ISOLATION AND USE OF PLURIPOTENT STEM CELL POPULATION FROM ADULT NEURAL CREST-DERIVED TISSUES

• US 20150044180A1
• Filed: 03/01/2013
• Published: 02/12/2015
• Est. Priority Date: 03/01/2012
• Status: Active Grant

• Current Assignee: US Department of Veterans Affairs (Government of the United States of America), University of Miami
• Original Assignee: University of Miami
• Inventors: Cheung, Herman S., Pelaez, Daniel, Huang, C-Y Charles
• Granted Patent: US 10,106,773 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• 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 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/062 Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

The present invention relates to methods of isolating a substantially homogeneous population of pluripotent stem cells from adult neural crest tissue (e.g., periodontal ligament) as well as pharmaceutical compositions comprising such isolated pluripotent stem cells. Methods of inducing the isolated pluripotent stem cells into specific cell lineages, such as neurogenic and retinogenic lineages, are also described. The isolated pluripotent stem cells find use in various regenerative medicine applications and the treatment of degenerative diseases.

Claims

1. A method of isolating pluripotent stem cells comprising:

extracting cells from tissue derived from the neural crest;

culturing said extracted cells under adherent conditions; and

isolating said cultured cells that express Connexin-43, wherein said isolated cells are pluripotent stem cells.

2. The method of claim 1, further comprising screening said extracted cells for expression of Connexin-43 and/or at least one stem cell marker prior to isolation.

3. The method of claim 2, wherein said at least one stem cell marker is Oct4, Nanog, Sox2, Klf4, or combinations thereof.

4. The method of claim 1, further comprising screening said extracted cells for expression of at least one neural crest marker prior to isolation.

5. The method of claim 4, wherein said at least one neural crest marker is p75 neurotrophin receptor, Nestin, Sox10, N-Cadherin, Notch1, BMP2, Snail, Slug, or combinations thereof.

6. The method of claim 1, wherein said pluripotent stem cells are isolated by fluorescence activated cell sorting or magnetic bead cell sorting.

7. The method of claim 1, further comprising expanding said isolated cells in culture.

8. The method of claim 1, wherein said tissue derived from the neural crest is adult periodontal ligament.

9. The method of claim 1, wherein said isolated stem cells are capable of differentiating into cells of the ectoderm, endoderm, and mesoderm lineages.

10. The method of claim 9, wherein said isolated stem cells generate teratomas when injected into immunodeficient mice.

11. A population of pluripotent stem cells isolated by the method of claim 1.

20. A method of making a pharmaceutical composition comprising suspending the stem cell population of claim 11 or claim 12 in a pharmaceutically acceptable carrier.

21. A pharmaceutical composition made by the method of claim 20.

22. A method of repairing damaged tissue in a subject in need thereof comprising administering to said subject the stem cell population of claim 11 or claim 12.

23. The method of claim 22, wherein the stem cells are autologous.

24. The method of claim 22, wherein said damaged tissue is neural tissue, retinal tissue, skeletal muscle tissue, cardiac tissue, bone, or cartilage.

25. The method of claim 22, wherein said stem cell population is induced to differentiate into a specific tissue lineage prior to administration.

26. The method of claim 25, wherein said specific tissue lineage is a neural or retinal lineage.

12. A substantially pure population of human adult pluripotent stem cells, wherein said stem cells are positive for at least one stem cell marker, express Connexin-43, are capable of differentiating into cells of the ectoderm, endoderm, and mesoderm lineages, and are isolated from human tissue derived from the neural crest.

13. The population of claim 12, wherein said at least one stem cell marker is Oct4, Nanog, Sox2, Klf4, or combinations thereof.

14. The population of claim 13, wherein Oct4 is detected in the nucleus of said stem cells.

15. The purified population of claim 12, wherein said stem cells are positive for at least one neural crest marker.

16. The purified population of claim 15, wherein said neural crest marker is p75 neurotrophin receptor, Nestin, Sox10, N-Cadherin, Slug, or combinations thereof.

17. The purified population of claim 12, wherein said human tissue derived from the neural crest is adult human periodontal ligament.

