Non-viral gene transfer by nucleofection allows stable gene expression in human neural progenitor cells.
Human neural progenitor cells (hNPCs) are a promising source to treat various neurodegenerative diseases. Potential applications are to use such cells for reprogramming to induce pluripotent stem cells or for secretion of proteins into the brain. These applications usually involve expression of heterologously expressed genes which is difficult to achieve in hNPCs. We tested several protocols for non-viral gene transfer and different promoters. Nucleofection and the cytomegalovirus enhancer/chicken beta-actin promoter allowed expression of foreign genes in hNPCs for up to 6 months. Treatment with the antibiotic G418 enabled us to select stably transfected cells which were subcloned and continued to express the NPC marker nestin. Differentiation of stably nucleofected hNPCs revealed that multipotency was maintained following long-term expansion of subcloned hNPCs. After differentiation for 3 weeks in vitro or in vivo following striatal transplantations transfected hNPCs expressed voltage-gated sodium channels suggesting the development of functional properties during neuronal maturation. In conclusion, stably nucleofected hNPCs can be isolated, subcloned, and expanded for up to 6 months without loss of their differentiation potential. These data provide a basis for future studies using hNPCs to investigate the neuronal differentiation in vivo after transplantation, the feasibility as a vector for gene (protein) therapy, and the induction of pluripotent stem cells.
Dieterlen
Maja-Theresa
MT
Department of Neurology, University of Leipzig, Liebigstrasse 22a, 04103 Leipzig, Germany.
Wegner
Florian
F
Schwarz
Sigrid C
SC
Milosevic
Javorina
J
Schneider
Barbara
B
Busch
Maria
M
Römuss
Ute
U
Brandt
Annett
A
Storch
Alexander
A
Schwarz
Johannes
J
D001921:Brain / Q000166:cytology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005333:Fetus; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D033581:Stem Cell Transplantation / Q000379:methods; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D009419:Nerve Tissue Proteins; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor