Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

The goal of this procedure is to precisely measure the effect that Clostridial neurotoxins have on synaptic transmission in networked cultures of neurons derived from mouse embryonic stem cells. This is accomplished by first adapting embryonic stem cells to a feeder cell-free suspension culture and maintaining adapted cells in a pluripotent state. The second step is to differentiate suspension adapted stem cells into neurons or ESNs, using a variation of the four four differentiation technique.

Next, ESNs are treated with a series of media to promote maturation into synaptically active networked neurons. The final step is to treat ESNs with clostridial, neurotoxins or other neurotoxins and measure the effects on monos synaptic activity using whole cell patch clamp electrophysiology. Ultimately, changes in the spontaneous production of monos synaptic activity are quantified from electrophysiological recordings normalized to age and lot matched control neurons, and compared among different conditions such as doses, times, or drug treatments.

The main advantage of this technique is that ESNs are the first cell-based model to replicate the functional pathologies responsible for the clinical manifestations of botulism and tetanus. We first had the idea for this method when we found that ESNs were highly susceptible to all clostridial neurotoxins based on snare protein cleavage, and also demonstrated spontaneous synaptic activity in emergent network behavior. Demonstrating the procedure will be Ms.Megan Lyman, a technician from my laboratory Begin by transferring feeder cell-free suspension culture adapted.

ESC aggregates to a 15 milliliter conical tube and allow the aggregates to settle into a compact pellet. Once the aggregates have settled, aspirate the SUP natant while being careful not to disrupt the cell pellet. Then add five milliliters of PBS to wash the cells and centrifuge at 100 times G for 2.5 minutes.

Carefully aspirate the PBS and then add 500 microliters of trypsin and invert the tube several times to gently disrupt the cell pellet. Then place the tube in the 37 degree Celsius water bath and incubate for three minutes. After the incubation time has elapsed, return the tube to the tissue culture hood and add 500 microliters of ESC medium to dilute the trypsin.

Then itrate the CEL suspension five to 10 times with the P 1000 pipette to achieve a single cell suspension. Count an eloqua of the cells using a hemo, cytometer and centrifuge the remainder of the cell suspension at 200 times G for three minutes. After aspirating the supinate, we suspend the cells to a final concentration of one times cent, the seven cells per milliliter with ESC medium.

Next transfer, 150 microliters of the suspension to 10 milliliters of ESC medium in a 10 centimeter bacterial dish and incubate at 37 degrees Celsius and 5%carbon dioxide. E ESCs are routinely passaged using this protocol. Every 48 hours differentiation of ESCs into neurons starts during a routine cell passage.

Dissociate the ESCs as before, but include an additional plate for neuronal differentiation. Transfer 350 microliters of the cell suspension into a 10 centimeter low attachment dish containing 25 milliliters of differentiation.Medium. Place the dish on an orbital shaker set to 30 to 45 RPM inside a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide and incubate for 48 hours.

After 48 hours, use a 25 milliliter pipette to transfer the differentiating cell aggregates to a 50 milliliter conical tube. Immediately after add 25 milliliters of fresh differentiation. Medium to the Petri dish.

Allow the aggregates to settle for two to five minutes, producing a visible pellet that is one to two millimeters deep. Carefully aspirate the medium, ignoring any single cells or small aggregate still in suspension, and use a P 1000 pipette to transfer the cell palette back to the Petri dish. Return the dish to the rotary shaker in the incubator.

After a further 48 hours, repeat the media change procedure. This time at 30 milliliters of differentiation medium supplemented with six micromolar all trans retinoic acid. The pellet formed will now be two to four millimeters deep after incubating the cells.

As before, repeat the media change procedure with retinoic acid supplementation for the final time. The pellet will be four to eight millimeters deep at this point the following day. Prepare the plating surfaces as outlined in the written portion of the protocol on the afternoon of the day of plating, or five milliliters each of pre coated MPC trypsin medium and 0.1%Soybean tryin inhibitor at 37 degrees Celsius.

Then use a 25 milliliter pipette to transfer differentiating aggregates from the culture plate to a 50 milliliter conical tube. Allow the aggregates to settle for three to five minutes and then carefully aspirate the medium without disturbing the pellet. Next, wash the pellet with five to 10 milliliters of PBS and after aggregates have settled.

Aspirate the PBS and repeat the wash. After the second PBS wash. Add five milliliters of MPC ization medium to the pellet and incubate it 37 degrees Celsius for five minutes.

Gently flicking the tube two to three times during the incubation. Next, add five milliliters of 0.1%soybean tripsin inhibitor to the cells and quickly mix by inverting. Then gently tritrate the cells 10 to 15 times with a 10 milliliter pipette until a relatively homogenous cell suspension is produced.

Slowly filter the cell suspension through a 40 or 70 micron cell strainer placed on top of a 50 milliliter conical tube. Then once all of the suspension has been filtered at one milliliter of N two, medium to the filter to wash the remaining cells through the strainer. Transfer the cell suspension to a 15 milliliter conical tube and centrifuge for six minutes at 200 times G.After aspirating the medium without disturbing the pellet triturate in two milliliters of N two medium, then add N two medium to a total volume of 10 milliliters.

