Directing Cholangiocyte Morphogenesis in Natural Biomaterial Scaffolds

  1. Quinton Smith
  2. Jennifer Bays
  3. Linqing Li
  4. Haniyah Shareef
  5. Christopher S. Chen
  6. Sangeeta N. Bhatia

  • Curated by eLife

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    Evaluation Summary:

    This paper is of interest for cell biologists, developmental biologists and tissue engineers. The authors identify a combination of natural extracellular matrix and growth factors that enables to grow cholangiocytes as branched three-dimensional ducts in culture. The work is physiologically relevant and represents an interesting step forward in the study of bile duct formation and disease, although the cultured ducts could be characterized more in depth.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch‐dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue.

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  1. eLife

    Evaluation Summary:

    This paper is of interest for cell biologists, developmental biologists and tissue engineers. The authors identify a combination of natural extracellular matrix and growth factors that enables to grow cholangiocytes as branched three-dimensional ducts in culture. The work is physiologically relevant and represents an interesting step forward in the study of bile duct formation and disease, although the cultured ducts could be characterized more in depth.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    This manuscript describes the capacity of primary human cholangiocytes to form ductal structures by self-organisation in progressively more complex culture conditions, including prefabricated channels. It reveals the importance of ECM components CollagenI and Fibrin and growth factor signalling by EGF and HGF for duct differentiation and branching. This analysis further shows an epistatic role of Notch signalling in this process. This work represents an important study of in vitro differentiation of commercial cholangiocytes into bilary ducts with a potential to contribute and advance tissue engineering approaches in the liver field. The presented work should be of interest to scientists in the stem cell differentiation and tissue engineering fields, as well as liver organogenesis.

    Strength: Overall, the conclusions and interpretation are supported by the analysis. With the increasing complexity of the conditions, it provides a unique angle and valuable insights into culturing strategies to generate intricate ductal structures.

    Weakness: My major concern is the statement that hierarchical intrahepatic bile ducts can be differentiated. The study shows exciting and promising data, however they require systematic characterisation of ductal differentiation in the various conditions. For instance, while 'hierarchical intrahepatic biliary ducts' are described for the chip configuration, neither cholangiocyte gene expression nor open lumens have been tested.

  3. eLife

    Reviewer #2 (Public Review):

    The lack of suitable in vitro culture system recapitulating bile duct formation and branching slows the analysis of the mechanisms driving biliary morphogenesis. Here, the authors used adult human primary intrahepatic cholangiocytes and first confirmed the biliary phenotype of the cells by analysing the expression of cholangiocyte-specific markers and measuring enzyme activity. The cholangiocytes formed limited branched structures in Matrigel, but sprouting was significantly increased when Matrigel was mixed with collagen type I and when the medium was supplemented with EGF. Functional metabolite transport into the biliary lumen is demonstrated. The biological relevance of this culture system is supported by experiments showing that the effects of collagen plus EGF is abrogated by genetic or pharmacological inhibition of Notch signaling, a pathway which is essential in vivo for normal bile duct morphogenesis. Finally, the authors show that the culture conditions can be adapted into a microfluidic chip that should be amenable to the introduction of flow.

    Strengths
    The paper is clearly written and illustrated, and the conclusions of the experiments are supported by the data. The biological relevance and biomedical interest of the new culture system combining Matrigel/collagen and EGF is well demonstrated by the experiments targeting Notch signaling and by the cultured ducts' capacity to transport a metabolite into the lumen.

    Weaknesses
    The main weakness is in the limited morphological, functional and transcriptomic characterization of the cultured bile ducts, which is in part underlined by the authors in the discussion. To convince the reader of the wide applicability of the culture system and on the advantages of this system over existing ones mentioned in the paper, data on its stability over time should ideally be provided. Knowledge on regional specificities of cholangiocytes have accumulated recently, and providing a more detailed gene expression profile of the cultured cholangiocytes would also help in evaluating the potential usefulness of the system. Although the microfluidic chip with cholangiocytes is very appealing, prove of functionality, not only morphogenesis, would considerably improve the paper.

