Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications
- 17 December 2015
- journal article
- research article
- Published by Springer Science and Business Media LLC in Cell and Tissue Banking
- Vol. 17 (2), 289-302
- https://doi.org/10.1007/s10561-015-9541-6
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 quality 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 mm2) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % 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 successfully 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 analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after 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.Keywords
Funding Information
- Ministry of Science and Technology (DTDL.2012-G/23)
This publication has 52 references indexed in Scilit:
- Umbilical cord mesenchymal stem cell transplantation significantly improves neurological function in patients with sequelae of traumatic brain injuryBrain Research, 2013
- Transplantation of human cord blood mononuclear cells and umbilical cord-derived mesenchymal stem cells in autismJournal of Translational Medicine, 2013
- Platelet Lysates Produced from Expired Platelet Concentrates Support Growth and Osteogenic Differentiation of Mesenchymal Stem CellsPLOS ONE, 2013
- A Xenogeneic-Free Protocol for Isolation and Expansion of Human Adipose Stem Cells for Clinical UsesPLOS ONE, 2013
- GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSCPLOS ONE, 2012
- Isolation of three important types of stem cells from the same samples of banked umbilical cord bloodCell and Tissue Banking, 2011
- TRANSPLANTATION AND CELLULAR ENGINEERING: Adipose tissue mesenchymal stem cell expansion in animal serum‐free medium supplemented with autologous human platelet lysateTransfusion, 2009
- Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone MarrowThe International Journal of Cell Cloning, 2009
- Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statementCytotherapy, 2006
- Searching for Alternative Sources of Postnatal Human Mesenchymal Stem Cells: Candidate MSC‐Like Cells from Umbilical CordThe International Journal of Cell Cloning, 2003