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the hairs in all species are important , not only from the aesthetic angle , but also as a protective barrier from environment .
human hair follicles stem cells ( hfscs ) are primarily cd200 + ( cluster of differentiation 200 positive ) keratinocyte stem cells . in close proximity to these , melanocyte stem cells ( melscs ) are located .
it is now well documented that melscs are located in the lower permanent portion of the hair follicle to replenish the pool of melanocytes and loss of melscs is thought to be responsible for hair graying.[36 ] herewith , we present data on comparison of cd200 + hfscs of keratinocyte lineage in pigmented and prematurely gray human hair follicle .
both gray and pigmented hair follicles were obtained using the follicular unit extraction method from three patients with premature graying of hair after taking their informed consent as recommended by the institutional ethics committee and institutional committee for stem cell research and therapy .
to obtain follicular units , 1-mm punch was rotated till mid - dermis in the direction of the hair follicle .
approximately 1020 pigmented and gray follicles were extracted per subject and collected separately in collection media containing dulbecco s modified eagle s medium ( dmem ) ( sigma , st .
louis , mo , usa ) , supplemented with penicillin , streptomycin and amphotericin - b ( gibco , gaithersburg , md , usa ) .
preparation of single cell suspension was carried out by a method described by us previously . for flow cytometry
, cells were labelled with pe ( phycoerythrin ) mouse anti - human cd200 antibody ( bd biosciences , ca , usa ) . marker for the negative population was set with the use of pe mouse igg1 antibody ( bd biosciences , ca , usa ) and unstained cell population .
after labelling , cells were acquired on a becton dickinson lsrii ( bd lsrii ) flow cytometer and analysed by bd facsdiva software version 6.1.2 . for each sample , at least duplicate acquisitions were performed . one explants / sq cm area with a total of five follicle of each group , gray and pigmented hair , were cultured according to the modified rheinwald - green system consisting of 3:1 , dmem and ham s f12 nutrient mix ( sigma , mo , usa ) , supplemented with 10% fetal bovine serum ( hyclone , nj , usa ) , 10 ng / ml epidermal growth factor ( peprotech , nj , usa ) , 2.8 g / ml hydrocortisone , 5 g / ml insulin , 10 g / ml transferrin ( sigma , mo , usa ) , 10 ng / ml cholera toxin ( millipore , ma , usa ) and 100 u / ml penicillin and 100 g / ml streptomycin ( gibco brl , gaithersburg , md , usa ) over fibronectin ( sigma , mo , usa ) coated culture dish ( bd falcon , md , usa ) .
for keratinocyte stemness , k19 and 1-integrin , ( millipore , ma , usa ) antibodies were used .
texas red ( bd biosciences , ca , usa ) conjugated secondary antibodies were used to develop fluorescence signal .
both gray and pigmented hair follicles were obtained using the follicular unit extraction method from three patients with premature graying of hair after taking their informed consent as recommended by the institutional ethics committee and institutional committee for stem cell research and therapy .
to obtain follicular units , 1-mm punch was rotated till mid - dermis in the direction of the hair follicle .
approximately 1020 pigmented and gray follicles were extracted per subject and collected separately in collection media containing dulbecco s modified eagle s medium ( dmem ) ( sigma , st .
louis , mo , usa ) , supplemented with penicillin , streptomycin and amphotericin - b ( gibco , gaithersburg , md , usa ) .
preparation of single cell suspension was carried out by a method described by us previously . for flow cytometry
, cells were labelled with pe ( phycoerythrin ) mouse anti - human cd200 antibody ( bd biosciences , ca , usa ) .
marker for the negative population was set with the use of pe mouse igg1 antibody ( bd biosciences , ca , usa ) and unstained cell population .
after labelling , cells were acquired on a becton dickinson lsrii ( bd lsrii ) flow cytometer and analysed by bd facsdiva software version 6.1.2 .
