Generation of human vascularized brain organoids

Abstract

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously, iPSCs from the same patient were differentiated into ECs. On day 34, the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54, and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible.

Fig. 1

At day 54, the day of transplantation, there is some penetration of capillaries into the outer layers of the organoid in vitro (a–d). Two weeks later, which coincides with the termination of the in-vivo transplantation experiment, organoids without endothelial coating do not contain any blood vessels in vitro (e–h). At the same time, organoids with endothelial coating show robust ingrowth of CD31-positive blood vessels into the organoid in vitro (i–l; hCD31, red; STEM121, green; DAPI, blue; all scale bars: 1 mm).

Fig. 2

Magnification of Fig. 1l. There is robust penetration of the outer, more organized layers of the organoid in vitro. There is some ingrowth of blood vessels into the STEM121-positive core of the organoid (white arrow). Some parts of the outer layer of the organoid are not vascularized which may be due to detachment of the Matrigel in affected areas during the incubation (hCD31, red; STEM121, green; DAPI, blue).

Fig. 3

A coronal section of the transplanted NSG mouse brain shows that the vascularized organoid measures almost 2 mm in largest diameter 2 weeks after transplantation and 68 days after embryoid body formation. The human brain stem cell marker STEM121 (green) exclusively stains structures inside the organoid (a). Magnified images of the coronal section of the organoid show that human blood vessels (hCD31, red, multiple white arrows to point to examples) have formed in several regions of the human brain organoid (b; STEM121, green; DAPI, blue). Regions of interest are delineated with boxes for Fig. 4a (white frame) and Fig. 4b (no frame). The nonvascularized organoid did not survive after 2 weeks in vivo (c; STEM121, green).

Fig. 4

A blood vessel has penetrated the center of a rosette inside the organoid in vivo. (a) A magnification of the white frame in Fig. 3b. Blood vessels can also be found on the periphery of rosettes (b; hCD31, red, white arrows to delineate a blood vessel; STEM121, green; DAPI, blue). (b) A rotated magnification of the box with no frame in Fig. 3b.