Differentiation of ciliated human midbrain-derived LUHMES neurons

Abstract

Many human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing.LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain.In LUHMES, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

Roles of SNORD115 and SNORD116 ncRNA clusters in neuronal differentiation

“…3B; genes are colored by cluster). The results are consistent with previous data on LUHMES cells and other analyses of neurogenesis (Lauter et al ., 2020). The biggest cluster, CL0 (25.2 % of all genes) contains genes that are stably expressed throughout differentiation.…”

“…However, we detected expression in human Lund human mesencephalic (LUHMES) cells following differentiation to neurons. LUHMES are an embryonic, mid-brain derived human cell line that can be induced to synchronously differentiate into polarized dopaminergic neurons (Lauter et al , 2020; Smirnova et al , 2016) (Fig. S1A).…”

“…S1A). Neuronal markers are expressed by day 6 of differentiation; including β3-tubulin (TUBB3), postsynaptic density protein 95 (PSD95, postsynaptic marker), kinesin 17 (KIF17, neuronal transport marker) and tyrosine hydroxylase (TH) (Lauter et al ., 2020; Smirnova et al ., 2016). This high synchrony was of particular importance for biochemical analyses during differentiation time courses.…”

“…LUHMES cells are frequently used as a model of dopaminergic neurons. Changes in the transcriptome (Lauter et al ., 2020) and proteome (Tüshaus et al , 2021) were reported up to day 6 of neuronal differentiation, at which point they were considered mature. In agreement with that, in our data most changes to the transcriptome occur between D00 and D06 of LUHMES differentiation (Fig.…”

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway

“…Here we investigate cilia in a human neuronal cell line, LUHMES [ 38 ], where cells proliferate as neuronal precursors or can be induced to differentiate and mature into neurons. Upon induction to differentiate, ciliation occurs asynchronously, is transient during the early differentiation stages and not every neuron ciliates.…”