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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Anti-TUBB3 [Tuj1], mouse	BioLegend	801201	1:1,000
Anti-GFAP, rabbit	GeneTex	GTX108711	1:500
Anti-rabbit AlexaFluor 488, goat.	Invitrogen	A-21206	1:1,000
Anti-mouse AlexaFluor 594, goat	Jackson ImmunoResearch	715-585-150	1:500
Fertilized White Leghorn chicken (Gallus gallus) eggs	Granja Gibert (Cambrils, Spain)
DAPI	Invitrogen	D1306	1:10,000
DPX	Sigma	100579	xylene-based mounting medium
Gentle Dissociation Solution	CreativeBiolabs	ITS-0622-YT187	cell dissociation solution
Matrigel	BD Biosciences	356234
Mowiol 4-88 mounting media	Merk	81381
Paper towel, lab-grade	Sigma-Aldrich	Z188956
ROCK inhibitor Y27632	Millipore	SCM075	10 nM
Sharp-Point Surgical Scissors	VWR	470106-340
Superfrost Plus Adhesion Microscope Slides	Epredia	J1800AMNZ