Inside the Making of Stem Cell Vascularized Organoids

Researchers at Stanford University have successfully cultured human pluripotent stem cells (hPSCs) to differentiate into vascularized cardiac and hepatic organoids, which can be used to study cardiac and hepatic development, as well as the effects of drug exposure on human organ development. The results were published in Science.

hPSCs, including human embryonic and induced pluripotent stem cells, can develop into a range of specialized cell types in the body, such as cardiomyocytes, hepatocytes, and various vascular cells. In addition, hPSCs can be used to generate organoids, which are self-organizing, three-dimensional structures that closely resemble key structural and functional aspects of their corresponding in vivo organs.

Multiple strategies have been investigated to produce organoids with functional vasculature, thereby avoiding necrosis in the center of organoids due to low oxygen levels. This enables greater organoid growth, thereby improving their ability to model development, disease, and drug responses. In addition, it enhances the survival of transplanted organoids for regenerative applications.

Nevertheless, hPSCs have demonstrated the ability to recapitulate key aspects of early development, including primitive streak formation, gastrulation, germ layer specification, and the generation of organ-specific cell types. Furthermore, geometric micropatterning of hPSCs has made it possible to reproduce and model to scale these developmental processes in vitro.

Building on this foundation, the researchers established an in vitro model that mimics the earliest stages of cardiac and hepatic organoid vascularization, corresponding to the first 3 weeks of human development and Carnegie Stages 9 and 10.

Using four hPSC fluorescent reporter lines and spatially micropatterned hPSCs, the researchers successfully generated cardiac vascularized organoids (cVOs) in a scalable and reproducible manner. The reporter systems allowed them to track the formation of gastruloids, progenitors, and cardiovascular cell types directly within developing cVOs.

They identified a combination of growth factors and small molecules that, when applied to micropatterned hPSCs, produced a spatially organized, branching, and lumenized vascular network alongside a multilineage cVO composed of endocardial, myocardial, epicardial, and neuronal cell types. Single-cell transcriptomic, high-resolution three-dimensional imaging, and functional assays demonstrated that cVOs closely resemble a 6.5-postconception week human embryonic heart at Carnegie Stages 19 and 20. However, they also observed some differences that warrant further exploration.

Furthermore, they found that NOTCH and BMP signaling were required for proper vascularization in cVOs, with BMP inhibition having a more pronounced impact than NOTCH. To show the broader applicability of this approach, they applied the same vascular-inducing cocktail to generate hepatic vascularized organoids, which also developed a well-organized, branching, and lumenized vasculature integrated with multilineage hepatic cells.

“Our in vitro model represents a technical advance for addressing questions regarding de novo organ vascularization. Furthermore, our results suggest that a conserved developmental program is involved in creating the vasculature within different organ systems,” the researchers concluded.