Human adherent cortical organoids in a multiwell format

Reviewer #1 (Public Review):

Summary:

Kroeg et al. describe a novel method for 2D culture human induced pluripotent stem cells (hiPSCs) to form cortical tissue in a multiwell format. The method claims to offer a significant advancement over existing developmental models. Their approach allows them to generate cultures with precise, reproducible dimensions and structure with a single rosette; consistent geometry; incorporating multiple neuronal and glial cell types (cellular diversity); avoiding the necrotic core (often seen in free-floating models due to limited nutrient and oxygen diffusion). The researchers demonstrate the method's capacity for long-term culture, exceeding ten months, and show the formation of mature dendritic spines and considerable neuronal activity. The method aims to tackle multiple key problems of in vitro neural cultures: reproducibility, diversity, topological consistency, and electrophysiological activity. The authors suggest their potential in high-throughput screening and neurotoxicological studies.

Strengths:

The main advances in the paper seem to be: The culture developed by the authors appears to have optimal conditions for neural differentiation, lineage diversification, and long-term culture beyond 300 days. These seem to me as a major strength of the paper and an important contribution to the field. The authors present solid evidence about the high cell type diversity present in their cultures. It is a major point and therefore it could be better compared to the state of the art. I commend the authors for using three different IPS lines, this is a very important part of their proof. The staining and imaging quality of the manuscript is of excellent quality.

Weaknesses:

(1) The title is misleading: The presented cultures appear not to be organoids, but 2D neural cultures, with an insufficiently described intermediate EB stage. For nomenclature, see: doi: 10.1038/s41586-022-05219-6. Should the tissue develop considerable 3D depth, it would suffer from the same limited nutrient supply as 3D models - as the authors point out in their introduction.

(2) The method therefore should be compared to state-of-the-art (well-based or not) 2D cultures, which seems to be somewhat overlooked in the paper, therefore making it hard to assess what the advance is that is presented by this work.

(3) Reproducibility is prominently claimed throughout the manuscript. However, it is challenging to assess this claim based on the data presented, which mostly contain single frames of unquantified, high-resolution images. There are almost no systematic quantifications presented. The ones present (Figure S1D, Figure 4) show very large variability. However, the authors show sets of images across wells (Figure S1B, Figure S3) which hint that in some important aspects, the culture seems reproducible and robust.

(4) What is in the middle? All images show markers in cells present around the center. The center however seems to be a dense lump of cells based on DAPI staining. What is the identity of these cells? Do these cells persist throughout the protocol? Do they divide? Until when? Addressing this prominent cell population is currently lacking.

(5) This manuscript proposes a new method of 2D neural culture. However, the description and representation of the method are currently insufficient.
(a) The results section would benefit from a clear and concise, but step-by-step overview of the protocol. The current description refers to an earlier paper and appears to skip over some key steps. This section would benefit from being completely rewritten. This is not a replacement for a clear methods section, but a section that allows readers to clearly interpret results presented later.
(b) Along the same lines, the graphical abstract should be much more detailed. It should contain the time frames and the media used at the different stages of the protocol, seeding numbers, etc.

Reviewer #2 (Public Review):

Summary:

In this manuscript, van der Kroeg et al have developed a method for creating 3D cortical organoids using iPSC-derived neural progenitor cells in 384-well plates, thus scaling down the neural organoids to adherent culture and a smaller format that is amenable to high throughput cultivation. These adherent cortical organoids, measuring 3 x 3 x 0.2 mm, self-organize over eight weeks and include multiple neuronal subtypes, astrocytes, and oligodendrocyte lineage cells.

Strengths:

(1) The organoids can be cultured for up to 10 months, exhibiting mature dendritic spines, axonal myelination, and robust neuronal activity.

(2) Unlike free-floating organoids, these do not develop necrotic cores, making them ideal for high-throughput drug discovery, neurotoxicological screening, and brain disorder studies.

(3) The method addresses the technical challenge of achieving higher-order neural complexity with reduced heterogeneity and the issue of necrosis in larger organoids. The method presents a technical advance in organoid culture.

(4) The method has been demonstrated with multiple cell lines which is a strength.

(5) The manuscript provides high-quality immunostaining for multiple markers.

