Biology Reference
In-Depth Information
indispensable for maintaining a cell in an undifferentiated state. Other important
biological characteristics are low immunogenicity and immunoregulatory features
that have been observed in vitro and in vivo that together allow for the use of these
cells for allo/xenografts. Amniotic cells also have anti-inflammatory, antimicrobial,
and antifibroblastic features, and do not show any evidence of tumor formation.
Unlike embryonic stem cells, amniotic cells do not present ethical problems for
their recovery from humans (Insausti et al.
2010
). For these reasons, particular
attention has been directed to stem cells derived from amniotic fluid and
membranes as an alternative source of mesenchymal stem cells (MSC) that are
useful in the field of regenerative therapy.
In recent studies, amniotic cells have been used in preliminary tests with animal
models to test their ability to regenerate damaged tissue following injuries of the
neuronal system (i.e., Parkinson's disease), kidney, bone marrow, or myocardium.
However, preclinical studies are still necessary to demonstrate the regenerative
capacity and to ensure the long-term safety of the treatment.
In this context, it is relevant that the animal model has morphofunctional
characteristics similar to humans. For this reason, sheep are considered an optimal
model for studying bone, skeletal muscle, and tendon diseases. To assess the
regenerative effect of transferred stem cells into a pathologically or experimentally
damaged tissue, it is necessary to have cells that express a marker, such as green
fluorescent protein (GFP), which allows for their identification following transfec-
tion. There are different methods for transferring foreign DNA into a cell. Viral
methods that use modified virus (adenovirus, lentivirus, or retroviruses) are the
most efficient techniques and are also technically demanding because they require
specific safety conditions. In contrast, nonviral-based methods, including chemical
systems, such as lipofection, and physical systems, such as electroporation, are less
efficient than viral methods, especially since they trigger higher levels of cell
mortality.
Recently, a new nonviral method based on electroporation, called nucleofection,
has been described. This technique consists of a combination of cell-specific
solutions and optimized electrical parameters that lead to increased efficiency of
gene transfer to the cell nucleus (Zaragosi et al.
2007
). In addition, this method is
effective on many primary cell lines that are typically difficult to transfect by
nonviral methods.
The aim of the present study was to optimize a nucleofection program that was
already tested on human MSC for ovine stem cells isolated from amniotic fluids
(AFSCs). Optimization was evaluated by examining cell viability, efficiency of
gene transfer in the short and long term, maintenance of stemness characteristics,
and osteo-plasticity in vitro.
