Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Podsumowanie

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Streszczenie

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

Wprowadzenie

Human pluripotent stem cell (hiPSC)-derived neurons are increasingly relevant in the areas of basic research, drug development, and regenerative medicine. Workflows and procedures to optimize their culture and maintenance, and improve the efficiency of differentiation into specific neuronal subtypes, are evolving rapidly^1,2. To improve the utility and cost-effectiveness of human stem cell-derived neurons as model systems amenable to high-content analyses in drug discovery and target validation, it is useful to decrease the culturing time required to generate mature, functional neurons, while retaining maximum robustness, reproducibility, and phenotype relevance. Although 3-dimensional organoid cultures are driving breakthroughs in neurodevelopment research³, 2-dimensional monolayer cultures are especially compatible with automated imaging-based applications due to their minimal tissue thickness.

However, the adaptation of imaging-based screening methods to models of human neurological and neurodevelopmental disease faces a major challenge. The protracted timeframe over which the human nervous system matures in vivo necessitates extended time in culture to accommodate natural programs of gene expression and achieve neuronal maturation.

One practical consequence of the lengthy neuronal differentiation program is that the maintenance of hiPSC-derived monolayer cultures must be sustained for many weeks to achieve adequate synapse maturity. During this time, neural progenitors that remain undifferentiated continue to divide. These can quickly overgrow the culture and usurp the nutrient content required to maintain viable postmitotic neurons. Vigorously dividing neural progenitor cells (NPCs) can also compete with neurons for the growth substrate. This can render such cultures subject to problems of poor adhesion, a condition unsuitable for imaging-based assays. Moreover, many investigators find that the smaller the culture volume, the greater the difficulty in maintaining healthy populations of differentiated neurons long enough to observe the late stages of neuronal differentiation. In other words, assays of synapse maturation using high content screening (HCS) approaches can be very challenging for human-derived neurons.

To circumvent some of these problems, a procedure of resuspending and replating previously differentiated hiPSC-derived neurons has been used. Firstly, it allows the study of neurite outgrowth (or, more accurately, neurite regeneration) in a population of fully committed neurons. Secondly, the replating of previously differentiated neurons from a large volume format (like 10 cm plates or larger), down to small volume formats (like HCS-compatible 96- or 384-well microtiter plates) enables a significant reduction in total culturing time in the small volume condition. This facilitates the study of synapse assembly and maturation over subsequent weeks in vitro.

However, the replating of mature neurons that have already established long neurites and a complex connectivity network presents several challenges, one of which is the sometimes high and variable rate of cell death. Here, we describe a replating procedure that results in excellent cell survival and reproducibility. Commonly, neurons are exposed to proteolytic enzymes for short incubation periods (typically ~3-10 min) in order to detach cells from the substrate prior to trituration. This brief proteolysis time is customarily used for resuspending and passaging many types of dividing cells, including non-neuronal cells and undifferentiated progenitors^4,5,6. However, for differentiated neurons bearing long, interconnected neurites, it is essential not only to detach cells from the substrate but also to disrupt the dendritic and axonal network in order to isolate individual cells while minimizing damage. Indeed, a thick meshwork of neurons usually tends to detach from the substrate as a single sheet, rather than as individual cells. If care is not taken to loosen the thick network of neurites, neurons not only become irreversibly damaged during trituration, but many of them fail to pass through the filter used to remove clumps, resulting in poor cell yield. Below we describe a simple modification to a widely-used protease incubation procedure to counter these difficulties.

In the protocol described below, neurons are incubated for 40-45 min with a mild protease, such as the proteolytic enzyme (e.g., Accutase). During the first 5-10 min after adding the enzyme, the neuronal network lifts off from the substrate as a sheet. Incubation with the protease proceeds for an additional 30-40 min before proceeding with gentle trituration and filtering. This extra incubation time helps ensure that the digestion of the material relaxes the intercellular network, thereby ensuring that subsequent trituration produces a suspension of individual cells. This procedure maximizes the uniformity of cell distribution upon replating while minimizing cell death. We have successfully applied this replating method to hiPSC-derived neuronal cultures generated by various differentiation protocols^7,8 and from various lines of hiPSCs. The procedure is nominally suitable for use with most or all lines of stem cell-derived neurons. We have observed that an extended protease incubation time is not absolutely essential for replating cultures from small format plates (e.g., 35 mm diameter); however, as we show here, it provides a significant benefit when replating from large diameter plates (e.g., 10 cm diameter or larger), probably because neurites in such plates can extend very long processes and form a densely interconnected array.

