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    Tissue Engineering

    Collective Behaviors Drive Self-Correcting Vascular Network Formation in Fetal Liver Organoids

    (K-402) Collective Behaviors Drive Self-Correcting Vascular Network Formation in Fetal Liver Organoids

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row K - Poster # 402
    Introduction:: Induced pluripotent stem cell (iPSC)-derived organoids have the potential to revolutionize regenerative medicine and developmental biology through the re-creation of human tissue, but a functional and realistic organoid suitable for many of these applications must contain a vascular network to transport oxygen, nutrients, and waste throughout the tissue [1]. When fetal liver organoids (FeLOs) are generated from iPSCs using a doxycycline (dox)-inducible GATA6 gene circuit, a subset of cells differentiate and spontaneously self-organize into a similar such network, producing intricate endothelial cell microvasculature [2]. This study investigated how these organized vascular networks emerge and converge on an optimal configuration despite variations in initial seeding conditions. To address this, we utilized dox-inducible GATA6-EGFP iPSCs (iGATA6 cells) and RFP-labeled iPSCs with dox-inducible ETV2 (iETV2 cells), where ETV2 promotes endothelial fates [3]. Differentiating these cells in co-culture produced FeLOs with red fluorescent endothelial cells, enabling spatiotemporal tracking of individual cell morphology and collective, inter-cell behaviors throughout the course of vascular network development and refinement as well as a mature vascular network upon maturation of the organoid.

    Materials and Methods::

    To produce the FeLOs, 25k iGATA6 cells were seeded in co-culture with varying numbers (2k-16k) of iETV2 cells on Matrigel-coated coverslips and provided with 1000 ng/mL dox and pluripotency media (mTeSR1) for five days before being switched to general differentiation media (STEMdiff APEL). Fluorescence images were taken daily to determine the locations of the iETV2 cells based on their RFP signature and to track changes in cell morphology and behavior over time. Day 14 (abbreviated as D14) immunofluorescence stains of endothelial cells were also examined to study differences in the extensiveness and characteristics of vascular networks from samples with varying levels of iETV2 supplementation. Images were processed using ImageJ, and AngioTool was used to quantify vascular coverage.



    Results, Conclusions, and Discussions:: Figure 1 reveals that the FeLO vascular network, appearing in red, forms through a series of collective cell behaviors. ETV2 cells migrate toward each other to form aggregates from day 1 to day 2 (abbreviated as D1-D2), which then compress (D3-D4) before re-dispersing into isolated endothelial precursor cells (D5-D7). On D8, there is a culture-wide shift toward a more interconnected vascular network. Thus, rather than differentiating in place, the ETV2 cells are interacting with each other and their environment to move into these aggregates and interconnect with each other after endothelial precursor dispersal. Thus, collective cell behaviors shape vascular network formation in FeLOs. Furthermore, Figure 2 demonstrates that these behaviors are not restricted to samples with 8k ETV2 cells, as all conditions display the same sequence of events. Figure 3 illustrates that, just as the collective behaviors are resistant to changes in iETV2 number, the coverage of the final vascular network is also not proportional to the starting number of iETV2 cells. Thus, the emerging microvascular network may have the innate ability to self-correct, producing similar levels of vasculature for the culture, regardless of the number of cells initially destined to become endothelial. Current experiments are evaluating the possibility of a causal relationship between collective behavior robustness and microvasculature coverage convergence. Understanding and modeling how endothelial networks arise in FeLOs will enable the optimization of vascularization in engineered organoids, increasing their scientific and therapeutic relevance through faithful modeling of and interfacing with human tissue.

    Acknowledgements (Optional): :

    I would like to thank Professor Mo Ebrahimkhani, Kamyar Keshavarz, and Joshua Hislop for mentorship and support in experimental design and execution as well as Jeremy Velazquez and Ryan LeGraw for initial generation of and experimentation with the iETV2 line. Funding was provided by the University of Pittsburgh Swanson School of Engineering, the Office of the Provost, the Department of Bioengineering, and the NIH R01 EB grant (EB028532). 



    References (Optional): : [1] Zhao, X. Front Bioeng Biotechnol, 2021, 9. [2] Guye, P. Nat Commun, 2016, 7. [3] Sumanas, S. Curr Top Dev Biol, 2016.