3D engineered neural tissue from human induced

15th BIANNUAL CONFERENCE OF THE HUNGARIAN NEUROSCIENCE SOCIETY
3D engineered neural tissue from human induced pluripotent
stem cells
Tamás Bellák1, Abinaya Chandrasekaran1,2, Anna Ochalek1,2, Viktor Szegedi1, Eszter Varga1, Csilla Nemes1,
Shuling Zhou1,3, Hasan Avci1, Julianna Kobolák1 and András Dinnyés1,2,4
1BioTalentum
Ltd., Gödöllő, Hungary
2Molecular Animal Biotechnology Laboratory, Szent István University, Gödöllő, Hungary
3Institute for Veterinary Science, University of Copenhagen, Denmark
4Departments of Equine Sciences and Farm Animal Health, Faculty of Veterinary Medicine,
Utrecht University, The Netherlands
Three-dimensional cell cultures have shown more physiological relevance (improved cell
survival, enhanced neuronal differentiation, better cell–cell and cell–matrix interaction)
compared to traditional two-dimensional systems.
Differentiation of patient-derived pluripotent stem cells into a three-dimensional (3D)
engineered neural tissue (ENT) provides advantages to study the pathophysiology of
neurodegenerative disorders such as Alzheimer’s disease, frontotemporal dementia or
spinocerebellar ataxia. 3D hENTs also allow preclinical analyses of selected drug candidates
and could be an efficient tool for toxicity screenings.
In this study fibroblasts and mononuclear blood cells were isolated from genetically and
clinically well-characterized patients, reprogrammed into induced pluripotent stem cell
state (iPSCs), differentiated into neurons and generated a compact 3D neural tissue using
an air-liquid interface-based system (Krause et al., 2009).
NMDA
MK801+NMDA
MK801+NMDA
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Change of fluorescence (%)
Introduction
For intracellular [Ca2+] measurements, the Fluo-4 NW Calcium Assay Kit was used according
to the manufacturer’s instructions. The kinetics of the change in the fluorescence intensity
was measured in a 24-well plate using a fluorescence plate reader. The 3D hENTs were
treated with NMDA using the built-in liquid micropipettor of the plate-reader, at an injection
speed of 100 µl/s.
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Figure 3. Representative curves showing the
changes of intracellular Ca2+ level after NMDA
ejection. Blocking NMDA receptors prevents the
rise of fluorescence which indicates the
specificity of the signal.
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Material and methods
Generation of 3D hENTs Fibroblasts and mononuclear blood cells from well-characterized patients were isolated
and reprogrammed into iPSC state using a Sendai virus reprogramming method. The cells were maintained in a
highly enriched media under feeder free condition. For efficient neuronal induction, iPSCs were induced with
Noggin and small molecule inhibitors SB431542 for 8 days under non-adherent suspension culture. Small neuronal
rosette enriched cultures were maintained in NMM media supplemented with 10ng/ml of EGF and bFGF for an
extra 5 days. Thereafter, the cells were dissociated and plated as small clumps in Poly-L-Ornithine/Laminin plates
for an additional 5 days. Upon efficient neural stem cells derivation, the cells structures were dissociated and
plated on a hydrophilic polytetrafluoroethylene (PTFE) membrane (6 mm diameter, 0.4 μm pore size; BioCellInterface). PTFE membrane was deposited on a Millicell-CM (0.4 μm) culture plate insert (Millipore). Media was
supplied below the inserts and changed every 2 days.
Electrophysiological recordings and immunocytochemistry After 6 weeks of air-liquid interface based
differentiation the engineered nervous tissue was characterized with electrophysiology (MEA: multi-electrode
array, Multi Channel System) and calcium imaging technique (from Invitrogen). Additional samples were fixed with
4% PFA for 30 min, cryosectioned (10-μm thickness, parallel sections) and analysed with immunocytochemical
method using different neuronal and glial markers. Photos were taken with a Zeiss Axio Imager Z1 fluorescence
microscope using Apotome mode and 40x magnification.
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Time (sec)
Results – Neuronal and glial differentiation
Results - MEA recordings and calcium imaging
Electric activity was recorded by a commercially available MEA setup. Briefly, the 3D hENTs
were placed on a chip containing 60 electrodes. Spontaneous spikes were recorded with 25
kHz, and evoked fEPSPs were recorded at 15 kHz sampling frequency.
Figure 1. Representative train of action potentials recorded from an 3D hENT (left). Right panel shows the
superimposed spikes.
TTX
fEPSP
Figure 2. Representative fEPSP recorded from a 3D
hENT. Black curve shows the evoked response after
TTX treatment.
Figure 4. 6-week old 3D hENTs had 80-100 μm thickness, a homogenous aspect and contained high
number of nestin positive precursors. High majority of the cells differentiated to neurons confirmed by βIIItubulin and NF200kD (specific for mature axons) positivity, but strong GFAP (glial fibrillaric acidic protein)
immunreactive zones were found close to the PTFE membrane (arrows). The presynaptic marker
synaptophysin was denser in the middle of the culture and closer to the air-culture interface (asterisk),
however, OSP (oligodendrocyte specific protein) immunreactive cells dispersed in the whole culture.
Dotted lines indicate the borders of PTFE membrane. Transversal sections. Scale bar: 50 μm.
Conclusions - Future directions
Compact 3D neural structure was successfully generated from human iPSC derived-NPCs
using an air-liquid interface-based method.
3D hENTs had spontaneous firing activity and the evidence of functional synapses.
Immunocytochemistry analyses confirmed these results with the presence of various
markers of maturity such as βIII-tubulin, NF200kD, Synaptophysin, GFAP and OSP.
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Acknowledgements
This work was supported by grants from EU FP7 projects (STEMMAD, PIAPP-GA-2012-324451; EpiHealth,
HEALTH-2012-F2-278418;
EpiHealthNet,
PITN-GA-2012-317146;
D-BOARD,
FP7-HEALTH-2012INNOVATION-1-305815).
We expect that patient specific iPSC-derived 3D ENTs will express the molecular
characteristics of a given disorder on a more complex way, therefore providing an effective
tool for studying the disease pathogenesis.
Our overall aim to develop patient-specific in vitro systems that will allow us customized
treatment for complex neurodegenerative disorders such as Alzheimer’s disease,
frontotemporal dementia (FTD) or spinocerebellar ataxia (SCA).