Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.

This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.

No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Human brain organoids are an emerging technique that allows us to recapitulate the early development of the human brain in vitro^1,2,3. Nevertheless, one of the major limitations of this model is the lack of vascularization, which plays indispensable roles not only in brain homeostasis but also in brain development⁴. In addition to the delivery of oxygen and nutrients, accumulating evidence suggests that the vascular system of the brain regulates neural differentiation, migration, and synaptogenesis during development⁵

The White Leghorn chicken (Gallus gallus) embryos were treated by following the Guide for the Care and Use of Laboratory Animals from the Institute of Laboratory Animals Resources, Commission of Life Sciences, National Research Council, USA, and the experiments were approved by the Council for Care and Use of Experimental Animals from the University of Barcelona.

1. Non-guided brain organoid preparation

1. Maintain H9 human embryonic stem cells (hESCs) in mTE.......

Selecting the embryo maturation schedule for the transplant
The experiment begins at D0 when fertilized eggs are incubated at 38 °C and 60% relative humidity. The chorioallantoic membrane (CAM) is a highly vascularized extraembryonic membrane that develops after egg incubation. It is formed by the fusion of the allantois and chorion. At D1, after 24 h of incubation, the air chamber is punctured to prevent the CAM from attaching to the inner shell membrane. Puncturing the air chamber at D1 impr.......

In this study, we describe a detailed protocol with numerous key steps that provide favorable growth and development of human brain organoids upon grafting without perturbing the survival of the chicken embryos. We recommended the use of sterile needles to puncture the air chamber of the egg after 24 h of incubation (day 1). Additionally, we also tried to make the puncture at day 4 (after checking through the eggshell by light to test the development of the vasculature to be sure that we were working only with healthy em.......

We thank Dr. Alcántara and Dr. Ortega from UB and the rest of the members in Dr. Acosta's lab for the insightful discussions. S.A. is Serra-Hunter fellow assistant professor from the Generalitat de Catalunya at Universitat de Barcelona.