ACCELERATED MATURATION OF HUMAN IPSC-DERIVED CEREBRAL CORTICAL AND PERIPHERAL SENSORY NEURONS

Broadbent, S1., Gillotin, S1., Rock, D1., Prime, S1.1Axol Bioscience Ltd., Cambridge, UK, support@axolbio.com;

METHODSCulture of the hiPSC-derived sensory neuron progenitorsAxol’s Human iPSC-derived sensory neuron progenitors (ax0055) were thawed, plated and cultured at 37oC in a 5%CO2/95% air atmosphere. Briefly, sensory neuron progenitor were plated in NeuralPlating-XF media (ax0033). On Days In Vitro (DIV) 1 the media was changed to Sensory Neuron Maintenance Media (ax0060) and BDNF (10ng/ml), GDNF (25ng/ml), NGF (25ng/ml) and NT-3(10ng/ml) (Complete SNMM) . On DIV3 the cells were treated with Mitomycin C for two hours. Following Mitomyicn C treatment the cells were placed in Complete SNMM; with or without Axol’snewly-developed media supplement, Sensory Neuron Maximizer (ax0058) to mimic the in vivo environment during development. Half media changes were performed every three days and the cellswere cultured up to DIV32.Culture of the hiPSC-derived cortical neuronsAxol’s Human iPSC-derived neuronal stem cells (NSCs, ax0016) were thawed, plated and differentiated into cerebral cortical neurons at 37oC in a 5%CO2/95% air atmosphere. Briefly NSCs wereplated in Neural Maintenance Media (NMM, ax0031) and ROCKi (10mM). On DIV2 the media was changed to NMM. From DIV3 half media changes into Neural Differentiation-XF-based media(ax0034) according to an Axol in-house defined process (Diff Media I, recipe available on request) were carried out every two days till DIV9. From DIV9 half-media changes into an NMM-based media(Diff Media II) were carried out until DIV23. From DIV23 half-media changes into different electrophysiological recording solutions were carried out every two days. The different solutions wereNeurobasal (Life Technologies Corp., UK), BrainPhys (Stemcell Technologies Inc., Canada), Axol’s NMM (ax0031) or NMM-XF (ax0032). Cells were cultured up to DIV64. Cortical neuron maturation wasaccelerated through changing media composition, co-culture and the transfection of pro-neural factors.Co-culture of hiPSC-derived cortical neurons with hiPSC-derived astrocytes and cortical interneuronsThe NSCs were also co-cultured with either Axol’s hiPSC-derived astrocytes (ax0665), hiPSC-derived cortical interneuron progenitor (ax0667) or both. When the NSCs were plated; astrocytes andinterneurons were also plated at an NSC:cell ratio of 9:1 (8:1:1 for the three cell-type co-cultures). Cortical neuron and astrocyte co-cultures were differentiated and cultured in the same medias asthe cortical neuron mono-culture. Co-cultures containing interneurons additionally had Glutamax (1x), BDNF (10ng/ml), GDNF (10ng/ml), ascorbic acid (200mM) and dibutyryl-cAMP (200mM) in theirDiff II media.TransfectionNSCs were plated at 300,000 cells/well and transfected on DIV3 with the pro-neural factors: Neurog2, Neurod1 and Neurod4 (8.33mg/well each) using the jetMESSENGER system (PolyplusTransfection, France). Cells were either fixed for ICC or harvested for PCR on DIV6 or DIV9.Immunofluorescent ImagingImmunostains to TUJ1, Nav1.7, Nav1.8 and VR1 along with DAPI counterstains (sensory neurons) or MAP2 with DAPI counterstains (cerebral cortical neurons) were applied to the fixed culturedneurons at different DIVs. Immunofluorescent imaging using an EVOS FL Auto (Life Technologies Corp., UK) was used to obtain images of the neurons to assess maturity and receptor expression.PCRAt different timepoints cortical neurons were harvested for PCR quantification. Briefly, cells were lyzed and their RNA extracted, isolated and purified using the RNeasy Minikit (Qiagen, Germany).The RNA was quantified using the Qubit RNA Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies Corp., UK). DNA was created and amplified by RT-PCR using a PTC-200 Gradient Cycler (Bio-RadLaboratories, US) and then quantified by qPCR using a QuantStudio3 and using the QuantStudio Design and Analysis Software v.1.4.2 (Life Technologies Corp., UK). PCR was used to investigate levelsof TUJ1, MAP2, TBR1, CTIP2, PAX6, PSD95, vGLUT1, FEZF2 and SATB2 in the cortical neurons.Multi-Electrode Arrays (MEA)Extracellular field potentials were acquired at 37oC using a high-throughput MEA system, here we simultaneously recorded extracellular potentials from 16 electrodes per well across 24-wells plates(MED64 Presto, Alpha MED Scientific Inc., Japan) at a sampling rate of 20kHz/channel and stored on a personal computer for later off-line analysis.MEA Electrophysiological AnalysisRaw data was filtered (Butterworth Low Pass = 3000Hz, Butterworth High Pass = 100 Hz) and acquired using MEA Symphony (Alpha MED Scientific Inc.). Trace plotting, spike extraction, waveformcreation and waveform characterisation were produced in Clampfit (Molecular Devices, LLC, US). Spikes were automatically extracted using a negative detection threshold of -9mV compared to amaximum baseline noise of ±6mV. Waveforms were created by Clampfit from 5ms pre-trigger to 8ms post-trigger and were averaged from at least 60s of recording. Average waveform characteristicsand their SEMs were calculated in Excel (Microsoft Corp., US) from the individual values calculated by Clampfit. Graphs were created by GraphPad Prism (GraphPad Software Inc., US).MEA Drug ApplicationDrugs were made-up in PBS and directly applied to the wells using a pipette while recording was paused. Sterility was maintained by applying the drug inside a horizontal laminar flow hood.MicroscopyMorphology was assessed using phase contrast microscopy on a Nikon Diaphot (Nikon Corp., Japan) using digiCamControl software (digicamcontrol.com).Calcium ImagingThe sensory neurons functionality was also assessed using calcium imaging. Sensory neurons were incubated in a cell-loading solution of 3mM Fluo4 AM in 0.02% Pluronic F-127 for 1 hour. The cellswere then washed in fresh media and stimulated by rising concentrations of capsaicin. Activity was captured by EVOS FL Auto (Life Technologies Corp., UK), recorded on ICY Bioimaging Software(France-Bioimaging, France) and analysed using FluoroSNNAP (University of Pennsylvania, US).

