Abstract
Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids). These organoids exhibit remarkable self-organization that resembles key aspects of in vivo organ development. However, organoids have an unpredictable anatomy, and poorly reflect the topography of the dorsoventral, mediolateral, and anteroposterior axes. In vivo the temporal and the spatial patterning of the developing tissue is orchestrated by signaling molecules called morphogens. Here, we used morphogen-soaked beads to influence the spatial identities within hESC-derived brain organoids. The morphogen- and synthetic molecules-soaked beads were interpreted as local organizers, and key transcription factor expression levels within the organoids were affected as a function of the distance from the bead. We used an on-chip imaging device that we have developed, that allows live imaging of the developing hESC-derived organoids. This platform enabled studying the effect of changes in WNT/BMP gradients on the expression of key landmark genes in the on-chip human brain organoids. Titration of CHIR99201 (WNT agonist) and BMP4 directed the expression of telencephalon and medial pallium genes; dorsal and ventral midbrain markers; and isthmus-related genes. Overall, our protocol provides an opportunity to study phenotypes of altered regional specification and defected connectivity, which are found in neurodevelopmental diseases.
Highlights
Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells into organ-like structures [1,2,3]
To follow the development of human neuroepithelium (NE) organoids [1,3,6], human embryonic stem cells (hESCs) aggregates were embedded in drops of collagen-laminin-based hydrogel (MatrigelTM) and inserted into a micro-fabricated compartment (h = 250 ± 10 μm, Figure 1A and Figure S1A–D; details of the device assembly are found in the methods section)
We explored the role of WNT and BMP4 gradients in establishing dorsoventral (DV) and anteroposterior (AP) fates in human brain organoids
Summary
Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids) [1,2,3] (reviewed by Di Lullo and Kriegstein, 2017 [4]) These organoids exhibit self-organization and developmental trajectories, which mimics key features of known in vivo organ development as well as gene expression patterns [5]. The initial positional information across the neural plate includes mediolateral gradients of BMP and Shh, together with an anteroposterior gradient of Wnt activity These conserved Wnt gradients mediate eventually the formation of the forebrain, midbrain, hindbrain, and the spinal cord along the anterior-posterior axis [15] (reviewed in Green et al 2014 [16]). It has been recently demonstrated that human hESCs grown in microfluidic devices and subjected to WNT gradients exhibited rostro-caudal identities from forebrain to midbrain to hindbrain, including the formation of isthmic organizer-like characteristics [22]. Spontaneous polarization of spinal cord organoids was observed in rotary cultures, where the organoids elongated as their differentiation progressed [29]
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