Vascularized human cortical organoids (vOrganoids) model cortical development in vivo

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Yingchao Shi ,

Le Sun ,

Mengdi Wang ,

Jianwei Liu ,

Suijuan Zhong,

Rui Li,

Peng Li,

Lijie Guo,

Ai Fang,

Ruiguo Chen,

Woo-Ping Ge,

Qian Wu ,

Xiaoqun Wang

Yingchao Shi, 

Le Sun, 

Mengdi Wang, 

Jianwei Liu, 

Suijuan Zhong, 

Rui Li, 

Peng Li, 

Lijie Guo, 

Ai Fang, 

Ruiguo Chen


Published: May 13, 2020

?This is an uncorrected proof.

AbstractModeling 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.

Citation: Shi Y, Sun L, Wang M, Liu J, Zhong S, Li R, et al. (2020) Vascularized human cortical organoids (vOrganoids) model cortical development in vivo. PLoS Biol 18(5):
e3000705. Editor: Bing Ye, University of Michigan, UNITED STATESReceived: July 10, 2019; Accepted: April 17, 2020; Published: May 13, 2020Copyright: © 2020 Shi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the paper and its Supporting Information files.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.Competing interests: The authors have declared that no competing interests exist.Abbreviations:
artificial cerebral spinal fluid; AIF1,
allograft inflammatory factor 1; AMPA,
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; Ang1,
angiopoietin-1; APV,
DL-2-Amino-5-phosphonopentanoic acid; BBB,
blood–brain barrier; BF,
bright field; BMI,
bicuculline methiodide; BRN2,
POU class 3 homeobox 2; CMV,
cytomegalovirus; CNQX,
6-Cyano-7-nitroquinoxaline-2,3-dione; CNS,
central nervous system; CP,
cortical plate; CR,
calretinin; CSF,
cerebrospinal fluid; CTIP2,
chicken ovalbumin upstream promoter transcription factor (COUP-TF)–interacting protein 2; DEG,
differentially expressed gene; DGC,
dodt gradient contrast; DLX1,
distal-less homeobox 1; dpi,
days postimplantation; EB,
embryonic body; EC,
endothelial cell; EOMES,
eomesodermin; ExN,
excitatory neuron; GABAA,
gamma-aminobutyric acid A; GAD1,
glutamate decarboxylase 1; GFAP,
glial fibrillary acidic protein; GFP,
green fluorescent protein; GO,
Gene Ontology; GW,
gestational week; HBMEC,
human brain microvascular endothelial cell; hESC,
human embryonic stem cell; HIF1α,
hypoxia induciable factor 1 subunit alpha; hiPSC,
human-induced pluripotent stem cell; HOPX,
homeodomain only protein X; HUN,
human nuclear; HUVEC,
human umbilical vein endothelial cell; IB4,
isolectin I-B4; ImN,
immature neuron; IPC,
intermediate progenitor cell; iPSC,
induced pluripotent stem cell; KCC2,
potassium-chloride transporter member 5; KSR,
Knockout Serum Replacement; LHX6,
LIM homeobox 6; MAP2,
microtubule associated protein 2; MBP,
myelin basic protein; MGE,
medial ganglionic eminence; MGE-div,
MGE dividing cell; Mural,
mural cells; NEAA,
nonessential amino acids; NeuN,
RNA binding fox-1 homolog 3; NEUROD2,
neuronal differentiation 2; NKX2-1,
NK2 homeobox 1; NMDA,
N-methyl-D-aspartate; NOD-SCID,
nonobese diabetic severe combined immunodeficient; NSC,
neural stem cell; OPC,
oligodendrocyte progenitor cell; oRG,
outer radial glia; P-gp,
P-glycoprotein; PAGA,
partition-based graph abstraction; PAX6,
paired box 6; PC,
principal component; PCA,
principal component analysis; PSD95,
postsynaptic density protein-95; RELN,
reelin; RG,
radial glia; SATB2,
SATB homeobox 2; scRNA-seq,
single-cell RNA sequencing; sEPSC,
spontaneous excitatory postsynaptic current; sIPSC,
spontaneous inhibitory postsynaptic current; SOX2,
SRY-box transcription factor 2; SST,
somatostatin; SYB2,
synaptobrevin 2; TBR2,
eomesodermin; TTX,
tetrodotoxin; UMAP,
Uniform Mainfold Approximation and Projection; VEGF,
vascular endothelial growth factor; vOrganoid,
vascularized organoid; vRG,
ventricular radial glia; VZ/SVZ,
ventricular zone/subventricular zone

IntroductionIn 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.
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.
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 may 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.
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.

