Developing Biliary Atresia-like Model by Treating Human Liver Organoids with Polyinosinic:Polycytidylic Acid (Poly (I:C))

Round 1

Reviewer 1 Report

In this article, Chung and colleagues analyzed the effects of Poly (I:C) treatment, in order to mimic the effect produced by a viral infection, on human hepatic organoids, comparing the results with biliary atresia organoids. (BA). This study aims to generate a 3D models for the study of BA starting from non-BA cholangiocytes, to overcome the scarce availability of BA patients from which to obtain cell cultures. BA is in fact a rare pediatric pathology whose triggering cause has been hypothesized to be an occult infection, probably by rotavirus, of the pregnant woman during the early stages of gestation. The main results obtained are: 1) that the treatment with Poly (I:C) induces phenotypic and growth changes in the organoids obtained from non-BA patients that recall those of the organoids from BA patients. 2) transcriptomic analysis of RNA shows that there are 19 genes showing a similar expression between organoids obtained from BA and Poly (I:C)-treated non-BA patients.

The data obtained are promising, but they are quite preliminary and several experiments are still needed to validate the authors' hypothesis and to reinforce the concept of the use of Poly (I:C)-treated non-BA organoids, to mimic the biology of BA patients.

In particular:

1) Treatment with Poly (I:C) primarily stimulates a response mediated by the TLR3 signal pathway. To verify that this effect is pure and that there is no interference, the Reviewer suggests to challenge Poly (I:C)-treated non-BA organoids with inhibitors of the TLR3 signaling pathway or inhibitors of the TLR3/ligand interaction and verifying their effects on the readouts analyzed in figure 1, as well as in gene expression. Conscious of the cost and difficulties of transcriptomics analysis, it would be sufficient to, at least, evaluate its effects (through real time PCR or Western Blot) on some of the 19 inflammatory mediators identified in the Venn diagram (figure 2).

2) As indicated by several seminal studies by Desmet VJ and by other subsequent studies, BA can be divided into two macro-categories, the prenatal (or early severe) forms, which are rarer and characterized by greater aggressiveness, an earlier onset and generally by genetic mutations (for example Inv, ADD3, ...) and perinatal ones, in which virosis and/or hepatotoxins seems to have a greater impact. This paper would therefore generate models of perinatal BA and to confirm this, it would be interesting to evaluate whether there are differences in expression between the 19 genes (or at least part of them) between prenatal and perinatal BA samples. In the absence of adequate cell cultures, it might be interesting to evaluate at least the immunohistochemical expression on archival sections.

Minor concerns:

Figure 1: The significance and number of organoids counted by cell type are not indicated.

The writings in figure 2 are small and difficult to read. I therefore suggest splitting the figure into two parts: one with panels A and B and a second with only the box plots, in order to enlarge the writings.

Along the text, a sort of "vortex" appears instead of some symbols, please correct.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript, the authors reported that “Polyinosinic:Polycytidylic acid [Poly (I:C)] treated human liver organoid as a novel research model for biliary atresia”

 

They showed that Poly I:C treated human liver organoids exhibit morphology and genetic signature compatible to organoids developed from BA liver samples

 

This study aim is interesting, however there are several concerns below .

 

1. The authors did not show whether this Poly I:C treated human liver organoids could be utilized as a novel research model for biliary atresia or not, thus, the title should be revised
2. Please show the data, other than poly I:C, whether inflammatory cytokine like, IL-6 or TNF-alpha could introduce human liver organoid like biliary atresia or not
3. in Abstract, please delete the numbering of 1), 2),3), and 4)

 

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Given the limited time for reviewing the comments and for the experiments, the authors made every effort to respond. The Reviewer, however, hopes that the comments suggested by both reviewers will be helpful for future work.

Reviewer 2 Report

The authors revised properly.