Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino

Abstract

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P<0.001) expression of the markers GFAP and β3 tubulin at the end of P2 compared to P1. The ability of MSCs to generate cell types related to neural lineage is complex and multifactorial, depending not only of inducing agents, but also the environment in which these cells will be cultivated. Thus a greater number of studies are necessary to better understand the process of neural transdifferentiation of MSCs from equine.INDEX TERMS: Horse, neural differentiation, adult stem cells, neural markers.

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

“…The cell population with the fusiform appearance described here in samples from both adipose tissue and bone marrow were morphologically similar to mesenchymal stem cells from human (Zuk et al 2001, You et al 2009), sheep (Fadel et al 2011), horse (Fortier et al 1998, Smith et al 2003, Hegewald et al 2004, Vidal et al 2006, 2007, Maia et. al 2012, De Vita et al 2013, and other animals reported in many studies, in which these cells were described as star-shaped or fibroblast-like.…”

“…In a current study about the potential of neural differentiation of mesenchymal stem cells from equine bone marrow, Maia et al (2012) surprisingly found that control cells culture that have not undergone any treatment, also expressed neural markers. These researchers suggested that FBS present in the culture medium may have influenced the differentiation in the control group, because it is known that FBS is rich in steroids, such as glucocorticoids and estrogen, which may have interacted with other growth factors, also present in the serum, driving a cascade of events to cell differentiation.…”

“…Fortier et al (1998) were able to complete the removal of blood cells and non-adherent hematopoietic stem cells of the mesenchymal stem cell culture by the ninth day of culture, when the population of predominant cells showed a similar morphology as the cells found in the present work. Maia et al (2012) had adherent cells from bone marrow mononuclear fraction between 24 and 48 hours of culture, and fibroblast-like cells morphology by the fourth day of culture. Therefore, in this study, it appears that the non-adherent cells were successfully removed from the adipose tissue cultures in the first exchange of medium, while the cell cultures from bone marrow still showed non-adherent cells, although in decreasing amounts, up to the last evaluation, thus explaining the heterogeneous appearance of the culture.…”

“…Mesenchymal stem cell cultures with DMEM medium supplemented with fetal bovine serum (FBS) are often used today (De Vita et al 2013, Maia et. al 2012.…”