Immortalized Human Dopaminergic Neuronal Precursor Cells (LUHMES)

Improved LUHMES Differentiation Protocol Generates More TH-positive Neurons

Human-derived neuronal cell lines, particularly the LUHMES cell line, are valuable for neurobiology and preclinical research. The LUHMES cell line can differentiate into post-mitotic neurons but currently only 10% of cells express tyrosine hydroxylase (TH), a marker for dopaminergic neurons. To address this, Harischandra et al. optimized a differentiation protocol using a cocktail of neurotrophic factors, cytokines, and antioxidants.

The initial LUHMES maintenance and differentiation protocol by Scholz et al. used 40 ng/mL bFGF for proliferation and 1 µg/mL tetracycline for differentiation (Fig. 1, top panel). Our DM-I protocol used growth factors and dibutyryl-cyclic AMP, yielding about 10% TH+ neurons. After literature review and optimization, they developed DM-II, incorporating BDNF, ascorbic acid, TGF-β III, and LIF (Fig. 1, bottom panel). LUHMES cells from the same passage were differentiated using DM-I or DM-II for up to 7 days. At 3, 5, and 7 days post-induction, cells were probed for dopaminergic markers. The new protocol (DM-II) consistently produced more TH+ neurons over 7 days (Fig. 2A). Quantification showed 1% TH+ cells at day 3 and 25% at day 5 with DM-II, compared to 0% and 7% with DM-I (Fig. 2B). By day 7, TH+ cells decreased to 12% with DM-II and 3% with DM-I.

Lund Human Mesencephalic (LUHMES) Neuronal Cell Line Supports Herpes Simplex Virus 1 Latency In Vitro

Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be differentiated into postmitotic neurons. These cells can be infected with herpes simplex virus 1 (HSV-1), making them a potential model for studying viral latency and reactivation. Unlike rodent neuron cultures, LUHMES cells are scalable and can maintain latency without continuous antiviral inhibitors. Edawrds et al. demonstrated the utility of LUHMES cells as a scalable model for HSV-1 latency and reactivation, mirroring in vivo properties.

The LUHMES cell line, known for its fully differentiated dopaminergic neuron properties, is a valuable tool for studying neurological diseases like Parkinson's disease. Engineered to express v-myc in a tetracycline-regulatable system, LUHMES cells can proliferate and then differentiate upon tetracycline addition. LUHMES cultures display neurite-like projections (Fig. 3A) and can be differentiated to express mature neuron markers within 5 days using tetracycline, GDNF, and dibutyryl cAMP. For our experiments, LUHMES cells were plated on coverslips, differentiated, and analyzed for neuron-specific markers via immunofluorescence. After 7 days, neuronal filament projections up to 200 µm long were observed (Fig. 3B and C). The differentiated cells contained neurofilament H-positive intermediate filaments (Fig. 3D to F), βIII-tubulin (Fig. 3G), and synaptophysin (Fig. 3H and I).