Журнал исследований и терапии стволовых клеток

Абстрактный

Trans-Differentiation of Rat Mesenchymal Stem Cells into Dopaminergic Neurons for Cell Transplantation

Ryan M Welchko, Travis D Hulse, Sabrina S Dieffenbach, Gabrielle P Shall, Huo Wangjing, Leslie R Siegal, Jared R Watters, Leveque T Xavier, Ming Lu, Julien Rossignol, Michael I Sandstrom and Gary L Dunbar

Objective: Transplantation of human embryonic dopaminergic progenitors within the striata of PD patients has provided encouraging results, but ethical concerns and tissue availability limit this approach. The use of mesenchymal stem cells (MSCs) provides a readily available source of cells, as they are derived from adult tissue. This in vitro study explored the use of MSCs as a cell source for DA neuronal induction utilizing a single adenovirus.
Methods: Our lab developed a novel adenovirus expressing multiple viral 2A genes allowing for the polycistronic expression of multiple genes (Ascl1, Lmx1a, and Nurr1) for transcription factors that are involved in DA neuron differentiation and used the gene for green fluorescent protein (gfp) to track transfection. MSCs were cultured with the adenovirus, monitored morphological changes as well as expression of gfp as evidenced by fluorescence microscopy. The presence of the viral DNA within the transfected cells was confirmed with PCR, Immunocytochemistry and RTPCR.
Results: MSCs cultured with the adenovirus, resulted in morphological changes as well as expression of gfp as evidenced by fluorescence microscopy. The presence of the viral DNA within the transfected cells was confirmed with PCR. Immunocytochemistry and RT-PCR analyses revealed that, cells expressing gfp have nuclear co-labeling of translated transcription factors LMX1a, and NURR1, as well as an up-regulation of these genes, along with an up-regulation of downstream gene targets, such as tyrosine hydroxylase (TH), and the dopamine transporter (DAT).