Engineering Morphogen Gradients to Grow Larger, Spatially Patterned Human Cortical Organoids

Scientific rationale and importance:

Organoids are human-induced pluripotent stem cell (iPSC)-derived 3D in vitro structures that model complex cellular interactions among various subpopulations and recapitulate aspects of human anatomy^1,2. Organoids have recently emerged as powerful modeling tools for developmental and disease-specific cellular interactions^1,3–6. However, organoid models exhibit high variability, both within and between batches, limiting their usefulness^1,3,5–7. Critically, organoids also do not present defined spatial patterning, which is specifically concerning for brain/cortical organoids^2,6,8–11. The cerebral cortex exhibits anteroposterior and medial-lateral patterning, established by morphogenetic gradients¹², and without this patterning, cortical organoids cannot acquire the area patterning specifications essential for cortical function. While specific tools and strategies have been established to mitigate variability¹³, little has been done to address regional patterning in cortical organoids. We propose to draw on classical experiments in developmental biology^14,15 and quickly and effectively generate in vitro gradients to create facsimiles of cortical regional identity. Our proposal will establish a rapid, cost-effective proof-of-concept demonstration that large, yet viable, brain organoids can be patterned to acquire specific regional contexts and will enable the routine generation of larger, more spatially defined cortical organoids, fully aligned with Project Gigabrain’s Track 1 emphasis on scaling organoid size and viability. Our patterning approach will potentiate a new technological leap in organoid modeling, enabling us to address region-specific pathologies and developmental questions.

Funding/facilities/ethics approvals:

The Pereira lab has state-of-the-art facilities for organoid culture, including standard tissue culture equipment (two laminar flow hoods, two incubators, centrifuges) and non-standard equipment such as two Clinostar II incubators (CellVivo, one of them with hypoxia) that allow for constant, vertical, centrifugal spinning of organoids, greatly reducing intra- and inter-batch variability, and an ImageXpress HT.ai high-throughput confocal (Molecular Devices) and a MuVie SPIM Lightsheet microscope (Bruker), all optimized for organoid work. We have a generous start-up and external funding from foundations for neurodegenerative disease-related work, and are supported by core facilities at the University of Alabama at Birmingham, the Killion Center for Neurodegeneration and Experimental Therapeutics, and the Department of Neurology at the Heersink School of Medicine. No additional equipment will be required for this project. All proposed work is pending IRB approval, but iPSC work is typically exempt.

Estimated Timeline:

It is estimated that this project will reach completion within a year; see Table 1 below for specific milestones.

Table 1: Gantt Chart of specific milestones.

2. Introduction:

Cortical organoid cultures are 3D, non-adherent in vitro tissue cultures. This free-floating aspect is essential: it enables cellular aggregation and internal patterning that, in the presence of external cues, are permissive of neuronal differentiation^2,8,16. Starting from iPSCs, the first step in differentiation is a push towards an ectodermal lineage, and the formation of neuroepithelium, typically detected at 15-20 days in vitro (d.i.v.), followed by neuronal differentiation, which typically follows the in vivo temporal order, with deep layers of the cortex (6 and 5) being formed first, followed by the upper layers (4-2), and astrogliogenesis². However, although the temporal order follows corticogenesis, the spatial order is inherently disorganized^2,10,11. Early organoid models would aggregate in culture, generating large clumps that were not larger organoids but rather the result of the fusion of smaller organoids through physical contact. Areas of individually differentiated neuroepithelium were generated throughout the organoids, resembling many small versions of a developing cerebral cortex². The use of specialized equipment, such as organoid incubators, enabled the growth of organoids in disposable bioreactors that prevent intra-organoid contact and greatly standardize size and reduce variability within and between individual batches. Despite this advancement, the issue remains: multiple distinct neuroepithelia are present, with no form of guided regional patterning within each individual organoid. The lack of spatial organization is particularly striking in disease models that require regional identity (e.g., motor cortex), and organoids cannot deliver on the promise of complex disease models until the diversity of area-specific cell types is fully recapitulated within a dish^3,6,7,16. Complex approaches to solving this issue, such as microfluidics-based morphogen gradient generation, while elegant, lack scalability and are resource-intensive, posing significant barriers to widespread use¹⁷.

