Mammalian neural crest stem cells

• US 5,589,376 A
• Filed: 08/15/1994
• Issued: 12/31/1996
• 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): Knode, Marian C.
• Assistant Examiner(s): DUFFY, PATRICIA ANN
• Application Number:
  US08/290,228
• Time in Patent Office: 869 Days
• Field of Search: 435/240.2, 435/240.21, 435/240.23, 435/29, 435/7.21, 435/240.1
• US Class Current: 435/325
• 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 methods for the isolation and clonal propagation of mammalian neural crest stem cells and isolated cellular compositions comprising the same. The methods employ a novel separation and culturing regimen and bioassays for establishing the generation of neural crest stem cell derivatives. These methods result in the production of non-transformed neural crest stem cells and their progeny. The invention demonstrates, at the clonal level, the self regeneration and asymmetrical division of mammalian neural crest stem cells for the first time in feeder cell-independent cultures. Lineage restriction is demonstrated within a developing clone and is shown to be sensitive to the local environment. Neural crest stem cells cultured on a mixed substrate of poly-D-lysine and fibronectin generate PNS neurons and glia, but on fibronectin alone the stem cells generate PNS glia but not neurons. The neurogenic potential of the stem cells, while not expressed, is maintained over time on fibronectin. The invention further includes transplantation assays which allow for the identification of mammalian neural crest stem cells from various tissues. It also includes methods for transplanting mammalian neural crest stem cells and/or neural or glial progenitors into mammals.

Claims

1. An isolated population of mammalian neural crest stem cells, wherein said cells are capable of self-renewal in a feeder cell-independent culture medium and capable of differentiation to peripheral nervous system neuronal or glial cells, wherein said neural crest stem cells express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express glial fibrillary acidic protein (GFAP), wherein at least one of said isolated 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 cell of said isolated population is capable of differentiation to a peripheral nervous system glial cell that expresses LNGFR, nestin and GFAP.

2. The population according to claim 1, wherein said neural crest stem cells do not express sulfatide, myelin protein P_o, peripherin, high-polysialic acid neural adhesion molecule (PSA-NCAM) or neurofilament.

3. The population according to claim 1, wherein said peripheral nervous system neuronal cells express peripherin.

4. The population according to claim 1, wherein said peripheral nervous system neuronal cells express PSA-NCAM.

5. The population according to claim 1, wherein said peripheral nervous system neuronal cells express neurofilament-68.

6. The population according to claim 1, wherein said peripheral nervous system neuronal cells express neurofilament-160.

7. The population according to claim 1, wherein said peripheral nervous system neuronal cells express neurofilament-200.

8. The population according to claim 1, wherein said peripheral nervous system neuronal cells express sulfatide.

9. The population according to claim 1, wherein said peripheral nervous system neuronal cells express myelin protein P_o.

10. An isolated population of mammalian neural crest stem cells capable of self-renewal in a feeder cell-independent culture medium and capable of differentiation to peripheral nervous system neuronal or glial cells, wherein said neural crest stem cells express low-affinity nerve growth factor receptor (LNGFR) and nestin, but do not express glial fibrillary acidic protein (GFAP), sulfatide, O₄, peripherin, high-polysialic acid neural adhesion molecule (PSA-NCAM), or neurofilament-160, wherein at least one of said isolated 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 cell of said isolated population is capable of differentiation to a peripheral nervous system glial cell that expresses LNGFR, nestin and GFAP, and sulfatide.