In vivo development and single-cell transcriptome profiling of human brain organoids

Abstract

Objectives: Human brain organoids can provide not only promising models for physiological and pathological neurogenesis but also potential therapies in neurological diseases. However, technical issues such as surgical lesions due to transplantation still limit their applications.

Materials and methods: Instead of applying mature organoids, we innovatively developed human brain organoids in vivo by injecting small premature organoids into corpus striatum of adult SCID mice. Two months after injection, single-cell transcriptome analysis was performed on 6131 GFP-labeled human cells from transplanted mouse brains.

Results: Eight subsets of cells (including neuronal cells expressing striatal markers) were identified in these in vivo developed organoids (IVD-organoids) by unbiased clustering. Compared with in vitro cultured human cortical organoids, we found that IVD-organoids developed more supporting cells including pericyte-like and choroid plexus cells, which are important for maintaining organoid homeostasis. Furthermore, IVD-organoids showed lower levels of cellular stress and apoptosis.

Conclusions: Our study thus provides a novel method to generate human brain organoids, which is promising in various applications of disease models and therapies.

FIGURE 1

In vivo development of human brain organoids in mouse brain. (A) Experiment procedure for premature organoid generation and injection into mouse corpus striatum. (B) IVD‐organoids were immunostained for GFP and DAPI. Left, coronal section showing IVD‐organoids survived in mouse corpus striatum. Scale bar, 1000 μm. Middle, enlarged image showing the ventricle zone‐like structure. Scale bars, 50 μm. Right, enlarged image showing the choroid plexus epithelium‐like structure. Scale bar, 100 μm. (C) Comparation of grafted cells (left) and IVD‐organoids (right). Scale bars, 500 μm. (D) Immunostaining of MAP2 in IVD‐organoids. Scale bars, 20 μm. (E) Immunostaining of GFAP and STEM121 in IVD‐organoids. Scale bars, 50 μm and 10 μm (high‐magnification images). (F) Immunostaining of IBA1 in IVD‐organoids. Scale bars, 50 μm. (G) Immunostaining of vascular marker CD31 in IVD‐organoids. Scale bars, 50 μm

FIGURE 2

Single‐cell transcriptome analysis of IVD‐organoids. (A) UMAP dimension reduction plots of IVD‐organoids. Colored by cell‐type annotation. BRC, BMP signal‐related cell; ChP, choroid plexus; RGC, radial glial cell; AS, astrocyte; NPC, neural progenitor cell. (B) Heatmap showing relative expression levels of cell‐type markers. (C) Expression patterns of marker genes in neuron‐related clusters. (D) Expression patterns of marker genes in supporting cells. (E) Expression patterns of marker genes in pericyte‐like cells. (F) Immunostaining of PDGFRβ in IVD‐organoids, dissociated cells and D60 hCOs. Scale bars, 10 μm. Quantification results are presented as mean ± s.e.m. (n = 3 for transplanted mice and cultured hCOs). (G) Expression patterns of striatal markers. (H) Immunostaining of ZBTB20 in IVD‐organoids, dissociated cells and D60 hCOs. Scale bars, 50 μm. Quantification results are presented as mean ± s.e.m. (n = 3 for transplanted mice and cultured D60 hCOs). ∗∗p < 0.01, ∗p < 0.05, unpaired two‐tailed t‐test

FIGURE 3

ChP cells are efficiently generated in IVD‐organoids. (A) Immunostaining of TTR in IVD‐organoids, dissociated cells and day 60 hCOs. Scale bars, 50 μm. Quantification results are presented as mean ± s.e.m (n = 3 for transplanted mice, dissociated cells and cultured hCOs). (B) Integrated UMAP plots showing joint clustering of IVD‐organoids and hCOs. Colored by samples (left) and cell types (right). (C) Violin plots showing expression levels of mature ChP markers in hCOs and IVD‐organoids. (D) Violin plots showing expression levels of tight junction markers in hCOs and IVD‐organoids. (E) PCA analysis of the ChP cell cluster in IVD‐organoids and various cell clusters in ChP organoids

FIGURE 4

IVD‐organoids exhibit improved cell survival. (A) Glycolysis signature levels in IVD‐organoids and published hCOs. (B) Violin plots showing expression levels of glycolysis‐related genes. (C) ER stress signature levels in IVD‐organoids and published hCOs. (D) Violin plots showing expression levels of ER stress‐related genes. (E) Hypoxia signature levels in IVD‐organoids and published hCO. (F) Violin plots showing expression levels of hypoxia‐related genes. (G) Apoptosis signatures between IVD‐organoids and published hCOs. (H) Violin plots showing expression levels of apoptosis‐related genes. (I) Immunostaining of cleaved caspase 3 in IVD‐organoids and D60 hCOs. Scale bars, 200 μm. Quantification results are presented as mean ± s.e.m. (n = 3 for transplanted mice and cultured D60 hCOs). ∗∗p < 0.01, unpaired two‐tailed t‐test