Here we demonstrate this method and briefly illustrate its application in assays for early neuritogenesis and for synapse maturation, which involves clustering of pre- and postsynaptic proteins along the dendrites and axons, followed by their later colocalization at synaptic sites. The examples highlight the advantages this protocol offers in preserving cell viability and reproducibility. First, it permits investigators to study early steps in human neuritogenesis. The experimental setting is similar to the commonly used primary cultures of rodent cortical or hippocampal neurons, where cells are extracted from late fetal or early postnatal brain, dissociated by trituration after gentle protease treatment, and allowed to initiate neurites or to regenerate neurites that were severed in the procedure^9,10. Similar to such rodent primary neurons, hiPSC-derived neurons begin to form or regenerate their neurites within hours after replating, allowing imaging of growth cones and neurite morphology in an environment optimal for high spatiotemporal imaging with fewer surrounding undifferentiated cells. We have observed that neurite outgrowth is more synchronized compared to the variable delays and different outgrowth rates seen when neurons first begin to differentiate from a progenitor population. In addition, replating enables assays of neurons expressing neuronal subtype markers that typically appear later in neural development, such as the cortical layer 5/6 transcription factor CTIP2 (Chicken ovalbumin upstream promoter transcription factor-interacting protein 2), or the pan-neuronal marker NeuN¹¹. An especially useful feature of the replating approach is that synaptogenesis proceeds within a time frame compatible with HCS.

Protokół

1. Differentiation Period Prior to Replating

1. Differentiate neurons on 10 cm dishes, using a protocol of choice^7,8 until neurons have formed a thick network with their processes and express not only early neuronal markers such as MAP2 or TuJ1, but also late markers such as NeuN.
2. Change half the medium of choice every 4 days during the neuronal differentiation process.
   NOTE: More extensive or frequent medium changes dilute essential trophic factors, and could disfavor maturation.
   1. For the iPSC-derived WT126 neurons, use the following post-differentiation culturing media: 5 mL 100x N2 supplement, 10 ng/mL BDNF, 10 ng/mL GDNF, 1 µg/mL laminin, 200 µM ascorbic acid, 1 µM dibutyryl-cAMP and 10 mL SM1 for 500 mL Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12). Gradually, using half-media changes, replace with neural basal medium (Table of Materials) and the same supplements.
   2. For the iPSC-derived CVB WT neurons, use the following post-differentiation culturing medium: 500 mL neural basal A medium (Table of Materials) and 500 mL DMEM/F12 medium with 2 µg/mL laminin, 10 mL glutamine supplement (Table of Materials), 0.75 mg/mL sodium bicarbonate, 5 mL minimum essential medium (MEM) nonessential amino acids, 0.2 mM ascorbic acid, 10 ng/mL BDNF, 20 mL 50x B27, and 10 mL 100x N2 supplement.

2. Coating Multiwells

1. The day before replating, coat with poly-L-ornithine (PLO). Dissolve PLO in sterile water to make a stock solution (10 mg/mL). Store this stock at -20 °C. Dilute PLO 1:100 in water to yield a concentration of 50 µg/mL when coating glass and 1:1,000 ratio (10 µg/mL) when coating plastic.
2. Apply the coating directly to the target plates. Use the volume of coating appropriate to the plate size (i.e., for a 24-well plate apply 500 µL of PLO solution per well).
3. Allow plates to sit in the dark overnight at room temperature.
4. Retrieve coated plates on the day of replating and transfer to a sterile biosafety cabinet.
5. Aspirate the PLO solution and rinse twice with sterile water.
6. Dilute laminin (1.15 mg/mL) in phosphate-buffered saline (PBS) at 1:400 dilution.
   NOTE: Thaw laminin at 4 °C and quickly add to PBS to avoid aggregation of laminin and uneven coating.
7. Aspirate sterile water and apply 500 µL of laminin coating to wells previously coated with PLO.
8. Place plates in a 37 °C incubator for a minimum of 4-6 h. Use longer incubations, up to 16 h, for glass surfaces. Use consistent incubation times.

