MULTIPOTENT NEURAL CELLS

• US 20110135696A1
• Filed: 06/16/2009
• Published: 06/09/2011
• Est. Priority Date: 06/17/2008
• Status: Abandoned Application

• Current Assignee: Mclean Hospital (Mass General Brigham, Inc.)
• Original Assignee: Mclean Hospital (Mass General Brigham, Inc.)
• Inventors: Isacson, Ole, Pruszak, Jan
• Application Number:
  US12/995,988
• Publication Number:
  US 20110135696A1
• Time in Patent Office: Days
• US Class Current: 424/400
• 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 substantially homogenous population of cells, wherein the cells of said population are characterized as CD24^HI and express at least one marker selected from the group consisting of TuJ1, MAP2, and doublecortin.

2. The population of claim 1, wherein said cells are further characterized as CD29^LO.

3. The population of claim 2, wherein said cells are further characterized as CD15⁻

.

4. The population of claim 3, wherein said cells further express tyrosine hydroxylase.

5. The population of claim 4, wherein said cells adopt a neuronal morphology when cultured in the presence of at least one growth factor.

6. The population of claim 5, wherein said growth factor is selected from the group consisting of fibroblast growth factor 8 (FGF8), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), transforming growth factor type beta-3 (TGF-β

3), and glial cell line-derived neurotrophic factor (GDNF).

7. The population of claim 5, wherein said cells are characterized as CD15⁻

/CD24^HI/CD29^LO.

11. A therapeutic composition comprising a population of claim 1.

12. The therapeutic composition of claim 11, wherein said population of cells is suspended in a physiologically compatible solution.

13. The therapeutic composition of claim 11, wherein said population of cells is encapsulated.

14. A method for treating a neurodegenerative disease in a patient, comprising administering to the brain of said patient a therapeutic composition of claim 11.

8. A substantially homogenous population of cells, wherein the cells of said population are characterized as CD24^LO and CD29^HI.

9. The population of any one of claim 8, wherein said cells are further characterized as CD15⁻

.

10. The population of any one of claim 8, wherein said cells are further characterized as CD15⁺.

15. A method for isolating early neurons and neuroblasts from in vitro culture comprising:

(i) culturing pluripotent cells in the presence of a growth factor 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; and

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

16. The method of claim 15, wherein said growth factor is selected from the group consisting of sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8), basic fibroblast growth factor (bFGF), and brain-derived neurotrophic factor (BDNF).

17. The method of claim 16, wherein CD15⁺ cells are removed from said subpopulation.

18. The method of claim 17, wherein cells expressing relatively high levels of CD29 are removed.

19. The method of claim 15, wherein the cells obtained in step (ii) represent not more 75% of the CD15⁻

cells obtained from step (i).

20. The method of claim 15, wherein the cells obtained from step (ii) represent not more 25% of the CD15⁻

cells from the cells obtained from step (i).

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

(i) culturing pluripotent cells in the presence of a growth factor 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;

(ii) removing CD15⁺ cells from the cells obtained from step (i); and

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

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

22. The method of claim 21, wherein said growth factor is selected from the group consisting of sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8), basic fibroblast growth factor (bFGF), and brain-derived neurotrophic factor (BDNF).

23. The method of claim 21, wherein the cells obtained from step (iii) represent not more than 75% of the CD15⁻

cells obtained from step (i).

24. The method of claim 21, wherein the cells obtained from step (iii) represent not more than 25% of the CD15⁻

cells obtained from step (i).

25. The method of claim 21, wherein said method further comprises selecting cells that express relatively low levels of CD24.

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

(i) culturing pluripotent cells in the presence of a growth factor 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;

(ii) removing CD15⁻

cells from obtained from step (i); and

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

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

27. The method of claim 26, wherein said growth factor is selected from the group consisting of sonic hedgehog (SHH), fibroblast growth factor-8 (FGF-8), basic fibroblast growth factor (bFGF), and brain-derived neurotrophic factor (BDNF).

28. The method of claim 26, wherein the cells obtained from step (iii) represent not more than 75% of the CD15⁺ cells from said population cultured in step (i).

29. The method of claim 26, wherein the cells obtained from step (iii) represent not more than 25% of the CD15⁺ cells from said population cultured in step (i).

30. The method of claim 26, wherein said method further comprises selecting cells that express relatively low levels of CD24.

31. A method for preparing substantially homogenous populations of cells having a stellate morphology, comprising:

(i) culturing in vitro cells having a stellate morphology;

(ii) preparing a fluid composition containing detached cells obtained from the culture of step (i); and

(iii) selecting said cells by flow cytometry to generate substantially homogenous cell populations, wherein the flow cytometer fluid pressure is less than about 50 psi and the fluid ejection nozzle has a diameter of greater than about 75 μ

m.

32. The method of claim 31, wherein said fluid pressure is between about 20 psi and about 50 psi.

33. The method of claim 31, wherein said ejection nozzle has a diameter of between about 75 μ

m and about 125 μ

m.

34. The method of claim 31, wherein said cells having a stellate morphology are neurons or neuronal precursor cells.