Generation and assembly of human brain region-specific three-dimensional cultures

Abstract

The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain-specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region-specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.

Figure 1.. General schematic for generating human forebrain spheroids from hPSC.

(a) Scheme illustrating the main stages of the method for generating dorsal (hCS) and ventral (hSS) forebrain spheroids from hPSCs. (b) Coronal section from GW18–20 human brain outlining dorsal (pallial) and ventral (subpallial) markers and their domains. (c) Examples of applications and functional assays that can be carried out using region-specific and assembled neural spheroids.

Figure 2.. Outline of the human forebrain spheroid protocol.

(a) Recommendations for growing hPSC colonies prior to aggregation and spheroid formation. Representative image of ideal hPSC colonies prior to passaging or enzymatic lifting to form spheroids. Scale bar, 600 μm. (b) Details of how to lift hPSC colonies to form spheroids using the enzyme dispase. Representative image of colonies immediately following dispase treatment or after transfer to ultra-low attachment plates. Scale bar, 150 μm. (c) Neural induction and patterning protocols and timeline for generating dorsal and ventral forebrain-specific 3D cultures, including hSS-ISRA (option C that includes exposure to RA and AlloP). The results shown here are from experiments using hPSC that conformed to institutional and federal regulations. IRB panel approval and appropriate informed consent were obtained for these studies.

Figure 3.. Images of forebrain spheroid formation.

Images of spheroids growing optimally (left hand images) and suboptimally (right hand images) are shown.(a) Representative images of hiPSC colonies grown on MEFs. Note that ideal colonies are large and sparsely distributed without evidence of central differentiation. Scale bar, 500 μm. (b) Representative images of spheroid formation during and immediately following dispase treatment. Note that intact colonies are separated without dislodging the layer of MEFs. Scale bar, 500 μm. (c) Representative images of ideal and abnormal neural spheroids during the neural induction period (days 3–14). Scale bar, 500 μm. (d) Representative images of ideal and abnormal neural spheroids during the maturation phase (after day 25). Note the accelerated growth in size of neural spheroids at this stage. Scale bar, 500 μm. The results shown here are from experiments using hPSC that conformed to institutional and federal regulations. IRB panel approval and appropriate informed consent were obtained for these studies.

Figure 4.. Expression of region-specific markers and fusion of dorsal and ventral neural spheroids into assembloids.

(a) Scheme illustrating expression patterns of dorsal and ventral forebrain markers in a coronal slice of GW18–20 human fetal brain. (b) Dorsal forebrain marker expression (PAX6) and ventral forebrain marker expression (NKX2.1) in pallial (hCS) and subpallial (c) spheroids (hSS) at day 25 of differentiation. Scale bar, 50 μm. (d) Pattern of expression of cortical markers in hCS at day 131 of differentiation in vitro. Scale bar, 100 μm. Inset shows expression pattern of CTIP2 (also known as BLC11B) and SATB2. Scale bar, 50 μm. (e) Pattern of expression of ventral forebrain markers in hSS at day 109 of differentiation. Scale bar, 50 μm. Inset shows details of GABA-ergic neuron. Inset scale bar, 12 μm. (f) Scheme and photos showing how spheroids are fused in the bottom of a 1.5 ml Eppendorf on day 0 of forebrain assembloid formation. Eppendorf tubes are held upright in a standard microtube rack. Inset scale bar, 1 cm. (g) Phase contrast image of forebrain assembloids at day 0 and at day 2. Scale bar, 500 μm. (h) Images following transfer of forebrain assembloids to a 96-well plate for live imaging. Scale bar, 4 cm. Inset scale bar, 1 cm. (i) Use of forebrain assembloids to monitor migration of virally labeled (lentivirus, Dlxi½b::eGFP) GABA-ergic neurons from hSS into hCS throughout first 24 days after fusion (daf). Scale bar, 200 μm. The results shown here are from experiments involving the use of hPSCs that conformed to institutional and federal regulations. IRB panel approval and appropriate informed consent were obtained for these studies.