3. Replating Differentiated Neurons

1. Rinse plate of differentiated neurons with PBS once gently. Disperse PBS gently down the wall of the plate, and not directly onto the cells, to avoid disrupting them.
2. Gently aspirate PBS, being careful to avoid touching the cells directly but to aspirate from the edge of the dish while tipping it towards the researcher.
3. Apply at minimum 1 mL of the proteolytic enzyme (Table of Materials) per 10 cm plate and return cells to incubator. Add slightly higher volumes if the tissue culture room exhibits a high evaporation rate due to low humidity.
4. Incubate cells with proteolytic enzyme for 40-45 min in order to detach them from the plate and to detach them from other neurons within the neuronal network.
   NOTE: Timing at this step is critical. Quenching the protease too early can lead to increased cell death after replating. The proteolytic enzyme manufacturer recommends that temperatures much lower than 37 °C be used with longer incubation periods for passaging cell lines (e.g., overnight at 4 °C). However, handling neurons at 4 °C should be avoided, as they often show poor survival after cold exposure. The manufacturer also states that a 60 min incubation with the enzyme at 37 °C leads to its enzymatic inactivation. However, in authors' experience, a 40-45 min incubation of hiPSC-derived neuronal cultures at 37 °C is sufficient for efficient dissociation and excellent neuronal survival upon replating.
5. Check neurons on a phase-contrast microscope during the incubation time and allow protease treatment to continue until the neural network completely detaches from the plate and starts to break apart in smaller sheets upon briefly shaking the plate under the microscope.
6. Quench the protease activity using 5 mL of fresh DMEM media per 1 mL of protease in the 10 cm plate to stop the digestion. Gently triturate cells against plate 5-8 times to disrupt network, using serological pipettes. Be careful not to apply too much pressure when triturating, as differentiated neurons are fragile. Do not use a P1000 tip, as the end is too sharp and narrow, and thus can sheer or damage the neurons.
7. Apply solution with cells through a cell strainer with 100 µm diameter mesh into a fresh 50 mL conical tube drop by drop.
8. Rinse strainer with an additional 5 mL of fresh DMEM media, after cells have filtered through.
9. Spin cells in benchtop centrifuge at 1,000 x g for 5 min.
10. Return conical tube to the biosafety cabinet and aspirate most of the media, leaving around 250 µL to ensure cells retain moisture.
11. Resuspend cells gently in 2 mL of fresh DMEM media. Do not pipette the pellet against the side of the tube. Instead, gently invert conical 2-3 times and pass cells through the end of a 5 mL serological pipet to dislodge them.
12. Apply 10 µL of resuspended neurons onto the edge of a hemocytometer.
13. Add 8-10 µL of trypan blue to droplet of cells to assess cell viability during this resuspension step. Apply 10 µL of this mixture to the hemocytometer or other automatic cell counters. Assess as viable those cells that are phase bright and exclude the trypan blue dye.
14. Determine amount of viable cells/mL, and prepare to dilute the contents of the conical tube according to the desired cell density.
15. Plate ~10,000 cells per well for a 384-well plate; plate ~150,000-200,000 cells per well for a 24-well plate.
16. Add fresh DMEM to the conical tube of resuspended cells to achieve appropriate dilution and add additional appropriate supplements such as B27 and/or BDNF, depending on the requirements of the specific cell line.
17. Gently tilt conical tube to mix 2-3 times.
18. Aspirate laminin coating from the 24-well plate, or from the 384-well multiwell plate using a 16-channel pipet and rinse once with PBS.
19. Aspirate PBS using a P1000 for the 24-well plate, or the 16-channel pipette for 384-well plates.
20. Apply cell solution to each well in a figure eight motion to avoid clumping. Plating uniformity might also be optimized by using automated liquid handling devices.
    NOTE: The addition of laminin to the media starting 2-4 days post-replating also helps maintain a homogenous distribution of the cells.
21. Repeat steps 3.19 and 3.20 for each well.
22. Return the plate to incubator set at 37 °C and 5% CO₂.
23. After 2 days, start changing half the medium every 4 days using the post-differentiation culturing media described in section 1.2. After the desired maturation time, fix cultures using 3.7% formaldehyde at 37 °C and stain cells according to the experimental requirements.

