Genetically engineered mammalian neural crest stem cells

• US 5,654,183 A
• Filed: 01/28/1994
• Issued: 08/05/1997
• Est. Priority Date: 07/27/1992
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Anderson, David J., Stemple, Derek L.
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/188,286
• Time in Patent Office: 1,285 Days
• Field of Search: 435/69.1, 435/172.3, 435/240.2, 435/320.1, 424/93.21, 514/44
• US Class Current: 435/456
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/35 Polyols, e.g. glycerin, inositol
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2500/80 from animals
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/02 Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/86 Melanocyte-stimulating hormone [MSH]
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/56 Fibrin; Thrombin
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

The invention includes mammalian multipotent neural stem cells and their progeny and methods for the isolation and clonal propagation of such cells. At the clonal level the stem cells are capable of self regeneration and asymmetrical division. Lineage restriction is demonstrated within developing clones which are sensitive to the local environment. The invention also includes such cells which are transfected with foreign nucleic acid, e.g., to produce an immortalized neural stem cell. The invention further includes transplantation assays which allow for the identification of mammalian multipotent neural stem cells from various tissues and methods for transplanting mammalian neural stem cells and/or neural or glial progenitors into mammals. A novel method for detecting antibodies to neural cell surface markers is disclosed as well as a monoclonal antibody to mouse LNGFR.

Claims

1. An isolated cellular composition of genetically-engineered mammalian neural crest stem cells which are capable of self-renewal in a feeder cell-independent culture medium or glial cells, wherein said genetically engineered neural crest stem cells 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 neural crest stem cell of said isolated composition is capable of differentiation to a peripheral nervous system neuronal cell that does not express LNGFR or nestin but does express neurofilament-160, and wherein at least one neural crest stem cell of said isolated composition is capable of differentiation to a peripheral nervous system glial cell that expresses LNGFR, nestin and GFAP.

2. The cellular composition of claim 1 wherein said neural crest stem cells are isolated from the neural crest.

3. A cellular composition according to claims 1 wherein said neural crest stem cells do not express sulfatide, myelin protein P_o, peripherin, high polysialic acid neural cell adhesion molecule (high PSA-NCAM) or neurofilament.

4. A method for generating an isolated cellular composition of genetically-engineered neural crest stem cells which 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 neural crest stem cells is capable of differentiation to a peripheral nervous system neuronal cell that does not express LNGFR or nest in but does express neurofilament-160, and wherein at least one of said neural crest stem cells is capable of differentiation to a peripheral nervous system glial cell that expresses LNGFR, nestin and GFAP, said method comprising contacting an isolated neural crest stem cell with foreign nucleic acid under conditions permissive for the uptake of said foreign nucleic acid into said neural crest stem cell.

5. The method according to claim 4, wherein conditions permissive for the uptake of said foreign nucleic acid comprise calcium phosphate-mediated transfection.

6. The method according to claim 4, wherein conditions permissive for the uptake of said foreign nucleic acid comprise retroviral infection.

7. The method according to claim 4 wherein said neural crest stem cells are isolated from the neural crest.

8. The method according to claim 4 wherein said neural crest stem cells do not express sulfatide, myelin protein P_o, peripherin, high polysialic acid neural cell adhesion molecule (high PSA-NCAM) or neurofilament.

9. A clonal population of genetically-engineered mammalian neural crest stem cells which are capable of self-renewal in a feeder cell-independent culture medium wherein said genetically engineered neural crest stem cells 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 of said clonal population is capable of differentiation to a peripheral nervous system neuronal cell that does not express LNGFR or nestin but does express neurofilament-160, and wherein at least one of said stem cells of said clonal population is capable of differentiation to a peripheral nervous system glial cell that expresses LNGFR, nestin and GFAP.

10. The clonal population of cells according to claim 9, wherein said peripheral nervous system neuronal cells express peripherin.

11. The clonal population of cells according to claim 9, wherein said peripheral nervous system neuronal cells express high-polysialic acid neural cell adhesion molecule (PSA-NCAM).

12. The clonal population of cells according to claim 9, wherein said peripheral nervous system neuronal cells express neurofilament-68.

13. The clonal population of cells according to claim 9, wherein said peripheral nervous system neuronal cells express neurofilament-160.

14. The clonal population of cells according to claim 9, wherein said peripheral nervous system neuronal cells express neurofilament-200.

15. The clonal population of cells according to claim 9, wherein said peripheral nervous system glial cells express sulfatide.

16. The clonal population of cells according to claim 9, wherein said peripheral nervous system glial cells express myelin protein P_o.

17. A clonal population of genetically-engineered mammalian neural crest stem cells which are capable of self-renewal in a feeder cell-independent culture medium wherein said genetically engineered multipotent neural stem cells express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express neuronal lineage or glial lineage markers including glial fibrillary acidic protein (GFAP), sulfatide, myelin protein P_o, peripherin, high-polysialic acid neural cell adhesion molecule (PSA-NCAM), or neurofilament-160, wherein at least one of said stem cells of said clonal population is capable of differentiation to a peripheral nervous system neuronal cell that does not express LNGFR or nestin but does express peripherin, PSA-NCAM and neurofilament-160, and wherein at least one of said stem cells of said clonal population is capable of differentiation to a peripheral nervous system glial cell finer expresses LNGFR, nestin, GFAP and sulfatide.