Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient

Abstract

The study of brain development in humans is limited by the lack of tissue samples and suitable in vitro models. Here, we model early human neural tube development using human embryonic stem cells (hESCs) cultured in a microfluidic device. The approach, named microfluidic-controlled stem cell regionalisation (MiSTR), exposes pluripotent stem cells to signalling gradients that mimic developmental patterning. Using a WNT-activating gradient, we generated neural tissue that exhibits progressive caudalisation from forebrain to midbrain to hindbrain, including formation of isthmic organiser characteristics. Single-cell transcriptomics revealed that rostro-caudal organisation was already established at 24 hours of differentiation, and that the first markers of a neural-specific transcription program emerged in the rostral cells at 48 hours. The transcriptomic hallmarks of rostro-caudal organisation recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. Thus, MiSTR will facilitate research on the factors and processes underlying rostro-caudal neural tube patterning.

Neural tube patterning: from a minimal model for rostrocaudal patterning towards an integrated 3D model

“…FB off / MB on. Our multiple toggle switch model was able to recapitulate data from in vitro studies on hPSCs cultured at different WNT signalling levels (8) and exhibited similar dynamics as recent experiments done on a synthetic in vitro setup (26). The toggle switch motif has the capability of controlling the boundaries of gene expression domains (27,40).…”

“…Neural tube rostrocaudal patterning is mainly governed by WNT-signalling, emerging from the isthmic organizer (25). It has been shown in vitro that controlling the gradient of the WNT-signalling, using GSK3 inhibitors either in different cell cultures (11) or in microfluidic devices (26), acting on differentiating human embryonic stem cells can lead to progressive caudalisation from forebrain to hindbrain. However, the gene regulatory network acting downstream of WNT-signalling that regulate this patterning processes has not been elucidated.…”

Accelerated differentiation of human pluripotent stem cells into neural lineages via an early intermediate ectoderm population

“…It is possible that neural induction described in previous studies also occurs via the primal ectoderm now described in this study. In their recent publication generating neural tissue from hESCs following dual SMAD inhibition, Rifes et al define a “pre‐ectodermal” population as an LHX5‐positive intermediate, 60 a marker seen in the populations produced in our current study. Additional evidence for a previously undefined, primal ectoderm may also be seen in the original dual SMAD inhibition protocol 9 .…”

“…The identity of the “primal” ectoderm described in this study now requires further investigation. Recent studies describe that both epiblast cells in early embryonic differentiation and human embryonic stem cells (hESCs) undergoing neural differentiation in vitro, acquire axial identity prior to overt neural induction and suggest that the in vitro patterned, “pre‐ectodermal” populations generated in this study may indeed have an in vivo counterpart 59,60 . A “pre‐ectodermal” designation is used because early ectoderm has been classically defined as a PAX6‐expressing primitive neuroepithelium.…”

Actuation Enhances Patterning in Human Neural Tube Organoids

“…For example, contractile canine kidney (MDCK) cells have been engineered to pull on a collagen substrate to generate origami-like folds 26 in a cell type-specific method relying on cell-mediated tractions which cannot be temporally specified or controlled.. In the context of modeling neural development, microfluidic ligand gradients have been used to generate millimeter-scale patterns in hPSC-derived cell monolayers 27,28 , and neuroepithelial cells have been made to adhere to mechanically activated micropatterns leading to perturbed patterns of gene expression 29 . These techniques have remained confined to 2D however, with limited flexibility in actuation mode.…”