Methods for obtaining adult human olfactory progenitor cells

• US 7,838,292 B1
• Filed: 03/29/2002
• Issued: 11/23/2010
• Est. Priority Date: 03/29/2001
• Status: Expired due to Fees

• Current Assignee: University of Louisville
• Original Assignee: University of Louisville
• Inventors: Lu, Chengliang, Klueber, Kathleen M., Roisen, Fred J.
• Primary Examiner(s): Kolker; Daniel E
• Application Number:
  US10/112,658
• Time in Patent Office: 3,161 Days
• Field of Search: None
• 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: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

An isolated human olfactory stem cell can be prepared by culturing human tissue from olfactory neuroepithelium to form neurospheres.

Claims

1. A method for preparing cultured cells from human olfactory neuroepithelium, comprising:

dispersing adult human tissue comprising olfactory neuroepithelium to form a cell population comprising separated cells;

culturing said cell population in a culture medium as an adherent cell population to induce formation of a suspended cell population, wherein said suspended cell population comprises neurospheres, wherein said suspended cell population comprises one or more cells that are each immunoreactive for both nestin and beta-tubulin isotype III; and

subculturing said suspended cell population, wherein said subculturing comprises collecting said suspended cell population comprising neurospheres;

dispersing said neurospheres into separated cells; and

culturing said separated cells, wherein said suspended cell population doubles every 18-24 hours in Olfactory Epithelial Medium (OEM) at 37°

C. in an atmosphere of humidified 5% CO₂, wherein said OEM consists of 90% Minimal Essential Medium with Hanks'"'"' salts with L-glutamine and 10% FBS; and

detecting the presence of cells in the suspended cell population that are each immunoreactive for both nestin and beta-tubulin isotype III with antibodies or antigen-binding fragments thereof that bind nestin and beta tubulin isotype III.

2. The method of claim 1, wherein the adult human tissue is from a cadaver.

3. The method of claim 1, wherein the adult human tissue is collected from a living donor.

4. The method of claim 3, wherein the collecting is carried out by endoscopic removal.

5. The method of claim 1, wherein the subculturing is carried out for more than three weeks.

6. The method of claim 1, wherein the subculturing is carried out for more than six weeks.

7. The method of claim 1, wherein said suspended cell population is subculturable for at least 200 passages in said OEM at 37°

C. in an atmosphere of humidified 5% CO₂.

8. The method of claim 1, further comprising freezing the suspended cell population.

9. A method of treating a neurological disorder, comprising:

transplanting cells prepared from human olfactory neuroepithelium into a patient, wherein said cells are prepared by a method comprising the steps of;

collecting human olfactory neuroepithelium tissue from a living donor by endoscopic removal;

dispersing the human olfactory neuroepithelium tissue to form a cell population comprising separated cells;

culturing said cell population in a culture medium as an adherent cell population to induce formation of a suspended cell population, wherein said suspended cell population comprises neurospheres, wherein said suspended cell population comprises one or more cells that are each immunoreactive for both nestin and beta-tubulin isotype III; and

subculturing said suspended cell population, wherein said subculturing comprises collecting said suspended cell population comprising neurospheres;

dispersing said neurospheres into separated cells; and

culturing said separated cells, wherein said suspended cell population doubles every 18-24 hours in Olfactory Epithelial Medium (OEM) at 37°

C. in an atmosphere of humidified 5% CO₂, wherein said OEM consists of 90% Minimal Essential Medium with Hanks'"'"' salts with L-glutamine and 10% FBS; and

detecting the presence of cells in the suspended cell population that are each immunoreactive for both nestin and beta-tubulin isotype III with antibodies or antigen-binding fragments thereof that bind nestin and beta tubulin isotype III.

10. The method of claim 9, wherein the neurological disorder is selected from the group consisting of spinal cord injury, Parkinson'"'"'s disease, amyotrophic lateral sclerosis and multiple sclerosis.

11. The method of claim 9, wherein the patient has a spinal cord injury and the transplanting is into the site of the spinal cord injury.

12. The method of claim 9, wherein the living donor is the patient.

13. The method of claim 9, further comprising contacting the cells prepared from human olfactory neuroepithelium with a differentiation factor prior to said transplanting.

14. The method of claim 13, wherein the differentiation factor is a growth factor.