Establishment of Intestinal Organoids from Common Marmosets
Round 1
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsThe authors have systematically revised the manuscript to address many of the concerns in the previous version, which is appreciated. However, there are still some issues which must be addressed, which are as follows:
1) The information in Table S1 is very useful. Please also include a sentence in the methods section to indicate the labels/names of the the sample lines are provided in the supplemental table. Also, similar to Figure 1 legend, for all the figures please indicate which sample line was utilized for that experiment, not just in the supplemental table. For example, in Figure 2, organoid samples from what 'individual' was used? In Figure 3, organoids from which 'individual' was used for staining? Please indicate this information in every figure legend.
2) Please indicate whether 'N' implies individual cells or wells, one single passage of cells, or multiple independent passages.)
3) Table 1 - the primer sequences for ACTB is missing.
Author Response
Comment 1:
The information in Table S1 is very useful. Please also include a sentence in the methods section to indicate the labels/names of the sample lines are provided in the supplemental table. Also, similar to Figure 1 legend, for all the figures please indicate which sample line was utilized for that experiment, not just in the supplemental table. For example, in Figure 2, organoid samples from what 'individual' was used? In Figure 3, organoids from which 'individual' was used for staining? Please indicate this information in every figure legend.
Answer 1:
We added a sentence in section 2.1 “Animals”. We also added which sample line was utilized for that experiment in the legends of Figure 2 and 3
Comment 2:
Please indicate whether 'N' implies individual cells or wells, one single passage of cells, or multiple independent passages.)
Answer 2:
We changed the phrase “N = 4” to “N = 4 individuals”.
Comment 3:
3) Table 1 - the primer sequences for ACTB is missing.
We are sorry for missing the information. We added the sequences of ACTB primers in the supplementary table 2.
Reviewer 2 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsI would like to thank the authors for all their adaptations to the original manuscript. My previous comments have been addressed as far as I can tell and I feel this has massively increased the quality of the manuscript. I do, however, have some additional comments that will further improve the quality and importance of this research (please see below). Overall, I consider this study worthwhile of being published as a communication article.
- Line 43: “All cell types in intestinal epithelial cells” does not make sense. It should rather read “All cell types in the intestinal epithelium…”
- Line 83: The correct plural of cecum is ceca
- Line 86: IEC has been abbreviated before and does not need to explain the abbreviation again
- Line 96: Could you please elaborate the age of your samples? It seems a lot of your marmosets are 0 months old. Did they die shortly after birth/at birth? Could this affect your study because of not fully developed intestines and an almost fetal stage of stem cells?
- Line 143: “Immunstaining” should read “Immunostaining”
- Lines 174-175: Please state clearly which dissociation method was used. It sometimes makes huge differences if common trypsin, TrypLE or for instance Accutase was used for dissociation.
- Figure 1B is not referenced within the main text
- Figure 2: Is there any evidence of Paneth cells? Was this checked? If not, I would highly recommend checking the expression of LYZ and one alpha defensin gene in addition. Since some species seem to lack Paneth cells, evidence about the situation in marmosets would be valuable. Even though Paneth cells are not expected in large intestine, if they are found, this might be a very nice finding. Was it surprising to see higher enteroendocrine and goblet cell abundance in organoids?
- Lines 264 ff.: It might be worthwhile to look for other examples using the Fuji medium for intestinal organoid culture. One example is Kramer et al. (https://pubmed.ncbi.nlm.nih.gov/32231153/) who called it refined medium. It seems they also observe a lot more secretory cells (almost as many as in differentiation medium) but still preserve self-renewal.
Comments on the Quality of English LanguageI would advise to get everything spell-checked by an English expert as there are still multiple typos and some grammatical inconsistencies/mistakes.
Author Response
Comment 1:
- Line 43: “All cell types in intestinal epithelial cells” does not make sense. It should rather read “All cell types in the intestinal epithelium…”
Answer 1：
We changed the phrase “All cell types in intestinal epithelial cells” to “All cell types in the intestinal epithelium”.
Comment 2:
- Line 83: The correct plural of cecum is ceca
Answer 2:
We corrected “enlarged cecum” to “enlarged ceca”.
Comment 3:
Line 86: IEC has been abbreviated before and does not need to explain the abbreviation again
Answer 3:
We deleted the explanation here.
Comment 4:
- Line 96: Could you please elaborate the age of your samples? It seems a lot of your marmosets are 0 months old. Did they die shortly after birth/at birth? Could this affect your study because of not fully developed intestines and an almost fetal stage of stem cells?