INTRODUCTIONThe development of neurons from IPSCs provides research models for academia and industry which offer the potential of being more representative of the human nervous system than existinganimal and cell-line based models. The potential of IPSC-derived cells is currently undermined by two major drawbacks; the length of time required to differentiate and culture them and an oftenimmature phenotype. The ability to produce more phenotypically relevant cells in a shorter time-frame would be a major boon for and would allow IPSC-derived cells to truly achieve their potentialas a game-changing research platform. To this end a number of approaches have been explored to produce more rapid maturation of IPSC-derived cells, particularly cardiomyocytes and neurons,including electrical and mechanical stimulation, changes in culture media composition, transfection, 3D-culture, scaffolding and co-culture with other cell-types.Here we present morphological, molecular and electrophysiological data obtained from Axol’s Human IPSC-derived cortical and sensory neurons showing how we have used a number of theseapproaches to produce a more rapid maturation and more physiological-relevant phenotypes than produced via conventional cell-culturing protocols. The maturation and functionality of the neuronswas assessed from their morphology, PCR, immunocytochemistry (ICC) and electrophysiology using calcium imaging and multielectrode (MEA) arrays.With the methods described here Axol Bioscience demonstrate the ability to produce more functionally relevant cortical and sensory neurons in a shorter time-frame than currently typical. Thesefindings should be translatable to other cell-types and this will greatly improve the utility of iPSC-derived cells to research and industry.

