[The isolation, subculture and identification of human adipose derived endothelial progenitor cells].
OBJECTIVE
To investigate the methods of isolating and identifying human adipose derived EPCs.


METHODS
The cells obtained from human lipoaspirates were plated on culture dishes coated with human fibronectin and were cultured in DMEM containing 2% FBS. Cells of passage 2 cultured in EGM-2 (2% FBS) served as the induced cells (experimental group), with cells cultured in DMEM (2% FBS) as the non-induced cells (control group) . Immunofluorescence was used to detect the expression of cell markers, including CD34, vWF and PECAM-1. FACS (fluorescence activated cell sorter) was used to quantitatively analyze the expression rate of cell markers (CD34, CD45, CD133 and PECAM-1). Fluorescence microscope was used to observe the function of taking up DiI-ac-LDL by the induced cells. To determine the ability of forming capillary-like structure in three-dimensional matrices, the induced cells were also cultured in methylcellulose.


RESULTS
The induced cells of passage 2 exhibited cobblestone morphology, similar to that of the endothelial cells. In contrast, these morphological changes were not observed in non-induced cells. Immunofluorescence detected expression of vWF, PECAM-1 in induced cells and CD34 in non-induced cells. FACS analysis showed (67.41 +/- 13.35)% of the induced cells expressed PECAM-1 and (6.73 +/- 2.21)% of the non-induced cells expressed PECAM-1 (P < 0.01), while (72.39 +/- 13.45)% of the non-induced cells expressed CD34 and (16.06 +/- 3.86)% of the induced cells expressed CD34 (P < 0.01). Fluorescence microscopy observed the induced cells took up low-density lipoprotein (LDL). The formation of "branch-like" structure confirmed their functional activity.


CONCLUSIONS
EPCs derived from human adipose may serve as another source of seeding cells for vascular tissue engineering.
Liu
Bo
B
Department of Plastic and Reconstructive Surgery, the 9th People Hospital of Shanghai, Shanghai 200011, China.
Cui
Lei
L
Fu
Quan-hua
QH
Yang
Ping
P
Xu
Zhi-cheng
ZC
Yin
Shu
S
Wang
Hao-fu
HF
Liu
Guang-peng
GP
Liu
Wei
W
Cao
Yi-lin
YL
D017667:Adipocytes / Q000166:cytology*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D013234:Stem Cells / Q000166:cytology*