Single-Cell Assessment of Human Stem Cell-Derived Mesolimbic Models and Their Responses to Substances of Abuse

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A very nice study comparing different protocols for 2D and 3D cultures that could/should contain MSN. The author then move on to look at the transcriptional responses to treatment in two different 3D models. The study is well done but poorly described. Overall, when the analysis and results are described, the manuscript is clear.  However, the description of the logic behind the study and the experimental design is convoluted and sometime contradictory. I am left to infer the purpose of the experiments from the Figures. The data presentation in Figures 1-3 is well done.  The display of the differential gene expression analysis needs to be improved.

Major:

-              Lack of clarity in description of the study.

-              The assembloid model is never described or characterized clearly. The untreated assembloid scRNAseq should be merged with the 7 protocols RNAseq and compared for cell types and subtypes ect. Some cryosections with staining to characterize the cell types should be performed.

-              Immunohistology should also be done on the 7 cultures characterized by scRNAseq and compared to the scRNAseq results.

-              The referencing in the introduction needs to be changed so that references are matched to the fact they are referencing, not just making several claims, and adding 5 references to the end of a sentence. In some places one reference would be sufficient. The referencing appears to be correct in the results section.

 

Specific issues:

Abstract:

-              The final sentence of the abstract is very confusing final sentence in abstract

-              As in the entire study what is done is not clear: are they making mixed cultures of iPSC derived DA and spiny neurons – spheroid?  Assembloids? The description on lines 64 – 67 of the introductions the authors summarize what they do in the manuscript. However, this description doesn’t completely match what is done in the manuscript.

Introduction:

-              On lines 40-50 “As an example, banked blood samples …” This sentences implys makes 100s of different cell lines are induced together

-              Line 48 – what do the authors mean by “variability in basal media compositions?”  Why is there no reference here?

-              Line 49-63 the references need to be split to match the facts referred to.

-              In the description of the models where it is unknown if there are how many MSN are there, they don’t mention striatal organoids specifically.  I find this odd as these are these models should have the MSN in abundance. It is also not clear if these other studies have not checked for MSN or were they just poorly characterized?

-              Are there clear reference transcriptional profiles to identify MSNs? Do they need to be identified by functional activity (ephys) and morphology? 

-              The authors say they are going to compare 7 2D and 3D protocols.  They should provide a simple table with the protocol reference, the culture type and the previously described proportion of MSN or other relevant detail, something to indicate why it was selected.  

-              The introduction implies that cerebral organoids have/may have MSN, are these included in the 7 protocols. They reference 15 2D protocols and 7 3D protocols.  What was the criteria for inclusion in the 7 for comparison?  Are the 7 protocols the two they develop and the 5 listed in Figure 1B?

Results:

-              In the results section the authors start of saying their goal is to compare protocols but instead they will develop their own.  I understand the difficulty in framing the study, but this round about is confusing to the reader.  Is the goal to develop a better protocol or to benchmark?  The focus needs to be improved/clarified/reframed.

-              In the introduction their goal is to characterize and compare but then suddenly in the results the goal is now to develop a model to have forebrain and midbrain organoids in culture, but early fusion of the two separate patterning protocols.

-              Is Figure 1 containing data from this paper? (I’m assuming yes, but this is not clear in the description) Did they grow organoids with 5 different protocols, perform IF and qPCR?  Figure 1 does not seem to show their own protocol at all but they description sounds as though this figure shows their protocol. If they are trying to show their protocol they should show a schematic of when they add what cells and reagents.  The labels in the bar charts in Figure 1 are the first author (accept Lancester, which I assume is whole brain) and do not seem to indicate an original protocol is being used.

-              I don’t understand why all 5 protocols are not tested in Figure 1 C-G there should be a complete comparison if this is supposed to be comprehensive.

-              The authors concluded that all the protocols are enriched for MSN and DN markers but only Figure 1E has all 5 protocols and the ratio of markers doesn’t indicate if they are enriched. 

-              Figure 1 the qPCR is labelled as relative gene expression, relative to which genes?

 

-              Good reporting of statistics Fig 1

 

-              Now the authors are indicating that the two new protocols are forebrain and midbrain are their protocols Rudibaugh1 and Rudibaugh2.  This is not at all clear in Figure 1.  The naming is also very confusing and could easily be clearer.  Now I understand that in results 2.2 they will compare their forebrain protocol to previously published 3D forebrain models (Xiang-Tanaka and Bagley), 90 days and four 2D striatal neuron protocols 45 Days in culture or in final differentiation. 

 

-              Figure 2 – legend has no “A”.

-              Are Figure 2 B  and C the merge of all 7 cultures?

-              Figure 2E has all the cells from all cultures with only GABAergic neurons from one culture.  This doesn’t show the proportions of GABA in the one culture (as in D) but instead the proportion out of all cells. This would be more informative to split by culture and then highlight the GABAergic neurons.  It would be even better to keep all cells in grey and have a light colour highlighting all for example “Bagely” cells and then a darker colour highlighting all “Bagely” GABAergic cells.

-              Description of the broad cell type annotation is good. The rest of this section is clear and described well.

