The HCN-1A cell line is a human cortical neuronal line that was initiated from tissue excised from a young female patient whose treatment for intractable seizures included a hemispherectomy. The cells are commonly utilized to model neuronal processes for scientific study.

HCN-1A cells test positive for somatostatin, glutamate, neuron specific enolase, tubulin, vimentin, vasoactive intestinal peptide, gamma aminobutyric acid, and cholecystokinin-8, but are negative for glial fibrillary acidic protein and myelin basis protein. By culturing the cortical neuronal cells with a cocktail of nerve growth factor, dibutyryl cyclic adenosine monophosphate, and 1-isobutyl-3-methylxanthine, their differentiation can be induced. When HCN-1A cells differentiate, they assume mature morphology and their growth rate slows considerably.

The patient whose tissue served as a source for the cells from which the HCN-1A cell line was developed was afflicted with unilateral megalencephaly, which is typified by the excessive growth of one, or a section of one, of the cerebral hemispheres. The condition is very serious, but its occurrence is rare. In some individuals, unilateral megalencephaly is evident at birth due to the appearance of the head, which may be unusually large and asymmetrical. The disorder may not become physically apparent in other patients until some time during the first few years of life. In addition to the physical signs, unilateral megalencephaly is commonly associated with delayed development, severe mental retardation, and untreatable seizures. The condition is thought to be related to an abnormality in the mechanism that typically regulates cell proliferation.

Immunofluorescence with mouse anti-alpha-tubulin was employed to visualize distribution of the microtubule network in the human cortical neuronal cell culture illustrated above. The secondary antibody (goat anti-mouse IgG) was conjugated to Cy2 and mixed with Alexa Fluor 568 conjugated to phalloidin to simultaneously image tubulin and the actin cytoskeleton. Nuclei were counterstained with Hoechst 33258. Images were recorded in grayscale with a 12-bit digital camera coupled to either a Nikon E-600 or Eclipse 80i microscope equipped with bandpass emission fluorescence filter optical blocks. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.

Human Cortical Neuronal Cells with MitoTracker Red CMXRos, Alexa Fluor 488, and Hoechst 33258 - Three fluorophores and a phallotoxin were utilized to triple label the culture of HCN-1A cells shown in this section. Mitochondria were targeted with MitoTracker Red CMXRos, the cytoskeletal F-actin network was localized with Alexa Fluor 488 conjugated to phalloidin, and DNA in the cell nucleus was counterstained with Hoechst 33258.

Distribution of Mitochondria and F-Actin in HCN-1A Cells - The culture of human cortical neuronal cells presented in this section was labeled with MitoTracker Red CMXRos and Alexa Fluor 488 conjugated to phalloidin to target the mitochondrial network and filamentous actin, respectively. The nucleic acid stain Hoechst 33258 was utilized to target cell nuclei.

Intracellular Relationship Between Neuronal Cell Nuclei and the Mitochondrial and Filamentous Actin Networks - Mitochondria and filamentous actin in a culture of HCN-1A cells were simultaneously stained with MitoTracker Red CMXRos and Alexa Fluor 488 conjugated to phalloidin. Nuclear DNA was counterstained with Hoechst 33258.

Triple Fluorophore Labeling of HCN-1A Cells - The monolayer human cortical neuronal cell culture presented in this section was labeled for the cytoskeletal filamentous actin and intracellular mitochondrial networks with Alexa Fluor 488 conjugated to phalloidin and MitoTracker Red CMXRos, respectively. Nuclei present in the cells were counterstained with the DNA-selective bisbenzimide dye, Hoechst 33258.