To address this gap in organoid biology, we turned to developmental biology, which faced similar questions when attempting to define morphogenetic gradients¹⁵. Isolating the molecules responsible for antero-posterior and medial-lateral gradients was not sufficient to prove the concept of a signaling molecule gradient that could propagate across tissue and define cellular and regional identity in a concentration-dependent manner. Chick embryos were exposed to beads bound to relevant molecules that released the proposed morphogens from a specific position relative to the embryo, thereby altering cellular identity depending on the beads' placement¹⁵. These elegant experiments helped define regional patterning and morphogenetic gradients and are key to what we propose to accomplish in organoids.

Hypothesis (H1): We propose that the induction of an external gradient of FGF-2/Activin A is sufficient to induce specification of a regional pattern in large cortical organoids. We hypothesize that the absence of regional patterning in organoids stems from the absence of morphogenetic gradients and that these gradients can be replicated in vitro, thereby increasing the spatial definition of cortical organoids. Further, we propose that improving spatial organization via morphogen gradients will reduce chaotic internal folding and hypoxic zones, and support the formation of larger, healthier organoids.

We will adapt a simple, standardized method for mounting zebrafish embryos for imaging during development to organoid cultures and induce an antero-dorsal gradient with slow-release beads¹⁸, using FGF-2 and Activin A as proof-of-concept. Then will measure the distribution of region-specific cell types and proteins by immunocytochemistry, and neural activity by calcium imaging.

Impact Statement: Our study will define the role of morphogenetic gradients in organoid biology and generate a technological leap forward in the complexity and disease relevance of cortical organoids.

3. Methods:

Participants/Sample

Model: We will establish a proof-of-concept using the well-characterized control iPSC line KOLF2.1J¹⁹. The KOLF2.1J line has been CRISPR-edited to generate lines with mutations associated with neurodegenerative diseases²⁰, and establishing this assay in the KOLF2.1J line will allow us, in the future, to characterize regionalized organoids within that existing framework. In addition, we will validate the final working conditions in n=3 independent control iPSC lines (Answer ALS) used for validation. N-per-condition and timepoint: 6-8 organoids. Although a traditional power analysis isn’t typically applied to iPSC studies, this n number has been sufficient to provide power for medium effects in spatial marker gradients using linear mixed-effect models.

Inclusion/Exclusion: Organoids will be generated using the Clinostar II incubators and bioreactors, which eliminate the risk of fusion. We will exclude organoids that deviate more than 20% in size from the average of their batch. Organoids with viability after cryosectioning 85% or lower than the average (quantified by cleaved caspase 3 immunostaining) or a necrotic core above 20% of organoid area will be excluded from downstream analysis.

Materials and Procedures

Gradients: To model FGF and WNT signaling gradients in vitro, we will use FGF-2 and Activin A Stembeads (Stem Cultures). Steambeads are slow-release microbeads designed for stem cell culture, in which growth factors are encapsulated in a PLGA hydrogel, thereby increasing their stability and enabling gradual release. Typical use of the FGF-2 Stembeads is 8 µl of bead mix per milliliter of medium to achieve a release of 10 ng/ml, with media exchanges every 3 days, whereas the Activin-A beads require 10 µl of mix per ml of medium for the same release. For both beads, the release rate is linear, allowing concentration adjustment. At d.i.v. 15, when the neuroepithelium begins to form, we will use low-melting agarose and our epMotion 96 pipetting robot (Eppendorf) to systematically and consistently generate three pedestal-type structures in each well of a six-well plate: two diametrically opposed pedestals and a third one in the center of the well. The organoids (in groups of 6-8) will be placed on the center pedestal, while the beads will be placed in one of the two other pedestals. For initial experiments, we will test FGF-2 only, in a concentration range (5, 10, 20, 40, 60, 80, 100 ng/ml), in cortical differentiation/maturation media without FGF. Once a minimum concentration that induces anterior patterning has been established, we will use Activin A beads to induce a posterior fate by placing them on the opposite pedestal to FGF, at the same concentration range. After two weeks of patterning, the organoids will be returned to Clinostar II incubators and bioreactors to support large-scale, uniform growth.