Vascularization in the 3D vOrganoid culture system
HUVECs, which are derived from the endothelium of veins from the umbilical cord, have been widely used to explore the function and pathology of ECs [31–33]. In addition, via coculturing with other cell types, HUVECs have been extensively used to characterize angiogenesis during tumorigenesis and other biological processes [34–36]. Due to the tube formation ability of HUVECs (S1A Fig), we generated vascularized cerebral organoids by coculturing hESCs or hiPSCs with HUVECs. Approximately 3 × 106 dissociated hESCs or hiPSCs and 3 × 105 HUVECs were plated onto low cell-adhesion plates, and uniformly sized tight embryoid body-like aggregates formed within the first 7 days. On day 18, the aggregates were transferred to petri dishes for neural induction culture. The resulting 3D aggregates were then replated for neural differentiation on day 35. Nonvascularized control organoids were generated by the same workflow except that no HUVECs were added (Fig 1A). vOrganoids differentiated and matured for up to approximately 200 days under the optimized culture conditions (Fig 1B, 1G and 1L).
Fig 1. Cerebral vOrganoids with vascular system recapitulate the cortical spatial organization.(A) Schematic diagram of the 3D culture methods for generating cerebral organoids with complicate vascular systems. (B) Representative bright field (BF) images of vOrganoids at different stages. Scale bar, 200 μm. (C) Whole mount imaging of vOrganoid on day 42. The elaborate mesh-like vascular systems in vOrganoids were displayed by immunofluorescence staining for LAMININ and IB4. Areas 1 and 2 outlined in boxes were magnified and reconstructed in 3D to depict the complexity of vasculature in vOrganoids. Scale bar, 100 μm (upper left), 50 μm (in box 1), 50 μm (in box 2). The arrowheads pointed out the hollows in the vascular systems that are permeable at different views. (D) Representative immunofluorescence staining figure for TBR2, SOX2, and IB4 to reveal that the vasculogenesis in vOrganoids is synchronous to the neurogenesis at early stage. Scale bar, 50 μm. (E) Representative immunofluorescence staining figure for HOPX, SOX2, and IB4 to demonstrate that the HOPX+ SOX2+oRG cells could be detected in the vOrganoids at day 65. Scale bar, 50 μm. (F) Representative immunofluorescence staining figure for CTIP2/IB4/PAX6 at day 65 to demonstrate that the IB4+ vascular structures would progressively extend into newborn neurons (CTIP2+) with the development of vOrganoids. Scale bar, 50 μm. (G) Representative IB4 and LAMININ staining figure in vOrganoid at day 210 to demonstrate that the vascular system could be maintained for over 200 days. Scale bars, 100 μm. (H) The spatial organization of vOrganoids was illustrated by immunofluorescence staining for TBR2/CTIP2 (left panel) and SATB2/CTIP2 (right panel) at day 65. Scale bar, 50 μm. (I) Representative immunofluorescence staining figure for the SATB2 and SOX2 to illustrate that SATB2+ cells are mainly located above the SOX2+ progenitor cells (left panel); SST and CR staining illustrated the emergence of interneurons in vOrganoids at day 65 (right panel). The “#5” and “#6” labelled in the upper left represent the number of continuous sections of vOrganoids. Scale bars, 50 μm. (J-L) Representative immunostaining figure for the pyramidal layer markers and interneuron markers in the continuous cryosections of vOrganoids at day 92 (J), day 128 (K), and day 210 (L). Scale bars, 50 μm. (M) The percentage of SATB2+, BRN2+, and CTIP2+ cells in the vOrganoids of day 128 and day 210, respectively. n=3 organoids from three independent experiments. All data are presented as means ± SEM, independent-samples t test, *p 
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