Biology Reference
In-Depth Information
showed after only 7 days of differentiation in vitro the presence of Alizarin Red-
positive crystals in GFP-positive cells and in nonnucleoporated AFSCs, while cells
maintained in growth medium continued to proliferate without increased calcium
deposits.
Nucleofected cells were proliferated in vitro in selective medium to assess the
stability of the transfection over time and to select for individual clones. After serial
dilutions were made, a single clone containing a high percentage of fluorescent cells
was selected using fluorescent microscopy and flow cytometry for each
nucleofection program. The clone obtained using the U-23 program had a 95.49%
rate of fluorescence with an intensity of 164, and the clone obtained using the C-17
program had a 93% rate of fluorescence with an intensity of 112.
1.4 Discussion
Nucleofection was a very efficient method for a primary cell line. The two
nucleofection programs optimized for human MSCs were also suitable for ovine
amniotic stem cells. The C-17 program was less efficient at transfecting the cells
compared to the U-23 program; however, C-17 resulted in a lower percentage of
cell mortality, the highest percentage viability, and the highest percentage of
transfected ovine AFSCs. Furthermore, by serial dilution in 96-well plates, single
colonies were isolated that were more morphologically homogeneous than the
original population, and nearly all cells expressed GFP.
Nucleofected AFSCs have been cultured for more than a month confirming that
nucleofection is a transfection method that is useful for long-term expression
because the exogenous plasmid is integrated into the genomic DNA of the host
cell and continues to be replicated during cell proliferation. Immunocytochemistry
and RT-PCR analyses indicated that nucleofected cells were unchanged based on
their expression of stemness markers and their ability to osteo-differentiate.
The availability of stable single clones of pluripotent stem cells expressing the
GFP
reporter gene opens the possibility of developing preclinical models to both
demonstrate the regenerative capacity of amniotic stem cells and investigate the
mechanisms underlying their integration in experimentally injured tissues.
References
Insausti CL, Blanquer M, Bleda P (2010) The amniotic membrane as a source of stem cells. Histol
Histopathol 25:91-98
Parolini O, Soncini M, Evangelista M, Schmidt D (2009) Amniotic membrane and amniotic fluid-
derived cells: potential tools for regenerative medicine? Regen Med 4:275-291
Zaragosi LE, Billon N, Ailhaud G, Dani C (2007) Nucleofection is a valuable transfection method
for transient and stable transgene expression in adipose tissue-derived stem cells. Stem Cells
25:790-797
