Quantifying differentiation of progenitor populations using cerebral organoid models for neurodevelopmental disorders

Summary

Neurodevelopmental disorders are characterized by complex phenotypes that often result from concomitant dysregulation of cell proliferation, differentiation, or other crucial developmental processes. Here, we present a protocol to quantify differentiation of progenitor populations during early stages of neurogenesis in induced pluripotent stem cell (iPSC)-derived cerebral organoids. We describe steps for organoid differentiation and maturation, sample preparation, immunofluorescence, and imaging and analysis using epifluorescence microscopy. This protocol can be used to compare cerebral organoids from control and patient-derived iPSCs.

For complete details on the use and execution of this protocol, please refer to Rakotomamonjy et al. (2023).¹

Graphical abstract

Highlights

• •Instructions to prepare cerebral organoid samples for immunofluorescence
• •Detection of neural progenitor and early neuron populations using immunofluorescence
• •Quantification of progenitor population ratios in cerebral organoids

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Neurodevelopmental disorders are characterized by complex phenotypes that often result from concomitant dysregulation of cell proliferation, differentiation, or other crucial developmental processes. Here, we present a protocol to quantify differentiation of progenitor populations during early stages of neurogenesis in induced pluripotent stem cell (iPSC)-derived cerebral organoids. We describe steps for organoid differentiation and maturation, sample preparation, immunofluorescence, and imaging and analysis using epifluorescence microscopy. This protocol can be used to compare cerebral organoids from control and patient-derived iPSCs.

Before you begin

This protocol details the visualization and analysis of the differentiation of neural progenitor populations in iPSC-derived cerebral organoids. Preparation includes culturing cerebral organoids and preparing samples for immunofluorescence. As referenced in Rakotomamonjy et al., cerebral organoids are prepared using the STEMDiff Cerebral Organoid Kit from STEMCELL Technologies.^1,3 Organoids are collected at days 21 and 40 of culture and then follow established protocols to fix, cryoprotect, embed, snap freeze, and cryosection slices for immunofluorescence.^2,3,5 Here we give an overview for making cerebral organoids. Our main protocol picks up with immunofluorescence to visualize and quantify aspects of progenitor populations and differentiation in organoids.

Note: This protocol is written with the assumption that those performing it have experience in culturing iPSCs. iPSC cultures must be healthy to achieve optimal results. Healthy iPSC cultures grow in colonies with soft edges, have no or minimal areas of differentiation, and are routinely checked for pluripotency. Pluripotency can be assessed by either fixing and staining for transcription factors OCT4, SOX2, NANOG and/or cytoplasmic markers SSEA-4, TRA-1-60, or by following a protocol for trilineage differentiation.

Grow cerebral organoids using STEMDiff Cerebral Organoid Kit

Timing: 20–40+ days

Here we give a brief overview of the main steps for the preparation of cerebral organoids using the STEMDiff Cerebral Organoid Kit from STEMCELL Technologies. For more detail, please refer to the original protocol.^2,4

• 1.Formation of Embryoid Bodies (Days 0–5).
  Day 0.

  • a.
    Dissociate iPSC cultures (ideal confluence 65%–80%) into single cells, collect and wash.
  • b.
    Count cells and resuspend in Seeding Media at concentration of 90,000 cells/mL.
  • c.
    Plate 100 μL of cell suspension into ultra-low attachment round bottom 96-well plates (9,000 cells/well).
  • d.
    Incubate plate(s) at 37°C; make sure cells aggregate into embryoid bodies.
  Days 2 & 4.

  • e.
    Add 100 μL EB Formation Media to each well gently.
  • f.
    Incubate at 37°C.
• 2.Induction (Days 5–7)
  Day 5.

  • a.
    Add 500 μL of Induction Media to ultra-low attachment 24-well plates.
  • b.
    Add 1–2 embryoid bodies to each well using a wide-bore p200 tip.
  • c.
    Incubate for 48 h at 37°C.
• 3.Expansion (Day 7–10).
  Day 7.

