Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Feb 26, 2020; 12(2): 123-138
Published online Feb 26, 2020. doi: 10.4252/wjsc.v12.i2.123
Clonal isolation of endothelial colony-forming cells from early gestation chorionic villi of human placenta for fetal tissue regeneration
Kewa Gao, Siqi He, Priyadarsini Kumar, Diana Farmer, Jianda Zhou, Aijun Wang
Kewa Gao, Siqi He, Jianda Zhou, Department of Burns and Plastic Surgery, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China
Kewa Gao, Siqi He, Priyadarsini Kumar, Diana Farmer, Aijun Wang, Surgical Bioengineering Laboratory, Department of Surgery, University of California Davis, Sacramento, CA 95817, United States
Kewa Gao, Siqi He, Priyadarsini Kumar, Diana Farmer, Aijun Wang, Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children, Sacramento, CA 95817, United States
Aijun Wang, Department of Biomedical Engineering, University of California Davis, Davis, CA 95817, United States
Author contributions: This study was designed and supervised by Wang A, Zhou J and Farmer D; experiments were performed by Gao K, He S and Kumar P; data analysis was conducted by Gao K and Wang A; funding was obtained by Farmer D and Wang A; the manuscript was written and revised by Gao K, He S, Kumar P, Farmer D, Zhou J and Wang A.
Supported by the Shriners Hospital for Children Postdoctoral Research Fellowship award, No. 84704-NCA-19; UC Davis School of Medicine Dean’s Fellowship award and funding from the NIH, No. 5R01NS100761-02 and No.R03HD091601-01; the California Institute of Regenerative Medicine, No. PC1-08103 and No. CLIN1-11404; Shriners Hospitals for Children, No. 85120-NCA-16, No. 85119-NCA-18, No. 85108-NCA-19 and No. 87200-NCA-19; March of Dimes Foundation, No. 5FY1682.
Institutional review board statement: The study was submitted to the UCD Institutional Review Board (IRB) and determined to be exempt from review.
Conflict-of-interest statement: Authors of this manuscript have no conflicts of interest to disclose.
Data sharing statement: We agree to share the research methods and data in this study to public.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Aijun Wang, PhD, Associate Professor, Surgical Bioengineering Laboratory, Department of Surgery, University of California, Davis School of Medicine, 4625 2nd Ave., Room 3005, Sacramento, CA 95817, United States.
Received: July 5, 2019
Peer-review started: July 17, 2019
First decision: August 23, 2019
Revised: December 3, 2019
Accepted: December 23, 2019
Article in press: December 23, 2019
Published online: February 26, 2020

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.


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.


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 CV-ECFCs 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.


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.


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.

Keywords: Placenta, Endothelial colony forming cells, Chorionic villi, Angiogenesis, Tissue engineering

Core tip: We established a magnetic sorting-assisted clonal isolation protocol to derive chorionic villus endothelial colony-forming cells (CV-ECFCs) from early gestation placentas. Using our protocol, a substantial number of CV-ECFCs can be obtained from chorionic villus sampling specimens within a short time frame, making it feasible for autologous fetal treatment. CV-ECFCs are comparable to umbilical cord blood-derived ECFCs in terms of surface marker expression, tube formation capability, transducibility, and compatibility with biomaterial delivery vehicles. CV-ECFCs represent a novel autologous source of cells for fetal or postnatal treatment of congenital anomalies or defects.