Protocol for generating human cortical organoids enriched in outer radial glia by guided differentiation

Abstract

The generation of human pluripotent stem cell (hPSC)-derived brain organoids is continuously refined, enhancing their reproducibility and complexity. Here, we present a guided differentiation protocol for generating cortical forebrain organoids and cortico-pericyte (CP)assembloids composed of a robust outer radial glia (oRG) population and an expanded outer subventricular zone (oSVZ). We describe the steps to generate hPSC-derived cortical organoids (COs), cortical pericytes, and CP assembloids. Moreover, we outline the procedures to characterize the organoids by immunostaining and to perform single-cell dissociation. For complete details on the use and execution of this protocol, please refer to Walsh et al.¹.

Keywords: Cell Biology; Cell Differentiation; Cell culture; Cell isolation; Developmental biology; Flow Cytometry; Neuroscience; Organoids; Stem Cells.

Graphical abstract

Figure 1

Generation of cortical organoids (COs) from hPSCs (A) Representative bright field phase image showing the morphology of a hPSC colony. Scale bars: left panel, 400 μm; right panel, 300 μm. (B) Schematical summary of the protocol used for the generation of COs. (C) Bright field images showing the progressive changes in morphology of neural spheroids during the first 14 days of differentiation into COs. Scale bar: 400 μm. (D) Bright field images of COs at day 32 and 45 of differentiation. Neuronal rosettes are evident at these stages. Scale bar: 500 μm.

Figure 2

Generation of neural crest (NC)-derived brain pericytes from hPSCs (A) Schematic representation of the protocol for the differentiation of hPSCs into NC-derived brain pericytes. (B) Bright field images showing the progressive changes in cell morphology and the generation of “bridge-like” (indicated by white arrows) structures during the NC differentiation. Scale bar: 250 μm. (C) Bright field images of the final part of the differentiation of NC into pericytes after the sorting step. The pericytes progenitor tends to progressively acquire a more mature morphology during the differentiation. Scale bar: 300 μm.

Figure 3

Generation of cortico-pericyte (CP) assembloids (A) Bright field pictures of the pericytes microtissue (left panel), at the moment of addition of day 14 CO to the pericyte microtissue (middle panel) and the fusion of pericyte microtissue and CO to create the CP assembloid (right panel). Scale bar: 300 μm. (B) Bright field images showing the progressive growth of CP assembloids during the differentiation. Scale bar: 500 μm.

Figure 4

Immunofluorescence (IF) analyses of organoids (A) Immunofluorescence images of an entire CP assembloid at day 60. The assembloids is stained for SOX2 (neural stem cells, gray), EOMES (intermediate progenitors [IPs], yellow) and TBR1 (deep-layer neurons, red). The single channels are shown. Scale bar: 500 μm. (B) Comparison by immunofluorescence staining of the neuronal rosette structure of organoids with and without LIF treatment and CP assembloids. The treatment with LIF or the incorporation of pericytes determines the increase of the oSVZ area (SOX2 positive cells outside the ventricular zone and the layer of EOMES positive cells). The neuronal rosettes are marked with SOX2 (in gray) and EOMES (in yellow), and deep-layer neurons (TBR1 positive) are marked in red. The single channels are shown. Scale bar: 200 μm. (C) Immunofluorescence analysis of the oRG in the neuronal rosettes. oRG cells are positive for SOX2 (gray), pVIM (green), GFAP (cyan) and HOPX (red) and are found in the oSVZ (SOX2 positive area surrounding the VZ). The single channels are shown. Scale bar: 100 μm. The image of the rosette shown here is a different perspective and magnification of the same rosette shown in the Figure 1F from Walsh et al.