Stable neural stem cell lines

• US 9,465,025 B2
• Filed: 04/15/2009
• Issued: 10/11/2016
• Est. Priority Date: 05/23/1996
• Status: Expired due to Fees

• Current Assignee: Neuralstem Incorporated (City of Tianjin (China))
• Original Assignee: Neuralstem Incorporated (City of Tianjin (China))
• Inventors: Yang, Renji, Johe, Karl K.
• Primary Examiner(s): HAYES, ROBERT CLINTON
• Application Number:
  US12/424,238
• Publication Number:
  US 20090263901A1
• Time in Patent Office: 2,736 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/148 Transforming growth factor alpha [TGF-a]
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/16 Animal cells
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 : Stem cells
• CPC Class Codes: G01N 33/9413 : Dopamine

Abstract

A systematic and efficient method for establishing stable neural stem cell lines and neuronal progenitor lines is described. The resulting cell lines provide robust, simple, and reproducible cultures of human and other mammalian neurons in commercially useful mass quantities while maintaining normal karyotypes and normal neuronal phenotypes.

Claims

1. A method for producing a stable cell line of human neural precursor cells in vitro, comprising the steps of:

a) preparing a culture of neural precursor cells in a serum-free medium;

b) culturing the neural precursor cells in the presence of a first mitogen, wherein said first mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα and

combinations thereof;

c) introducing into the neural precursor cells a recombinant DNA construct comprising a receptor ligand-regulated c-myc gene, wherein the c-myc gene is fused with DNA encoding a ligand-binding domain of a nuclear receptor;

d) contacting the neural precursor cells with an agent that binds the ligand-binding domain of the nuclear receptor, wherein the agent comprises a myc-activating chemical selected from the group consisting of β

-estradiol, RU38486, dexamethasone, thyroid hormones, retinoids, and ecdysone;

e) further culturing the neural precursor cells in a medium containing the first mitogen and a second mitogen to produce a stable cell line of neural precursor cells, wherein said second mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα

, serum and combinations thereof, with the proviso that the second mitogen is other than the first mitogen, and

f) expanding the neural precursor cells of the stable cell line beyond thirty cell doublings in the medium containing the first mitogen and the second mitogen,

wherein the expanded neural precursor cells of the stable cell line of neural precursor cells are capable of differentiating into neurons, astrocytes, and oligodendrocytes after removal of the first and second mitogen from the medium.

2. The method of claim 1, wherein the c-myc gene is fused with a DNA sequence coding for a ligand-binding domain of a nuclear receptor selected from the group consisting of a ligand binding domain for an estrogen receptor, an androgen receptor, a progesterone receptor, a glucocorticoid receptor, a thyroid hormone receptor, a retinoid receptor, and an ecdysone receptor.

3. The method of claim 1, wherein the human neural precursor cells are derived from an in vitro culture of pluripotent embryonic stem cells.

4. A cell line produced according to the method of claim 1.

5. The cell line of claim 4, wherein the neural precursor cells of the produced stable cell line maintain a multipotential capacity beyond thirty cell doublings to differentiate into neurons, astrocytes and oligodendrocytes.

6. The cell line of claim 4, wherein the neural precursor cells of the produced stable cell line maintain a bipotential capacity beyond thirty cell doublings to differentiate into neurons and astrocytes.

7. The cell line of claim 4, wherein the neural precursor cells of the produced stable cell line maintain a bipotential capacity beyond thirty cell doublings to differentiate into astrocytes and oligodendrocytes.

8. The cell line of claim 4, wherein the neural precursor cells of the produced stable cell line maintain a capacity beyond thirty cell doublings to differentiate into neurons.

9. The cell line of claim 4, wherein the neural precursor cells of the produced stable cell line maintain a capacity beyond thirty cell doublings to differentiate into astrocytes.

19. The cell line of claim 4, wherein the neural precursor cells of the produced stable cell line maintain a capacity beyond thirty cell doublings to differentiate into oligodendrocytes.

10. A method for producing a stable human neural precursor cells clone in vitro, comprising the steps of:

a) preparing a culture of neural precursor cells in a serum-free medium;

b) culturing the neural precursor cells in the presence of a first mitogen, wherein said first mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα and

combinations thereof;

c) introducing into the neural precursor cells a recombinant DNA construct comprising a retroviral vector containing the neomycin-resistance gene and a fusion gene of human c-myc cDNA and human estrogen receptor cDNA;

d) further culturing the neural precursor cells in a medium containing the first mitogen and a second mitogen, wherein said second mitogen is selected from the group consisting of aFGF, bFGF, EGF, TGFα

, serum and combinations thereof, with the proviso that the second mitogen is other than the first mitogen, wherein the medium includes G418 to select for the neural precursor cells containing the recombinant DNA construct, wherein the medium includes β

-estradiol;

e) plating the neural precursor cells obtained from step d) at a cell density of 0.5×

10⁶ to 1.0×

10⁶ cells per 100 mm plate by supplementing the neural precursor cells with unmodified primary cells in the continued presence of G418 in a medium containing the first mitogen and the second mitogen, and

f) expanding the neural precursor cells of the stable cell line beyond thirty cell doublings in the continued presence of G418 in the medium containing the first mitogen and the second mitogen, and

g) isolating a stable neural precursor clone from the plated neural precursor cells.

11. The method of claim 10, wherein the human neural precursor cells are derived from an in vitro culture of pluripotent embryonic stem cells.

12. A cell line produced according to the method of claim 10.

13. The cell line of claim 12, wherein the neural precursor cells of the produced stable cell line maintain a multipotential capacity beyond thirty cell doublings to differentiate into neurons, astrocytes and oligodendrocytes.

14. The cell line of claim 12, wherein the neural precursor cells of the produced stable cell line maintain a bipotential capacity beyond thirty cell doublings to differentiate into neurons and astrocytes.

15. The cell line of claim 12, wherein the neural precursor cells of the produced stable cell line maintain a bipotential capacity beyond thirty cell doublings to differentiate into astrocytes and oligodendrocytes.

16. The cell line of claim 12, wherein the neural precursor cells of the produced stable cell line maintain a capacity beyond thirty cell doublings to differentiate into neurons.

17. The cell line of claim 12, wherein the neural precursor cells of the produced stable cell line maintain a capacity beyond thirty cell doublings to differentiate into astrocytes.

18. The cell line of claim 12, wherein the neural precursor cells of the produced stable cell line maintain a capacity beyond thirty cell doublings to differentiate into oligodendrocytes.