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©2019 BY NEUCYTE, INC.

SynFire® iNs are generated using a patented procedure for direct reprogramming and exhibit the main characteristics of human primary neurons, such as expression of typical pan-neuronal markers and complex electrophysiology, including spontaneous and evoked action potentials and synchronized network activity. Neuronal subtype identities have been confirmed by staining and patch clamping.

Pure populations of human neural cell types NeuCyte uses in our culture include:

  • Glutamatergic excitatory neurons

  • GABAergic inhibitory neurons

  • Astroglia

The advantages of the SynFire iNs include:

  • Real human biology: these cells more closely resemble real human biology, resulting in better ability to predict responses to compounds

  • Rapid maturation: produced through a direct reprogramming approach leading to rapid and homogeneous maturation, SynFire iNs exhibit mature synaptic network activity, such as synchronous bursting phenotypes resembling those in rodent primary cultures appearing within three to four weeks

  • Reliable, robust and ready-to-use: this reprogramming approach also results in lot-to-lot consistency, providing reproducible results

  • Flexible modular system: the user can control subtype to subtype relative seeding density and ratio to fit individual projects, allowing for tracking,analyzing and manipulating specific cell types

View the following characterization data of the SynFire iNs:

Expression of pan-neuronal and subtype specific markers, rapidly mature to form complex networks and cellular morphologies 
 
Pan-Neuron and Subtype Specific Markers
E1
E2
TUJ-1 / GABA / Hoechst
TUJ-1 / GFAP / Hoechst
Elaborate Networks
tdTomato TUJ-1 DAPI
TUJ-1 / DAPI
Synapsin 1 / MAP2
A
B
D
C
Pan-Neuron and Subtype Specific Markers
E1
E2
TUJ-1 / GABA / Hoechst
TUJ-1 / GFAP / Hoechst
Elaborate Networks
F1
TUJ-1 / DAPI
F2
Synapsin 1 / MAP2
G
tdTomato TUJ-1 DAPI

SynFire iNs exhibit mature neuronal characteristics through immuno-staining. The modular aspect of SynFire neural cells allow for defined co-culture conditions and specific ratios of mixed neuronal subtypes, including inhibitory GABAergic neurons. SynFire iNs show expression of neuronal markers within 3 days, formation of spontaneous action potentials within 6-8 days and synchronized neuronal network activity within 21-28 days.

(A) Pan-neuronal marker Map2 / Astroglia marker GFAP / Nuclear staining Dapi. (B) Pan-neuronal marker Map2 / Synaptic marker Synapsin1 / Nuclear staining Dapi. (C) Pan-neuronal marker β3-Tubb (Tuj1) / Inhibitory neuron GABA-A receptor, α1 / Nuclear staining Dapi. (D) Pan-neuronal marker Map2 / Vesicular GABA transporter VGat/ Nuclear staining Dapi.

(E-G) NeuCyte’s iNs Mature Very Fast to Form Complex Network, including mature synapses (F) and spine-like structures (G). (E1) Pan-neuronal marker Tuj1 / Astroglia marker GFAP / Nuclear staining Hoeschst. (E2) Pan-neuronal marker β3-Tubb (Tuj1) / Inhibitory neuron GABA-A receptor, α1 / Nuclear staining Hoeschst. 3-4 week old co-cultures exhibit complex neuronal networks, morphologies and show mature synaptic markers. (F1) Pan-neuronal marker Tuj1 / Nuclear staining Dapi. (F2) Pan-neuronal marker Map2 / Synaptic marker Synapsin1 / Nuclear staining Dapi. (G) Zoom in of spine-like formations on tdTomato labeled glutamatergic excitatory neuron.

 
SynFire iNs demonstrate principal neurophysiological properties

Patch-clamp studies show mature intrinsic and extrinsic properties in SynFire neural cultures including (A) voltage-dependent K+ and Na+ currents (i, step depolarization from -90 mV to +50 mV; ii, I/V curves from different iPSC lines) and (B) action potential firing (evoked).

SynFire iNs show (C) bursting of single neurons and (D) large postsynaptic currents indicating advance synaptic competence. Pure SynFire® subtype cultures (E) of either (i) only excitatory iNs or (ii) only inhibitory iNs exclusively show glutamate-mediated excitatory postsynaptic currents (EPSCs) or GABA-mediated inhibitory postsynaptic currents (IPSCs), respectively.

