A More Rapid Method for Culturing LUHMES-Derived Neurons Provides Greater Cell Numbers and Facilitates Studies of Multiple Viruses
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1263893 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The ability to study mature neuronal cells ex vivo is complicated by their nature as non-dividing and difficulty in obtaining large numbers of primary cells from organisms. Thus, numerous transformed progenitor models have been developed that can be routinely cultured then scaled and differentiated to mature neurons. Here, we present a new method for differentiating one such model, the LUHMES dopaminergic neurons. This method is two days faster than some established protocols, results in nearly five times greater numbers of mature neurons, and involves fewer handling steps that could introduce technical variability. Moreover, it overcomes the problem of cell aggregate formation that commonly impedes high-resolution imaging, cell dissociation and downstream analysis. While recently established for herpes simplex virus type 1, we demonstrate that LUHMES neurons can facilitate studies of other herpesviruses, as well as RNA viruses associated with childhood encephalitis and hemorrhagic fever. This protocol provides an improvement in the generation of large-scale neuronal cultures, which may be readily applicable to other neuronal 2D cell culture models and provides a system for studying neurotrophic viruses. We name this method the Streamlined Protocol for Enhanced Expansion and Differentiation Yield, or SPEEDY method.
2025-07-30 | GSE297466 | GEO
Project description:To study mechanisms of neurodegenerative diseases, neuronal cell lines are important model systems and are often differentiated into postmitotic neuron-like cells to resemble more closely primary neurons obtained from brains. One such cell line is the Lund Human Mesencephalic (LUHMES) cell line which can be differentiated into dopamine-like neurons and is frequently used to study mechanisms of Parkinson’s disease (PD) and neurotoxicity. Neuronal differentiation of LUHMES cells is commonly verified by measurement of selected neuronal markers, but little is known about proteome-wide protein abundance changes during differentiation. Using mass spectrometry and label-free quantification (LFQ) we compared the proteome of differentiated and undifferentiated LUHMES cells as well as of cultured primary murine midbrain neurons, which are mainly dopaminergic. Neuronal differentiation induced substantial changes of the LUHMES cell proteome (18.4% reveal protein abundance changes of more than 4-fold), with proliferation-related proteins (e.g. MCMs) being strongly down-regulated and neuronal and dopaminergic proteins being up to 1000-fold upregulated, such as L1CAM and SNCA. Several of these proteins, including MAPT and SYN1, may be useful new markers to experimentally validate neuronal differentiation of cultured LUHMES cells. Primary midbrain neurons were more closely related to differentiated than to undifferentiated LUHMES cells with respect to the abundance of proteins related to neurodegeneration or to genetic forms of PD. In summary, our comparative proteomic analysis demonstrates that differentiated LUHMES cells are a suitable model for studies on PD and neurodegeneration and provides a resource of the proteome-wide changes during neuronal differentiation.
2020-09-24 | PXD020044 | Pride
Project description:Differential transcriptome analysis in LUHMES cells overexpressing human wild-type alpha-synuclein or GFP
2022-03-31 | GSE191302 | GEO
Project description:we performed lentiviral CRISPR interference (CRISPRi) by recruiting dCas9 fused with the KRAB domain to the CSMD1 enhancer (fam3) in the neuronal precursor cell line – Lund human mesencephalic (LUHMES). Given that the expression of CSMD1 was not detectable in LUHMES cells we differentiated these cells into neurons. Differentiated neurons with CRISPRi of CSMD1 enhancer showed significantly higher expression of CSMD1 than control.
2022-06-30 | E-MTAB-8027 | biostudies-arrayexpress
Project description:LUHMES cells share many characteritics with human dopamingeric neurons in the substantia nigra, the cells whose demise is responsible for the motor symptoms in Parkinson’s disease (PD). LUHMES cells can therefore be used bona fide as a model to study pathophysiological processes involved in PD. Previously, we showed that LUHMES cell degenerate after six days upon overexpression of wild type alpha-synuclein. In the present study we performed a transcriptome and proteome expression analysis in alpha-synuclein-overexpressing cells and GFP-expressing control cells in order to identify genes and proteins that are differentially regulated upon overexpression of alpha-synuclein. The analysis was performed four days after the initiation of alpha-synuclein or GFP overexpression, before the cells died in order to identify processes that preceded cell death.
2022-05-19 | PXD028322 | Pride
Project description:Retrospective analysis of RNAi hits derived from Wang et. al. has found that genes with greater transcripts-per-million (TPM) in S. mansoni are more likely to produce physical phenotypes. To create the best rate-of-return in our large-scale RNAi screen to identify essential schistosome genes that produce physical phenotypes, we quantified TPM of each gene expressed in male parasites after culturing them with female parasites for 72 hours.
2025-03-10 | GSE290988 | GEO
Project description:Study of MeCP2 in LUHMES-derived neurons
| PRJNA517116 | ENA
Project description:greater flamingo cloacal microbiome Targeted loci environmental
| PRJNA485732 | ENA
Project description:Identification of toxicants that underlie neurological diseases is a neglected area awaiting a valid strategy to identify such toxicants. We sought biomarkers that respond to known neurotoxicants in LUHMES immortalized neurons and evaluated these biomarkers for use in screening libraries of environmental toxicants. LUHMES immortalized human dopaminergic neurons were surveyed by RNA sequencing following challenge with Parkinsonian toxicants rotenone, 6-hydroxydopamine, MPP+, and ziram (zinc dimethyldithiocarbamate; Zn2+DDC2), as well as additional toxicants paraquat, MS275, and methylmercury. The metallothionein gene MT1G was the most dynamic gene expression response to all seven toxicants. Multiple toxicants also increased transcripts for SLC30A1 and SLC30A2 zinc secretion transporters, the SLC7A11 xCT cystine/glutamate antiporter important for glutathione synthesis, DNA damage Inducible Transcript 3 (DDIT3), and secreted growth factors FIBIN and CXCL12, whereas several toxicants decreased expression of the Apelin growth factor (APLN). These biomarker genes showed advantages in sensitivity by responding to many toxicants at sub-cytotoxic concentrations. Since several of these biomarker genes and prior neurological disease studies implicated disruption of metal distribution, we tested metal chelator thiram (dimethyldithiocarbamate, DDC), Zn2+DDC2, and several other metals and metal chelates for cytoxicity and induction of MT1G expression. Metals and chelators that caused dynamic increases in MT1G expression caused cytotoxicity, whereas Ni2+DDC2 caused MT1G increase, but no cytotoxicity. These results bolster prior work suggesting that neurons are characteristically sensitive to depletion of glutathione or to disruption of cellular metal distribution and provide biomarkers to search for such neurotoxicants in chemical libraries.
2020-08-11 | GSE150005 | GEO
Project description:To date, most in vitro toxicity testing has focused on acute effects of compounds at high concentrations. This testing strategy does not reflect real-life exposures, which might contribute to long-term disease outcome. We used a 3D-human dopaminergic in vitro LUHMES cell line model to determine whether effects of short-term rotenone exposure (100 nM, 24 h), are permanent or reversible. A decrease in complex I activity, ATP, mitochondrial diameter and neurite outgrowth were observed acutely. After compound removal, complex I activity was still inhibited, however, ATP levels were increased, cells were electrically active and aggregates restored neurite outgrowth integrity and mitochondrial morphology. We identified significant transcriptomic changes after 24 h which were not present 7 days after wash-out. Our results suggest that testing short-term exposures in vitro may capture many acute effects which cells can overcome, missing adaptive processes and long-term mechanisms. Additionally, to study cellular resilience, cells were re-exposed to rotenone after wash-out. Pre-exposed cells maintained higher metabolic activity than controls and presented a different expression pattern in genes previously shown to be altered by rotenone. NEF2L2, ATF4 and EAAC1 were downregulated upon single hit on day 15, but unchanged in pre-exposed aggregates. DAT and CASP3 were only altered after re-exposure to rotenone while TYMS and MLF1IP were downregulated in both single-exposed and pre-exposed aggregates. In summary, our study shows that a human cell-based 3D model can be used to assess cellular adaptation, resilience and long-term mechanisms relevant to neurodegenerative research. In this study acute and delayed effects of rotenone on transcriptome in 3D LUHMES were assessed by microarray. The goal was to characterize perturbations in gene expression immediately after exposure to rotenone and identify whether these perturbations persist after compound wash-out and recovery period
2018-06-27 | GSE116280 | GEO