Nucleofection is the most efficient non-viral transfection method for neuronal stem cells derived from ventral mesencephali with no changes in cell composition or dopaminergic fate

¹ Hannover Medical School, Department of Neuroanatomy (OE 4140) & Center for Systems Neurosciences Hannover (ZSN), Hannover, Germany
² Hannover Medical School, Institute of Biochemistry, Hannover, Germany
³ Hannover Medical School, Department of Trauma Surgery, Hannover, Germany

^* To whom correspondence should be addressed. E-mail: grothe.claudia{at}mh-hannover.de.

	Abstract

Neuronal progenitor cells (NPC) play an important role in potential regenerative therapeutic strategies for neurodegenerative diseases, like Parkinson's disease. However, survival of transplanted cells is yet limited and the identification of grafted cells in situ remains difficult. The use of NPC could be more effective with regard to a better survival and maturation when transfected with (a) neurotrophic factor(s). Therefore, we investigated the possibilities to transfect mesencephalic neuronal progenitors with different constructs carrying neurotrophic factors or the expression reporters EGFP and DsRed. Different techniques for transfection were compared, the highest transfection rate of up to 47% was achieved by nucleofection. Mesencephalic neuronal progenitors survived the transfection procedure, 6 hours after transfection viability was about 40%, and the transfected cells differentiated into e.g. tyrosine hydroxylase positive neurons. Within the group of transfected cells, many progenitors and several neurons were found. In order to provide the progenitor cells with a neurotrophic factor, different isoforms of FGF-2 were introduced. To follow the behaviour of the transfected cells in vitro, functional tests like the cell viability assay (WST-1) and the cell proliferation assay (BrdU-ELISA) were performed. In addition, these transfected NPC were viable after transplantation, expressed tyrosine hydroxylase in vivo and could easily be detected within the host striatum due to their EGFP expression. This study shows that genetic modification of neural progenitors could provide attractive perspectives for new therapeutic concepts in neurodegenerative diseases.

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