Biology Reference
In-Depth Information
embryonic stem (ES) cells, which have ethical limitations, and adult stem cells,
which are less available and more difficult to grow in in vitro culture. However,
despite their therapeutic potential, both cell types have specific limitations.
Although adult stem cells can be directly isolated from the patient and are therefore
immunologically compatible with the patient, they are generally difficult to isolate
and grow in culture, and their recovery involves invasive interventions. In contrast,
ES cells can proliferate rapidly in culture and differentiate into cells of all adult
tissues; however, in addition to the need to resolve the ethical issues surrounding
their use, they may lead to tumor formation after transplantation. Recently, the
plasticity of amnion-derived cells was reported, and these cells have attracted much
attention for their regenerative potential.
Amnion-derived stem cells, available in large quantities (as they can be retrieved
from placentae at term), display clear characters of staminality, do not undergo
oncogenic deviation, can differentiate into all three germ layers, and have no ethical
concerns (Miki et al
2005
). Moreover, since the placenta is where fetal-maternal
immunotolerance is developed, these cells have immunomodulatory properties and
appear to be well tolerated after allotransplantation. Collectively, these cells repre-
sent a promising candidate for use in regenerative medicine.
Despite these promising properties, the mechanisms that condition the
staminality of these cells throughout expansion in vitro and differentiation are
still largely unknown. Therefore, this research has been designed to investigate
the properties of stemness of ovine amnion-derived stem cells and, in particular, of
amniotic epithelial stem cells and their ability to undergo osteogenic differentiation
after expansion in vitro. The data reported indicate that sheep amniotic epithelial
cells (AEC) display interesting characteristics of staminality, and their osteogenic
potential represents an alternative to bone marrow mesenchymal stem cells for the
development of osteogenic regenerative strategies.
2.2 Materials and Methods
AECs were retrieved from the amniotic membranes of fetuses between 25 and
35 cm in length (i.e., at 3 months of development). After amniotic membrane
isolation, the epithelial layer was mechanically peeled under a stereomicroscope
and incubated in 0.25% trypsin in 0.5 mM Na-EDTA at 37
C for 20 min. The cells
released were collected and seeded at a concentration of 3
10
3
cells/cm
2
in 175-cm
2
flasks in
-MEM supplemented with 20% fetal calf serum, 1% ultraglutamine, 1%
penicillin/streptomycin, and 10 ng/ml epidermal growth factor. At 80% confluence,
the cells were dissociated by 0.05% trypsin at 37
C for 5 min and plated again as
described above. These procedures were repeated for 12 expansion passages.
Aliquots of cells obtained from the 1st, 6th, and 12th expansion passages were
used to evaluate the expression of surface adhesion markers (CD29 and CD49) and
the nuclear stemness markers (Oct4, SOX2, Nanog, and TERT) by immunohisto-
chemistry, and to investigate the level of cell-wide DNA methylation. The cells
a
