Directed differentiation of induced pluripotent stem cells to basal forebrain cholinergic neurons
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1
University of Lübeck, Institute of Neurogenetics, Germany
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2
University of Lübeck, Department of Neurology, Germany
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3
Hannover Medical School, Department of Neurology, Germany
Basal forebrain cholinergic neurons (BFCN) arise from the medial ganglionic eminence (MGE) and migrate into different regions of the forebrain. Their dysfunction is believed to play a major role in the development of dyskinesias and dystonias. Differentiating this cell type from induced pluripotent stem cells (iPSC) might help providing in vitro models for a wide variety of diseases. Previous protocols rely on embryoid body formation, we therefore sought for better culture handling and reproducibility by establishing a protocol based on adherent conditions.
IPSC reprogrammed retrovirally from human adult fibroblasts were plated down and deprived from serum and fibroblast growth factor. Neural induction started by addition of the small molecules SB 431542 and LDN 193189. For rostro-ventral patterning the small molecules purmorphamine and XAV 939 were added in a timed manner for 20 days. RNA was isolated at day 20 for qPCR analysis. Cells were plated on poly-d-lysine/laminine coated coverslips and differentiated for 30 to 60 days by addition of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), insuline-like growth factor 1 (IGF-1), dbcAMP, and the notch-inhibitory small molecule DAPT. Thereafter cells either underwent electrophysiological analysis by whole-cell recording or were fixed and stained for immunofluorescence.
After 20 days of patterning cells demonstrated a robust up-regulation of markers associated with the MGE and the cholinergic lineage (NKX2.1, LHX8). After differentiation a considerable amount of mature neurons with a large soma size (30 - 40 µm) expressed markers of BFCN as choline acetyltransferase (CHAT), vesicular acetylcholine transporter (VACHT), ISL-1, P75 and NKX2.1. Differentiated cells showed neuronal electrophysiological properties as fast inward sodium and slow outward potassium currents and spontaneous generation of action potentials. Some cells even showed an after-hyperpolarization “sag”, typically exhibited by BFCN.
It can be concluded that the differentiation protocol possesses the ability of deriving neurons exhibiting features of BFCN on a morphological, expression and electrophysiological level.
Keywords:
cholinergic interneurons,
iPS cells,
Directed differentiation,
medial ganglionic eminence,
Electrophysiology
Conference:
5th Biennial Workshop on Dystonia: “Controversies in Dystonia and Parkinsonism” | Nobile Collegio Chimico Farmaceutico, Rome, May 29-30, 2015, Rome, Italy, 29 May - 30 May, 2015.
Presentation Type:
Poster presentation
Topic:
Dystonia
Citation:
Capetian
P,
Krajka
V,
Pauly
MG,
Stengel
F,
Meier
B,
Stanslowsky
N,
Naujock
M,
Seibler
P,
Wegner
F and
Klein
C
(2015). Directed differentiation of induced pluripotent stem cells to basal forebrain cholinergic neurons.
Front. Neurol.
Conference Abstract:
5th Biennial Workshop on Dystonia: “Controversies in Dystonia and Parkinsonism” | Nobile Collegio Chimico Farmaceutico, Rome, May 29-30, 2015.
doi: 10.3389/conf.fneur.2015.57.00005
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Received:
28 Apr 2015;
Published Online:
29 Apr 2015.
*
Correspondence:
Dr. Philipp Capetian, University of Lübeck, Institute of Neurogenetics, Lübeck, 23562, Germany, Capetian_P@ukw.de