Organoid characterization and readouts: Organoids will be collected at D30, D45, D60, D120, D200. Readouts will include: cryosectioning (sagittal, oriented per FGF2/Activin A gradient) and immunostaining for PAX6, FOXG1, EMX2 (anterior/dorsal); NR2F1/NR2F2 (posterior); CTIP2 (layer 5 and 6), TBR1 (layer 6), SATB2 (layers 2-3), Ki67 (cycling progenitors), and cleaved caspase 3 (apoptosis).

Calcium imaging: Live organoids will be sectioned in a vibratome (D45-D60), and live slices will be infected with GCAMP6s. Calcium imaging will be performed in our HT.ai confocal microscope, as previously established, and analyzed in our ImageJ/FIJI pipeline.

Controls: We will use no-bead controls, empty beads, and beads with a single factor (FGF-2 or Activin A).

Planned Analyses

Pre-processing: Organoids will be released from low-melting agarose by gentle heating to 37-42°C, as for zebrafish embryos. Organoids will be collected with a plastic Pasteur pipette, with care to preserve orientation (FGF-2 side facing up). Organoids will be cryoprotected in a 10-30% sucrose gradient to preserve orientation and embedded in OCT in a plastic mold, with care to mark the orientation of FGF-2. Sectioning will be performed in the sagittal plane (parallel to the gradient) at 40 μm thickness intervals. Distance bins for quantification will be set at 100 μm intervals.

Primary outcomes (H1): Primary outcomes will be the differential and gradual expression of markers, such as high EMX2 near the FGF-2 source and high NR2F2 near the Activin-A source, with intensity decreasing as distance from the source increases.

Statistical tests: For immunocytochemistry, we will use linear mixed-effects models (LMMs) with random effects for Line and Batch to quantify specific markers relative to the distance from FGF-2 and Activin-A sources, with Benjamini-Hochberg adjustment for false discovery rate. For calcium imaging, we will quantify event rate, burst synchronicity, and propagation velocity depending on distance bins using LMMs.

If-Then contingencies:

· IF necrosis is over 20% at day 30, then reduce the bead concentration to one side or increase the bead distance from the center pedestal.

· IF FGF-2 biases towards proliferation but not patterning THEN shorten exposure (48h pulses) and increase washout.

· IF Activin-A induces a mesoderm fate (tested by expression of TBXT, the mesodermal transcription factor Brachyury) THEN drop dose by 50%.

Expected outcomes:

· Larger, more robust, cortical organoids with smaller necrotic cores (due to incubation in Clinostar biorreactors).

· Formation of opposing gradients in regional markers (anterior/posterior).

· Improved cytoarchitectural organization and region-biased network activity.

Caveats/Alternatives:

· Overpatterning and toxicity: we will titrate the beads to determine the minimum concentrations required for FGF-2 and Activin-A and quantify cell viability.

· Bead drift: This is unlikely, but we will confirm immobilization by imaging and exclude it if there is drift of> 100 μm.

· If the data support it, we will apply for a bridge grant to perform spatial transcriptomics.

Ethics and Data Management

iPSC studies are exempt from IRB approval, although they will be submitted for revision if the grant is awarded. There is no patient identifying information associated with this study. Raw and processed data will be stored on an institutional server and made publicly available in GEO. The code will be made available on GitHub, and any publications will be made available as preprints.

4. Pilot Data (Optional):

We have not generated pilot data for this proposal. However, we have an established pipeline for generating reproducible cortical organoids, infrastructure specialized for organoid growth and imaging, and prior experience with the proposed cellular markers and calcium-imaging workflows.

5. Budget:

Personal: Post-doctoral fellow (10% including fringe): $9,473.00

Reagents: FGF-2 and Activin-A beads: $2,500.00.

iPSC and Organoid culture media: $4,500.00 (including reagents for basic iPSC culture such as Matrigel)

Labware: Standard tissue culture plasticware: $1,000.00.