CONCLUSIONThe molecular, pharmacological and electrophysiological data here demonstrate that the functional maturity of hiPSC-derived sensoryand cortical neurons can be significantly hastened through a number of methods, including the use of media supplements, the design ofmedia composition, transfection and the use of co-culture. This addresses one of the major barriers to the wider use of hiPSC-derivedneuronal cells in research and industry and has wider applications for a range of iPSC-derived cell-types.

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Fig 6: Electrophysiology of Axol’s Cerebral Cortical Neurons when co-cultured with Axol’sAstrocytes and Interneurons. Co-culture of cortical neurons with interneurons and astrocytes produced more mature firing characteristics than a monoculture of cortical neurons alone. All data from DIV30, 10 minute traces A. Representative MEA trace and extracted average waveform for cortical neuron monoculture. Firing is unsynchronised without burst firing. B. Representative MEA trace and extracted average waveform for cortical neuron co-cultured with interneurons. Firing is highly synchronised with a reduced frequency but increased burst firing. The waveform is unchanged. C. Representative MEA trace and extracted average waveform for cortical neuron co-cultured with interneurons and astrocytes. Firing is highly synchronised with an increased frequency and amplitude and increased burst firing. The waveform shape is unchanged but the amplitude is increased.

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Fig 1: Changes in Morphology and Immunocytochemistry of Axol’s Sensory Neurons when cultured with Sensory Neuron Maturation Maximizer Supplement When cultured with Sensory Neuron Maturation Maximizer Supplement, sensory neurons have improved neuriteoutgrowth and express functional markers of mature sensory neurons. A. Phase contrast images of sensory neuronscultured in either Complete SNMM or Complete SNMM supplemented with Sensory Maturation Maximizer(ax0058, 1x) on DIV28. With the supplement there are more neurites present, they are thicker and more evenlyspread. The cell bodies are also less clustered. Morphological changes were apparent in cells treated with theMaximizer by DIV8 ± 1.5 (SEM, n=4) B. Immunocytochemistry data showed the expression of markers of maturefunctional sensory neurons in Maximizer-treated neurons. TUJ1 antibody (red) reacts with neuron-specific class IIIbeta-tubulin, a component of tubulin a major component of microtubules within the cytoskeleton of neuronal cell-bodies and axons. Nav1.7 antibody (green) and Nav1.8 antibody (red) reacts to the Nav1.7 and Nav1.8 sodiumchannels, respectively, required for nociception in sensory neurons. VR1 antibody (green) reacts to the VanilloidReceptor 1 a receptor in sensory neurons which reacts to heat and capsaicin. DAPI counterstain (blue). Thiscompares to 6+ weeks when using conventional tissue culture, with a minimum 30% increase in numbers ofneuronal bodies expressing the above biomarkers by DIV28.

1. ACCELERATED MATURATION OF HIPSC-DERIVED SENSORY NEURONS

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Fig 2: Calcium Imaging of the response to 1mM Capsaicin of Sensory Neurons when cultured with Maximizer compared to control cells.When cultured with Sensory Neuron Maturation Maximizer Supplement, sensory neurons respond to lowconcentrations of Capsaicin indicating the presence of functional TRPV1 channels. A. Average deltaF/Fo values forthe control and Maximizer-cultured sensory neurons treated with vehicle control and 1mM capsaicin on DIV20. B &C. Pie Chart and Table showing capsaicin threshold concentrations for sensory neurons treated with Maximizersupplement compared to sensory neurons normally cultured in Complete SNMM. 91% of Regions-Of-Interest (ROIs)show a response to the lowest concentration of capsaicin tested (1mM) compared to only 19% of control cells andwhile 99% of the ROIs of treated sensory neurons respond to the highest concentration of capsaicin (100mM), 57%of non-treated sensory neurons remain non-responsive. This shows that even by DIV20 the Maximizer-treatedsensory neurons show enhanced capsaicin responses compared to non-treated.