18. The purified population of claim 12, wherein 80% or greater of said stem cells are positive for Oct4.

19. The purified population of claim 12, wherein 90% or greater of said stem cells are positive for Oct4.

27. A method of differentiating pluripotent stem cells isolated from adult periodontal ligament to neural progenitor cells or neural cells comprising:

incubating said pluripotent stem cells in a culture media comprising epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for a period of time, wherein a plurality of said pluripotent stem cells are differentiated into neural progenitor cells or neural cells at the end of the period of time.

28. The method of claim 27, wherein EGF is present in the media at a concentration of about 25 ng/ml to about 150 ng/ml.

29. The method of claim 28, wherein EGF is present in the media at a concentration of about 50 ng/ml.

30. The method of claim 27, wherein bFGF is present in the media at a concentration of about 25 ng/ml to about 150 ng/ml.

31. The method of claim 30, wherein bFGF is present in the media at a concentration of about 50 ng/ml.

32. The method of claim 27, wherein the period of time is about 4 to about 12 days.

33. The method of claim 32, wherein the period of time is about 8 days.

34. The method of claim 27, wherein a plurality of the neural progenitor cells or neural cells express one or more markers selected from nestin, β

-III tubulin (TUBB3), neurofilament medium (NEFM), SOX1, synaptophysin, and glial fibrillary acidic protein (GFAP).

35. The method of claim 27, wherein a plurality of the neural progenitor cells or neural cells exhibit a voltage-gated sodium current and/or a delayed, rectifying potassium current.

36. The method of claim 27, wherein the neural cells are neurons and/or glia.

37. The method of claim 27, wherein the pluripotent stem cells are human pluripotent stem cells.

38. A method of differentiating pluripotent stem cells isolated from adult periodontal ligament to retinal progenitor cells or retinal cells comprising:

culturing said pluripotent stem cells under non-adherent conditions in a first induction media comprising a first concentration of Noggin, a first concentration of Dkk-1, and optionally insulin-like growth factor-1 (IGF-1) for a first period of time to obtain neurospheres; and

plating the neurospheres on a substrate in a second induction media comprising a second concentration of Noggin and a second concentration of Dkk-1 and optionally IGF-1 for a second period of time, wherein a plurality of said pluripotent stem cells are differentiated into retinal progenitor cells or retinal cells at the end of the second period of time.

39. The method of claim 38, wherein the second concentration of Noggin is ten-fold higher than the first concentration of Noggin.

40. The method of claim 38, wherein the second concentration of Dkk-1 is ten-fold higher than the first concentration of Dkk-1.

41. The method of claim 38, wherein the first concentration of Noggin is about 1 ng/ml to about 10 ng/ml.

42. The method of claim 38, wherein the first concentration of Dkk-1 is about 1 ng/ml to about 10 ng/ml.

43. The method of claim 38, wherein IGF-1 is present in a concentration of about 5 ng/ml to about 50 ng/ml.

44. The method of claim 38, wherein the second concentration of Noggin is about 10 ng/ml to about 100 ng/ml.

45. The method of claim 38, wherein the second concentration of Dkk-1 is about 10 ng/ml to about 100 ng/ml.

46. The method of claim 38, wherein the first induction media comprises 1 ng/ml Noggin, 1 ng/ml Dkk-1, and 5 ng/ml IGF-1.

47. The method of claim 38, wherein the second induction media comprises 10 ng/ml Noggin and 10 ng/ml Dkk-1.

48. The method of claim 38, wherein the first period of time is about 3 days.

49. The method of claim 38, wherein the second period of time is about 7 days to about 50 days.

50. The method of claim 38, wherein the second period of time is at least about 25 days.

51. The method of claim 38, wherein the substrate is coated with matrigel.

52. The method of claim 38, wherein the neurospheres express one or more neuronal progenitor cell or neural markers selected from Nestin, p75 neurotrophin receptor, ABCG2, Pax6, and TUBB3.

53. The method of claim 38, wherein a plurality of the retinal progenitor cells or retinal cells express one or more eye field specification genes selected from Lhx2, DCX, Chx10, Rx, Sox2, and Otx2.

54. The method of claim 38, wherein a plurality of the retinal progenitor cells or retinal cells are Pax6^nuclear, Rx positive.

55. The method of claim 38, wherein a plurality of the retinal progenitor cells or retinal cells express Nrl, rhodopsin, and/or rhodopsin kinase.

56. The method of claim 38, wherein a plurality of the retinal progenitor cells or retinal cells exhibit an increase in intracellular calcium in response to glutamate stimulation.