4. Cell Viability Assays Post-replating

1. Add the early cell death reporter (VivaFix: 0.5 µL of the 50 µL stock solution per 500 µL culture media per well) after briefly vortexing the stock solution.
2. Image the live and dead cells cultured on glass-bottom multiwells, after 20 min, without any washing step, since the dye fluoresces only once inside the cells, or fix and co-stain with 4',6-diamidino-2-phenylindole (DAPI) and/or other antibodies before imaging using a confocal microscope.

5. Immunostaining

1. Fix cultures with 3.7% formaldehyde in PBS plus 120 mM sucrose for 20 min at 37 °C.
2. Rinse and permeabilize with 0.2% Triton X-100 for 5 min at room temperature, and then block for 30 min with 2% bovine serum albumin (BSA).
3. Aspirate the BSA without rinsing and incubate for 1 h at room temperature with rabbit anti-MAP2 antibody 1:1,000, mouse anti-β3 tubulin (TuJ1) antibody 1:2,000, chicken anti-NeuN antibody (1:100) and rat anti-CTIP2 antibody (1:500), or with mouse anti-PSD95 (1:200), rabbit anti-synapsin 1 (1:200) and chicken anti-MAP2 antibody for synaptic staining.
4. Rinse with PBS, and incubate with Alexa Fluor-conjugated secondary antibodies plus DAPI for 45 min at 37 °C.
5. Finally, wash twice with PBS before imaging.

6. Calcium imaging

1. Infect cultured cells with AAV8-syn-jGCAMP7f-WPRE (The Salk Institute for Biological Studies, GT3 Core Facility) at 10 days post-replating and image to assess spontaneous calcium transients at 3-4 weeks post-replating.

7. Image acquisition and Analysis

NOTE: For details on the acquisition system please refer to Calabrese et al.¹².

1. Use an imaging module for manual or automated image acquisition, and an image analysis software module, such as CellProfiler¹³, for morphometric measurements.
2. Calculate average length of TuJ1 positive neurites and MAP2 positive dendrites by measuring total length per field of view divided by the number of neurons (DAPI + MAP2 or TuJ1 positives cells) within the same field.

Wyniki

The replating of hiPSCs-derived neurons that have been differentiated for multiple weeks offers several advantages. However, detaching and replating differentiated neurons that have long, interconnected dendrites and axons (Figure 1A) can result in a high fraction of irreversibly damaged neurons.

As described in the protocol section, we used incubation with a proteolytic enzyme to detach the neurons from the substrate. Typically, due to their thic...

Dyskusje

We have demonstrated a straight-forward procedure for the resuspension and replating of human neuronal cultures that optimizes viability, differentiation success, and subcellular imaging in a manner that is compatible with high content screening platforms, and other assays relevant to drug discovery. Figure 6 illustrates the overall workflow and examples of such applications.

Although here we focused on hiPSC-derived neurons that are directed toward a cortical neu...