  • a.
    Embed each individual embryoid body at the center of a 15 μL of Matrigel droplet.
  • b.
    Incubate at 37°C for 30 min.
  • c.
    Use Expansion Media to move embryoid bodies in Matrigel into ultra-low attachment 6-well plates (3 mL per well, 12-16 embryoid bodies per well).
  • d.
    Incubate at 37°C for 3 days.
• 4.Maturation (Days 10–40+).
  Day 10.

  • a.
    Replace media carefully with Maturation Media (3 mL per well).
  • b.
    Place plates on an orbital shaker at 65 rpm.
  • c.
    Incubate at 37°C.
  • d.
    Change media every 3–4 days until samples are ready to collect.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-N Cadherin polyclonal antibody (recommended dilution 1:200)	Sigma-Aldrich	Cat #: SAB5700641
Phospho-Vimentin (Ser56) polyclonal antibody (recommended dilution 1:100)	Cell Signaling Technology	Cat #: 3877, RRID: AB_2216265
Anti-Histone H3 (phospho S28) monoclonal antibody (HTA28) (recommended dilution 1:500)	Abcam	Cat #: ab10543, RRID: AB_2295065
Anti-SOX2 polyclonal antibody (recommended dilution 1:200)	MilliporeSigma	Cat #: AB5603, RRID: AB_2286686
Pax-6 monoclonal antibody (recommended dilution 1:200)	Santa Cruz Biotechnology	Cat #: sc-81649, RRID: AB_1127044
Anti-TBR2/Eomes polyclonal antibody (recommended dilution 1:200)	Abcam	Cat #: ab23345, RRID: AB_778267
Anti-Ctip2 monoclonal antibody (25B6) (recommended dilution 1:500)	Abcam	Cat #: ab18465, RRID: AB_2064130
Anti-TBR1 polyclonal antibody (recommended dilution 1:200)	Abcam	Cat #: ab31940, RRID: AB_2200219
Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (recommended dilution 1:200)	Thermo Fisher Scientific	Cat #: A-21203, RRID: AB_141633
Donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (recommended dilution 1:200)	Thermo Fisher Scientific	Cat #: A-31571, RRID: AB_162542
Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (recommended dilution 1:200)	Thermo Fisher Scientific	Cat #: A-21206, RRID: AB_2535792
Donkey anti-rat IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (recommended dilution 1:200)	Thermo Fisher Scientific	Cat #: A-21208, RRID: AB_2535794
Donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (recommended dilution 1:200)	Thermo Fisher Scientific	Cat #: A-31573, RRID: AB_2536183
Chemicals, peptides, and recombinant proteins
Matrigel hESC-qualified matrix, LDEV-free	Corning	Cat #: 354277
Y-27632 2HCl	Selleck Chemicals	Cat #: S1049
Accutase	MilliporeSigma	Cat #: SCR005
Paraformaldehyde	MilliporeSigma	Cat #: 158127
Normal donkey serum	SouthernBiotech	Cat # 0030-01
Bovine serum albumin	MilliporeSigma	Cat #: A2153
Hoechst 33342	Thermo Fisher Scientific	Cat #: H3570
ProLong Gold antifade mountant	Thermo Fisher Scientific	Cat #: P36930
Critical commercial assays
STEMdiff Cerebral Organoid Kit	STEMCELL Technologies	Cat #: 08570
STEMdiff Cerebral Organoid Maturation Kit	STEMCELL Technologies	Cat #: 08571
Costar ultra-low attachment round bottom 96-well plate	Corning	Cat #: 7007
Nunclon Sphera ultra-low attachment 24-well plate	Thermo Fisher Scientific	Cat #: 174930
Costar ultra-low attachment 6-well plate	Corning	Cat #: 3471
Axygen 200 μL wide bore pipette tips	Corning	Cat #: TF-205-WB-R-S
Experimental models: Cell lines
iPSC lines	-	-
Software and algorithms
GraphPad Prism 9.5.1	GraphPad	https://www.graphpad.com/
ImageJ	National Institutes of Health	https://imagej.net/ij/index.html

Materials and equipment

4% Paraformaldehyde

Reagent	Final concentration	Amount
96% Paraformaldehyde	4% Paraformaldehyde	32 g
D-PBS w/o Ca2+, Mg2+	-	400 mL
1 mM NaOH	-	-

CRITICAL: Operate in a laboratory fume hood for safety.

