Motoneuron differentiation of immortalized human spinal cord cell lines

Abstract

Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM ϩ /neurofilament ϩ precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM ϩ / neurofilament ϩ cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuronspecific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChATϩ motoneurons and GABA ϩ interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

Identification and characterization of neuronal precursors and their progeny from human fetal tissue

“…Furthermore, phenotypically restricted lineages have also been reported for human brain tissue. These include glial restricted precursors (GRPs), which have been isolated from adult human brain tissue (Scolding et al, 1999;Roy et al, 1999), and recent reports have suggested the existence of neuronal restricted precursors (NRPs) in both fetal (Raymon et al, 1999;Li et al, 2000) and adult (Roy et al, 2000a,b;Wang et al, 2000) human tissue.…”

“…Maturation was rapid (2 weeks) compared to that of human NEP cell cultures (>4 weeks; Piper et al, 2000) but was much slower than that in rodent cultures (5 days; Kalyani et al, 1998;Mujtaba et al, 1999) or in immortalized human cell lines (4 days; Li et al, 2000). Rodent NRPs expressed nestin, and virtually all BrdU-incorporating NRPs were nestin + (Mayer-Proschel et al, 1997).…”

“…Furthermore, phenotypically restricted lineages have also been reported for human brain tissue. These include glial restricted precursors (GRPs), which have been isolated from adult human brain tissue (Scolding et al, 1999;Roy et al, 1999), and recent reports have suggested the existence of neuronal restricted precursors (NRPs) in both fetal (Raymon et al, 1999;Li et al, 2000) and adult (Roy et al, 2000a,b;Wang et al, 2000) human tissue.NSCs or neuroepithelial precursor cells (NEPs) can generate neurons in vitro (Carpenter et al, 1999;Piper et al, 2000) as well as in vivo (Svendsen et al, 1998;Vescovi et al, 1999b). In vitro, neurons derived from less committed, NEP-like cell populations develop slowly and do ★ Correspondence to: Mary T. Lucero, PhD, Department of Physiology, 410 Chipeta Way, Room 155, Salt Lake City, UT 84108-1297., mary.lucero@m.cc.utah.edu.…”

“…In contrast, only occasional HNPs coexpressed nestin, and in triple-labeling experiments we showed that nestin expression was not seen in dividing HNPs. Unlike the case with rodent NRP cultures (Mayer-Proschel et al, 1997;Kalyani et al, 1998;Mujtaba et al, 1999), HNPs have proved difficult to grow at low densities without genetic intervention (Raymon et al, 1999;Li et al, 2000) and have remained refractory to strict clonal analyses. HNPs could be isolated by immunopanning and would adhere to laminin/fibronectin but would divide only once or twice in culture.…”

“…The limited evidence available suggests that this may be true for human precursor cells as well. Immortalized human neuronal precursors (HNPs) mature more rapidly than NSCs from stem cell cultures; for example, immortalized HNPs derived from the spinal neuroepithelium develop action potentials and antigenic phenotypes consistent with spinal cord motor neurons after 4 days (Raymon et al, 1999;Li et al, 2000). Recently, HNPs were isolated from the adult ventricular zone and hippocampus using fluorescence-activated cell sorting (FACS) of brain cells transfected with plasmid DNA in which a neuronal precursor-selective Tα1 tubulin promoter was used to drive the expression of GFP (Roy et al, 2000a,b;Wang et al, 2000).…”