In vitro generation of differentiated neurons from cultures of mammalian multipotential CNS stem cells

• US 6,040,180 A
• Filed: 05/07/1997
• Issued: 03/21/2000
• Est. Priority Date: 05/23/1996
• Status: Expired due to Term

• Current Assignee: Neuralstem Incorporated (City of Tianjin (China))
• Original Assignee: NeuralStem Biopharmaceuticals, Ltd.
• Inventors: Johe, Karl K.
• Primary Examiner(s): Allen, Marianne P.
• Application Number:
  US08/919,580
• Time in Patent Office: 1,049 Days
• Field of Search: 435/325, 435/375, 435/377, 435/347, 435/352, 435/363, 435/366, 435/368
• US Class Current: 435/377
• 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: 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 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0623 Stem 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

The present invention reveals in vitro cultures of region-specific, terminally differentiated, mature neurons derived from cultures of mammalian multipotential CNS stem cells and an in vitro procedure by which the differentiated neurons may be generated. The procedure involves the culturing of multipotential CNS stem cells from a specific region in a chemically defined serum-free culture medium containing a growth factor; replacing the medium with growth factor-free medium; harvesting the stem cells by trypsinization; plating the stem cells at a density of between 100,000 to 250,000 cells per square centimeter; and culturing the stem cells in a glutamic acid-free chemically defined serum-free culture medium.

Claims

1. A method for in vitro generation of neurons, astrocytes and oligodendrocytes from an adhesion culture of mammalian multipotential CNS stem cells, wherein the mammalian multipotential CNS stem cells maintain the multipotential capacity to differentiate into neurons, astrocytes and oligodendrocytes, divide in serum-free medium supplemented with a mitogen and differentiate upon withdrawal of mitogen, comprising the steps of:

a) culturing multipotential CNS stem cells in a chemically defined serum-free culture medium containing a growth factor by;

1) dissociating cells from central nervous system tissue by mechanical trituration in the absence of divalent cations,

2) culturing the dissociated cells adhered onto a plate in a chemically defined serum-free culture medium,

3) plating dissociated cells at a density not exceeding 20,000 cells/cm² and, in subsequent passages, replating the cultured cells at a density not exceeding 10,000 cells/cm²,

4) adding daily to the cultured cells a growth factor selected from the group consisting of

i) bFGF at a concentration of at least 10 ng/ml,

ii) EGF at a concentration of at least 10 ng/ml,

iii) TGF-alpha at a concentration of at least 10 ng/ml, and

iv) aFGF at a concentration of at least 10 ng/ml plus 1 μ

g/ml heparin,

5) replacing the culture medium with fresh medium within every two days,

6) passaging the cultured cells within four days after plating so as not to exceed 50% confluence, and

7) passaging the cultured cells by treating the cultured cells with saline solution free of divalent cations and scraping cells from the plate;

b) replacing the medium with growth factor-free, serum-free medium;

c) harvesting the stem cells;

d) plating the stem cells at a density of between 100,000 to 250,000 cells per square centimeter; and

e) culturing the cells in a glutamic acid-free chemically defined serum-free culture medium.

2. The method of claim 1, wherein said mammalian multipotential CNS stem cells are isolated from fetal central nervous system tissue selected from the group consisting of cortex, olfactory tubercle, retina, septum, lateral ganglionic eminence, medial ganglionic eminence, amygdala, hippocampus, thalamus, hypothalamus, ventral and dorsal mesencephalon, brain stem, cerebellum, and spinal cord.

3. The method of claim 1 wherein said chemically defined serum-free culture medium of step a) or step e) is N2 or N2-modified media.

4. The method of claim 1 wherein the glutamic acid-free chemically defined serum-free culture medium of step e) is supplemented with between 10-100 ng/ml of brain-derived neurotrophic factor.

5. The method of claim 1, wherein the mammalian multipotential CNS stem cells are derived from central nervous system tissue from a rat.

6. The method of claim 1, wherein the mammalian multipotential CNS stem cells are derived from central nervous system tissue from a human.