Generation of human-induced pluripotent-stem-cell-derived cortical neurons for high-throughput imaging of neurite morphology and neuron maturation

Summary High-throughput imaging allows in vitro assessment of neuron morphology for screening populations under developmental, homeostatic, and/or disease conditions. Here, we present a protocol to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis. We describe the use of a notch signaling inhibitor to generate homogeneous neuronal populations at densities amenable to individual neurite identification. We detail neurite morphology assessment via measuring multiple parameters including neurite length, branches, roots, segments and extremities, and neuron maturation.


SUMMARY
High-throughput imaging allows in vitro assessment of neuron morphology for screening populations under developmental, homeostatic, and/or disease conditions. Here, we present a protocol to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis. We describe the use of a notch signaling inhibitor to generate homogeneous neuronal populations at densities amenable to individual neurite identification. We detail neurite morphology assessment via measuring multiple parameters including neurite length, branches, roots, segments and extremities, and neuron maturation.

BEFORE YOU BEGIN
The pluripotent stem cell-derived cortical neural progenitors used in the following protocol were generated according to the methods described by us 1

and others. 2
Note: The cortical neural progenitors and mature neurons should be cultured in a humidified 37 C incubator with 5% CO 2 .
Note: All procedures should be performed in a sterile environment. All waste materials should be considered potentially biohazardous and disposed according to the laboratory's waste disposal policy.

Institutional permission
The experiments involving human iPS cells were approved by the Northern Sydney Local Health District Human Research Ethics Committee, Australia (Reference number: RESP/15/314). b. Do not wash the mouse laminin coating. Aspirate the mouse laminin immediately before adding the cell suspension in culture media to avoid air drying the coated surface.
CRITICAL: Throughout the coating period ensure the coating solution covers the entire surface of the well. Unevenly coated surface area will result in poor attachment and maturation.

Preparing cortical base media
Timing: 30 min 5. Prepare media as outlined in the materials and equipment section Tables 3 and 4.

MATERIALS AND EQUIPMENT
Note: The cortical base and maturation media described in Tables 3 and 4 can be stored up to a week at 4 C.
Note: The growth factor stocks described in Table 5 can be stored up to 2 weeks at À20 C. For long term-storage, the stocks should be stored at -80 C.

STEP-BY-STEP METHOD DETAILS
Seeding cortical neural progenitor cells for maturation This section outlines how to thaw and seed cryopreserved cortical neural progenitors in cell culture plates to differentiate them to mature cortical neurons.
1. Pre-warm cortical base media in a 37 C water bath.
2. To thaw a frozen vial of neural progenitors, remove the frozen vial of cells from storage and quickly thaw cells in a water bath at 37 C by gently swirling the vial for 1-2 min.
Note: Thaw one frozen vial of cells at a time to prevent prolonged exposure to toxic DMSO present in freezing media at higher temperatures.
3. In a 15 mL conical tube, dilute the 1 mL thawed cell suspension slowly with 7 mls of pre-warmed cortical base media supplemented with ROCK inhibitor (Y27632, 10 mM).
Note: Y27632 significantly enhances recovery of neural progenitor cells from cryopreserved stocks.
4. Centrifuge the cell suspension at 300 3 g for 5 min at room temperature (20 C-25 C). 5. Gently remove the supernatant using a vacuum suction leaving behind an undisturbed cell pellet. 6. Resuspend the cell pellet in 1 mL of cortical base media supplemented with Y27632 (10 mM).
CRITICAL: Gentle titration of the cell pellet is critical for optimal cell survival and necessary for homogenous distribution of the cells in solution. This is important to ensure an accurate cell count and an even distribution of cells upon seeding into wells.
7. Perform cell counting using 10 mL of cell suspension using an automated cell counter or a hemocytometer to calculate cell number and prepare the cell suspension at the desired cell density for seeding.
Note: For this protocol, prepare a cell suspension at 100,000 cells/mL media.
Note: Using an automated cell counter system (for example, Invitrogen Countess 3 Automated Cell Counter) can help identify the proportion of live and dead cells while performing cell counting. One freeze-and-thaw cycle is expected to cause about 5%-10% cell death.  8. Seed 100 mL resuspended cell solution with a density of 100,000 cells/mL media into one well of a 96-well plate.