    The materials and methods section does not describe the experimental procedures with sufficient detail.

  4. eLife

    Reviewer #3 (Public Review):

    The manuscript from Smith et al. describes an in vitro platform to study bile duct cells morphogenesis and validate the role of Notch in this process. The authors use commercially available cholangiocytes, which they place in 3D culture and assess the capacity of the cells to form branching networks in the presence or absence of Notch signaling. Finally, they seed the cells on microfluidic chips. The resulting structures do not remain patent in chips incorporating Matrigel/collagen gels, but do so when fibrin-based gels are used.

    Strengths
    Branching morphogenesis plays an important role both for biliary development and disease. Therefore, in principle, developing an in vitro platform to model this process could be beneficial both for groups working on developmental biology and for labs focusing on infantile cholangiopathies, such as Alagille syndrome.

    Weaknesses
    Despite the usefulness of a modeling platform for tubulogenesis, the manuscript fails to illustrate the strengths of the system described and the analysis performed is insufficient to support the authors' claims. In particular:

    The authors claim that their system differs from existing in vitro platforms for growing cholangiocytes in various aspects. However, these claims are not substantiated by a direct comparison between systems and, in some cases, they are contradicted by the references in the manuscript. For example, Sampaziotis et al. claim to grow mature and not adult-stem cell derived cholangiocytes. Although, the system by Huch et al. is based on adult stem cells, the characterization of cholangiocytes in the manuscript is very limited and does not provide evidence of marker expression or functional properties that are not demonstrated by adult stem cell-derived cholangiocytes. I agree about the difficulties in perfusing organoid systems and this is an important point; however, densified collagen tubes seeded with human cholangiocytes have also described and referenced in the paper (Ref 11).

    Furthermore, the authors are using adult cholangiocytes for studying an embryological process (tubulogenesis) that happens at the level of the ductal plate and is initiated by the interaction between hepatoblasts and the ductal plate mesenchyma. Conceptually, adult frozen primary cholangiocytes expanded in monolayer and then cultured in Matrigel fail to recapitulate this setup; which renders the system not physiologically relevant. Therefore, it is difficult to assess how the mechanisms observed in this setting translate to human development or disease without further validation in vivo, or in patient tissue.

    Importantly, the characterization of the cells and the platform lacks context and depth. Comparison to fresh primary cholangiocytes is required to assess the value of the system. A very limited number of markers is assessed, often with a single modality (e.g. only IF or only QPCR). Moreover, the data shown does not always support the claims of interpretation of the authors, but I have made specific recommendations on how to improve these aspects in the next section. The main concern here is the lack of convincing evidence for the presence of a lumen inside the branching structures, which recapitulates the lumen of bile ducts in vivo, ie surrounded by cells circumferentially with dimensions corresponding to the terminal branches of the intrahepatic ducts.

    The use of microfluid chips could be an exciting aspect of the paper, as this has not been extensively described in the literature with human cells (although it has been achieved with mouse cells, PMID: 31465556) and the authors have a track record in this area. However, the matrix that was used for all the cells' characterization throughout the paper seems not to be working very well for maintaining a patent lumen and the authors switch to a fibrin-based ECM. There are 3 issues with this approach. First, although fibrin is present in the body, it is usually present in injury, which renders it an even less physiological matrix than Matrigel to study morphogenesis. Second, the human cells are not characterized in this new matrix, which could impact on marker expression or function. Third, and most importantly, the authors do not demonstrate how this chip could be used by the community, which functional assays could be performed, whether there is any benefit to the profile of the cells from perfusion or what additional insight it provides the system provides compared to Matrigel.

  5. Version published to 10.1002/advs.202102698
  6. Version published to 10.1101/2021.04.09.439196v1 on bioRxiv