Studies On The Location, Ultrastructure And Culture Of Melanocytes From Fetal Hair Follicles

Objective: To explore the location of melanocytes in fetal scalp and observe theirultrastructures under transmission electron microscope (TEM). To isolate and culturehuman follicle melanocytes （HFM）in vitro and then investigate the effects of threesynthetic oligonucleotides containing CpG motif (CpG-ODN) on tyrosinase activity andmorphology of the cultured melanocytes.This study will provide a novel measure andclue to treat vitiligo and partly illuminate the mechanisms of pigmentation afterinflammation.Methods: Scalp tissue was collected from the lifeless human embryos and fetuses fromsuction curettages (due to developmental abnormity). The specimens were provided bythe department of gynaecology and obstetrics in the first affiliated hospital of BengbuMedical college. The fetal age, which extended from120-180days, was estimated bythe crown-rump length, foot length, and menstrual age. The specimens were processedfor immunohistology, immunofluorescence, electron microscopy and culture in vitro,respectively.(1)Immunohistochemical study: the streptavidin-peroxidase method wasperformed. For staining using NKI/beteb or HMB45, hair samples without sectioning orcryostat sections were fixed with cold acetone, followed by blocking intrinsicperoxidase with3%H2O2. Samples were incubated with primary antibodies for1h atroom temperature, followed by a30-min incubation with biotinylated anti-mouseimmunoglobulins.(2) Immunofluorescence: all assays were performed on4μmfrozen sections. Samples were fixed in10%formalin then blocked with5%normalbovine serum albumin in TBS-T blocking solution. Sections were incubated in primaryantibody overnight at4°C and detected with FITC conjugated anti-rat. Primaryantibodies included: mouse anti-NKI/beteb, rat-anti-HMB-45, rat-anti-CD34.(3)Electron microscopy: Tissue samples were fixed for2-4hours in2.5%glutaraldehyde,then washed in buffer and postfixed in1%OsO4in distilled water for an additional hour,they were then flooded with1%aqueous uranyl acetate for1h to stain en bloc.Dehydration was carried out in a graded series of alcohols, transferred into propyleneoxide, and embedded in Epon. Thin sections were cut at70nm thickness, stained withuranyl acetate and lead citrate, and view in a JEM-1010transmission electron microscope.(4) Flow cytometry: Hair follicles in the remaining dermis were isolated bycollagenase Ⅱ treatment. The cells suspension from the hair follicles were prepared bytrypsin/ethylenediamine tetraacetic acid treatment, and accepted routine immunoassaydouble-labeled with CD34-PE/CD3-APC and CD34-PE/CD45-perCP, respectively,and were assayed by flow cytometry.(4) The culture in vitro: The follicle melanocyteswere isolated and cultured by two steps digestion of collagenase and trypsin. In order toinvestigate the effects of various CpG-ODN (CPG2006, CPG1668and CPG1826) ontyrosinase activity and morphology of the melanocytes from fetal scalp hair follicles,tyrosinase activity was measured by Dopa oxidation and cell morphology was detectedby silver nitrate staining.Results:(1) The positive cells stained by NKI/beteb antibody were observed in theouter root sheath (ORS) of hair follicle, and some in epidermal base part. HMB-45positive cells were present in the hair bulb, hair matrix and epidermal basilar part, butnot in ORS.(2) CD34positive cells mainly present in the outer root sheath bugle area ofhair follicle, a few were seen in the hair bulb and the basilar part of epidermis. Thefindings from the flow cytometry demonstrated that the CD34positive cells accountedfor8.54-13.04percentages of isolated follicular cells.(3) Melanocytes with clearcytoplasm and heterochromatic nuclei are located in the epidermis in touch with thebasement membrane. These cells consisted of various stages of melanosomes, most ofthem were immature. Melanosomes transferred to neighbouring keratinocytes wereevident. The melanocytes located in the developing hair follicle, e.g., in the ORS, hadfew dendrites, contained most immature melanosomes. In this study, we also observedthe melanosomes which presenting as degradative conglomerates within phagocyticvacuoles of the keratinocytes. Neither individual premelanosomes nor melanosomesoccurring free in the keratinocytes were observed.(4) The significant changes weredetected in cell morphology in group CpG2006and CpG1668, with big cell body,obvious pigmentation, and many dendrites.The melanocyte tyrosinase activity waspromoted significantly by CpG2006, CpG1826and CpG1668syntheticoligonucleotides.Conclusions:(1) There are melanocytes with different status in hair follicle. Themelanocytes at the bulges of ORS were immature. Those located in hair bulbs and. epidermal basilar part were mature.(2) Most CD34-positive cells localized at the bulgearea of hair follicle, which was presumed as the residence of follicle stem cells. CD34positive cells accounted for the8.54-13.04percentages of total isolated follicle cells.(3)The ultrastructure of melanocytes in fetal hair follicles is different from that in epidermis,the former showed more immature and maybe melanocytic stem cells.(4) Somesynthetic oligonucleotides containing CpG motif were able to promote tyrosinaseactivity and increase cell dendrite, in the future, could be used to treat vitiligo.