Maturation of cortical neural progenitors
Timing: 15 days Note: Figure 1 outlines the cortical neuron differentiation protocol.
9. At 24 h after cell seeding, using a light microscope confirm cells have survived, attached to the plate, and are evenly distributed within the well.
CRITICAL: Low cell densities will have a significant impact on neuron maturation. If the cell cultures have more than 20-25% cell death, then it is best to discard and repeat the experiment.
10. Day 1: remove the cortical base media with Y27632 and gently wash each well once with PBS À/À to remove residual Y27632. Replace media with cortical differentiation base media without Y27632.
Note: On day 1 and 3, perform a 100% media change i.e., replace all 100 mL of media. The media used on these two days are different.
11. Days 3-15: Replace media with cortical maturation media supplemented with growth factors BGDAL (growth factor concentrations and abbreviations and detailed in Table 5) and Y-secretase complex/notch pathway inhibitor (DAPT).
CRITICAL: the growth factors should be added to the media on the day of media change to minimize degradation.
Note: Media change should be performed every second day. As neurons mature, they are more sensitive to total media aspiration.
Note: From days 5 to 15, perform 90% media change i.e., remove 90 mL media and add 90 mL fresh media. The differentiation media on days 5-15 is the same. So, the 10% left-over media will not impact the concentration of the growth factors.
Note: At the end of step 11, cortical neurons are day 40 of maturation, noting cells are cryopreserved at day 25 after the onset of iPSc cortical differentiation, plus a additional 15 days in culture (according to the aforementioned steps).

Immunostaining of mature cortical neurons
Timing: 5 h This section outlines how to perform immunostaining 3 of mature cortical neurons.
Note: A ready to use kit, Cytofix/Cytoperm TM Fixation/Permeabilization Kit was used for the fixation, blocking, antibody incubations and wash steps involved in the immunofluorescence staining protocol.
Note: The volume added to each well of the 96-well plate is 100 mL in steps 12 to 26.  and image (Figures 2A and 2B). c. Analyze images of neurons using image analysis pipeline, detailed in steps 32 and 33 below ( Figure 2C). d. Segment nuclei and neurites (Figures 2D and 2E) and measure neurite outgrowth parameters ( Figures 2F-2J) 33. Measure parameters of neurite outgrowth including neurite length, branching, extremities, and segments using the ''Find neurites'' building block.
Note: The ''Find neurites'' building block is tailored to detect neurites and provides a set of interactive illustrations and tuning dialogues to visualize the neurite detection results and adjust the parameters if required. Figure 2C shows the parameters we used. We measured the neurite outgrowth parameters for the early (Day1) and mature (Day 15) neurons. As expected, Day 15 mature neurons had relatively longer and more complex MAP2 positive neurites ( Figures 2F-2J).
Note: TBR1 and CTIP2 mature cortical markers are expressed in the nucleus 4 ( Figures 3B and 3E).
b. Analyze the images using image analysis pipeline presented in Figure 3G (for TBR1) and 3H (for CTIP2). c. Use the ''find nuclei'' building block to segment the image and find nuclei (as described in step 32). d. Use the ''Calculate Intensity Properties'' building block to determine the fluorescence intensity of TBR1/CTIP2 markers in the nucleus region.
Note: Nucleus expressing the TBR1/CTIP2 markers will have a high fluorescence intensity. Set an intensity threshold using an isotype control to identify positively stained nuclei.

OPEN ACCESS
Note: Percentage of other mature cortical markers such BRN2, SATB2 and CUX1 can also be measured using the same image analysis pipeline.

