Methods for preparation of human hair-follicle derived multipotent adult stem cells

• US 8,999,706 B2
• Filed: 04/12/2006
• Issued: 04/07/2015
• Est. Priority Date: 04/12/2005
• Status: Active Grant

• Current Assignee: The Wistar Institute
• Original Assignee: The Wistar Institute, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Xu, Xiaowei, Fang, Douglas Dong, Herlyn, Meenhard, Yu, Hong
• Primary Examiner(s): Wilson, Michael C.
• Application Number:
  US11/911,400
• Publication Number:
  US 20090130065A1
• Time in Patent Office: 3,282 Days
• Field of Search: 435/325, 435/455
• US Class Current: 435/325
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• 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 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0628 Hair stem cells; Hair progenitors mesenchymal stem cells from hair follicles C12N5/0666

Abstract

Isolated human multipotent adult stem cell and isolated populations of cells that include human multipotent adult stem cells are disclosed. Human hair-follicle derived multipotent adult stem cells and methods of preparing isolated populations of cells that include human multipotent adult stem cells are disclosed. Isolated human hair-follicle derived multipotent adult stem cell that can differentiate in culture into a neuronal cell, a glial cell, a melanocyte cell, a muscle cell, an osteocyte, a chondrocyte, and a lymphocyte. Isolated human hair-follicle derived multipotent adult stem cell that can grow in cell culture in spheres are disclosed. Human pancreas derived multipotent adult stem cells, human liver derived multipotent adult stem cells, human kidney derived multipotent adult stem cells, human heart derived multipotent adult stem cells, human neural derived multipotent adult stem cells and methods of preparing isolated populations of cells that include such human multipotent adult stem cells are disclosed. Method of treating an individual who has diabetes, cardiac muscle damage, muscle damage and disease, neurodegenerative disease or nerve damage or injury, bone loss, damage and/or disease, cartilage loss, damage and/or disease, hair loss and immune disorders, are disclosed.

Claims

1. A method of preparing an isolated population of human hair-follicle derived multipotent adult stem cells comprising the steps of:

a) incubating a human scalp sample with dispase to free hair follicles from dermal tissue;

b) washing the free hair follicles of a);

c) treating the follicles of b) with trypsin such that follicular epithelium is dissociated from hair shaft

d) obtaining a single cell suspension from the dissociated follicular epithelium treated in step c;

e) culturing said single cells in conditioned human embryonic stem cell (hESC) media wherein said hESC media is selected from the group consisting of

i) DMEM/F-12 at 3 parts conditioned DMEM/F-12 medium to 1 part fresh medium, 20 ng/ml EGF and 40 ng/ml bFGF or

ii) hESC medium consisting of about 80% Knockout DMEM/F-12 medium, about 20% Knockout serum replacer, about 200 mM L-glutamine, about 0.1 mM β

-mercaptoethanol, about 1% non-essential amino acids, and about 4 ng/ml bFGF,

said hESC media being conditioned via use as growth medium for mouse embryonic fibroblasts and being mixed with fresh hESC medium at about 3 parts conditioned hESC to 1 part fresh hESC, and supplemented with additional bFGF at about 4 ng/ml, such that hairspheres consisting of adhesive aggregates form from said single cells, wherein cells isolated from said hair spheres so cultured are multipotent stem cells which express Oct4 and Nanog but lack expression of SSEA-3 and SSEA-4 and

(f) isolating said multipotent adult stem cells.

2. A method of preparing an isolated population of human hair-follicle derived multipotent adult stem cells comprising the steps of:

a) incubating a human scalp sample with dispase to free hair follicles from dermal tissue;

b) washing the free hair follicles of a);

c) treating said hair follicles with trypsin, thereby dissociating follicular epithelium from hair shaft;

d) obtaining a single cell suspension from the dissociated epithelium of step b);

e) culturing said single cells in minimal serum free media supplemented with human basic fibroblast growth factor and human epidermal growth factor, such that hairspheres consisting of adhesive aggregates form from said single cells, wherein cells isolated from said hairspheres are multipotent stem cells which express Oct4 and Nanog but lack expression of SSEA-3 and SSEA-4.