Methods for isolating early neurons and neuroblasts

• US 9,249,389 B2
• Filed: 09/26/2013
• Issued: 02/02/2016
• Est. Priority Date: 06/17/2008
• Status: Active Grant

• Current Assignee: Mclean Hospital (Mass General Brigham, Inc.)
• Original Assignee: Mclean Hospital (Mass General Brigham, Inc.)
• Inventors: Isacson, Ole, Pruszak, Jan
• Primary Examiner(s): Kolker, Daniel E
• Assistant Examiner(s): MACFARLANE, STACEY NEE
• Application Number:
  US14/037,475
• Publication Number:
  US 20140087464A1
• Time in Patent Office: 859 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61B 17/1695 Trepans or craniotomes, i.e. specially adapted for drilling thin bones such as the skull
• 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: A61K 35/54 Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
• CPC Class Codes: A61K 9/0085 Brain, e.g. brain implants; Spinal cord
• CPC Class Codes: A61K 9/5068 Cell membranes or bacterial membranes enclosing drugs with additional exogenous lipids A61K9/127; virus envelopes A61K9/5184
• CPC Class Codes: A61M 5/178 Syringes
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

The inventions disclosed herein are based on the identification of novel cell populations derived from human embryonic stem cells and other pluripotent cells. The inventive cell populations may be used for cell therapies for the treatment of various neurological diseases and as substrates in pharmacological assays.

Claims

1. A method for isolating early neurons and neuroblasts of human origin from in vitro culture comprising:

(i) culturing human pluripotent neural cells in the presence of a growth factor combination comprising sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8), basic fibroblast growth factor (bFGF), and brain-derived neurotrophic factor (BDNF) that induces at least some of said pluripotent cells to increase expression of TuJ1 relative to pluripotent cells cultured in the absence of said growth factor combination;

(ii) further culturing the cells cultured under the growth factor combination under a second growth factor combination comprising SHH, FGF-8, and BDNF and not bFGF; and

(iii) selecting the cells obtained from step (i) that express high levels of CD24, wherein said subpopulation is identified as early neurons and neuroblasts.

2. The method of claim 1 further comprising a step of removing CD15⁺ cells from said subpopulation by subjecting the cells from said subpopulation to fluorescent assisted cell sorting (FACS).

3. The method of claim 2 further comprising a step of removing cells expressing high levels of CD29 by subjecting the cells from said subpopulation to fluorescent assisted cell sorting (FACS).

4. The method of claim 1, wherein the cells obtained in step (iii) represent not more 75% of the CD15⁻

cells obtained from step (i).

5. The method of claim 1, wherein the cells obtained from step (iii) represent not more 25% of the CD15⁻

cells from the cells obtained from step (i).

6. The method of claim 1, further comprising the step of

culturing the cells culture under the second growth factor combination under a third growth factor combination consisting of FGF-8 and BDNF and not bFGF or SHH.

7. A method for isolating human neural crest cells from in vitro culture comprising:

(i) culturing human pluripotent neural cells in the presence of a growth factor combination sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8), basic fibroblast growth factor (bFGF), and brain-derived neurotrophic factor (BDNF) that induces at least some of said pluripotent cells to increase expression of TuJ1 relative to pluripotent cells cultured in the absence of said growth factor combination;

(ii) further culturing the cells cultured under the growth factor combination under a second growth factor combination comprising SHH, FGF-8, and BDNF and not bFGF;

(iii) removing CD 15⁺ cells from the cells obtained from step (ii) by fluorescent assisted cell sorting (FACS); and

(iv) selecting cells that express relatively high levels of CD29 from the cells obtained from step (ii),

wherein the cells obtained following steps (iii) and (iv) are identified as neural crest cells.

8. The method of claim 7, wherein the cells obtained from step (iv) represent not more than 75% of the CD15⁻

cells obtained from step (i).

9. The method of claim 7, wherein the cells obtained from step (iv) represent not more than 25% of the CD15⁻

cells obtained from step (i).

10. The method of claim 7, wherein said method further comprises selecting cells that express low levels of CD24.

11. A method for isolating human neural precursor cells from in vitro culture comprising:

(i) culturing human pluripotent neural cells in the presence of a growth factor combination consisting of sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8), basic fibroblast growth factor (bFGF), and brain-derived neurotrophic factor (BDNF) that induces at least some of said pluripotent cells to increase expression of TuJ1 relative to pluripotent cells cultured in the absence of said growth factor combination;

(ii) further culturing the cells cultured under the growth factor combination under a second growth factor combination comprising SHH, FGF-8, and BDNF and not bFGF,

(iii) removing CD15⁻

cells from obtained from step (ii); and

(iv) selecting cells that express high levels of CD29 from the cells obtained from step (ii),

wherein the cells obtained following steps (iii) and (iv) are identified as early neurons and neuroblasts.

12. The method of claim 11, wherein the cells obtained from step (iv) represent not more than 75% of the CD15⁺ cells from said population cultured in step (i).

13. The method of claim 11, wherein the cells obtained from step (iv) represent not more than 25% of the CD15⁺ cells from said population cultured in step (i).

14. The method of claim 11, wherein said method further comprises selecting cells that express low levels of CD24.