Dissolve 32 g of PFA in 300 mL of D-PBS on a hot plate. PFA will dissolve at 60°C–62°C, pay close attention to not let the temperature rise above 65°C. Add NaOH until pH reaches 7.0. Filter through a coffee filter and top volume to 400 mL using D-PBS. Aliquot and freeze for up to 1 year at -20°C.

30% sucrose solution

Weigh 300 g of sucrose and dissolve in D-PBS to a final volume of 1 L. To improve shelf life, filter sterilize before storing. Store solution at 4°C for 2–4 weeks.

Gelatin Solution

Reagent	Final concentration	Amount
Sucrose	10%	10 g
D-PBS w/o Ca2+, Mg2+	-	100 mL
Gelatin	7.5%	7.5 g

Add 10 g of sucrose to 100 mL of D-PBS and dissolve. Add 7.5 g of gelatin to the sucrose solution and mix thoroughly. Keep gelatin solution in a water bath at 55°C until ready to use to avoid solidification. This is made fresh for each use; do not store.

PBS-T

Add 1 mL of Tween 20 to 1 L of D-PBS and mix thoroughly. This can be stored at a temperature between 15°C and 25°C for an extended period of time.

Citrate Buffer

Reagent	Final concentration	Amount
Tri-sodium citrate	10 μM	2.94 g
dH2O	-	1 L
HCl	-	-
NaOH	-	-
Tween 20	0.5%	0.5 mL

Add 2.94 g of Tri-sodium citrate (dihydrate) to 950 mL of dH₂O and dissolve completely. Adjust pH to 6.0 with HCl or NaOH. Add 0.5 mL of Tween 20 and top the entire volume up to 1 L using dH₂O. Mix thoroughly. Store at a temperature between 15°C and 25°C for up to 3 months, or at 4°C for longer.

Blocking Solution

Dilute Normal Donkey Serum in PBS-T to a final concentration of 5%. Make fresh for each use.

Primary Dilution Buffer

Add BSA to PBS-T to a final concentration of 5%. Mix thoroughly. Make fresh for each use.

Nuclear Staining Solution

Make a 0.5 μg/mL Nuclear Staining Solution by diluting 0.5 μL of Hoechst 33342 in 10 mL of 1x PBS. Mix thoroughly and wrap container in foil to protect from light exposure. Store at 4°C indefinitely.

Cryostat

Leica CM1860 UV Cryostat.

Orbital shaker

New Brunswick Scientific Innova 2000 Platform Shaker.

Steamer

Hamilton Beach digital food steamer, 5.5 quart; model 37530Z.

Epifluorescence microscope

Keyence BZ-X700 Series.

Step-by-step method details

Prepare organoids for immunofluorescence

Timing: 2–3 days

A proper histology protocol is essential to capture and analyze the complex cytoarchitecture of 3D cerebral organoids. Adequate embedding, fixation, cryoprotection and cryosectioning is key to minimize tissue damage prior to immunofluorescence. This protocol is adapted from STEMCELL Technologies.^2,3,5

• 1.
  Fix cerebral organoids (17 h).

  • a.
    Cut a transfer pipette tip to create a wider opening (option to use a p1000 wide-bore tip). Transfer organoids to a glass jar.
  • b.
    Wash organoids for 10 min in 1x PBS. Repeat twice.
  • c.
    Add 5 mL of 4% PFA per organoid. Incubate for 16 h or overnight.

    Note: Fixation time may need to be optimized for antibodies not included in this protocol.

    CRITICAL: While handling PFA, work in a chemical fume hood with proper PPE. Any solid materials used while handling PFA must be disposed of properly for chemical waste pickup.

  • d.
    Remove PFA and wash with 1x PBS for 10 min. Repeat twice.

    CRITICAL: Liquid PFA waste must be collected for proper disposal via chemical waste pickup.

  • e.
    Samples can be stored in PBS-T at 2°C–8°C for up to one week.
• 2.
  Cryoprotect organoids (16 h).

  • a.
    Remove PBS-T from organoids. Add 5 mL of 30% sucrose per organoid. Organoids will float at top of 30% sucrose solution.
  • b.
    Equilibrate overnight at 2°C–8°C. Cryoprotection is complete once organoids have sunk to the bottom of the glass jar.
• 3.
  Embed organoids (1 h).

  • a.
    Warm gelatin solution to 37°C.
  • b.
    Discard sucrose solution and add enough gelatin to completely cover the organoids.
  • c.
    Incubate at 37°C for 1 h.
  • d.
    Transfer organoids and gelatin solution to embedding mold. Use gelatin solution to fill up embedding mold while avoiding creating bubbles; center organoids before gelatin solution hardens.
• 4.
  Snapfreezing (1 h 30 min).

  • a.
    In an appropriate container mix dry ice and 100% ethanol.
  • b.
    Once the mixture stops boiling, add the molds containing the embedded sample so that they are in contact with the cold ethanol.
  • c.
    Keep the sample in the slurry until frozen. The gelatin will turn white and opaque.
  • d.
    Samples should be kept at –80°C for long-term storage.
• 5.
  Cryosectioning (2–3 h).

  • a.
    Remove frozen samples from storage and allow them to acclimate to a temperature in between –26 and –30°C in the cryostat chamber.
  • b.
    Section organoids at a thickness of 10 μm.

    CRITICAL: Be cautious to not to tear samples while cryosectioning; only an intact sample will yield data representative of the organoids’ cytoarchitecture.

  • c.
    Slides should be stored at –80°C.
    After sectioning, immunostaining is conducted following an adaptation of the protocol by STEMCELL Technologies.⁵ Here we will cover the steps prior to primary antibody incubation.
• 6.Antigen retrieval (50 min).
  Antigen retrieval must be done before any immunostaining can be performed on cryosectioned organoid samples.

  CRITICAL: Avoid the temperature reaching 100°C during antigen retrieval; this can cause damage to the tissue.

  • a.
    Add 800 μL of citrate buffer to cryosectioned samples, laying the slides flat on a movable surface, such as a wire rack.
  • b.
    Place slides in a food steamer, steam for 20 min carefully monitoring that the temperature in the steaming chamber does not rise above 99°C.
  • c.
    Remove slides and place in a Coplin staining jar. Let them cool down for 5 min.
  • d.
    Wash slides in the Coplin staining jar for 5 min with double-distilled water.
  • e.
    Wash slides in the Coplin staining jar for 10 min with PBS-T using gentle agitation. Repeat twice.
• 7.
  Blocking (1 h and 10 min).

  • a.
    Lay slides flat in a humidified chamber and pipette 200 μL of Blocking Solution on the samples.
  • b.
    Incubate at a temperature between 15°C and 25°C for 1 h.

Immunofluorescence

Timing: 1–2 days

All the following protocols rely on the same immunofluorescence protocol, detailed below. Please follow the steps of this protocol, and modify antibodies based on the desired assay.

CRITICAL: Never dry out your sample(s) while immunostaining. Keep samples in the dark when incubating with light sensitive reagents. Avoid air bubbles when mounting slides.

• 8.
  Incubate with primary antibody.

  • a.
    Following blocking, make dilution(s) of your antibody of interest in Primary Dilution Buffer. 200 μL of primary antibody solution is usually sufficient per slide. Antibodies and dilutions are detailed in respective protocols.
  • b.
    Add primary antibody to sample(s) and incubate in a humidified chamber at 4°C for 16 h or overnight.
• 9.
  Wash samples.

  • a.
    The following day, wash samples by adding glass slides with samples to a Coplin jar filled with PBS-T.
  • b.
    Incubate for 10 min with slight agitation on an orbital shaker. Repeat twice.
• 10.
  Incubate with secondary antibody.

  • a.
    Dilute Donkey anti Host secondary antibody at 1:200 in PBS-T; 200 μL of PBS-T is sufficient to cover each slide. Host and color of antibody is dependent on protocol.
  • b.
    Incubate secondary antibody for 2 h at a temperature between 15°C and 25°C in a dark humidified chamber.
• 11.Wash samples by repeating Step 2.
• 12.
  Stain nuclei.

  • a.
    Add Nuclear Staining Solution to samples; 200 μL per slide is sufficient.
  • b.
    Incubate for 10 min at a temperature between 15°C and 25°C in the dark.
• 13.
  Mount samples.

  • a.
    Pipette 80 μL of Prolong Gold Antifade onto the slide with sample(s). With the help of forceps, gently lower a coverslip to avoid air bubbles.
  • b.
    Let dry in a dark chamber for at least 2 h, or until completely dry.

Assay proliferative population of Day 21 cerebral organoids

Timing: 2–3 days

By Day 21 of cerebral organoid culture, neural rosettes have formed and begun the process of expansion and differentiation. In this period of rapid growth, assaying different properties of proliferation can reveal differences between organoids derived from distinct conditions (i.e., patient cell lines). The number and organization of neural rosettes in organoids can reveal the intrinsic ability to aggregate, organize and begin the differentiation process in cell lines of interest. The number of neural rosettes can be detected using N-cadherin staining, which outlines the ventricles of each rosette (Step 1). The overall size and organization of the neural rosettes can be used as an indicator of the size of the proliferative population in the ventricular zone; this can be done by staining with nuclear marker SOX2 (Step 2). Lastly, comparing the number of mitotic cells in neural rosettes can indicate differences in the proliferative population; this is done by staining with mitosis specific phosphorylated histone H3 and phosphorylated Vimentin (Step 3).

• 14.
  Detect number of neural rosettes using N-cadherin.

  • a.
    Follow immunofluorescence protocol detailed above. The primary antibody for this protocol is Rabbit anti N-cadherin (1:200); the secondary antibody is Donkey anti Rabbit 594 (1:200).
  • b.
    Image samples using an epifluorescence microscope with a 4x objective (Figure 1A).
  • c.
    Count the number of N-cadherin lined rosettes per organoid using the Cell Counter plugin in FIJI.

    Note: Rosettes are defined as having a lumen; count N-cadherin lined rosettes that have a lumen for a given cross-section of the sample(s). Refer to the example in Figure 1A and Cell Counter plug-in illustrated in Figure 1B.

  • d.
    Repeat for at least 3 organoids per condition and/or genotype. Assemble data for analysis using GraphPad Prism.

    CRITICAL: For each organoid select the widest cross-section for analysis.

• 15.
  Quantify average size of ventricular zone using SOX2.

  • a.
    Follow immunofluorescence protocol detailed above. The primary antibody for this protocol is Rabbit anti SOX2 (1:200); the secondary antibody is Donkey anti Rabbit 594 (1:200).
  • b.
    Image samples using an epifluorescence microscope with a 10x objective (Figure 2A).
  • c.
    Using FIJI, measure the thickness of 4-5 rosettes with clear lumens per organoid at 3 points; average these measurements together to obtain the average thickness of each rosette.

    Note: Refer to examples in Figures 2B and 2C.

  • d.
    Repeat for at least 3 organoids per condition and/or genotype. Assemble data for analysis using GraphPad Prism.

    CRITICAL: For each organoid select the widest cross-section for analysis.

• 16.
  Quantify number of mitotic cells using p-Vimentin and p-Histone H3 per neural rosette.

  • a.
    Follow immunofluorescence protocol detailed above. The primary antibodies for this protocol are Rat anti p-Histone H3 (1:500) and Rabbit anti p-Vimentin (1:100) Secondary antibodies are Donkey anti Rat 488 (1:200) and Donkey anti Rabbit 594 (1:200).
  • b.
    Image samples using an epifluorescence microscope with a 4x objective (Figure 3A).
  • c.
    Count the number of anchored mitotic cells (+p-Vim and +p-HH3) or purely mitotic cells (+p-HH3) per rosette using the Cell Counter plugin in FIJI.

    Note: The example in Figures 3B–3D shows three neural rosettes with cells stained with p-Histone H3, p-Vimentin, and Hoechst.

  • d.
    Repeat for at least 3 organoids per condition and/or genotype. Assemble data for analysis using GraphPad Prism.

    CRITICAL: For each organoid select the widest cross-section for analysis.

Figure 1.

Detection and quantification of neural rosettes

(A) Immunofluorescence of cerebral organoid cross-section, co-stained with N-cadherin in red and Hoechst 33342 nuclear stain in blue. All counted neural rosettes have lumens, numbered 1–21. Scale bar 100 μm.

(B) Image of Cell Counter plug-in in FIJI/ImageJ.

Figure 2.

Quantification of ventricular zone

(A) Immunofluorescence of cerebral organoid cross-section, co-stained with SOX2 in red and Hoechst 33342 nuclear stain in blue. Yellow boxes outline example neural rosettes used to quantify the average size of the ventricular zone. Scale bar 100 μm.

(B) and (C) Depiction of measurements taken to evaluate the thickness of rosettes boxed in (A). Scale bar 20 μm in both.

Figure 3.

Detection and quantification of mitotic progenitor cells

(A) Immunofluorescence of cerebral organoid cross-section, co-stained with p-Histone H3 in green, p-Vimentin in red, and Hoechst 33342 nuclear stain in blue Scale bar 100 μm. Yellow box is zoomed in in (B) with scale bar 100 μm. Individual channels are shown in (C) p-Histone in green, and (D) p-Vimentin in red. All counts made using Cell Counter.

Assay differentiation in Day 40 cerebral organoids

Timing: 2–3 days

By day 40, cerebral organoids have developed a layered architecture and begun the process of neuronal differentiation. By immunostaining for transcription factors specific to different cell fates such as PAX6, TBR2, TBR1, and CTIP2, we can discern different cell lineages as well as their arrangement within the organoid, which tells us about differentiation patterns. There are two different modes of neurogenesis seen in early neural development. Indirect neurogenesis occurs when a progenitor (PAX6+ radial glia cell) divides asymmetrically, giving rise to both a radial glia (PAX6+) and intermediate progenitor daughter cell (TBR2+).⁶ The intermediate progenitor then goes on to divide and make neurons. Direct neurogenesis occurs when a radial glia cell division skips the intermediate progenitor generation, with a daughter cell becoming a neuron directly.^6,7 Measuring the ratio of radial glia (PAX6+ nuclei) and intermediate progenitors (TBR2+ nuclei) in cerebral organoids can elucidate whether cerebral organoids derived from a cell line or condition of interest has a propensity towards direct or indirect neurogenesis (Step 1). TBR1 is a marker for post-mitotic neurons, while CTIP2 is a marker for neurons specified for layer V of the cortex. The colocalization of TBR1 and CTIP2 indicates a transition in neuron fate from newborn neuron to layer V; neurons solely expressing TBR1 have yet to acquire a cortical layer fate, and neurons solely expressing CTIP2 have committed to layer V of the cortex.^6,7 By measuring the colocalization of these two markers, the progression of early neurogenesis can be assessed (Step 2).

• 17.
  Quantify propensity towards indirect neurogenesis using the ratio of TBR2/PAX6

  • a.
    Follow immunofluorescence protocol detailed above. The primary antibodies for this protocol are Mouse anti PAX6 (1:200) and Rabbit anti TBR2 (1:200). Secondary antibodies are Donkey anti Mouse 594 (1:200) and Donkey anti Rabbit 488 (1:200).
  • b.
    Image samples using an epifluorescence microscope with a 4x objective.
  • c.
    Count the number of radial glia (PAX6+ nuclei) and intermediate progenitor cells (TBR2+ nuclei) within a defined region of interest (ROI), a 50 μm-wide column placed at the center of each rosette. Use the Cell Counter plugin in FIJI.

