Neuroectodermal differentiation in vitro and in vivo of rat bone marrow-derived multipotent adult progenitor cells

Resumen

Multipotent adult progenitor cells (MAPC) have been previously described as bone marrow (BM)-derived stem cells capable of differentiating in vitro into cells of the three germ layers, mesoderm, endoderm and ectoderm (Reyes et al. 2001; Jiang et al. 2002). MAPC have been proven to be difficult to isolate and culture. Nevertheless, they have been isolated from the BM of different mammals such as human and mouse. In the present thesis we aimed to evaluate the neuroectodermal differentiation potential of rat-derived MAPC (rMAPC) in vitro and in vivo, upon transplantation into neurogenic and non-neurogenic regions of adult rat brain. rMAPC were isolated from the BM of adult Sprague Dawley rats. rMAPC bulk cultures were heterogeneous and comprised by two main subpopulations of cells, small and large cells. Only cells in the small-size subpopulation showed MHC class I- and CD44-negative phenotype. Culture at low densities was mandatory for the maintenance of these cells. rMAPC showed extensive proliferation potential and certain multilineage differentiation potential, based on the acquisition of morphological and phenotypic characteristics typical of cells from diverse tissues (endothelial, hepatic). Moreover, rMAPC expressed some of the genetic markers of embryonic stem cells, namely OCT4 and SSEA-1, albeit at low levels. However, rMAPC were not able to differentiate towards neuroectodermal phenotypes on a persuasive basis in vitro, by any of the protocols utilized (addition of specific cytokines, astrocyte co-cultures, retrovirally-mediated overexpression of Pax6 and Ngn2, transfection with a dominant negative form of RE1-silencing transcription factor -REST-). Although differentiated rMAPC acquired some phenotypic characteristics of neural cells (expression of nestin and ß-III-tubulin proteins), neither the expression of more mature neural markers nor functional characteristics typical of neuronal cells could be observed. Regarding in vivo transplantation experiments, rMAPC injected into neurogenic SVZ of the adult rat brain showed a pronounced migratory capacity along the rostral migratory stream, but grafted cells failed to differentiate into neuronal, astroglial or oligodendroglial cell types. Nevertheless, grafted rMAPC displayed pro-angiogenic/vasculogenic effects into adult brain, suggesting that, independently of their potential to differentiate into the neural lineage, they might be useful therapeutic tools for certain diseases, such as brain ischemia.