Isolation and use of pluripotent stem cell population from adult neural crest-derived tissues

• US 10,106,773 B2
• Filed: 03/01/2013
• Issued: 10/23/2018
• 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
• Primary Examiner(s): Wehbe, Anne Marie S
• Application Number:
  US14/382,287
• Publication Number:
  US 20150044180A1
• Time in Patent Office: 2,062 Days
• Field of Search: 435368, 435378, 435 71, 435 721, 424 937
• 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, wherein the tissue derived from the neural crest is adult periodontal ligament or dental pulp from exfoliated deciduous teeth;

culturing said extracted cells under adherent conditions; and

isolating said cultured cells that express Connexin-43, wherein said isolating is

performed using a reagent with an affinity for said Connexin-43 to obtain a population of said cultured cells that express Connexin-43, wherein said population comprises at least 80% pluripotent stem cells.

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

3. The method of claim 2, wherein said at least one stem cell marker or neural crest marker is Oct4, Nanog, Sox2, Klf4, p75 neurotrophin receptor, Nestin, Sox10, N-Cadherin, Notch1, BMP2, Snail, Slug, or combinations thereof.

4. The method of claim 1, wherein said isolated stem cells are capable of differentiating into cells of the ectoderm, endoderm, and mesoderm lineages and generating teratomas when injected into immunodeficient mice.

5. A method of repairing damaged tissue in a subject in need thereof comprising administering to said subject pluripotent stem cells isolated using the method of claim 1.

6. The method of claim 5, wherein the stem cells are autologous.

7. The method of claim 5, wherein said damaged tissue is neural tissue, retinal tissue, skeletal muscle tissue, cardiac tissue, bone or cartilage.