Weaknesses:

(1) Direct head-to-head comparison with standard organoid culture seems to be missing and may be valuable for benchmarking, ie what can be done with the new method that cannot be done with standard culture and vice versa, ie what are the aspects in which new method could be inferior to the standard.

(2) It would be important to further benchmark the throughput, ie what is the success rate in filling and successfully growing the organoids in the entire 384 well plate?

(3) For each NPC line an optimal seeding density was estimated based on the proliferation rate of that NPC line and via visual observation after 6 weeks of culture. It would be important to delineate this protocol in more robust terms, in order to enable reproducibility with different cell lines and amongst the labs.

Reviewer #3 (Public Review):

Summary:

Kroeg et al. have introduced a novel method to produce 3D cortical layer formation in hiPSC-derived models, revealing a remarkably consistent topography within compact dimensions. This technique involves seeding frontal cortex-patterned iPSC-derived neural progenitor cells in 384-well plates, triggering the spontaneous assembly of adherent cortical organoids consisting of various neuronal subtypes, astrocytes, and oligodendrocyte lineage cells.

Strengths:

Compared to existing brain organoid models, these adherent cortical organoids demonstrate enhanced reproducibility and cell viability during prolonged culture, thereby providing versatile opportunities for high-throughput drug discovery, neurotoxicological screening, and the investigation of brain disorder pathophysiology. This is an important and timely issue that needs to be addressed to improve the current brain organoid systems.

Weaknesses:

While the authors have provided significant data supporting this claim, several aspects necessitate further characterization and clarification. Mainly, highlighting the consistency of differentiation across different cell lines and standardizing functional outputs are crucial elements to emphasize the future broad potential of this new organoid system for large-scale pharmacological screening.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Kroeg et al. describe a novel method for 2D culture human induced pluripotent stem cells (hiPSCs) to form cortical tissue in a multiwell format. The method claims to offer a significant advancement over existing developmental models. Their approach allows them to generate cultures with precise, reproducible dimensions and structure with a single rosette; consistent geometry; incorporating multiple neuronal and glial cell types (cellular diversity); avoiding the necrotic core (often seen in free-floating models due to limited nutrient and oxygen diffusion). The researchers demonstrate the method's capacity for long-term culture, exceeding ten months, and show the formation of mature dendritic spines and considerable neuronal activity. The method aims to tackle multiple key problems of in vitro neural cultures: reproducibility, diversity, topological consistency, and electrophysiological activity. The authors suggest their potential in high-throughput screening and neurotoxicological studies.

Strengths:

The main advances in the paper seem to be: The culture developed by the authors appears to have optimal conditions for neural differentiation, lineage diversification, and long-term culture beyond 300 days. These seem to me as a major strength of the paper and an important contribution to the field. The authors present solid evidence about the high cell type diversity present in their cultures. It is a major point and therefore it could be better compared to the state of the art. I commend the authors for using three different IPS lines, this is a very important part of their proof. The staining and imaging quality of the manuscript is of excellent quality.

We thank the reviewer for the positive comments on the potential of our novel platform to address key problems of in vitro neural culture, highlighting the longevity and reproducibility of the method across multiple cell lines.

Weaknesses:

(1) The title is misleading: The presented cultures appear not to be organoids, but 2D neural cultures, with an insufficiently described intermediate EB stage. For nomenclature, see: doi: 10.1038/s41586-022-05219-6. Should the tissue develop considerable 3D depth, it would suffer from the same limited nutrient supply as 3D models - as the authors point out in their introduction.

We appreciate the opportunity to clarify this point. We respectfully disagree that the cultures do not meet the consensus definition of an organoid. In fact, a direct quote from the seminal nomenclature paper referenced by the reviewer states: “We define organoids as in vitro-generated cellular systems that emerge by self-organization, include multiple cell types, and exhibit some cytoarchitectural and functional features reminiscent of an organ or organ region. Organoids can be generated as 3D cultures or by a combination of 3D and 2D approaches (also known as 2.5D) that can develop and mature over long periods of time (months to years).” (Pasca et al, 2022 doi10.1038/s41586-022-05219-6). Therefore, while many organoid types indeed have a more spherical or globular 3D shape, the term organoid also applies to semi-3D or non-globular adherent organoids, such as renal (Czerniecki et al 2018, doi.org/10.1016/j.stem.2018.04.022) and gastrointestinal organoids (Kakni et al 2022, doi.org/10.1016/j.tibtech.2022.01.006). Accordingly, the adherent cortical organoids described in the manuscript exhibit self-organization to single radial structures consisting of multiple cell layers in the z-axis, reaching ~200um thickness (therefore remaining within the limits for sufficient nutrient supply), with consistent cytoarchitectural topology and electrophysiological activity, and therefore meet the consensus definition of an organoid.

