Dubonyte, U. | bioRxiv Science

bioRxiv2025

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

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.

neuroscience↗

bioRxiv2022

Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality.

The human brain is a complex, three-dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human specific midbrain organoids. Human iPSC-derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuro-inflammation related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC-derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC-derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress- related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuro- inflammation. Main Points- Macrophage precursors can be efficiently co-cultured with midbrain organoids, they integrate into the tissue and differentiate into microglia in 3D. - Organoids containing microglia have a smaller size and show a down-regulation of oxidative stress-related genes. - Organoids co-cultured with microglia show differences in genes related to synaptic remodeling and action potential, as well as a more spontaneous action potential firing.

neuroscience↗