The procedure of obtaining qualified
endothelial progenitor cells (EPCs) is still unclear
and there has been some controversy on their biological
properties and time of emergence. In this study,
we used long-term culture of Adipose Derived Stem
Cells (ADSCs) in an endothelial induction medium to
obtain endothelial progenitor-like cells, and investigated
the features of a few surface markers and the
physiologic functions of the cells produced. In order to
achieve our aim, rat ADSCs were isolated and cultured
in an endothelial basal medium (EBM2), supplemented
with an endothelial growth medium (EGM2).
The cells were cultured 1 week for short-time,
2 weeks for mid-time, and 3 weeks for long-time
cultures. Morphological changes were monitored by
phase contrast and electron microscopy. The expressions
of a few endothelial progenitor cells markers
were analyzed by real-time RT-PCR. Low-density
lipoprotein uptake and lectin binding assay were also
performed for functional characterization. After
induction, ADSCs showed changes in morphology
from spindle-shaped in the first week to cobblestoneshaped
during the next 2 weeks. Then, endothelial cell
phenotype was defined by the presence of Weibel–
Palade bodies in the cytoplasm and tube formation,
without the use of Matrigel in the third week. In
keeping with gene expression analysis, VEGFR-2
showed significant expression during early stages of
endothelial differentiation for up to 3 weeks. A
significantly increased expression of Tie2 was
observed on day 21. Likewise, VE-Cadherin, CD34,
CD133, WVF and CD31 were not significantly
expressed within the same period of time. Endothelial
differentiated cells also showed little LDL uptake and
little to no lectin binding during the first 2 weeks of
induction. However, high LDL uptake and lectin
binding were observed in the third week. It appears
that long term culture of ADSCs in EGM2 leads to
significantly increased expression of some endothelial
progenitor cells markers, strong DiI-ac-LDL uptake,
lectin binding and tube-like structure formation in
endothelial differentiated cells. Therefore, selection of
an appropriate culture time and culture medium is
crucial for establishing an efficient route to obtain sufficient numbers of EPCs with optimized quantity
and quality.

EB00000002939K

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• Long term culture and differentiation of endothelial progenitor like cells from rat adipose derived stem cells