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Vascularized human cortical organoids (vOrganoids) model cortical development in vivo [1]

['Yingchao Shi', 'State Key Laboratory Of Brain', 'Cognitive Science', 'Cas Center For Excellence In Brain Science', 'Intelligence Technology', 'Institute Of Brain-Intelligence Technology', 'Shanghai', 'Institute Of Biophysics', 'Chinese Academy Of Sciences', 'Beijing']

Date: 2023-05

Modeling the processes of neuronal progenitor proliferation and differentiation to produce mature cortical neuron subtypes is essential for the study of human brain development and the search for potential cell therapies. We demonstrated a novel paradigm for the generation of vascularized organoids (vOrganoids) consisting of typical human cortical cell types and a vascular structure for over 200 days as a vascularized and functional brain organoid model. The observation of spontaneous excitatory postsynaptic currents (sEPSCs), spontaneous inhibitory postsynaptic currents (sIPSCs), and bidirectional electrical transmission indicated the presence of chemical and electrical synapses in vOrganoids. More importantly, single-cell RNA-sequencing analysis illustrated that vOrganoids exhibited robust neurogenesis and that cells of vOrganoids differentially expressed genes (DEGs) related to blood vessel morphogenesis. The transplantation of vOrganoids into the mouse S1 cortex resulted in the construction of functional human-mouse blood vessels in the grafts that promoted cell survival in the grafts. This vOrganoid culture method could not only serve as a model to study human cortical development and explore brain disease pathology but also provide potential prospects for new cell therapies for nervous system disorders and injury.

Funding: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020601, XDB32010100) to XW, National Basic Research Program of China (2019YFA0110101 and 2017YFA0103303 to XW; 2017YFA0102601 to QW), the National Natural Science Foundation of China (31671072 to QW; 31771140 and 81891001 to XW), and the Grants of Beijing Brain Initiative of Beijing Municipal Science & Technology Commission (Z181100001518004) to XW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we developed a 3D culture protocol to generate vOrganoids by coculturing hESCs or hiPSCs with human umbilical vein endothelial cells (HUVECs) in vitro. In our studies, HUVECs were connected and formed a well-developed mesh-like or tube-like vascular system in the cerebral organoids. vOrganoids recapitulated neocortical development, exhibiting different cell types and a neural circuit network, in vitro. In addition, single-cell RNA sequencing (scRNA-seq) analysis verified that vOrganoids shared similar molecular properties and cell types with the human fetal telencephalon. Finally, we intracerebrally implanted vOrganoids into mice and observed that the grafted vOrganoids survived and integrated into the host cortical tissue in vivo. Importantly, the vessels in vOrganoid grafts connected well with the native blood vessels in the rodents to build a new functional vascularization system. This vOrganoid culture system serves as a model for studying human cortical development and provides new potential therapeutic strategies for treating brain disorders or injuries.

Previous studies have successfully established suitable approaches for generating cerebral organoids from human embryonic stem cells (hESCs) or hiPSCs that can recapitulate in vivo human cortical development and a well-polarized ventricle neuroepithelial structure that consists of ventricular radial glia (vRGs), outer radial glia (oRGs), and intermediate progenitor cells (IPCs) and the production of mature neurons within layers [ 12 , 13 , 15 , 17 , 18 , 20 ]. However, a major limitation of current culture approaches that prevents truly in vivo–like functionality is the lack of a microenvironment, such as vascular circulation. Previous studies have reported that the development of the nervous system and the vascular system in the brain is synchronous [ 21 – 23 ]. Vascularization is specifically required for oxygen, nutrient, and waste exchange and for signal transmission in the brain. Additionally, blood vessels around neural stem cells (NSCs) serve as a microenvironment that maintains homeostasis, and they play essential roles in NSC self-renewal and differentiation during embryonic development [ 24 , 25 ]. A lack of vascular circulation can induce hypoxia during organoid culture and accelerate necrosis, which consequently hinders the normal development of neurons and their potential migration [ 26 ]. To overcome these limitations, some studies have tried to generate vascularized organoids (vOrganoids) by coculturing hESC- or hiPSC-derived cerebral organoids with endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) from the same patient [ 27 ]. In addition, recent studies have established a robust method for generating vascularized human cortical organoids by introducing hESCs that ectopically express ETV2 into organoids [ 28 ]. Mansour and colleagues showed that transplanting human cerebral organoids into the adult mouse brain can result in the formation of a vascularized and functional brain organoid model in vivo [ 29 ]. In addition, other reports have demonstrated that compared to transplanting dissociating neural progenitor cells, engrafting cerebral organoids into the lesioned mouse cortex induces enhanced survival and robust vascularization [ 30 ]. All of these studies indicate that vascularization is one of the feasible methods to improve organoid survival. In addition to the methods reported in these studies, other stable and reproducible methods are required for establishing vascularized cerebral organoids to model human brain development in vitro and to perform in vivo transplantation.

Organoids have recently been used to study the development of and pathological changes in different tissue types, such as pancreas, liver, kidney, and retina tissues [ 3 – 8 ]. In addition, several different methods involving the differentiation of human-induced pluripotent stem cells (hiPSCs) have been developed to generate organoids that mimic nervous system development [ 9 – 19 ]. Three-dimensional brain organoids are comprised of multiple cell types that collectively exhibit cortical laminar organization, cellular compartmentalization, and organ-like functions. Therefore, compared to conventional 2D culture, organoids are advantageous because they can recapitulate embryonic and tissue development in vitro and are better at mirroring the functionality, architecture, and geometric features of tissues in vivo.

In contrast to the rodent lissencephalic cortex, the human neocortex has evolved into a highly folded gyrencephalic cortex with enormous expansion of the cortical surface and increases in cell type and number [ 1 , 2 ]. Animal models, particularly rodents, have provided significant insight into brain development, but the complexity of the human neocortex cannot be fully captured with these models. Therefore, understanding the genetic changes as well as the mechanistic steps that underpin the evolutionary changes that occur during the development of the neocortex in primates may require new model systems.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000705

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