Mammalian multipotent neural stem cells

A method for creating immortalized neural crest stem cells in vitro that contain recombinase target sites flanking an immortalization gene in the genome of said immortalized cells, said method comprising transforming neural crest stem cells with exogeneous nucleic acid comprising:

• a) a first recombinase site;
• b) an immortalization gene;
• and
• c) a second recombinase site;
• such that in the absence of a recombinase said exogeneous nucleic acid is incorporated into the genome of said cell, wherein said neural crest stem cells are capable of self-renewal in a feeder cell-independent culture medium, express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express neuronal or glial lineage markers including glial fibrillary acidic protein (GFAP), wherein at least one of said stem cells is capable of differentiation to a peripheral nervous system (PNS) neuronal cell that does not express LNGFR or nestin but does express neurofilament-160, and wherein at least one of said stem cells is capable of differentiation to a PNS glial cell that expresses LNGFR, nestin and GFAP.

A method for the in vitro disimmortalization of immortalized neural crest stem cells comprising:

• a) incorporating exogeneous nucleic acid comprising:
• i) a first recombinase target site;
• ii) an immortalization gene;
• iii) a negative selection marker gene;
• and
• iv) a second recombinase target site;
• into the genome of neural crest stem cells to produce immortalized cells which contain an excisable immortalization gene in an orientation such that excision of the sequence between the recombinase target sites excises the immortalization gene and the negative selection marker;
• and
• b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene and said negative selection marker gene are excised,
• wherein said neural crest stem cells are capable of self-renewal in a feeder cell-independent culture medium, express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express neuronal or glial lineage markers including glial fibrillary acidic protein (GFAP), wherein at least one of said stem cells is capable of differentiation to a peripheral nervous system (PNS) neuronal cell that does not express LNGFR or nestin but does express neurofilament-160, and wherein at least one of said stem cells is capable of differentiation to a PNS glial cell that expresses LNGFR, nestin and GFAP.

A method for the in vitro disimmortalization of immortalized neural crest stem cells comprising:

• a) incorporating exogeneous nucleic acid comprising:
• i) a first recombinase target site;
• ii) an immortalization gene;
• iii) a selection marker gene;
• and
• iv) a second recombinase target site;
• into the genome of neural crest stem cells to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that excision of the sequence between the recombinase target sites excises the immortalization gene, resulting in the expression of the selection marker;
• and
• b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene is excised,
• wherein said neural crest stem cells are capable of self-renewal in a feeder cell-independent culture medium, express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express neuronal or glial lineage markers including glial fibrillary acidic protein (GFAP), wherein at least one of said stem cells is capable of differentiation to a peripheral nervous system (PNS) neuronal cell that does not express LNGFR or nestin but does express neurofilament-160, and wherein at least one of said stem cells is capable of differentiation to a PNS glial cell that expresses LNGFR, nestin and GFAP.

A method for the in vitro disimmortalization of immortalized neural crest stem cells comprising:

• a) incorporating exogeneous nucleic acid comprising:
• i) a first recombinase target site;
• ii) an immortalization gene;
• iii) a first selection marker gene;
• iv) a second recombinase target site;
• and
• v) a second selection marker gene;
• into the genome of neural crest stem cells to produce immortalized cells which contain an excisable immortalization gene in an orientation such that when the immortalization gene is expressed, the first selection marker gene is also expressed and the second selection marker gene is not expressed and when said immortalization gene is excised said second selection marker is expressed;
• and
• b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene is excised,
• wherein said neural crest stem cells are capable of self-renewal in a feeder cell-independent culture medium, express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express neuronal or glial lineage markers including glial fibrillary acidic protein (GFAP), wherein at least one of said stem cells is capable of differentiation to a peripheral nervous system (PNS) neuronal cell that does not express LNGFR or nestin but does express neurofilament-160, and wherein at least one of said stem cells is capable of differentiation to a PNS glial cell that expresses LNGFR, nestin and GFAP.