Materiały

Name	Company	Catalog Number	Comments
Post Replating Media
L-Ascorbic Acid	Sigma	A4403	Add 1 mL of 200 mM stock to 1 L of N2B27 media
Dibutyryl-cAMP	Sigma	D0627	Add 1 µM
Human BDNF	Peprotech	450-02	10 ng/ml final concentration
B27 (50x)	Thermofisher Scientific	17504044	Add 20 mL to 1 L N2B27 media
DMEM/F12 with Glutamax	Thermofisher Scientific	31331093	Add N2 and distribute in 50 mL conicals; parafilm wrap lids
Human GDNF	Peprotech	450-10	10 ng/mL final concentration
Glutamax	Thermofisher Scientific	35050038	Add 10 ml to 1 L N2B27 media; glutamine supplement
Mouse Laminin	Sigma	P3655-10mg	Add 100 µL to 50 mL N2B27
MEM Nonessential Amino Acids	Thermofisher Scientific	11140035	Add 5 mL to 1 L N2B27 media
N2 (100x) Supplement	Life Technologies	17502048	Add 5 mL to 500 mL media
Neurobasal A Media	Thermofisher Scientific	10888022	Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media
Neurobasal Media	Thermofisher Scientific	21103049	for WT126 cells; neural basal media
SM1 Supplement	StemCell Technologies	5711	Add 1:50 to media
Sodium bicarbonate	Thermofisher Scientific	25080-094	Add 10 mL to 1 L N2B27 media
Plate Preparation
10 cm Tissue Culture Dishes	Fisher Scientific	08772-E	Plastic TC-treated dishes
6-well Tissue Culture Dishes	Thomas Scientific	1194Y80	NEST plates
Mouse Laminin	Life Technologies	23017-015	Add 1:400 on plastic
Poly-Ornithine	Sigma	P3655-10mg	Add 1:1,000 on plastic
UltraPure Distilled Water	Life Technologies	10977-015	To dilute Poly-L-Ornithine
Replating Reagents
100 mM Cell Strainer	Corning	431752	Sterile, individually wrapped
384-well plate, uncoated	PerkinElmer	6007550	Coat with PLO and Laminin
DPBS	Life Technologies	14190144	Dulbecco's phosphate-buffered saline
Poly-D-Lysine-Precoated 384-well Plates	PerkinElmer	6057500	Rinse before coating with laminin
StemPro Accutase	Life Technologies	A1110501	Apply 1 mL/10 cm plate for 30-45 min; proteolytic enzyme
Fixation Materials
37% Formaldehyde	Fisher Scientific	F79-1	Dissolved in PBS
Sucrose	Fisher Scientific	S5-12	0.8 g per 10 mL of fixative
Immunostaining Materials
Alexa Fluor 488 Goat anti-mouse	Invitrogen	A-11001	secondary antibody
Alexa Fluor 568 Goat anti-chicken	Invitrogen	A-11041	secondary antibody
Alexa Fluor 647 Goat anti-chicken	Invitrogen	A-21449	secondary antibody
Alexa Fluor 561 Goat anti-rat	Invitrogen	A-11077	secondary antibody
DAPI	Biotium	40043	visualizes DNA
Mouse antibody against b3-tubulin (TuJ-1)	Neuromics	MO15013	early stage neuronal marker
Rat antibody against CTIP2	Abcam	ab18465	layer 5/6 cortical neurons
Chicken antibody against MAP2	LifeSpan Biosciences	LS-B290	early stage neuronal marker
Chicken antibody against NeuN	Millipore	ABN91	late stage neuronal marker
Rabbit antibody against MAP2	Shelley Halpain	N/A	early stage neuronal marker
Mouse antibody against PSD-95	Sigma	P-246	post-synaptic marker
Rabbit antibody against Synapsin 1	Millipore	AB1543	pre-synaptic marker
Bovine serum albumin (BSA)	GE Healthcare Life Sciences	SH30574.02	10% in PBS for blocking
Titon X-100	Sigma	9002931	Dilute to 0.2% on PBS for permeabilization
Viability Markers
Vivafix 649/660	Biorad	135-1118	cell death marker
Calcium Imaging
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
AAV8-syn-jGCAMP7f-WPRE	THE SALK INSTITUTE, GT3 Core Facility	N/A	calcium reporter in a viral delivery system
hiPSC-derived NPCs
WT 126 (Y2610)	Gage lab	N/A	Marchetto et al., 2010
CVB WT24	Yeo and Goldstein labs	N/A	unpublished