In the initial phase of the study will be based on scalp sample obtained from cadavers kept in the department anatomy. Tissue samples will be processed routinely for H&E and trichrome stained sections. The volume densities of the epidermis, dermis, and hair follicles will be obtained by point counting on histological section. The volume densities of the HHFSCs, melanocytes, keratinocytes, Langerhans cells, and Merkel cells will be depicted utilizing immunohistochemically stained sections utilizing specific markers for HHFSCs and the functional cells.

 

In the 2nd phase fresh scalp samples will be obtained from volunteers under informed consent by a dermatologist. Samples will be transported appropriately in an ice-cold transport medium to college labs where excess fat is trimmed off, and the sample diced into small pieces. The diced samples will be subjected to enzymatic dissociation in 12.5 mg/ml dispose (Invitrogen, Carlsbad, CA) in Dulbecco’s modified Eagle’s medium (DMEM) for 24 hours at 4°C. After treatment, the epidermis will be peeled off from the dermis, and hair follicles plucked from the dermis. Hair follicles will then be rinsed thoroughly with phosphate-buffered saline to prevent contaminating epidermal or dermal cells and will be examined under an inverted microscope. To confirm the integrity of plucked hair follicles, some of the hair follicles will be fixed in 10% buffered formalin, embedded in paraffin, and processed for H&E sections.

 

In the 3rd phase cell separation from the follicle will be done by enzymatic (collagenase, dispose, trypsin) digestion, filtering and centrifugation. The cells will be cultured in the HHFSC culture medium (Human Hair Follicle Stem Cell Complete Media with Serum) and checked by the growth curve and flow cytometry to analyze cell populations based on specific marker expression to quantify the differentiated cell. HHFSCs will be seeded into the non-cell-adhesive microwell array device at different cell densities and checked with a phase contrast microscope and colony forming assay and confirmed by RT-PCR.

In phase 4 the HHFSCs will be induced to differentiate into functional cells by culturing them in culture media containing growth factors and signaling molecules specific to keratinocytes and melanocytes. For differentiation into keratinocytes epidermal growth factor or keratinocyte growth factor can be used. Melanocyte differentiation will be achieved by using a melanocyte differentiation medium.

 

In the 5th phase, skin wound healing animal models will be used to evaluate the capability of HHFSCS in promoting skin wound healing. The healing process will be assessed histologically and electron microscopically. Emphasis will be on histomorphometric analysis of acute epidermal wound healing and wound closure.