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

Kewa Gao, Siqi He, Jianda Zhou, Priyadarsini Kumar, Diana Farmer, Aijun Wang

Research output: Contribution to journalArticle

Abstract

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 x 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.

Original languageEnglish (US)
Pages (from-to)123-138
Number of pages16
JournalWorld Journal of Stem Cells
Volume12
Issue number2
DOIs
StatePublished - Feb 26 2020

Keywords

  • Angiogenesis
  • Chorionic villi
  • Endothelial colony forming cells
  • Placenta
  • Tissue engineering

ASJC Scopus subject areas

  • Histology
  • Molecular Biology
  • Genetics
  • Genetics(clinical)
  • Cell Biology

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