(2) The method therefore should be compared to state-of-the-art (well-based or not) 2D cultures, which seems to be somewhat overlooked in the paper, therefore making it hard to assess what the advance is that is presented by this work.

It was not our intention to benchmark this model quantitatively against other culture systems. Rather, we have attempted to characterize the opportunities and limitations of this approach, with a qualitative contrast to other culture methods. Compared to state-of-the-art 2D neural network cultures, adherent cortical organoids provide distinct advantages in:

(1) Higher order self-organized structure formation, including segregation of deeper and upper cortical layers.

(2) Longevity: adherent cortical organoids can be successfully kept in culture up to 1 year where 2D cultures typically deteriorate after 8-12 weeks.

(3) Maturity, including the formation of dendritic mushroom spines and robust electrophysiological activity.

(4) Cell type diversity including a more physiological ratio of inhibitory and excitatory neurons (10% GAD67+/NeuN+ neurons in adherent cortical organoids, vs 1% in 2D neural networks) and the emergence of oligodendrocyte lineage cells.

On the other hand, limitations of adherent cortical organoids compared to 2D neural network cultures are:

(1) Culture times for organoids are much longer than for 2D cultures and the method can therefore be more laborious and more expensive.

(2) Whole cell patch clamping is not easily feasible in the organoids because of the restricting dimensions of the 384well plates.

(3) Reproducibility is prominently claimed throughout the manuscript. However, it is challenging to assess this claim based on the data presented, which mostly contain single frames of unquantified, high-resolution images. There are almost no systematic quantifications presented. The ones present (Figure S1D, Figure 4) show very large variability. However, the authors show sets of images across wells (Figure S1B, Figure S3) which hint that in some important aspects, the culture seems reproducible and robust.

We made considerable efforts to establish quantitative metrics to assess reproducibility. We applied a quantitative scoring system of single radial structures at different time points for multiple batches of all three lines as indicated in Figure S1D. This figure represents a comprehensive dataset in which each dot represents the average of a different batch of organoids containing 10-40 organoids per batch. To emphasize this, we will adapt the graph to better reflect the breadth of the dataset. Additional quantifications are given in Figure S2 for progenitor and layer markers for Line 1 and in Figure S5 for interneurons across all three lines, showing relatively low variability. That being said, we acknowledge the reviewer’s concerns and will modify the text to reduce the emphasis of this point, pending more extensive data addressing reproducibility across a wide range of parameters.

(4) What is in the middle? All images show markers in cells present around the center. The center however seems to be a dense lump of cells based on DAPI staining. What is the identity of these cells? Do these cells persist throughout the protocol? Do they divide? Until when? Addressing this prominent cell population is currently lacking.

A more comprehensive characterization of the cells in the center remains a significant challenge due to the high cell density hindering antibody penetration. However, dye-based staining methods such as DAPI and the LIVE/DEAD panel confirm a predominance of intact nuclei with very minimal cell death. The limited available data suggest that a substantial proportion of the cells in the center are proliferative neural progenitors, indicated by immunolabeling for SOX2 and Ki67. We will add additional figures to support these findings. Furthermore, we are currently optimizing the conditions to perform single cell / nuclear RNA sequencing to further characterize the cellular composition of the organoids.

(5) This manuscript proposes a new method of 2D neural culture. However, the description and representation of the method are currently insufficient.
(a) The results section would benefit from a clear and concise, but step-by-step overview of the protocol. The current description refers to an earlier paper and appears to skip over some key steps. This section would benefit from being completely rewritten. This is not a replacement for a clear methods section, but a section that allows readers to clearly interpret results presented later.

We will revise the manuscript to include a more detailed step-by-step overview of the protocol.

(b) Along the same lines, the graphical abstract should be much more detailed. It should contain the time frames and the media used at the different stages of the protocol, seeding numbers, etc.

As suggested, we will also adapt the graphical abstract to include more detail.

Reviewer #2 (Public Review):

Summary:

In this manuscript, van der Kroeg et al have developed a method for creating 3D cortical organoids using iPSC-derived neural progenitor cells in 384-well plates, thus scaling down the neural organoids to adherent culture and a smaller format that is amenable to high throughput cultivation. These adherent cortical organoids, measuring 3 x 3 x 0.2 mm, self-organize over eight weeks and include multiple neuronal subtypes, astrocytes, and oligodendrocyte lineage cells.

Strengths:

(1) The organoids can be cultured for up to 10 months, exhibiting mature dendritic spines, axonal myelination, and robust neuronal activity.

(2) Unlike free-floating organoids, these do not develop necrotic cores, making them ideal for high-throughput drug discovery, neurotoxicological screening, and brain disorder studies.

(3) The method addresses the technical challenge of achieving higher-order neural complexity with reduced heterogeneity and the issue of necrosis in larger organoids. The method presents a technical advance in organoid culture.

(4) The method has been demonstrated with multiple cell lines which is a strength.

(5) The manuscript provides high-quality immunostaining for multiple markers.

We appreciate the reviewer’s acknowledgement of the strengths of this novel platform as a technical advance in organoid culture that reduces heterogeneity and shows potential for higher throughput experiments.

Weaknesses:

(1) Direct head-to-head comparison with standard organoid culture seems to be missing and may be valuable for benchmarking, ie what can be done with the new method that cannot be done with standard culture and vice versa, ie what are the aspects in which new method could be inferior to the standard.

In our opinion, it would be extremely difficult to directly compare methods because of substantial differences. Most notably, whole brain organoids grow to large and irregular globular shapes, while adherent cortical organoids have a highly standardized shape confined by the limits of a 384-well. Moreover, it was not our intention to benchmark this model quantitatively against other culture systems. Rather, we have attempted to characterize the opportunities and limitations of this approach, with a qualitative contrast to other culture methods.

(2) It would be important to further benchmark the throughput, ie what is the success rate in filling and successfully growing the organoids in the entire 384 well plate?

Figure S1D shows the success rate of organoid formation and stability of the organoid structures over time. In addition, we will add the number of wells that were filled per plate.

(3) For each NPC line an optimal seeding density was estimated based on the proliferation rate of that NPC line and via visual observation after 6 weeks of culture. It would be important to delineate this protocol in more robust terms, in order to enable reproducibility with different cell lines and amongst the labs.

Figure S1C provides the relationship between proliferation rate and seeding density, allowing estimation of seeding densities based on the proliferation rate of the NPCs. However, we appreciate the reviewers feedback and will modify the methods to provide more detail.

Reviewer #3 (Public Review):

Summary:

Kroeg et al. have introduced a novel method to produce 3D cortical layer formation in hiPSC-derived models, revealing a remarkably consistent topography within compact dimensions. This technique involves seeding frontal cortex-patterned iPSC-derived neural progenitor cells in 384-well plates, triggering the spontaneous assembly of adherent cortical organoids consisting of various neuronal subtypes, astrocytes, and oligodendrocyte lineage cells.

Strengths:

Compared to existing brain organoid models, these adherent cortical organoids demonstrate enhanced reproducibility and cell viability during prolonged culture, thereby providing versatile opportunities for high-throughput drug discovery, neurotoxicological screening, and the investigation of brain disorder pathophysiology. This is an important and timely issue that needs to be addressed to improve the current brain organoid systems.

We thank the reviewer for highlighting the strengths of our novel platform. We appreciate that all three reviewers agree that the adherent cortical organoids presented in this manuscript reliably demonstrate increased reproducibility and longevity. They also commend its potential for higher throughput drug discovery and neurotoxicological/phenotype screening purposes.

Weaknesses:

While the authors have provided significant data supporting this claim, several aspects necessitate further characterization and clarification. Mainly, highlighting the consistency of differentiation across different cell lines and standardizing functional outputs are crucial elements to emphasize the future broad potential of this new organoid system for large-scale pharmacological screening.

We appreciate the feedback and will add more detail on consistency and standardization of functional outputs.