-              The authors say they can’t completely conclude which is the best protocol from the scRNAseq and I agree.  However, there is not any histology or immunofluorescence characterization.  MSN will have a distinctive morphology, it would be useful to have the correlation of the IF with the scRNAseq to characterize these neurons.  A co-labelling with a striatal markers, GAD1, and Tuj1 or maybe phalloidin would be very revealing.

-              If there is as much diversity in the 2D cultures as the 3D cultures why do the authors now believe that the 3D are more representative than the 2D cultures?

-              The experimental set up for the Dopamine, Cocaine, Morphine treatments is unclear: what culture protocols are tested with which treatments? 

-              The assembloids are not introduced or characterized.

-              Are different experimental paradigms performed on the Bagely forebrain organoids and the assembloids? In Figure A it seems everything is matched as expected. However, in Figure 3 B we don’t see each model with each treatment – why?  The same is the case in Figure 4.

-              Figure 3C could be better represented with an upset plots.

-              Figure 3D should have cell type annotation matching Figure 3B.

-              Here the authors a trying to do a comprehensive analysis to identify patterns common across the 3 treatments.  It isn’t clear if this is analysis from both 3D models together, separately, or just one of the two, as they state there are 20 contrast it implies either the two were combined or only one model was used.

-              They show in Figure 4A that synaptic long-term potentiation is identified once.  Does this mean no other pathways are identified at all?

-              Figure B, C, D should be represented in a better way. Perhaps a heat map by cell type the normalized expression levels with two conditions compared.

-              The authors perform pseudobulk analysis aggregating gene expression.  Why do they use all the cells?  This analysis can be easily performed by aggregating each cell type separately, maintaining the cell type specificity, although losing the inflated power and huge variability of single cell, cell type specificity can be maintained.  Also, this analysis can only be done if there are technical replicates, which is not described.

-              Figure 5 should be presented in a more compelling way.

Author Response

Please see the attached file

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to the Author:

 

1.      Do you have representative images of these striatal and midbrain organoids organoids?

2.      Are there any epigenetic mechanisms involved in the addictions?

3. Some of the latest literature on organoid research is missing and must be discussed and referenced in their work. This is important so that readers are aware of the current literature. They are:

1)         Organoids. Nature Reviews Methods Primers 2022, 2(1): 94.

2）      Rational Design of Oral Drugs Targeting Mucosa Delivery with Gut Organoid Platforms. Bioactive Materials 2023, 30, 116-128.

Author Response

Do you have representative images of these striatal and midbrain organoids?
R31. Representative images are located in Figure S1.

Are there any epigenetic mechanisms involved in the addictions?
R32. Yes, histone modifications such as acetylation, methylation, and even dopamine and serotonin
themselves, have been implicated in addiction literature. However, due to resource limitations, we focused
efforts on the transcriptional effects of substances of abuse.

Some of the latest literature on organoid research is missing and must be discussed and referenced in their
work. This is important so that readers are aware of the current literature. They are:
1) Organoids. Nature Reviews Methods Primers 2022, 2(1): 94.
2） Rational Design of Oral Drugs Targeting Mucosa Delivery with Gut Organoid Platforms. Bioactive
Materials 2023, 30, 116-128.

R33. These references have been added to the introduction.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The abstract and introduction now clearly explain the aim of the work. The other changes have also improved the manuscript and I believe it is much stronger. I still have a few minor concerns and larger concern.

1.        Figure 1: All protocols are labelled by the protocol name except Lancaster is labelled as “Whole Brain” – this should be changed to match the labeling scheme.

2.        Supplementary figure 1 –The image quality needs to be higher, when I zoom in it is very pixelated and I can’t see cells.  There are scale bars, but the size isn’t written, this must be indicated in the figures or in the legends.  I believe we are seeing parts of organoids and then a zoomed in section, this should be clear in the legend.

3.        Figure 5A the scale is z-score, but a z-score of what? Relative mRNA expression or genes in the pathway?  Number of ratio of genes in the pathway?

There is one larger problem in my opinion; the authors description of the data in the results section is not supported by the results shown in Figure 5. The authors claim that genes in specific pathways are differentially expressed after treatment compared to control across all cells but the plots do not reflex this.  The author need to remidy this mis-match. They could change their claims/description of the data, or change the genes selected to one that are altered in an obvious way or change the data representation to reflex the gene expression changes if they truely exist. I was not specific in my previous review of this figure.

These are the specific problems with this figure. 

1.        Figure 5B: EIF2AK3 is described as up regulated in both morphine and dopamine treated samples but this visually does not appear to be the case in the violin plots.  In the bar chart dopamine and control are identical in the forebrain organoids, morphine is slightly increase compared to the matching control organoid, but the changes are clearly not significant as the variation is extensive.

 

2.    For all violin plots in Figure 5C the expression doesn’t look increased except for MAP3K20 where it is completely gone, MAP3K2 looks very slightly increased. Perhaps a different type of plot would make these changes more visually apparent.  At least in the bar chart all the MAP gene expression trend toward an increase in expression in the dosed cells. 

 

Comments on the Quality of English Language

There was a sentence with a grammatical error or just unclear phrasing.  I didn't go back to find the sentense or check the whole document. You should double check the revised manuscript. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf