An immunosuppressive animal model induced by physical stress that forced mice to swim in cold water(14±1℃)and the restorative effect of Tremella polysac charide(TP)on the suppressed immune function by stress were studied in mice.It was found that the spleen plaque forming cell(PFC)response to sheep red blood cells,delayed cuta- neous hypersensitivity(DCH)induced by dinitrochlorobenzene and the lymphocyte prolifer- ation stimulated by concanavalin A(Con A)were significantly decreased in stressed mice. In addition.the maximal decrease of PFC was reached in 9-12 days after stress.A- drenolectomy could not affect the decrease of PFC in stressed mice.TP(200.400mg/kg) ig for 8-14 days significantly restored the PFC.DCH and lymphocyte proliferation to nor- mal level in stressed mice.

Immunopotentiating Effects of Cucurbitacin B in Mice

Cucurbitacin B(CUB)is a major active principle contained in the calyx melo of Cucumis melo L.The immunopotentiating effects of CuB(im,qd×5)were studied.At lower doses, CuB increased the number of peripheral blood T lymphocytes(0.1 mg/kg),the rate of PHA-induced lymphocyte transformation(0.2 mg/kg),the number of plaque forming cells(PFC)of the spleen(0.2 mg/kg)and the level of serum hemolysin(0.4 mg/kg).The phagocytosis of macrophages and the clearance rate of charcoal particles were enhanced only by a large dose(0.8 mg/kg).The results indicate that CuB can potentiate both cellular and humoral immune function.