Answer 4:
We added the elaborated age and the date of birth and death in supplementary table 1.
0 day old individuals died after birth, not at birth. We sampled intestines from marmosets that had been euthanized for reasons unrelated to our study; therefore, we were unable to select their age.
All the organoid lines we established showed expression of marker genes of all the major cell types in the intestinal epithelium, so we believe the organoids we have established can be used to model functions of intestinal epithelium of marmosets. However, the age of the marmosets may have affected the characteristics of the established organoids. We should be aware of that point when utilizing the organoids in further studies.
Comment 5:
- Line 143: “Immunstaining” should read “Immunostaining”
Answer 5:
We corrected the typo.
Comment 6:
- Lines 174-175: Please state clearly which dissociation method was used. It sometimes makes huge differences if common trypsin, TrypLE or for instance Accutase was used for dissociation.
Answer 6:
We added the information that we used TrypLE Express.
Comment 7:
- Figure 1B is not referenced within the main text
Answer 7:
The fourth sentence in the section 3.1 is the explanation of fig.1B. We cited Fig.1B here.
Comment 8:
- Figure 2: Is there any evidence of Paneth cells? Was this checked? If not, I would highly recommend checking the expression of LYZ and one alpha defensin gene in addition. Since some species seem to lack Paneth cells, evidence about the situation in marmosets would be valuable. Even though Paneth cells are not expected in large intestine, if they are found, this might be a very nice finding. Was it surprising to see higher enteroendocrine and goblet cell abundance in organoids?
Answer 8:
Thank you for your suggestion to check Paneth cell evidence. As you mentioned, some studies report absence of Paneth cells in some species (e.g. Li et al. Cell Regeneration (2022) 11:19). The markers of Paneth cell had not been checked.
We tried RT-qPCR of LYZ and DEFA5 on jejunal, cecal and colonic organoids and tissues of Cj768. As a result, the expression pattern of these two genes differed greatly, so currently it is difficult to conclude whether Paneth cells exist in marmoset intestine including colon.
For Paneth cells are important in host-microbiome interaction, studying this point will be interesting. We will investigate this point further in the future, but we would like to not include this data in the manuscript this time.
High expression of EEC and goblet cell markers were not very surprising considering the single cell RNAseq data Fujii et al. has reported. The important point we propose is that the condition was similar in non-human primates.
Comment 9:
- Lines 264 ff.: It might be worthwhile to look for other examples using the Fuji medium for intestinal organoid culture. One example is Kramer et al. (https://pubmed.ncbi.nlm.nih.gov/32231153/) who called it refined medium. It seems they also observe a lot more secretory cells (almost as many as in differentiation medium) but still preserve self-renewal.
Answer 9:
Thank you for your suggestion. We cited Kramer et al. as an example that report the characteristics of intestinal organoids cultured in Fujii medium.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors of the manuscript entiteled "Establishment of intestinal organoids from common marmosets",
I appreciate your very concise description of the establishment of marmoset intestinal organoids, however, I came across several shortcomings of your study. Alongside the very limited number of samples (1) and the missing statistical evaluation, the discussion also lacks important points such as the comparison to other studies using cecal organoids from other species. The point about intestinal adaptation to different feed is a very interesting one and could have been explored more in this study, at least via RT-qPCR analysis of certain genes. Please see more specific comments below:
Major comments:
- Lines 46-48: The authors state that they have intestinal organoids from marmoset intestines, but only cecal organoids have been characterised in more detail. The authors try to explain why cecum is of particular interest in their introduction, but perhaps the emphasis on this should be even clearer for the reader to understand why all the other organoids have not been investigated further.
- Lines 54-58: Similar to the previous comment it is stated that organoids were established from six animals but only one has been analysed further. What is the reason behind this?
- You have used reverse transcriptase quantitative PCR (using RNA as a starting material = RT-qPCR) and not quantitative PCR on DNA; please correct this throughout the manuscript (qPCR alone would be misleading)
- Figure 1: all photos are too small for the reader to see them clearly. Especially scale bars are incredibly thin and small. They all show different white balance and many are too bright. It might be a good idea to choose different photos with the same properties.
- Figure 1C: Please explain clearly what is meant by the information "Day 7 P 7" etc.
- Figure 2/Lines 154-156: There are two main issues here: 1) Your standard deviations seem to be incorrectly displayed. A standard deviation has the same difference from a mean both up and down of the mean. This is definitely not correctly annotated for ASCL2, OLFM4, STMN1, VIL1, MUC2, TFF3 and NEROG3. The second problem is your statement that you state higher or comparable gene expression compared to tissue. However, your standard deviations are much too big to perform meaningful comparisons. Even though the differences might not be statistically significant in most cases, SDs like these only from technical replicates do not seem to be reliable. In the end, no statistics were performed at all. The authors should think about increasing statistical power by increasing their sample size and improving protocols to minimize SD from technical replicates.
- Figure 3: Scale bars are too small.
- Lines 185-186: Both cited studies (6&20) used organoids from the small intestine. Many other studies show that organoids from the small and large intestine show very different morphologies. Also, study 6 used a proliferation vs. a differentiation medium for their organoid cultures and shows that when organoids differentiate, they tend to show more budding behavior. Therefore, I believe a direct comparison to these other model systems (even from a different species) should be made with more caution.
- Lines 189-192: The authors' statement about marmoset organoids not responding to nicotinamide is peculiar. Why is this data not shown here is the experiment was carried out? The manuscript could have gone with more figures and data, so a page/figure limit cannot be the reason for not showing this data if the fact marmoset organoids do not respond to it is even stressed in the discussion.
- Lines 201-204: The authors' talk about an EGF withdrawal experiment and figure S1, which does not seem to be included in the provided supplementary data. Please provide all necessary information. It is also not mentioned in the list of supplementary materials at the end of the manuscript.
Minor comments:
- Line 35: this should say "in vitro" instead of "in vivo"
- Lines 102-103: This sentence is a duplication of the previous sentences about fixation and embedding.
- Lines 139-142: The meaning of the last sentence of the figure description seems a bit hard to grasp. Please rephrase this sentence.
- Lines 148-149: Please cite literature for your statement that budding structures are indicative of a heterogeneous cell population.
- Line 168 ff.: Please do not use italicized letters for protein names (MKI67)
- Line 220: here it says "primary antibody list", even though this list also includes secondary antibodies
Reviewer 2 Report
Comments and Suggestions for Authors1) Introduction - It would be good to have a little more elaboration on what work already exists in common marmoset vs. what is novel in this new work that has not been previously described. Are common marmosets well-studied for gut/intestinal research? This needs to be expanded on more to support the value of this new model development.
2) More elaboration on the precise ages of the marmosets at time of acquisition of cell lines is needed. What are the ages of the "infant" group, vs. the "adult" group? The paper also requires more details on how the primary tissue was obtained from each marmoset. Were the isolation methods consistent?
3) It is unclear from the manuscript why 6 marmoset tissues were utilized to establish primary lines but only one group adult-3 was used to establish organoid lines, but only one male line was further characterized. At least 2 lines of adult, both male and female, would have been ideal to have. Or one line of adult and one line of infant. Please explain why only adult-3 from marmoset male was chosen for organoid development? The criteria for this selection must also be included in Figure 1.
4) Figure 1 - What do P0, P4, D7 etc. mean? Please provide this clearly in the figure legend as well as in the written text in results section.
5) Multiple minor typos are present throughout the manuscript, for example: Line 89 in 'quantitative PCR' the 'Q' must be capitalized; Line 117 'marmoset' is misspelled as 'marmost', Line 145 'organoids' is misspelled as 'orgnoids', and many more. Not all typos are provided here, please go through the manuscript systematically and correct all typos.
6) Figure 2 - (i) are the n=3 technical replicates all from the same passage? Or were three different preparations of organoids done? This needs to be explained. If all are from the same passage, the organoids need to be prepared from at least n=3 passages and assessed. (ii) please provide the individual 'n' as dots in the graph to allow accurate visualization of the variability; the graphs need to be scatter+bar graph, not just bar graph. (iii) the variability is quite high in the graphs. what was statistical analysis results? conclusions cannot be made without statistical significance.
7) Results section of Figure 2 needs to have citations to validate why these markers were chosen. Were the markers chosen based on marmoset studies or human studies data? This also needs to be explained, as this is a unique model and the expression pattern may not be the same in indication.
8) Figure 3 - Please provide both zoomed out view as well as 20 micron view (as given), to allow for visualization of the whole morphology of the organoid. Also, add what serotonin marker is used to identify in the figure legend (for example: 'hormone produced by enteroendocrine cell'.
9) Figure 3 - Also, could CHGA+serotonin costaining be done and quantification of costaining done, to validate that enteroendocrine cells are producing the serotonin?
10) Figure 3 - Please provide some form of quantitative assessment of the expression. For example, counting fluorescence staining, or assessing fluorescence intensity.
11) How big is the size of the average organoid? Some basic morphometric characterization of the organoids would be appropriate to round out this study.
