Human LUHMES and NES cells as models for studying primary cilia in neurons

Coschiera, Andrea; Watts, Michelle E.; Kere, Juha; Tammimies, Kristiina; Swoboda, Peter

doi:10.1016/bs.mcb.2022.12.012

Cited by 2 publications

(8 citation statements)

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“…We surmise that some of the truncated IFT88 and IFT172 KD cilia are microscopically indistinguishable from those oriented vertically in 3D space (cf. [ 42 ]). Like in RFX2 -/- (and in non-ciliated) neurons, altered cilia in IFT88 and IFT172 KD neurons led to fewer neurons in the population being able to reach differentiation stage 3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Our complete differentiation time course experiments from precursor to mature neuron revealed a clear pattern: the presence of cilia steadily increased and peaked at d3 of differentiation (around 70% ciliation) before decreasing during later differentiation stages (d4-d6). Cilia are dynamic organelles during the cell cycle [ 71 ], during neuron differentiation both in vivo and in vitro [ 24 , 31 , 42 ] and during the differentiation of non-neuronal cell types [ 57 , 72 ]. Similarly, ciliation in our post-mitotic, differentiating LUHMES neurons is dynamic and transient, suggesting a ciliary time window that appears to “open” after newborn neurons undergo the first centrosome-associated differentiation steps (polarization, growth cone development, initiation of neurites) [ 36 , 37 ] and to “close” (disassembly of cilia) before the emergence of large numbers of fully functional synapses as signaling hubs during the later neuron differentiation and maturation stages [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

“…Lu nd h uman mes encephalic (LUHMES) cells, a v-myc immortalized neuronal precursor cell line, was obtained from the ATCC ( https://www.atcc.org/products/crl-2927 ). LUHMES cells were cultured in a standard incubator (37 °C, 5% CO 2 ) as previously described [ 26 , 42 , 70 ]. LUHMES cells were grown in DMEM/F-12 Ham growth medium (Sigma-Aldrich D6421) supplemented with L-glutamine solution (Sigma-Aldrich G7513; 2.5 mM), N-2 supplement (Gibco 17502048; 1 ×) and human heat stable basic Fibroblast Growth Factor (bFGF) (Thermo Fisher Scientific PHG0369; 20 ng/ml) in vessels pre-coated with poly-L-ornithine hydrobromide (Sigma-Aldrich P3655; 50 μg/ml) and fibronectin from human plasma (Sigma-Aldrich F1056; 1 μg/ml).…”

Section: Methodsmentioning

confidence: 99%

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

Coschiera,

Yoshihara,

Lauter

et al. 2024

BMC Biol

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Background Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell’s immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. Results We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation “ciliary time window” during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. Conclusions We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in “mild” impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Coschiera,

Yoshihara,

Lauter

et al. 2024

BMC Biol

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“…Lund human mesencephalic (LUHMES) cells, a v-myc immortalized neuronal precursor cell line, was obtained from the ATCC (https://www.atcc.org/products/crl-2927). LUHMES cells were cultured in a standard incubator (37°C, 5% CO2) as previously described (Scholz et al, 2011;Lauter et al, 2020;Coschiera et al, 2023). LUHMES cells were grown in DMEM/F-12 Ham growth medium (Sigma-Aldrich D6421) supplemented with L-glutamine solution (Sigma-Aldrich G7513; 2.5 mM), N-2 supplement (Gibco 17502-048; 1×) and human heat stable basic Fibroblast Growth Factor (bFGF) (Thermo Fisher Scientific (which was not certified by peer review) is the author/funder.…”

Section: Cell Culture and Growth Conditionsmentioning

confidence: 99%

“…Immunocytochemistry was performed as previously described (Lauter et al, 2020;Coschiera et al, 2023). 7.5×10 4 LUHMES cells were seeded 24h prior to differentiation into pre-coated 35 mm dishes (Corning 353001) containing round borosilicate cover glasses (VWR 631-0150P), pre-treated with hydrochloric acid fuming 37%, rinsed with dH2O and absolute ethanol and finally stored in 70% ethanol.…”

Section: Immunocytochemistry and Fluorescence Intensity Quantificationmentioning

confidence: 99%

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

Coschiera¹,

Yoshihara²,

Lauter³

et al. 2023

Preprint

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Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and a mutant with altered cilia (RFX2 -/-), we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

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Human LUHMES and NES cells as models for studying primary cilia in neurons

Cited by 2 publications

References 34 publications

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

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

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

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