    Note: Refer to the example in Figure 4. Yellow rectangles in Figures 4B and 4C are 50 μm wide ROI.

    Note: Alternatively, the entire area of the rosette can be used for analysis.

  • d.
    Repeat for at least 3 organoids per condition and/or genotype. Assemble data for analysis using GraphPad Prism.

    CRITICAL: For each organoid select the widest cross-section for analysis.

• 18.
  Quantify colocalization of TBR1/CTIP2 neurons.

  • a.
    Follow immunofluorescence protocol detailed above. The primary antibodies for this protocol are Rat anti CTIP2 (1:500) and Rabbit anti TBR1 (1:200). Secondary antibodies are Donkey anti Rat 488 (1:200) and Donkey anti Rabbit 594 (1:200).
  • b.
    Image samples using an epifluorescence microscope with a 10x objective (Figures 5A–5C).
  • c.
    Select a ROI of 200 μm × 200 μm that covers both neuronal populations.

    Note: Refer to the example with the yellow box ROI shown in Figure 5A, and zoomed in in Figures 5D–5F.

  • d.
    Quantify the colocalization coefficient of TBR1+ and CTIP2+ neurons by using the JACoP plug-in FIJI (Figure 5G).

    CRITICAL: Make sure to check the settings in the red labeled tab for proper thresholding.

  • e.
    Repeat for at least 3 organoids per condition and/or genotype. Assemble data for analysis using GraphPad Prism.

    CRITICAL: For each organoid select the widest cross-section for analysis.

Figure 4.

Quantification of radial glia and intermediate progenitor cells

(A) Neural rosette of cerebral organoid cross-section, co-stained with PAX6 in red, TBR2 in green, and Hoechst 33342 nuclear stain in blue. Dashed yellow line traces border of rosette. Scale bar 50 μm. Individual channels are shown in (B) PAX6 in red, and (C) TBR2 in green. Yellow rectangle defines the region of interest for counting PAX6+ and TBR2+ positive nuclei using Cell Counter.

Figure 5.

Quantification of early neuron fate progression

(A) Immunofluorescence of cerebral organoid cross-section, co-stained with TBR1 in red, CTIP2 in green, and Hoechst 33342 nuclear stain in blue. Scale bar 100 μm. Individual red and green channels shown in (B) and (C), respectively. Yellow box 200 μm by 200 μm is zoomed in in (D) merged, (E) red channel, and (F) green channel.

(G) Path and conditions to use the JACoP plug-in in FIJI/ImageJ, used to quantify colocalization.

Expected outcomes

The progenitor population of the cerebral cortex gives rise to all excitatory neurons. Evaluation of progenitor populations and the various stages of differentiation across progenitor populations in cerebral organoids can help us infer the stage of development of the organoid and inform on molecular mechanisms of neurodevelopmental disorder (NDD) pathogenesis. By assaying and quantifying the number of neural rosettes, the thickness of the ventricular zone, and the number of actively mitotic cells per neural rosette, proliferative potential of the progenitor population can be inferred. In instances of decreased proliferative potential, one possible explanation could be an increase in asymmetrical cell division. A higher ratio of TBR2+ intermediate progenitor cells to PAX6+ radial glia cells is indicative of asymmetrical cell division favoring neurogenesis. Lastly, TBR1 and CTIP2 colocalization can be used to quantify the progression of newborn neurons as they begin to acquire cortical layer fate.

Quantification and statistical analysis

Appropriate statistical analysis dependent on experimental condition(s) can be done using the GraphPad Prism software. For example, if comparing the average number of neural rosettes across cerebral organoids from 3 different genotypes, use an unpaired one-way ANOVA (or Kruskall-Wallis if data are non-parametric).

Limitations

The major limitations of the described protocols are that they are completed using fixed tissues and an iPSC derived neural model. Therefore, these assays only detail a snapshot of the development of cerebral organoids from iPSC lines of interest, from which the data is expected to model early neural development in humans.