one explants / sq cm area with a total of five follicle of each group , gray and pigmented hair , were cultured according to the modified rheinwald - green system consisting of 3:1 , dmem and ham s f12 nutrient mix ( sigma , mo , usa ) , supplemented with 10% fetal bovine serum ( hyclone , nj , usa ) , 10 ng / ml epidermal growth factor ( peprotech , nj , usa ) , 2.8 g / ml hydrocortisone , 5 g / ml insulin , 10 g / ml transferrin ( sigma , mo , usa ) , 10 ng / ml cholera toxin ( millipore , ma , usa ) and 100 u / ml penicillin and 100 g / ml streptomycin ( gibco brl , gaithersburg , md , usa ) over fibronectin ( sigma , mo , usa ) coated culture dish ( bd falcon , md , usa ) . cells grown over the coverslips or cytospin preparations were taken for the immunofluoresence . for keratinocyte stemness , k19 and 1-integrin , ( millipore , ma , usa ) antibodies were used .
texas red ( bd biosciences , ca , usa ) conjugated secondary antibodies were used to develop fluorescence signal .
the mean  sd cd200 + hfscs population were 9.4  1.4% and 3.5  0.5% for pigmented and gray hair follicles , respectively ( p = 0.002 ) [ table 1 ] .
there was a marked visible difference in the spread of cell sheet in culture from the pigmented and gray hair follicle .
the growth of the cells from the gray hair follicle stopped at around day 20 - 22 , whereas the growth of the cells continued from the pigmented follicle . at day 35 , only few cells could be observed in the gray hair follicle culture .
a sheet of cells was observed from the pigmented follicle culture at day 35 [ figure 1a and b ] . because of poor cell number
percentage of ors cells from gray and pigmented hair follicles positive for hair follicle stem cell marker cd200 ( a ) patchy residual pigmentation on face , ( b ) after treatment with q - switched nd : yag laser immunofluorescence studies were carried out for the hfscs cultured from the pigmented hair follicle .
cells cultured from the pigmented hair follicle stained positive for hfscs markers k19 and 1 integrin antibody [ figure 1c and d ] .
hair graying is the most obvious sign of aging in humans , yet its mechanism is largely unknown . thumb rule of hair graying is that by 50 years of age , 50% of people have 50% gray hair .
analysis of melscs in mice at different ages has revealed that melscs population gets depleted with aging and this results in hair graying .
melscs give rise to the progenitor cells , which differentiate into pigment - containing melanocytes .
melanoblasts in the human hair follicle bulge area also show a similar decrease in number and ectopic differentiation in the bulge - subbulge area with aging .
melscs have direct contact with hfscs marker expressing keratinocytes in the bulge area . surrounding hfscs of keratinocyte lineage secrete transforming growth factor- , which is critical for the maintenance of melscs by promoting their quiescence and immaturity .
our findings suggest that cd200 + hfscs are depleted from outer root sheath ( ors ) of gray phenotype of the hair follicle , which may result in secondary loss of melanocyte stem cell pool .
the size of the cell sheet in culture was small in case of gray hair follicle as compared to pigmented follicle . due to fewer cells obtained after in vitro expansion of gray hair follicle cells
the depletion of hfscs pool could be the reason behind the small size of the colony obtained from the culture of the gray hair follicle .
however , the growth of the hair follicle is not affected in comparison to black follicle in real life . in in vivo condition
, there may be other niches of the hair follicle stem cells such as sebaceous gland and hair bulb , which might be contributing towards the hair growth .
the findings of our study are preliminary and carried out on a small sample size . nevertheless , the decrease in hfscs was clearly observed in grey hair follicle .
introduction : melanocyte stem cells ( melscs ) are known to be depleted in gray hair follicles . 
 hair follicle stem cells ( hfscs ) are important for maintenance of stemness of melscs.methods:we compared the proportion of cd200 + ( cluster of differentiation 200 positive ) stem cells in the outer root sheath cell suspension of gray and pigmented hair follicles of three patients with the premature graying of hair . in addition , explants culture for hfscs was also carried out from gray and pigmented hair follicles . 
 cultured hfscs were also differentiated into melanocytes.results:the mean  sd cd200 + hfscs population were 9.4  1.4% and 3.5  0.5% for pigmented and gray hair follicles , respectively ( p = 0.002 ) . in explants culture , the growth of hfscs from the gray hair follicle stopped at around day 2022 , whereas the growth of the cells from the pigmented follicle continued.conclusion:cd200 + hfscs are depleted in prematurely gray hair in the humans . 
 cd200 + hair follicle stem cell yield is poorer in gray hair explant culture than pigmented hair explant culture .