Single-use Bioreactor plastic vessels (for CellVivo Clinostar II incubators): $4,000.00

Viral particles: GCAMP6s (Addgene) $1,500.00

Antibodies: $2027.00. A considerable number of antibodies are already available in the laboratory.

Of note, the high-throughput HT.ai confocal and light sheet microscopes are located in the Pereira laboratory and will be available for free use.

6. References

1. Chang, Y., Kim, J., Park, H., Choi, H. & Kim, J. Modelling neurodegenerative diseases with 3D brain organoids. https://doi.org/10.1111/brv.12626 doi:10.1111/brv.12626.

2. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

3. Papaspyropoulos, A., Tsolaki, M., Foroglou, N. & Pantazaki, A. A. Modeling and Targeting Alzheimer’s Disease With Organoids. Frontiers in Pharmacology vol. 11 396 Preprint at https://doi.org/10.3389/fphar.2020.00396 (2020).

4. Qian, X. et al. Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation. Cell Stem Cell 26, 766-781.e9 (2020).

5. Sreenivasamurthy, S., Laul, M., Zhao, N., Kim, T. & Zhu, D. Current progress of cerebral organoids for modeling Alzheimer’s disease origins and mechanisms. Bioeng. Transl. Med. 8, e10378 (2022).

6. Quadrato, G., Brown, J. & Arlotta, P. The promises and challenges of human brain organoids as models of neuropsychiatric disease. Nat. Med. 22, 1220–1228 (2016).

7. Di Lullo, E. & Kriegstein, A. R. The use of brain organoids to investigate neural development and disease. Nat. Rev. Neurosci. 18, 573–584 (2017).

8. Chiaradia, I. & Lancaster, M. A. Brain organoids for the study of human neurobiology at the interface of in vitro and in vivo. Nature Neuroscience vol. 23 1496–1508 Preprint at https://doi.org/10.1038/s41593-020-00730-3 (2020).

9. Sherwood, J., Quadrato^*^, G., Sherwood^*^, J. L. & Arlotta, P. Long-term culture and electrophysiological characterization of human brain organoids. Protoc. Exch. https://doi.org/10.1038/protex.2017.049 (2017) doi:10.1038/protex.2017.049.

10. Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017).

11. Velasco, S. et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 570, 523–527 (2019).

12. Appan, D., Hsu, S. M., Hsu, W. H. & Chou, S. J. Patterning the cerebral cortex into distinct functional domains during development. Curr. Opin. Neurobiol. 80, 102698 (2023).

13. Pereira, J. D. et al. Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions. Nature Communications 2021 12:1 12, 1–17 (2021).

14. Vieira, C., Garda, A. L., Shimamura, K. & Martinez, S. Thalamic development induced by Shh in the chick embryo. Dev. Biol. 284, 351–363 (2005).

15. Simsek, M. F. & Özbudak, E. M. Patterning principles of morphogen gradients. Open Biol. 12, (2022).

16. Lancaster, M. A. & Knoblich, J. A. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science (1979). 345, 1247125–1247125 (2014).

17. Dissecting how morphogens shape cell fates in human neural organoids. Nature Methods 2025 23:2 23, 297–298 (2025).

18. Graeden, E. & Sive, H. Live imaging of the zebrafish embryonic brain by confocal microscopy. Journal of Visualized Experiments https://doi.org/10.3791/1217 (2009) doi:10.3791/1217.

19. Pantazis, C. B. et al. A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell Stem Cell 29, 1685-1702.e22 (2022).

20. Ramos, D. M., Skarnes, W. C., Singleton, A. B., Cookson, M. R. & Ward, M. E. Tackling neurodegenerative diseases with genomic engineering: A new stem cell initiative from the NIH. Neuron 109, 1080–1083 (2021).

Engineering Morphogen Gradients to Grow Larger, Spatially Patterned Human Cortical Organoids

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Engineering Morphogen Gradients to Grow Larger, Spatially Patterned Human Cortical Organoids

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Proposal

Proposal

Funding Progress

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Peer Reviews

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Funding Progress

Nonprofit Recipient

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Peer Reviews

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