Characterization of 3D organotypic epithelial tissues reveals tonsil-specific differences in tonic interferon signaling
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Abstract
Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e. , cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data ( https://viz.datascience.arizona.edu/3DEpiEx/ ).
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The current study uses 3D organotypic rafts to culture primary keratinocytes from Foreskin, Tonsil and Cervix. Further the authors looked at the transcriptomic profiles of each tissue types to study similarities and differences depending on the tissue of origin as well as show the similarity in the tissue specific gene signatures and the ex-vivo samples (data from GTEx). As mentioned by authors Skin and Cervix keratinocytes have been previously cultured on collagen rafts however extending it to Tonsil provides resource and possibility of growing more tissue specific epithelial cells in 3D.
Majo…
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The current study uses 3D organotypic rafts to culture primary keratinocytes from Foreskin, Tonsil and Cervix. Further the authors looked at the transcriptomic profiles of each tissue types to study similarities and differences depending on the tissue of origin as well as show the similarity in the tissue specific gene signatures and the ex-vivo samples (data from GTEx). As mentioned by authors Skin and Cervix keratinocytes have been previously cultured on collagen rafts however extending it to Tonsil provides resource and possibility of growing more tissue specific epithelial cells in 3D.
Major comments
- As the papers focus is to culture epithelial/ epidermal cells on 3D rafts, methods section needs more details about the raft composition, preparation, fibroblast embedding what was the plate size used for raft preparation and culturing of cells on those rafts. What culture media was used for epithelial raft cultures?
We have a detailed published protocol that highlight these details. However, we will expand on some of these details in the manuscript
Results: Figure 1, authors show IF staining's for COL17A1 as marker for basal cells and cornulin for differentiated layers. However, it is important to show how many cells in the basal layer are proliferative? (or how many layers of proliferative cells are present in different epithelia analysed here?) after 14 days majority of cells might already start losing their stemness potential (maybe staining for at least ki67 if staining for basal stem cell marker not possible? Along with loricrin or Involucrin might be good idea).
We will stain for ki67 as suggested. However, based on published data using these raft cultures, we do not expect that many cells will be positive.
This is also important as from supp fig 3 you can see F1 has higher expression of Loricrin, filaggrin etc as compared to all other samples indicating higher diff in this sample. Also, if authors can comment on what was the passage of cells used? And have they observed any difference in the re-epithelization in early passage versus late passage of keratinocytes?
We will expand on this is the updated manuscript. Importantly, we grow these cells in a rho-kinase inhibitor that ‘conditionally’ immortalizes these cells as described (DOI: 10.1172/JCI42297).
It is interesting to see Tonsillar 3D epithelia recapitulate the crypt and surface epithelia and authors also show this with gene expression profile, if possible (Optional), can authors show staining for crypt specific and surface specific markers.
We agree that this is an important control. This will be included.
For all the Supplementary tables where only Ensembl ids are represented, please add gene Id column alongside (it is easier to get biological context from gene id for the reader rather than looking up Ensembl ids). Rename the file names to include the Supplementary file 1, 2, 3?
Since there is 1-to-1 conversion for Ensembl to Gene Id, we elected to not include these. The online app does try tp accommodate this as much as possible. We propose to include two versions of each table. 1 with Ensemble ids only and one with both IDs.
Its excellent to see that in vitro tissue signature matched the in vivo tissue samples (Figure 8) but it will be interesting to show the gene expression differences if found any between the in vitro and in vivo samples that will give insight on the changes as result of in vitro system.
Since the in vivo data will be a mixture of epithelial cells and stroma, these comparisons are not straightforward. However, we are currently examining the use of existing scRNA-Seq data to begin addressing these concerns. This data will be included in the next revision.
Minor comments
- Abstract: Give sample number (n?) and brief results about the genes that had tissue specific expression pattens.
- Gene names needs to be in Italics throughout.
- Introduction: page 5 line 9, authors claim that they based on comparisons they can "identify potential therapeutic targets for various disease" I think this statement either needs experimental evidence or statement / claim needs to be modified.
- Data submission to GEO???
- Typo (page 15, line 16 should be "HFK-down", same on page 23 "ectocervix", "endocervix", "uterus", so on, please correct, comma needs to be placed after "
- Page 24 last line is the heatmap referred here Fig 9B?
