ETD system

Electronic theses and dissertations repository


Tesi etd-07032013-080303

Thesis type
Tesi di laurea specialistica
Corso di studi
relatore Prof.ssa Batistoni, Renata
Parole chiave
  • embryoid bodies
  • neural potential
  • amniotic fluid cell
Data inizio appello
Data di rilascio
Riassunto analitico
Human embryonic stem cells research is ethically and politically controversial because it involves the destruction of human embryos, whereby research on adult stem cells has opened the door to exciting therapeutic advances.
Adult stem cells have been recently shown to differentiate into several lineages and are becoming an attractive source of multipotent stem cells for transplantation, tissue engineering and gene therapy given their apparent advantages of accessibility, renewal capacity, multipotentiality and the reduced possibility of immune rejection after transplantation. Furthermore, adult stem cells with same potential of embryonic stem cell allow to overcome many of the ethical and political hurdles in stem cells research.
The amniotic fluid contains stem cells derived from embryonic and extra-embryonic tissues whose properties are being studied. These cells are routinely obtained for prenatal diagnosis of a wide range of fetal abnormalities and can be expanded in vitro. Recently, it has been suggested that amniotic fluid contain an heterogeneous population of cell types and several findings suggest that it contains pluripotent stem cells that can differentiate into derivatives of all three embryonic germ layers.
Amniotic fluid–derived stem cells have been shown to differentiate into several mesenchymal lineages and have been proposed as an important therapeutic multipotent stem cell source. Nevertheless, their ability to undergo terminal neuronal differentiation and to form functional neurons has been a matter of contention. Whereby, the aim of this study was to establish the neuronal differentiation ability of the c-Kit-positive population from GFP-transgenic rat amniotic fluid, called rat amniotic fluid stem cells (rAFSCs), a stable line with high proliferative capacity.
Because, undifferentiated rat GFP-AFS cells are proliferative and not yet committed to a neural lineage, our hypothesis was that, under appropriate conditions, rat amniotic fluid stem cells, grown as embryoid body-like (EB like), could differentiate into the neural lineage in vitro.
Immunocytochemical analysis was performed to characterize undifferentiated
c-kit-positive population of rAFSCs grown in Chang Medium demonstrating that they were positive for Nestin, GFAP, DCX, NF200,Vimentin and negative for βIII-tubulin (Tuj-1), PAX6 and OCT4. Furthermore, to assess the AFS cell neural potential it was investigated the expression of neural stem cell markers and markers of neural differentiation into undifferentiated rAFSCs by RT-PCR that showed the expression of the neural stem cell markers Sox2, Pax6, nestin and the expression of the neuronal differentiation marker βIII-tubulin.
The aim of the project was also to investigate whether, the Embryoid Body-like structures that these cells form in culture on different substrates, such as Poly-L-lysine/Laminin, Poly-L-ornithine or by Hanging Drop method, were true Embryoid Bodies (EBs).
Since, EBs have the potential to differentiate into somatic cells making up the three germ layers, RT-PCR was performed on rAFS-derived EB-like structures showing the expression of Sm22, Pax6, and βIII-tubulin. Furthermore, to assess whether these EB-like structures were able to differentiate into neurons, hanging drop derived EB-like structures were induced into neural differentiation using two different neural media and immunostaining was performed to check for expression of NF200, GFAP, DCX and βIII-tubulin.
Lastly, we tested on rAFSCs the cytotoxic effect of small molecules, known to induce neuronal differentiation, in order to identify the best treatment concentrations to be used in future studies.