SynFire iNs are suited for studying short and long term plasticity and show (F) fast synaptic depression upon 10 Hz stimulus as well as (E) robust NMDA currents starting at 5 weeks in culture. In part adopted and modified from (Zhang et al., 2013) and (Yang et al., 2017)). SynFire® iNs are suited for studying short term plasticity and show (F) fast synaptic depression upon 10 Hz stimulus. In mature SynFire® neural cultures, starting at 5 weeks post plating, robust NMDA currents can be measured and (H) isolated by blocking GABA- and glutamate-mediated postsynaptic currents (PTX and CNQX). Moreover, extra-synaptic NMDA currents can be specifically analyzed by (G) co-application of activating glutamate and glycine in the presence of the NMDA inhibitor D-AP5. SynFire® neural cultures rapidly mature within 5 weeks (I) reaching a resting membrane potential <-60 mV and showing stable excitability (action potential threshold and overshoot).

 
Rapid maturation observed through ontogeny of neural network activitiy

These co-cultures contain 70% Glutamatergic, 30% GABAergic neurons and human astrocytes. (A) Representative raster plots from MEA recordings at weeks 1-4. Axion 48 well MEA plates were used to assess activity. (B) Graphs quantifying multiple MEA parameters. i. number of active electrodes; ii mean firing rate; iii. ISI coefficient of variation.

 
SynFire co-cultures demonstrate predictive responsiveness to GABA and AMPA modulators

Neuronal firing and network activity were assessed in SynFire co-cultures after dosing with the GABA-A blockers Bicuculline (BIC 3 uM) and Picrotoxin (PTX 10 uM) or the AMPA blocker CNQX (30 uM).  Changes in weighted mean firing rate (wFMR), burst frequency, network burst frequency and synchrony index were measured using Axions’ MEA plates. GABA blockers have an organizing effect on the network firing.  Meanwhile, AMPA blockers cause a break-down in synchronous firing.

 
Independent comparison of SynFire neural cells to other iPSC derived neurons demonstrates superior functionality

A

(A) Raster plots show neural network activity of induced neural co-cultures and other commercially available neurons. Data was recorded on Axion MEA plates. Recording: Maestro/Axis, burst/network burst detector ON, detault setting for spontaneous firing; rat neurons on 96 wells; others on 48 wells.

(B) Plot shows the mean firing rate (MFR) of SynFireinduced neural co-cultures and other commercially available neurons. MFR was assessed using Axion MEA plates. Axion Maestro Axis software Default setting for spontaneous neuron firing was used.

B

NeuCyte’s platform is ideal for a wide range of applications

  • Drug discovery and pre-clinical testing

    • Custom in vitro neural disease modeling

    • Development of neural cell based assays

    • Phenotypic and targeted drug screening

    • Neural subtype specific biochemistry

    • Target identification and validation in biologically relevant tissues

  • CNS safety/ Neurotoxicity

    • Cell death, apoptosis, autophagy and mitochondrial activity assays

    • Cell stress tests

    • Neural network physiology assessment (MEA)

    • Compound seizurogenic potential testing

    • Neurite outgrowth and morphology evaluations

    • Mechanism of action prediction by gene expression profiling

View the following application data of the SynFire iN co-cultures:
 
Neurite outgrowth assay using SynFire iN co-cultures

(A) SynFire neural cultures were treated with the actin filament disruptive toxin Latrunculin A (100 nM).  Neurite length was assessed and quantified over a period of 44 hrs using a live imaging Incucyte system. (B) Representative images of the neurite traces from both excitatory and inhibitory neurons.  

 
Seizure liability assay with compounds from the HESI NeuTox MEA seizure prediction initiative using SynFire iN co-cultures

For each MEA parameter, measurements from vehicle- or compound-treated wells were normalized to their respective baseline values. All parameters are expressed as percent change.  Significance for Bicuculline (positive control) relative to DMSO (Vehicle) was determined via Student's T-test (n = 4, p<0.05). Significance for test compounds relative to DMSO (Vehicle) was determined via One-Way ANOVA (n = 4, p<0.05). Pilocarpine and acetaminophen (negative control) are shown here.

 
SynFire neural cultures serve to test anti-epileptic drugs (AED) efficacy

NeuCyte’s iNs/MEA platform measures quantifiable effects of drugs on neuronal activity. Chemical induced seizure-like activity can be reversed in a dose dependent manner by several AEDs. Assays performed with mixed excitatory/inhibitory iN co-cultures.