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

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.