Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials.
OBJECTIVE
Adult bone marrow (BM) is the major source of mesenchymal stem cells (MSC) for cell therapy. However, aspiration of BM involves invasive procedures. We isolated MSC from human full term umbilical cord tissues (UC). The biological characteristics of MSC derived from UC (UC-MSC) were further determined and compared with normal adult bone marrow-derived MSC (BM-MSC).


METHODS
MSC were isolated from UC by enzyme digestion and cultured in appropriate growth medium. The isolation efficiency, cell yield, colony-forming unit-fibroblast (CFU-F) frequency, growth kinetics, phenotypic characteristics, multi-lineage differentiation capacity, cytokine spectrum as well as hematopoiesis-supportive function of UC-MSC were determined and compared with those of BM-MSC.


RESULTS
MSC were successfully isolated from all 36 UC and six BM samples we collected for this study. The mean number of nucleated cells isolated from UC was 1yen106/cm and the yield of adherent cells was 8.6yen105/cm. UC-MSC shared most of the characteristic of BM-MSC, including fibroblastic-like morphology, immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials, and hematopoiesis-supportive function. The CFU-F frequency was higher in UC nucleated cells (1:1609 +/- 0.18) than in BM nucleated cells (1:35700 +/- 0.01) (p < 0.05). Furthermore, in comparison with BM-MSC, the UC-MSC had a higher proliferation capacity and lower levels of expression of CD106 and HLA-ABC (p < 0.05). Immunofluoresent and western blot assays revealed that UC-MSC had a higher percentage of neuron specific enolase-positive cells than had BM-MSC after neuronal induction. Finally, reverse transcriptase polymerase chain reaction analysis showed that UC-MSC had a cytokine spectrum very similar to that of BM-MSC, including expression of the mRNA of stem cell factor, leukemia inhibitor factor, macrophage-colony stimulating factor, Flt3-ligand, interleukin-6, vascular endothelial growth factor and stromal-derived factor-1, but UC-MCS additionally expressed mRNA of granulocyte macrophage and granulocyte colony-stimulating factors. After co-culture with CD34+ cord blood cells for 5 weeks, no significant difference in colony-forming cells was observed between the CD34+ cells/UC-MSC and CD34+ cells/BM-MSC co-cultures (p > 0.05).


CONCLUSIONS
We have established a protocol to isolate abundant MSC from human umbilical cords with a 100% success rate. The comparative study indicates that UC is an excellent alternative to BM as a source of MSC for cell therapies.
Lu
Lu-Lu
LL
National Engineering Research Center of Cell Products, AmCellGene Co. Ltd.
Liu
Yong-Jun
YJ
Yang
Shao-Guang
SG
Zhao
Qin-Jun
QJ
Wang
Xin
X
Gong
Wei
W
Han
Zhi-Bo
ZB
Xu
Zhen-Shu
ZS
Lu
Yong-Xin
YX
Liu
Delong
D
Chen
Zhi-Zhe
ZZ
Han
Zhong-Chao
ZC
D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D002448:Cell Adhesion; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D003114:Colony-Forming Units Assay / Q000379:methods; D016207:Cytokines / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology*; D006410:Hematopoiesis; D006801:Humans; D007202:Indicators and Reagents; D007231:Infant, Newborn; D008071:Lipoprotein Lipase / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology*; D009474:Neurons / Q000166:cytology; D053495:Osteopontin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics
D016207:Cytokines; D007202:Indicators and Reagents; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D008071:Lipoprotein Lipase