EXPECTED OUTCOMES
The differentiation protocol described above should result in mature cortical neurons (Figure 4). Notch signaling pathway regulates the proliferation of cortical progenitors. 5 DAPT, an inhibitor of notch signaling, is often used to promote cortical progenitors exit cell-cycle in vitro resulting in neuron maturation. Reflective of the impact of prolonged Notch inhibition, our neuronal cultures had high proportion of mature cortical neurons, seen with the expression of mature cortical neuron markers TBR1 and CTIP2. By day 15 after seeding neural progenitors, the neurons formed long and complex neurites (Figures 2 and 4). The neurons were uniformly spread out across the wells with minimum neuronal clustering allowing segmentation of individual neurites for image analysis (Figure 2). These outcomes (homogeneous cortical maturation and uniformly spread-out cultures) are ideal for imaging and analyzing neurite morphology. Although this protocol generates a high population of mature cortical neurons, it is possible that a small population of non-neurons may also be generated. Using cell type specific markers, such as GFAP for astrocytes can help identify any undesired nonneuronal cell types.
To further test if the neurons are mature, neuron functionality was tested using whole cell patch clamp ( Figure 5). The electrophysiological activities of neurons were measured in voltage clamp mode. Voltage pulses are delivered to step the membrane potential from -100 mV to +70 mV (four steps from +40 mV to +70 mv using 10 mV interval steps) and the current responses are recorded. Cells exhibited spontaneous excitatory inward and outward currents indicating the formation of functional excitatory neurons. The dataset for Figure 5 is available at https://doi.org/10.5281/ zenodo.7882388.

LIMITATIONS
The image analysis presented in this protocol is performed using Harmony Perkin Elmer, a licensed image analysis software. However, similar image analysis measuring neurite outgrowth can be performed using open source image analysis software's such as Cell Profiler 6 and ImageJ with NeuronJ PlugIn. 7 Both Cell Profiler and ImageJ support multiple image file formats such as JPEG and TIFF. Low neural progenitor to mature cortical neuron differentiation efficiency (relevant to steps 9-11).

Potential solution
The cortical neuron progenitors used in this study were differentiated from iPS cells using a dual SMAD neural induction protocol, as previously described. 1 The progenitors were then expanded and maintained using FGF2. At day 25, before cryopreserving the cells, we performed immunohistochemistry to confirm the emergence of basally dividing TBR2+ intermediate progenitor cells. Cryopreserving late/matured progenitors will lead to low survival rate during the thawing process. This has been tested in multiple cell lines.
It is essential to follow this iPS to neural progenitors differentiation protocol to successfully differentiate neural progenitors to mature cortical neurons.

Problem 2
Early and late cell loss during the differentiation protocol (relevant to steps 9-11).
Potential solution All differentiation protocols described in this manuscript use the same density of 10,000 per well of a 96-well plate or 0.32 cm 2 while setting up differentiation. Excessively low cell densities will significantly impact neuron maturation. Setting up replica (sister) plates will allow fixing and counting cells as you progress through the differentiation protocol. For example, our differentiation protocol is for Case 2. Late cell loss: Healthy neuronal cultures have a small proportion of aged apoptotic cells that may be lost during the course of the 15-day differentiation. This is estimated to be 5-10% cell loss. If there is a high cell loss, then this can indicate A) unhealthy neuronal cultures arising from-incorrectly maintained differentiation conditions or lack of differentiation ability of the iPS cell line or pathogenic disease associated effects B) As the neurons differentiate and mature, they make neuronal networks that can lift of relatively easily additional care should be taken to gently change media without disturbing the neuronal networks. We suggest using a motorized multichannel pipette for media change. This allows having low and consistent media suction speed and pressure across all wells of a plate and between plates. As mentioned in the CRITICAL note after step 11, perform a 90% media change (not 100%) to avoid the risk of lifting off neurons from the plate.

Problem 3
High cell proliferation during the differentiation protocol (relevant to steps 9-11).

Potential solution
As suggested above, setting up replica (sister) plates will allow fixing and counting cells as you progress through the differentiation protocol. It is well established that DAPT promotes cortical progenitors to exit cell-cycle and promotes neuron maturation. The same has been observed in our differentiation protocol. If the cells continue to proliferate highly after continued prolonged treated with DAPT, it would be advised to recheck DAPT stock preparation and storage conditions.

Potential solution
We have observed that neurons are highly sensitive to temperature changes. Disturbance by handing of the incubators with neurons should be kept to a minimum. Frequent opening/closing the incubators or having the neuron plate outside the incubator for longer durations can cause neurite retraction.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and regents should be directed to and will be fulfilled by the lead contact, Gautam Wali (g.wali@neura.edu.au).

Materials availability
This study did not generate new unique reagents, cell, or mouse lines.

Data and code availability
The protocol includes all data generated during the study.