Human adult stem cells and direct cell reprogramming for restoring cognitive function in the aging brain.

REGGIANI PC; MOREL GR; ZAPPA F; MAZZOLINI G; GOYA RG; LÓPEZ-LEÓN M ; GARCIA MG; LEHMANN M

Congreso; Congreso de la Federación de Sociedades de Neurociencia de Lationoamérica y el Caribe.; 2016

There is a growing interest in the implementation of Regenerative Medicine for the treatment of age-associated neurodegenerative diseases. Thus, adult bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) have been reported as promising candidates for the treatment of neurodegenerative disorders. We have used human BM-MSCs to restore hippocampal morphology and cognitive performance in aging rats which are considered a suitable animal model of age-related cognitive deficits. We also want to use direct cell reprogramming to convert adult fibroblasts into neural progenitor cells (NPCs) in order to set up a cell therapy strategy in the hippocampus suitable for personalized regenerative medicine in aging individuals. To this end, we have constructed a regulatable bidirectional plasmid vector harboring the four pluripotency genes, oct4, sox2, klf4, and c-myc (the Yamanaka genes), and the humanized green fluorescent protein (hGFP) reporter gene. The Yamanaka genes were cloned as a bicistronic tandem (STEMCCA cassette) in which each pair of genes, flanking the IRES element, are linked by a self-processing CHYSEL sequence (2A type). Both, the STEMCCA cassette and the gfp gene are under the control of a bidirectional Tet-Off promoter. While the original Kim study showed that the four Yamanaka genes allow an efficient direct reprogramming, the fact that c-Myc is oncogenic has made us consider the possibility of also employing a direct reprogramming protocol using a plasmid that only expresses the oct4, sox2 and klf4genes. The reason for constructing a plasmid vector instead of a retrovector is that theformer delivers the pluripotency genes in a non-integrative fashion. Since plasmids are less efficient than viral vectors for gene delivery, we are using magnetofection, a magnetic nanoparticle-based technology to optimize transfection of the Yamanaka genes. Fluorescently-labeled human BM-MSCs were intracerebroventricularly (bilaterally) injected to 27-month-old female rats. Experimental subjects were divided into 3 groups (N=8): Youngintact, Senile-intact and Senile-MSC. We used the Barnes maze test to assess hippocampusdependent learning and spatial memory before and after cell injection. Also, we assessed recognition memory with the Novel Object Recognition test. Using an unbiased stereological approach, we assessed immature cell number (DCX cells) in the hippocampal dentate gyrus and astrocyte number (GFAP cells) in the stratum radiatum. Additionally, we conducted timecourse studies to determine MSC integration and viability in the brain at different post-injection times. The results suggest that human BM-MSC therapy improves spatial memory (increases goal hole exploration activity) in MSC-treated senile rats as compared with intact senile counterparts although spatial performance remains significantly lower than that of the young rats. Neurogenesis (immature neuron number) in the hippocampal dentate gyrus was also improved by the treatment. Histological analysis revealed that part of the MSCs integrated into ependymal cell layer and to a lesser extent in the brain parenchyma. In parallel, we checked the structure of our STEMCCA plasmid by sequencing and restriction analysis. Also we have characterized the regulatability of our vector in cell culture by Doxycycline. We conclude that human BM-MSC therapy partially reverses the decline in cognitive performance that occurs in aging rats and improves a number of hippocampal morphologic parameters. We also conclude that adult stem cells are a suitable biological tool for the treatment of age-related cognitive decline.