- Fig. 1 legends please indicate what F1, F2, F3, C1--- T1--- represent. Fig 1C Please add axis range/ values for protein atlas data as well.
- Can authors comment in discussion how was current 3D cervix cells on raft method different from Meyers, C., 1996 3D system?
All these ‘minor’ comments will be addressed.
Reviewer #1 (Significance (Required)):
This article does extend and validate the 3D raft culture method to different epithelial tissues in addition to Skin and cervix. This will be useful for the researchers using co culture systems and interested in understanding epithelial cell and immune cell interactions or host pathogen interacts etc
Describe your expertise: establishing and maintaining primary skin and oral keratinocyte cultures on feeders and 3D cultures on DEDs, Organoid cultures from oral keratinocytes, Oral cancer biology, Histopathology, transcriptomics study, Immuno-oncology.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary Jackson et al.'s manuscript describes an experiment that directly compares 3D organotypic assays created with primary human epithelial cells from foreskin, cervix and tonsil using histological and bulk RNA sequencing approaches. The authors convincingly show the retention of site-specific histological and transcriptomic differences between the stratified epithelial tissues in culture. Differentially expressed genes are identified and pathway analyses suggest genes that might be involved in the different differentiation processes between these tissue sites and differential regulation of ECM and immune pathways. Differentially expressed genes are used to develop a classifier for tissue identification, which is tested using GTEx data.
Major Comments • The interferon stimulated genes of B cells and macrophages (from Mostafavi et al., 2016) are likely to be very different from those in epithelial cells, so the analysis presented in Figure 9 seems like a stretch to me.
We will include caveats to this interpretation. We are planning stimulation experiments of each tissue to compare IFN responses. However, depending on the outcomes, these may end up being outside of the scope of the current manuscript.
OPTIONAL: Further data comparing the nature and magnitude of the interferon responses of the three epithelia would improve interest in the manuscript but are not necessary for publication of the current dataset.
See above
Minor Comments • Details of n numbers and what each point represents should be added to Figure 1C. Are these points measurements from 25 um intervals of just one raft per donor? What are 'fields of view' here? Are measurements from one section or from multiple sections per raft? • Page 12 - provide a figure/panel citation for the "micrograph derived from a tonsillectomy" that is suggested for comparison. • In Figure 1 - Figure Supplement 1, how representative of the whole raft are these images? Does the extent of stratification change near to the edge of the collagen gel, for example? How well matched for location within a raft are the images shown? • Page 24 - clarify uses of the phrase "down-regulated in tonsils". Presumably this section refers to tonsil epithelium in 3D organotypic rafts.
Typos • Page 3 - "the cervix is lined with stratified squamous epithelia", should be epithelium. • "J.G. Rheinwald" in in text references. • Page 6 - 'or' not 'and' in first sentence of primary cell culture section.
All these ‘minor’ comments and typos will be addressed.
Reviewer #2 (Significance (Required)):
This highly descriptive study provides a detailed analysis of a bulk RNA sequencing experiment comparing foreskin, cervix and tonsil 3D organotypic rafts. Retained histological and transcriptional differences between epithelial tissues of different origins in organotypic assays are well documented in the literature (e.g., parmoplantar vs non-parmoplantar skin, PMID: 36732947; airway tract, PMID: 32526206) so the observed differences between these three very distinct anatomical tissues are unsurprising overall. The data have been made available via SRA and a shiny web app and are likely to be of interest and use to other researchers working on these tissues in culture. The experiment was performed in matched cell culture conditions so replicates are well-controlled, if limited in number (n=3).
We appreciate this feedback. We agree this is a descriptive study. Nonetheless, we believe there is value in formally demonstrating differences and similarities between these tissues. The provided references will be included to expand our discussion.
I am an epithelial cell biologist specializing in human cell culture models. I do not have sufficient computational background to comment in detail on the RNA sequencing methods or analysis within the manuscript.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
This is very carefully analysed and written study describing the transcriptional differences between in vitro models of epithelia derived from cervix, foreskin and tonsil tissues. Importantly, they compare the findings to in vivo samples using publicly available data. The findings are significant and will be of interest to the scientific community. I cannot fault the analysis pathways or the conclusions, and the manuscript is a pleasure to read. I recommend it is accepted for publication as is.
