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.

Expression and molecular mechanism of PCNA,Caspase-3,IL-6 and Survivin proteins in chorionic villi and decidual tissue of early embryo damage

Objective:This study aims to explore its role in embryo damage and the relationship between them by testing the expression of PCNA,Caspase-3,IL-6 and Survivin protein in the chorionic villi and decidual tissue of patients with unexplained early embryo damage and normal early pregnancy at the same time voluntarily requested uterine aspiration abortion in order to clarify the pathogenesis of early embryo damage from the cellular and molecular perspectives,and provide theoretical basis for the diagnosis and treatment of embryo damage.Methods:From July 2018 to July 2019,30 patients with unexplained embryo damage were selected as embryo damage group,thirty normal early pregnancy patients with aspiration abortion were selected as the normal early pregnancy group.The decidual tissue and chorionic villi of the two groups were collected to observe the structural and pathological changes.Immunohistochemical method was used to detect the distribution of PCNA,Caspase-3,IL-6 and Survivin in the chorionic villi and decidual tissue,as well as the expression changes of the above four proteins.Results:Compared with the normal early pregnancy group,the chorionic villi in the early embryo damage group were obviously dysplasia,degenerative changes,structural disorders,homogeneous destruction,obvious reduction or even disappearance of trophoblast cells,inflammatory cell infiltration,more neutrophils and lymphocytes,interstitial edema and cell atrophy,accompanied by hemorrhage;The decidual tissue of the intima was not good,most of the cells were spindle-shaped,the structure was disordered,the stroma has edema,inflammatory cells can be seen,and hemorrhage existed.The results of immunohistochemistry showed that the expression of PCNA protein in chorionic villi and decidual tissue of early embryo damage group decreased significantly(P<0.05),while the expression of Caspase-3 protein increased significantly(P<0.05).The expression of IL-6 protein decreased significantly(P<0.05),and the expression of Survivin protein decreased significantly(P<0.05).Conclusion:During early pregnancy,due to the down-regulation of the expression of proliferating cell nuclear antigen PCNA and apoptosis inhibitor protein Survivin in chorionic and decidual tissues,the balance between proliferation and apoptosis is broken,which has an increase in Caspase-3 expression and a decrease in IL-6 expression.It may be that the balance of Th1/Th2 in chorionic and decidual tissues inclines to Th1,which leads to excessive apoptosis of chorionic and decidual tissues and the cessation of early embryo damage.

The Influences of Mifepristone, Norethisterone and Tamoxifen on the Glycosph

By using the Ladisch partitioning and microscale-analysis of HPTLC,the comparative quantitative and qualitative studies of gangliosides (Gg)and neutral glycosphingolipids(N-GSL)compositions from human chorionic villi tissues of normal early pregnant women and women treated with mifepristone, norethisterone(NET)and tamoxifen(TAM)were reported in this paper.The patterns of Gg and N-GSL in three treated groups were similar to those in normal pregnant group.The total values of Gg from the chorionic villi tissues reduced significantly in three treated groups(P<0.01).In all treated groups,the amounts of NeuNAC-Gal-Glc-cer(GM3),NeuNAC-NeuNAC-Gal-Glc-cer(GD3)and Gal-GalNAC-(NeuNAC)-Gal-Glc-cer(GM1)were decreased significantly compared with those in normal(P<0.01orP<0.05).In NET and TAM groups,Neu NAC-Gal-GalNAC-(NeuNAC-NeuNAC)-Gal-Glc-cer(GT1b) was markedly lower than that in normal(P<0.01).The total values of N-GSL extracted from the chorionic villi tissues were obviously higher in mifepristone and TAM groups than those in normal(P<0.01).The Gal-Glc-cer(LacCer)(CDH)and Gal-Gal-Glc-cer(Gal-LacCer)(CTH)were greatly increased in mifepristone group as compared with normal P<0.05).Paragloboside:Gal-GalNAC-Gal-Glc-cer(PG)in NETgroup was significantly higher than that in normal(P<0.01).