Centrifuge for five minutes at 200 times. G.Aspirate the media and repeat the pellet washing process again. Resus suspending the pellet in 10 milliliters of N two medium.

Remove an aliquot of the cells and count with a hemo cytometer, and then repeat the centrifugation Reese. Suspend the cells in N two medium to a final concentration of one times 10 to the seven cells per milliliter and plate it a density of 150, 000 to 200, 000 cells per centimeter squared. Indic is prepared at DIV minus one and place in the tissue culture incubator at 37 degrees Celsius.

Maintain ESNs according to the instructions in the written protocol. First, dilute botulinum neurotoxin serotype A to 100 times the desired final concentration in ESN culture, medium and warm to 37 degrees Celsius. Then add an appropriate volume of toxin to DIV 21 plus ESN cultures.

Swell the culture dish and return to the incubator at the desired time. Point aspirate the ESN culture medium and wash twice with extracellular recording buffer or ERB. Then add four milliliters of ERB supplemented with five micromolar tetrodatoxin to block action potentials and 10 micromolar bi coline to antagonize gabaa receptor activity.

Next, transfer the dish to the electrophysiology rig. Neither perfusion nor temperature control is required for the measured inhibition of synaptic transmission or mist assay. After using a micro pipette puller to pull bo silicate pipettes with five to 10 mega ohms of resistance, backfill a pipette with intracellular recording buffer.

Then gently dip the pipette in silicon reagent. Secure the recording pipette onto the electrode holder and attach an air filled syringe to tubing that is ported into the electrode holder. Provide steady positive pressure via the syringe while lowering the recording pipette into the ERB.

After gently landing the recording pipette on the SOR of the neuron to be recorded. See positive pressure by breaking the seal on the syringe. Reconnect the syringe and apply sustained negative pressure through gentle inspiration to form a giga ohm seal.

Once the giga ome seal is formed, decrease the holding voltage to minus 70 millivolts. Then carefully apply short pulses of negative pressure using the syringe to break into whole cell configuration. Monitor capacitance spikes for 30 seconds to confirm that the patch is stable.

Cancel the capacitance spikes in the HEA software and switch to current clamp mode to monitor and record resting membrane potential without adjustment for liquid junction potentials. The resting membrane potential could be between minus 67 and minus 82 millivolts. Adjust the gain to two millivolts per pico amp.

Switch to voltage clamp mode and perform a continuous minus 70 millivolts recording for three to five minutes to detect miniature excitatory, post-synaptic currents. Analyze three to five minutes of recorded data to detect me PSCs using default settings for AMPA receptor EPCs in mini analysis, collect and save information on detected events. After collecting M-E-P-S-C frequencies for eight to 12 controls and eight to 12 botulinum neurotoxin treated samples for each exposure condition.

Analyze frequency against age and lot matched controls, then determine the statistical significance of percent inhibition of synaptic activity using appropriate statistical tests from div seven to div 49, ESNs express neurotropic compartments and form synaptic puncture. Taxo dendritic interfaces. ESNs undergo inhibition of synaptic activity when exposed to botulinum neurotoxin serotypes a TG and tetanus toxin.

These representative traces from ESNs were collected 20 hours after bath edition of botulinum neurotoxin serotypes, A TG tetanus, neurotoxin or vehicle. Each neurotoxin reduced synaptic activity by over 95%in comparison to controls. The following four images show the sensitivity of using mist to measure monos synaptic activity in botulinum neurotoxin serotype, A treated ESNs.

This first image shows representative traces from missed measurements of synaptic activity 20 hours after bath edition of botulinum neurotoxin serotype A.The voltage clamp trace segments exhibited a decrease in M-E-P-S-C frequency following exposure to botulinum neurotoxin serotype A.This image shows quantitation of M-E-P-S-C frequency and confirms a dose dependent decrease in M-E-P-S-C frequency. The median inhibitory concentration was determined with the least squares fit of a non-linear regression using a four parameter variable slope. Note, the limit of detection by mist in SNS is at least 0.005 picaMolar.

This bar graph shows cytometry quantitation of SNAP 25 cleavage measured by western blot. Note the reduced sensitivity of snare protein cleavage as a readout of intoxication. In comparison to mist, a single asterisk indicates a P value of less than 0.05.

A triple asterisk indicates a P value of less than 0.001. These findings demonstrate that networked populations of stem cell derived neurons offer a physiologically relevant cell-based model of clostridial neurotoxin poisoning. The use of SNS is expected to significantly reduce animal distress and death by serving as a suitable replacement for the mouse lethality assay with the added benefit of improved speed specificity and sensitivity.

This technique paves the way for researchers in the field of neurotoxicology to employ moderate throughput screening techniques based on neuronal network activity to facilitate therapeutic screening and toxin detection for for clostridial neurotoxins.