Biology Reference
In-Depth Information
transcriptase reactions and gene-specific PCRs were performed to evaluate mRNA
expression of
OCT4A
,
SOX2
, and
NANOG
.
1.2.5 Clonal Selection
The remaining cells were harvested by trypsinization and resuspended in growth
medium supplemented with antibiotics. Suspended cells (200
m
l for a total of
4
10
3
cells) were used for serial dilutions in 96-well flat-bottom plates. At
confluency, the wells with the highest percentage of fluorescent cells were
evaluated by fluorescent microscopy. Selected clones were plated in 12-well plates,
and successively into 90-mm dishes. GFP expression was analyzed on a flow
cytometer.
1.2.6
In Vitro Osteogenic Differentiation
AFSCs were induced to osteo-differentiate with differentiation medium (
MEM
a
supplemented with 10% FCS, 1 mM ascorbic acid, 1 M
-glycerol phosphate,
b
500
M dexamethasone, 1% of penicillin/streptomycin, and 1% of -glutamine).
Cells were incubated for 7 days in a humidified 38.5
C/5% CO
2
incubator. Miner-
alization was evaluated by Alizarin Red S staining.
m
1.3 Results
The viability of nucleofected AFSCs, calculated as the ratio of the number of
adhered cells to the total number of cells exposed to nucleofection, was 1.6 and
12% for cells nucleofected with the U-23 and C-17 programs, respectively. The
efficiency of gene transfer, measured as the percentage of GFP-positive cells by
flow cytometry, was found to be 28.5% and 37.2% for the U-23 and C-17 programs,
respectively.
To test if the nucleofection influenced the characteristics of amniotic stem cells,
we evaluated the expression of molecular markers specific to stem cells and the
ability of these cells to osteo-differentiate in vitro. Immunocytochemistry analysis,
performed before and after nucleofection, showed the presence of stemness markers
(SOX2, NANOG, and TERT) in both nucleofected and control cells. The localiza-
tion of the markers was nuclear in 40% of the AFSCs. Gene expression analysis of
stemness markers using RT-PCR showed only the presence of
NANOG
mRNA,
while
SOX2
and
OCT4A
were not expressed in nucleofected cells or in the control.
Osteogenic differentiation in vitro was determined by histological staining
(Alizarin Red) that indicated deposits of hydroxyapatite. Histological analysis
