Isolation and characterization of outer root sheath melanocytes of human hair follicles

Outer root sheath melanocytes (ORSM) are not yet routinely cultured and their biology is not known in detail because of their relatively low numbers in the hair follicle and their limited proliferative capacity in in vitro culture in routine media.

To develop a method for culturing ORSM more easily and to investigate the length of telomeres and antigenic characteristics of ORSM compared with epidermal melanocytes (EM).

Hair follicles were obtained from three Korean individuals during hair transplantation surgery. Single-cell suspensions of the outer root sheath were made and cultured in melanocyte growth medium with stem cell factor. After 21 days, second-passage outer root sheath keratinocytes (ORSK) (2 x 10(4) mL(-1) MGM) were added into the culture plates. We studied the proliferation pattern, morphological and antigenic characteristics of ORSM for each passage of cultured cells, and observed ORSM telomere length.

We established an ORSM culture method using ORSK. Two morphologically different ORSM types were obtained in the primary cultures. At the end of primary culture, ORSM appeared as whitish-cream pellets. The proliferation pattern of ORSM showed a sigmoidal shape, the accumulated numbers of population doublings showed a plateau after approximately 5 months, and senescence occurred at approximately 33 +/- 5 accumulated population doublings. The length of ORSM telomeres continued to shorten as the cells proliferated. In contrast, EM showed a marked proliferation from the early proliferation period which formed a plateau pattern towards the later period, and the number of accumulated population doublings was estimated to be 18 +/- 5 after 2 months. ORSM in the primary culture reacted variably with l-dihydroxyphenylalanine (DOPA): some cells were DOPA negative, some DOPA positive. There were some different antigenic expressions of microphthalmia-associated transcription factor (MITF) showing cytoplasmic expression in ORSM and nuclear expression in EM. By nuclear extraction and Western blotting, we showed that MITF expression of ORSM was marked in the cytoplasm and minimal in the nucleus. Antigenic expression of MITF and Bcl-2 gradually decreased with increasing passage number, whereas tyrosinase-related protein-1 expression did not change.

Culture of ORSM requires ORSK or ORSK-related factors; ORSM have greater proliferation potential and show different MITF antigenic expression compared with EM; and the length of ORSM telomeres shortens with repeated proliferation.