Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

Summary

The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton's jelly.

Abstract

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord Wharton's jelly, as well as from the umbilical arteries and umbilical vein. They are known as perivascular stem cells obtained from umbilical arteries (UCA-PSCs), perivascular stem cells obtained from the umbilical vein (UCV-PSCs), and mesenchymal stem cells obtained from Wharton's jelly (WJ-MSCs). UCA-PSCs and UCV-PSCs are pericytes derived from perivascular regions that are progenitors of MSCs. Isolation and culture of the three kinds of cells is an important source for studying stem cell transplantation and repair. The present protocol focuses on the isolation and culture of cells through mechanical separation, adherent culture, and cell crawling out. Through this technique, the three different types of stem cells can be derived. Cell surface markers were detected by flow cytometry. The stem cells were detected for multilineage differentiation potential by adipogenic, osteogenic, and neural-like differentiation, which is consistent with the phenotype of MSCs. This experimental protocol expands the source of UC-MSCs. In addition, the cell isolation method provides a basis for further study of regenerative medicine and other applications.

Introduction

Human umbilical cord mesenchymal stem cells (UC-MSCs) are widely used in regenerative medicine because of their noninvasive operation, low immunogenicity, and lack of ethical dispute¹. In many studies, UC-MSCs isolated from Wharton's jelly (WJ) can attach to the wall, undergo multi-differentiation, and express markers of mesenchymal stem cells (MSCs)². However, almost all MSCs originate from the perivascular region³. Pericytes, as a subset of perivascular cells, are progenitor cells of MSCs⁴. Therefore, UC-MSCs can be isolated from the umbilical cord WJ, umbilical arteries ....

Protocol

All experimental procedures were approved by the Clinical Research Ethics Committee, Third Affiliated Hospital, Soochow University. Informed written consent was obtained from the human subjects. Individuals with full-term vaginal delivery or cesarean section were included in the present study to obtain the umbilical cord. The umbilical cord comes from healthy newborns without gender bias. The neonate had an Apgar score of 8-10. The Apgar score is a quick test for newborns given soon after their birth. This test checks a .......

Discussion

This study isolated three different kinds of cells from the umbilical cord arteries, vein, and Wharton's jelly. The umbilical cord was delivery waste, and its use was simple, safe, and without ethical dispute⁵. UC-MSCs are original and have strong differentiation ability¹. Previous studies have shown that the amount of UC-MSCs isolated from umbilical cords by the collagenase, trypsin, and hyaluronidase digestion method was not abundant; the stem cells cannot be passaged man.......

Materials

Name	Company	Catalog Number	Comments
Adipogenic differentiation kit	Gibco	A1007001	Multidirectional differentiation
Alizarin red staining solution	Sigma	A5533	Multidirectional differentiation
Antibody against CD13	Thermo Fisher Scientific	MA1-12034	flow analysis
Antibody against CD34	BD Biosciences	560942	flow analysis
Antibody against CD45	BD Biosciences	561865	flow analysis
Antibody against CD73	BD Biosciences	940294	flow analysis
Antibody against HLA-DR	BD Biosciences	555560	flow analysis
Anti-fluorescence quenching agent	Abcam	AB103748	Immunofluorescence
Anti-Mouse IgG H&L (Alexa Fluor 488)	abcam	ab150113	Multidirectional differentiation
ATRA	STEMCELL Technologies	302-79-4	cell culture
bFGF	Gibco	13256029	Multidirectional differentiation
BSA	Sigma	V900933	Immunofluorescence
Cell incubator	Thermo Fisher Scientific	HERAcell 240i	cell culture
Cell-counting kit-8	Dojindo	CK04	cell proliferation
Centrifuge	Thermo Fisher Scientific	Sorvall™ MTX-150	cell culture
DAPI	Sigma	10236276001	Immunofluorescence
DMSO	Sigma	D1435	cell culture
FBS	Gibco	10099141	cell culture
FITC Mouse Anti-Human IgG	BD Biosciences	560952	flow analysis
Flow Cytometer	Thermo Fisher Scientific	A24864	flow analysis
Fluorescence microscope	Thermo Fisher Scientific	IM-5	flow analysis
Gelatin	Sigma	48722	Multidirectional differentiation
Leica Microscope	Leica	DM500	Multidirectional differentiation
LG-DMEM medium	Gibco	11-885-084	cell culture
Microplate reader	Thermo Fisher Scientific	A51119500C	cell proliferation
Neurogenic induction	Gibco	A1647801	Multidirectional differentiation
Oil red O solution	Sigma	O1516	Multidirectional differentiation
Osteogenic induction	Cyagen	HUXXC-90021	Multidirectional differentiation
Paraformaldehyde	Sangon Biotech	30525-89-4	Immunofluorescence
Pasteur pipette	Biosharp	BS-XG-03L	cell culture
PBS (phosphate buffered saline)	Hyclone	SH30256.LS	cell culture
Penicillin streptomycin	Hyclone	SV30010	cell culture
Primary antibody against NSE	Santa Cruz Biotechnology	sc-292097	Multidirectional differentiation
SPSS 22.0	IBM	SPSS 22.0	Statistical analysis
The cell climbing sheets	CITOTEST Scientific	80346-0910	Multidirectional differentiation
TritonX-100	Sangon Biotech	9002-93-1	Immunofluorescence
Trypsin	Gibco	25300120	cell culture