Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells

Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1+ cells that infiltrate the neural tissue in a microglia-like manner.

: Full length blots for the detection of b-Actin and Brachyury (T). Bry(T) expression was demonstrated performing immunoblot analyses (blot on the right side). As loading control b-Actin is detected (blot on the left side). Three independent inductions of MPCs from iPS cells (MPCs 1 -MPCs 3) are depicted.    To demonstrate that the protocol for the generation of vascularized human tumor organoids is applicable for different independently generated iPS cell lines, we generated tumor organoids from STEMCCA NHDF iPSCs and Sendai NHDF iPSCs. (A) Pictures of 20 randomly picked organoids at day 2 and day 7 are depicted. Organoids look undistinguishable regarding size and morphology between the different experiments. (B) CD31 + vascular networks can be detected in tumor organoids derived from Sendai NHDF iPSCs as well as from STEMCCA NHDF iPSCs.  To investigate variations between vascularized tumor organoids from the same experiment, we randomly picked 20 organoids at day 4, performed CD31 immunofluorescence analyses and measured the organoid surface as well as the area covered by CD31 + cells. (A) Immunofluorescence analyses of 20 organoids are depicted. Control spheroids consisting of tumor cells only do not show any CD31 + cells. (B) The total organoid surface was quantified as well as the percentage of CD31 + area. We measured a mean surface area of ca. 500 µm 2 (+/-71.5 µm 2 ) and a percentage of CD31 + area of 80% (+/-5.3%). Analyses were performed at whole mount stained organoids.           (2) A neural aggregate is transferred to a well already containing a mesodermal aggregate and both are co-cultured. (3) Attached aggregates are embedded into basement membrane extract (BME). (4) Embedded aggregates are transferred to a 6well plate and cultured on a rocking table in the humidified incubator. (B) Neural and mesodermal aggregate attached to each other forming a dumbbell-like structure. (C) Fused aggregates embedded in BME. (D) Vascularized neural organoids generated from STEMCCA NHDF iPSCs 30 days after BME embedding. (E-G) Immunofluorescence analyses of vascularized neural organoids at day 30 after BME embedding. E shows an overview about the whole organoid, revealing CD31 + vascularized mesodermal parts (red) and TUJ1 + neural parts (green). Higher magnifications are depicted in F-G. (H) A vascularized neural organoid from Sendai NHDF iPSCs is shown.

Figure S6
Movie S1: Distribution of the endothelial network within tumor organoids.
The movie shows the distribution of the CD31 + endothelial network (red) within a tumor organoid. Tumor cells are GFP + (green). Images were obtained by confocal laser scanning microscopy of a whole mount stained tumor organoid. The organoid morphology is flattened due to squashing of the whole-mount under the cover slip.
Vessel-like structures can be observed around a core of GFP-positive tumor cells.
Some endothelial sprouts are found penetrating the tumor cell mass.

Movie S2: Blood flow towards tumor organoid.
A tumor organoid (green) transplanted to the chorioallantoic membrane of a chicken embryo is shown at day 3 after transplantation.

Movie S3: Vascular network within the mesodermal part of a neural organoid.
The vascular network within the mesodermal part of the neural organoid is visualized using immunofluorescence analyses with a specific antibody targeted against CD31.
A whole mount staining was performed and the tissue was subsequently cleared using ethylcinnamate. The video shows different planes of a confocal z-stack.