TY  - JOUR
A1  - Schenke, Maren
A1  - Schjeide, Brit-Maren
A1  - Püschel, Gerhard Paul
A1  - Seeger, Bettina
T1  - Analysis of motor neurons differentiated from human induced pluripotent stem cells for the use in cell-based Botulinum neurotoxin activity assays
JF  - Toxins
N2  - Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.
KW  - Botulinum neurotoxin
KW  - motor neurons
KW  - cell-based in vitro assay
KW  - potency
KW  - assessment
KW  - induced pluripotent stem cells
Y1  - 2020
U6  - https://doi.org/10.3390/toxins12050276
SN  - 2072-6651
VL  - 12
IS  - 5
PB  - MDPI
CY  - Basel
ER  - 
TY  - JOUR
A1  - Nie, Yan
A1  - Wang, Weiwei
A1  - Xu, Xun
A1  - Zou, Jie
A1  - Bhuvanesh, Thanga
A1  - Schulz, Burkhard
A1  - Ma, Nan
A1  - Lendlein, Andreas
T1  - Enhancement of human induced pluripotent stem cells adhesion through multilayer laminin coating
JF  - Clinical hemorheology and microcirculation : blood flow and vessels
N2  - Bioengineered cell substrates are a highly promising tool to govern the differentiation of stem cells in vitro and to modulate the cellular behavior in vivo. While this technology works fine for adult stem cells, the cultivation of human induced pluripotent stem cells (hiPSCs) is challenging as these cells typically show poor attachment on the bioengineered substrates, which among other effects causes substantial cell death. Thus, very limited types of surfaces have been demonstrated suitable for hiPSC cultures. The multilayer coating approach that renders the surface with diverse chemical compositions, architectures, and functions can be used to improve the adhesion of hiPSCs on the bioengineered substrates. We hypothesized that a multilayer formation based on the attraction of molecules with opposite charges could functionalize the polystyrene (PS) substrates to improve the adhesion of hiPSCs. Polymeric substrates were stepwise coated, first with dopamine to form a polydopamine (PDA) layer, second with polylysine and last with Laminin-521. The multilayer formation resulted in the variation of hydrophilicity and chemical functionality of the surfaces. Hydrophilicity was detected using captive bubble method and the amount of primary and secondary amines on the surface was quantified by fluorescent staining. The PDA layer effectively immobilized the upper layers and thereby improved the attachment of hiPSCs. Cell adhesion was enhanced on the surfaces coated with multilayers, as compared to those without PDA and/or polylysine. Moreover, hiPSCs spread well over this multilayer laminin substrate. These cells maintained their proliferation capacity and differentiation potential. The multilayer coating strategy is a promising attempt for engineering polymer-based substrates for the cultivation of hiPSCs and of interest for expanding the application scope of hiPSCs.
KW  - Polymeric substrate
KW  - surface coating
KW  - induced pluripotent stem cells
KW  - cell adhesion
Y1  - 2019
U6  - https://doi.org/10.3233/CH-189318
SN  - 1386-0291
SN  - 1875-8622
VL  - 70
IS  - 4
SP  - 531
EP  - 542
PB  - IOS Press
CY  - Amsterdam
ER  -