HUMAN ADULT STEM CELLS AND DIRECT CELL REPROGRAMMING FOR RESTORING COGNITIVE FUNCTION IN THE AGING BRAIN

MICAELA LÓPEZ-LEÓN; MARIANA G. GARCÍA; MARIANNE LEHMANN; MARIA F. ZAPPA VILLAR; GUILLERMO MAZZOLINI; RODOLFO GOYA; PAULA C. REGGIANI; GUSTAVO R. MOREL

CABA

Congreso; 2nd FALAN congress; 2016

IBRO-FALAN

There is a growing interest in the implementationof Regenerative Medicine for the treatment of age-associated neurodegenerativediseases. Thus, adult bone marrow-derived Mesenchymal Stem Cells (BM-MSCs) havebeen reported as promising candidates for the treatment of neurodegenerativedisorders. We have used human BM-MSCs to restore hippocampal morphology andcognitive performance in aging rats which are considered a suitable animal modelof age-related cognitive deficits. We also want to use direct cellreprogramming to convert adult fibroblasts into neural progenitor cells (NPCs)in order to set up a cell therapy strategy in the hippocampus suitable forpersonalized regenerative medicine in aging individuals. To this end, we haveconstructed a regulatable bidirectional plasmid vector harboring the four pluripotencygenes, oct4, sox2, klf4, and c-myc (the Yamanaka genes),and the humanized green fluorescent protein (hGFP) reporter gene. The Yamanakagenes were cloned as a bicistronic tandem (STEMCCA cassette) in which each pairof genes, flanking the IRES element, are linked by a self-processing CHYSELsequence (2A type). Both, the STEMCCA cassette and the gfp gene are under thecontrol of a bidirectional Tet-Off promoter. While the original Kim study (*) showedthat the four Yamanaka genes allow an efficient direct reprogramming, the factthat c-Myc is oncogenic has made us consider the possibility of also employing a direct reprogramming protocol usinga plasmid that only expresses the oct4, sox2 and klf4genes. The reason for constructing a plasmid vectorinstead of a retrovector is that the former delivers the pluripotency genes ina non-integrative fashion. Since plasmids are less efficient than viral vectorsfor gene delivery, we are using magnetofection, a magnetic nanoparticle-based technology to optimize transfectionof the Yamanaka genes. Fluorescently-labeled human BM-MSCs wereintracerebroventricularly (bilaterally) injected to 27-month-old female rats.Experimental subjects were divided into 3 groups (N=8):Young-intact, Senile-intact and Senile-MSC. We used theBarnes maze test to assess hippocampus-dependent learning and spatial memorybefore and after cell injection. Also, we assessed recognition memory with theNovel Object Recognition test. Using an unbiased stereological approach, weassessed immature cell number (DCX cells) in the hippocampal dentate gyrus andastrocyte number (GFAP cells) in the stratum radiatum. Additionally, weconducted timecourse studies to determine MSC integration and viability in thebrain at different post-injection times.The results suggest that human BM-MSC therapyimproves spatial memory (increases goal hole exploration activity) inMSC-treated senile rats as compared with intact senile counterparts althoughspatial performance remains significantly lower than that of the young rats.Neurogenesis (immature neuron number) in thehippocampal dentate gyrus was also improved by the treatment. Histologicalanalysis revealed that part of the MSCs integrated into ependymal cell layerand to a lesser extent in the brain parenchyma. In parallel, we checked thestructure of our STEMCCA plasmid by sequencing and restriction analysis. Alsowe have characterized the regulatability of our vector in cell culture byDoxycycline.We conclude that human BM-MSC therapy partiallyreverses the decline in cognitive performance that occurs in aging rats andimproves a number of hippocampal morphologic parameters.We also conclude that adult stem cells are asuitable biological tool for the treatment of age-related cognitive decline.* Kim J,Efe JA, Zhu S, Talantova M, Yuan X, Wang S, Lipton SA, Zhang K, Ding S. Directreprogrammingof mouse fibroblasts to neural progenitors. Proc natl Acad Sci USA 2011;108:7838-7843.