Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord.

Multipotent neural stem cells (NSCs) have the potential to differentiate into neuronal and glial cells and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages. In particular, when grafted into the adult spinal cord, NSCs are restricted to the glial lineage, indicating that the host spinal cord environment is not permissive for neuronal differentiation. To identify the stage at which neuronal differentiation is inhibited we examined the survival, differentiation, and integration of neuronal restricted precursor (NRP) cells, derived from the embryonic spinal cord of transgenic alkaline phosphatase rats, after transplantation into the adult spinal cord. We found that grafted NRP cells differentiate into mature neurons, survive for at least 1 month, appear to integrate within the host spinal cord, and extend processes in both the gray and white matter. Conversely, grafted glial restricted precursor cells did not differentiate into neurons. We did not observe glial differentiation from the grafted NRP cells, indicating that they retained their neuronal restricted properties in vivo. We conclude that the adult nonneurogenic CNS environment does not support the transition of multipotential NSCs to the neuronal commitment stage, but does allow the survival, maturation, and integration of NRP cells.

Stable expression of the alkaline phosphatase marker gene by neural cells in culture and after transplantation into the CNS using cells derived from a transgenic rat.

Multipotent stem cells and more developmentally restricted precursors have previously been isolated from the developing nervous system and their properties analyzed by culture assays in vitro and by transplantation in vivo. However, the variety of labeling techniques that have been used to identify grafted cells in vivo have been unsatisfactory. In this article we describe the characteristics of cells isolated from a transgenic rat in which the marker gene human placental alkaline phosphatase (hPAP) is linked to the ubiquitously active R26 gene promoter. We show that hPAP is readily detected in embryonic neuroepithelial stem cells, neuronal-restricted precursor cells, and glial-restricted precursor cells. Transgene expression is robust and can be detected by both immunocytochemistry and histochemistry. Furthermore, the levels of hPAP on the cell surface are sufficient for live cell labeling and fluorescence-activated cell sorting. Expression of hPAP is stable in isolated cells in culture and in cells transplanted into the spinal cord for at least 1 month. We submit that cells isolated from this transgenic rat will be valuable for studies of neural development and regeneration.

Isolation of a glial-restricted tripotential cell line from embryonic spinal cord cultures.

Neuroepithelial stem cells (NEPs), glial-restricted precursors (GRPs), and neuron-restricted precursors (NRPs) are present during early differentiation of the spinal cord and can be identified by cell surface markers. In this article, we describe the properties of GRP cells that have been immortalized using a regulatable v-myc retrovirus construct. Immortalized GRP cells can be maintained in an undifferentiated dividing state for long periods and can be induced to differentiate into two types of astrocytes and into oligodendrocytes in culture. A clonal cell line prepared from immortalized GRP cells, termed GRIP-1, was also shown to retain the properties of a glial-restricted tripotential precursor. Transplantation of green fluorescent protein (GFP)-labeled subclones of the immortalized cells into the adult CNS demonstrates that this cell line can also participate in the in vivo development of astrocytes and oligodendrocytes. Late passages of the immortalized cells undergo limited transdifferentiation into neurons as assessed by expression of multiple neuronal markers. The availability of a conditionally immortalized cell line obviates the difficulties of obtaining a large and homogeneous population of GRPs that can be used for studying the mechanism and signals for glial cell differentiation as well as their application in transplantation protocols.