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ACKNOWLEDGEMENTSWe thank our collaborators at AlphaMED for the use of the Presto and all their technical assistance. This research was partfunded by the MESO-BRAIN Project, an EU Horizon 2020 Research and Innovation Programme under grant agreement No. 713140.

Fig 4: Changes in Morphology, Immunocytochemistry and RNA expression of Axol’s CerebralCortical Neurons when transfected with three pro-neural factors.Co-transfection of Axol’s iPSC-derived neuronal stem cells with the three pro-neural factors, Neurog2, Neurod1 and Neurod4 rapidlyproduced clear differences in the morphology, immunocytochemistry and RNA expression levels of the resulting cortical neurons. A. Phasecontrast (top) and immunocytochemistry (bottom) taken on DIV15 of cortical neurons which are either Untransfected (left) or Transfectedwith the three pro-neural factors (right). MAP2 antibody (green) reacts with Microtubule Associated Protein 2, a neuron-specific proteinthat promotes assembly and stability of the microtubule network. DAPI counterstain (blue). Transfection has produced increased neuriteoutgrowth and increased expression levels of MAP2. B & C. PCR confirmed an increase in expression levels of several key cortical markersby DIV3 (B. Increases in TUJ1, MAP2, TBR1 & PAX6, n=3) and by DIV6 (C. Increases in PSD95, vGLUT1 and FEZF2) in transfected cells vsuntransfected. SATB2 levels were unchanged or reduced suggesting they have a Layer 5/6 phenotype. Accelerated functional maturity wasconfirmed by MEA recordings with the earlier appearance of spontaneous spike firing (DIV16 vs DIV34; 178.3 ± 6.3 vs 57.6 ± 7.5spikes/min), burst firing (DIV16 vs DIV34; 8.1 ± 0.9 vs 0.90 ± 0.48 bursts/min) and synchronised burst firing (DIV35 vs DIV64+), compared tocontrol conditions (data not shown)

2. ACCELERATED MATURATION OF HIPSC-DERIVED CORTICAL NEURONS

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Fig 5: Electrophysiology of Axol’s CorticalNeurons when cultured in different neuronalmaintenance medias.Untransfected iPSC-derived neuronal stem cells were differentiated intocortical neurons in monoculture under identical conditions. On DIV23the cells were changed into one of four different maintenance medias;Neurobasal, BrainPhys, Axol’s NMM (ax0031) or NMM-XF (ax0032) andcultured till DIV47. Cells maintained in Neurobasal or NMM failed todemonstrate any significant spontaneous activity. BrainPhys-maintainedcells did eventually produce significant spontaneous activity (1546 ± 48spikes/well, n=10) but only from DIV33. NMM-XF produced the earliestand most sustained levels of spontaneous activity displaying 1350 ± 50spikes/well (n=10) from DIV26 increasing to 7544 ± 285 spikes/well(n=10) by DIV46.

Fig 3: Electrophysiology of Axol’s Sensory Neurons when cultured with Sensory NeuronMaturation Maximizer SupplementRepresentative MEA traces of sensory neurons maintained in Complete SNMM (left) or Complete SNMM with ax0058 (right) All traces are60s and compare the same electrodes in each figure. A. Spontaneous activity. Treated cells showed significant spontaneous activity byDIV21 (top) compared to DIV28 (bottom) B. Response to capsaicin on DIV22. Rising concentrations of capsaicin produced significantlyincreased activity in treated cells with minimal effect on untreated. This indicates the presence of functional TRPV1 channels in the treatedsensory neurons C. Response to menthol on DIV27. Rising concentrations of menthol produced significantly increased activity in treatedcells with minimal effect on untreated. This indicates the presence of functional TRPM8 channels in the treated sensory neurons

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