The mammalian central nervous system is developmentally derived from neuroepithelial cells in the neural plate. These neuroepithelial cells grow and differentiate in response to signals from their surrounding environment. Many of those signals have been well characterized and others remain to be discovered. In cell culture, a conditioned medium, a feeder cell layer, or a tissue extract has been used as supplement in addition to those factors well characterized for maintaining the multipotent status of neural progenitor cells. To date, there have been many types of neural progenitor cells established in culture from various stages of development and from different regions of the nervous system of various species. This chapter will provide a brief introduction to those cultures and a detailed method for culturing rat neural epithelial cells at embryonic stage E9 and characterizing them in vitro and in vivo.

Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/ neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific 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 ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABAf(+) 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. (C) 2000 Wiley-Liss, Inc.

Sensory and motor neuron-derived factor (SMDF) is a member of the neuregulin family of proteins. SMDF is structurally characterized by a novel N-terminal domain. Using the signal sequence and N-terminal 28 amino acids (the "epitope") of herpes simplex virus type 1 glycoprotein D (gD), we have expressed SMDF as an epitope-tagged protein (gD-SMDF) in 293 cells, and purified it to > 98% homogeneity on a monoclonal anti-gD column. gD-SMDF stimulates human Schwann cell growth and 3H-thymidine incorporation in MCF-7 and T47D human breast tumor cells in vitro. The biological activity of gD-SMDF is consistent with its ability to compete with 125I-labeled heregulinbeta1 peptide (rHRGbeta1(177-244)) to bind to soluble dimeric ErbB receptor-IgG fusion proteins. gD-SMDF binds with low affinity to homodimeric ErbB3-IgG and ErbB4-IgG but with higher affinity to heterodimeric ErbB2/ErbB3-IgG and ErbB2/ErbB4-IgG. Using a SMDF-IgG(Fc) fusion protein we generated a monoclonal antibody (3G11) which binds SMDF, crossreacts with rHRGbeta1(177-244), and neutralizes the in vitro activities of gD-SMDF and rHRGbeta1(177-244) in human Schwann cells.

With recent advances in cell culture techniques it is possible to isolate human SCs from adult peripheral nerves, expand and purify their number in cell culture, and construct a cellular prosthesis from the cultured cells. The current study was designed to ascertain whether these techniques could be used to repair nonhuman primate nerve injuries. In 12 adult femalecynomologousmonkeys, the musculocutaneous (msk) nerve was divided and prevented from regenerating and the brachioradialis nerve (brach) was exposed bilaterally (n=24 nerves) and injured so that a 15-mm gap existed within the nerve. The brach nerves were either repaired with sural nerve autografts (n=6), guidance channels which contained monkey SCs (120×106cells/ml;n=6), or guidance channels without SCs (n=6). The remaining brach nerves (n=6) had either no injury or an injury to the nerve without a repair. Autologous expanded primate SCs were increased in number at least 10-fold over a 2-week period at which time the SC purity exceeded 99.9%. Monkeys in each group, including the control group, regained some degree of elbow flexion after 3 months despite sectioning both the mask nerve and the brach nerve; therefore, we were unable to determine simply on clinical grounds which repair was the most effective in promoting functional recovery. Brach nerves repaired with sural nerve grafts were superior to both the channels which contained SCs and empty channels in regards to the number of myelinated axons proximal, within, and distal to the repair site (P<0.05). Electrophysiologic results closely paralleled the histologic data with evidence of reinnervation of the brachioradialis muscle based on the compound muscle action potential in both sural nerve graft and monkey SC channel repair groups.