Reviewer #3 (Significance (Required)):
This is an important study that is highly significant for researchers interested in epithelia tissue and infection. The data are clearly presented and the analysis is thorough. The authors state that they will make the data publicly available. This will be an important resource for the community.
We appreciate the kind words
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
This is very carefully analysed and written study describing the transcriptional differences between in vitro models of epithelia derived from cervix, foreskin and tonsil tissues. Importantly, they compare the findings to in vivo samples using publicly available data. The findings are significant and will be of interest to the scientific community. I cannot fault the analysis pathways or the conclusions, and the manuscript is a pleasure to read. I recommend it is accepted for publication as is.
Significance
This is an important study that is highly significant for researchers interested in epithelia tissue and infection. The data …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
This is very carefully analysed and written study describing the transcriptional differences between in vitro models of epithelia derived from cervix, foreskin and tonsil tissues. Importantly, they compare the findings to in vivo samples using publicly available data. The findings are significant and will be of interest to the scientific community. I cannot fault the analysis pathways or the conclusions, and the manuscript is a pleasure to read. I recommend it is accepted for publication as is.
Significance
This is an important study that is highly significant for researchers interested in epithelia tissue and infection. The data are clearly presented and the analysis is thorough. The authors state that they will make the data publicly available. This will be an important resource for the community.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary
Jackson et al.'s manuscript describes an experiment that directly compares 3D organotypic assays created with primary human epithelial cells from foreskin, cervix and tonsil using histological and bulk RNA sequencing approaches. The authors convincingly show the retention of site-specific histological and transcriptomic differences between the stratified epithelial tissues in culture. Differentially expressed genes are identified and pathway analyses suggest genes that might be involved in the different differentiation processes between these tissue sites and differential regulation of ECM and immune pathways. Differentially …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary
Jackson et al.'s manuscript describes an experiment that directly compares 3D organotypic assays created with primary human epithelial cells from foreskin, cervix and tonsil using histological and bulk RNA sequencing approaches. The authors convincingly show the retention of site-specific histological and transcriptomic differences between the stratified epithelial tissues in culture. Differentially expressed genes are identified and pathway analyses suggest genes that might be involved in the different differentiation processes between these tissue sites and differential regulation of ECM and immune pathways. Differentially expressed genes are used to develop a classifier for tissue identification, which is tested using GTEx data.
Major Comments
- The interferon stimulated genes of B cells and macrophages (from Mostafavi et al., 2016) are likely to be very different from those in epithelial cells, so the analysis presented in Figure 9 seems like a stretch to me.
- OPTIONAL: Further data comparing the nature and magnitude of the interferon responses of the three epithelia would improve interest in the manuscript but are not necessary for publication of the current dataset.
Minor Comments
- Details of n numbers and what each point represents should be added to Figure 1C. Are these points measurements from 25 um intervals of just one raft per donor? What are 'fields of view' here? Are measurements from one section or from multiple sections per raft?
- Page 12 - provide a figure/panel citation for the "micrograph derived from a tonsillectomy" that is suggested for comparison.
- In Figure 1 - Figure Supplement 1, how representative of the whole raft are these images? Does the extent of stratification change near to the edge of the collagen gel, for example? How well matched for location within a raft are the images shown?
- Page 24 - clarify uses of the phrase "down-regulated in tonsils". Presumably this section refers to tonsil epithelium in 3D organotypic rafts.
Typos
- Page 3 - "the cervix is lined with stratified squamous epithelia", should be epithelium.
- "J.G. Rheinwald" in in text references.
- Page 6 - 'or' not 'and' in first sentence of primary cell culture section.
Significance
This highly descriptive study provides a detailed analysis of a bulk RNA sequencing experiment comparing foreskin, cervix and tonsil 3D organotypic rafts. Retained histological and transcriptional differences between epithelial tissues of different origins in organotypic assays are well documented in the literature (e.g., parmoplantar vs non-parmoplantar skin, PMID: 36732947; airway tract, PMID: 32526206) so the observed differences between these three very distinct anatomical tissues are unsurprising overall. The data have been made available via SRA and a shiny web app and are likely to be of interest and use to other researchers working on these tissues in culture. The experiment was performed in matched cell culture conditions so replicates are well-controlled, if limited in number (n=3).
