Single-cell characterization of neovascularization using hiPSC-derived endothelial cells in a 3D microenvironment

  1. Simon Rosowski
  2. Caroline Remmert
  3. Maren Marder
  4. Misao Akishiba
  5. Judith Bushe
  6. Annette Feuchtinger
  7. Alina Platen
  8. Siegfried Ussar
  9. Fabian Theis
  10. Sandra Wiedenmann
  11. Matthias Meier

  • Curated by eLife

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    eLife Assessment:

    A comprehensive single-cell transcriptomic study was performed in this study to gain insight into the development of endothelial cells and other co-developing mural cells derived from human induced pluripotent stem cells (hiPSCs) in 3D environment. This study gave us important information about signature genes, trajectories, and cell-cell interactions at various stages of vessel formation. Accordingly, the results of this study could potentially be of valuable interest to scientists working in the fields of stem cells, regenerative medicine, and tissue engineering.

    (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. Reviewer #3 agreed to share their name with the authors.)

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Abstract

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  1. Version published to 10.1016/j.stemcr.2023.08.008
  2. eLife

    eLife Assessment:

    A comprehensive single-cell transcriptomic study was performed in this study to gain insight into the development of endothelial cells and other co-developing mural cells derived from human induced pluripotent stem cells (hiPSCs) in 3D environment. This study gave us important information about signature genes, trajectories, and cell-cell interactions at various stages of vessel formation. Accordingly, the results of this study could potentially be of valuable interest to scientists working in the fields of stem cells, regenerative medicine, and tissue engineering.

    (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. Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This manuscript provided descriptive information about the development of endothelial cells and co-developing mural cells. The researcher induced hiPSCs into mesodermal cells before furthermore differentiating the cells into endothelial cells by chemical induction. To investigate in vitro neovascularization, the researchers employed single-cell RNA sequencing to analyze the differentiation process of hiPSCs into endothelial cells under various 3D microenvironments at several time points. Utilization of single cell RNA sequencing in this manuscript provides insight about signature process and molecules in various time points and conditions, where cell-cell interaction between endothelial cells and pericytes, angiogenic signaling and roles of cytokine signaling during tubulogenesis are highlighted. This study also compared the 2D environment previously employed by another study to the 3D microenvironment and found interesting differences in endothelial cells proliferation pattern, extracellular matrix production, and expression of mesenchyme-angioblast markers. In conclusion, this study gave us a lot of important data for improving strategies for future vascular tissue engineering.

    Strength
    This study is well designed with a robust method, convincing data, and in-depth analysis of various possible conditions for endothelial cells differentiation. This study could provide additional information about the dynamics of vascularization and serve as a benchmark for future hIPSC-derived endothelial cells-based experiments.

    Weakness
    As several studies have been published before about single-cell sequencing profiling of endothelial cell derived from hIPSCs, the novelty and merit of this study are impacted accordingly.

  4. eLife

    Reviewer #2 (Public Review):

    This paper describes the use of a 3D system for neovascularization of pluripotent derived human vascular cells, focusing on endothelial cell maturation and function.

    This is a single cell transcriptomic study with associated informatics.

    The data is relevant and interesting but there are too many assumptions and few validations that undermine the importance of the work and the advance that it might create.

  5. eLife

    Reviewer #3 (Public Review):

    This work provides a detailed single-cell transcriptomic analysis of endothelial cell (EC) differentiation from induced pluripotent stem cells in a suspension culture. The data demonstrates that the protocol produces a large number of both endothelial cells and mural cells, which is comparable to a 2D monoculture, with differences observed mainly in the expression of ECM genes. This first part of the study shows that EC differentiation works well both in 2D monolayer and in suspension cultures, with some key differences in their gene expression patterns, which has been shown before. Here, a detailed transcriptomic landscape of single cells during differentiation is provided. The second part of the paper examines how the transcriptome of ECs and pericytes (PC) that are differentiated in a suspension culture is remodeled in a 3D hydrogel environment, when cells sprout and form tubular structures. Based on the single-cell transcriptomes, the present study allows detection of different EC and PC populations during tube formation and cell maturation, and the identification of the genes and transcription factors, which regulate cellular behavior and phenotype, such as coalescing and sprouting ECs.

    Strengths:
    The study is an extensive description of remodeling of the transcriptomic landscape in endothelial cells and mural cells during differentiation from human induced pluripotent stem cells. It demonstrates the potential of endothelial cell and pericyte differentiation in suspension culture, which allows larger yields compared to 2D monolayer cultures.
    The novelty in the study is the detailed transcriptomic characterization of the different cell clusters formed during tube formation and maturation in 3D hydrogel. This provides important information for future experiments studying the mechanisms of vasculogenesis and angiogenesis in vitro and for designing vascularization for organ-on-chip approaches.
    The data gathered here provides a great possibility to identify unknown interaction mechanisms between PC and EC during vascular development and maturation. Especially identification of PC subpopulations, which seem to produce both common and individual ligands for ECs is intriguing and creates several new research questions.

    Weaknesses:
    The paper has a lot of transcriptomic data from different cell culture conditions and time points, making it difficult to identify the key observations and novel findings of the study. The paper provides a resource for future studies, but does not answer a clear biological question or test a hypothesis.
    One of the concerns is the large number of mural cells in maturing 3D hydrogel cultures (66-85%), indicating that the protocol directs the cells more towards mesenchymal than endothelial cells or ECs lose their identity over time. It would be important to show the localization and organization of these two cell types in the 3D cultures and to demonstrate if the different EC and pericyte subclusters localize to certain parts of the network.

  6. Version published to 10.1101/2022.02.15.480506 on bioRxiv