Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications

 http://repository.vnu.edu.vn/handle/VNU_123/27744

Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. 

Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2.

UC– MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and qual-ity control for their use in clinical applications.

This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors.

Blood vasculature was removed from UC.

UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm 2 ) of UC membrane and Whar-ton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 Cin5 %CO2.

The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice.

Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS).

All UC-MSC (n=30) samples were success-fully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles.

Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes.

The anal-ysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenesNanog andOct4after long term culture but failed to transfer tumors in NOD/SCID mice.

Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications

Title: 

	Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications
Authors:	Pham, Phuc Van Truong, Nhat Chau Le, Phuong Thi-Bich
Keywords:	Activated platelet rich plasma Clinical application of mesenchymal stem cells Umbilical cord Umbilical cord derived mesenchymal stem cells Good manufacturing practice UC–MSCs
Issue Date:	2016
Publisher:	H. : ĐHQGHN
Citation:	ISIKNOWLEDGE
Abstract:	Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC– MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and qual-ity control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm 2 ) of UC membrane and Whar-ton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 Cin5 %CO2. The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n=30) samples were success-fully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The anal-ysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenesNanog andOct4after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications
Description:	CELL AND TISSUE BANKING Volume: 17 Issue: 2 Pages: 289-302 ; TNS06455
URI:	http://repository.vnu.edu.vn/handle/VNU_123/27744
Appears in Collections:	Bài báo của ĐHQGHN trong Web of Science