I am an epithelial cell biologist specializing in human cell culture models. I do not have sufficient computational background to comment in detail on the RNA sequencing methods or analysis within the manuscript.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The current study uses 3D organotypic rafts to culture primary keratinocytes from Foreskin, Tonsil and Cervix. Further the authors looked at the transcriptomic profiles of each tissue types to study similarities and differences depending on the tissue of origin as well as show the similarity in the tissue specific gene signatures and the ex-vivo samples (data from GTEx). As mentioned by authors Skin and Cervix keratinocytes have been previously cultured on collagen rafts however extending it to Tonsil provides resource and possibility of growing more tissue specific epithelial cells in 3D.
Major comments
- As the papers focus is to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The current study uses 3D organotypic rafts to culture primary keratinocytes from Foreskin, Tonsil and Cervix. Further the authors looked at the transcriptomic profiles of each tissue types to study similarities and differences depending on the tissue of origin as well as show the similarity in the tissue specific gene signatures and the ex-vivo samples (data from GTEx). As mentioned by authors Skin and Cervix keratinocytes have been previously cultured on collagen rafts however extending it to Tonsil provides resource and possibility of growing more tissue specific epithelial cells in 3D.
Major comments
- As the papers focus is to culture epithelial/ epidermal cells on 3D rafts, methods section needs more details about the raft composition, preparation, fibroblast embedding what was the plate size used for raft preparation and culturing of cells on those rafts. What culture media was used for epithelial raft cultures?
- Results: Figure 1, authors show IF staining's for COL17A1 as marker for basal cells and cornulin for differentiated layers. However, it is important to show how many cells in the basal layer are proliferative? (or how many layers of proliferative cells are present in different epithelia analysed here?) after 14 days majority of cells might already start losing their stemness potential (maybe staining for at least ki67 if staining for basal stem cell marker not possible? Along with loricrin or Involucrin might be good idea). This is also important as from supp fig 3 you can see F1 has higher expression of Loricrin, filaggrin etc as compared to all other samples indicating higher diff in this sample. Also, if authors can comment on what was the passage of cells used? And have they observed any difference in the re-epithelization in early passage versus late passage of keratinocytes?
- It is interesting to see Tonsillar 3D epithelia recapitulate the crypt and surface epithelia and authors also show this with gene expression profile, if possible (Optional), can authors show staining for crypt specific and surface specific markers.
- For all the Supplementary tables where only Ensembl ids are represented, please add gene Id column alongside (it is easier to get biological context from gene id for the reader rather than looking up Ensembl ids). Rename the file names to include the Supplementary file 1, 2, 3?
- Its excellent to see that in vitro tissue signature matched the in vivo tissue samples (Figure 8) but it will be interesting to show the gene expression differences if found any between the in vitro and in vivo samples that will give insight on the changes as result of in vitro system.
Minor comments
- Abstract: Give sample number (n?) and brief results about the genes that had tissue specific expression pattens.
- Gene names needs to be in Italics throughout.
- Introduction: page 5 line 9, authors claim that they based on comparisons they can "identify potential therapeutic targets for various disease" I think this statement either needs experimental evidence or statement / claim needs to be modified.
- Data submission to GEO???
- Typo (page 15, line 16 should be "HFK-down", same on page 23 "ectocervix", "endocervix", "uterus", so on, please correct, comma needs to be placed after "
- Page 24 last line is the heatmap referred here Fig 9B?
- Fig. 1 legends please indicate what F1, F2, F3, C1--- T1--- represent. Fig 1C Please add axis range/ values for protein atlas data as well.
- Can authors comment in discussion how was current 3D cervix cells on raft method different from Meyers, C., 1996 3D system?
Significance
This article does extend and validate the 3D raft culture method to different epithelial tissues in addition to Skin and cervix. This will be useful for the researchers using co culture systems and interested in understanding epithelial cell and immune cell interactions or host pathogen interacts etc
Describe your expertise: establishing and maintaining primary skin and oral keratinocyte cultures on feeders and 3D cultures on DEDs, Organoid cultures from oral keratinocytes, Oral cancer biology, Histopathology, transcriptomics study, Immuno-oncology.
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