Generation of Trophoblast Organoids from Chorionic Villus Sampling

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes a method for generation of trophoblast organoids from CVS. The methods and technical details are clearly described. I am concerned about the limitatons the the proposed methods when it comes to the usefulness of this organoid model. The last section of the manuscript explores major limitations related to these organoids in replicating the placental environment. Do authors have any suggestions how to overcome some of these drawbacks?

Best regards,

Author Response

The authors would like to thank the reviewer for the provided suggestion. Careful consideration was given to the reviewer’s comment after which we tried to adjust the manuscript accordingly. In the section ‘Limitations’, we have added the following statement (lines 440-445): “Further development of CVS-derived trophoblast organoid models might include co-culturing with (matched) populations of stromal, endothelial or immune cells, all of which can be potentially isolated from residual CVS tissue. Also, integration with microfluidic cell culture devices might increase the complexity and fidelity of CVS-derived trophoblast organoids, emulating physiologically-relevant interactions at higher levels of tissue organization.”

Furthermore, we elaborate on the statements regarding trophoblast organoid polarity (lines 458-462): “For this, trophoblast organoids were transferred to suspension cultures, a method previously shown to control epithelial polarity (Reference to Co et al. Nature Methods 2021). We anticipate that CVS-derived trophoblast organoids are amenable to this methodology, however, future research should examine potential effects on organoid derivation efficiency and long-term expansion.”  

Reviewer 2 Report

Comments and Suggestions for Authors

In this report, Rijn et al. present a protocol for generating trophoblast organoids from chorionic villus samples. The work is timely as trophoblast organoids are an emerging in vitro model for placental research. The authors nicely describe the unique benefits of using chorionic villus biopsies, specifically, for organoid generation. The authors address decidual cell contamination and the inversion of the cell orientation, which are relevant and important points. Providing some additional clarifications would improve the comprehensiveness of the protocol. Individual comments and requests are included below.

 

1.     It would be helpful to add clarifying statements in the introduction regarding how trophoblast enrichment is performed during enzymatic digestion step (Page 2 Line 63).

2.     Provide a reference, if applicable, for the TOM formulation.

3.     EVT differentiation is described in step 26. It would be informative to see characterization of the EVT by phase contrast imaging, ICC, and qRT-PCR analyses.

4.     Inclusion of time course analyses for CVS trophoblast organoids would be informative. How do the morphology, transcript and protein expression profiles change over the 14-day culture?

Author Response

The authors would like to thank the reviewer for the provided suggestions. Careful consideration was given to the reviewer’s comments after which we tried to adjust the manuscript accordingly. Please find every point addressed in detail below:

1. Simple enzymatic digestion seemed, in our hands, to be the most efficient method for enrichment of trophoblast suspensions from chorionic villi. Additional separation methods did not result in better yield or viability. To clarify this, we have added the following statement to section 2.2 Dissociation (lines 97-101): “Enzymatic digestion followed by removal of large tissue fragments and cell aggregates using cell strainers is sufficient for enrichment of viable cytotrophoblast suspensions, whereas additional separation techniques, including immunomagnetic cell sorting and density gradient centrifugation, did not result in improved cytotrophoblast yield or viability. “
2. We have added a reference for the original TOM formulation we have adjusted for this protocol (line 276-277).
3. Comprehensive characterization of EVTs is indeed valuable and certainly merits more detailed research. In our studies, we did not prioritize EVT characterization. Although briefly addressed in our original work, we acknowledge that EVT differentiation is a valuable element of trophoblast organoid models and have performed some additional experiments. We have added the section “2.6 EVT differentiation”, in which we include data on EVT differentiation, including brightfield and immunofluorescence microscopy (Figure 4).
4. To provide more information on time course changes, we have included a brightfield image of the morphology of trophoblast organoids during the formation stage in Figure 1. In this stage, small spheroid structures appear after 3-7 of culture. We have added the following information to the text:
   1. Lines 66-68: “Small spheroids appear within 3-7 days, that grow out to form villous-like organoid structures after 10-14 days.”
   2. Lines 107-109: “After initial seeding of the trophoblast, small spheroids with an average diameter of 100 µm appear after 3-7 days of culture. If these structures do not appear after 7 days, trophoblast organoid cultures will likely not develop.”

Furthermore, we have added qPCR data of day 7 organoids for the trophoblast markers genes in Figure 3.

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, Rijn et al. provided the protocol for generating trophoblast organoids from chorionic villi samples. The manuscript is well written, interesting and easy to follow. The experimental design, methodology and result presentation are convincing. However, there are concerns about the manuscript that have been detailed below:

 

Can this organoid system differentiate into extravillous trophoblast cells?

 

Did the author compare these organoids with organoids derived from human trophoblast stem cells?

Author Response

The authors would like to thank the reviewer for the provided suggestions. Careful consideration was given to the reviewer’s comments after which we tried to adjust the manuscript accordingly. Please find every point addressed in detail below:

Q1: Can this organoid system differentiate into extravillous trophoblast cells?

We acknowledge that EVT differentiation is a valuable element of trophoblast organoid models. Induction of EVT differentiation is described in section 4.3 ‘Maintenance of trophoblast organoids cultures’, however, we have added the section “2.6 EVT differentiation” in which we include data on EVT differentiation, including brightfield microscopy and immunofluorescence microscopy (Figure 4). 

Q2: Did the author compare these organoids with organoids derived from human trophoblast stem cells?

We have added the section “2.7 Comparison with hTSC-derived trophoblast organoids” in which we compare CVS-derived trophoblast organoids with human trophoblast stem cell (hTSC)-derived trophoblast organoids on a transcriptomic level, utilizing previously published, publicly available single-cell RNA sequencing data. For this, we performed comprehensive quality control and integration to remove potential batch effects, followed by differential expression testing. The latter showed enrichment of syncytiotrophoblast-specific transcripts in CVS-derived trophoblast organoids compared to hTSC-derived trophoblast organoids. These data are presented in Figure 5 and Supplementary table S1. Although getting a grasp, we would like to accentuate that further functional analyses are required to make definitive claims regarding potential differences between trophoblast organoids derived from CVS tissue and trophoblast stem cells.       

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Responses and revisions are satisfactory.

Figure 5 Legend--Volcano misspelled.