Rapid, complete and large‐scale generation of post‐mitotic neurons from the human LUHMES cell line

Abstract

We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as neuronal model system. Neurodevelopmental profiling of the proliferation stage (d0, day 0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with down-regulation of some precursor markers and further up-regulation of neuronal genes. Neurite network formation involved the outgrowth of 1-2, often > 500 lm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-overexpressing cells. Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake-and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP and glial cell derived neurotrophic factor were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP. In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology.

Microfluidic construction of minimalistic neuronal co-cultures

“…The use of PEGylated regions allows proteins to be subsequently added which is preferred to the application of a PL-protein mixture. For example, fibronectin can be added for the differentiation and maintenance of LUHMES cells 36 and laminin for promoting outgrowths from primary cultures. 37 The novel water masking method for in situ biomaterial patterning by plasma stencilling is simple, reliable and widely applicable to the variety of adhesion materials appropriate to the different neuron types.…”

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

“…Cell differentiation was generally performed following the published protocol [14] . Briefly, 4×10 6 LUHMES cells were seeded onto a pre-coated 100-mm dish.…”

“…Following similar differentiation protocol as described in the study published by Scholz et al [14] , LUHMES cells were differentiated in the culture medium containing tetracycline (to suppress v-myc gene expression) and cAMP/ GDNF (to promote dopaminergic features). The cells were differentiated in 100-mm dishes for two days and then re-plated in 6-well plates for another 4 d of differentiations ( Figure 1A).…”

“…They can be easily cultured and maintained, similar to other cell lines. After several-day differentiation using tetracycline, cyclic AMP (cAMP) and glial derived neurotrophic factor (GDNF), these cells exit cell cycle and gradually develop into dopaminergic-like neurons, which express dopaminergic neuron specific markers biochemically and develop extensive neurites morphologically [13,14] . Functionally, these post-mitotic cells display spontaneous electrical activities and are able to release/uptake dopamine, which suggests a high relevance between differentiated LUHMES cells and human dopaminergic neurons [14] .…”

Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes

“…In this study, we apply LUHMES human neuronal cells that can be differentiated within 6 days into mature neurons [26]. Here we compare toxic effects of organic MeHgCl and thiomersal as well as inorganic HgCl 2 in human astrocytes (CCF-STTG1) and differentiated human neurons (LUHMES) within one study for the first time.…”