Troubleshooting

Problem 1

Variability among cerebral organoids. It is important to distinguish between good and bad differentiations. If you are using this protocol to compare control and patient-derived cerebral organoids, it is also important to identify if variability is a part of the phenotype.

Potential solution

• •
  Variability from organoid to organoid is to be expected. The STEMCELL Technologies STEMdiff Cerebral Organoid Kit protocol details the expected size of organoids at Day 5, how embedding in Matrigel should induce blebbing of the organoids, and other expected milestones of early organoid differentiation. While there are always outliers, the majority of your control cerebral organoids should develop along this trajectory; if they do not, consider:

  • ○
    Checking pluripotency of iPSCs
  • ○
    Karyotyping iPSCs (especially if CRISPR technology was used to develop cell lines)
  • ○
    Double counting your cells as you plate for initial embryoid body formation
  • ○
    Using a multichannel pipettor during initial plating step to minimize variability
  • ○
    Checking the quality of your hESC Matrigel
• •If you are working with patient-derived iPSCs and notice deviation from the expected trajectory of cerebral organoid differentiation across multiple biological replicates from multiple clones and/or patient-derived cell lines, this may be a phenotype of the patient genotype. If the iPSCs are healthy in culture (no differentiation, confirmed pluripotency), and the quality of the technique being used to culture organoids is confirmed by low variability and success with control iPSCs, it is possible the deviation from expected results is indicative of the impact the genotype of interest has on this process of differentiation. Some aspects that may be observed include smaller embryoid bodies (sometimes multiple per 96 well), differences in circularity of embryoid bodies, and reduced or increased blebbing of the neuroectoderm, among others.

Problem 2

Inconsistent development of neural rosettes in organoids. Neural rosette formation can vary based on genotype, especially those that impact cell migration and differentiation. Depending on the properties of the model being studied, there may be inconsistencies on the timeline of neural rosette formation.

Potential solution

• •A control cell line should always be included in the experiment to reference for expected rosette formation timelines as well as stage of progenitor cell differentiation.
• •You may find that neural rosettes are either not formed or further along in the process of differentiation than what is reported in this protocol for the mentioned timelines. Troubleshooting may require the incorporation of a pilot study to determine key time points that are representative of neurogenesis for the model being studied.

Problem 3

Sliced organoid samples have tears, crumbly quality, air bubbles, or other defects from technical error. (prepare organoids for immunofluorescence: Step 5).

Potential solution

• •If you notice tears in your organoid sections as you are cryosectioning them, try using a new razor blade or increasing the temperature of the cryostat.
• •Ensure that your samples are adequately cryoprotected in 30% sucrose. If you incubate fixed samples for 16 h or overnight in the sucrose solution and they have not sunk to the bottom of the tube, remove the sucrose solution and replace with fresh solution until they sink to the bottom. Once they sink to the bottom, they have been fully cryoprotected.
• •During the process of immunostaining, you must be very gentle with your slice organoid samples. Harsh pipetting can disturb the samples, even lifting parts off the slide. When mounting the samples, make sure you have no air bubbles! Air bubbles can look like lumens of neural rosettes and can skew data.

Problem 4

Low fluorescence signal in samples.

Potential solution

• •The process of antigen retrieval can damage sectioned organoid samples if not performed properly. Make sure that the temperature does not reach 100°C. Preparing fresh citrate buffer can also be a solution. (prepare organoids for immunofluorescence: Step 6).
• •We detail suggested dilutions of antibodies used in our experiments. If using other antibodies, or altering other aspects of the protocol (i.e., organoid section thickness), and struggling to see strong fluorescence, please play around with primary antibody dilutions.
• •Make sure light sensitive reagents are incubated in the dark. (immunofluorescence: Steps 3 through 6).
• •Increase exposure when imaging on the microscope.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Alicia Guemez-Gamboa (alicia.guemez@northwestern.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be fulfilled by the technical contact, Annika Schroder (annika.schroder@northwestern.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study does not report original code.