Abstract
The aim of the present work was to explore mesenchymal stem cells (MSCs) differentiation potential towards neural phenotype. MSCs are self-renewable multipotent cells shown to be able to support hematopoiesis and to improve functional outcomes in animal models of neurological disorders. MSCs were obtained by plastic adherence from iliac crest bone marrow of healthy donors for allogeneic transplantation and, for the in vitro studies, were cultured on laminin-coated dishes in a B27 Neurobasal medium with 3% to 10% FBS for 3 weeks. Few cell (11%) showing bipolar morphologies, expressed β-tubulin III and GFAP. Furthermore, only GAP43 expression was detected by RT-PCR. Addition of exogenous neurotrophins to cultures did not improve neural differentiation. To investigate the brain microenvironment effect on MSCs, cells were cultured on brain sections and supernatant of the cultures analyzed by ELISA. In this condition, MSCs were shown to release soluble human NT3/NT4 and NGF and to express p75 and TrkC receptors by immunocytochemistry. In order to improve these observations, we analyzed the human neurotrophin and receptor gene expression profile by GEArray technology. The expression patterns of human trkC, NT3, NT4, and NGF mRNA were consistent with the results of immunostaining.
To evaluate the in vivo MSCs differentiation potential, 50.000 cells labeled with a fluorescent dye (PKH26) were injected into the right parietal cortex of newborn Balb/C and nude mice (4 and 7 days old). Seven and 45 days later, immunocytochemistry and RT-PCR were performed on brain sections using the following neural specific markers: neurofilament-M, NSE, GFAP, b-tubulin III, MAP-2ab, nestin, Gal-C, and anti TrkC, TrkA and p75NFGR. FISH analysis was also performed using both Cy-3 labeled human Pan Centromeric and FITC labeled mouse Pan Centromeric probes. In 7 out of 52 Balb/C mice analyzed, fluorescent cells were detected 30 days post-injection but only one mouse showed NF and MAP-2ab expression by immunocytochemistry on FISH positive cells thirty days after transplantation. These data were confirmed by RT-PCR for the presence of human GAPDH. In nude mice, fluorescent cells were also detected away from the site of injection indicating cells migration throughout the brain. Moreover, 7 and 45 days post-injection, a high percentage of cells was shown to express the TrkC and p75 receptors. Isolation of the single human MSCs transplanted cells from brain sections was performed by laser microdissection analysis. ELISA analysis from these dissected areas showed the expression of human NT3/NT4 and NGF neurotrophin’s. Finally, several transplanted human MSCs expressing the Ve-cadherin were found close to blood vessels after 45 days of transplantation, whereas these cells were negative for human KDR and CD45. In addition, we determined the capability of conditioned MSCs media to regulate the angiogenesis in a tube formation assay. In conclusion, our data show an in vitro and in vivo capacity of MSCs to express neurotrophins under epigenetic stimuli rather than a real neural differentiation potential.
Neural differentiation of adipose-derived stem cells by indirect co-culture with Schwann cells