Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts.
Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at all time points.
Institute of Neurogenetics, University of LübeckLübeck, Germany; Department of Neurology, University of LübeckLübeck, Germany.
Philipp
P
Capetian
Department of Neurosurgery, University of Kiel Kiel, Germany.
Luis
L
Azmitia
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Martje G
MG
Pauly
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Victor
V
Krajka
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Felix
F
Stengel
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Eva-Maria
EM
Bernhardi
Laboratory of Stereotaxy and Interventional Neuroscience, Department of Stereotactic and Functional Neuroscience, University Medical Center Freiburg Freiburg im Breisgau, Germany.
Mariana
M
Klett
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Britta
B
Meier
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Philip
P
Seibler
Department of Neurology, Hannover Medical School Hanover, Germany.
Nancy
N
Stanslowsky
Department of Neurology, University of Lübeck Lübeck, Germany.
Andreas
A
Moser
Institute of Physiology, University of Kiel Kiel, Germany.
Andreas
A
Knopp
Institute of Human Genetics, University of Lübeck Lübeck, Germany.
Gabriele
G
Gillessen-Kaesbach
Department of Neurosurgery, University of Erlangen-Nuremberg Erlangen, Germany.
Guido
G
Nikkhah
Department of Neurology, Hannover Medical School Hanover, Germany.
Florian
F
Wegner
Laboratory of Stereotaxy and Interventional Neuroscience, Department of Stereotactic and Functional Neuroscience, University Medical Center Freiburg Freiburg im Breisgau, Germany.
Máté
M
Döbrössy
Institute of Neurogenetics, University of Lübeck Lübeck, Germany.
Christine
C
Klein
adult human fibroblasts; direct reprogramming; episomal vectors; induced neural stem cells; lineage conversion; plasmid based reprogramming