Mapping early patterning events in human neural development usingan in-vitro microfluidic stem cell model

Gaurav Singh Rathore; Matias Ankjær; Pedro Rifes; Erno Hänninen; Fátima Sanchís-Calleja; Charlotte Rusimbi; Janko Kajtez; Amalie Holm Nygaard; Louise Piilgaard; Ugne Dubonyte; Zehra Abay-Nørgaard; Jens Bager Christensen; Kristoffer Lihme Egerod; J. Gray Camp; Tune H. Pers; Barbara Treutlein; Agnete Kirkeby

doi:10.1101/2025.05.23.655442

NeurosciencebioRxivMay 23, 2025

Preprint

Mapping early patterning events in human neural development usingan in-vitro microfluidic stem cell model

Gaurav Singh Rathore iD, Matias Ankjær, Pedro Rifes iD, Erno Hänninen, Fátima Sanchís-Calleja iD, Charlotte Rusimbi iD, Janko Kajtez iD, Amalie Holm Nygaard iD, Louise Piilgaard iD, Ugne Dubonyte iD, Zehra Abay-Nørgaard iD, Jens Bager Christensen iD, Kristoffer Lihme Egerod iD, J. Gray Camp iD, Tune H. Pers iD, Barbara Treutlein iD, Agnete Kirkeby iD

Abstract

Stem cell models can provide insights into human brain development at embryonic stages which are normally inaccessible. We previously developed the Microfluidic Stem Cell Regionalisation (MiSTR) model, which recapitulates the rostro-caudal patterning of human neural tube through a WNT activation (WNTa) gradient. Through temporal single cell transcriptomics of rostro-caudal and dorso-ventral gradient-patterned MiSTR, we found that rostro-caudal subtypes were regionally specified and fate-determined already during the late epiblast stage, several days before onset of neuralisation at day 3-4. Rostral cells were characterised by expression of HESX1 and SHISA2 during pre-neuralisation and PAX6 during early neuralisation, whereas caudal cells expressed FST and HOXA1 during pre-neuralisation and SOX1 as the dominant neuralising factor. In contrast to the early rostro-caudal specification, response to ventralisation in telencephalic progenitors was developmentally delayed and occurred around day 9. We further uncovered temporal events in human midbrain-hindbrain boundary formation and ventral forebrain patterning, contributing new knowledge on early human neural region-specification.

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